EA043828B1 - ANTISENSE OLIGONUCLEOTIDES TARGETED AT ALPHA-SYNUCLEIN AND THEIR APPLICATION - Google Patents

Description

Link to the list of sequences presented in electronic form

Contents of the th2019e sequence listing presented in electronic form (name:

3338_107PC01_SequenceListing_ST25.txt, size: 10458 bytes; and date of creation: January 10, 2019) presented herein is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present invention provides an antisense oligomeric compound (ASO) that targets the junction of intron 1 and exon 2 of the alpha-synuclein transcript (SNCA) in a cell, resulting in decreased expression of alpha-synuclein protein (SNCA). Reducing SNCA protein expression may be beneficial for a range of diseases, such as multiple system atrophy, Parkinson's disease, Parkinson's disease dementia (PDD), and Lewy body dementia.

Prerequisites for creating the invention

Alpha-synuclein (SNCA), a member of the synuclein family of proteins, is a small soluble protein that is expressed predominantly in neural tissues. See Marques O. et al., Cell Death Dis., 19: e350 (2012). It is expressed in many cell types but is predominantly localized to the presynaptic terminals of neurons. Although the exact function has not yet been fully elucidated, it has been proposed that SNCA plays an important role in regulating synaptic transmission. For example, SNCA acts as a molecular chaperone in the formation of SNARE complexes that mediate docking of synaptic vesicles to the presynaptic membranes of neurons. SNCA can also interact with other proteins, such as microtubule-associated protein tau, which helps stabilize microtubules and regulate vesicular transport.

Because of SNCA's role in regulating synaptic transmission, changes in SNCA expression and/or function can disrupt important biological processes. Such disorders are thought to contribute to α-synucleinopathies, which are neurodegenerative diseases characterized by the abnormal accumulation of SNCA protein aggregates in the brain.

Accordingly, insoluble inclusions of misfolded, aggregated, and phosphorylated SNCA protein are a pathological feature of diseases such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). See Galvin J.E. et al., Archives of Neurology, 58: 186-190 (2001); and Valera E. et al., J. Neurochem., 139 Suppl 1: 346-352 (Oct. 2016).

α-Synucleinopathies, such as Parkinson's disease, are common progressive neurodegenerative diseases of the brain, especially among the elderly. See Recchia A. et al., FASEB J., 18: 617-26 (2004). An estimated 7-10 million people worldwide live with these disorders, with approximately 60,000 new cases reported each year in the United States alone. Treatment costs for an individual can easily exceed $2,500 per year, and therapeutic surgery can cost up to $100,000 per patient. Thus, more reliable and cost-effective treatment options are urgently needed.

Brief description of the invention

The present invention relates to an antisense oligonucleotide (ASO) containing, consisting essentially of, or consisting of the contiguous nucleotide sequence AtTcctttacaccACAC (SEQ ID NO: 4), where the capital letter represents beta-D-hydroxy-LNA and the lowercase letter represents DNA. In other embodiments, the ASO contains an internucleotide linkage selected from the group consisting of a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoramidate linkage, a phosphorothioate linkage, and combinations thereof. In some embodiments, the internucleotide linkage is a phosphorothioate linkage.

In some embodiments, the ASO comprises, consists essentially of, or consists of OxyAs DNAts OxyTs DNAcs DNAcs DNAts DNAts DNAts DNAas DNAcs DNAas DNAcs DNAcs OxyAs OxyMCs OxyAs OxyMC, wherein OxyA, OxyT, and OxyMC are beta-D-oxy-LNA containing adenine, beta-D-hydroxy-LNA containing thymine and beta-D-hydroxy-LNA containing methylcytosine, respectively, and where DNAt, DNAc and DNAa are DNA thymine, DNA cytosine and DNA adenine, respectively. In some embodiments, the ASO of the present invention has the molecular formula C ₁₇₁ H ₂₁₄ N ₅₆ O ₉₀ P ₁₆ S ₁₆ and the structure shown in FIG. 1B, where M+ is the counterion. In some embodiments, the counterion is selected from the group consisting of H+, Na ⁺ , _NH4 ⁺ , and any combination thereof. In certain embodiments, the counterion is Na ⁺ .

The present invention also provides a conjugate containing an ASO as disclosed herein, wherein the ASO is covalently attached to at least one non-nucleotide or non-polynucleotide moiety. In some embodiments, the non-nucleotide or non-polynucleotide fragment comprises a protein, a fatty acid chain, a sugar moiety, a glycoprotein, a polymer, or any combination thereof.

Also provided herein is a pharmaceutical composition containing ASO or

- 1 043828 conjugate as described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a therapeutic agent. In certain embodiments, the therapeutic agent is an alpha-synuclein antagonist. In some embodiments, the alpha-synuclein antagonist is an anti-alpha-synuclein antibody or fragment thereof.

The present invention further provides a kit containing an ASO, conjugate or composition as disclosed herein. Also disclosed is a diagnostic kit containing an ASO, conjugate or composition of the present invention.

The present invention also provides a method of inhibiting or reducing the expression of SNCA protein in a cell, the method comprising introducing an ASO, conjugate or composition as disclosed herein into a cell expressing SNCA protein, wherein expression of SNCA protein in the cell is inhibited or reduced after introduction. In some embodiments, the ASO inhibits or reduces SNCA mRNA expression in the cell upon administration. In some embodiments, SNCA mRNA expression is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least at least about 70%, at least about 80%, at least about 90%, or about 100% after administration compared to a cell not exposed to ASO. In other embodiments, ASO reduces SNCA protein expression in a cell upon administration by at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to a cell not exposed to ASO. In some embodiments, the cell is a neuron.

Provided herein is a method of treating synucleinopathy in a subject in need thereof, comprising administering an effective amount of an ASO, conjugate or composition of the present invention. In some embodiments, the synucleinopathy is selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), multiple system atrophy, dementia with Lewy bodies, and any combinations thereof.

Also provided herein is the use of an ASO, conjugate or composition of the present invention for the manufacture of a drug. The present invention also provides the use of an ASO, conjugate or composition for the manufacture of a medicament for the treatment of synucleinopathy in a subject in need thereof. In some embodiments, the ASOs, conjugates, or composition of the present invention are for use in the treatment of synucleinopathy in a subject in need thereof. In other embodiments, the ASOs, conjugates, or composition of the present invention are for use in therapy.

In some embodiments, the subject is a human. In some embodiments, the ASOs, conjugates, or compositions are administered orally, parenterally, intrathecally, intracerebroventricularly, pulmonaryly, topically, or intraventricularly.

Brief description of the figures

In fig. 1A shows the contiguous nucleotide sequence of ASO-005459. OxyA, OxyT and OxyMC are, respectively, beta-D-oxy-LNA containing adenine, beta-D-oxy-LNA containing thymine, and beta-D-oxy-LNA containing methylcytosine; DNAt, DNAc and DNAa are, respectively, DNA thymine, DNA cytosine and DNA adenine; and s represents a phosphorothioate bond.

In fig. 1B shows the molecular structure of ASO-005459 as disclosed herein. The structure shown has the molecular formula C ₁₇₁ H ₂₁₄ N ₅₆ O ₉₀ P ₁₆ S ₁₆ and each of M+ is a pharmaceutically acceptable counterion such as H+, Na ⁺ or NH4+.

In fig. 2A and 2B show the effect of ASO-005459 on SNCA and tubulin (Tub) protein expression in primary neurons isolated from A53T-PAC transgenic mice. Neurons were treated with 10-point titrations of ASO-005459 and the amounts of SNCA protein and tubulin were measured. The percentage inhibition of the α-Syn/Tub ratio (Fig. 2A) and Tub levels (Fig. 2B) are shown. Each data point represents a separate replicate.

In fig. Figure 3 shows the effect of ASO-005459 on the expression levels of SNCA (circle), protein S (alpha) (PROS1 (square)), and tubulin (TUBB3 (triangle)) mRNA in human neurons. Neurons were treated with varying concentrations of ASO-005459 for 6 days and then mRNA levels were measured using the QUANTIGENE® assay. The level of mRNA expression is shown as a percentage of control. Data shown represent the mean ± standard deviation (SD) of duplicate determinations.

In fig. Figure 4 shows SNCA mRNA expression levels in the hippocampus of A53T-PAC mice three days after administration of ICV 100 μg ASO-005459 (open square) or vehicle control (closed circle). SNCA mRNA expression levels were measured by qRT-PCR, normalized to GAPDH mRNA levels, and then expressed relative to the mean expression level of the vehicle group. The horizontal line marks the reference value of 1 (i.e. the value at which expression

- 2 043828 SNCA mRNA will be equivalent to the level of expression observed in the vehicle control group). Data shown are mean ± SD from replicate determinations. Statistical analysis was performed using one-way ANOVA test followed by Dunnett's test. *** p<0.001.

In fig. 5A and 5B show a comparison of the average body weight of mice treated with ASO-005459. In fig. 5A, A53T-PAC mice were dosed with 3.13, 12.5, 25, or 50 μg of ASO-005459 and their body weights were measured at 0, 1, and 2 weeks post-treatment. In fig. 5B, C57BL/6 mice were dosed with 100 μg of ASO-005459 and body weights were measured once per week over a 28-day course. In both figs. 5A and 5B animals treated with vehicle control were used as controls. Data shown represent mean ± SD from multiple animals (n=5). Statistical analysis was performed using a two-way ANOVA test.

In fig. 6A, 6B, and 6C show SNCA mRNA expression levels in the hippocampus (FIG. 6A), brainstem (FIG. 6B), and striatum (FIG. 6C) of A53T-PAC mice 14 days after administration of ICV ASO-005459 (3.13 , 12.5, 25, or 50 μg) or vehicle control. SNCA mRNA levels were measured by qRT-PCR, normalized to GAPDH mRNA levels, and then expressed relative to the mean of the vehicle group. Data shown represent mean ± SD from multiple animals (n=5). Each circle represents a different animal. The horizontal line marks a reference value of 1 (i.e., the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group). Statistical analysis was performed using one-way ANOVA test followed by Dunnett's test. *** p<0.001.

In fig. 7 shows the level of ASO-005459 detected in the hippocampus (black), brainstem (light gray), and striatum (dark gray) in A53T-PAC mice 14 days after treatment with ASO-005459. Mice received 3.13, 12.5, 25, or 50 μg of ASO-005459 via ICV administration. Data shown represent mean ± SD from multiple animals (n=5).

In fig. 8A, 8B, 8C, and 8D show the relationship between ASO-005459 exposure levels and SNCA mRNA expression in the hippocampus (FIG. 8A), brainstem (FIG. 8B), and striatum (FIG. 8C) of A53T-PAC mice 14 days after processing ASO-005459. In fig. Figure 8D shows data for the hippocampus (circle), brainstem (square), and striatum (triangle) combined. Each data point represents an individual animal. A four-parameter, nonlinear fit is shown for the hippocampus (Fig. 8A) and brainstem (Fig. 8B).

In fig. 9A and 9B are dose-response curves showing the effect of ASO-005459 on SNCA mRNA expression levels in A53T-PAC mice. Animals received (via ICV injection) 12.5 μg (circle), 25 μg (square), or 50 μg (triangle) ASO-005459 and were sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks after administration of doses. SNCA mRNA expression levels in the brainstem (Fig. 9A) and striatum (Fig. 9B) were assessed by qRT-PCR and then normalized to vehicle control. Average values shown. The horizontal line marks the reference value of 100% (ie, the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

In fig. 10A and 10B show the effect of ASO-005459 on SNCA mRNA expression levels in A53T-PAC mice 4 weeks after ASO-005459 administration. Animals received either vehicle control or various concentrations of ASO-005459 (12.5, 25, or 50 μg). The relative expression level of SNCA mRNA (normalized to vehicle control) is shown for the brainstem (Fig. 10A) and for the striatum (Fig. 10B). Each data point represents an individual animal. The mean ± SD from multiple animals is also shown. Statistical analysis was performed using one-way ANOVA test with Dunnett's correction for multiple comparisons. The horizontal line marks a reference value of 1 (i.e., the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

In fig. 11A and 11B show a dose-response curve showing the effect of ASO-005459 on the expression level of SNCA protein in brain tissues of A53T-PAC mice. Animals received (via ICV injection) 12.5 μg (circle), 25 μg (square), or 50 μg (triangle) ASO-005459 and were sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks after dosing. SNCA protein levels were measured in both the brainstem (Fig. 11A) and striatum (Fig. 11B) by ELISA and then normalized to vehicle control. Average data shown. The horizontal line marks the reference value of 100% (ie, the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

In fig. 12A and 12B show the effect of ASO-005459 on SNCA protein expression levels in A53T-PAC mice 8 weeks after administration of ASO-005459. Animals received either vehicle control or various concentrations of ASO-005459 (12.5, 25, or 50 μg). Relative levels of SNCA protein expression (normalized to vehicle control) are shown for the brainstem (Fig. 12A) and for the striatum (Fig. 12B). Each data point represents an individual animal. Horizontal

- 3 043828 line marks the reference value of 100% (ie the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

The mean ± SD from multiple animals is also shown. Statistical analysis was performed using one-way ANOVA test with Dunnett's correction for multiple comparisons.

In fig. 13A and 13B show the kinetics of SNCA mRNA and SNCA protein expression levels in cynomolgus monkeys following administration of ASO-005459. Each animal received either vehicle control or ASO-005459 (8 mg) and was then sacrificed at 24 hours, 3 days, 2, 4, 8, 13, or 20 weeks post-dose. At each time point, SNCA mRNA (Fig. 13A) and SNCA protein (Fig. 13B) expression levels were assessed in the following tissues: medulla (upper left panel), dorsal striatum (upper middle panel), pons (upper right panel), cerebellum (lower left panel), lumbar spinal cord (lower middle panel), and frontal lobe (lower right panel). Expression levels are shown as a percentage of vehicle control. Data for both individual animals and the average are shown. The horizontal line marks the reference value of 100% (ie, the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

In fig. 14A and 14B show the relative expression level (as a percentage of vehicle control) for both SNCA mRNA (FIG. 14A) and SNCA protein (FIG. 14B) in cynomolgus monkeys 2 weeks after administration of ASO-005459. Animals received vehicle control or various concentrations of ASO-005459 (2, 4, or 8 mg). Expression levels were assessed in the following tissues: medulla (upper left panel), dorsal striatum (upper middle panel), pons (upper right panel), cerebellum (lower left panel), lumbar spinal cord (lower middle panel), and frontal lobe (bottom right panel). Expression levels are shown as a percentage of vehicle control. Data for both individual animals and the average are shown. The horizontal line marks the reference value of 100% (ie, the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

Detailed Description of the Invention

I. Definitions.

It should be noted that the term refers to one or more objects; for example, by nucleotide sequence is meant one or more nucleotide sequences. Thus, the terms one or more and at least one may be used interchangeably herein.

In addition, the term and/or when used herein should be understood as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term and/or used in a phrase such as A and/or B is intended to include A and B, A or B, A (separately) and B (separately). Likewise, the term and/or used in a phrase such as A, B and/or C is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately); and C (separately).

It is understood that wherever aspects are described herein by the term comprising, they may be alternatively and similarly described using the terms consisting of and/or consisting substantially of.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one skilled in the art with a general vocabulary of many of the terms used in this disclosure.

Units, prefixes and symbols are indicated in their accepted Systeme International de Unites (SI) form. Numeric ranges include numbers that define a range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5'-3' orientation. Amino acid sequences are written from left to right in amino-carboxy orientation. The headings set forth herein are not intended to limit various aspects of the disclosure that may be made by reference to the description as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the description as a whole.

The term roughly is used herein to mean approximately, roughly, about, or in the area. When a term is roughly used in connection with a numeric range, it modifies that range by extending its boundaries beyond the upper and lower bounds of the specified numeric range. Typically, a term can roughly change the numerical value above and below a specified value with a deviation of, for example, 10% up or down (above or below). For example, if ASO is stated to reduce SNCA protein expression in a cell following ASO administration by at least about 60%, this implies that SNCA levels are reduced by an amount in the range of 50 to 70%.

The term antisense oligonucleotide (ASO) refers to an oligomer or polymer of nucleosides, such as naturally occurring nucleosides or modified forms thereof, that are covalently linked to each other through internucleotide bonds. An ASO useful for the invention includes at least one non-naturally occurring nucleoside. The ASO is complementary to the target nucleic acid such that the ASO hybridizes to the target nucleic acid sequence. The terms antisense ASO, ASO and oligomer as used herein are interchangeable with the term ASO.

The term nucleic acids or nucleotides is intended to cover a variety of nucleic acids. In some embodiments, the term nucleic acids or nucleotides refers to a target sequence, such as pre-mRNA, mRNA, or DNA, in vivo or in vitro. When the term refers to nucleic acids or nucleotides in a target sequence, the nucleic acids or nucleotides may be naturally occurring sequences within a cell. In other embodiments, the nucleic acids or nucleotides refer to the sequence in the ASO of the invention. When the term refers to a sequence in an ASO, the nucleic acids or nucleotides are non-naturally occurring, i.e. chemically synthesized, enzymatically produced, recombinantly produced, or any combination thereof. In one embodiment, the nucleic acids or nucleotides in the ASO are produced synthetically or recombinantly, but are not a naturally occurring sequence or fragment thereof. In another embodiment, the nucleic acids or nucleotides in the ASO are non-naturally occurring because they contain at least one nucleotide analogue that is not naturally occurring. The term nucleic acid or nucleoside refers to a single segment of nucleic acid, such as DNA, RNA, or an analog thereof, present in a polynucleotide. Nucleic acid or nucleoside includes naturally occurring nucleic acids or non-naturally occurring nucleic acids. In some embodiments, the terms nucleotide, unit, and monomer are used interchangeably. It will be understood that with respect to a sequence of nucleotides or monomers, what they refer to is a sequence of bases such as A, T, G, C or U, and the like.

As used herein, the term nucleotide refers to a glycoside containing a sugar group, a basic group, and a covalently linked group (linking group), such as a phosphate or phosphorothioate internucleotide linking group, and includes both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides containing a modified sugar and/or base. Herein, a single nucleotide (unit) may also be referred to as a monomer or nucleic acid unit.

As used herein, the term nucleoside is used to refer to a glycoside containing a sugar group and a basic group that can be covalently linked by internucleotide bonds between ASO nucleosides. In the field of biotechnology, the term nucleoside is commonly used to refer to a monomer or nucleic acid unit. In the context of ASO, the term nucleoside can only refer to a single base, i.e. to a nucleotide base sequence containing cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), which implies the presence of a sugar backbone and internucleotide bonds. Likewise, particularly in the case of oligonucleotides where one or more internucleotide linking groups are modified, the term nucleotide may refer to a nucleoside. For example, the term nucleotide can be used even when indicating the presence or nature of bonds between nucleosides.

One skilled in the art will appreciate that the 5' terminal nucleotide of an oligonucleotide does not contain a 5' internucleotide linking group, although it may contain a 5' terminal group.

The term downstream of a nucleotide sequence means that the nucleic acid or nucleotide sequence is located in the 3' direction from the reference nucleotide sequence. In some embodiments, downstream nucleotide sequences refer to sequences that follow the transcription start point. For example, the translation initiation codon of a gene is located downstream of the transcription start site.

Unless otherwise noted, sequences presented herein are listed from the 5' end (left) to the 3' end (right).

The term upstream refers to a nucleotide sequence that is located in the 5' direction from the reference nucleotide sequence.

As used herein, the term transcript refers to the primary transcript resulting from the transcription of DNA and, after processing, becomes messenger RNA (mRNA), i.e. the precursor of messenger RNA (pre-mRNA), and the processed mRNA itself. The term transcript can be used interchangeably with pre-mRNA and mRNA. After the DNA strands are transcribed into primary transcripts, the newly synthesized primary transcripts are modified in several ways to convert them into their mature functional forms, such as mRNA, tRNA, rRNA, lncRNA, miRNA, and others. Thus, the term transcript can include exons, introns, 5'UTR and 3'UTR.

- 5 043828

As used herein, the term expression refers to the process by which a polynucleotide produces a gene product, such as RNA or a polypeptide. It includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA) and translation of mRNA into a polypeptide. Expression produces a gene product. As used herein, the gene product may be a nucleic acid, such as messenger RNA, produced by transcription of a gene, or a polypeptide that has been translated from the transcript. Gene products described herein further include nucleic acids that have undergone post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides that have undergone post-translational modifications, such as methylation, glycosylation, addition of lipids, association with other protein subunits, or proteolytic cleavage.

The term naturally occurring SNCA polypeptide variant refers to variants of an SNCA polypeptide sequence or SNCA nucleic acid sequence (eg, transcript) that exist naturally within a particular taxonomic group, such as mammals such as mouse, monkey, and human. In general, when using naturally occurring SNCA polynucleotide variants, the term may also include any allelic variant of SNCA-encoding genomic DNA that is found at chromosome position 17q21 by chromosomal rearrangement or duplication, and RNA, such as mRNA, derived therefrom. Naturally occurring variants may also include variants resulting from alternative splicing of SNCA mRNA. With respect to a particular polypeptide sequence, for example, the term also includes naturally occurring forms of the protein, which may thus be processed, for example, by co- or post-translational modifications such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.

As used herein, the term complement refers to a sequence that is complementary to a reference sequence. It is well known that complementarity is a basic principle of DNA replication and transcription, and this property is inherent in two DNA or RNA sequences, for example, when they are aligned and antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, reminiscent of looking in a mirror and observing reflection of things. Therefore, for example, the complement of the sequence 5' ATGC 3' can be written as 3' TACG 5' or 5' GCAT 3'. The terms reverse complement, reverse complementary, and reverse complementarity as used herein are interchangeable with the terms complement, complementary, and complementarity. Thus, the sequence 5' attcctttacaccacac 3' (SEQ ID NO: 4) may be complementary to 5' gtgtggtgtaaaggaat 3'.

As used herein, a reference to a SEQ ID number (ie, SEQ ID NO: 4) includes a specific nucleotide sequence, but does not include any construct or complete chemical structure. When reference is made herein to a specific ASO number (ie, ASO-005459), the reference includes the sequence, the specific ASO construct, and the chemical structure.

Activity is usually expressed as an IC ₅₀ or EC ₅₀ value in µM, nM or pM unless otherwise stated. Activity can also be expressed as percentage inhibition. The IC ₅₀ represents the average inhibitory concentration of the therapeutic molecule. EC ₅₀ represents the average effective concentration of the therapeutic molecule relative to vehicle or control (eg, saline). In functional assays, the IC ₅₀ is the concentration of a therapeutic molecule that reduces a biological response, such as mRNA transcription or protein expression, by 50% of the biological response achieved by the therapeutic molecule. In functional assays, the EC ₅₀ is the concentration of a therapeutic molecule that produces 50% of the biological response, such as mRNA transcription or protein expression. IC ₅₀ or EC ₅₀ can be calculated by any number of methods known in the art.

By subject or individual or animal or patient or mammal is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, pets, farm animals, sporting animals and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears etc.

The term pharmaceutical composition refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered. Such a composition may be sterile.

An effective amount of ASO disclosed herein is an amount sufficient to accomplish the specifically stated purpose. The effective amount can be determined empirically and in conventional manner depending on the stated purpose.

Terms such as curative or treatment, or treat, or alleviate, or alleviate refer to (1) therapeutic measures that cure, slow, reduce symptoms and/or

- 6 043828 stop the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and/or slow down the development of the target pathological condition or disorder. Thus, those who need treatment include those who already have a disorder; those who are predisposed to the occurrence of a disorder; and those in whom the disorder is prevented. In some embodiments, a subject receives successful treatment of a disease or condition disclosed elsewhere herein, in accordance with the methods presented herein, if the patient demonstrates, for example, complete, partial, or temporary relief or elimination of symptoms associated with the disease or violation.

II. ASO-005459.

The ASO of the present invention (i.e., ASO-005459) contains a contiguous nucleotide sequence of 17 nucleotides in length that corresponds to the complement region (i.e., the junction between intron 1 and exon 2) of the SNCA transcript, i.e. nucleotides 7604-7620 from SEQ ID NO: 1. The ASO of the invention has the nucleotide sequence shown in SEQ ID NO: 4 (i.e. attcctttacaccacac) with the ASO design LDLDDDDDDDDDDLLLL (i.e. AtTcctttacaccACAC), where L denotes a nucleoside of the closed nucleic acid acids (ie LNA, eg beta-D-hydroxy-LNA), and D stands for deoxyribonucleic acid (DNA). Accordingly, the 1-, 3-, and 14-17th nucleotides from the 5' end of ASO-005459 are beta-D-hydroxy-LNA, and each of the other nucleotides is DNA. The ASO disclosed herein also has the following chemical structure: OxyAs DNAts OxyTs DNAcs DNAcs DNAts DNAts DNAts DNAas DNAcs DNAas DNAcs DNAcs OxyAs OxyMCs OxyAs OxyMC, where s denotes a phosphorothioate bond. The structural formula for ASO-005459 is shown in FIG. 1B, where M+ is a pharmaceutically acceptable counterion. As used herein, the term pharmaceutically acceptable counterion refers to an ion that accompanies the ionic forms to maintain electrical neutrality that is not biologically or otherwise undesirable and thus allows the preparation of a pharmaceutically acceptable salt form. Thus, in some embodiments, the pharmaceutically acceptable counterion may be H ⁺ , Na ⁺ , K ⁺ , NH4 ⁺ , Li ⁺ or any other cation with a charge of 1 ⁺ . In some embodiments, the pharmaceutically acceptable counterion is H ⁺ , Na ⁺ , NH4 ^{+ ,} and combinations thereof.

As used herein, the term pharmaceutically acceptable salts refers to derivatives of ASO-005459 wherein ASO-005459 is modified (eg, adding a cation disclosed herein) to form salts thereof. Such salts retain the desired biological activity of the ASO without producing undesirable toxicological effects. The ASO of the invention may be present in any salt form. In some embodiments, the ASO of the invention is in the form of a sodium salt. In other embodiments, the ASO is in the form of a potassium salt.

ASO-005459 can bind to the intron1/exon2 junction of SNCA mRNA and prevent translation of SNCA mRNA. In some embodiments, due to sugar modification, the ASO of the invention has a binding affinity for a target RNA sequence that is enhanced by at least 5%, at least 10%, at least 20%, at least 30% , at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to a control (eg ASO without such sugar modification).

The ASO monomers described herein are linked to each other through linking groups. Accordingly, each monomer is linked to the adjacent 3' monomer through a linking group.

One skilled in the art will appreciate that, in the context of the present disclosure, the 5' monomer at the end of the ASO does not contain a 5' linking group, although it may or may not contain a 5' terminal group.

The terms linking group and internucleotide bond are intended to refer to a group capable of covalently linking two nucleotides together. Examples include phosphate groups and phosphorothioate groups.

Examples of internucleotide linkages include a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoramidate linkage, a phosphorothioate linkage, and combinations thereof. See also WO 2007/031091, which is incorporated herein by reference in its entirety.

In one aspect of the ASO of the invention, the nucleotides are linked to each other via phosphorothioate groups.

It is recognized that the inclusion of phosphodiester linkages, such as single or double linkages, in phosphorothioate ASO, particularly between or adjacent to nucleotides, can modify the bioavailability and/or biodistribution of ASO; see WO 2008/113832, hereby incorporated by reference.

In some embodiments, such as the above embodiments, where applicable and not specifically stated, all remaining linking groups are phosphodiester or phosphorothioate, or a mixture thereof.

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In some embodiments, all internucleotide linking groups are phosphorothioate.

An LNA nucleoside is a 2'-modified nucleoside that contains a diradical linking the C2' and C4' of the ribose sugar ring of the nucleoside (also called a 2'-4' bridge), which restricts or blocks the conformation of the ribose ring. These nucleosides are also called bridging nucleic acid or bicyclic nucleic acid (BNA) in the literature. The ribose conformation block is associated with increased hybridization affinity (duplex stabilization) when LNA is introduced into an oligonucleotide for a complementary RNA or DNA molecule. This can usually be determined by measuring the melting temperature of the oligonucleotide/complement duplex.

Non-limiting examples of LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/0 36698, 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), p. 1569-81; Mitsuoka et al., Nucleic Acids Research, 2009, 37(4), 1225-1238; and Wan & Seth, J. Medical Chemistry, 2016, 59, 9645-9667.

Other non-limiting exemplary LNA nucleosides are shown in Scheme 1.

Scheme 1

In a specific embodiment, the LNA suitable for disclosure is betaD-okcu-LNA.

II.A. Conjugates.

As used herein, the term conjugate refers to ASO-005459 covalently linked to a non-nucleotide moiety.

Conjugation of ASO-005459 to one or more non-nucleotide moieties may improve the pharmacology of the ASO, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the ASO. In some embodiments, the conjugate moiety modifies or enhances the pharmacokinetic properties of the ASO by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the ASO. In particular, the conjugate can target an ASO to a specific organ, tissue or cell type and thereby enhance the effectiveness of the ASO in that organ, tissue or cell type. At the same time, the conjugate may serve to reduce ASO activity in non-target cell types, tissues or organs, eg, off-target activity or activity in non-target cell types, tissues or organs. WO 93/07883 and WO 2013/033230 propose suitable conjugate moieties. Additional suitable conjugate fragments are those that are capable of binding to the asialoglycoprotein receptor (ASGPr). In particular, trivalent N-acetylgalactosamine conjugates are suitable for coupling to ASGPr, see, for example, WO 2014/076196, WO 2014/207232 and WO 2014/179620.

- 8 043828

ASO conjugates and their synthesis have also been described in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., ch. 16, Marcel Dekker, Inc.,

2001, Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

In some embodiments, the non-nucleotide fragment (conjugate fragment) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (eg, bacterial toxins), vitamins, viral proteins ( for example, capsids) and their combinations.

II.B. Activated ASOs.

The term activated ASO as used herein refers to an ASO of the present invention that is covalently linked (i.e., functionalized) to at least one functional moiety that allows the ASO to covalently link to one or more conjugated moieties, i.e. fragments that are not themselves nucleic acids or monomers to form the conjugates described herein. Typically, the functional moiety will contain a chemical group that is capable of covalently binding to the ASO, for example through a 3'-hydroxyl group or an exocyclic NH ₂ group of the adenine base, a spacer that may be hydrophilic, and a terminal group that is capable of binding to the conjugated moiety (for example, an amino group, a sulfhydryl group, or a hydroxyl group). In some embodiments, this end group is unprotected, such as an NH ₂ group. In other embodiments, the end group is protected, for example, by any suitable protecting group, such as those described in Protective Groups in Organic Synthesis by Theodora W. Greene and Peter GM Wuts, 3rd edition (John Wiley & Sons, 1999).

In some embodiments, ASO-005459 is functionalized at the 5' end to provide covalent attachment of the conjugated moiety to the 5' end of the ASO. In other embodiments, the ASO of the invention may be functionalized at the 3' end. In still other embodiments, the ASO of the invention may be functionalized along the backbone or on a heterocyclic base moiety. In yet other embodiments, the ASO of the present invention may be functionalized at more than one position independently selected from the 5' end, 3' end, backbone, and base.

In some embodiments, the activated ASO of the invention is synthesized by incorporating during synthesis one or more monomers that are covalently linked to a functional group. In other embodiments, the activated ASO of the present invention is synthesized using monomers that have not been functionalized, and the ASO is functionalized after the synthesis is complete.

III. Pharmaceutical compositions and routes of administration.

ASO-005459 can be used in pharmaceutical formulations and compositions. Accordingly, such compositions contain a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

ASO-005459 may be included in a dosage unit, for example, a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver a therapeutically effective amount to a patient without causing serious side effects in the patient being treated. However, in some forms of therapy, serious side effects may be acceptable while ensuring a positive therapeutic outcome.

The formulated drug may contain pharmaceutically acceptable binders and adjuvants. Capsules, tablets or pills may contain, for example, the following compounds: microcrystalline cellulose, gum or gelatin as binders; starch or lactose as excipients; stearates as lubricants; various sweeteners or flavorings. In the case of capsules, the dosage unit may contain a liquid carrier such as fatty oils. Likewise, part of such a dosage unit may be a sugar or enteric coating. Oligonucleotide formulations can also be emulsions consisting of active pharmaceutical ingredients and a lipid forming a micellar emulsion.

The pharmaceutical compositions of the present invention can be administered in several ways depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration may be (a) oral;

(b) pulmonary, for example, by inhalation or insufflation of powders or aerosols, including administration by inhaler; intratracheal, intranasal;

(c) topical, including epidermal administration, transdermal administration, ophthalmic administration and mucosal administration, including vaginal and rectal administration; or (d) parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, for example, intrathecal, intracerebroventricular or intraventricular administration.

In one embodiment, ASO-005459 is administered intravenously, intraperitoneally, orally,

- 9 043828 locally or as a bolus injection or injected directly into the target organ. In some embodiments, ASO-005459 is administered intrathecally or intracerebroventricularly as a bolus injection.

Pharmaceutical and topical compositions may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders. Standard pharmaceutical carriers, aqueous powder or oily bases, thickeners, and the like may be necessary or desirable. Examples of topical formulations include formulations in which ASO-005459 is present in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Compositions and formulations for oral administration include, but are not limited to, powders or granules, microparticles, nanoparticles, suspensions or solutions in water or anhydrous media, capsules, gel capsules, sachets, tablets or mini-tablets. Compositions and formulations for parenteral, intrathecal, intracerebroventricular or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or auxiliaries substances.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be prepared from a variety of components, which include, but are not limited to, preformulated liquids, self-emulsifying solids and self-emulsifying semi-solids. Drug delivery to target tissue can be enhanced by carriers including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles, and microspheres (Dass C.R., J. Pharm. Pharmacol., 2002, 54(l):3-27).

The pharmaceutical compositions of the present invention, which for convenience may be presented in unit dosage form, can be prepared in accordance with conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredients with pharmaceutical carrier(s) or excipient(s). Typically, the formulations are prepared by uniformly and thoroughly mixing the active ingredients with liquid carriers or finely divided solid carriers, or both, and then, if necessary, molding the resulting product.

For parenteral, subcutaneous, intradermal or topical administration, the formulation may include a sterile diluent, buffers, tonicity regulators and antibacterial agents. Active ASOs can be formulated in combination with carriers that protect against degradation or rapid elimination from the body, including implants or microcapsules with controlled release properties. For intravenous administration, the carriers may be saline or phosphate buffered saline. International publication WO 2007/031091 (A2), published March 22, 2007, further provides suitable pharmaceutically acceptable diluents, carriers and adjuvants, which are incorporated herein by reference.

IV. Diagnostics.

This disclosure further provides a diagnostic method that can be used in the diagnosis of SNCA-related diseases, such as synucleinopathy. Non-limiting examples of synucleinopathy include, but are not limited to, Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies, and multiple system atrophy.

ASO-005459 can be used to measure SNCA transcript expression in tissue or body fluids obtained from an individual and compare the measured level of expression to a standard level of SNCA transcript expression in normal tissue or body fluids, wherein an increase in expression level over the standard is indicative of a disorder , treatable with ASO-005459.

ASO-005459 can be used to analyze SNCA transcript levels in a biological sample using any methods known to those skilled in the art (Touboul et al., Anticancer Res. (2002), 22(6A): 3349-56; Verjout et al., Mutat Res (2000), 640: 127-38); Stowe et al., J. Virol. Methods (1998), 75(1): 93-91).

By biological sample is meant any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing an SNCA transcript. Methods for obtaining tissues by biopsy and biological fluids from mammals are well known in the art.

V. Sets containing ASO.

This disclosure further provides kits that contain ASO-005459 described herein and that can be used to perform the methods described herein

- 10 043828 document. In certain embodiments, the kit contains at least ASO-005459 in one or more containers. In some embodiments, the kits contain all components necessary and/or sufficient to perform the detection assay, including all controls, instructions for performing the assays, and any necessary software for analyzing and reporting the results. One skilled in the art will readily understand that ASO-005459 can be readily included in one of the established kit formats that are well known in the art.

VI. Methods of application.

ASO-005459 can be used for therapeutic and prophylactic purposes. SNCA is a 140 amino acid protein predominantly expressed in neurons at presynaptic terminals, where it is thought to play a role in the regulation of synaptic transmission. It is predicted to exist in vivo as both an unfolded monomer and a stable tetramer composed of α-helices and has been shown to undergo several post-translational modifications. One modification that has been extensively studied is the phosphorylation of SNCA at amino acid serine 129 (S129). Typically, only a small percentage of SNCA is constitutively phosphorylated at S129 (pS129), whereas the vast majority of SNCA found in pathological intracellular inclusions are SNCA pS129. These pathological inclusions consist of aggregated insoluble accumulations of misfolded SNCA proteins and are a characteristic feature of a group of neurodegenerative diseases collectively known as synucleinopathies (or α-synucleinopathies).

In synucleinopathies, SNCA can form abnormal aggregates in neurons known as Lewy bodies, which are characteristic of both Parkinson's disease (PD), Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB). Thus, ASO-005459 may reduce the number of pathological SNCA aggregates or prevent the formation of pathological SNCA aggregates. In addition, oligodendrocytes exhibit SNCA-rich lesions called glial cytoplasmic inclusions (GCIs), which are a hallmark of the rapidly progressive, fatal synucleinopathy known as multiple system atrophy (MSA). In some embodiments, ASO-005459 reduces the amount of GCI or prevents the formation of GCI. Reports of undetectable or low levels of SNCA mRNA expression in oligodendrocytes suggest that some pathological form of SNCA spreads from highly expressed neurons to oligodendrocytes. In some embodiments, ASO-005459 reduces or prevents the spread of SNCA, such as a pathological form of SNCA, from neurons.

ASO-005459 can be used in research, for example, to specifically inhibit SNCA protein synthesis (usually by degrading or inhibiting mRNA and thereby preventing protein formation) in cells and experimental animals, thereby facilitating functional analysis of the target or assessment of its utility as a target for therapeutic intervention. Also provided are methods of inhibiting the expression of SNCA mRNA and/or SNCA protein in cells or tissues, comprising contacting the cells or tissues in vitro or in vivo with an effective amount of ASO-005459, conjugates or compositions of the invention.

With respect to therapeutic agents, an animal or human suspected of having a disease or disorder that can be treated by modulating the expression of SNCA transcript and/or SNCA protein is treated by administering ASO-005459 in accordance with the present disclosure. In addition, methods are provided for treating a mammal, for example, treating a human suspected of having or being susceptible to a disease or condition associated with expression of SNCA transcript and/or SNCA protein, by administering a therapeutically or prophylactically effective amount of ASO-005459 or compositions of the invention. The ASO, conjugate or pharmaceutical composition of the invention is typically administered in an effective amount. In some embodiments, an ASO or conjugate of the invention is used in therapy.

The invention further provides ASO-005459 for use in the treatment of one or more diseases mentioned herein, such as a disease selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), Lewy body dementia, multiple system atrophy, and any combinations of them.

The invention further provides a method of treating α-synucleinopathies, the method comprising administering an effective amount of ASO, conjugates, or pharmaceutical compositions thereof to an animal in need thereof (such as a patient in need thereof).

In some embodiments, the disease, disorder, or condition is associated with overexpression of a SNCA gene transcript and/or SNCA protein.

The invention also provides methods of inhibiting (for example, by reducing) the expression of a SNCA gene transcript and/or SNCA protein in a cell or tissue, the method comprising contacting the cell or tissue in vitro or in vivo with an effective amount of ASO, conjugates or pharmaceuticals thereof. ceutical compositions of the invention to achieve a decrease in the expression of the SNCA gene transcript, thereby reducing the level of SNCA protein.

In some embodiments, ASO-005459 is used to reduce SNCA mRNA expression in one or more brain regions, such as the hippocampus, brainstem, striatum, or any combination thereof. In other embodiments, ASO-005459 reduces SNCA mRNA expression, e.g., in the brainstem and/or striatum by less than 70%, less than 60%, less than 50%, less than 40%, less than 30 %, less than 20%, less than 10%, or less than 5% compared to SNCA mRNA expression after administration or exposure to vehicle (without ASO) on days 3, 5, 7, 10, 14, 15, 20, 21 or 25. In some embodiments, SNCA mRNA expression is maintained at levels below 70%, below 60%, below 50%, below 40%, below 30%, below 20%, below 10%, or below 5% relative to mRNA expression SNCA after administration or exposure to vehicle (without ASO) for up to 28, 30, 32, 35, 40, 42, 45, 49, 50, 56, 60, 63, 70, or 75 days.

In other embodiments, ASO-005459 reduces the expression of SNCA mRNA and/or SNCA protein in the medulla, dorsal striatum, pons, cerebellum, lumbar spinal cord, frontal lobe, and/or any combination thereof.

The invention also provides the use of ASO-005459 or a conjugate of the invention for the manufacture of a drug. The invention also provides a composition containing ASO-005459 or a conjugate thereof for use in treating a disorder as defined herein, or a method for treating a disorder as defined herein. The present invention also provides ASO-005459 or conjugates for use in therapy. The present invention further provides ASO-005459 or conjugates for use in the treatment of synucleinopathy.

The invention further provides a method of inhibiting SNCA protein in a cell that expresses SNCA, comprising introducing ASO-005459 or a conjugate in accordance with the invention into the cell to thereby effect inhibition of SNCA protein in the cell.

The invention includes a method of reducing, attenuating, preventing or treating neuronal hyperexcitability in a subject in need thereof, comprising administering ASO-005459 or a conjugate in accordance with the invention.

The invention also provides a method of treating a disorder as described herein, the method comprising administering ASO-005459 or a conjugate in accordance with the invention as described herein and/or a pharmaceutical composition in accordance with the invention to a patient in need thereof.

ASO-005459 and other compositions of the invention can be used to treat conditions associated with overexpression or expression of a mutated version of the SNCA protein.

The invention provides ASO-005459 or a conjugate in accordance with the invention for use as a drug, for example, for the treatment of α-synucleinopathies. In some embodiments, α-synucleinopathy is a disease selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), Lewy body dementia, multiple system atrophy, and any combinations thereof.

The invention further provides the use of ASO-005459 in the manufacture of a medicament for treating a disease, disorder or condition as defined herein. In some embodiments, ASO-005459 or a conjugate thereof is used for the manufacture of a medicament for the treatment of α-synucleinopathy, epilepsy, or a combination thereof.

In general, one aspect of the invention relates to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of SNCA (ie, α-synucleinopathy), comprising administering to the mammal a therapeutically effective amount of ASO, as described herein.

A disease or disorder as defined herein, in some embodiments, may be associated with a mutation in the SNCA gene or a gene whose protein product is associated with or interacts with the SNCA protein. Thus, in some embodiments, the target mRNA is a mutated form of the SNCA sequence.

An interesting aspect of the invention is directed to the use of ASO-005459, as defined herein, or a conjugate, as defined herein, for the manufacture of a medicament for the treatment of a disease, disorder or condition, as defined herein.

The methods of the present invention can be used to treat or prevent diseases caused by abnormal levels of SNCA protein. In some embodiments, diseases caused by abnormal levels of SNCA protein are α-synucleinopathies. In some embodiments, α-synucleinopathies include Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies, and multiple system atrophy.

Alternatively, in some embodiments, the invention further provides a method for treating abnormal levels of SNCA protein, the method comprising administering ASO-005459 or a conjugate of the invention, or a pharmaceutical composition of the invention to a patient in need thereof.

The invention also relates to ASO-005459, a composition or conjugate as defined herein, for use as a drug.

The invention further relates to the use of a compound, composition or conjugate, as defined herein, for the manufacture of a medicament for the treatment of abnormal levels of SNCA protein or expression of mutant forms of SNCA protein (such as allelic variants, for example those associated with one of diseases mentioned in this document).

A patient in need of treatment is a patient suffering or likely to suffer from a disease or disorder.

In carrying out the present invention, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology known to those skilled in the art will be used. Such techniques are described in detail in the literature. See, for example, Sambrook et al., ed. (1989), Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992), Molecular Cloning: A Laboratory Manual (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985), DNA Cloning, vol. I and II; Gait, ed. (1984), Oligonucleotide Synthesis; Mullis et al., U.S., pat. No. 4683195; Hames and Higgins, eds. (1984), Nucleic Acid Hybridization; Hames and Higgins, eds. (1984), Transcription And Translation; Freshney (1987), Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984), A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987), Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, vol. 154 and 155; Mayer and Walker, eds. (1987), Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds. (1986), Handbook of Experimental Immunology, vol. I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); Crooke, Antisense drug Technology: Principles, Strategies and Applications, 2nd ed., CRC Press (2007); and in Ausubel et al. (1989), Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

The following examples are offered as illustrations and not as limitations.

Examples

Example 1: Construction of ASO-005459.

The ASO described herein (ie, ASO-005459) was designed to target the junction between intron 1 and exon 2 of the SNCA pre-mRNA (ie, nucleotides 7604-7620 SEQ ID NO: 1). ASO-005459 was designed as a gapmer (eg, alternating gapmer) and contains the closed nucleic acids LNA (capital letters), beta-deoxy LNA at the 5' end and 3' end, and a phosphorothioate backbone. But the backbone may be other types of backbone (eg, phosphodiester linkage, phosphotriester linkage, methylphosphonate linkage, phosphoramidate linkage, or combinations thereof).

The ASOs of the present invention were synthesized using methods well known in the art. Exemplary methods for preparing such ASOs are described in Barciszewski et al., chapter 10, Locked Nucleic Acid Aptamers in Nucleic Acid and Peptide Aptamers: Methods and Protocols, vol. 535, Gunter Mayer (ed.) (2009), the entire contents of which are incorporated herein by reference.

Example 2. Multiparameter analysis (High Content Assay) to measure the decrease in SNCA protein levels in primary neurons.

ASO-005459 was tested for its ability to reduce SNCA protein expression in primary mouse neurons. Primary neuronal cultures were prepared from the forebrain of PAC-Tg (SNCA ^A53t ) ^+/+ mice; SNCA ^- / ^- (PAC-A53T) carrying the fully human SNCA gene with the A53T mutation in a SNCA knockout mouse background. See Kuo Y. et al., Hum. Mol. Genet., 19: 1633-50 (2010). All procedures involving mice were performed in accordance with Animal Test Methods (ATM) approved by the Bristol-Myers Squibb Animal Care and Use Committee (ACUC). Primary neurons were generated by papain digestion according to the manufacturer's protocol (Worthington Biochemical Corporation, LK0031050). Isolated neurons were washed and resuspended in Neurobasal medium (NBM, Invitrogen) supplemented with B27 (Gibco), 1.25 μM Glutamax (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B.

Cells were seeded into poly-D-lysine-coated multiwell plates at 5400 cells/cm ² (eg, 384-well plates at 6000 cells/well in 25 μl NBM). ASO was diluted in water and added to cells at DIV01 (i.e., 1 day after seeding). ASO was added to 2x the final concentration in the medium, then delivered to the cells manually. Alternatively, ASO in water was dispensed using a Labcyte ECHO acoustic dispenser station. For ECHO distribution, 250 nL of ASO in water was added to cells in media, followed by the addition of an equal volume aliquot representing a fresh aliquot of NBM. For primary screening, ASO was added to final concentrations of 5, 3.3, 1, 200 nM, or 40 nM. To determine the activity, we prepared

- 13 043828

8-10 point titrations of ASO from a 0.75 mM stock solution were then delivered to cultured cells to produce final concentrations ranging from 2.7-4000 or 4.5-10000 nM. ASO-000010 (TCTgtcttggctTTG, SEQ ID NO: 5) and ASO-000838 (AGAaataagtggtAGT, SEQ ID NO: 6) (5 μM) were included in each plate as reference control inhibitors for tubulin and SNCA, respectively.

Cells were incubated with ASO for 14 days to achieve a sustained reduction in mRNA.

After 14 days of incubation, cells were fixed by adding fixative to final concentrations of 4% formaldehyde (J.T. Baker) and 4% sucrose (Sigma) to the wells. Cells were fixed for 15 min, and then the fixative was aspirated from the wells. Cells were then permeabilized for 20 min with phosphate-buffered saline (PBS) containing 0.3% Triton-X 100 and 3% bovine serum albumin (BSA) or 3% normal goat serum. The permeabilization buffer was then aspirated from the wells and the cells were washed once with PBS. Primary antibodies were then diluted in PBS containing 0.1% Triton X-100 and 3% BSA. Dilutions of 1:1000 rabbit anti-SNCA antibody (Abcam) and 1:500 chicken anti-tubulin antibody (Abcam) were used. Cells were incubated with primary antibodies for 2 h to overnight. After incubation, the primary antibody staining solution was aspirated and the cells were washed 2 times with PBS. A secondary staining solution containing a 1:500 dilution of goat antibodies to chicken immunoglobulins conjugated to the fluorescent dye Alexa 567, goat antibodies to rabbit immunoglobulins conjugated to Alexa 488, and Hoechst (10 μg/ml) in PBS containing 0 .1% Triton X-100 with 3% BSA, and the plates were incubated for 1 hour. After this, the secondary staining solution was aspirated from the wells and the cells were washed 3 times with PBS. After washing the cells, 60 μl of PBS was added to each well. The plates were then stored in PBS until imaging.

For imaging, plates were scanned on a Thermo-Fisher (Cellomics) CX5 system using Spot Detector Bio-Application (Cellomics) to quantify nuclei (Hoechst dye, channel 1), tubulin extensions (Alexa 567, channel 2), and SNCA (Alexa 488 , channel 3). The number of objects (nuclei) was observed but not published in the database. The total area covered by tubulin was quantified as a function of SpotTotalAreaCh2, and the total intensity of SNCA staining was quantified as SpotTotalIntenCh3. Tubulin measurement was included to monitor toxicity. To determine the reduction in SNCA protein levels, the ratio of SNCA intensity to tubulin staining area was calculated and the results were normalized as the median % inhibition, using the median of vehicle-treated wells as the overall value and wells ASO-000010 or ASO-000838 as the most inhibited wells for tubulin or SNCA, respectively.

Example 3 QUANTIGENE® Assay (96-well assay) to measure mRNA reduction in human neurons.

The ability of ASO-005459 to reduce human SNCA mRNA and/or possible off-target human mRNA species was measured in vitro using the QUANTIGENE® assay. Human neurons (Cellular Dynamics Inc., iNeurons) were thawed, plated, and cultured according to the manufacturer's specifications. These iNeurons are a high-purity population of human neurons derived from induced pluripotent stem (iPS) cells using Cellular Dynamic's proprietary differentiation and purification protocols.

Lysis. Cells were seeded in 96-well plates coated with polyX-ornithine/laminin at 50,000–10,000 cells per well (depending on the expression of the non-target of interest) and maintained in neurobasal medium supplemented with B27, Glutamax and penicillin-streptomycin. ASO was diluted in water and added to DIV01 cells (i.e., 1 day after seeding). For single measurements, a final ASO concentration of 0.5 μM was typically used. To determine _IC50, neurons were treated with 1:4 dilutions to generate a seven-point concentration-response curve with the highest concentration being 5 μM. Cells were then incubated at 37°C and 5% CO ₂ for 6 days to achieve a sustained decrease in mRNA levels.

After incubation, the medium was removed and the cells were washed 1x in DPBS and lysed as follows. Messenger RNA content in the lysate was measured using the QUANTIGENE® 2.0 Reagent System (AFFYMETRIX®), which quantifies RNA using a signal amplification method (branched DNA method) based on a specially designed set of RNA capture probes. Cell lysis working buffer solution was prepared by adding 50 μl of proteinase K to 5 ml of prewarmed (37°C) Lysis mix and diluting in dH ₂ O to a final dilution of 1:4. The working buffer for lysis was added to the plates (from 100 to 150 μl/well, depending on the expression of the non-target being studied), ground 10 times, hermetically sealed and incubated for 30 min at 55°C. After lysis, the wells were ground 10 more times and the plates were stored at -80°C or analyzed immediately.

Analysis. Depending on the specific capture probe used (i.e., SNCA, PROS1, or tubulin), lysates were diluted (or undiluted) in the lysis mixture. Lysates were then added to capture plates (96-well polystyrene plates coated with capture probes) in a total volume of 80 μl/well.

- 14 043828

Working probe set reagents were prepared by combining nuclease-free water (12.1 µl), lysis mixture (6.6 µl), blocking reagent (1 µl) and custom probe set 2.0 (0.3 µl) (human SNCA , part number SA-50528, human PROS1, part number SA-10542, or human beta-3 tubulin, part number SA-15628) according to the manufacturer's instructions (QUANTIGENE® 2.0 AFFYMETRIX®). 20 μL of probe working set reagents were then added to 80 μL of lysate dilution (or 80 μL of lysis mix for original samples) on the capture plate. The plates were centrifuged at 240 g for 20 s and then incubated for 16–20 h at 55°C for hybridization (target RNA capture).

Signal amplification and target RNA detection began by washing the plates 3 times with buffer (300 μl/well) to remove any unbound material. Pre-Amplifier 2.0 Hybridization Reagent 2.0 was then added (100 μl/well), incubated at 55°C for 1 hour, then aspirated and wash buffer was added, and aspirated 3 times. The 2.0 Amplifier hybridization reagent (100 μl/well) was then added as described, incubated for 1 h at 55°C, and the washing step was repeated as previously described. 2.0 Label Probe Hybridization Reagent (100 μl/well) was then added, incubated for 1 h at 50°C, and the washing step was repeated as previously described. The plates were again centrifuged at 240 g for 20 s to remove any excess wash buffer, and then 2.0 Substrate (100 μl/well) was added to the plates. The plates were incubated for 5 min at room temperature, and then the plates were imaged on a PerkinElmer Envision multichannel reader in luminometer mode for 15 min.

Definition of data. For the gene of interest, the mean background signal in the assay was subtracted from the mean signal of each technical replicate. The average background-subtracted signals for the gene of interest were then normalized to the average background-subtracted signal for the housekeeping RNA of tubulin. The percent inhibition for the treated sample was calculated relative to the lysate of the control treated sample. The results of QUANTIGENE® assays for ASO-treated cells are shown, for example, in FIG. 3.

Example 4 QUANTIGENE® Assay (96-well assay) to measure the reduction of mRNA levels in Ramos cells.

Ramos cells (a human lymphocyte cell line) were used to measure a possible decrease in the mRNA level of the non-target IKZF3 (IKAROS family zinc finger 3). Because Ramos cells do not express SNCA, RB1 (RB, transcriptional corepressor 1), which is expressed in Ramos cells, was used as a positive control to evaluate ASO-mediated knockdown of IKZF3 mRNA expression. Two ASOs were synthesized to bind and knockdown human RB1 mRNA expression. Beta-2-microglobulin (β2Μ) was used as a housekeeping control gene. Ramos cells were grown in suspension in RPMI medium supplemented with FBS, glutamine, and penicillin/streptomycin.

Lysis. Cells were seeded in 96-well plates coated with poly-L-ornithine/laminin at 20,000 cells per well and maintained in neurobasal medium containing B27, Glutamax, and penicillin-streptomycin. ASO was diluted in water and added to cells 1 day after plating (DIV01) to a final concentration of 1 μM. After ASO treatment, cells were incubated at 37°C for 4 days to achieve a sustained reduction in mRNA levels. After incubation, the medium was removed and the cells were lysed as follows. Measurement of messenger RNA in the lysate was performed using the QUANTIGENE® 2.0 Reagent System (AFFYMETRIX®), which quantifies RNA using a signal amplification method (branched DNA method) based on a specially designed set of RNA capture probes. The lysis mixture (QUANTIGENE® 2.0 AFFYMETRIX®) was preheated in an incubator at 37°C for 30 min. To lyse cells in suspension, 100 μl of 3x lysis buffer (containing 10 μl/ml proteinase K) was added to 200 μl of cells in suspension. The cells were then ground 10 times to lyse, the plate was sealed and incubated for 30 min at 55°C. Lysates were then stored at −80°C or analyzed immediately.

Analysis. Depending on the specific capture probe used (i.e., IKZF3, RB1, and β2Μ), lysates were diluted (or undiluted) in the lysis mixture. Lysates were then added to a capture plate (96-well polystyrene plate coated with capture probes) in a total volume of 80 μl/well. Reagents for working probe sets were prepared by combining 12.1 µl nuclease-free water, 6.6 µl lysis mixture, 1 µl blocking reagent, 0.3 µl specific probe set 2.0 (human IKZF3, catalog number SA-17027, Human RB1, part number SA-10550 or beta-2 microglobulin, part number SA-10012) according to the manufacturer's instructions (QUANTIGENE® 2.0 AFFYMETRIX®). 20 μL of probe working set reagents were then added to 80 μL of lysate dilution (or 80 μL of lysis mix for original samples) on the capture plate. The plates were then incubated for 16–20 h at 55°C for hybridization (target RNA capture). Signal amplification and target RNA detection began by washing the plates with buffer 3 times (300 μl/well) to remove any unbound material. Hybridization reagent was then added

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2.0 Pre-Amplifier 2.0 (100 µl/well), incubated at 55°C for 1 hour, then aspirated and wash buffer added, and aspirated 3 times. The 2.0 Amplifier hybridization reagent (100 μl/well) was then added as described, incubated for 1 h at 55°C, and the washing step was repeated as previously described. 2.0 Label Probe Hybridization Reagent (100 μl/well) was then added, incubated for 1 h at 50°C, and the washing step was repeated as previously described. The plates were again centrifuged at 240 g for 20 s to remove excess wash buffer, and then 2.0 Substrate (100 μl/well) was added to the plates. The plates were incubated for 5 min at room temperature and then imaged on a PerkinElmer Envision multichannel reader in luminometer mode for 15 min.

Definition of data. For the gene of interest, the average background signal in the assay (i.e., no lysate, 1× lysis buffer only) was subtracted from the average signal of each technical replicate. The average background-subtracted signals for the gene of interest were then normalized to the average background-subtracted signal for housekeeping RNA (for Ramos cells, beta-2-microglobulin). The percent inhibition for the treated sample was calculated relative to the mean value of the untreated sample lysate. The results of QUANTIGENE® assays for cells treated with ASO-005459 are shown in Table. 4.

Example 5 qPCR assay to measure the decrease in SNCA mRNA levels in SK-N-BE(2) cells.

ASO-005459, which targets SNCA, was tested for its ability to reduce SNCA mRNA expression in an ATCC-derived human neuroblastoma cell SK-N-BE(2) (CRL-2271).

SK-N-BE(2) cells were grown in cell culture medium (MEM [Sigma, catalog no. M2279], supplemented with 10% fetal bovine serum [Sigma, catalog no. F7524], 1x GlutamaxTM [Sigma, catalog no. 3050 -038], 1x MEM solution of essential amino acids [Sigma, catalog number M7145] and 0.025 mg/ml gentamicin [Sigma, catalog number G1397]). Cells were trypsinized every 5 days by washing with phosphate-buffered saline (PBS) [Sigma, catalog number 14190-094] followed by the addition of 0.25% trypsin in EDTA (Sigma, T3924), incubating for 2-3 min at 37°C and grinding before cell seeding. Cells were maintained in culture for up to 15 passages.

For experimental use, 12,500 cells per well were seeded in 96-well plates (Nunc, cat. no. 167008) in 100 μl of growth medium. Oligonucleotides were prepared from a 750 μM stock solution. ASO dissolved in PBS was added approximately 24 h after cells were seeded to a final concentration of 25 μM for single-point assays. Cells were incubated for 4 days without any media replacement. To determine the activity, 8 concentrations of ASO were prepared in a final concentration range of 16–50,000 nM. ASO-004316 (CcAAAtcttataataACtAC, SEQ ID NO: 7) and ASO-002816 (TTCctttacaccACAC, SEQ ID NO: 8) were included as controls.

After incubation, cells were harvested by removing the medium followed by the addition of 125 μl of PURELINK® Pro 96 Lysis buffer (Invitrogen 12173.001 A) and 125 μl of 70% ethanol. RNA was purified according to the manufacturer's instructions and eluted in a final volume of 50 μl of water, resulting in an RNA concentration of 10–20 ng/μl. The RNA was diluted 10-fold with water before the one-step qPCR reaction. For a one-step qPCR reaction, a qPCR mix (qScript TMXLE 1-step RT-qPCR TOUGHMIX® Low ROX from QauntaBio, catalog number 95134-500) was mixed with two Taqman probes in a ratio of 10:1:1 (qPCR mix: probe 1: probe 2) to generate a master mix. Taqman probes were purchased from LifeTechnologies: SNCA: Hs01103383_m1; PROS1: Hs00165590_m1: TBP: 4325803; GAPDH 4325792. Master mix (6 µl) and RNA (4 µl, 1-2 ng/µl) were then mixed in a qPCR plate (MICROAMP® optical 384-well, 4309849). Once sealed, the plate was quickly spun, 1000g for 1 min at room temperature, transferred to a ViiaTM 7 system (Applied Biosystems, Thermo) and the following PCR conditions were used: 50°C for 15 min; 95°C for 3 min; 40 cycles: 95°C for 5 s, followed by a decrease in temperature at a rate of 1.6°C/s, and then 60°C for 45 s. Data were analyzed using QuantStudioTM Real_time PCR software.

Example 6 In Vitro Assay of ASO-005459 for Reduction of Human SNCA mRNA.

Activity of ASO-005459 in mouse neurons.

Using the methods described above in Example 2, ASO-005459 was tested for its ability to reduce SNCA protein expression by reducing SNCA mRNA levels. Briefly, primary neurons obtained from PAC-A53T mice were treated with ASO-005459 or control ASOs for 14 days. The cells were then fixed and SNCA protein and tubulin protein levels were measured using the High Content Imaging multiparameter assay. Tubulin levels were measured to monitor toxicity and normalize for decreased SNCA protein levels.

As shown below table. 1, incubation of cells with 4 nM ASO-005459 resulted in a 21% decrease in SNCA protein expression. At 40 nM and 5 μM ASO-005459, SNCA protein expression was reduced by 84 and 86%, respectively. In contrast, ASO-005459 had minimal or no effect on tubulin protein expression levels.

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Table 1

Activity of ASO-005459 in A53T-PAC neurons ASO-005459 concentration aSyn/tub %inhibit. SD N Tube %inhibit. SD N 4 nM 21.11 33.71 3 -8.60 12.07 4 40 nM 84.28 13.21 8 4.32 28.65 7 5 µM 86.45 7.91 2 29.01 18.06 2 8О=standard deviation. N=number of tests.

To further evaluate activity, A53T-PAC neurons were treated with 10-point titrations of ASO-005459 as described above in Example 2A, and _IC50 values for the effect on SNCA and tubulin proteins were determined. As shown in FIG. 2A, 2B and in table. 2 (below), a concentration-dependent decrease in the a-Syn/tubulin ratio was observed with an average _IC50 value of 7.4 nM. This observation is consistent with the single-point activity data shown in Table. 1 (above). Moreover, ASO-005459 had little effect on Tub levels. Taken together, these results indicate that ASO-005459 effectively reduces SNCA protein levels with minimal impact on overall cell viability.

Table 2 Activity and selectivity assessed for ASO-005459 for SNCA and tubulin protein in A53T-PAC neurons

aSyn/tub IC50 (nM) SD N tube IC50 (nM) SD N 7.42 2.73 6 >4000 N.A. 5 8О=standard deviation. N=Konn4ecTBO tests.

Efficacy of ASO-005459 in SK-N-BE(2) cells.

Using the method described in Example 5, ASO-005459 was tested for its ability to reduce SNCA mRNA levels in SK-N-BE(2) after 4 days of treatment.

Incubation of cells with 25 μM ASO-005459 resulted in a 92% decrease in SNCA mRNA levels in SK-N-BE(2) cells.

Activity of ASO-005459 in human neurons.

The SNCA activity of ASO-005459 was confirmed using primary human neurons as described above in Example 3. Briefly, human neurons were derived from induced pluripotent stem (iPS) cells. Cells were treated with ASO-005459 or control ASOs for 6 days and then mRNA levels were measured using the QUANTIGENE® assay. Because human neurons also express PROS1, a potential non-target of ASO-005459, PROS1 mRNA levels were also measured to assess the effect of ASO-005459 on non-target genes. Additionally, tubulin mRNA (TUBB) was measured to monitor toxicity.

As shown in FIG. Ziv tab. 3 (below), ASO-005459 induced a concentration-dependent decrease in SNCA expressed in human neurons, with an average IC value of _50-100 nM. This _1C50 value is ~13 times lower than the _1C50 value for reducing SNCA protein levels in PAC-A53T neurons (Table 2, above). The reason for this shift in activity is unclear but may be due to differences in ASO uptake, metabolism, or SNCA knockdown kinetics. Although ASO-005459 also demonstrated a concentration-dependent decrease in PROS1 mRNA levels in human neurons; the IC50 value was found to be 30-50 times lower than the IC50 value for SNCA. These results confirm the SNCA activity of ASO-005459 in human neurons and indicate that ASO-005459 is 30- to 50-fold more selective for SNCA compared to PROS1.

Table 3 Activity and selectivity of ASO-005459 for SNCA and PROS1 mRNA in human neurons

Date of analysis The consignment SNCA 1C ₅₀ (nM) PROS1 IC50 (nM) tivvz 1S ₅₀ (nM) PROS1/SAG4 ratio IC50 8/26/2016 01-001 42 2127 >5000 51 12/8/2016 01-004 157 >5000 >5000 >32

Lot=The specific lot number of the ASO-005459 used.

Activity of ASO-005459 in Ramos cells.

IKZF3 is another potential non-target of ASO-005459, but unlike PROS1, it is not expressed in human neurons. Instead, IKZF3 is stably expressed in Ramos cells, a human lymphocytic cell line. Ramos cells were treated with 1 µM

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ASO-005459 and measured the effect on IKZF3 mRNA expression. Beta-2 microglobulin mRNA was also measured to monitor toxicity. Because Ramos cells do not express SNCA, another gene, RB1 (RB transcriptional corepressor 1), was used as a positive control for ASO-mediated knockdown. ASO-006754 (GGTgaggtttggtaGA, SEQ ID NO: 9) and ASO-006755 (GGTgaggtttggtagaAG, SEQ ID NO: 10) target RB1 and were included in the study. As shown in table. 4 (below), at a concentration of 1 μM, ASO-005459 had no effect on IKZF3 mRNA expression, demonstrating the specificity of ASO-005459 for SNCA. ASO-005459 also had no effect on RB1 and e2M mRNA expression, indicating that ASO-005459 is not toxic to Ramos cells. In contrast, ASO-006754 and ASO-006755 (control ASOs) reduced RB1 levels by 87 and 83%, respectively, confirming ASO activity in Ramos cells.

Table 4

Effect of 1 µM ASO-005459 on IKZF3 in Ramos cells

ASO Target The consignment IKZF3 (%con) RB1 (%con) β2Μ (%con) ASO-005459 SNCA 01-004 102 99 94 ASO-006754 RB1 01-001 104 13 87 ASO-006755 RB1 01-001 116 17 83

Lot=The specific lot number of the ASO being used.

Taken together, the results presented herein demonstrate that ASO-005459 is active and highly selective in reducing SNCA mRNA levels, which in turn mediates a reduction in SNCA protein levels. The results also show that ASO-005459 is well tolerated in both mouse and human neurons. These data support the further development of ASO-005459 as a disease-modifying therapeutic for the treatment of synucleinopathies.

Example 7 In Vivo Tolerance and Reduction of SNCA mRNA Levels in Vivo.

The in vivo tolerability of the ASOs of the present invention was tested to understand how the ASOs are tolerated when injected into various animal models (ie mice and cynomolgus monkeys).

Mice.

Subjects. Male and female (2-3 months old) PAC-Tg (SNCA ^A53T ) ^+/+ mice; SNCA ^−/− (PAC-A53T), carrying the fully human SNCA gene with the A53T mutation in an SNCA knockout mouse genetic background, was used for short-term, long-term, and PK/PD in vivo efficacy studies. In some cases, wild-type (WT) C57B/6 mice were used for long-term (i.e., 4 weeks) health assessments. Mice were housed in groups of 4 or 5 in a temperature-controlled room with unlimited access to food and water. All procedures involving mice were performed in accordance with Animal Test Methods (ATM) approved by the Bristol-Myers Squibb Animal Care and Use Committee (ACUC).

Preparation of solution for dosing ASO. Sterile saline syringes (1 mL) equipped with 0.2 μm Whatman filters and nuclease-free centrifuge tubes were used to prepare dosing solutions. The specified volume of water or saline solution was added to the ASO powder and vortexed (~1 min) to dissolve the ASO powder. The solution was then left for 10 min and vortexed again for ~1 min. The tubes were centrifuged briefly to return all liquid to the bottom of the tube, and then the solution was filtered through a sterile 0.2 μm filter into a second RNase-free tube. A small aliquot of the stock solution was diluted to 1 mg/mL for concentration analysis using Nanodrop. The analytical sample was vortexed three times with manual inversion to ensure thorough mixing. The UV absorbance of the sample was then measured twice at 260 nm using Nanodrop (the stand was rinsed and wiped three times before applying the sample). The test sample was discarded after completion of the analysis. The sample was considered ready for dispensing if the UV absorbance was between 90 and 110% of the sample. If the UV absorbance exceeded 110% of the sample, a secondary dilution was prepared; if absorbance was <90%, the sample was prepared with a higher initial concentration and similar steps as described above were followed. Samples were stored at 4°C until use.

Intracerebroventricular injection (ICV) using the freehand method. ICV injection was performed using a Hamilton microsyringe equipped with a 27- or 30-gauge needle according to the Haley and McCormick method. The needle was provided with a polyethylene guard at a distance of 2.5–3 mm from the tip to limit its penetration into the brain. Mice were anesthetized using the anesthetic isoflurane (1–4%). After sufficient anesthesia, mice were held by the flaccid skin of the neck on the back with the thumb and first finger of one hand. By applying gentle but firm pressure, the animal's head was then immobilized by pressing it against a hard, flat surface. Dosing was performed using 10 μl Hamilton syringes fitted with a 27/4 gauge needle. The needle tip was then inserted through the scalp and skull, approximately 1 mm lateral and 1 mm caudal from the bregma level (i.e., to the right of the midline,

- 18 043828 approximately 3 mm back, measured from the eye line). After positioning, the ASO needle was added in a volume of 5 μL to vehicle saline and injected for ∼30 s. The needle was left in place for 5–10 s before removal. Mice were returned to their home cage and allowed to recover for ~2–4 min. Mice were observed continuously for 30 min immediately after dosing to identify adverse behavioral effects of drug and/or dosing. During this time, any mouse that experienced a seizure more than 3 times was immediately euthanized and given an automatic score of 20. Drug tolerability was assessed 1 h ± 15 min after dosing. Animals that demonstrated intolerance after administration of compounds (tolerance score >4) were sacrificed immediately after the 1-h assessment.

ASO tolerability assessment. Animals dosed with ASO were assessed immediately after dosing and observed for 2 hours for any adverse effects. For short-term tolerability (AT) studies, mice were assessed at the time of dosing and again 3 days after ASO injection. For long-term health assessment, mice were weighed weekly and observed for any health or behavioral problems until completion of the experiment. Mice that lost more than 15% of their initial body weight or exhibited tolerance problems were excluded from the studies and euthanized. Health status and tolerability assessments were performed according to the following table.

Table 5

Tolerability assessment systema

Category Point 1 Point 2 Point 3 Point 4 Hyperactive behavior, stereotypies, behavior in the home cage •Very little increase in home cage exploration or vertical locomotor activity compared to controls •Increased exploration of the home cage (e.g. digging, burrowing, etc.) •Increased grooming •Moderately increased home cage exploration •Detectable stereotypies (e.g. circling, repetitive movements, etc.) •Significant hyperactivity •Significant stereotypies Reduced vigilance, exploration and responsiveness •Some decrease in exploratory activity •Respond normally to stimulation •Drowsiness •Slightly decreased response to touch or manipulation •Stupor (decreased response, decreased corneal reflex) •Coma (no response to stimulation, e.g. compression), no corneal reflex Motor coordination and strength •Moderate change in gait and grip strength (fall through 510) *No falls, normal righting response •Decreased grip strength (fall in less than 5 sec) •Moderate ataxia (eg, slow righting response, rocking) •Highly reduced grip strength (fall in less than 2 sec) •Ataxia (eg, staggering, falling, impaired gait) •Severe anaxia (eg, crawling, falling from a beam) •Unable to straighten up Posture, appearance, breathing •Very slightly abnormal posture (mild deviation) • Slightly abnormal posture (eg, hunched over, stretched out, low posture, tail position, Straub tail) •Piloerection or ptosis “untidy outer covering •Moderately abnormal posture (eg, the animal lies on its stomach and cannot stand up) •Shallow breathing •Significantly abnormal posture (e.g. animal lying on its side) •Facial paralysis (e.g. drooling, protruding tongue) •Difficulty breathing Tremor, hyperactivity, convulsions •Detectable tremor •Hyperresponsiveness to stimuli (eg noise) •Significant tremor •Minor or partial, rearing and falling as part of convulsions •Repetitive or convulsive seizures (running, jumping, clonic and/or tonic) ^a Normal is assessed as ' 0. Animals were assessed on an individual basis

- 19 043828 at successive time points after dosing. Observation was carried out after 1 hour ± 15 minutes, then after 24 ± 2 hours, then after 7 days (if necessary).

Convulsions were counted at the 1-h time point, even if they occurred before the observation window. An overall tolerability score was calculated based on the sum of the individual category scores, with a maximum possible score of 20.

Collection of tissues. After final behavioral and health assessments, mice were decapitated by guillotine and the brains were quickly removed. Each brain was divided into two hemispheres and

a) the hippocampus was dissected for mRNA measurements in 3-day short-term tolerance studies;

b) the hippocampus, brainstem, and striatum from one hemisphere were dissected for mRNA measurements, while the same areas were dissected from the second hemisphere for protein/PK measurements in PK/PD dose-response studies.

In some studies, blood and cerebrospinal fluid (CSF) were also collected for PK (blood) and PK/protein (CSF) measurements. To collect blood and CSF, mice were deeply anesthetized with isoflurane (4%). Blood was collected by cardiac puncture using a 23-gauge needle. Once removed, the blood was transferred to 2 mL BD Microtainer tubes (K2EDTA BD #365974) and placed on ice until processing. To process blood, tubes were centrifuged at 4500xg for 10 min at 4°C. The plasma was then removed and placed in 0.5 ml Eppendorf tubes and stored at -80°C until use. To collect CSF, the chest cavity was opened to expose the heart, and as much blood as needed was collected to avoid CSF contamination. CSF samples were collected from the magna tank using micropipettes and placed in Eppendorf tubes, Protein LoBind. The tubes were then centrifuged at 4500xg for 15 min at 4°C. CSF was carefully transferred into clean 0.5 ml LoBind Eppendorf tubes and stored at -80°C until further use.

Cynomolgus macaque data.

Subject. Male cynomolgus macaques weighing 3.5–10.0 kg at baseline were used. Each was implanted with an intrathecal catheter to access cerebrospinal fluid (CSF), inserted at the L3 or L4 vertebral level. The distal tip of the polyurethane catheter extended into the intrathecal space to approximately the L1 vertebra. The proximal end was connected to a subcutaneous access port located in the animal's lower back. Animals were allowed to heal for at least two weeks before the start of the study. Laboratory animals were cared for in accordance with the Public Health Policy on the Humane Care and Use of Laboratory Animals and the NRC Guide for the Care and Use of Laboratory Animals (2011) (National Research Council: Guide for the Care and Use of Laboratory Animals (The National Academies Collection: Reports) funded by National Institutes of Health. National Academies Press (US), Washington (DC)). The protocol was approved by the Wallingford Animal Care and Use Committee of the Bristol-Myers Squibb Company.

CSF and blood sampling: The CSF port was accessed subcutaneously using aseptic techniques, and CSF sampling was performed from awake animals sitting upright in a primate restraint chair. Approximately 0.1 mL of CSF was discarded at the beginning of collection to clear dead space in the catheter and port. CSF was collected by gravity flow to a maximum of 0.5 ml CSF per sample. CSF was spun at 2000 g at 4°C for 10 min. The supernatant was frozen on dry ice or liquid nitrogen and stored at −90°C until analysis.

Blood samples were taken from an accessible vein, usually the saphenous vein. Blood samples were prepared in several procedures depending on the specific indicator. For plasma, blood was collected in EDTA-treated tubes. For serum, blood was collected into serum separation tubes and allowed to clot for at least 30 min before centrifugation. To measure coagulation and clotting factors, blood was collected into citrate tubes, and for RNA analysis, blood was later collected into tubes containing RNA. After processing, samples were frozen on dry ice or liquid nitrogen and kept frozen until analysis.

Intrathecal dosing. Animals were trained to dosage while awake and using modified commercially available restraint chairs, the animals were maintained in a supine position. Antisense oligonucleotides (ASOs) targeting SNCA were dissolved in saline, filter sterilized, and injected at a rate of 0.33 mL/min in a volume of 1.0 mL, followed by a rinse with 0.5 mL of sterile water. The total infusion time was 4.5 minutes. Animals remained in a supine position for 30 min after infusion.

Opening. Cynomolgus monkeys were administered an appropriate volume of commercially available euthanasia solution under ketamine and/or isoflurane anesthesia. Immediately after autopsy, tissue was obtained and the brain was transferred to wet ice for excision. Areas of interest were excised using 4–6 mm sections in the ASI Cyno Brain Matrix as well as free-hand techniques. Samples were placed fresh in RNAlater® or frozen on dry ice for later analysis. CNS tissue was quickly excised from cynomolgus cacaques, and pieces no longer than 4 mm in any axis were collected and placed later in 5 ml of RNA.

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Samples were stored at 4°C overnight, then at -20°C for storage until analysis.

Brain regions analyzed included medulla, pons, midbrain, cerebellum, dorsal striatum (left and right), hippocampus (left and right), frontal cortex (left and right), temporal cortex (left and right), parietal cortex (left and right), occipital cortex (left and right), and cortical white matter. In addition, the spinal cord was collected from the cervical, thoracic and lumbar regions. Samples were also collected from the liver, kidneys and heart. In some cases, trigeminal, tibial, and aortic nuclei samples were collected to study untargeted pharmacology in these areas.

Quantification by ELISA of ASO concentrations in mouse or monkey tissue, plasma, and CSF.

The tissue was homogenized with plasma and water in a 1:1 ratio. The standard curve was generated by 2-fold serial dilution from 5000 to 4.9 nM in plasma (for plasma and CSF) and plasma:water (for tissue samples), and then further diluted to a 5000-fold dilution using 5x taken separately SSCT (750 mM NaCl and 75 mM sodium citrate, pH 7.0, containing 0.05% (v/v) Tween-20) and in 5x SSCT containing capture reagents at a concentration of 35 nM and detection reagents at a concentration 35 nM to give a standard range of 1-1000 pM. The dilution factor used varied depending on the expected concentration range of the sample. The capture probe was AAAGGAA with 3′ Biotin (Exiqon), and the detection probe was 5′ DigN-isopropyl 18 linker—GTGTGGT (Exiqon).

Experimental samples and standards were added to Clarity Lysis Buffer (Phenomenex, cat. no. AL0-8579) at a 1:1 ratio before dilution with capture and detection buffer and before transfer to the ELISA plate. CSF samples were diluted in plasma (2-fold) before adding lysis buffer. The streptavidin-coated plate (Thermo 15119) was washed 3 times with 5x SSCT buffer. 100 μl of samples were added and incubated for 60 min at room temperature. Detection probe, 100 μl anti-Dig-AP Fab fragment diluted 1:4000 in PBS containing 0.05% Tween-20 (Roche Applied Science, catalog number 11093274910), was added and incubated for 60 min at room temperature. temperature. After washing the plate with 2x SSCT buffer for 30 min at room temperature, 100 μl of Tropix CDP-star Sapphire II substrate (Applied Biosystems) was added. ASO concentrations were measured using luminescence (Enspire-PerkinElmer).

Alpha-synuclein protein measurements.

Brain tissue samples were homogenized at 10 ml/g tissue in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) using a Qiagen Tissuelyser bead homogenizer II for 25 cycles/s with a 5 mm stainless steel ball for 2 min. Homogenized samples were incubated for 30 min on ice. Aliquots of 50 μL of each sample were retained for PK analysis. The remaining samples were centrifuged at 20800 g for 60 min at 4°C. The supernatant was saved and used for analysis. Total protein was measured using the Pierce BCA Protein Assay Kit (23227).

Brain tissue extracts.

SNCA protein was measured using the MJFR1+4B12 ELISA. Briefly, ELISA plates (Costar) were coated with 100 μl of anti-SNCA antibody MJFR1 (Abcam) at a concentration of 0.1 μg/ml, diluted in BupH carbonate-bicarbonate buffer, pH 9.4 (Thermo Scientific) overnight (O/ N) at 4°C. The next day, plates were washed 4 times with Dulbecco's PBS (Life Technologies) and blocked with 3% BSA (protease-free bovine serum albumin, fraction V, Roche Diagnostic) in PBS for 2–3 h at room temperature (RT) or nights at 4°C. Both standards and brain samples were diluted in 1% BSA/0.05% Tween/PBS containing Roche protease inhibitor (Roche 11836145001, 1 bead/25 ml) and phosphatase inhibitor 2 and 3 (Sigma, 1:100). Wild-type SNCA (rPeptide) was used as a standard. Samples were loaded in duplicate (50 μl/well) and incubated overnight (O/N) at 4°C. After equilibrating the plates to room temperature, 50 μl of detection antibody 4B12 (Biolegend) (diluted 1:4000 in 1% BSA/0.1% Tween/DPBS) was added to each well and co-incubated with the samples at room temperature for ∼2 h. The detection antibody was preconjugated to alkaline phosphatase (AP kit from Novus Biologicals). The plates were then washed 4 times with 0.05% Tween/PBS and developed with 100 μl of alkaline phosphatase substrate (Tropix CDP Star Ready-to-Use with Sapphire II, T-2214, Life Technologies) for 30 min. Luminescence intensity was measured using a Perkin Elmer EnVision (2102 Multilabel Reader). The plates were shaken continuously (Titer shaker, speed 3) during analysis. Data were analyzed using GraphPad Prism. Total protein in brain tissue was measured using a Micro protein assay kit (Thermofisher #23235) according to the manufacturer's instructions.

Cerebrospinal fluid (CSF). SNCA protein was measured using the U-PLEX Human SNCA Kit (cat. no. K151WKK-2, Meso Scale Discovery) according to the manufacturer's instructions. CSF samples were diluted 10-fold. Hemoglobin was measured in CSF samples using

- 21 043828 Abcam mouse Hemoglobin ELISA kit (ab157715). CSF samples were diluted 40-fold to measure hemoglobin.

Measurements of mRNA using qRT-PCR.

Brain regions were harvested and placed in 1.5 ml RNA-later Tissue Protect tubes (Qiagen, part number 76514) that were pre-filled with RNA-later, an RNA stabilization solution. Tissue in RNA-later solution can be stored at 4°C for 1 month or at -20 or -80°C indefinitely.

RNA isolation. RNeasy Plus Mini Kit: RNA from mouse hippocampus and cerebral cortex was isolated using the RNeasy Plus Mini Kit (Qiagen, cat. no. 74134). Tissue samples were homogenized in a volume of 600 or 1200 μl of RLT Plus buffer containing 10 μl/ml 2-mercaptoethanol and 0.5% Dx reagent. 600 μL lysis buffer was used if the tissue sample was <20 mg, 1200 μL lysis buffer was used for tissue samples >20 mg. For homogenization, the tissue sample was transferred to a 2.0 ml round bottom Eppendorf Safe-Lock tube (Eppendorf, part number 022600044) containing 600 µl RLT Plus Buffer (plus 10 µl/ml 2-mercaptoethanol and 0.5% Dx reagent) , and 5 mm stainless steel balls (Qiagen, part number 69989). Samples were homogenized using a Qiagen TissueLyser II instrument. Samples were processed for 2.0 min at 20 Hz, samples were rotated 180° and processed for an additional 2.0 min at 20 Hz. The samples were then treated for 2.0 min at 30 Hz, the samples were rotated 180° and processed for another 2.0 min at 30 Hz. Homogenization was used longer and/or at a higher frequency if treatment was not completed. 600 μl of tissue lysate was then transferred to a gDNA Eliminator spin column in a 2.0 ml collection tube and samples were centrifuged for 30 s at 10,000<i>g . All centrifugation steps were carried out at room temperature. The flow stream was collected and an equal volume of 70% ethanol was added and mixed. 600 μL was transferred to the RNeasy spin column in a 2.0 mL collection tube, and the samples were centrifuged for 15 s at 10,000 g. The flow was discarded and the remaining 600 μL of sample was added to the spin column. The spin columns were centrifuged and the flow path was discarded. The columns were washed with 700 μl of wash buffer RW1, centrifuged for 15 s at 10,000g, and the flow path was discarded. The columns were then washed 2 times with 500 μl RPE buffer containing 4 volumes of ethanol as described in the kit protocol. The columns were first centrifuged for 15 s at 10,000 g for the first wash, and then for 2.0 min at 10,000 g for the second wash. After the second wash, the columns were centrifuged once for 1.0 min at 10,000 g to dry the membranes. The columns were then transferred to a new 1.5 mL collection tube and 30 μL of water-free RNase was added directly to the center of the membrane. The membranes were left to incubate for 10 min at room temperature. The columns were then centrifuged for 1.0 min at 10,000g to elute the RNA. The RNA-containing eluate was collected and stored on ice until RNA concentrations were determined by UV absorbance using a NanoDrop spectrophotometer (Thermo). RNA samples were stored at -80°C.

RNA isolation. RNEASY® Plus Universal Mini Kit: RNA from all other tissue samples from cynomolgus monkey, mouse and rat was isolated using the RNEASY® Plus Universal Mini Kit (Qiagen, catalog number 73404). For homogenization, 50 μg or less of tissue sample was transferred to a 2.0 mL Round Bottom Eppendorf Safe-Lock Tube (Eppendorf, part number 022600044) containing 900 μL of QIAZOL® Lysis Reagent and 5 mm stainless steel beads (Qiagen , catalog number 69989). Samples were homogenized using a TissueLyser II homogenizer, Qiagen. Samples were processed for 2.0 min at 20 Hz, samples were rotated 180° and processed for an additional 2.0 min at 20 Hz. The samples were then processed for 2.0 min at 30 Hz, the samples were rotated 180° and processed for another 2.0 min at 30 Hz. Homogenization for a longer time and/or at a higher frequency was used if the treatment was not completed. The homogenized tissue lysate was then transferred to a new 2.0 mL Round Bottom Eppendorf Safe-Lock Tube and left at room temperature for 5.0 min. 100 μL of gDNA Eliminator solution was added to each tube and the tubes were shaken vigorously for 30 s. 180 μl of chloroform (Sigma, cat. no. 496189) was added to each tube and the tubes were shaken vigorously for 30 s. The tubes were left at room temperature for 3 minutes. The tubes were centrifuged at 12000 g for 15 min at 4°C. After centrifugation, the upper aqueous phase was transferred to a new 2.0 mL, ∼500 μL Eppendorf Round Bottom Safe-Lock Tube. An equal volume of 70% ethanol was added and mixed. All subsequent centrifugation steps were carried out at room temperature. 500 μl was transferred to an RNeasy spin column placed in a 2.0 ml collection tube, and samples were centrifuged for 15 s at 10,000 g. The filtrate was discarded and the remaining 500 μL of sample was added to the spin column. The spin columns were centrifuged, the filtrate was discarded, and the columns were washed with 700 μl of Wash Buffer RWT containing 2 volumes of ethanol. The columns were centrifuged for 15 s at 10,000 g, and the filtrate was discarded. The columns were then washed twice with 500 μL RPE buffer containing 4 volumes of ethanol as described in the kit protocol. The columns were first centrifuged for 15 s at 10,000 g for the first wash, and then for 2.0 min at 10,000 g for the second wash.

- 22 043828 washing. After the second wash, the columns were centrifuged once for 1.0 min at 10,000 g to dry the membranes. The columns were then transferred to a new 1.5 mL collection tube and 30 μL of RNase-free water was applied directly to the center of the membrane. The membranes were left to incubate for 10 min at room temperature. The columns were centrifuged for 1.0 min at 10,000 g to elute the RNA. Eluates containing RNA were collected and stored on ice until RNA concentration was determined by UV absorbance using a NanoDrop spectrophotometer (Thermo). RNA samples were stored at -80°C.

cDNA synthesis using reverse transcription. 300 ng of RNA was diluted to a final volume of 10.8 μl using nuclease-free water (Invitrogen, part number 10977-015) in a PCR-96-AB-C microplate (Axygen, part number 321-65-051). To each well, 6.0 μl of reaction mixture 1 was added containing the following components: 2.0 μl of random decamers at a concentration of 50 μM (Ambion, catalog number AM5722G) and 4.0 μl of 1x dNTP mixture (Invitrogen, catalog number 10297- 018). The plate was sealed with optical sealing tape (Applied Biosystems, part number 4360954) and centrifuged for 1.0 min at 1000 × g at room temperature. The plate was then heated for 3.0 min at 70°C using a 96-well Thermal Cycler GeneAmp PCR System 9700 (Applied Biosystems). The plate was then completely cooled on ice. Then 3.25 μl of reaction mixture 2 (containing 2 μl of 10x cDNA synthesis buffer, 1.0 μl of MMLV-RT reverse transcriptase enzyme 200 U/μl (Ambion, catalog number 2044) and 0.25 µl of RNase inhibitor 40 units/µl (Ambion, catalog number AM2682)). The plate was sealed with optical sealing tape and centrifuged for 1.0 min at 1000xg at room temperature. Using a 96-well Thermal Cycler, the plate was heated at 42°C for 60 min, then at 95°C for 10 min. The plates were then cooled on ice. The cDNA plates were stored at 20°C until ready to be used for PCR analysis.

qPCR for amplification and quantification of SNCA and GAPDH mRNA expression: cDNA was diluted 5-fold in nuclease-free water in a PCR-96-AB-C microplate. A 16 µl Master Mix solution consisting of the following: 10 µl Taqman Gene Expression Master Mix (Applied Biosystems, part number 4369016), 1.0 µl 20x Taqman Primer Probe Set (Applied Biosystems) and 5.0 µl nuclease-free water was added into each well of a 384-well optical PCR plate (Applied Biosystems, part number 4483315). 4.0 μL of diluted cDNA was added to each well of a 384-well optical PCR plate. The plate was sealed with optical sealing tape and centrifuged for 1.0 min at 100xg at room temperature. PCR was performed on an Applied Biosystems 700 HT Fast Real-Time PCR System using the following parameters in standard mode: 50°C for 2.0 min, 95°C for 10 min, then 40 cycles at 95°C for 15 s and 60°C for 1.0 min.

Primer-probe sets for qRT-PCR. Primer probe sets from Applied Biosystems (Thermo Fisher) included the following:

1) human alpha-synuclein (catalog number Hs01103383_m1) with a FAM tag;

2) human PROS1 (catalog number HS00165590_m1) with FAM tag;

3) Cynomolgus macaque alpha-synuclein (catalog number Mf02793033_m1) with a FAM tag;

4) Cynomolgus macaque GAPDH (catalog number Mf04392546_g1) with FAM tag;

5) Cynomolgus GAPDH (cat. no. Mf04392546_g1) with Primer Limited VIC tag;

6) rat alpha-synuclein (catalog number Rn01425141_m1) with FAM tag;

7) rat GAPDH (catalog number Rn01775763-g1) with FAM tag;

8) rat GAPDH (cat. no. 4352338E) with Primer Limited VIC tag;

9) mouse GAPDH (catalog number Mm99999915-g1) with FAM tag;

10) mouse GAPDH (cat. no. 4352339E) with Primer Limited VIC tag.

Example 8: In Vivo Activity and Tolerability Analysis of ASO-005459 in Mice.

ASO-005459 is an LNA-modified ASO specific for human SNCA. The in vitro results (described above) demonstrate that ASO-005459 is potent and selective in reducing SNCA mRNA levels in primary neurons. The in vitro results also suggest that ASO-005459 is well tolerated.

A53T-PAC mice.

To evaluate whether these results are also valid in vivo, 100 μg of ASO-005459 was administered to A53T-PAC mice via ICV injection, and tolerance and knockdown (KD) of SNCA mRNA in the hippocampus were assessed 3 days after injection as described in Example 4 ( higher).

As shown in table. 6 and 7 (below), ASO-005459 was well tolerated with an overall mean tolerability score of 1. In addition, ASO-005459 significantly reduced SNCA mRNA levels by >90% in the hippocampus 3 days after administration (Figure 4).

- 23 043828

Table 6

Toxicity rating @1D 3D mRNA %KD Toxicity rating @28D Toxicity assessment WT @28D ASO-005459 0.50 90.83 0.00

Table 7

Tolerability of ASO-005459 in A53T-PAC mice, 3-day study

Animal# Hyperactivity Vigilance Motor strength and coordination Posture/breathing Tremors/convulsions Total score 31 0 2 1 2 0 5 32 0 0 0 0 0 0 33 0 0 0 0 0 0 34 0 0 0 0 0 0 35 0 0 0 0 0 0 average 1.00 S.E.M. 1.00

To evaluate in vivo activity, A53T-PAC mice were dosed with ASO-005459 at 3.13, 12.5, 25, or 50 μg via ICV injection. Tolerability was assessed by measuring body weight on days 7 and 14 after dosing. SNCA mRNA expression was assessed 14 days after dosing, when animals were sacrificed and their tissues were collected. SNCA mRNA knockdown was measured in three brain regions: the hippocampus, brainstem, and striatum. The brainstem and striatum are the two regions that are most affected in the brain of patients with MSA and PD. In a separate study, C57BL/6 mice were dosed with 100 μg of ASO-005459 and tolerability was assessed by measuring the animals' body weight on days 7, 14, 21, and 28 post-dosing. Wild-type (WT) C57BL/6 mice were administered ICV 100 μg ASO-005459 and body weight and behavior were monitored over a 4-week period. The reduction in SNCA mRNA expression was not measured in these animals because ASO-005459 does not target mouse SNCA.

As shown in FIG. 5A and 5B, there were no significant differences in body weight between mice (both A53T-PAC and C57BL/6 mice) treated with ASO-005459 (all concentrations) and mice treated with vehicle control. Animals (C57BL/6) did not exhibit any abnormal behavior throughout the experiment. See table. 8 (below). These results indicate that ASO-005459 is well tolerated. Additionally, mice treated with ASO-005459 showed a significant and dose-dependent reduction in SNCA mRNA expression in all three brain regions tested. See fig. 6A-6C. In the hippocampus, SNCA mRNA expression was reduced by 53, 73, 80, and 96% for 3.13, 12.5, 25, and 50 μg of ASO-005459, respectively. Similar dose-dependent knockdowns were also observed in the brainstem. In the striatum, SNCA mRNA knockdowns were more variable and less robust compared to other regions (Figures 6A-6C): 75 and 46% knockdown was observed with 50 and 25 μg of ASO-005459, respectively. However, there was no significant reduction in SNCA mRNA expression when using 12.5 and 3.13 μg of ASO-005459. Possible explanations for the lower activity observed in the striatum are differences in ASO levels or the kinetics of SNCA mRNA knockdown.

Table 8

Tolerability of ASO-005459 in a 28-day study

Animal# Time point (day) Hyperactivity Vigilance Move. strength and coordination Posture/breathing Tremors/convulsions Total score 26 1 0 0 0 0 0 0 26 28 0 0 0 0 0 0 27 1 0 0 0 0 0 0 27 28 0 0 0 0 0 0 28 1 0 0 0 0 0 0 28 28 0 0 0 0 0 0 29 1 0 0 0 0 0 0 29 28 0 0 0 0 0 0 thirty 1 0 0 0 0 0 0 thirty 28 0 0 0 0 0 0

Levels of ASO-005459 were measured in all three brain regions of A53T-ASD mice. As shown in Fig. 24043828 7 and in table. 9 (below), brain effects were approximately proportional to dose and similar in all three regions, including the striatum. The effects of ASO-005459-response for different brain regions are shown in Fig. 8A-8D. Similar exposure-response relationships were observed in the hippocampus and brainstem, with estimated _IC50 values of 179 and 206 nM, respectively. In comparison, the exposure-response relationship in the striatum was relatively steep, indicating slower kinetics of decline in SNCA mRNA levels in this region. See fig. 8C.

Table 9

Summary of effects on the brain

ASO-005459 in the 14-day A53T-PAC study

Dose (mcg) Average (nM) SD Regarding the hippocampus Hippocampus 3.13 214 43 12.5 411 140 25 600 223 50 1916 807 Brain stem 3.13 160 47 0.7 12.5 266 47 0.6 25 440 102 0.7 50 707 202 0.4 Striatum 3.13 212 38 1.0 12.5 438 103 1.1 25 586 109 1.0 50 1332 302 0.7

To obtain more extensive dose-response/time data, ASO-005459 was reintroduced (0, 12.5, 25, or 50 μg) directly into the cerebral ventricles of A53T-PAC mice by freehand ICV injection. Animals were sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks post-dosing, and SNCA mRNA expression in the brainstem and striatum was assessed.

As shown in FIG. 9A and 9B (and consistent with the data presented above), administration of ASO-005459 to A53T-ASD mice resulted in a significant reduction in SNCA mRNA expression (relative to vehicle controls) in both the brainstem and striatum. The reduction appears to be both time- and dose-dependent, with a peak reduction (~90%) observed in the brainstem at 50 μg ASO-005459 approximately 4 weeks post-dosing (Figure 9A). In the striatum, a peak reduction (~55%) was observed with 50 μg of ASO-005459 approximately 3 days after dosing (Fig. 9B). SNCA mRNA expression levels remained significantly reduced compared to vehicle controls 4 weeks post-dosing (Figures 10A and 10B) and returned to baseline levels approximately 16 weeks post-dosing (Figures 9A and 9B).

As shown in FIG. 11A and 11B, administration of ASO-005459 to animals also resulted in a time- and dose-dependent decrease in SNCA protein levels in both brainstem and striatal brain tissue. A peak reduction (~75%) was observed in the brainstem at the 50 μg dose at 8 weeks post-dosing (Figure 11A). For striatal brain tissue, a peak reduction (~75%) was also observed at the 50 μg dose, but 4 weeks after dosing (Fig. 11B). Expression levels for individual mice 8 weeks post-dosing are presented in FIG. 12A (brain stem) and FIG. 12B (striatum). Although SNCA protein levels returned close to baseline levels approximately 12 weeks post-dosing in the striatum, expression levels in the brainstem were still significantly reduced (~25%) up to 16 weeks post-dosing.

Example 9. Analysis of the activity and in vivo tolerance of antisense oligonucleotides (ASOs) targeting SNCA in cynomolgus monkeys.

To further evaluate the activity and tolerability of ASO in vivo, a cynomolgus monkey model with an intrathecal port (Cyno IT) was developed. This model allows us to evaluate ASO-005459-mediated SNCA knockdown as well as potential 1-base pair mismatch knockdown of the nontargets PROS1 and IKZF3. The ASO-005459 target sites in SNCA, PROS1 and IKZF3 are completely conserved between humans and cynomolgus monkeys.

As described above in Example 6, each animal was implanted with an intrathecal cerebrospinal fluid (CSF) catheter inserted at the L3 or L4 vertebral level. ASO-005459 was dissolved in saline and administered to animals (8 mg/animal), infused over 4.5 min using the IT port (2 animals per dosing group). Animals were then euthanized 24 hours and 3 days after dosing, when tissues were collected for analysis of ASO exposure and activity. Analyze

- 25 043828 Brain regions targeted included medulla (Med), pons (V-Pons), midbrain (VMB), cerebellum (CBL), dorsal striatum (left and right) (CauP), hippocampus (left and right) ( femur), frontal cortex (left and right) (FrC), temporal cortex (left and right) (TeC), parietal cortex (left and right) (PaC), occipital cortex (left and right) (Occ) and cortical white matter (WM). In addition, spinal cords were collected from the cervical (CSC), thoracic (TSC), and lumbar (LSC) regions. Samples were also collected from the liver, kidney, heart, trigeminal nucleus, tibial nerve, and aorta to study off-target pharmacology in these areas.

Also, as observed in mice, ASO-005459 was well tolerated in cynomolgus monkeys without any adverse effects (data not shown). And as shown in table. 10 (below), administration of ASO-005459 showed robust knockdown of SNCA mRNA in various regions of the cynomolgus monkey brain.

Table 10

Effect of ASO-005459 on SNCA mRNA levels in the cynomolgus monkey brain

ASO No. Dose (mg) Time, point (weeks and) Me d NE L Fr C Ra S Sai R Those C Os with Hi P Vm in VPo ns CS with TS with LS C W m ASO- 0054 59 8 24 hours 174 140 14 6 149 137 73 8 24 hours 129 108 13 8 133 145 90 8 3 days 162 69 69 112 62 19 8 3 days 127 67 72 114 87 31 2 2 133 140 10 9 161 104 21 2 2 117 127 100 126 180 111 4 2 62 86 22 116 49 4 4 2 81 72 50 145 97 13 8 2 31 97 7 124 66 14 8 2 thirty 38 15 26 75 12 29 29 86 29 13 6 2 79 8 4 26 50 4 12 53 3 13 9 24 21 4 6 4 87 8 4 38 61 9 120 thirty 10 8 8 121 85 40 98 78 5 8 8 93 52 14 61 36 eleven 8 13 thirty 63 8 81 27 1 8 13 25 28 8 49 22 1 8 20 40 38 35 76 63 9 8 20 22 52 35 76 26 eleven

- 26 043828

To further characterize the reduction in SNCA mRNA described above, cynomolgus monkeys were dosed with ASO-005459 (total 8 mg per animal) and sacrificed 24 h, 3 days, 2, 4, 8, 13, or 20 weeks after dosing to assess SNCA mRNA expression levels. in various tissues. As shown in FIG. 13A, the maximum reduction was observed between 2 weeks and 13 weeks after dosing. Maximum reductions of 70, 65, 75, 35, 94 and 99% were observed in the medulla, cerebellum, pons, dorsal striatum, frontal and lumbar spinal cord, respectively. This decrease in SNCA mRNA expression level also correlates with a time-dependent decrease in SNCA protein expression level (Fig. 13B).

Next, to evaluate whether the reduction in expression levels in cynomolgus monkeys was also dose dependent, animals received 2, 4, or 8 mg of ASO-005459 and were then euthanized 2 weeks after dosing. As shown in FIG. 14A and 14B, the reduction in SNCA mRNA and SNCA protein expression levels was also dose dependent, with the greatest reduction observed at 8 mg of ASO-005459.

The results presented herein demonstrate that ASO-005459 is potent and selective in reducing SNCA mRNA and that ASO-005459 is well tolerated in neurons and in preclinical species in vivo. Moreover, the results obtained in A53T-PAC neurons confirm that ASO-005459-mediated mRNA reduction leads to decreased SNCA protein levels in vitro and in vivo. Additionally, results obtained in A53T-PAC mice and cynomolgus monkeys demonstrate that ASO-005459 reduces SNCA mRNA and SNCA protein levels in the brain at doses that are well tolerated. Taken together, these results support the continued development of ASO-005459 as a disease-modifying therapeutic for the treatment of synucleinopathies.

This PCT application claims priority to US Provisional Application 62/616937, filed January 12, 2018, which is incorporated herein by reference in its entirety.

Sequence Listing <110> BRISTOL-MYERS SQUIBB COMPANY

ROCHE INNOVATION CENTER COPENHAGEN A/C <120> Antisense oligonucleotides targeting alpha-synuclein and their applications <130> 3338.107PC01/ELE/C-K/DKC <150> 62/616,937 <151> 2018-01-12 <160> 10 <170> PatentIn version 3.5 <210>1 <211>2400 <212> DNA <213> Homo sapiens <400>1 gagataggga cgaggagcac gctgcaggga aagcagcgag cgccgggaga ggggcgggca60 gaagcgctga caaatcagcg gtgggggcgg agagccgagg agaaggagaa ggaggaggac120 taggaggagg aggacggcga cgaccagaag gggcccaaga gagggggcga gcgaccgagc180 gccgcgacgc ggaagtgagg tgcgtgcggg ctgcagcgca gaccccggcc cggcccctcc240 gagagcgtcc tgggcgctcc ctcacgcctt gccttcaagc cttctgcctt tccaccctcg300 tgagcggaga actgggagtg gccattcgac gacaggttag cgggtttgcc tcccactccc360 ccagcctcgc gtcgccggct cacagcggcc tcctctgggg acagtccccc ccgggtgccg420 cctccgccct tcctgtgcgc tccttttcct tcttctttcc tattaaatat tatttgggaa480 ttgtttaaat tttttttttta aaaaaagaga gaggcgggga ggagtcggag ttgtggagaa540

- 27 043828

gcagagggac tcaggtaagt acctgtggat ctaaacggggc gtctttggaa atcctggaga 600 acgccggatg ggagacgaat ggtcgtgggc accggggaggg ggtggtgctg ccatgaggac 660 ccgctgggcc aggtctctgg gaggtgagta cttgtccctt tggggagcct aaggaaagag 720 acttgacctg gctttcgtcc tgcttctgat attcccttct ccacaagggc tgagagatta 780 ggctgcttct ccgggatccg cttttccccg ggaaacgcga ggatgctcca tggagcgtga 840 gcatccaact tttctctcac aaaaatctg tctgcccgct ctcttggttt ttctctgtaa 900 agtaagcaag ctgcgtttgg caaataatga aatggaagtg caaggaggcc aagtcaacag 960 gtggtaacgg gttaacaagt gctggcgcgg ggtccgctag ggtggaggct gagaacgccc 1020 cctcgggtgg ctggcgcggg gttggagacg gcccgcgagt gtgagcggcg cctgctcagg 1080 gtagatagct gagggcgggg gtggatgttg gatggattag aaccatcaca cttggggcctg 1140 ctgtttgcct gagtttgaac cacaccccga gtgagcagtt agttctgttg cctacgcctt 1200 tccaccatca acctgttagc cttcttctgg gattcatgtt aaggatacc ctgaccctaa 1260 gcctccagct tccatgcttc taactcatac tgttaccctt tagaccccgg gaatttaaaa 1320 aaggggttaa tcttttcatg caactccact tctgaaatgc agtaataaca actcagga 1380 ttcatcctaa tccgtggtta ggtggctaga cttttactag ccaagatgga tggggagatgc 1440 taaattttta atgccagagc taaaaatgtc tgctttgtcc aatggttaaa tgagtgtaca 1500 cttaaaagag tctcacactt tggagggttt ctcatgattt ttcagtgttt tttgtttatt 1560 tttccccgaa agttctcatt caaagtgtat tttatgtttt ccagtgtggt gtaaaggaat 1620 tcattagcca tggatgtatt catgaaagga ctttcaaagg ccaaggaggg agttgtggct 1680 gctgctgaga aaaccaaaca gggtgtggca gaagcagcag gaaagacaaa aggggtgtt 1740 ctctatgtag gtaggtaaac cccaaatgtc agtttggtgc ttgttcatga gtgatggggtt 1800 aggataatca atactctaaa tgctggtagt tctctctctt gattcatttt tgcatcattg 1860 cttgtcaaaa aggtggactg agtcagaggt atgtgtaggt aggtgaatgt gaacgtgtgt 1920 atttgagcta atagtaaaaa atgcgactgt ttgcttttcc agatttttaa ttttgcccta 1980 atatttatga ctttttaaaa atgaatgttt ctgtacctac ataattctat ttcagagaac 2040 agttttaaaa actcatagtc ttttaaaaaa taatcaagaa tattcttaag aatcaaaatc 2100 attgatggat ctgtgatttc ttttaccatc atgaaaaatg tttgtcaatt ttaatccatt 2160 ctgattttta aaatatgact ttgatatgcc cctgtgatgt gtataaagag acctatttgt 2220 ggccctaaaa tggaaagaac agattagtct ttgatagagt tacttcatgt gatcatttgg 2280 tctctgtgaa cactgaggac agagaaaagt gcttgagggc tgctactaat ctctcagaaa 2340 catttgtata gttcatccat caaatgacac acatactaaa agaataaaga aattgatgct 2400

- 28 043828 <210> 2 <211> 3215 <212> DNA <213> Homo sapiens

<400> 2 aggagaagga gaaggaggag gactaggagg aggagaggacgg cgacgaccag aaggggccca 60 agagagggg cgagcgaccg agcgccgcga cgcggaagtg aggtgcgtgc gggctgcagc 120 gcagaccccg gccccggcccc tccgagagcg tcctgggcgc tccctcacgc cttgccttca 180 agccttctgc ctttccacc tcgtgagcgg agaactggga gtggccattc gacgacagtg 240 tggtgtaaag gaattcatta gccatggatg tattcatgaa aggactttca aaggccaagg 300 aggggagttgt ggctgctgct gagaaaacca aacagggtgt ggcagaagca gcaggaaaga 360 caaaagagg tgttctctat gtaggctcca aaaccaagga ggggagtggtg catggtgtgg 420 caacagtggc tgagaagacc aaagagcaag tgacaaatgt tggagggagca gtggtgacgg 480 gtgtgacagc agtagcccag aagacagtgg agggagcagg gagcattgca gcagccactg 540 gctttgtcaa aaaggaccag ttggggcaaga atgaagaagg agccccacag gaaggaattc 600 tggaagatat gcctgtggat cctgacaatg aggcttatga aatgccttct gaggaagggt 660 atcaagacta cgaacctgaa gcctaagaaa tatctttgct cccagtttct tgagatctgc 720 tgacagatgt tccatcctgt acaagtgctc agttccaatg tgccagtca tgacatttct 780 caaagttttt acagtgtatc tcgaagtctt ccatcagcag tgattgaagt atctgtacct 840 gccccccactc agcatttcgg tgcttccctt tcactgaagt gaatacatgg tagcagggtc 900 tttgtgtgct gtggattttg tggcttcaat ctacgatgtt aaaacaaatt aaaaacacct 960 aagtgactac cacttatttc taaatcctca ctattttttt gttgctgttg ttcagaagtt 1020 gttagtgatt tgctatcata tattataaga tttttaggtg tcttttaatg atactgtcta 1080 agaataatga cgtattgtga aatttgttaa tatatataat acttaaaaat atgtgagcat 1140 gaaactatgc acctataaat actaaatatg aaattttacc attttgcgat gtgttttatt 1200 cacttgtgtt tgtatataaa tggtgagaat taaaataaaa cgttatctca ttgcaaaaat 1260 attttatttt tatcccatct cactttaata aaaaaatca tgcttataag caacatgaat 1320 taagaactga cacaaaggac aaaaatataa agttattaat agccatttga agaaggaga 1380 attttagaag aggtagagaa aatggaacat taaccctaca ctcggaattc cctgaagcaa 1440 cactgccaga agtgtgtttt ggtatgcact ggttccttaa gtggctgtga ttaattattg 1500 aaagtggggt gttgaagacc ccaactacta ttgtagagtg gtctatttct cccttcaatc 1560 ctgtcaatgt ttgctttacg tattttgggg aactgttgtt tgatgtgtat gtgtttataa 1620 ttgttataca tttttaattg agccttttat taacatatat tgttattttt gtctcgaaat 1680

- 29 043828

aattttttag ttaaaatcta ttttgtctga tattggtgtg aatgctgtac ctttctgaca 1740 aaaataata ttcgaccatg aataaaaaaa aaaaaaaagt gggttcccgg gaactaagca 1800 gtgtagaaga tgattttgac tacaccctcc ttagagagcc ataagacaca ttagcacata 1860 ttagcacatt caaggctctg agagaatgtg gttaactttg tttaactcag cattcctcac 1920 tttttttttt taatcatcag aaattctctc tctctctctc tctttttctc tcgctctctt 1980 tttttttttt tttttacagg aaatgccttt aaacatcgtt ggaactacca gagtcacctt 2040 aaaggagatc aattctctag actgataaaa atttcatggc ctcctttaaa tgttgccaaa 2100 tatatgaatt ctaggatttt tccttaggaa aggtttttct ctttcaggga agatctatta 2160 actccccatg ggtgctgaaa aaaacttga tggtgaaaaa ctctgtataa attaatttaa 2220 aaattatttg gtttctcttt ttaattattc tggggcatag tcatttctaa aagtcactag 2280 tagaaagtat aatttcaaga cagaatattc tagacatgct agcagtttat atgtattcat 2340 gagtaatgtg atatatattg ggcgctggtg aggaaggaag gaggaatgag tgactataag 2400 gatggttacc atagaaactt ccttttttac ctaattgaag agagactact acagagtgct 2460 aagctgcatg tgtcatctta cactagag aaatggtaag tttcttgttt tatttaagtt 2520 atgtttaagc aaggaaagga tttgttattg aacagtatat ttcaggaagg ttagaagtg 2580 gcggttagga tatattttaa atctacctaa agcagcatat tttaaaaatt taaaagtatt 2640 ggtattaaat taagaaatag aggacagaac tagactgata gcagtgacct agaacaattt 2700 gagattagga aagttgtgac catgaattta aggatttatg tggatacaaa ttctccttta 2760 aagtgtttct tcccttaata tttatctgac ggtaattttt gagcagtgaa ttactttata 2820 tatcttaata gtttatttgg gaccaaacac ttaaacaaaa agttctttaa gtcatataag 2880 ccttttcagg aagcttgtct catattcact cccgagacat tcacctgcca agtggcctga 2940 ggatcaatcc agtcctaggt ttattttgca gacttacatt ctcccaagtt attcagcctc 3000 atatgactcc acggtcggct ttaccaaaac agttcagagt gcactttggc acacaattgg 3060 gaacagaaca atctaatgtg tggtttggta ttccaagtgg ggtctttttc agaatctctg 3120 cactagtgtg agatgcaaac atgtttcctc atctttctgg cttatccagt atgtagctat 3180 ttgtgacata aaaatatat acatatatga aaata 3215

<210>3 <211>140 <212> PROTEIN <213> Homo sapiens <400>3

Met Asp Val Phe Met Lys Gly Leu Ser Lys Ala Lys Glu Gly Val Val

5 1015

- 30 043828

Ala Ala Ala Glu Lys Thr Lys Gln 20

Gly Val Ala Glu Ala Ala Gly Lys 25 30

Thr Lys

Glu Gly Val Leu Tyr Val Gly

40

Val His

Gly Val Ala Thr Val Ala Glu

Asn Val 65

Gly Gly Ala Val Val Thr Gly 70

Ser Lys Thr Lys Glu Gly Val 45

Lys Thr Lys Glu Gln Val Thr 60

Val Thr Ala Val Ala Gln Lys 75 80

Thr Val Glu Gly

Ala Gly Ser Ile 85

Ala Ala Ala Thr

Gly Phe Val Lys 95

Lys Asp Gln Leu

100

Gly Lys Asn Glu

Glu Gly Ala Pro 105

Gln Glu Gly Ile 110

Leu Glu Asp Met

115

Pro Val Asp Pro

120

Asp Asn Glu Ala

Tyr Glu Met Pro 125

Ser Glu Glu Gly Tyr

130

Gln Asp Tyr Glu Pro Glu Ala

135 140 <210> 4 <211> 17 <212> DNA <213> Artificial sequence <220>

<223> Antisense oligonucleotide <400> 4 attcctttac accacac <210> 5 <211> 15 <212> DNA <213> Artificial sequence <220>

<223> Antisense oligonucleotide <400> 5 tctgtcttgg ctttg <210> 6 <211> 16 <212> DNA <213> Artificial sequence <220>

-

Claims (2)

<223> Antisense oligonucleotide <400>6 agaaataagt ggtagt <210>7 <211>20 <212> DNA <213> Artificial sequence <220> <223> Antisense oligonucleotide <400>7 ccaaatctta taataactac <210>8 <211>16 <212> DNA <213> Artificial sequence <220> <223> Antisense oligonucleotide <400>8 ttcctttaca cccac16 <210>9 <211>16 <212>DNA <213>Artificial sequence <220> <223> Antisense oligonucleotide <400>9 ggtgaggttt ggtaga16 <210>10 <211>18 <212> DNA <213> Artificial sequence <220> < 223 > Antisense oligonucleotide < 400 > 10 ggtgaggttt ggtagaag CLAIM 1. An antisense oligonucleotide (ASO) containing or consisting of the contiguous nucleotide sequence AtTcctttacaccACAC (SEQ ID NO: 4), where the capital letter represents beta-D-hydroxy-LNA and the lowercase letter represents DNA. 2. ASO according to claim 1, which contains an internucleotide linkage selected from the group consisting of a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoramidate linkage, phos -