EA046118B1 - ANTISENSE OLIGONUCLEOTIDES TARGETING ALPHA-SYNUCLEIN AND THEIR APPLICATIONS - Google Patents

Description

Link to sequence listing provided in electronic form

The contents of the electronically submitted sequence listing (title: 3338.109PC01_SequenceListing_ST25.txt, size: 13817 bytes; and creation date: January 10, 2019) provided in this application are incorporated herein by reference in their entirety.

Field of technology to which the present invention relates

The present disclosure relates to antisense oligomeric compounds (ASOs) that target the intron-exon junction of the alpha-synuclein (SNCA) transcript in a cell, resulting in decreased expression of the alpha-synuclein (SNCA) protein. Decreased expression of the SNCA protein may have beneficial effects on a number of medical disorders, such as multiple system atrophy, Parkinson's disease, Parkinson's disease dementia (PDD), and dementia with Lewy bodies.

Prior art of the present invention

Alpha-synuclein (SNCA), a member of the synuclein family of proteins, is a small soluble protein that is expressed predominantly in tissues of the nervous system. 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 its exact function remains to be elucidated, SNCA is thought to play an important role in the regulation of synaptic transmission. For example, SNCA functions as a molecular chaperone in the formation of SNARE complexes that mediate the docking of synaptic vesicles to presynaptic neutron membranes. SNCA can also interact with other proteins, such as the microtubule-associated protein tau, to help stabilize microtubules and regulate vesicle trafficking.

Because of SNCA's role in regulating synaptic transmission, alterations in SNCA expression and/or function can disrupt critical biological processes. Such abnormalities are thought to contribute to the α-synucleopathies, which are neurodegenerative diseases characterized by the abnormal accumulation of SNCA protein aggregates in the brain. Accordingly, insoluble inclusion bodies of misfolded, aggregated, and phosphorylated SNCA protein are a pathological marker for diseases such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). See Galvin JE et al., Archives of Neurology 58: 186–190 (2001); and Valera E et al., J Neurochem 139 Suppl 1: 346–352 (Oct. 2016).

α-Synucleopathies, such as Parkinson's disease, are common progressive neurodegenerative disorders of the brain, particularly in older adults. See, Recchia A et al., FASEB J. 18: 617-26 (2004). It is estimated that approximately seven to ten million people worldwide live with these disorders, with approximately 60,000 new cases reported each year in the United States alone. The cost of treatment for an individual can easily exceed $2,500 per year, and therapeutic surgery can cost as much as $100,000 per patient. Thus, there is a great need for more reliable and cost-effective treatment options.

Brief summary of the present invention

The present disclosure is directed to an antisense oligonucleotide (ASO) comprising a contiguous nucleotide sequence of 10-30 nucleotides in length that is complementary to a nucleic acid sequence within an alpha-synuclein (SNCA) transcript, wherein the nucleic acid sequence comprises nucleotides 7592-7637 of SEQ ID NO: 1. According to some embodiments, the nucleic acid sequence comprises nucleotides 7602-7627 of SEQ ID NO: 1. According to other embodiments, the nucleic acid sequence corresponds to nucleotides 7603-7620 of SEQ ID NO: 1. According to certain embodiments, the nucleic acid sequence corresponds to nucleotides 7604-7620 of SEQ ID NO: 1. According to some embodiments, the SNCA transcript comprises SEQ ID NO: 1. According to some embodiments, the contiguous nucleotide the sequence comprises a sequence represented by any of SEQ ID NOs: 4-21, with one, two, three or four mismatches. According to other embodiments, the contiguous nucleotide sequence comprises a sequence represented by any of SEQ ID NOs: 4-21. According to certain embodiments, the contiguous nucleotide sequence comprises SEQ ID NO: 15 or SEQ ID NO: 7.

According to some embodiments, an antisense oligonucleotide of the present disclosure is capable of inhibiting the expression of a human SNCA transcript in a cell that expresses a human SNCA transcript.

According to some embodiments, the continuous nucleotide sequence comprises at least one nucleotide analogue. According to some embodiments, the antisense oligonucleotide is a gapmer. According to other

- 1 046 118 In other embodiments, the antisense oligonucleotide is a gapmer with alternating nucleotides in the flanking regions. According to some embodiments, the antisense oligonucleotide as disclosed herein has the formula 5'-A-B-C3', wherein (i) region B is a continuous sequence of at least 6 DNA units that are capable of attracting RNase; (ii) region A is a first wing sequence of 1-10 nucleotides, wherein the first wing sequence comprises one or more nucleotide analogues and, optionally, one or more DNA units, and wherein at least one of the nucleotide analogues is located at the 3' end of A; and (iii) region C is a second wing sequence of 1-10 nucleotides, wherein the second wing sequence comprises one or more nucleotide analogs and, optionally, one or more DNA units, and wherein at least one of the nucleotide analogs is located at the 5' end of C. According to some embodiments, region A comprises a combination of nucleotide analogs and DNA units selected from (i) 1-10 nucleotide analogs and 0 DNA units; (ii) 1-9 nucleotide analogs and 1 DNA unit; (iii) 1-8 nucleotide analogs and 1-2 DNA units; (iv) 1-7 nucleotide analogs and 1-3 DNA units; (v) 1-6 nucleotide analogs and 1-4 DNA units; (vi) 1-5 nucleotide analogs and 1-5 DNA units; (vii) 1-4 nucleotide analogs and 1-6 DNA units; (viii) 1-3 nucleotide analogs and 1-7 DNA units; (ix) 1-2 nucleotide analogs and 1-8 DNA units; and (x) 1 nucleotide analog and 1-9 DNA units. According to some embodiments, region C comprises a combination of nucleotide analogs and DNA units selected from (i) 1-10 nucleotide analogs and 0 DNA units; (ii) 1-9 nucleotide analogs and 1 DNA unit; (iii) 1-8 nucleotide analogs and 1-2 DNA units; (iv) 1-7 nucleotide analogs and 1-3 DNA units; (v) 1-6 nucleotide analogs and 1-4 DNA units; (vi) 1-5 nucleotide analogs and 1-5 DNA units; (vii) 1-4 nucleotide analogs and 1-6 DNA units; (viii) 1-3 nucleotide analogs and 1-7 DNA units; (ix) 1-2 nucleotide analogs and 1-8 DNA units; and (x) 1 nucleotide analog and 1-9 DNA units. According to some embodiments, region A is a first wing pattern selected from any of the ASOs in Figs. 2 and 3 and/or region C is a second wing pattern selected from any of the ASOs in Figs. 2 and 3, wherein the uppercase letter represents a nucleoside analog and the lowercase letter represents DNA.

According to some embodiments, an antisense oligonucleotide of the present disclosure comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleotide analogs. According to certain embodiments, the nucleotide analog or analogs are one or more sugar-modified nucleosides selected from the group consisting of locked nucleic acid (LNA); 2'-O-alkyl RNA; 2'-amino DNA; 2'-fluoro DNA; arabino nucleic acid (ANA); 2'-Φτορ-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), a conformationally hindered ethyl nucleoside analog (cEt), 2'-O-methyl nucleic acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), and any combination thereof. In some embodiments, the nucleotide analog or analogs comprise a bicyclic sugar. In certain embodiments, the bicyclic sugar comprises cEt, 2',4'-sterically hindered 2'-O-methoxyethyl (cMOE), LNA, α-L-LNA, P-D-LNA, 2'-O,4'-C-ethylene bridged nucleic acids (ENA), amino-LNA, oxy-LNA, or thio-LNA. According to some embodiments, the nucleotide analog or analogs comprise LNA.

According to some embodiments, the antisense oligonucleotide comprises one or more 5'-methylcytosine nucleobases. According to some embodiments, the antisense oligonucleotide comprises two to five LNAs in the 5' region of the antisense oligonucleotide. According to some embodiments, the antisense oligonucleotide comprises two to five LNAs in the 3' region of the antisense oligonucleotide.

According to some embodiments, the antisense oligonucleotide has an in vivo tolerability of less than or equal to a total score of 4, wherein the total score is the sum of the scores of five categories that represent 1) hyperactivity; 2) decreased activity and agitation; 3) motor dysfunction and/or ataxia; 4) abnormal body posture and breathing; and 5) tremors and/or convulsions, and wherein the score for each category is measured on a scale of 0-4. According to certain embodiments, the in vivo tolerability is less than or equal to a total score of 3, a total score of 2, a total score of 1, or a total score of 0.

According to some embodiments, the antisense oligonucleotide reduces the expression of SNCA mRNA in a cell by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, compared to a cell not exposed to the antisense oligonucleotide. According to

- 2 046118 in other embodiments, the antisense oligonucleotide reduces the expression of SNCA protein in a cell by at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a cell not exposed to the antisense oligonucleotide.

According to some embodiments, the antisense oligonucleotide comprises nucleotides A, T, C, and G and at least one analog of nucleotides A, T, C, and G and has a sequence score greater than or equal to 0.2, wherein the sequence score is calculated using formula I:

number of nucleotides C and their analogues - number of nucleotides G and their analogues (I) total length in nucleotides.

According to some embodiments, the antisense oligonucleotide is from 10 to 24 nucleotides in length or is from 14 to 21 nucleotides in length.

According to other embodiments, the antisense oligonucleotide has a length of 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

According to some embodiments, the nucleotide sequence of the antisense oligonucleotide comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NOs: 4-21, with a pattern selected from the group consisting of the patterns in Figs. 2 and 3, wherein the uppercase letter represents a nucleoside with a modified sugar and the lowercase letter represents DNA. According to certain embodiments, the nucleotide sequence comprises, consists essentially of, or consists of SEQ ID NO: 15 with the pattern ASO-005459 and SEQ ID NO: 7 with the pattern ASO-005578 or ASO-005584. According to other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NOs: 4-21, with the corresponding chemical structure in Fig. 2 and 3. According to certain embodiments, the nucleotide sequence comprises, consists essentially of, or _consists of ASO-005459, ASO005578, and ASO _{- 005584} . According to some embodiments _, the _{ASO of} the present disclosure _has the molecular formula _{C171H214N56O90P16S16 . According} to some embodiments _, the ASO has the molecular formula _{C182H229N58O96P17S17} . According to _other embodiments, the ASO _{has the} molecular formula _{C181H227N58O96P17S17 .} According to some embodiments, an antisense oligonucleotide of the present disclosure comprises an internucleoside bridge selected from: a phosphodiester bridge, a phosphotriester bridge, a methylphosphonate bridge, a phosphoramidate bridge, a phosphorothioate bridge, and combinations thereof. According to certain embodiments, the internucleoside bridge comprises one or more stereochemically defined modified phosphate bridges.

The present disclosure also relates to a conjugate comprising an antisense oligonucleotide as disclosed herein, wherein the antisense oligonucleotide is covalently attached to at least one non-nucleotide moiety or moiety other than a polynucleotide. According to some embodiments, the non-nucleotide moiety or moiety other than a polynucleotide comprises a protein, a fatty acid chain, a sugar moiety, a glycoprotein, a protein, or any combination thereof.

Also provided herein is a pharmaceutical composition comprising an antisense oligonucleotide or conjugate as disclosed 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 alpha-synuclein antibody or fragment thereof.

The present disclosure further provides a kit comprising an antisense oligonucleotide, conjugate or composition as disclosed herein. Also disclosed is a diagnostic kit comprising an antisense oligonucleotide, conjugate or composition according to the present disclosure. The present disclosure is also directed to a method of inhibiting or reducing the expression of a SNCA protein in a cell, wherein the method comprises administering an antisense oligonucleotide, conjugate or composition as disclosed herein to a cell expressing a SNCA protein, wherein the expression of the SNCA protein in the cell is inhibited or reduced upon administration. According to some embodiments, the antisense oligonucleotide inhibits or reduces the expression of SNCA mRNA in the cell upon administration. According to certain 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 about 70%, at least about 80%, at least about 90%, or approximately

- 3 046 118 by 100% after administration compared to a cell not exposed to the antisense oligonucleotide. According to other embodiments, the antisense oligonucleotide reduces the expression of the SNCA protein in the cell after administration by at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to a cell not exposed to the antisense oligonucleotide. According to some embodiments, the cell is a neuron.

Provided herein is a method of treating a synucleopathy in a subject in need thereof comprising administering an effective amount of an antisense oligonucleotide, conjugate or composition of the present disclosure. According to some embodiments, the synucleopathy is selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), multiple system atrophy, Lewy body dementia and any combinations thereof. Also provided herein is the use of an antisense oligonucleotide, conjugate or composition of the present disclosure for the manufacture of a medicament. The present disclosure also relates to the use of an antisense oligonucleotide, conjugate or composition for the manufacture of a medicament for the treatment of a synucleopathy in a subject in need thereof. According to some embodiments, an antisense oligonucleotide, conjugate or composition of the present disclosure is for use in the therapy of a synucleopathy in a subject in need thereof. According to other embodiments, the antisense oligonucleotide, conjugate, or composition of the present disclosure is for use in therapy. According to some embodiments, the subject is a human. According to some embodiments, the antisense oligonucleotide, conjugate, or composition is administered orally, parenterally, intrathecally, intraventricularly, pulmonarily, locally, or intraventricularly. According to some embodiments, the nucleotide analog comprises a nucleoside with a modified sugar. According to some embodiments, the nucleoside with a modified sugar is an affinity enhancing nucleoside with a modified sugar.

Brief description of the drawings

Fig. 1A, 1B and 1C show the genomic sequence (partial) of alpha-synuclein, the mRNA and the protein sequence of alpha-synuclein. The sequence in Fig. 1A is the partial genomic sequence of SNCA (i.e., residues 6001 to 8400 in SEQ ID NO: 1). SEQ ID NO: 1 is identical to the sequence of SNCA pre-mRNA except that nucleotide t in SEQ ID NO: 1 is shown as u in pre-mRNA. SEQ ID NO: 2 in Fig. 1B is the sequence of SNCA mRNA except that nucleotide t in SEQ ID NO: 2 is shown as u in mRNA. Fig. 1C shows the sequence of SNCA protein (SEQ ID NO: 3).

Fig. 2 shows exemplary ASOs that target the intron-exon junction in SNCA pre-mRNA. Each column in Fig. 2 shows a sequence ID number (SEQ ID No.) assigned only to that sequence, the positions of the start and end of the target region on the SNCA pre-mRNA sequence, the positions of the start and end of the target region on the SNCA mRNA sequence, a design number (DES No.), an ASO sequence with a design, an ASO number (ASO No.), and an ASO sequence with a chemical structure.

Figure 3 shows an ASO targeting the intron-exon junction of SNCA pre-mRNA with an illustrative wing pattern modification. Each column in Figure 3 shows a sequence ID number (SEQ ID No.) assigned only to that sequence, the positions of the start and end of the target region on the SNCA pre-mRNA sequence, a pattern number (DES No.), an ASO sequence with pattern, an ASO number (ASO No.), and an ASO sequence with the chemical structure and wing pattern modification identified. DES-316392, DES-316393, DES-316394, and DES-316395 show different ASO schemes possible for SEQ ID NO: 7. DES-287031, DES287957, DES-287959, DES-287962, DES-288906, and DES-313413 show different ASO schemes possible for SEQ ID NO: 12. DES-319536, DES-319537, DES-325058, DES-325059, DES-325060, DES325061, DES-325062, DES-325063, DES-325064, DES-325065, DES-325066, DES-325067, DES-325068, and DES-325069 show different ASO patterns possible for SEQ ID NO: 14. DES-324636, DES324637, DES-324638, DES-324639, DES-324640, DES-324641, DES-324642, DES-324643, DES-324644, DES-324645, DES-324646, and DES324647 show different ASO patterns possible for SEQ ID NO: 15. For the ASO patterns, uppercase letters denote nucleotide analogs (e.g., LNA or 2'O-methyl (OMe)) and lowercase letters denote DNA nucleotides. Capital letters with or without underlining indicate that the two letters may represent different nucleotide analogs, such as LNA and 2'-O-methyl. For example, capital letters with underlining may represent 2'-O-methyl, while capital letters without underlining represent LNA. In the chemical structure column of ASO, OMe represents 2'-O-methyl nucleoside, L represents LNA, D represents DNA, and the numbers after L or D indicate the number of nucleotides in LNA or DNA.

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Figure 4 shows the percentage reduction in SNCA protein expression in both the SK-N-BE(2) human neuroblastoma cell line (SK cells) and primary neurons isolated from A53T-RAS transgenic mice (RAS neurons) after in vitro culture with different ASOs as described in Example 2A for RAS neurons and in Example 2E for SK cells. For SK cells, cells were treated with 25 μM ASO, and SNCA mRNA expression (normalized to GAPDH) is shown as a percentage of the control value. For RAS neurons, cells were treated with either 40 nM or 5 μM ASO, and SNCA protein expression (normalized to tubulin) is shown as a percentage inhibition. Where a value is not shown, the particular ASO was not tested under the particular conditions.

Figure 5 shows the activity of various ASOs in reducing SNCA protein expression in primary neurons isolated from A53T-RAS transgenic mice in vitro. As described in Example 2A, RAS neurons were cultured in vitro with 10 successively decreasing concentrations of various ASOs, and the activity (IC ₅₀ ) of the ASOs is shown as the ratio of SNCA to tubulin expression (μM).

Figure 6 shows the tolerance score (Tox Score) and the percentage reduction (or knockdown KD) of SNCA mRNA and SNCA protein expression in ASO-treated A53T-PAC transgenic or WT mice (see Example 4). Tolerance scores are shown for 1 day (ID) and 28 days (28D) after ASO administration. The percentage reduction of SNCA mRNA and SNCA protein expression is shown for 3 days and 28 days after ASO administration in the hippocampus (Hippo), brainstem (BS), and striatum (Str).

Figures 7A and 7B show the effects of ASO-005459 on SNCA and tubulin (Tub) protein expression in primary neurons isolated from A53T-PAC transgenic mice. Neurons were treated with 10 sequentially decreasing concentrations of ASO-005459 and SNCA and tubulin protein amounts were measured. Percent inhibition as α-synuclein/tubulin ratio (Figure 7A) and tubulin levels (Figure 7B) are shown. Each data point in the graph represents an individual replicate.

Figure 8 shows the effects of ASO-005459 on the mRNA expression levels of SNCA (circle), S protein (alpha) (PROS1 (square)) and tubulin (TUBB3 (triangle)) in human neurons. Neurons were treated with varying concentrations of ASO-005459 for 6 days and mRNA levels were then measured using the QUANTIGENE® assay. mRNA expression levels are shown as a percentage of the control value. Data shown are the mean ±SD of duplicate determinations.

Figure 9 shows SNCA mRNA expression levels in the hippocampus of A53T-PAC mice three days after ICV administration of 100 μg ASO-005459 (open square) or vehicle control (filled circle). SNCA mRNA expression levels were measured by qRT-PCR, normalized to GAPDH mRNA, and then expressed relative to the mean expression level in the vehicle group. The horizontal line represents the 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). Data shown are the mean ± SD of duplicate determinations. Statistics shown are based on one-way ANOVA with Dunnett's post hoc test. ***p<0.001.

Figures 10A and 10B show a comparison of the mean body weights of mice treated with ASO-005459. In Figure 10A, A53T-PAC mice were dosed with 3.13 μg, 12.5 μg, 25 μg, or 50 μg of ASO-005459 and their body weights were measured at 0, 1, and 2 weeks post-treatment. In Figure 10B, C57BL/6 mice were dosed with 100 μg of ASO-005459 and their body weights were measured once weekly for 28 days. In both Figures 10A and 10B, vehicle control-treated animals served as controls. Data shown are the mean ±SD from multiple animals (n=5). Statistics shown are based on two-way analysis of variance.

Figures 11A, 11B, and 11C show SNCA mRNA expression levels in the hippocampus (Figure 11A), brainstem (Figure 11B), and striatum (Figure 11C) of A53T-PAC mice 14 days after ICV administration of ASO-005459 (3.13 μg, 12.5 μg, 25 μg, or 50 μg) or vehicle control. SNCA mRNA levels were measured by qRT-PCR, normalized to GAPDH mRNA, and then expressed relative to the mean of the vehicle group. Data shown are the mean ± SD of multiple animals (n=5). Each circle represents an individual animal. Horizontal lines represent the 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). Statistics shown are based on one-way ANOVA with Dunnett's post hoc test. ***p<0.001.

Figure 12 shows the level of ASO-005459 detected in the hippocampus (black), brainstem (light gray), and striatum (dark gray) of A53T-PAC mice 14 days after ASO-005459 treatment. Mice received 3.13 μg, 12.5 μg, 25 μg, or 50 μg of ASO-005459 via ICV administration. Data shown represent the mean ±SD from multiple animals (n=5).

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Figures 13A, 13B, 13C, and 13D show the relationship between ASO-005459 exposure levels and SNCA mRNA expression in the hippocampus (Figure 13A), brainstem (Figure 13B), and striatum (Figure 13C) of A53T-PAC mice fourteen days after ASO-005459 treatment. Figure 13D shows data from the hippocampus (circle), brainstem (square), and striatum (triangle) in combination. Each data point in the graph represents an individual animal. Four-parameter nonlinear fits are shown for the hippocampus (Figure 13A) and brainstem (Figure 13B).

Figures 14A and 14B show dose-response curves for the effects of ASO-005459 on SNCA mRNA expression levels in A53T-PAC mice. Animals were given (via ICV injection) 12.5 μg (circle), 25 μg (square), or 50 μg (triangle) of ASO-005459 and sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks post-dose. SNCA mRNA expression levels in both the brainstem (Figure 14A) and striatum (Figure 14B) were assessed by qRT-PCR and then normalized to vehicle controls. The mean of the data is shown. The horizontal line represents the reference value of 100% (i.e. the value at which SNCA mRNA expression would be equivalent to the expression level observed in the medium control group).

Figures 15A and 15B show the effects of ASO-005459 on SNCA mRNA expression levels in A53TPAC mice 4 weeks after ASO-005459 administration. Animals received either vehicle control or variable concentrations of ASO-005459 (12.5, 25, or 50 μg). Relative SNCA mRNA expression levels (normalized to vehicle control) are shown for both the brainstem (Figure 15A) and striatum (Figure 15B). Each data point represents an individual animal. The mean ±SD from multiple animals is also shown. Statistics shown are based on comparisons with the vehicle group using one-way ANOVA with Dunnett's correction for multiple comparisons. The horizontal lines represent the reference value of 1 (i.e., the value at which SNCA mRNA expression would be equivalent to the expression level observed in the medium control group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figures 16A and 16B show dose-response curves for the effects of ASO-005459 on SNCA protein expression levels in brain tissue of A53T-PAC mice. Animals were given (via ICV injection) 12.5 μg (circle), 25 μg (square), or 50 μg (triangle) of ASO-005459 and sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks post-dose. SNCA protein levels were measured in both the brainstem (Figure 16A) and striatum (Figure 16B) by ELISA and then normalized to vehicle control. The mean of the data is shown. The horizontal line represents the reference value of 100% (i.e. the value at which SNCA mRNA expression would be equivalent to the expression level observed in the medium control group).

Figures 17A and 17B show the effects of ASO-005459 on SNCA protein expression levels in A53TPAC mice 8 weeks after ASO-005459 administration. Animals received either vehicle control or variable concentrations of ASO-005459 (12.5, 25, or 50 μg). Relative SNCA protein expression levels (normalized to vehicle control) are shown for both the brainstem (Figure 17A) and striatum (Figure 17B). Each data point in the graph represents an individual animal. The horizontal line represents the 100% reference value (i.e., 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. Statistics shown are based on comparisons to the vehicle group using one-way ANOVA with Dunnett's correction for multiple comparisons.

Figures 18A and 18B show the effects of ASO-005578 and ASO-005584 on SNCA mRNA expression levels in cynomolgus monkey brain tissue. In Figure 18A, animals received one of vehicle control (circle), ASO-005578 (square, 4 mg), or ASO-005584 (triangle, 4 mg). The animals were then sacrificed 2 weeks after dosing. In Figure 18B, animals received either vehicle control or ASO-005584 (8 mg per animal) and were then sacrificed 2 weeks (square) and 4 weeks after dosing (triangle). In both Figures, 18A and 18B were used to assess SNCA mRNA expression levels in the following tissues: medulla oblongata (upper left section), caudate putamen (upper middle section), pons (upper right section), cerebellum (upper left section), lumbar spinal cord (lower middle section), and frontal cortex (lower right section). SNCA mRNA expression levels shown in Figs. 18A and 18B were normalized to GAPDH and are presented relative to the mean of the vehicle control group. Both individual animal data and the mean are shown. The horizontal line represents the 100% reference value (i.e., the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control group).

Figures 19A and 19B show the dynamics of SNCA mRNA and SNCA protein expression levels in cynomolgus monkeys following ASO-005459 administration. Each animal received either vehicle control or ASO-005459 (8 mg) and was then sacrificed at 24 h, 3 days, 2, 4, 8, 13, or 20 weeks after administration.

- 6,046,118 doses. At each time point, SNCA mRNA (Fig. 19A) and SNCA protein (Fig. 19B) expression levels were assessed in the following tissues: medulla (upper left section), dorsal striatum (upper middle section), pons (upper right section), cerebellum (upper left section), lumbar spinal cord (lower middle section), and frontal cortex (lower right section). Expression levels are presented as percentages relative to vehicle control. Both individual animal data and the mean are shown. The horizontal line represents the 100% reference value (i.e., the value at which SNCA mRNA expression would be equivalent to the expression level observed in the vehicle control).

Figures 20A and 20B show the relative expression levels (as percentages relative to vehicle control) of both SNCA mRNA (Figure 20A) and SNCA protein (Figure 20B) in cynomolgus monkeys 2 weeks after administration of ASO-005459. Animals received either vehicle control or variable concentrations of ASO-005459 (2, 4, or 8 mg). Expression levels were assessed in the following tissues: medulla (upper left section), dorsal striatum (upper middle section), pons (upper right section), cerebellum (upper left section), lumbar spinal cord (lower middle section), and frontal cortex (lower right section). Expression levels are presented as percentages relative to vehicle control. Both individual animal data and mean values are shown. The horizontal line represents the reference value of 100% (i.e. the value at which SNCA mRNA expression would be equivalent to the expression level observed in the medium control group).

Fig. 21A shows the contiguous nucleotide sequence of ASO-005459. OxyA, OxyT, and OxyMC represent adenine-beta-D-oxy-LNA, thymine-beta-D-oxy-LNA, and methylcytosine-beta-Doxy-LNA, respectively; DNAt, DNAc, and DNAa represent DNA, cytosine DNA, and adenine DNA, respectively; and s represents a phosphorothioate bridge.

Fig. 21B shows the molecular structure of ASO-005459, which is disclosed herein. The disclosed structure has a _{molecular formula} of _{Ci7iH2i4N56O90Pi6Si6} , and each M+ is a pharmaceutically acceptable counterion such as H ⁺ , Na ^+, or _NH4 ⁺ .

Detailed disclosure of the present invention

I. Definitions.

It should be noted that the singular form of a term refers to one or more units of the term; for example, the term "nucleotide sequence" should be understood to represent one or more nucleotide sequences. In this regard, the terms "one" (or some), "one or more" and "at least one" may be used interchangeably herein. Moreover, when and/or is used herein, it should be understood as specifically disclosing each of two specified features or components, with or without the other feature or component. Thus, as used herein, the term "and/or" when used in a phrase such as "A and/or B" is intended to include A and B, "A" or "B", "A" (alone) and "B" (alone). Similarly, the term "and/or" when used in a phrase such as "A, B" and/or "C" is intended to cover each of the following: "A, B" and "C"; "A, B" or "C"; "A" or "B"; "B" or "C"; "A" and "B"; "A" and "B"; "A" (alone); In (alone) and C (alone). It should be understood that in all cases where aspects are described in this document with the expression comprising, other similar aspects described by the terms consisting of and/or consisting essentially of are also intended.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

Units, prefixes, and symbols are in International System of Units (SI) form. Numeric ranges include the numbers defining the range. Unless otherwise noted, nucleotide sequences are written left to right in a 5'-3' orientation. Amino acid sequences are written left to right in an amino- to carboxy-terminal orientation. The section headings provided herein are not limitations on the various aspects of the disclosure as they might have when applied to this specification as a whole. Accordingly, terms defined immediately below are more fully defined by reference to this specification in its entirety.

The term approximately, as used in the context of this document, means approximately, rounded, about, or in the vicinity of. When the term approximately is used in conjunction with a numerical range, it modifies the range by extending its boundaries above and below the stated numerical values. Generally, the term approximately may modify a numerical value above and below the stated value by a deviation of, for example, 10 percent, up or down (above or below)

- 7 046118 below). For example, if it is stated that an ASO reduces the expression of SNCA protein in a cell after administration of the ASO by at least about 60%, it is meant 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 via internucleoside bridges. ASOs useful in the present disclosure include at least one non-naturally occurring nucleoside. The ASO is complementary to a 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 terms nucleic acids or nucleotides are intended to include a plurality of nucleic acids. According to some embodiments, the terms nucleic acids or nucleotides refer 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. According to other embodiments, nucleic acids or nucleotides refer to a sequence in an ASO according to the present disclosure. When the term refers to a sequence in an ASO, the nucleic acids or nucleotides are non-naturally occurring, i.e., chemically synthesized, produced by enzymatic reactions, produced by recombinant DNA technology, or any combination thereof. According to one embodiment, the nucleic acids or nucleotides in the ASO are produced by synthesis or by recombinant DNA technology and are not a naturally occurring sequence or fragment thereof. According to another embodiment, the nucleic acids or nucleotides in the ASO are not naturally occurring because they comprise at least one nucleotide analog that is not naturally occurring. The terms nucleic acid or nucleoside refer to a single segment of a nucleic acid, such as DNA, RNA, or an analog thereof, present in a polynucleotide. A nucleic acid or nucleoside includes naturally occurring nucleic acids or non-naturally occurring nucleic acids. According to 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, which is referred to as a sequence of bases such as A, T, G, C, or U, and analogs thereof. The term "nucleotide" as used herein refers to a glycoside comprising a sugar moiety, a base moiety, and a covalently linked group (bridging group), such as phosphate or phosphorothioate internucleotide bridging groups, and encompasses both naturally occurring nucleotides such as DNA or RNA and non-naturally occurring nucleotides comprising a modified sugar and/or base, which are also referred to herein as nucleotide analogs. As used herein, a single nucleotide (unit) may also be referred to as a monomer or unit of a nucleic acid. According to certain embodiments, the term nucleotide analogs refers to nucleotides having modified sugar moieties. Non-limiting examples of nucleotides having modified sugar moieties (e.g., LNA) are disclosed elsewhere herein. According to other embodiments, the term nucleotide analogs refers to nucleotides having modified nucleotide base moieties. Nucleotides having modified nucleotide base fragments include, but are not limited to, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine.

The term nucleoside, as used in the context of this document, refers to a glycoside comprising a sugar moiety and a base moiety that may be covalently linked via internucleotide bridges between nucleosides in an ASO. In the field of biotechnology, the term nucleoside often refers to a monomer or unit of a nucleic acid. In the context of an ASO, the term nucleoside may refer only to a base, i.e., a sequence of nucleotide bases comprising 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 bridges. Likewise, particularly in the case of oligonucleotides in which one or more of the internucleotide bridge groups are modified, the term nucleotide may refer to a nucleoside. For example, the term nucleotide may even be used when specifying the presence or nature of bridges between nucleosides.

The term nucleotide length as used in the context of this document means the total number of nucleotides (monomers) in a given sequence. For example, the sequence AtTcctttacaccACAC (SEQ ID NO: 15) has 17 nucleotides; therefore, the nucleotide length of the sequence is

- 8 046118 tids is 17. Thus, the term length in nucleotides is used in this document interchangeably with the number of nucleotides.

As will be appreciated by one skilled in the art, the 5' terminal nucleotide in an oligonucleotide does not contain a 5' internucleotide bridging group, although it may contain a 5' terminal end group.

As used herein, a coding region or coding sequence is a portion of a polynucleotide that consists of codons that are translated into amino acids. Although a stop codon (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, untranslated regions (UTRs), etc., are not part of the coding region. The boundaries of the coding region are typically defined by a start codon at the 5' end, encoding the amino terminus of the resulting polypeptide, and a translational stop codon at the 3' end, encoding the carboxyl terminus of the resulting polypeptide.

The term non-coding region, as used herein, means a nucleotide sequence that does not constitute a coding region. Examples of non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcription terminators, introns, untranslated regions (UTRs), non-coding exons, and the like. Some of the exons may comprise, or be part of, the 5' untranslated region (5' UTR) or 3' untranslated region (3' UTR) of each transcript. The untranslated regions are important for efficient translation of the transcript and for controlling the rate of translation and half-life of the transcript.

The term region, when used in the context of a nucleotide sequence, refers to a portion of that sequence. For example, the phrase region in a nucleotide sequence or region in the complementary strand of a nucleotide sequence refers to a sequence that is shorter than the nucleotide sequence but longer than at least 10 nucleotides located within the particular nucleotide sequence or complementary strand of the nucleotide sequence, respectively. The terms subsequence or subsequence may also refer to a region in a nucleotide sequence.

The term "downstream" when applied to a nucleotide sequence means that the nucleic acid or nucleotide sequence is located at the 3' end of the nucleotide sequence in question. According to certain embodiments, downstream nucleotide sequences refer to sequences that are located after the transcription start point. For example, the translation initiation codon of a gene is located downstream of the transcription start site.

The term above refers to a nucleotide sequence that is located 5' of the referenced nucleotide sequence. Unless otherwise indicated, sequences provided herein are listed from 5' (left) to 3' (right).

As used herein, the term regulatory region refers to nucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region and that influence the transcription, processing, stability, or translation of the RNA of the associated coding region. Regulatory regions may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector molecule binding sites, UTRs, and stem-loop structures. If the coding region is intended for expression in a eukaryotic cell, the polyadenylation signal and transcription termination sequence will typically be located 3' to the coding sequence. The term transcript as used in the context of this document can refer to the primary transcript that is synthesized during DNA transcription and becomes messenger RNA (mRNA) after processing, i.e., the messenger RNA precursor (pre-mRNA), and to the processed mRNA itself. The term transcript can be used interchangeably with pre-mRNA and mRNA. After 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.

The term expression, as used herein, refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. It includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA) and the translation of mRNA into a polypeptide. Expression produces a gene product. As used herein, a gene product may be either a nucleic acid, such as messenger RNA, produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described

- 9 046118 defined herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

The terms identical or percent identity, in the context of two or more nucleic acids, refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned (with gaps introduced if necessary) for maximum correspondence, disregarding any conservative amino acid substitutions within the limits of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to produce amino acid or nucleotide sequence alignments. One such non-limiting example of a sequence alignment is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and is included in the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). According to certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA, USA), or Megalign (DNASTAR) are additional publicly available computer programs that can be used to align sequences. According to certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using the NWSgapdna.CMP template and a gap opening penalty of 40, 50, 60, 70, or 90 and a gap extension penalty of 1, 2, 3, 4, 5, or 6). According to certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), can be used to determine the percent identity between two amino acid sequences (e.g., using either the BLOSUM 62 matrix or the PAM250 matrix and a gap opening penalty of 16, 14, 12, 10, 8, 6, or 4 and a gap extension penalty of 1, 2, 3, 4, 5). Alternatively, according to certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using the PAM120 residue table, a gap extension penalty of 12, and a gap opening penalty of 4. A person skilled in the art can determine appropriate parameters for maximizing alignment using specific alignment software. In certain embodiments, default parameters in the alignment software are used.

According to certain embodiments, the percent identity X of the first nucleotide sequence to the second nucleotide sequence is calculated as 100x(Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (which are aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Each of the different regions within one target polynucleotide sequence that is aligned with the reference polynucleotide sequence may have its own percent sequence identity. It is worth noting that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded down to 80.2. It should also be noted that the length value will always be an integer. In the context of this document, the terms homologous and homology are used interchangeably with the terms identity and identical.

The term naturally occurring variant thereof refers to variants of an SNCA polypeptide sequence or an SNCA nucleic acid sequence (e.g., a transcript) that exist naturally within a particular taxonomic group, such as mammals, such as mice, monkeys, and humans. Generally, when applied to naturally occurring variants of a polynucleotide, the term also includes any allelic variant of the SNCA-encoding genomic DNA that is located at chromosomal position 17q21 by chromosomal translocation or duplication, and RNA, such as mRNA, derived therefrom. Naturally occurring variants may also include variants derived

- 10 046118 resulting from alternative splicing of SNCA mRNA. When applied to a specific polypeptide sequence, for example, the term also includes naturally occurring forms of the protein, which may therefore be subject to processing, for example, by co-translational or post-translational modifications such as cleavage, proteolytic cleavage, glycosylation, etc. In determining the degree of complementarity between the ASOs of the present disclosure (or portions thereof) and a target region of a nucleic acid that encodes a mammalian SNCA protein (e.g., an SNCA gene) such as those disclosed herein, the degree of complementarity (also homology or identity) is expressed as the percent identity (or percent homology) between the sequence of the ASO (or portion thereof) and the sequence of the target region (or the reverse complement of the target region) that best aligns with it. The percentage is calculated by counting the number of aligned bases that are identical in the two sequences, dividing by the total number of contiguous monomers in the ASO, and multiplying by 100. In such a comparison, if gaps exist, it is preferred that such gaps be only mismatches, rather than regions in which the number of monomers within the gap differs between the ASOs of the present disclosure and the target region.

The term complementary sequence, as used in the context of this document, means a sequence that is complementary to a reference sequence. It is well known that complementarity is a basic principle of DNA replication and transcription, since it is a property shared by two DNA or RNA sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking into a mirror. Thus, for example, the complementary sequence for the sequence 5'ATGC3' may be written as 3'TACG5' or 5'GCAT3'. The terms reverse complementary sequence, reverse complementary, and reverse complementarity, as used in the context of this document, are interchangeable with the terms complementary sequence, complementary, and complementarity.

The terms corresponding and corresponds, when applied to two separate nucleic acid sequences or nucleotide sequences, may be used to specify regions in the sequences that correspond or are similar to each other based on homology and/or functional properties, although the nucleotides in the particular sequences may be numbered differently. For example, different isoforms of a gene transcript may have similar or conserved portions of the nucleotide sequences, the numbering of which may differ between the corresponding isoforms due to alternative splicing and/or other modifications. It is also contemplated that when characterizing a nucleic acid or nucleotide sequence (e.g., a gene transcript), different numbering systems may be used and either sequence numbering may begin at the translational start codon or include the 5'UTR. It is also contemplated that the nucleic acid or nucleotide sequence of different variants of a gene or gene transcript may vary. However, in the context of this document, regions in variants that have nucleic acid or nucleotide sequence homology and/or have common functional properties are considered to correspond to each other. For example, a nucleotide sequence of a SNCA transcript corresponding to nucleotides X through Y in SEQ ID NO: 1 (the reference sequence) refers to a SNCA transcript sequence (e.g., SNCA pre-mRNA or mRNA) that has an identical sequence or a similar sequence to nucleotides X through Y in SEQ ID NO: 1. A person skilled in the art will be able to identify the corresponding X and Y residues in the SNCA transcript sequence by aligning the SNCA transcript sequence with SEQ ID NO: 1. The terms corresponding nucleotide analog and corresponding nucleotide are intended to indicate that the nucleotide base in the nucleotide analog and the naturally occurring nucleotide has the same base pairing or hybridization ability. For example, when a 2-deoxyribose unit of a nucleotide is linked to adenine, the corresponding nucleotide analog comprises a pentose unit (other than 2-deoxyribose) linked to adenine. The term DES number or DES No., as used herein, refers to a unique number assigned to a nucleotide sequence having a specific pattern of nucleosides (e.g., DNA) and nucleoside analogs (e.g., LNA). As used herein, an ASO pattern is shown by a combination of uppercase and lowercase letters. For example, DES005459 refers to the ASO sequence AtTcctttacaccACAC (SEQ ID NO: 15) with the ASO pattern LDLDDDDDDDDDDLLLL (i.e., AtTcctttacaccACAC), where L (i.e., an uppercase letter) denotes a nucleoside analog (e.g., LNA), and D (i.e., a lowercase letter) denotes a nucleoside (e.g., DNA).

The term ASO number or ASO No., as used in the context of this document, refers to a unique number assigned to a nucleotide sequence having a detailed chemical structure of constituents, such as nucleosides (e.g. DNA), nucleoside analogues (e.g.

- 11 046118 example, beta-D-OKCu-LNA), nucleotide bases (e.g., A, T, G, C, U, or MC), and backbone structure (e.g., phosphorothioate or phosphodiester). For example, ASO-005459 refers to OxyAs DNAts OxyTs DNAcs DNAcs DNAts DNAts DNAts DNAas DNAcs DNAas DNAcs DNAcs OxyAs OxyMCs OxyAs OxyMC.

Activity is typically expressed as an IC ₅₀ or EC ₅₀ value in μM, nM, or pM unless otherwise stated. Activity can also be expressed as percent inhibition. The IC ₅₀ is the median inhibitory concentration of the therapeutic molecule. The EC ₅₀ is the median effective concentration of the therapeutic molecule compared to a medium or control (e.g., saline). In functional assays, the IC ₅₀ is the concentration of the 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 EC50 is the concentration of the therapeutic molecule that generates 50% of the biological response, such as mRNA transcription or protein expression. The IC50 or EC50 can be calculated by any number of means known in the art.

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

The term pharmaceutical composition refers to a preparation that is present in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that have unacceptable toxicity to the subject to which the composition is to be administered. Such a composition may be sterile.

An effective amount of an ASO as disclosed herein is an amount sufficient to achieve a specific intended purpose. An effective amount can be determined empirically and routinely depending on the intended purpose.

Terms such as treating, or treating, or curing, or alleviating, or ameliorating refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and/or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and/or slow the progression of a pathological condition or disorder of interest. Thus, subjects in need of treatment include subjects who already have a disorder; subjects predisposed to a disorder; and subjects in whom a disorder is to be prevented. According to certain embodiments, a subject successfully receives treatment for a disease or condition disclosed elsewhere herein in accordance with the methods presented herein if the patient exhibits, for example, complete, partial, or temporary alleviation or elimination of symptoms associated with the disease or disorder.

II. Antisense oligonucleotides.

The present disclosure utilizes antisense oligonucleotides for use in modulating the function of nucleic acid molecules encoding mammalian α-Syn, such as a SNCA nucleic acid, e.g., a SNCA transcript, including SNCA pre-mRNA and SNCA mRNA, or naturally occurring variants of such nucleic acid molecules encoding mammalian α-Syn. The term ASO, as used herein, refers to a molecule formed by a covalent linkage of two or more nucleotides (i.e., an oligonucleotide).

An ASO comprises a continuous nucleotide sequence of about 10 to about 30 nucleotides in length, such as 10-20, 16-20, or 15-25 nucleotides. The terms antisense ASO, antisense oligonucleotide, and oligomer, as used herein, are interchangeable with the term ASO.

A reference to a SEQ ID number includes a specific sequence of nucleotide bases, but does not include any pattern or complete chemical structure shown in Fig. 2 or 3. Moreover, the ASOs disclosed in the figures herein show a typical pattern, but are not limited to the specific pattern shown in the figures, unless otherwise indicated. As used herein, one nucleotide (unit) may also be referred to as a monomer or a unit. When this description refers to a specific ASO number, the reference includes the sequence, the specific pattern of the ASO, and the chemical structure. When this description refers to a specific DES number, the reference includes the sequence and the specific pattern of the ASO. For example, when a claim (or this description) refers to SEQ ID NO: 15, it includes only the nucleotide sequence attcctttacaccacac. When a claim (or this description) refers to DES-005459, it includes the nucleotide sequence attcctttacaccacac with the ASO pattern,

- 12 046118 shown in the figure (i.e., AtTcctttacaccACAC). Alternatively, the pattern of ASO-005459 may be written as SEQ ID NO: 15, wherein each of the first nucleotide, the third nucleotide, and the 14th ^{through 17th} nucleotides from the 5' end is a modified nucleotide, such as an LNA, and each of the other nucleotides is an unmodified nucleotide (e.g., DNA). The ASO number includes the sequence and pattern of the ASO, as well as specific details of the ASO. Thus, ASO-005459, as referenced herein, means OxyAs DNAts OxyTs DNAcs DNAcs DNAts DNAts DNAas DNAcs DNAas DNAcs DNAcs OxyAs OxyMCs OxyAs OxyMC, wherein s denotes a phosphorothioate bridge. According to various embodiments, the ASOs of the present disclosure do not comprise RNA (units). According to some embodiments, the ASO comprises one or more DNA units. According to one embodiment, the ASO of the present disclosure is a linear molecule or is synthesized as a linear molecule. According to some embodiments, the ASO is a single-stranded molecule and does not comprise short stretches of, for example, at least 3, 4 or 5 contiguous nucleotides that are complementary to equivalent stretches within the same ASO (i.e., duplexes), and therefore, the ASOs are not (substantially) double-stranded. According to some embodiments, the ASO is not substantially double-stranded. According to some embodiments, the ASO is not an siRNA. According to various embodiments, the ASOs of the present disclosure may consist entirely of a contiguous nucleotide stretch. Thus, according to some embodiments, the ASO is not substantially self-complementary.

According to one embodiment, the ASOs of the present disclosure may be in the form of any pharmaceutically acceptable salts. The term pharmaceutically acceptable salts, as used herein, refers to derivatives of the ASOs of the present disclosure, wherein the ASOs are modified (e.g., by adding a cation) to form salts thereof. Such salts retain the desired biological activity of the ASOs without providing undesirable toxic effects. According to some embodiments, the ASOs of the present disclosure are in the form of a sodium salt. According to other embodiments, the ASOs are in the form of a potassium salt.

II.A. Target.

Accordingly, the ASO of the present disclosure is capable of down-regulating (e.g., reducing or eliminating) the expression of SNCA mRNA or protein. In this regard, the ASO of the present disclosure may have the effect of indirectly inhibiting SNCA protein by reducing SNCA mRNA levels, typically in a mammalian cell, such as a human cell, such as a neuron. In particular, the present disclosure is directed to ASOs that target one or more regions of SNCA pre-mRNA. Synonyms of SNCA are known and include NACP, the non-A-beta component of AD amyloid, PARK1, PARK4, and PD1. The sequence for the SNCA gene can be found under Public Accession Number NC_000004.12, and a partial sequence for the SNCA pre-mRNA transcript (residues 6001-8400) can be found under Public Accession Number NG_011851.1 (SEQ ID NO: 1). The sequence for the SNCA protein can be found under Public Accession Numbers: P37840, A8K2A4, Q13701, Q4JHI3, and Q6IAU6, each of which is incorporated herein by reference in its entirety. Naturally occurring variants of the SNCA gene product are known. For example, naturally occurring variants of the SNCA protein can comprise one or more amino acid substitutions selected from: A30P, E46K, H50Q, A53T, and any combinations thereof. Thus, an ASO of the present disclosure can be designed to reduce or inhibit the expression of naturally occurring variants of the SNCA protein.

Mutations in SNCA are known to cause one or more pathological conditions. The ASO of the present disclosure can be used to reduce or inhibit the expression of a SNP or, alternatively, a spliced SNCA transcript containing one or more mutations, and thus reduce the production of mutated SNCA protein. Examples of SNCA protein mutants include, but are not limited to, a SNCA protein containing one or more mutations selected from: D2A, E35K, Y39F, H50A, E57K, G67_V71del, V71_V82del, A76_V77del, A76del, V77del, A78del, A85_F94del, Y125F, Y133F, Y136F, and any combination thereof. The ASO of the present disclosure can be designed to reduce or inhibit the expression of any SNCA protein mutants.

An example of an ASO target nucleic acid sequence is SNCA pre-mRNA. SEQ ID NO: 1 in Fig. 1A is a partial genomic sequence of SNCA (residues 60018400). SEQ ID NO: 1 is identical to the SNCA pre-mRNA sequence except that the t nucleotide in SEQ ID NO: 1 is shown as a u in pre-mRNA. According to certain embodiments, the target nucleic acid comprises an intron region of SNCA protein-encoding nucleic acids or naturally occurring variants thereof and RNA nucleic acids derived therefrom, such as pre-mRNA. According to other embodiments, the target nucleic acid comprises an exon region of SNCA protein-encoding nucleic acids

- 13,046,118 acids or naturally occurring variants thereof and RNA nucleic acids derived therefrom. According to some embodiments, for example, when used in research or diagnostics, the target nucleic acid can be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA target nucleic acids. According to one embodiment, the partial genomic sequence of SNCA is shown as GenBank accession number NG_011851.1 (residues 6001-8400) (SEQ ID NO: 1). The mRNA encoding the SNCA protein is shown as SEQ ID NO: 2. See, Fig. 1B. The sequence of the SNCA protein encoded by the SNCA mRNA is shown as SEQ ID NO: 3. See, Fig. 1C.

According to one embodiment, an ASO of the present disclosure comprises a contiguous nucleotide sequence of 10-30 nucleotides in length that is complementary to a nucleic acid sequence within a SNCA transcript, such as a region corresponding to the intron-exon junction of SEQ ID NO: 1, wherein the nucleic acid sequence corresponds to nucleotides 7602-7627 of SEQ ID NO: 1.

According to certain embodiments, the ASOs hybridize to or are complementary to a region within the SNCA transcript, such as nucleotides 7602-7627 of SEQ ID NO: 1, and have a sequence score equal to or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0. Methods for calculating a sequence score are disclosed elsewhere herein.

According to some embodiments, the ASOs of the present disclosure hybridize to an intron-exon junction in a SNCA transcript, such as a region corresponding to the junction between intron 1 and exon 2 in SEQ ID NO: 1. According to some embodiments, the ASOs of the present disclosure comprise a contiguous nucleotide sequence that hybridizes to a nucleic acid sequence of a SNCA transcript or a region within a SNCA transcript sequence (the target region), wherein the nucleic acid sequence corresponds to nucleotides 7602-7627 in SEQ ID NO: 1. According to another embodiment, the ASOs of the present disclosure comprise a contiguous nucleotide sequence that hybridizes to a nucleic acid sequence of a SNCA transcript or a region within a SNCA transcript sequence, wherein the nucleic acid sequence corresponds to nucleotides 7602-7627 in SEQ ID NO: 1. NO: 1, and wherein the ASO has one of the schemes described herein (e.g., in Section II, e.g., a gapmer scheme, e.g., a gapmer scheme with alternating nucleotides in flanking regions), or a chemical structure shown elsewhere herein (e.g., Figs. 2 and 3). According to some embodiments, the target region corresponds to nucleotides 7604-7620 of SEQ ID NO: 1. According to other embodiments, the target region corresponds to nucleotides 7603-7620 of SEQ ID NO: 1. According to other embodiments, the target region corresponds to nucleotides 7602-7619 of SEQ ID NO: 1. According to certain embodiments, the ASOs hybridize to a region within an exon of the SNCA transcript, such as SEQ ID NO: 1, and have a sequence score equal to or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0. Methods for calculating a sequence score are disclosed elsewhere herein.

According to certain embodiments, the target region corresponds to nucleotides 7602-7627 of SEQ ID NO: 1 ± 10 nucleotides at the 3' end, the 5' end, or both. According to other embodiments, the target region corresponds to nucleotides 7604-7620 of SEQ ID NO: 1 ± 1, ± 2, ± 3, ± 4, ± 5, ± 6, ± 7, ± 8, ± 9, or ± 10 nucleotides at the 3' end, the 5' end, or both.

According to certain embodiments, an ASO of the present disclosure is capable of hybridizing to a target nucleic acid (e.g., a SNCA transcript) under physiological conditions, i.e., in vivo. According to some embodiments, an ASO of the present disclosure is capable of hybridizing to a target nucleic acid (e.g., a SNCA transcript) in vitro. According to some embodiments, an ASO of the present disclosure is capable of hybridizing to a target nucleic acid (e.g., a SNCA transcript) in vitro under stringent conditions. Stringent conditions for in vitro hybridization depend on, among other things, productive cellular uptake, RNA availability, temperature, free energy of association, salt concentration, and time (see, e.g., Stanley T Crooks, Antisense Drug Technology: Principles, Strategies and Applications, 2 ^nd Edition, CRC Press (2007)). Typically, high-moderate stringency conditions are used for in vitro hybridization to allow hybridization between substantially similar nucleic acids but not between dissimilar nucleic acids. Examples of stringent hybridization conditions include hybridization in 5x sodium citrate buffered saline (SSC) (0.75 M sodium chloride/0.075 M sodium citrate) for 1 hour at 40°C, followed by washing the sample 10 times in 1xSSC at 40°C and 5 times in 1xSSC buffer at room temperature. In vivo hybridization conditions consist of in vitro conditions

- 14 046118 cells (e.g., physiological pH and ionic strength conditions inside the cell) that determine the hybridization of antisense oligonucleotides to target sequences. In vivo conditions can be mimicked in vitro by using relatively low stringency conditions. For example, hybridization can be performed in vitro in 2xSSC (0.3 M sodium chloride/0.03 M sodium citrate), 0.1% SDS at 37°C. A wash solution containing 4xSSC, 0.1% SDS can be used at 37°C with a final wash in 1xSSC at 45°C.

P.V. ASO sequences.

The ASOs of the present disclosure comprise a contiguous nucleotide sequence that corresponds to the complementary sequence of a region of the SNCA transcript, such as a nucleotide sequence corresponding to SEQ ID NO: 1.

According to certain embodiments, the present disclosure relates to an ASO that comprises a contiguous nucleotide sequence having a total length of 10-30 nucleotides, such as 10-15 nucleotides, 10-20 nucleotides, or 10-25 nucleotides, wherein the contiguous nucleotide sequence is at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to a region within the complementary sequence of a mammalian SNCA transcript, such as SEQ ID NO: 1 or SEQ ID NO: 2. ASOs can comprise a contiguous nucleotide sequence that is completely complementary (perfectly complementary) to an equivalent region of a nucleic acid that encodes a mammalian SNCA protein (e.g., SEQ ID NOs: 1 and 2). ASOs may comprise a contiguous nucleotide sequence that is fully complementary (perfectly complementary) to a nucleic acid sequence or region within the sequence corresponding to nucleotides X-Y in SEQ ID NO: 1, wherein X and Y represent the pre-mRNA start site and the pre-mRNA end site of NG011851.1, respectively, as shown in Fig. 2. Moreover, ASOs may have a design described elsewhere herein (e.g., in Section II.G, e.g., a gapmer design, e.g., a gapmer design with alternating nucleotides in flanking regions), or a chemical structure shown elsewhere herein (e.g., Figs. 2 and 3). According to some embodiments, the ASO comprises a contiguous nucleotide sequence that is fully complementary (perfectly complementary) to a nucleic acid sequence or a region within the sequence corresponding to nucleotides X-Y in SEQ ID NO: 2, wherein X and Y represent an mRNA start site and an mRNA end site, respectively. According to certain embodiments, the nucleotide sequence of an ASO of the present disclosure or a contiguous nucleotide sequence is at least about 80% identical to a sequence selected from SEQ ID NO: 4-21 (i.e., the sequences in FIGS. 2 and 3), such as at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, such as about 100% identical (homologous). In some embodiments, the ASO has a design described elsewhere herein (e.g., in Section II.G.I, e.g., a gapmer design, e.g., a gapmer design with alternating nucleotides in flanking regions), or a nucleoside chemical structure shown elsewhere herein (e.g., Figs. 2 and 3).

According to some embodiments, an ASO of the present disclosure comprises at least one ASO with a pattern (e.g., DES number) disclosed in Figs. 2 and 3. According to some embodiments, an ASO of the present disclosure comprises at least one ASO with a pattern (e.g., DES number) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 3′ end than the ASOs disclosed in Figs. 2 and 3. According to other embodiments, an ASO of the present disclosure comprises at least one ASO with a pattern (e.g., DES number) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 5′ end than the ASOs disclosed in Figs. 2 and 3. According to other embodiments, an ASO of the present disclosure comprises at least one ASO with a pattern (e.g., DES number) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 5' end and/or at the 3' end than the ASOs disclosed in Figs. 2 and 3. According to other embodiments, an ASO of the present disclosure comprises at least one ASO with a chemical structure (e.g., ASO number) disclosed in Figs. 2 and 3. According to some embodiments, an ASO of the present disclosure comprises at least one ASO with a chemical structure (e.g., DES number) disclosed in Figs.

- 15 046 118 rom ASO) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 3' end than the ASOs disclosed in Figs. 2 and 3. According to other embodiments, an ASO of the present disclosure comprises at least one ASO with the chemical structure (e.g., ASO number) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 5' end than the ASOs disclosed in Figs. 2 and 3. According to other embodiments, an ASO of the present disclosure comprises at least one ASO with the chemical structure (e.g., ASO number) disclosed in Figs. 2 and 3, wherein the ASO is one nucleotide, two nucleotides, three nucleotides, or four nucleotides shorter at the 5' end and/or at the 3' end than the ASOs disclosed in Figs. 2 and 3.

According to some embodiments, the ASO (or a portion of contiguous nucleotides thereof) is selected from or comprises one of the sequences selected from the group consisting of SEQ ID NO: 4-21 and a region of at least 10 contiguous nucleotides therein, wherein the ASO (or a portion of contiguous nucleotides thereof) may optionally comprise one, two, three or four mismatches compared to the corresponding SNCA transcript.

According to one embodiment, the ASO comprises a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21. According to some embodiments, the ASO comprises a sequence selected from the group consisting of SEQ ID NO: 7 (e.g., sequence ASO-005578 and ASO-005584) and SEQ ID NO: 15 (e.g., sequence ASO-005459).

In some embodiments, an ASO of the present disclosure binds to a target nucleic acid sequence (e.g., a SNCA transcript) and is capable of inhibiting or reducing the expression of the SNCA transcript by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in tissue (e.g., a brain region) of a mouse expressing the human SNCA gene (e.g., A53T-PAC), when administered in vivo at doses of 3.13 μg, 12.5 μg, 25 μg, 50 μg, or 100 μg, compared to a control (e.g., an internal control such as GADPH or tubulin, or with a mouse treated with only the vehicle control) when measured using an assay, such as quantitative PCR or the QUANTIGENE® assay disclosed herein.

In some embodiments, the ASOs are capable of reducing SNCA protein expression by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in tissue (e.g., a brain region) of a mouse expressing a human SNCA gene (e.g., A53T-PAC) when administered in vivo at doses of 3.13 μg, 12.5 μg, 25 μg, 50 μg, or 100 μg, compared to a control (e.g., an internal control such as GADPH or tubulin, or a mouse administered with a vehicle control only) when measured by an assay, e.g., the high content assay disclosed in this document (see example 2A).

In some embodiments, the ASOs of the present disclosure bind to a target nucleic acid sequence (e.g., a SNCA transcript) and are capable of inhibiting or reducing the expression of the SNCA transcript by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in tissue (e.g., a brain region) of a cynomolgus monkey expressing a wild-type SNCA gene when administered once or twice in vivo at doses of 2 mg, 4 mg, or 8 mg, compared to a control (e.g., an internal control such as GADPH or tubulin, or a cynomolgus monkey administered a vehicle control only) at measured using an assay such as quantitative PCR or the QUANTIGENE® assay disclosed herein.

According to some embodiments, the ASOs are capable of reducing SNCA protein expression by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in tissue (e.g., a brain region) of a cynomolgus monkey expressing

- 16 046118 containing the wild-type SNCA gene, when administered once or twice in vivo at doses of 2 mg, 4 mg, or 8 mg, compared to a control (e.g., an internal control such as GADPH or tubulin, or a cynomolgus monkey administered a vehicle control only) when measured by an assay, e.g., the high content assay disclosed herein (see Example 2A).

According to other embodiments, the ASOs of the present disclosure are capable of reducing SNCA mRNA expression in vitro by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in mouse primary neurons expressing the full-length human SNCA gene (e.g., ASD neurons) when the neurons are in contact with 5 μM, 3.3 μM, 1 μM, 4 nM, 40 nM, or 200 nM of the antisense oligonucleotide, compared to a control (e.g., an internal control such as GADPH or tubulin, or mouse primary neurons expressing the full-length human SNCA gene upon exposure to saline only) when measured using an assay, such as the QUANTIGENE® assay disclosed herein.

According to other embodiments, the ASOs are capable of reducing SNCA protein expression in vitro by at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% in mouse primary neurons expressing a full-length human SNCA gene (e.g., ASD neurons) when the neurons are in contact with 5 μM, 3.3 μM, 1 μM, 4 nM, 40 nM, or 200 nM of an antisense oligonucleotide, compared to a control (e.g., an internal control such as GADPH or tubulin, or mouse primary neurons expressing a full-length human SNCA gene (when exposed to saline alone) when measured using an assay, such as the high content assay disclosed herein (see Example 2A).

In some embodiments, the ASOs are capable of reducing SNCA mRNA expression in vitro by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in a human neuroblastoma cell line (e.g., SK-N-BE(2)) expressing the full-length human SNCA gene when the neuroblastoma cells are exposed to 25 μM of the antisense oligonucleotide, compared to a control (e.g., an internal control such as GADPH or tubulin, or neuroblastoma cells expressing the full-length human SNCA gene when exposed to saline alone) when measured by an assay such as, quantitative PCR disclosed in this document.

According to some embodiments, the ASOs disclosed herein are capable of reducing SNCA protein expression in vitro by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% in a human neuroblastoma cell line (e.g., SK-N-BE(2)) expressing the full-length human SNCA gene when the neuroblastoma cells are in contact with 25 μM of the antisense oligonucleotide, compared to a control (e.g., an internal control such as GADPH or tubulin, or neuroblastoma cells expressing the full-length human SNCA gene when exposed to saline alone) when measured with using analysis, such as the high content analysis disclosed in this document (see Example 2A).

According to certain embodiments, the ASOs of the present disclosure bind to a SNCA transcript and inhibit or reduce the expression of SNCA mRNA by at least about 10% or about 20% compared to a normal (i.e., control) expression level in a cell, such as at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to a normal expression level (such as the expression level in the absence of the ASO or conjugate(s)) in a cell. In certain embodiments, the ASO reduces the expression of SNCA protein in a cell following administration of the ASO by at least 60%, at least 70%, at least 80%, or at least 90%, compared to a cell not exposed to the ASO (i.e., a control). In some embodiments, the ASO reduces the expression of SNCA protein in a cell following administration of the ASO by at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to

- 17 046118 with a cell not exposed to ASO (i.e. control).

According to certain embodiments, an ASO of the present disclosure has at least one property selected from the following: (1) reduces SNCA mRNA expression in a cell compared to a control cell that has not been exposed to the ASO; (2) does not significantly reduce calcium fluctuations in the cell; (3) does not significantly reduce tubulin staining intensity in the cell; (4) reduces α-Syn protein expression in the cell; and (5) any combinations thereof compared to a control cell that has not been exposed to the ASO.

According to some embodiments, an ASO of the present disclosure does not significantly reduce calcium fluctuations in a cell, such as in neurons. If an ASO does not significantly reduce calcium fluctuations in a cell, this property of the ASO corresponds to reduced neurotoxicity of the ASO. According to some embodiments, the calcium fluctuations are greater than or equal to 95%, greater than or equal to 90%, greater than or equal to 85%, greater than or equal to 80%, greater than or equal to 75%, greater than or equal to 70%, greater than or equal to 65%, greater than or equal to 60%, greater than or equal to 55%, or greater than or equal to 50% of the fluctuations in a cell not exposed to the ASO. Calcium fluctuations are important for the proper functioning of neurons. It has been shown that networks of cortical neurons undergo spontaneous calcium fluctuations, resulting in the release of the neurotransmitter glutamate. Calcium fluctuations can also regulate the interactions of neurons with associated glia in addition to other associated neurons in the network, releasing other neurotransmitters in addition to glutamate. Regulated calcium fluctuations are required for homeostasis of neural networks for normal brain function. (See Shashank et al., Brain Research, 1006(1): 8–17 (2004); Rose et al., Nature Neurosci., 4:773–774 (2001); Zonta et al., J Physiol Paris., 96(34):193–8 (2002); Pasti et al., J Neurosci., 21(2): 477–484 (2001).) Glutamate also activates two separate ion channels, the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the N-methyl-D-aspartate (NMDA) receptor.

In some embodiments, the calcium fluctuations measured in these methods are AMPA-dependent calcium fluctuations. In some embodiments, the calcium fluctuations are NMDA-dependent calcium fluctuations. In some embodiments, the calcium fluctuations are gamma-aminobutyric acid (GABA)-dependent calcium fluctuations. In some embodiments, the calcium fluctuations may be a combination of two or more AMPA-dependent, NMDA-dependent, or GABA-dependent calcium fluctuations.

According to certain embodiments, the calcium fluctuations measured in these methods are AMPA-dependent calcium fluctuations. In order to measure AMPA-dependent calcium fluctuations, the calcium fluctuations can be measured in the presence of ^Mg2 + ions (e.g., MgCl2). According to certain embodiments, the method further comprises adding Mg2 ⁺ ions (e.g., MgCl2) in an amount that allows the AMPA-dependent calcium fluctuations to be detected. According to some embodiments, the effective ion concentration that allows the AMPA-dependent calcium fluctuations to be detected is at least about 0.5 mM. According to other embodiments, the effective concentration of ions for inducing AMPA-dependent calcium fluctuations is at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 1 mM, at least about 1.5 mM, at least about 2.0 mM, at least about 2.5 mM, at least about 3.0 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. According to a particular embodiment, the concentration of Mg2 ⁺ ions (e.g., MgCl2) useful in the methods is 1 mM. According to certain embodiments, the concentration of Mg2 ⁺ ions (e.g., MgCl2) useful for the methods is from about 1 mM to about 10 mM, from about 1 mM to about 15 mM, from about 1 mM to about 20 mM, or from about 1 mM to about 25 mM. Mg2 ⁺ ions can be added by adding magnesium salts such as magnesium carbonate, magnesium chloride, magnesium citrate, magnesium hydroxide, magnesium oxide, magnesium sulfate, and magnesium sulfate heptahydrate.

According to some embodiments, the fluctuations in calcium levels are measured in the method by using fluorescent probes that detect fluctuations in intracellular calcium levels. For example, detection of intracellular calcium flux can be achieved by staining cells with fluorescent dyes that bind to the ion

- 18 046118 calcium (known as fluorescent calcium indicators) that result in a detectable change in fluorescence (e.g., Fluo-4 AM and Fura Red AM dyes, available from Molecular Probes, Eugene, OR, USA).

According to other embodiments, the ASOs of the present disclosure do not significantly reduce the intensity of tubulin staining in a cell. According to some embodiments, the intensity of tubulin staining is greater than or equal to 95%, greater than or equal to 90%, greater than or equal to 85%, greater than or equal to 80%, greater than or equal to 75%, greater than or equal to 70%, greater than or equal to 65%, greater than or equal to 60%, greater than or equal to 55%, or greater than or equal to 50% of the intensity of tubulin staining in a cell not exposed to the ASO (or exposed to a saline solution).

According to some embodiments, such a property is observed when using a concentration of the ASO of the present disclosure from 0.04 nM to 400 μM. In the same or a different embodiment, inhibition or reduction of SNCA mRNA and/or SNCA protein expression in a cell results in mRNA or protein levels of less than 100%, such as less than 98%, less than 95%, less than 90%, less than 80%, such as less than 70%, compared to cells not exposed to the ASO. Modulation of the expression level can be determined by measuring SNCA protein levels, such as by methods such as SDS-PAGE followed by Western blotting using appropriate antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring SNCA mRNA levels, such as by Northern blot or quantitative RT-PCR. When measuring inhibition based on mRNA levels, the level of downregulation when using an appropriate dosage, such as a concentration of about 0.04 nM to about 400 μM, in accordance with some embodiments, is typically a level of about 10-20% of normal levels in the cell in the absence of ASO.

According to certain embodiments, an ASO of the present disclosure has an in vivo tolerability score of less than or equal to a total score of 4, wherein the total score is the sum of five category scores that represent 1) hyperactivity; 2) decreased activity and agitation; 3) motor dysfunction and/or ataxia; 4) abnormal body posture and breathing; and 5) tremor and/or convulsions, and wherein the score for each category is measured on a scale of 0-4. According to certain embodiments, the in vivo tolerability score is less than or equal to a total score of 3, a total score of 2, a total score of 1, or a total score of 0. According to one embodiment, the in vivo tolerability score is determined as described in the examples below.

In some embodiments, the ASO may tolerate 1, 2, 3, or 4 (or more) mismatches when hybridized to a target sequence and still bind sufficiently to the target to exhibit the desired effect, i.e., down-regulation of the target mRNA and/or protein. For example, mismatches may be compensated for by an increased length of the ASO nucleotide sequence and/or an increased number of nucleotide analogs, as disclosed elsewhere herein.

According to some embodiments, an ASO of the present disclosure comprises no more than 3 mismatches when hybridized to a target sequence. According to other embodiments, the contiguous nucleotide sequence comprises no more than 2 mismatches when hybridized to a target sequence. According to other embodiments, the contiguous nucleotide sequence comprises no more than 1 mismatch when hybridized to a target sequence.

According to some embodiments, an ASO of the present disclosure comprises a nucleotide sequence or a region within the sequence according to any of SEQ ID NOs: 4-21, an ASO sequence with a scheme as described in Figs. 2 and 3, and an ASO sequence with a chemical structure as described in Figs. 2 and 3.

However, it is contemplated that, in some embodiments, the nucleotide sequence of an ASO may comprise additional nucleotides at the 5' or 3' end, such as, independently, 1, 2, 3, 4, or 5 additional nucleotides at the 5' and/or 3' end that are non-complementary to the target sequence. In this regard, the ASOs of the present disclosure may, in some embodiments, comprise a continuous nucleotide sequence that is flanked by additional nucleotides at the 5' and/or 3' end. In some embodiments, the additional nucleotides at the 5' and/or 3' end are naturally occurring nucleotides, such as DNA or RNA.

According to some embodiments, an ASO of the present disclosure has a sequence score greater than or equal to 0.2, wherein the sequence score is calculated using Formula I:

- 19 046118 number of nucleotides C and their analogues - number of nucleotides G and their analogues (I) total length in nucleotides.

According to other embodiments, an ASO according to the present disclosure has a sequence score greater than or equal to 0.2, wherein the sequence score is calculated using formula IA:

number of C nucleotides and 5-methylcytosine nucleotides - number of G PA nucleotides) total length of nucleotides.

In these embodiments, a sequence score greater than or equal to a threshold value corresponds to reduced neurotoxicity of the ASO. According to certain embodiments, an ASO of the present disclosure has a sequence score greater than or equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. According to one embodiment, an ASO of the present disclosure comprises a contiguous nucleotide sequence that hybridizes to a non-coding region in a SNCA transcript, wherein the ASO sequence score is greater than or equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. According to another embodiment, an ASO of the present disclosure comprises a contiguous nucleotide sequence that hybridizes to an intron region of an SNCA transcript, wherein the ASO sequence score is greater than or equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. According to another embodiment, an ASO of the present disclosure comprises a contiguous nucleotide sequence that hybridizes to an intron-exon junction in a SNCA transcript, wherein the sequence score of the ASO is greater than or equal to about 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0. According to all of these embodiments, when the sequence score is greater than or equal to the threshold, the ASO is considered to have reduced neurotoxicity.

I.S. Length of ASO.

ASOs may comprise a contiguous nucleotide sequence with a total length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides.

According to some embodiments, ASOs comprise a contiguous nucleotide sequence with a total length of about 10-22, such as 10-21, or 12-18, such as 13-17, or 12-16, such as 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides.

According to some embodiments, ASOs comprise a contiguous nucleotide sequence with a total length of 16, 17, 18, 19, or 20 (16-20) contiguous nucleotides.

According to some embodiments, an ASO according to the present disclosure consists of no more than 22 nucleotides, such as no more than 21 or 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. According to some embodiments, an ASO according to the present disclosure comprises less than 22 nucleotides. It should be understood that when a range is given for the length of an ASO or a continuous nucleotide sequence, the range includes the lower and upper length values represented in the range, for example from (or from and to) 10-30, includes both 10 and 30.

II.D. Nucleosides and nucleoside analogs.

According to one aspect of the present disclosure, the ASOs comprise one or more non-naturally occurring nucleotide analogs. Nucleotide analogs as used herein are variants of natural nucleotides, such as DNA or RNA nucleotides, produced by modifications to the sugar moiety and/or base moiety. The analogs may, in principle, simply be silent or equivalent to the natural nucleotides in the context of the oligonucleotide, i.e., do not functionally affect the way in which the oligonucleotide works to inhibit expression of the target gene. However, such equivalent analogs may be useful, for example, if they are easier or cheaper to manufacture, or are more stable under storage or manufacturing conditions, or are a marker or label. However, according to some embodiments, the analogs will functionally affect the way in which the ASO works to inhibit expression; for example, by providing increased binding affinity for the target, and/or increased resistance to intracellular nucleases, and/or improved facilitation of transport into the cell. Specific examples of nucleoside analogs are described, for example, by Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443; and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1.

II.D.1. Nucleotide base.

The term nucleotide base includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds when nucleic acids hybridize. In the context of the present disclosure, the term nucleotide base also encompasses modified nucleotide bases that may differ from naturally occurring nucleotide bases.

- 20 046118 but are functional in nucleic acid hybridization. According to some embodiments, the nucleotide base moiety is modified by modifying or substituting the nucleotide base. In this context, the nucleotide base refers to naturally occurring nucleotide bases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research vol 45 page 2055, and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

According to some embodiments, the nucleotide base moiety is modified by replacing a purine or pyrimidine with a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleotide base selected from isocytosine, pseudoisocytosine, 5-methyl-cytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

The nucleotide base moieties may be designated by a letter code for each corresponding nucleotide base, such as A, T, G, C, or U, where each letter may optionally include modified nucleotide bases with equivalent function. For example, in exemplary oligonucleotides, the nucleotide base moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, 5-methylcytosine nucleosides of LNA may be used for LNA gapmers.

II.D.2. Modification of sugar.

The ASOs of the present disclosure may comprise one or more nucleosides that have a modified sugar moiety, i.e., a modification of the sugar moiety compared to the ribose sugar moiety found in DNA and RNA. Numerous nucleosides have been prepared with a modified ribose sugar moiety, primarily for the purpose of improving certain properties of the oligonucleotides, such as affinity and/or resistance to nucleases.

Such modifications include those in which the structure of the ribose ring is modified, for example by substitution with a hexose ring (HNA) or a bicyclic ring, which typically has a diradical bridge between the C2' and C4' carbons at the ribose end (LNA), or a broken ribose ring, which typically lacks a bond between the C2' and C3' carbons (e.g. UNA). Other nucleosides with a modified sugar include, for example, nucleic acids with a bicyclohexose (International Application WO2011/017521) or nucleic acids with tricyclic sugars (International Application WO2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a moiety other than a sugar, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

Sugar modifications also include modifications made by replacing substituent groups on the ribose ring with groups other than hydrogen or the 2'-OH group found naturally in RNA nucleosides. For example, substituents can be introduced at the 2', 3', 4', or 5' positions. Nucleosides with modified moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. Indeed, much attention has been devoted to the development of 2'-substituted nucleosides, and numerous 2'-substituted nucleosides have been found to have useful properties when incorporated into oligonucleotides, such as increased nucleoside stability and increased affinity.

In some embodiments, the sugar modification comprises an affinity enhancing sugar modification, such as an LNA. The affinity enhancing sugar modification increases the binding affinity of the ASO to a target RNA sequence (e.g., the intron 1/exon 2 junction of SNCA mRNA). In some embodiments, an ASO comprising a sugar modification disclosed herein has a binding affinity to 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 (e.g., an ASO without such sugar modification).

II.D.2.a. 2'-modified nucleosides.

A 2'-modified sugar is a nucleoside that has a substituent other than H or -OH at the 2' position (2'-substituted nucleoside) or contains a 2'-linked diradical, and includes 2'-substituted nucleosides and LNA (2'-4'-diradically bridged) nucleosides. For example, a 2'-modified sugar can provide increased binding affinity and/or increased nuclease resistance of the oligonucleotide. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and the nucleoside 2'-F-ANA. For additional examples, see, e.g.

- 21 046118 mer, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429–4443; and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293–213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2'-substituted modified nucleosides.

II.D.2.b. Locked nucleic acid (LNA) nucleosides.

LNA nucleosides are modified nucleosides that contain a linker group (called a diradical or bridge) between the C2' and C4' atoms of the ribose sugar ring of the nucleotide. These nucleosides are also referred to in the literature as bridged nucleic acid or bicyclic nucleic acid (BNA).

According to some embodiments, the modified LNA nucleoside or nucleosides in the ASO of the present disclosure have the general structure of formula II or III:

or

Formula II Formula III wherein W is selected from -O-, -S-, -N(R ^a )-, -C(R ^a R ^b )-, such as, in some embodiments, -O-; B is a moiety of a nucleobase or a modified nucleobase; Z is an internucleoside bridge to an adjacent nucleoside or a 5'-terminal group; Z* is an internucleoside bridge to an adjacent nucleoside or a 3'-terminal group; and X is a group selected from the group consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, -C(R ^a )=N-, -O-, -Si(R ^a )2-, -S-, -SO2-, -N(R ^a )- and >C=Z. According to some embodiments, X is selected from the group consisting of: -O-, -S-, NH-, NR ^a R ^b , -CH2-, CR ^a R ^b , -C(=CH2)-, and -C(=CR ^a R ^b )-. According to some embodiments, X is -O-. According to some embodiments, Y is a group selected from the group consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, -C(R ^a )=N-, -O-, -Si(R ^a )2-, -S-, -SO2-, -N(R ^a )-, and >C=Z. According to some embodiments, Y is selected from the group consisting of: -CH2-, -C(R ^a R ^b )-, -CH2CH ₂ -, -C(R ^a R ^b )-C(R ^a R ^b )-, -CH ₂ CH ₂ CH ₂ -, -C(R ^a R ^b )C(R ^a R ^b )C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, and -C(R ^a )=N-.

According to some embodiments, Y is selected from the group consisting of: _-CH2- , -CHRa-, -CHCH3-, CR ^a R ^b - and -XY- together denote a divalent linking group (also referred to as a radical), together define a divalent linking group consisting of 1, 2, 3, or 4 groups/atoms selected from the group consisting of -C(R ^a R ^b )-, -C(R ^a )=C(R ^b )-, C(R ^a )=N-, -O-,

-Si(R ^a )2-, -S-, -SO2-, -N(R ^a )- and >C=Z.

According to some embodiments, -XY is a diradical selected from the groups consisting of: -X-CH2-, -X-CR ^a R ^b -, -X-CHR ^a -, -XC(HCH3)-, -OY-, -O-CH2-, -S-CH2-, -NH-CH2-, -O-CHCH3-, -CH2-O-CH2, -O-CH(CH3CH3)-, -O-CH2-CH2-, OCH2-CH2-CH2-, -O-CH2OCH2-, -O-NCH2-, -C(=CH2)-CH2-, -NR ^a -CH2-, NO-CH2, -S-CR ^a R ^b - and -S-CHR ⁸ -. According to some embodiments, -XY- is -O-CH ₂ - or -O-CH(CH ₃ )-.

According to certain embodiments, Z is selected from -O-, -S-, and -N(R ^a )-, and each of Ra and, if present, R ^b , is independently selected from hydrogen, optionally

- 22 046118 optionally substituted C _1-6 -alkyl, optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkylyl, hydroxy, optionally substituted C _1-6 -alkoxy, C _2-6 -alkoxyalkyl, C _2-6 -alkenyloxy, carboxy, C _1-6 -alkoxycarbonyl, C _1-6 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C _1-6 -alkyl)amino, carbamoyl, mono- and di(C _1-6 -alkyl)-amino-carbonyl, amino-C _1-6 -alkylaminocarbonyl, mono- and di(C _1-6 -alkyl)amino-C1-6 _- alkyl-aminocarbonyl, C1-6 _- alkyl-carbonylamino, carbamido, _C1-6- alkanoyloxy, sulfono, _C1-6 -alkylsulfonyloxy, nitro, azido, sulfanyl, _C1-6 alkylthio, halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two substituents R ^a and R ^b attached to the same atom together may define an optionally substituted methylene (= _CH2 ), wherein for all chiral centers the asymmetric groups may be in either the R or S orientation.

According to some embodiments, R ¹ , R ² , R ³ , R ⁵ and R ⁵ * are independently selected from the group consisting of: hydrogen, optionally substituted Cj-6 alkyl. optionally substituted C _2-6 -alkenyl, optionally substituted C _2-6 -alkynyl, hydroxy, C _1-6 -alkoxy, C _2-6 alkoxyalkyl, C _2-6 -alkenyloxy, carboxy, C _1-6 -alkoxycarbonyl, C _1-6 -alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C _{1-6 -alkyl)amino, carbamoyl, mono- and di(C 1-6 -alkyl} )amino-carbonyl, amino-C _1-6 -alkyl-aminocarbonyl, mono- and di(C _1-6 -alkyl)amino-C _1-6 -alkylaminocarbonyl, C _1-6 -alkyl-carbonylamino, carbamido, C _1-6 -alkanoyloxy, sulfono, C _1-6 alkylsulfonyloxy, nitro, azido, sulfanyl, C _1-6 -alkylthio and halogen, wherein aryl and heteroaryl may be optionally substituted, and wherein two substituents attached to the same atom together may define oxo, thioxo, imino or optionally substituted methylene.

According to some embodiments, R ¹ , R ² , R ³ , R ⁵ and R ⁵ * are independently selected from C _1-6 alkyl, such as methyl, and hydrogen.

According to some embodiments, all of R ¹ , R ² , R ³ , R ⁵ , and R ⁵ * are hydrogen.

According to some embodiments, all of R ¹ , R ² , R ³ are hydrogen, and one of R ⁵ and R ⁵ * is also hydrogen and the other of R ⁵ and R ⁵ * is other than hydrogen, such as C _1-6 alkyl, such as methyl.

According to some embodiments, R ^a is either hydrogen or methyl. According to some embodiments, R ^b , if present, is either hydrogen or methyl.

According to some embodiments, one or both of R ^a and R ^b are hydrogen.

According to some embodiments, one of R ^a and R ^b is hydrogen and the other is other than hydrogen.

According to some embodiments, one of R ^a and R ^b is methyl and the other is hydrogen.

According to some embodiments, both R ^a and R ^b are methyl.

According to some embodiments, the diradical -XY- is _-OCH2- , W is O, and ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are all hydrogen. Such LNA nucleosides are disclosed in International Applications WO99/014226, WO00/66604, WO98/039352, and WO2004/046160, all of which are hereby incorporated by reference, and include those commonly known as beta-D-oxy-LNA and alpha-L-oxy-LNA nucleosides.

According to some embodiments, the diradical -XY- is _-SCH2- , W is O, and all of ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are hydrogen. Such thio-LNA nucleosides are disclosed in International Applications WO99/014226 and WO2004/046160.

According to some embodiments, the diradical -XY- is _-NHCH2- , W is O, and all of ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are hydrogen. Such amino-LNA nucleosides are disclosed in International Applications WO99/014226 and WO2004/046160.

According to some embodiments, the diradical -XY- is -OCH2 _{- CH2-} or -O- _CH2 - _CH2 - _CH2- , W is O, and ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are all hydrogen. Such LNA nucleosides are disclosed in International Application WO00/047599 and Morita et al., Bioorganic & Med. Chem. Lett. 12 73-76, which are hereby incorporated by reference, and include those commonly known as 2'-O-4'C-ethylene-bridged nucleic acids (ENA).

According to some embodiments, the diradical -XY- is -OCH2-, W is O, and all of ^R1 , ^R2 , ^R3 and one of ^R5 and ^R5 * are hydrogen, and the other of ^R5 and ^R5 * is often other than hydrogen, such as _C1-6 alkyl, such as methyl. Such 5'-substituted LNA nucleosides are disclosed in International Application WO2007/134181.

According to some embodiments, the diradical -XY- is -OCR ^a R ^b -, wherein one or both of R ^a and R ^b are other than hydrogen, such as methyl, W is

- 23 046118 is O, and all of R ¹ , R ² , R ³ and one of R ⁵ and R ⁵ * are hydrogen and the other of R ⁵ and R ⁵ * is other than hydrogen, such as C _1-6 alkyl, such as methyl. Such bis-modified LNA nucleosides are disclosed in International Application WO2010/077578.

In some embodiments, the diradical -XY- is a divalent linker group -O-CH(CH ₂ OCH ₃ )- (2'O-methoxyethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). In some embodiments, the diradical -XY- is a divalent linker group -O-CH(CH ₂ CH ₃ )- (2'O-ethyl bicyclic nucleic acid - Seth at al., 2010, J. Org. Chem. Vol 75(5) pp. 1569-81). According to some embodiments, the diradical -XY- is -O-CHR ^a -, W is O, and all of R ¹ , R ² , R ³ , R ⁵ , and R ⁵ * are hydrogen. Such 6'-substituted LNA nucleosides are disclosed in International Applications WO10036698 and WO07090071.

In some embodiments _{, the diradical -XY- is -OCH(CH2OCH3 )} -, W is O, and ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are all hydrogen. Such LNA nucleosides are also known in the art as cyclic MOEs (cMOEs) and are disclosed in International Application WO07090071. In some embodiments, the diradical -XY- is a divalent linker group -O-CH( _CH3 )-, in either the R- or S configuration. In some embodiments, the diradical -XY- collectively is a divalent linker group -O- _CH2 -O- _CH2- (Seth et al., 2010, J. Org. Chem). According to some embodiments, the diradical -XY- is -O-CH( _CH3 )-, W is O, and ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are all hydrogen. Such 6'methyl-LNA nucleosides are also known in the art as cET nucleosides, and they can be either the (S)cET or (R)cET stereoisomers, which are disclosed in International Applications WO07090071 (beta-D) and WO2010/036698 (alpha-L)).

In some embodiments, the diradical -XY- is -OCR ^a R ^b -, wherein neither R ^a nor R ^b is hydrogen, W is O, and all of R ¹ , R ² , R ³ , R ⁵ , and R ⁵ * are hydrogen. In some embodiments, both of R ^a and R ^b are methyl. Such 6' di-substituted LNA nucleosides are disclosed in International Application WO 2009006478.

According to some embodiments, the diradical -XY- is -SCHR ^a -, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ⁵ * are hydrogen. Such 6'-substituted thio-LNA nucleosides are disclosed in International Application WO11156202. According to some embodiments of the 6'-substituted thio-LNA, R ^a is methyl.

According to some embodiments, the diradical -XY- is -C(= _CH2 )-C( ^{RaRb )} -, such as -C(= _CH2 ) _-Ch2- or -C(= _CH2 )-CH( _CH3 )-, W is O, and ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are all hydrogen. Such vinyl-carbo-LNA nucleosides are disclosed in International Applications WO08154401 and WO09067647.

In some embodiments, the diradical -XY- is -N(OR ^a )-, W is O, and R ¹ , R ² , R ³ , R ⁵ , and R ⁵ * are all hydrogen. In some embodiments, ^Ra is C 1-6 alkyl, such as methyl. Such LNA nucleosides are known as N-substituted LNAs and are disclosed in International Application WO2008/150729. In some embodiments, the diradical -XY- collectively represents the divalent linker group -O-NR ^a -CH3- (Seth et al., 2010, J. Org. Chem). In some embodiments, the diradical -XY- is -N(R ^a )-, W is O, and R ¹ , R ² , R ³ , R ⁵ , and R ⁵ * are all hydrogen. According to some embodiments, R ^a is C1-6alkyl, such as methyl. According to some embodiments, one or both of R ⁵ and R ⁵ * are hydrogen, and if substituted, the other of R ⁵ and R ⁵ * is C1-6alkyl, such as methyl. In such an embodiment, all of R ¹ , R ² , R ³ can be hydrogen, and the diradical -XY- can be selected from -O-CH ₂ - or -O-CH(CRa)-, such as from -O-CH(CH ₃ )-.

According to some embodiments, the diradical is -CR ^a R ^b -OCR ^a R ^b -, such as CH2-O-CH2-, W is O, and all of R ¹ , R ² , R ³ , R ⁵ and R ⁵ * are hydrogen. According to some embodiments, R ^a is C 1-6 alkyl, such as methyl. Such LNA nucleosides are also known as conformationally constrained nucleotides (CRN) and are disclosed in International Application WO2013036868.

In some embodiments, the diradical is -O- ^{CRaRbO - CRaRb-} , such as O- ^CH2 -O-CH2-, W is O, and all of ^R1 , ^R2 , ^R3 , ^R5 , and ^R5 * are hydrogen. In some embodiments, ^Ra is C1-6alkyl, such as methyl. Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238.

It will be understood that in cases where this is not specified, LNA nucleosides may be present in the beta-D or alpha-L stereoisoform.

Specific examples of LNA nucleosides are shown in Scheme 1.

- 24 046118

Scheme 1

As illustrated in the examples, in accordance with some embodiments of the present disclosure, the LNA nucleosides in the oligonucleotides are beta-O-oxyLNA nucleosides.

II.E. Nuclease-mediated degradation.

Nuclease-mediated degradation refers to an oligonucleotide that is capable of mediating the degradation of a complementary nucleotide sequence upon formation of a duplex with that sequence.

According to some embodiments, the oligonucleotide may function through nuclease-mediated degradation of a target nucleic acid, wherein the oligonucleotides of the present disclosure are capable of recruiting a nuclease, in particular an endonuclease, preferably an endoribonuclease (RNase), such as RNase H. Examples of oligonucleotide designs that function through nuclease-mediated mechanisms are oligonucleotides that typically comprise a stretch of at least 5 or 6 DNA nucleosides and are flanked on one or both sides by affinity-enhancing nucleosides, such as gapmers.

II.F. Activity and recruitment of RNase H.

The RNase H activity of an antisense oligonucleotide refers to its ability to attract RNase H when it is in duplex with a complementary RNA molecule and induce cleavage and subsequent degradation of the complementary RNA molecule. International application WO01/23613 provides in vitro methods for determining RNase H activity which can be used to determine the ability to attract RNase H. Generally, an oligonucleotide is considered to be capable of attracting RNase H if, when provided with a complementary target nucleic acid sequence, it has an onset rate, measured in pmol/L/min, which is at least 5%, such as at least 10% or more than 20%, of the onset rate determined using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested but comprising only DNA monomers with phosphorothioate bridges between all monomers in the oligonucleotide, and using the technique provided in Examples 91-95 of International application WO01/23613.

According to some embodiments, an oligonucleotide is considered to be substantially incapable of recruiting RNase H if, when provided with a complementary target nucleic acid, the initial rate for RNase H, measured in pmol/L/min, is less than 20%, such as less than 10%, such as less than 5%, of the initial rate determined using an oligonucleotide having the same base sequence but comprising only DNA monomers with no 2' substitutions and with phosphorothioate bridges between all monomers in the oligonucleotide, and using the methodology set forth in Example 91-95 of the International Patent Application.

- 25 046118 native application WO01/23613.

II.G. ASO structure.

The ASO of the present disclosure may comprise a nucleotide sequence that comprises both nucleotides and nucleotide analogs and may be in the form of a gapmer. Examples of gapmer configurations that may be used with the ASO of the present disclosure are described in U.S. Patent Application Publication No. 2012/0322851.

The term gapmer as used in the context of this document refers to an antisense oligonucleotide that comprises a region with RNase H-attracting oligonucleotides (a gap) that is flanked at the 5' and 3' end by one or more affinity-enhancing modified nucleosides (side moieties). Various gapmer designs are described in this document. The term LNA gapmer is a gapmer oligonucleotide in which at least one of the affinity-enhancing modified nucleosides is an LNA nucleoside. The term "mixed-wing gapmer" refers to an LNA gapmer in which the flanking regions comprise at least one LNA nucleoside and at least one DNA nucleoside or modified nucleoside other than LNA, such as at least one 2'-substituted modified nucleoside, such as, for example, the nucleoside(s) 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA. According to some embodiments, the mixed-wing gapmer has one flanking region that comprises LNA nucleosides (e.g., 5' or 3') and the other flanking region (3' or 5', respectively) comprises 2'-substituted modified nucleoside(s).

According to some embodiments, in addition to enhancing the affinity of the ASO for the target site, some nucleoside analogs also mediate RNase binding and RNase (e.g., RNase H) cleavage.

II.G.1. Gapmer diagram.

According to one embodiment, an ASO of the present disclosure is a gapmer. An ASO gapmer is an ASO that comprises a continuous stretch of nucleotides that is capable of attracting an RNase, such as RNase H, such as a region of at least 6 nucleotides of DNA, referred to herein as a B region (B), wherein the B region is flanked at both the 5' and 3' end by regions of affinity-enhancing nucleotide analogs, such as from 1-10 nucleotide analogs at both the 5' and 3' end relative to the continuous stretch of nucleotides that is capable of attracting an RNase, these regions being referred to as A regions (A) and C regions (C), respectively.

In certain embodiments, the gapmer is a gapmer with alternating nucleotides in the flanking regions, examples of which are discussed below. In certain embodiments, the gapmer with alternating nucleotides in the flanking regions exhibits less off-target binding than a traditional gapmer. In certain embodiments, the gapmer with alternating nucleotides in the flanking regions exhibits better long-term tolerability than a traditional gapmer.

A gapmer with alternating nucleotides in the flanking regions may comprise a (poly)nucleotide sequence of the formula (from 5' to 3'), A-B-C, wherein: region A (A) (the 5' region or first wing sequence) comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-10 nucleotide analogues, such as LNA units, and region B (B) comprises at least six consecutive nucleotides that are capable of attracting RNase (upon duplex formation with a complementary RNA molecule, such as a pre-mRNA or mRNA target), such as DNA nucleotides, and region C (C) (the 3' region or second wing sequence) comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-10 nucleotide analogues, such as LNA units; wherein regions A and C may include, at any position in A and C, 1-3 insertions of regions of DNA nucleotides (for example, DNA insertions), wherein each of these DNA insertions may have a length of 1-6 DNA links.

According to certain other embodiments, a gapmer, e.g. a gapmer with alternating nucleotides in the flanking regions, comprises a (poly)nucleotide sequence of the formula (from 5' to 3'), A-B-C or, optionally, A-B-C-D or D-A-B-C, wherein: region A (A) (the 5' region) comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-10 nucleotide analogues, such as LNA units, and region B (B) comprises at least five consecutive nucleotides that are capable of attracting RNase (upon duplex formation with a complementary RNA molecule, such as a target mRNA), such as DNA nucleotides, and region C (C) (the 3' region) comprises at least one nucleotide analogue, such as at least one LNA unit, such as from 1-10 nucleotide analogues, such as LNA units, and region D (D), when present, comprises 1, 2, or 3 nucleotide units, such as DNA nucleotides. According to some embodiments, region A comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide analogs, such as LNA units, such as 2-5 nucleotide analogs, such as 2-5 LNA units, such as 2-5 nucleotide analogs, such as 3-5 LNA units; and/or the C region consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide analogs, such as LNA units, such as from 2-5 nucleotide analogs, such as 2-5 LNA units, such as 2-5 nucleotide analogs, such as 3-5 LNA units.

According to some embodiments, B comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that are capable of attracting RNase, or 6-14, 7-14, 8-14, or 7-10, or 7-9, such as 8, such as 9, such as 10, or such as 14 consecutive nucleotides that are capable of attracting RNase. According to some embodiments, region B comprises at least five DNA nucleotide units, such as 5-23 DNA units, such as 5-20 DNA units, such as 5-18 DNA units, such as 6-14 DNA units, such as 8-14 DNA units, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 DNA units.

According to some embodiments, region A comprises 3, 4 or 5 nucleotide analogs such as LNA, region B consists of 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 DNA units, and region C consists of 3, 4 or 5 nucleotide analogs such as LNA. Such patterns include (A-B-C) 5-10-5, 3-14-3, 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4 and 4-7-3 and may further include region D, which may have from one to 3 nucleotide units such as DNA units.

According to some embodiments of an ASO according to the present disclosure, for example, a gapmer with alternating nucleotides in the flanking regions has the formula 5'-A-BC-3', wherein (i) region B is a contiguous sequence of at least 5, 6, 7, 8, 9 or 10, such as 5-18 DNA units, that are capable of attracting RNase; (ii) region A is a first wing sequence of 1-10 nucleotides, wherein the first wing sequence comprises one or more nucleotide analogues and, optionally, one or more DNA units (e.g., a DNA insert), and wherein at least one of the nucleotide analogues is located at the 3' end of A; and (iii) region C is a second wing sequence of 1-10 nucleotides, wherein the second wing sequence comprises one or more nucleotide analogues and, optionally, one or more DNA units (e.g., a DNA insert), and wherein at least one of the nucleotide analogues is located at the 5' end of C.

According to some embodiments, the first wing sequence (region A in the formula) comprises a combination of nucleotide analogs and DNA units selected from (i) 1-10 nucleotide analogs and 0 DNA units; (ii) 1-9 nucleotide analogs and 1 DNA unit; (iii) 1-8 nucleotide analogs and 1-2 DNA units; (iv) 1-7 nucleotide analogs and 1-3 DNA units; (v) 1-6 nucleotide analogs and 1-4 DNA units; (vi) 1-5 nucleotide analogs and 1-5 DNA units; (vii) 1-4 nucleotide analogs and 1-6 DNA units; (viii) 1-3 nucleotide analogs and 1-7 DNA units; (ix) 1-2 nucleotide analogs and 1-8 DNA units; (x) 1 nucleotide analogue and 1-9 DNA links.

According to certain embodiments, the second wing sequence (region C in the formula) comprises a combination of nucleotide analogs and DNA units selected from (i) 1-10 nucleotide analogs and 0 DNA units; (ii) 1-9 nucleotide analogs and 1 DNA unit; (iii) 1-8 nucleotide analogs and 1-2 DNA units; (iv) 1-7 nucleotide analogs and 1-3 DNA units; (v) 1-6 nucleotide analogs and 1-4 DNA units; (vi) 1-5 nucleotide analogs and 1-5 DNA units; (vii) 1-4 nucleotide analogs and 1-6 DNA units; (viii) 1-3 nucleotide analogs and 1-7 DNA units; (ix) 1-2 nucleotide analogs and 1-8 DNA units; (x) 1 nucleotide analogue and 1-9 DNA links.

According to some embodiments, region A of the ASO formula has a subformula selected from the scheme of the first wing of any ASOs in Figs. 2 and 3, and/or region C of the ASO formula has a subformula selected from the structure of the second wing of any ASOs in Figs. 2 and 3, wherein the uppercase letter represents a nucleotide analog (e.g., an analog with a modified sugar, which can also be written as L), and the lowercase letter represents DNA (which can also be written as D). According to certain embodiments of the ASO, for example, a gapmer with alternating nucleotides in the flanking regions has the formula 5' A-B-C 3', wherein region B is a contiguous sequence of 5-18 DNA units, region A has the formula LLDLL, LDLLL, or LLLDL, and region C has the formula LLDLL or LDLDLL, and wherein L is an LNA unit and D is a DNA unit. According to some embodiments, the ASO has the formula 5' A-B-C 3', wherein region B is a contiguous sequence of 10 DNA units, region A has the formula LDL, and region C has the formula LLLL, wherein L is an LNA unit, and D is a DNA unit.

Additional gapmer designs are disclosed in International Application WO2004/046160, which is hereby incorporated by reference in its entirety. International Application WO2008/113832, which is hereby incorporated by reference in its entirety, relates to ASO gapmers in the form of short oligonucleotides (shortmers). According to some embodiments, the ASOs presented herein may be such gapmer shortmers. According to some embodiments of the ASO, for example, a gapmer with alternating nucleotides in the flanking regions comprises a contiguous nucleotide sequence of a total of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units, wherein the contiguous nucleotide sequence has the formula (5'-3'), A-B-C or, optionally, A-B-C-D or D-A-B-C, wherein region A consists of 1, 2, 3, 4 or 5 nucleotide analog units, such as LNA units; region B consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous nucleotide units that are capable of recruiting RNase when forming a duplex with a complementary RNA molecule (such as a target mRNA); and region C consists of 1, 2, 3, 4, or 5 nucleotide analog units, such as LNA units. If present, region D consists of a single DNA unit. In some embodiments, A comprises 1 LNA unit. In some embodiments, region A comprises 2 LNA units. In some embodiments, region A comprises 3 LNA units. In some embodiments, region A comprises 4 LNA units. In some embodiments, region A comprises 5 LNA units. In some embodiments, region C comprises 1 LNA unit. According to some embodiments, C comprises 2 LNA units. According to some embodiments, region C comprises 3 LNA units. According to some embodiments, region C comprises 4 LNA units. According to some embodiments, region C comprises 5 LNA units. According to some embodiments, region B comprises 6 nucleotide units. According to some embodiments, region B comprises 7 nucleotide units. According to some embodiments, region B comprises 8 nucleotide units. According to some embodiments, region B comprises 9 nucleotide units. According to certain embodiments, region B comprises 10 nucleoside units. According to certain embodiments, region B comprises 11 nucleoside units. According to certain embodiments, region B comprises 12 nucleoside units. According to certain embodiments, region B comprises 13 nucleoside units. According to certain embodiments, region B comprises 14 nucleoside units. According to certain embodiments, region B comprises 7-23 DNA monomers or 518 DNA monomers. According to some embodiments, region B comprises 623 DNA units, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 DNA units. According to some embodiments, region B consists of DNA units. According to some embodiments, region B comprises at least one LNA unit that is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 LNA units in the alpha-L configuration. According to some embodiments, region B comprises at least one alpha-L-oxy-LNA unit, or wherein all of the LNA units in the alpha-L configuration are alpha-L-oxy-LNA units. According to some embodiments, the number of nucleotides present in A-B-C is selected from (nucleotide analog units - region B - nucleotide analog units): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, or 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 49-1, 1-9-4, or 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3 and 3-10-1. According to some embodiments, the number of nucleotides in A-B-C is selected from: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. According to other embodiments, the ASO comprises 10 DNA units in B, LDLLL in A (first wing), and LLDLL in C (second wing). According to other embodiments, the ASO comprises 9 DNA units in B, LDDLL in A, and LDLDLL in C. According to other embodiments, the ASO comprises 10 DNA units in B, LLDLL in A, and LLDLL in C. According to further embodiments, the ASO comprises 9 DNA units in B, LLLLL in A, and LDDLL in C. According to certain embodiments, each of regions A and C comprises three LNA monomers, and region B consists of 7, 8, 9, 10, 11, 12, 13, or 14 nucleoside monomers, such as DNA monomers. According to some embodiments, each of A and C consists of two LNA units, and B consists of 7, 8, or 9 nucleotide units, such as DNA units. According to various embodiments, other gapmer arrangements include those in which regions A and/or C are composed of 3, 4, 5, or 6 nucleoside analogs, such as monomers containing a 2'O-methoxyethyl ribose sugar (2'-MOE) or monomers containing a 2'-fluoro-deoxyribose sugar, and region B is composed of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleosides, such as DNA monomers, wherein regions A-B-C have 3-8-3, 3-9-3, 3-10-3, 5-10-5, or 4-12-4 monomers. Additional gapmer circuits are disclosed in International Application No. WO 2007/146511A2, which is hereby incorporated by reference in its entirety.

According to some embodiments, an ASO with alternating nucleotides in the flanking regions has at least 10 contiguous nucleotides comprising a region A, a region B, and a region C (A-B-C), wherein region B comprises at least 5 contiguous nucleoside units and is flanked at the 5' end by a region A of 1-8 contiguous nucleoside units and at the 3' end by a region

- 28 046118 com C from 1-8 adjacent nucleoside units, wherein region B, when forming a duplex with complementary RNA, is capable of attracting RNase H, and wherein region A and region C are selected from the group consisting of the following:

(i) region A comprises a 5' LNA nucleoside unit and a 3' LNA nucleoside unit, and at least one DNA nucleoside unit between the 5' LNA nucleoside unit and the 3' LNA nucleoside unit, and region C comprises at least two 3' LNA nucleosides;

(ii) region A comprises at least one 5' LNA nucleoside, and region C comprises a 5' LNA nucleoside unit, at least two terminal 3' LNA nucleoside units, and at least one DNA nucleoside unit between the 5' LNA nucleoside unit and the 3' LNA nucleoside unit, and (iii) region A comprises a 5' LNA nucleoside unit, and a 3' LNA nucleoside unit, and at least one DNA nucleoside unit between the 5' LNA nucleoside unit and the 3' LNA nucleoside unit; and region C comprises a 5' LNA nucleoside unit, at least two terminal 3' LNA nucleoside units and at least one DNA nucleoside unit between the 5' LNA nucleoside unit and the 3' LNA nucleoside unit.

According to some embodiments, region A or region C comprises 1, 2, or 3 DNA nucleoside units. According to other embodiments, region A and region C comprise 1, 2, or 3 DNA nucleoside units. According to other embodiments, region B comprises at least five contiguous DNA nucleoside units. According to certain embodiments, region B comprises 6, 7, 8, 9, 10, 11, 12, 13, or 14 contiguous DNA nucleoside units. According to some embodiments, region B is 8, 9, 10, 11, or 12 nucleotides in length. According to other embodiments, region A comprises two 5' terminal LNA nucleoside units. According to some embodiments, region A has the formula 5'[LNA] _1-3 [DNA] _1-3 [LNA] _1-3 or 5'[LNA] _1-2 [DNA] _1-2 [LNA] _1-2 [DNA] ₁ 2[LNA]1-2. According to other embodiments, region C has the formula [LNA]1 ₃ [DNA] _1-3 [LNA] _2-3 3' or [LNA] _1-2 [DNA] _1-2 [LNA] _1-2 [DNA] _1-2 [LNA] _2-3 3'. According to other embodiments, region A has the formula 5'[LNA] _1-3 [DNA] _1-3 [LNA] _1-3 or 5'[LNA] _1-2 [DNA] _1-2 [LNA] ₁₂ [DNA] _1-2 [LNA _1-2 , and region C comprises 2, 3, 4, or 5 consecutive LNA nucleoside units. According to some embodiments, region C has the formula [LNA] _1-3 [DNA] ₁₃ [LNA] _2-3 3' or [LNA] _1-2 [DNA] _1-2 [LNA] _1-2 [DNA] _1-2 [LNA] _2-3 3', and region A comprises 1, 2, 3, 4, or 5 consecutive LNA nucleoside units. According to other embodiments, region A has a sequence of LNA and DNA nucleosides, 5'-3', selected from the group consisting of

L, LL, LDL, LLL, LLDL, LDLL, LDDL, LLLL,

LLLLL, LLLDL, LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLLL, LLLLDL, LLLDLL, LLLDLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL, LDDDLL, LLDDDL and LDLDLD, wherein L is an LNA nucleoside and D is a DNA nucleoside. According to other embodiments, region C has a sequence of LNA and DNA nucleosides, 5'-3', selected from the group consisting of

LL, LLL, LLLL, LDLL, LLLLL, LLDLL, LDLLL, LDDLL, LDDLLL, LLDDLL,

LDLDLL, LDDDLL, LDLDDLL, LDDLDLL, LDDDLLL and LLDLDLL.

According to a further embodiment, region A has a sequence of LNA and DNA nucleosides, 5'-3', selected from the group consisting of

LDL, LLDL, LDLL, LDDL, LLLDL,

LLDLL, LDLLL, LLDDL, LDDLL, LLDLD, LDLLD, LDDDL, LLLLDL, LLLDLL, LLDLLL, LDLLLL, LLLDDL, LLDLDL, LLDDLL, LDDLLL, LDLLDL, LDLDLL, LDDDLL, LLDDDL and LDLDLD, and region C has a sequence of LNA and DNA nucleosides, 5'-3', selected from the group consisting of

LDLL, LLDL, LLLLL, LLDLL, LDLLL, LDDLL, LDDLLL, LLDDLL,

LDLDLL, LDDDLL, LDLDDLL, LDDLDLL, LDDDLLL and LLDLDLL.

According to certain embodiments, an ASO with alternating nucleotides in the flanking regions has adjacent nucleotides comprising a nucleoside sequence, 5'-3', selected from the group consisting of

- 29 046118

LLDDDDDDDDDDDLLLL, LLLDDDDDDDDDDLDLL, LLLDDDDDDDDLDLDLL, LLLLDDDDDDDDDLDLL, LLLDDLDDDDDDDDDLL, LLLLLDDDDDDDDDDLL, LLLLLLDDDDDDDDDLL, LLLDLDDDDDDDDDLLL, LLDDLDDDDDDDDDLDLLL, LLLLLDDDDDDDDDDLDLL, LLDLLLDDDDDDDDDLDLL, LLLDDDDDDDDLDLDDLL, LLLLDDDDDDDDLDDDLL, LLLLDDDDDDDLDDLDLL, LLLLLLDDDDDDDDLDLL, LLLDLDDDDDDDDLDDLL, LLLLLDDDDDDDDDLDLLL, LLLLDDDDDDDDDDLDLLL,

LLDDDLLDDDDDDDDLL, LLLDDDDDDDDDLDDLL, LLLDLDDDDDDDDDLLL, LLLLDDDDDDDDLDDLL, LLLDDLLDDDDDDDDLL, LLLLLDDDDDDDDDDLL, LLDLLLDDDDDDDDDLL, LLLDLLDDDDDDDDDLL,

LLDDLDDDDDDDDDLLDLL, LLLDDDDDDDDDDDLDLL, LLLDDDDDDDDDDLDDLL, LLLLDDDDDDDDDLDDLL, LLLLDDDDDDDDDDLDLLLL, LLLDDDDDDDDDLDLDLL, LLLDLDDDDDDDDDDDLL, LLLDLDDDDDDDDDLDLL, LLLLLLDDDDDDDDDLDDLL, LLLLDDDDDDDDDDDLDDLL,

LLLLDLDDDDDDDDDLL, LLLDDDDDDDDDDDDLL, LLLDLDDDDDDDDDDLL, LLLDDDLDDDDDDDDLL, LLLDDLLDDDDDDDLLL, LDLLLDDDDDDDDDLDDLL, LLLDLDDDDDDDDDDDDLL, LLLLDDDDDDDDDDDLL, LLLLLLDDDDDDDDDLDLL, LLLLDDDDDDDDDDDLDLL, LLLLDDDDDDDDDDDDDLL, LLLLDDDDDDDDDLDLLL, LLLLDDDDDDDDLDLDLL, LLDLLDDDDDDDDDDDLL, LLLDLDDDDDDDDDLDLL, LLLLDDDDDDDDDLLDLL, LLLLDDDDDDDDDDDLLDLL, LLLDDDDDDDDDDDDLLLDLL,

LLLDDDDDDDDDDDLDLLL, LLLLLDDDDDDDDDLLDLL, LLLDDDDDDDDDDDLDDLL,

LLDLLDDDDDDDDDLDDLL, LLLDLDDDDDDDDDDLDLL, LLLDLDDDDDDDDDLDDLL,

LLLLDDDDDDDDDLDLDLL, LLLLDDDDDDDDLLDLDLL, LDLLLDDDDDDDDDDLLDLL,

LLDLLDDDDDDDDDDLLDLL, LLDDLDDDDDDDDDDDLLLL, LLDLDDDDDDDDDDDDDLLL, LLDDLDDDDDDDDDDDDLLL, LLLDLDDDDDDDDDDDDDLLLL, LLLLDDDDDDDDDDDDDLLLLL, LLLDDLDDDDDDDDDDDLLLLL, LLDDLLDDDDDDDDDDDLLLLL, LLDLLDDDDDDDDDLDDLLL, LLDLDDLDDDDDDDDDLLLL, LLLDLDDDDDDDDDLDDLLL, LLDLLDDDDDDDDDDLDLLL, LLDDLLDDDDDDDDDDLLLDLL, LLLDDLDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDDLDLLLL, LLDDLLDDDDDDDDDDLLLLLL, LLLLDDDDDDDDDDDDLDLLLL, LLLDLDDDDDDDDDDDLLLL,

LLDLDDDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDDLLLL, LLLDDDDDDDDDDDLDDLLL, LLDLLDDDDDDDDDDDDDLLLL, LLLLDDDDDDDDDDLLDDLL, LLDDLDLDDDDDDDDDLLLLLL, LLLDLDDDDDDDDDDDLDLLLL, LLLDLDDDDDDDDDDLLLLL, LLLLDDDDDDDDDLDLDDLL, LLLDLDDDDDDDDDDDLLLL, LLLDLDDDDDDDDDDLLDLL, LLLLDDDDDDDDDLDDLDLL, LLDLLDDDDDDDDDLLDDLL, LLLDLDDDDDDDDDLLDDLL, LLLDLDDDDDDDDDLDLDLL, LLLDDLDDDDDDDDDDLLLL, LLLLDDDDDDDDDDLDLDLL,

LLLLDDDDDDDDDDDLDLLL and LLDDLLDDDDDDDDDDLDLL;

wherein L is an LNA nucleoside and D is a DNA nucleoside. According to other embodiments, the LNA nucleoside is beta-D-hydroxy-LNA. According to other embodiments, the ASO with alternating nucleotides in the flanking regions has adjacent nucleotides comprising a sequence of alternating LNA and DNA nucleoside units, 5'-3', selected from the group consisting of:

- 30 046118

1-1-1-10-4, 1-2-1-9-2-1-2, 1-2-1-9-1-1-3, 2-3-2-8-2, 1- 1-2-1-1-9-2, 3-10-11-2, 3-9-1-2-2, 3-8-1-3-2, 3-8-1-1-1- 1-2, 3-1-1-9-3, 3-1-1-8-1-1-2, 4-9-1-1-2, 4-8-1-2-2, 3-3-1- 8-2, 32-1-9-2, 3-2-2-8-2, 3-2-2-7-3, 5-7-1-2-2, 1-1-3-10- 2, 1-1-3-7-1-2-2, 1-1-4-9-2, 2-1-3-9-2, 3-1-1-10-2, 3-1-1- 7-1-2-2, 3-1-2-9-2, 4-7-1-3-2, 5-9-1-1-2, 4-10-1-1-2, 3-11-1-1-2, 2-1-1-10-1-1-2, 1-1-3-9-1-12, 3-10-1-2-2, 3-9- 1-3-2, 3-8-1-1-1-2-2, 4-9-1-2-2, 4-9-1-1-3, 4-8-1-3-2, 4-8-1-2-3, 4-8-1-1-1-1-2, 47-1-2-1-1-2, 4-7-1-1-1-2-2, 2-1-2-11-2, 2-1-3-8-1-1-2, 3-1-1-11-2, 3-1-1-9-1-1-2, 3-1-1-8-1-2- 2, 3-11-7-1-1-1-1-2, 4-9-2-1-2, 4-7-1-3-3, 5-9-1-1-3, 5-9- 1-2-2, 4-10-2-1-2, 4-10-1-1-3, 4-10-1-2-2, 3-112-1-2, 3-11-1-1-3, 5-9-2-1-2, 3-11-1-2-2, 2-1-2-9-1-2-2, 3-1-1-10-1-1-2, 3-1-1-9-1-2- 2, 4-9-1-1-1-12, 4-8-2-1-1-1-2, 1-1-3-10-2-1-2, 2-1-2-10-2-1-2, 2-1-1-12-4, 2-2-1-11-4, 3-1-1-11-4, 2-1-1- 13-3, 21-2-11-4, 2-2-1-12-3, 3-11-1-2-3, 3-1-1-12-3, 2-1-2-12-3, 4-11-2-1-2, 4-10-2-2-2, 3-2-1-9-1-1-3, 22-1-1-1-9-4, 2-2-2-9-1-1-3, 3-1-1-9-1-1-1-1-2, 2-1-2-9-1-2-3, 3-1-1-10-1-1-3, 2-1-1-2-1-9-4, 4-9-11-1-2-2, 3-1-1-9-1-2-3, 2-1-1-1-1- 10-4, 2-1-2-10-1-1-3, 2-1-1-1-1-9-2-1-2, 2-2-2-9-2-1-2, 4-9-1-2-11-2, 3-2-1-9-2-1-2, 2-1-2-9-2-2-2, 2-1-1-1-1-9-1-1-3, 3-1-1-9-2-2-2, 2-2- 2-10-4, 2-1-2-9-1-1-1-1-2, 4-10-1-2-3,3-2-1-10-4, 3-1-1- 10-2-1-2, 4-10-1-1-1-1-2, 4-11-1-1-3 and 2-2-2-10-1-1-2;

The number represents the number of LNA links, the next one represents the number of DNA links, and after that are sections of alternating LNA and DNA.

According to other embodiments, the ASOs of the present disclosure are represented as any of the ASO numbers selected from Figs. 2 and 3. According to some embodiments, the ASOs of the present disclosure (i.e., ASO-005459) comprise a 17 nucleotide long contiguous nucleotide sequence that corresponds to the complementary sequence of a region (i.e., the junction between intron 1 and exon 2) in the SNCA transcript, i.e., nucleotides 7604-7620 of SEQ ID NO: 1. According to some embodiments, an ASO of the present disclosure has a nucleotide sequence as set forth in SEQ ID NO: 15 (i.e., attcctttacaccacac) with the ASO scheme LDLDDDDDDDDLLLL (i.e., AtTcctttacaccACAC), wherein L is a locked nucleic acid nucleoside (i.e., LNA, e.g., beta-D-oxy-LNA), and D is deoxyribonucleic acid (DNA). Accordingly, the ^1st , ^3rd, and 14th-17th nucleotides from the 5' end of ASO-005459 are beta-D-oxy-LNA, and each of the other nucleotides is DNA. According to other embodiments, the ASO disclosed herein also has the following chemical structure: OxyAs DNAts OxyTs DNAcs DNAcs DNAts DNAts DNAas DNAcs DNAas DNAcs DNAcs OxyAs OxyMCs OxyAs OxyMC, wherein s is a phosphorothioate bridge. The structural formula for ASO-005459 is shown in Fig. 21B, wherein M ⁺ is a pharmaceutically acceptable counterion. The term pharmaceutically acceptable counterion, as used herein, refers to an ion that complements the ionic species to maintain electroneutrality, which is not biologically or otherwise undesirable, and thereby allows for the preparation of a pharmaceutically acceptable salt form. Accordingly, in some embodiments, the pharmaceutically acceptable counterion may be H ⁺ , Na ⁺ , K ⁺ , NH4 ⁺ , Li ^+, or any other cation, such as a cation with a charge of ¹⁺ . In some embodiments, the pharmaceutically acceptable counterion is H ⁺ , Na ⁺ , NH4 ^+, or combinations thereof.

II.H. Internucleotide bridges.

The monomers of the ASOs described herein are linked together via bridging groups. Accordingly, each monomer is linked to the 3'-adjacent monomer via a bridging 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' bridging group, although it may or may not contain a 5' end group.

The terms bridging group and internucleotide bridge are intended to mean a group capable of covalently linking two nucleotides together. Specific and preferred examples include phosphate groups and phosphorothioate groups.

The nucleotides in an ASO according to the present disclosure, or sequences of adjacent nucleotides therein, are linked together via bridging groups. Accordingly, each nucleotide is linked to a 3' adjacent nucleotide via a bridging group.

Suitable internucleotide bridges include those disclosed in International Application WO2007/031091, for example the internucleotide bridges disclosed in the first paragraph on page 1 of International Application WO2007/031091 (incorporated herein by reference in its entirety).

- 31 046118

Examples of suitable internucleotide bridges that can be used with the present disclosure include a phosphodiester bridge, a phosphotriester bridge, a methylphosphonate bridge, a phosphoramidate bridge, a phosphorothioate bridge, and combinations thereof.

In some embodiments, it is preferred to modify the internucleotide bridge from a normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate - these two, being cleavable by RNase H, also provide the ability of this antisense inhibition pathway to reduce target gene expression.

Suitable sulfur (S) containing internucleotide bridges as provided herein may be preferred. Phosphorothioate internucleotide bridges are also preferred, particularly for the gap region (B) in gapmers.

Phosphorothioate bridges can also be used for flanking regions (A and C, and for linking A or C to D, and within the D region if needed).

The A, B and C regions may, however, contain internucleotide bridges other than phosphorothioate, such as phosphodiester bridges, particularly, for example, when the use of nucleotide analogues protects the internucleotide bridges within the A and C regions from degradation by endonucleases, such as when the A and C regions contain LNA nucleotides.

The internucleotide bridges in the ASO may be phosphodiester, phosphorothioate, or boranophosphate to allow RNase H cleavage of the target RNA. Phosphorothioate is preferred due to improved nuclease resistance and other reasons such as ease of manufacture. According to some embodiments, the internucleotide bridges comprise one or more stereochemically defined internucleotide bridges (e.g., such as stereochemically defined modified phosphate bridges, such as phosphodiester, phosphorothioate, or boranophosphate bridges with a defined stereochemistry). The term stereochemically defined internucleotide bridge is used interchangeably with the term chirally controlled internucleotide bridge and refers to an internucleotide bridge in which the stereochemical position of the phosphorus atom is controlled such that a specified number of Rp or _Sp is present within the ASO chain in the internucleotide bridge. The stereochemical position of the chiral bridge can be determined (controlled), for example, by asymmetric synthesis. An ASO having at least one stereochemically determined internucleotide bridge can be called a stereochemically determined ASO, which includes both a fully stereochemically determined ASO and a partially stereochemically determined ASO.

According to some embodiments, the ASO is completely stereochemically defined. A completely stereochemically defined ASO refers to a sequence of ASOs that has a defined chiral center (Rp or Sp) in each internucleotide bridge in the ASO. According to some embodiments, the ASO is partially stereochemically defined. A partially stereochemically defined ASO refers to a sequence of ASOs that has a defined chiral center (Rp or Sp) in at least one internucleotide bridge, but not in all of the internucleotide bridges. Thus, a partially stereochemically defined ASO can include bridges that are achiral or not stereochemically defined, in addition to the at least one stereochemically defined bridge. If an internucleotide bridge in the ASO is stereochemically defined, the desired configuration, either

Rp or Sp is present in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the ASO.

According to one aspect of the ASO of the present disclosure, the nucleotides and/or nucleotide analogs are linked to each other via phosphorothioate groups. It is understood that the inclusion of phosphodiester linkages, such as one or two linkages, in an ASO in which all other linkages are phosphorothioate, particularly between or adjacent to the nucleotide analog units (typically in the A and/or C region) can modify the bioavailability of the ASO and/or the distribution of the ASO in biological systems - see International Application WO2008/113832, incorporated herein by reference.

According to some embodiments, such as the embodiments mentioned above, where appropriate and not specifically stated, all other bridging groups are either phosphodiester or phosphorothioate or a mixture thereof.

According to some embodiments, all internucleotide bridging groups

- 32 046118 py are phosphorothioate.

With respect to particular gapmer oligonucleotide sequences such as those provided herein, it will be appreciated that, in various embodiments, where the bridges are phosphorothioate bridges, alternative bridges such as those disclosed herein may be used, for example, phosphate (phosphodiester) bridges may be used, particularly in the case of bridges between nucleotide analog units such as LNA. Similarly, with respect to particular gapmer oligonucleotide sequences such as those provided herein, where the C residues are annotated as 5-'methyl-modified cytosine, in various embodiments, one or more of the Cs present in the ASO may be unmodified C residues.

U.S. Patent Publication No. 2011/0130441, which was published June 2, 2011 and is incorporated herein by reference in its entirety, relates to ASO compounds having at least one bicyclic nucleoside attached to the 3' or 5' ends via a neutral internucleoside bridge. Thus, the ASOs of the present disclosure may have at least one bicyclic nucleoside attached to the 3' or 5' ends via a neutral internucleoside bridge such as one or more of a phosphotriester, a methylphosphonate, a MMI (3'- _Ch2- N( _CH3 )-O-5'), an amide-3 (3'- _CH2 -C(=O)-N(H)-5'), a formatetal (3'-O- _CH2 -O-5'), or a thioformacetal (3'-S- _CH2 -O-5'). The remaining bridges may be a phosphorothioate.

According to some embodiments, ASOs of the present disclosure have internucleotide bridges as described in Figs. 2 and 3. As used herein, for example in Figs. 2 and 3, phosphorothioate bridges are designated by s, and phosphodiester bridges are designated by the absence of s.

II.I. Conjugates.

The term conjugate, as used in the context of this document, refers to an oligonucleotide that is covalently linked to a non-nucleotide moiety (the conjugated moiety, or C site, or third site).

Conjugation of an oligonucleotide according to the present disclosure with one or more non-nucleotide moieties can improve the pharmacological properties of the oligonucleotide, for example, by affecting the activity, distribution in cells, uptake into cells or stability of the oligonucleotide. According to some embodiments, the conjugated moiety modifies or improves the pharmacokinetic properties of the oligonucleotide by improving the distribution in cells, bioavailability, metabolism, clearance, permeability and/or uptake of the oligonucleotide into cells. In particular, the conjugate can target the oligonucleotide to a specific organ, tissue or cell type and thereby increase the effectiveness of the oligonucleotide in this organ, tissue or cell type. At the same time, the conjugate can serve to reduce the activity of the oligonucleotide in non-target cell types, tissues or organs, for example, non-target activity or activity in non-target cell types, tissues or organs. Suitable conjugation moieties are disclosed in International Applications WO 93/07883 and WO2013/033230. Further suitable conjugation moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr). In particular, trivalent N-acetylgalactosamine conjugation moieties are suitable for binding to ASGPr, see, for example, International Applications WO 2014/076196, WO 2014/207232 and WO 2014/179620.

Oligonucleotide conjugates and their synthesis have also been described in comprehensive reviews in Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, ST. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001; and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

According to an embodiment, the non-nucleotide moiety (conjugated moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), and combinations thereof.

II. J. Activated ASOs.

The term "activated ASO" as used herein refers to an ASO of the present disclosure that is covalently linked (i.e., functionalized) to at least one functional moiety that allows the ASO to covalently bond to one or more conjugate moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates described herein. Typically, the functional moiety will comprise a chemical group that is capable of covalently bonding to the ASO, such as via a 3'-hydroxyl group or an exocyclic MH2 group in an adenine base, a spacer that may be hydrophilic, and a terminal group that is capable of bonding to a conjugated moiety (e.g., an amino, sulfhydryl, or hydroxyl group). In some embodiments, the terminal group is unprotected, such as an MH2 group. In accordance with

- 33 046118 in other embodiments, the terminal 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).

According to some embodiments, the ASOs of the present disclosure are functionalized at the 5' end to allow covalent attachment of a conjugation moiety to the 5' end of the ASO. According to other embodiments, the ASOs of the present disclosure may be functionalized at the 3' end. According to other embodiments, the ASOs of the present disclosure may be functionalized along the backbone or at the heterocyclic base moiety. According to other embodiments, the ASOs of the present disclosure may be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone, and the base.

In some embodiments, activated ASOs of the present disclosure are synthesized by incorporating during synthesis one or more monomers that are covalently attached to a functional moiety. In other embodiments, activated ASOs of the present disclosure are synthesized using monomers that have not been functionalized, and the ASOs are functionalized upon completion of the synthesis.

III. Pharmaceutical compositions and routes of administration.

The ASOs of the present disclosure can be used in pharmaceutical formulations and compositions. Accordingly, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt, or excipient.

The ASOs of the present disclosure may be included in a unit formulation, such as in a pharmaceutically acceptable carrier or diluent, in an amount sufficient to deliver a therapeutically effective amount to a patient without causing serious side effects to the patient receiving the treatment. However, in some forms of therapy, serious side effects may be acceptable in order to ensure a positive result of the therapeutic treatment. The formulated drug may contain pharmaceutically acceptable binders and excipients. Capsules, tablets or pills may contain, for example, the following compounds: microcrystalline cellulose, acacia or gelatin as binders; starch or lactose as excipients; stearates as lubricants; various sweetening or flavoring agents. In the case of capsules, the dosage unit may contain a liquid carrier like fatty oils. Likewise, sugar coatings or enteric coatings may form part of the dosage unit.

Oligonucleotide formulations may also be emulsions of active pharmaceutical ingredients and a lipid that forms a micellar emulsion.

The pharmaceutical compositions of the present disclosure can be administered by various routes depending on whether local or systemic treatment is desired and the area to be treated. Administration can be (a) oral, (b) pulmonary, such as by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, (c) topical, including epidermal, transdermal, ocular and mucosal, including vaginal and rectal delivery; or (d) parenteral administration, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, such as intrathecal, intracerebroventricular or intraventricular administration. According to one embodiment, the ASO is administered IV, IP, orally, topically, or as a bolus injection, or administered directly into the target organ. According to another embodiment, the ASO is administered intrathecally or intracerebroventricularly as a bolus injection.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oil bases, thickeners and the like may be necessary or desirable. Examples of formulations for topical administration include those in which the ASOs of the present disclosure are present in admixture with a local delivery vehicle 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 non-aqueous media, capsules, soft gel capsules, sachets, tablets or minitablets. 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 pharmaceutical

- 34 046118 physically acceptable carriers or excipients.

Pharmaceutical compositions according to the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be formed from a variety of components, which include, but are not limited to, prepared liquids, self-emulsifying solids, and self-emulsifying semi-solids. Delivery of the drug to the target tissue can be enhanced by carrier-mediated delivery, including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched dendrimers, polyethyleneimine polymers, nanoparticles, and microspheres (Dass CR. J Pham Pharmacol 2002; 54(1): 3-27).

The pharmaceutical compositions of the present disclosure, which for convenience may be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). Typically, the compositions are prepared by uniformly and intimately bringing the active ingredients into association with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into a defined form.

In case of parenteral, subcutaneous, intradermal or topical administration, the composition may include a sterile diluent, buffers, tonicity regulators and antibacterial agents. Active ASOs can be prepared with carriers that protect against degradation or immediate elimination from the body, including in the form of implants or microcapsules with controlled release properties. In case of intravenous administration, the carriers can be physiological saline or phosphate-buffered saline. International Application Publication No. WO2007/031091 (A2), published March 22, 2007, further discloses suitable pharmaceutically acceptable diluent, carrier and excipients, which are hereby incorporated herein by reference.

IV. Diagnostics.

The present disclosure further provides a diagnostic method useful in diagnosing SNCA-related diseases, such as synucleopathy. Non-limiting examples of synucleopathy include, but are not limited to, Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies, and multiple system atrophy.

The ASO of the present disclosure can be used to measure the expression of SNCA transcript in tissue or body fluid from an individual and compare the measured expression level to a standard expression level of SNCA transcript in normal tissue or body fluid, wherein an increase in the expression level compared to the standard is indicative of a disease treatable with the ASO of the present disclosure.

The ASO of the present disclosure 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).

A biological sample is any biological sample obtained from an individual, cell line, tissue culture, or other source of cells that potentially express an SNCA transcript. Methods for obtaining tissue and body fluid biopsies from mammals are well known in the art.

V. Sets containing ASO.

The present disclosure further provides kits that contain an ASO of the present disclosure described herein and that can be used to perform the methods described herein. According to certain embodiments, a kit contains at least one ASO in one or more containers. According to some embodiments, the kits contain all of the 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 appreciate that the disclosed ASO can be readily included in one of the recognized kit formats that are well known in the art.

VI. Methods of application.

The ASO of the present disclosure can be used for therapy and prophylaxis. SNCA is a 140 amino acid protein expressed predominantly in neurons at presynaptic terminals, where it is believed to play a role in the regulation of synaptic transmission. It has been proposed to exist primarily as both an unfolded monomer and a stable tetramer 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). Normally, only a small percentage of SNCA is permanently phosphorylated.

- 35 046118 folded at S129 (pS129), while the vast majority of SNCA found in pathological intracellular inclusions is pS129 SNCA. These pathological inclusions consist of aggregated insoluble collections of misfolded SNCA proteins and are a characteristic feature of a group of neurodegenerative diseases known collectively as synucleopathies. In synucleopathies, SNCA can form pathological 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, the ASOs of the present invention can reduce the amount of pathological SNCA aggregates or prevent the formation of pathological SNCA aggregates. In addition, abnormal SNCA-rich foci called glial cytoplasmic inclusions (GCIs) are found in oligodendrocytes and are a hallmark of a rapidly progressive fatal synucleopathy known as multiple system atrophy (MSA). According to some embodiments, ASOs of the present disclosure reduce the amount of GCIs or prevent the formation of GCIs. Reports of either undetectable or low levels of SNCA mRNA expression in oligodendrocytes suggest that some abnormal form of SNCA is spreading from neurons, where it is highly expressed, to oligodendrocytes. According to certain embodiments, ASOs of the present disclosure reduce or prevent the spread of SNCA, such as an abnormal form of SNCA, from neurons.

The ASO can be used in research, for example, to specifically inhibit SNCA protein synthesis (typically by disrupting or inhibiting mRNA, thereby preventing protein formation) in cells and experimental animals, thereby facilitating functional analysis of the target or assessing its usefulness as a target for therapeutic intervention. Additionally provided are methods for down-regulating SNCA mRNA and/or SNCA protein expression in cells or tissues comprising contacting the cells or tissues, in vitro or in vivo, with an effective amount of one or more of the ASOs, conjugates or compositions of the present disclosure.

In the case of therapy, 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 the ASO compounds of the present disclosure. Further provided are methods of treating a mammal, such as treating a human, suspected of having or predisposed to having a disease or condition associated with the expression of SNCA transcript and/or SNCA protein, by administering a therapeutically or prophylactically effective amount of one or more of the ASOs or compositions of the present disclosure. An ASO, conjugate, or pharmaceutical composition of the present disclosure is typically administered in an effective amount. According to some embodiments, an ASO or conjugate of the present disclosure is used in therapy.

The present disclosure further provides an ASO according to the present disclosure for use in the treatment of one or more of the diseases mentioned herein, such as a disease selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies, multiple system atrophy, and any combinations thereof.

The present disclosure further provides a method of treating α-synucleopathies, the method comprising administering an effective amount of one or more ASOs, conjugates, or pharmaceutical compositions thereof to an animal in need thereof (such as a patient in need thereof).

According to certain embodiments, the disease, disorder, or condition is associated with overexpression of the SNCA gene transcript and/or SNCA protein.

The present disclosure also provides methods for inhibiting (e.g., 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 one or more ASOs, conjugates, or pharmaceutical compositions thereof of the present disclosure to disrupt the expression of the SNCA gene transcript, thereby reducing the level of SNCA protein. According to certain embodiments, the ASO is used to reduce the expression of SNCA mRNA in one or more regions of the brain, such as the hippocampus, brainstem, striatum, or any combination thereof. In other embodiments, the ASOs reduce SNCA mRNA expression, such as 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 following administration or exposure to vehicle (without the ASO) on day 3, day 5, day 7, day 10, day 14, day 15, day 20, day 21, or day 25. In some embodiments, SNCA mRNA expression is maintained at below 70%, below 60%, below 50%, below 40%, below 30%, below 20%, below 10%, or below 5% compared to SNCA mRNA expression

- 36 046118 after administration or exposure to the environment (without ASO) up to 28 days, up to 30 days, up to 32 days, up to 35 days, up to 40 days, up to 42 days, up to 45 days, up to 49 days, up to 50 days, up to 56 days, up to 60 days, up to 63 days, up to 70 days or up to 75 days.

According to other embodiments, an ASO of the present disclosure reduces the expression of SNCA mRNA and/or SNCA protein in the medulla oblongata, dorsal striatum, pons, cerebellum, lumbar spinal cord, frontal cortex, and/or any combinations thereof. The present disclosure also provides the use of an ASO or conjugate described herein for the manufacture of a medicament. The present disclosure also provides a composition comprising an ASO or conjugate thereof for use in the treatment of a disorder referred to herein or in a method of treating a disorder referred to herein. The present disclosure also provides ASOs or conjugates for use in therapy. The present disclosure further provides ASOs or conjugates for use in the treatment of synucleopathy.

The present disclosure further provides a method of inhibiting an SNCA protein in a cell that expresses SNCA, comprising administering an ASO or conjugate of the present disclosure to the cell to provide inhibition of the SNCA protein in the cell.

The present disclosure includes a method of reducing, attenuating, preventing or treating neuronal hyperexcitability in a subject in need thereof, comprising administering an ASO or conjugate of the present disclosure. The present disclosure also provides a method of treating a disorder mentioned herein, the method comprising administering an ASO or conjugate of the present disclosure described herein and/or a pharmaceutical composition of the present disclosure to a patient in need thereof.

ASO and other compositions according to the present disclosure can be used to treat conditions associated with overexpression or expression of a mutated variant of the SNCA protein.

The present disclosure provides an ASO or conjugate of the present disclosure for use as a medicament, such as for treating α-synucleopathies. According to some embodiments, the α-synucleopathy 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 present disclosure further provides for the use of an ASO of the present disclosure in the manufacture of a medicament for treating a disease, disorder, or condition mentioned herein. According to some embodiments, an ASO or conjugate of the present disclosure is used to manufacture a medicament for treating an α-synucleopathy, a seizure disorder, or a combination thereof.

As outlined in general, one aspect of the present disclosure is directed to a method of treating a mammal suffering from conditions associated with or sensitive to abnormal levels of SNCA (i.e., α-synucleopathy), which comprises administering to the mammal a therapeutically effective amount of an ASO that specifically targets a SNCA transcript that contains one or more LNA units. The ASO, conjugate, or pharmaceutical composition of the present disclosure is typically administered in an effective amount.

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

An interesting aspect of the present disclosure is directed to the use of an ASO (compound) as defined herein, or a conjugate as defined herein, for the preparation of a medicament for the treatment of a disease, disorder or condition as mentioned herein.

The methods of the present disclosure can be used to treat or prevent diseases caused by abnormal levels of SNCA protein. According to some embodiments, the diseases caused by abnormal levels of SNCA protein are α-synucleopathies. According to certain embodiments, α-synucleopathies include Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies, and multiple system atrophy. As set forth in the alternative, according to some embodiments, the present disclosure is further directed to a method of treating abnormal levels of SNCA protein, the method comprising administering an ASO of the present disclosure, or a conjugate of the present disclosure, or a pharmaceutical composition of the present disclosure to a patient in need thereof.

The present disclosure also relates to an ASO, composition or conjugate as defined herein for use as a medicinal product. The present disclosure further

- 37 046118 particularly 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 the expression of mutant forms of SNCA protein (such as allelic variants, such as those associated with one of the diseases referred to herein).

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

Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., 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, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195 to Mullis et al.; 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., NY); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 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, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (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 of the sources cited above, as well as all sources cited in this document, are incorporated herein by reference in their entirety.

The following examples are provided for illustrative purposes and not for limiting purposes.

Examples

Example 1. Designing ASO.

The antisense oligonucleotides described herein were designed to target various sites within the SNCA pre-mRNA. See Fig. 1A for the SNCA genomic sequence and Fig. 1B for the SNCA cDNA. For example, ASOs were designed to target sites designated using the pre-mRNA start site and pre-mRNA end site for NG_011851.1, as shown in Fig. 2, and/or the mRNA start site and end site for its mRNA. Exemplary ASO sequences (e.g., SEQ ID numbers) are described in Figs. 2 and 3. In some embodiments, the ASOs are designed to have a gapmer structure (e.g., gapmers with alternating units). See DES numbers. Figs. 2 and 3 provide non-limiting examples of the ASO design for selected sequences. The same methods can be applied to any other sequences disclosed in this document. The gapmers were designed to contain locked nucleic acids - LNA (capital letters). For example, a gapmer can have a beta-deoxy-LNA at the 5' end and at the 3' end and have a phosphorothioate backbone. But the LNA can also be replaced by any other nucleotide analogs, and the backbone can be other types of backbones (e.g., a phosphodiester bridge, a phosphotriester bridge, a methylphosphonate bridge, a phosphoramidate bridge, or combinations thereof).

The ASOs 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 expressly incorporated herein by reference.

Example 2A: High-content assay to measure SNCA protein reduction in primary neurons.

ASOs targeting SNCA were examined for their ability to reduce SNCA protein expression in mouse primary neurons. Primary neuron cultures were prepared from the forebrain of PAC-T g(SNCA ^A53T ) ^+/+ ;SNCA ^-/- (PAC-A53T) mice carrying the complete human SNCA gene with the A53T mutation in a mouse SNCA knockout background. See Kuo Y et al., Hum Mol Genet, 19: 1633-50 (2010). All procedures involving mice were performed according to Animal Test Methods (ATM) approved by the Bristol-Myers Squibb Animal Care and Use Committee (ACUC). Primary neurons were prepared 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 units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml

- 38 046118 amphotericin B.

Cells were seeded in poly-E-lysine coated multiwell plates at 5400 cells/ ^cm2 (e.g., 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 at a concentration 2-fold higher than the final concentration in medium, then delivered to the cells manually. Alternatively, ASO in water was dispensed using the Labcyte ECHO acoustic dispenser. For ECHO dispensing, 250 nl ASO in water was added to cells in medium, followed by an aliquot volume of fresh BM. For primary screening, ASOs were added at final concentrations of 5 μM, 3.3 μM, 1 μM, 200 nM, or 40 nM. For activity determination, 8-10 sequentially decreasing concentrations of ASOs were prepared from a 0.75 mM stock solution and then delivered to cultured cells at final concentrations ranging from 2.7-4,000 nM or 4.5-10,000 nM. ASO-000010 (TCTgtcttggctTTG, SEQ ID NO: 22) and ASO000838 (AGAaataagtggtAGT, SEQ ID NO: 23) (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 steady-state reduction in mRNA levels.

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 using 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. A 1:1000 dilution of rabbit anti-SNCA antibody (Abcam) and a 1:500 dilution of 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 twice with PBS. Secondary antibody staining solution containing Alexa 567-conjugated goat anti-chicken antibody, Alexa 488-conjugated goat anti-rabbit antibody at a 1:500 dilution and Hoechst dye (10 μg/mL) in PBS containing 0.1% Triton X-100 with 3% BSA was added to the cells and the plates were incubated for 1 h. After this, the secondary antibody 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 visualization, plates were scanned on a Thermo-Fisher (Cellomics) CX5 using the Spot Detector bio-application (Cellomics) to quantify nuclei (Hoechst stain, channel 1), tubulin inclusions (Alexa 567, channel 2), and SNCA (Alexa 488, channel 3). The number of objects (nuclei) counted was tracked but not published in the database. The total area occupied by tubulin was defined as the total spot area for channel 2 (SpotTotalAreaCh2) and the total staining intensity for SNCA was defined as the total spot intensity for channel 3 (SpotTotalIntenCh3). Tubulin measurement was included for toxicity monitoring. To determine the reduction in SNCA protein levels, the ratio of the intensity for SNCA to the area of stained area for tubulin was calculated and the results were normalized as % median inhibition using the median value for medium-treated wells as the total value and ASO-000010 or ASO-000838 wells as the wells with maximum inhibition for tubulin or SNCA, respectively.

Example 2B: Measurement of spontaneous fluctuations in calcium levels.

Reduced intracellular fluctuations in unbound calcium concentrations (calcium swings) are consistent with increased neurotoxicity and may therefore indicate decreased in vivo tolerability. To measure spontaneous calcium swings in primary cortical neurons, rat primary cortical neurons were prepared from Sprague-Dawley rat embryos (E19). Briefly, cortices were dissected and incubated at 37°C for 30-45 min in papain/DNase/Earle's balanced salt solution (EBSS). After homogenization and centrifugation of the cell pellet, the reaction was stopped by incubation with EBSS containing protease inhibitors, bovine serum albumin (BSA) and DNase. The cells were then homogenized and washed with neurobasal medium (NB, Invitrogen) supplemented with 2% B-27, 100 μg/ml penicillin, 85 μg/ml streptomycin, and 0.5 mM glutamine.

Cells were seeded at 25,000 cells/well onto poly-E-lysine-coated 384-well fluorescence imaging plates (BD Biosciences) in a volume of 25 μl/well supplemented with neurobasal (NB) medium (containing B27 supplement and 2 mM glutamine). Cells were grown for 12 days at 37°C in 5% _CO2 and were supplied with 25 μl additional medium at DIV04 (i.e., 4 days after seeding) and DIV08 (i.e., 8 days after seeding) for use at DIV12 (i.e., 12 days after seeding). On the day of the experiment, NB medium was removed from the plate and the cells were washed once with assay buffer (Hanks balanced salt solution containing 2 mM _CaCl2 and 10 mM Hopes pH 7.4) at 37°C at 50 µl/well. The oscillations were examined both in

- 39 046118 in the presence and absence of 1 mM MgCl ₂ . Cells were loaded with a permanent fluorescent dye for calcium, Fluo-4-AM (Invitrogen, Molecular Probes F14201). Fluo-4-AM was prepared at a concentration of 2.5 mM in DMSO containing 10% pluronic F-127 and then diluted 1:1000 in assay buffer to a final concentration of 2.5 μM. Cells were incubated for 1 h with 20 μl of 2.5 μM Fluo4-AM at 37°C in 5% CO ₂ . After incubation, an additional 20 μl of room temperature assay buffer was added and cells were allowed to equilibrate to room temperature in the dark for 10 min.

Plates were read on an FDSS 7000 fluorescence plate reader (Hamamatsu) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The total fluorescence acquisition time was 600 s at an acquisition rate of 1 Hz for all 384 wells. The baseline background signal (measurement of intracellular calcium levels) was blocked for 99 s before addition of ASO. ASO was added using the 384-well head in the FLIPR in 20 μl of assay buffer at a concentration of 75 μM to give a final concentration of 25 μM. In some cases, ASOs targeting the tau protein, such as ASO-000013 (OxyAs OxyTs OxyTs DNAts DNAcs DNAcs DNAas DNAas DNAas DNAts DNAts DNAcs DNAas OxyMCs OxyTs OxyT; ATTtccaaattcaCTT, SEQ ID NO: 24) or ASO-000010 (TCTgtettggctTTG, SEQ ID NO: 22), were included as controls.

The fluorescence intensity time series measurements (described above) were exported from the Hamamatsu reader and transferred to a proprietary application in the IDBS E-Workbook suite for data processing and normalization. In each 384-well screening plate, up to 48 individual ASOs were assayed, in quadruplicate. Twelve wells were exposed to a positive control (ASO-000010), which significantly inhibits calcium swings measured over the 300 s recording period, and 12 wells were exposed to an inactive negative control ASO (ASO-000013), which does not inhibit the observed calcium swings. Finally, 24 wells were assigned to a control medium consisting of RNase-DNase-free water at the same concentration used to dilute the ASOs under study. The effects of the studied ASOs in single cells on calcium oscillation frequency (over a 300-second period) were expressed as % of control with the median number of calcium oscillations calculated in the 24 media control wells. Individual 384-well assay plates were QC'd if the positive and negative control ASOs (ASO-000010 and ASO-000013) exhibited well-characterized pharmacology in the Ca assay and if the media and pharmacology control wells generated a minimum of ~20 calcium oscillations over the 300-second period of the experiment.

Example 2C: QUANTIGENE® assay (96-well plate assay) for measuring mRNA reduction in human neurons.

The ability of ASO to reduce human SNCA mRNA levels and/or potential non-target human mRNA species was measured in vitro using the QUANTIGENE® assay. Human neurons (Cellular Dynamics Inc., iNeurons) were thawed, seeded, and cultured according to the manufacturer's instructions. These iNeurons cells represent a highly pure population of human neurons derived from induced pluripotent stem (iPS) cells using Cellular Dynamic's proprietary differentiation and purification protocols.

Lysis: Cells were seeded onto poly-E-ornithine/laminin-coated 96-well plates at 50,000 to 100,000 cells per well (depending on the expression of the non-target mRNA of interest) and maintained in neurobasal medium supplemented with B27, glutamax, and penicillin-streptomycin. ASO was diluted in water and added to cells at DIV01 (i.e., 1 day after seeding). For single concentration measurements, a final concentration of 0.5 μM ASO was typically used. For IC ₅₀ determinations, neurons were treated with seven concentrations to generate a seven-point concentration-response curve at a 1:4 dilution, with the highest concentration being 5 μM for IC ₅₀ determinations. Cells were then incubated at 37°C and 5% _CO2 for 6 days to achieve a steady-state reduction in mRNA levels.

Following incubation, the medium was removed and the cells were washed 1x in DPBS and subjected to subsequent lysis. MRNA in the lysate was measured using the QUANTIGENE® 2.0 Reagent System (AFFYMETRIX®), which quantifies RNA using a branched DNA signal amplification method dependent on a specially designed RNA capture probe set. Cell lysis working buffer was prepared by adding 50 µl proteinase K to 5 ml pre-warmed (37°C) lysis mixture and diluted in _dH2O to a final dilution of 1:4. The working lysis buffer was added to the plates (100 to 150 µl/well depending on the expression of the non-target mRNA being studied), homogenized 10 times, sealed and incubated for 30 min at 55°C. After lysis, the well contents were homogenized more than 10 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 tube

- 40 046118 line) lysates were diluted (or not) in lysis mix. Lysates were then added to capture plates (96-well polystyrene plates coated with capture probes) in a total volume of 80 µl/well. The working probe reagents in the kits were prepared by combining nuclease-free water (12.1 µl), lysis mix (6.6 µl), blocking reagent (1 µl), and specific Probe Kit 2.0 (0.3 µl) (human SNCA catalog #SA-50528, human PROS1 catalog #SA-10542, or human beta-3-tubulin catalog #SA-15628) according to the manufacturer's instructions (QUANTIGENE® 2.0 AFFYMETRIX®). Then 20 µl of the working probe reagents in the kit were added to 80 µl of the diluted lysate (or 80 µl of the lysis mix in the case of background samples) on a capture plate. The plates were centrifuged at 240 g for 20 s and then incubated for 16-20 h at 55°C for hybridization (capture of target RNA).

Signal amplification and detection of target RNA was initiated by washing the plates with buffer 3 times (300 µl/well) to remove any unbound material. Pre-Amplifier hybridization reagent 2.0 was then added (100 µl/well), incubated at 55°C for 1 h, aspirated, and wash buffer was added and aspirated 3 times. Amplifier hybridization reagent 2.0 was then added as described (100 µl/well), incubated for 1 h at 55°C, and the washing step was repeated as described above. Label Probe hybridization reagent 2.0 was then added (100 µl/well), incubated for 1 h at 50°C, and the washing step was repeated as described above. The plates were again centrifuged at 240g for 20 s to remove any excess wash buffer, and then Substrate 2.0 (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 multilabel plate reader in luminometer mode for 15 min.

Data Definition: For the gene of interest, the average background signal in the assay was subtracted from the average signal for each technical replicate. The average signals for the gene of interest minus the background signal were then normalized to the average signal for constitutive tubulin RNA minus the background signal. The percent inhibition for the treated sample was calculated relative to the lysate of the control-treated sample. The results of the QUANTIGENE® assays for ASO-treated cells are shown, for example, in Fig. 8.

Example 2D: QUANTIGENE® assay (96-well plate assay) for measuring mRNA reduction in Ramos cells.

To measure the possible reduction in levels of non-target human IKZF3 (IKAROS zinc finger protein 3) mRNA, Ramos cells (a human lymphocyte cell line) were used. Since Ramos cells do not express SNCA, RB1 (RB transcriptional corepressor 1), which is expressed in Ramos cells, was used as a positive control to assess ASO-mediated knockdown of IKZF3 mRNA expression. Two ASOs were synthesized that bind to human RB1 mRNA and knockdown human RB1 mRNA expression. Beta-2-microglobulin (P2M) was used as a control housekeeping gene. Ramos cells were grown in suspension in RPMI medium supplemented with FBS, glutamine, and penicillin/streptomycin.

Lysis: Cells were seeded in poly-L-ornithine/laminin-coated 96-well plates at 20,000 cells/well and maintained in neurobasal medium containing B27, glutamax, and penicillin-streptomycin. ASO was diluted in water and added to the cells 1 day after seeding (DIV01) to a final concentration of 1 μM. Following ASO treatment, cells were incubated at 37°C for 4 days to achieve steady-state reduction in mRNA levels. Following incubation, medium was removed and cells were further lysed. mRNA was measured using the QUANTIGENE® 2.0 Reagent System (AFFYMETRIX®), which quantifies RNA using a branched DNA signal amplification method dependent on a custom-designed RNA capture probe set. The lysis mixture (QUANTIGENE® 2.0 AFFYMETRIX®) was pre-warmed in an incubator at 37°C for 30 min. To lyse the cells in suspension, 100 µl of 3x lysis buffer (with 10 µl/ml proteinase K) was added to 200 µl of cells in suspension. The cells were then homogenized 10 times for lysis and the plate was sealed and incubated for 30 min at 55°C. The lysates were then stored at -80°C or analyzed immediately.

Assay: Depending on the specific capture probe used (i.e. IKZF3, RB1, and P2M), lysates were diluted (or not) in lysis mix. Lysates were then added to a capture plate (96-well polystyrene plate coated with capture probes) in a total volume of 80 µl/well. The working probe reagents in the kits were prepared by combining 12.1 µL of nuclease-free water, 6.6 µL of lysis mix, 1 µL of blocking reagent, 0.3 µL of the specific probe kit 2.0 (human IKZF3 catalog #SA-17027, human RB1 catalog #SA-10550, or human beta-2 microglobulin catalog #SA-10012) according to the manufacturer's instructions (QUANTIGENE® 2.0 AFFYMETRTX®). Then, 20 µL of the working probe reagents were

- 41 046118 probes in the kit were added to 80 µl of diluted lysate (or 80 µl of lysis mix for background samples) on a capture plate. The plates were then incubated for 16-20 h at 55°C for hybridization (capture of target RNA). Signal amplification and detection of target RNA was initiated by washing the plates with buffer 3 times (300 µl/well) to remove any unbound material. Pre-Amplifier hybridization reagent 2.0 was then added (100 µl/well), incubated at 55°C for 1 h, then aspirated and wash buffer was added and aspirated 3 times. Next, 2.0 Amplifier hybridization reagent was added as described (100 µl/well), incubated for 1 h at 55°C, and the washing step was repeated as described above. Next, 2.0 Label Probe hybridization reagent was added (100 µl/well), incubated for 1 h at 50°C, and the washing step was repeated again as described above. 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 visualized on a PerkinElmer Envision multilabel plate reader in luminometer mode for 15 min.

Data Definition: For the gene of interest, the mean background signal (i.e. no lysate, 1x lysis buffer only) was subtracted from the mean signal for each technical replicate. The mean signals for the gene of interest minus the background signal were then normalized to the mean signal for the constitutive mRNA minus the background signal (in the case of Ramos cells, this was beta-2-microglobulin mRNA). The percent inhibition for the treated sample was calculated relative to the mean value for the untreated sample lysate. The results of the QUANTIGENE® assays for ASO-treated cells are presented in Table 4.

Example 2E. qPCR assay to measure SNCA mRNA reduction in SK-N-BE(2) cells.

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

SK-N-BE(2) cells were grown in cell culture medium (MEM [Sigma, catalog #M2279] supplemented with 10% fetal bovine serum [Sigma, catalog #F7524], 1x GLUTAMAX™ [Sigma, catalog #3050-038], 1x MEM nonessential amino acid solution [Sigma, catalog #M7145], and 0.025 mg/mL gentamicin [Sigma, catalog #G1397]). Cells were trypsinized every 5 days by washing with phosphate-buffered saline (PBS) [Sigma, catalog #14190-094] followed by addition of 0.25% trypsin-EDTA (Sigma, T3924), 2-3 min of incubation at 37°C, and homogenization 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, catalog #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 cell seeding to a final concentration of 25 μM for single concentration studies. Cells were incubated for 4 days without any medium change. For activity measurements, 8 concentrations of ASO were prepared covering a final concentration range of 16-50,000 nM. ASO-004316 (CcAAAtcttataataACtAC, SEQ ID NO: 25) and ASO-002816 (TTCctttacaccACAC, SEQ ID NO: 12) were included as controls. Following incubation, cells were harvested by removing the medium followed by the addition of 125 µl PURELINK® Pro 96 Lysis Buffer (Invitrogen 12173.001 A) and 125 µl 70% ethanol. RNA was purified according to the manufacturer's instructions and eluted in a final volume of 50 µl water, resulting in an RNA concentration of 10-20 ng/µl. RNA was diluted 10-fold in water prior to the one-step qPCR reaction. For the one-step qPCR reaction, qPCR mix (qScript TMXLE 1-step RT-qPCR TOUGHMIX® Low ROX from QauntaBio, catalog #95134-500) was mixed with two Taqman probes at a ratio of 10:1:1 (qPCR mix:probe 1:probe 2) to form a master mix. Taqman probes were obtained from OTLifeTechnologies: SNCA: Hs01103383_ml; PROS1: Hs00165590_ml: TBP: 4325803; GAPDH 4325792. Master mix (6 µl) and RNA (4 µl, 1-2 ng/µl) were then mixed in a qPCR plate (MICROAMP® 384-well optically clear bottom plates, 4309849).

After sealing, the plate was briefly centrifuged at 1000 g for 1 min at RT (room temperature) and transferred to the 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 of 95°C for 5 s followed by a temperature decrease of 1.6°C/s followed by maintenance at 60°C for 45 s. Data were analyzed using QUANTSTUDIO™ Real-Time PCR Software.

Example 3. In vitro assay of ASO-005459 for reduction of human SNCA mRNA levels.

ASO-005459 is a 17-base LNA-modified ASO that targets the intron 1/exon 2 boundary of human SNCA pre-mRNA (see FIG. 2, SEQ ID NO: 15).

- 42 046118

Activity of ASO-005459 in mouse neurons.

Using the methods described above in Example 2A, ASO-005459 was examined for its ability to reduce SNCA protein expression as a further result of reducing SNCA mRNA levels. Briefly, primary neurons derived from PAC-A53T mice were treated with ASO005459 or control ASOs for 14 days. The cells were then fixed and SNCA protein and tubulin protein levels were measured using high content imaging. Tubulin levels were measured to monitor toxicity and to normalize the reduction in SNCA protein levels.

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

Table 1

ASO-005459 activity in A53T-RAS neurons

Concentration ASO-005459 Inhibition of a-synuclein/tubulin, % SD N Tubulin inhibition, % 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

SD = standard deviation N = number of tests

To further evaluate activity, A53T-RAS neurons were treated with 10 sequentially decreasing concentrations of ASO-005459 as described above in Example 2A and _IC50 values for the effects on SNCA and tubulin proteins were determined. As shown in Figs. 7A and 7B and Table 2 (below), a concentration-dependent decrease in the α-synuclein/tubulin ratio was observed, with an average _IC50 of 7.4 nM. This observation was consistent with the single concentration activity data presented in Table 1 (above). Moreover, ASO-005459 had negligible effects on tubulin levels. Collectively, these results indicate that ASO-005459 actively reduces SNCA protein levels with minimal effects on overall cell viability.

Table 2

Evaluation of the potency and selectivity of ASO-005459 for SNCA and tubulin protein in A53T-RAS neurons

IC50 for asinuclein/tubulin (nM) SD N IC50 for tubulin (nM) SD N 7.42 2.73 6 >4000 NA 5

SD = standard deviation N = number of tests

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

Using the method described in Example 2E, 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% reduction in SNCA mRNA levels in SK-N-BE(2) cells.

Activity of ASO-005459 in human neurons.

The activity of ASO-005459 against SNCA was confirmed using primary human neurons as described above in Example 2C. Briefly, human neurons were derived from induced pluripotent stem (iPS) cells. Cells were treated with ASO-005459 or control ASOs for 6 days, and mRNA levels were then measured using the QUANTIGENE® assay. Since human neurons also express PROS1, a potential off-target of ASO-005459, PROS1 mRNA levels were also measured to assess the effects of ASO-005459 on off-target genes. Additionally, tubulin (TUBB) mRNA was measured to monitor toxicity.

As shown in Fig. 8 and Table 3 (below), ASO-005459 induced a concentration-dependent reduction in SNCA expressed in human neurons, with a mean _IC50 of 100 nM. This _IC50 is -13-fold weaker than the _IC50 for the reduction in SNCA protein levels in PAC-A53T neurons (Table 2, above). The reason for this shift in potency is unclear, but may be due to differences in ASO uptake, metabolism, or the kinetics of SNCA knockdown. Although ASO-005459 also exhibited concentration-dependent reductions in PROS1 mRNA levels in human neurons; the _IC50 was 30-50-fold weaker than the _IC50 for SNCA. These results confirm the activity of ASO-005459 against SNCA in human neurons and indicate that ASO-005459 is

- 43 046118 is 30-50 times more selective for SNCA compared to PROS1. Table 3

Activity and selectivity of ASO-005459 towards SNCA and PROS1 mRNA in human neurons

Date of analysis Party SNCA 1C ₅₀ (nM) PROS1 1C ₅₀ (nM) TUBB3 1C ₅ o (nM) Kuo ratio for PROS1/5ML4 8.26.2016 01-001 42 2127 >5000 51 12.8.2016 01-004 157 >5000 >5000 >32

Batch=specific batch number of the applied ASO-005459

Activity of ASO-005459 in Ramos cells.

IKZF3 is another potential off-target for ASO-005459, but unlike PROS1, it is not expressed in human neurons. However, IKZF3 is highly expressed in Ramos cells, a human lymphocyte cell line. Ramos cells were treated with 1 μM ASO-005459 and the effect on IKZF3 mRNA expression was measured.

To monitor toxicity, beta-2-microglobulin mRNA levels were also measured. Since 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: 26) and ASO-006755 (GGTgaggtttggtagaAG, SEQ ID NO: 27) 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 P2M 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 the activity of ASOs in Ramos cells.

Table 4

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

ASO Target Party IKZF3 (% CONC.) RB1 (% CONC.) /!2M (% CONC.) ASO-005459 SNCA 01-004 102 99 94 ASO-006754 RB1 01-001 104 13 87 ASO-006755 RB1 01-001 116 17 83

Batch = specific batch number of the ASO used

Taken together, the results presented in this table demonstrate that ASO-005459 is potent and highly selective for reducing SNCA mRNA levels, which in turn mediates a reduction in SNCA protein levels. The results also demonstrate that ASO-005459 is well tolerated in both murine and human neurons. These data support the continued development of ASO-005459 as a disease-modifying therapeutic for the treatment of synucleopathies.

Example 4. In vivo tolerability and reduction of SNCA mRNA levels in vivo.

The in vivo tolerability of selected ASOs was investigated to see how the ASOs were tolerated when injected into different animal models (i.e. mice and cynomolgus monkeys).

Mice.

Subjects: Male and female PAC-Tg(SNCA ^A53T ) ^+/+ ; SNCA ^-/- (PAC-A53T) mice (2-3 months old) carrying the complete human SNCA gene with the A53T mutation on a mouse SNCA knockout background were used for short-term, long-term efficacy, and in vivo PK/PD 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 animal facility with access to food and water ad libitum. 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 ASO dosing solution: Syringes containing sterile saline (1 mL), syringes fitted with 0.2 µm Whatman filters, and nuclease-free centrifuge tubes were used to prepare dosing solutions. The specified volume of water or saline was added to the ASO powder and vortexed (~1 min) to dissolve the ASO powder. The solution was then allowed to settle for 10 min and vortexed again for ~1 min. The tubes were briefly centrifuged 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 2nd RNase-free tube. A small aliquot of the primary stock solution was diluted to 1

- 44 046118 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 pedestal was washed and wiped three times before sample application). The test sample was discarded after the analysis was complete. The sample was considered ready for dispensing if its UV absorbance was between 90 and 110% of the sample. If the UV absorbance was greater than 110% of the sample, a secondary dilution was prepared; if the absorbance was <90%, the sample was prepared at a higher stock concentration, followed by similar steps as described above. Samples were stored at 4°C until use.

Manual intracerebroventricular injection (ICV): ICV injections were performed using a Hamilton microsyringe fitted with a 27- or 30-gauge needle according to the method of Haley and McCormick. The needle was fitted with a polyethylene stopper 2.5-3 mm from the tip to limit its penetration into the brain. Mice were anesthetized with isoflurane anesthetic (1-4%). After sufficient anesthesia was achieved, mice were restrained by a fold of skin behind the neck between the thumb and forefinger of one hand. Using gentle but firm pressure, the animal's head was then immobilized by pressing it against a hard, flat surface. Doses were administered using 10-μl Hamilton syringes fitted with a 27 ¹ /2-gauge needle. The needle tip was then inserted through the scalp and cranium, approximately 1 mm lateral and 1 mm posterior to bregma (i.e., to the right of the midline, approximately 3 mm posterior when measured from eye line). Once the needle was in place, ASO was given in a volume of 5 μl in saline medium and injected over ~30 s. The needle was left in place for 5-10 s before being removed. Mice were returned to their home cages and allowed to recover for ~2-4 min. Mice were continuously monitored for 30 min immediately post-dose to assess for adverse drug and/or dosing effects on behavior. During this time period, any mouse that convulsed at more than 3 separate points was immediately sacrificed and automatically scored 20. Drug tolerability was assessed 1 h ±15 min post-dose. Animals that received doses of intolerable compounds (tolerance score >4) were sacrificed immediately after assessment within 1 h.

ASO Tolerability Assessment: Animals receiving ASO doses were assessed immediately post-dose and monitored for any adverse effects for 2 hours. For acute tolerance (AT) studies, mice were assessed at dosing and again at study exit, i.e., 3 days after ASO injection. For long-term health assessments, mice were weighed weekly and monitored for any health and behavioral issues until completion of the experiment. Mice that had weight losses greater than 15% of their initial body weight or demonstrated tolerance issues were excluded from the studies and sacrificed. Health and tolerance assessments were performed according to the following schedule.

- 45 046118

Table 5

Tolerability Assessment Systema

Category Rating 1 Rating 2 Rating 3 Rating 4 Hyperactivity, stereotypies, behavior in a permanent cage •Very slightly increased exploration or rearing compared to controls •Increased exploration of the permanent residence cage (e.g., digging, burying, etc.) •Increased grooming of the body surface •Moderately increased activity in the permanent cage •Revealed stereotypies (e.g., circling, pathologically cyclical actions, etc.) •Severe hyperactivity •Severe stereotypies Decreased alertness, exploratory behavior, and reactivity •Some decrease in exploratory activity •Responds normally to stimulation •Drowsiness •Slightly decreased responsiveness to touch or manipulation • Numbness (decreased reactivity, decreased corneal reflex) •Comatose state (does not respond to stimulation, such as a pinch), no corneal reflex Coordination of movements and strength •Slight change in gait or grip strength (falls in 510 seconds) •No falls, normal response in the form of recovery of body position •Reduced grip strength (falls in less than 5 seconds) •Mild ataxia (eg, slow recovery, rocking) •Severely decreased grip strength (falls in less than 2 seconds) •Ataxia (eg, staggering, abnormal gait with falling) •Severe ataxia (eg, crawling, inability to grasp a bar) •Unable to regain body position Posture, appearance, breathing •Very slightly expressed abnormal posture (barely noticeable) •Mild abnormal posture (e.g. hunched, stretched, low posture, tail carriage, Straubtail) •Piloerection or drooping of upper eyelid •Disheveled coat •Moderate abnormal posture (eg, prone position) •Shallow breathing • Marked abnormal posture (eg, lying on side) • Facial nerve paralysis (eg, drooling, protruding tongue) • Difficulty breathing

Category Rating 1 Rating 2 Rating 3 Rating 4 Tremors, hyperactivity, convulsions •Detectable tremor •Excessive response to stimuli (e.g. noise) •Noticeable tremors •Few or partial seizures, rearing up on hind legs and falling over during convulsions •Repetitive or continuous seizures (running, jerking, clonic seizures and/or tonic seizures)

and the normal state corresponds to a score of 0

Animals are assessed individually at successive time points after dosing. Observations are scheduled at 1 h ± 15 min, then 24 h ± 2 h, then 7 days (if necessary). Convulsions are counted over a 1-h period, even if they occurred before the observation period. An overall tolerability score is calculated from the sum of the individual category scores, with a maximum possible score of 20.

Tissue collection: Following final behavioral and health assessments, mice were decapitated

- 46 046118 were guillotined and the brains were rapidly removed. Each brain was divided into two hemispheres and a) the hippocampus was dissected out for mRNA measurements in a 3-day acute tolerance study; b) the hippocampus, brainstem and striatum from one hemisphere were dissected out for mRNA measurements, while the same regions were dissected out from the other hemisphere for protein/PK measurements in dynamic PK/PD dose-response studies.

In some studies, blood and cerebrospinal fluid (CSF) were also collected for PK (blood) and PK/protein (CSF) measurements. For blood and CSF collection, mice were deeply anesthetized with isoflurane (4%). Blood was collected via cardiac puncture using a 23G needle. After removal, blood was transferred to 2 mL BD Microtainer tubes (K2EDTA BD #365974) and placed on ice until processing. For blood processing, tubes were centrifuged at 4500 x g for 10 min at 4°C. Plasma was then removed and placed in 0.5 mL Eppendorf tubes and stored at -80°C until use. For CSF collection, the chest cavity was opened to expose the heart and as much blood as possible was drained to avoid CSF contamination. CSF samples were collected via the cerebellomedullary cistern using micropipettes and placed into lo-bind protein Eppendorf tubes. The tubes were then centrifuged at 4500 x g for 15 min at 4°C. CSF was carefully transferred into clean 0.5 ml lo-bind Eppendorf tubes and stored at -80°C until further use.

Data from Javanese macaques.

Subject: Male cynomolgus monkeys weighing 3.5-10.0 kg at baseline were used. Each had an intrathecal cerebrospinal fluid (CSF) catheter implanted into the L3 or L4 vertebrae. The distal end of the polyurethane catheter was passed within the intrathecal space to approximately the L1 vertebra. The proximal end was connected to a subcutaneous exit port located on the animal's lumbar region. Animals were allowed to heal for at least two weeks prior to study initiation. Laboratory animals were cared for in accordance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Guide for the Care and use of Laboratory Animals NRC (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 Sample Collection: The CSF collection port was accessed subcutaneously using aspect techniques and CSF samples were collected from awake animals seated upright in a primate restraint chair. Approximately 0.1 mL of CSF was drained at the beginning of collection to clear stagnant space in the catheter and port. CSF was collected by gravity flow to a maximum volume of 0.5 mL CSF per sample. CSF was centrifuged at 2000 g at 4°C for 10 min. The supernatant was frozen on dry ice or in liquid nitrogen and stored at -90°C until analysis.

Blood samples were collected from an accessible vein, typically the saphenous vein. Blood samples were prepared using a variety of procedures depending on the specific measurement being discussed. For plasma, blood was collected into EDTA-treated tubes. For serum, blood was collected into serum separator tubes and allowed to clot for at least 30 min before centrifugation. For coagulation and coagulation factor measurements, blood was collected into citrate-containing tubes, and for RNA analysis, blood was collected into tubes containing RNA later reagent. After processing, samples were frozen on dry ice or in liquid nitrogen and kept frozen until analysis.

Intrathecal Dosing: Animals were trained to receive the dose while awake and maintained in a supine position using modified commercially available restraint chairs. SNCA-targeting antisense oligonucleotides (ASOs) were dissolved in saline, filter sterilized, and infused at 0.33 mL/min in a volume of 1.0 mL followed by a 0.5 mL sterile water rinse. Total infusion time was 4.5 min. Animals were maintained in a supine position for 30 min post-infusion.

Necropsy: Cynomolgus monkeys were administered an appropriate volume of commercially available euthanasia solution under ketamine and/or isoflurane anesthesia. Tissues were obtained immediately after necropsy and brains were transferred to ice for dissection. Regions of interest were sectioned into 4-6 mm sections using the ASI Cyno Brain Matrix as well as manual techniques. Samples were mounted fresh in RNAlater or frozen on ice for later analysis. CNS tissue was rapidly dissected from cynomolgus monkeys and pieces no longer than 4 mm in any axis were collected and placed in 5 ml of RNAlater. Samples were stored at 4°C overnight, then transferred to -20°C for storage until analysis. Regions of interest were

- 47 046118 The brain regions included the medulla oblongata, 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 white matter of the cerebral cortex. In addition, spinal cord samples were collected from the cervical, thoracic, and lumbar regions. Samples were also collected from the liver, kidney, and heart. In some cases, samples of the trigeminal nuclei, tibial nerve, and aorta were collected to evaluate off-target pharmacological properties in these regions.

Quantitative determination of ASO concentration by ELISA in mouse or monkey tissues, plasma and CSF.

The tissue was homogenized with plasma and water at a 1:1 ratio. A calibration curve was constructed by 2-fold serial dilution from 5000 to 4.9 nM in plasma (for plasma and CSF) and in a plasma:water mixture (for tissue samples), and then further diluted to a total of 5000-fold dilutions in 5xSSCT alone (750 mM NaCl and 75 mM sodium citrate, pH 7.0, containing 0.05% (v/v) Tween-20) and in 5xSSCT containing 35 nM capture reagent and 35 nM detection reagent, giving 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' DigNisopropyl-18 linker-GTGTGGT (Exiqon).

Experimental samples and standards were added to Clarity Lysis Buffer (Phenomenex, catalog #AL0-8579) at a 1:1 ratio before dilution with capture buffer and detection buffer and before transfer to the ELISA plate. CSF samples were diluted with plasma (2-fold) before addition of lysis buffer. The streptavidin-coated plate (Thermo 15119) was washed 3 times with 5xSSCT buffer. Samples (100 µl) were added and incubated for 60 min at room temperature. Detection probe—100 μl of AP-Fab fragment against Dig diluted 1:4000 in PBS containing 0.05% Tween-20 (Roche Applied Science, catalog number 11 093 274 910) was added and incubated for 60 min at room temperature. After washing the plate with 2xSSCT buffer, 100 μl of Tropix CDP-star Sapphire II substrate (Applied Biosystems) was added for 30 min at room temperature. Antisense oligonucleotide concentrations were measured by luminescence (Enspire-PerkinElmer).

Measurements of the alpha-synuclein protein.

Brain tissue samples were homogenized at a volume of 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 II bead homogenizer at 25 cycles/sec with 5 mm steel beads for a total of 2 min. Homogenized samples were incubated for 30 min on ice. A 50 μl aliquot of each sample was saved for PK characterization analysis. The remaining samples were centrifuged at 20,800 g for 60 min at 4°C. The supernatant was saved and used for analysis. Total protein was measured using a 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 0.1 μg/ml MJFR1 anti-SNCA antibody (Abcam) diluted in VirH carbonate-bicarbonate buffer, pH 9.4 (Thermo Scientific), overnight (O/N) at 4°C. The next day, the plates were washed 4 times with PBS with Dulbecco's broth (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 overnight 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 standard. Samples were loaded in duplicate (50 μl/well) and incubated O/N at 4°C. After the plates were equilibrated to RT, 50 µl of 4B12 detection antibody (Biolegend) (diluted 1:4000 in 1% BSA/0.1% Tween/DPBS) was added to each well and co-incubated with samples at RT for ~2 h. The detection antibody was pre-conjugated with 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, T2214, Life Technologies) for 30 min. Luminescence counts were measured using a Perkin Elmer EnVision (2102 Multilabel Reader). Plates were maintained under constant shaking (trial plate shaker, speed 3) during analysis. Data were analyzed using GraphPad Prism software. Total protein in brain tissue was measured using the Micro protein assay kit (Thermofisher #23235) according to the manufacturer's instructions.

Cerebrospinal fluid (CSF): SNCA protein levels were measured using the U-PLEX kit

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Human SNCA Kit: (catalog number K151WKK-2, Meso Scale Discovery) according to the manufacturer's instructions. CSF samples were diluted 10-fold. Hemoglobin levels were measured in CSF samples using the Abcam mouse Hemoglobin ELISA kit (abl57715). For hemoglobin measurements, CSF samples were diluted 40-fold.

Measurement of mRNA levels by qRT-PCR.

Brain regions were collected and placed in tissue protection tubes containing 1.5 ml RNA-later reagent (Qiagen, catalog #76514), which were pre-filled with RNAlater reagent, an RNA stabilizing solution. Tissue in RNA-later solution can be stored at 4°C for 1 month or at -20°C or -80°C indefinitely. RNA isolation: RNeasy Plus Mini Kit: RNA from mouse hippocampus and cortex was isolated using the RNeasy Plus Mini Kit (Qiagen, catalog #74134). Tissue samples were homogenized in 600 µl or 1200 µl RLT Plus buffer containing 10 µl/ml 2-mercaptoethanol and 0.5% Dx reagent. 600 µl lysis buffer was used if the tissue sample weight was <20 mg, 1200 µl lysis buffer was used for tissue samples weight >20 mg. For homogenization, the tissue sample was transferred to a 2.0 ml round-bottomed Eppendorf Safe-Lock tube (Eppendorf, catalog #022600044) containing 600 µl RLT Plus buffer (plus 10 µl/ml 2-mercaptoethanol and 0.5% Dx reagent) and 5 mm stainless steel beads (Qiagen, catalog #69989). Samples were homogenized using Qiagen's TissueLyser II instrument. Samples were run for 2.0 min at 20 Hz, samples were rotated 180° and run for an additional 2.0 min at 20 Hz. Samples were then run for 2.0 min at 30 Hz, rotated 180°, and run for an additional 2.0 min at 30 Hz. Longer homogenization and/or higher frequency homogenization were used if processing was incomplete. 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 g. All centrifugation steps were performed at RT. The filtrate was collected and an equal volume of 70% ethanol was added and mixed. 600 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 600 μl sample was added to the spin column. The spin columns were centrifuged and the filtrate was discarded. The columns were washed with 700 μl RW1 wash buffer, centrifuged for 15 s at 10,000g 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 10,000g for the first wash and then for 2.0 min at 10,000g for the second wash. After the second wash, the columns were centrifuged once for 1.0 min at 10,000g for dry membranes. The columns were then transferred to a new 1.5 ml collection tube and 30 µl of RNase-free water was added directly to the center of the membrane. The membranes were allowed to incubate for 10 min at RT. The columns were then centrifuged for 1.0 min at 10,000 g to elute the RNA. The eluate containing RNA was collected and stored on ice until RNA concentrations could be 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 cynomolgus monkey, mouse, and rat tissue samples was isolated using the RNEASY® Plus Universal Mini Kit (Qiagen, catalog #73404). For homogenization, 50 μg or less of tissue sample was transferred to a 2.0 mL round-bottomed Eppendorf Safe-Lock tube (Eppendorf, catalog #022600044) containing 900 μL of QIAZOL® Lysis Reagent and a 5 mm stainless steel bead (Qiagen, catalog #69989). Samples were homogenized using Qiagen's TissueLyser II instrument. Samples were run for 2.0 min at 20 Hz, samples were rotated 180°, and run for an additional 2.0 min at 20 Hz. Samples were then run for 2.0 min at 30 Hz, samples were rotated 180°, and run for an additional 2.0 min at 30 Hz. Longer homogenization and/or homogenization at a higher frequency were used if processing was incomplete. The homogenized tissue lysate was then transferred to a new 2.0 mL Eppendorf Safe-Lock round bottom tube and maintained at RT for 5.0 min. 100 μL of gDNA Eliminator Solution was added to each tube and the tubes were vortexed vigorously for 30 s. 180 μL of chloroform (Sigma, catalog #496189) was added to each tube and the tubes were vortexed vigorously for 30 s. The tubes were maintained at RT for 3 min. The tubes were centrifuged at 12,000 g for 15 min at 4°C. After centrifugation, the upper aqueous phase was transferred to a new 2.0 mL Eppendorf Safe-Lock round bottom tube in a volume of ~500 µL. An equal volume of 70% ethanol was added and mixed. All future centrifugation steps were performed at RT. 500 µL was transferred to an RNeasy spin column placed in a 2.0 mL collection tube and the samples were centrifuged for 15 s at 10,000 g. The filtrate was discarded and the remaining 500 µL sample was added to the spin column. The spin columns were centrifuged and the filtrate was discarded and the columns were washed with 700 µL RWT wash buffer 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 of RPE buffer containing 4 vol.

- 49 046118 mA ethanol as described in the kit protocol. 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 for dry membranes. Columns were then transferred to a new 1.5 ml collection tube and 30 µl of RNase-free water was added directly to the center of the membrane. Membranes were allowed to incubate for 10 min at RT. Columns were centrifuged for 1.0 min at 10,000 g to elute 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 by reverse transcription: 300 ng of RNA was diluted to a final volume of 10.8 µl using nuclease-free water (Invitrogen, catalog #10977-015) in a PCR-96-AB-C microplate (Axygen, catalog #321-65-051). 6.0 µl was added to each well of Reaction Mix 1 containing the following: 2.0 µl of 50 µM random ten-monomer sequences (Ambion, catalog #AM5722G) and 4.0 µl of 1x dNTP mix (Invitrogen, catalog #10297-018). The plate was sealed using optically clear sealing tape (Applied Biosystems, catalog #4360954) and centrifuged for 1.0 min at 1000 x g at RT. The plate was then heated for 3.0 min at 70°C using a GeneAmp PCR System 9700 96-well Thermal Cycler (Applied Biosystems). The plate was then completely cooled on ice. Then 3.25 μl of reaction mix 2 (containing 2 μl of 10× Strain Synthesis Buffer, 1.0 μl of MMLV-RT reverse transcriptase enzyme at 200 U/μl (Ambion, cat. #2044), and 0.25 μl of RNase inhibitor at 40 U/μl (Ambion, cat. #AM2682)) was added to each well. The plate was sealed using optically clear sealing tape and centrifuged for 1.0 min at 1000 x g at RT. Using a 96-well thermal cycler, the plate was heated to 42°C for 60 min followed by 95°C for 10 min. The plates were then cooled on ice. cDNA plates were stored at 20°C until ready for use 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. 16 μl of a master mix solution consisting of the following: 10 μl of 2x Taqman Gene Expression Master Mix (Applied Biosystems, catalog #4369016), 1.0 μl of 20x Taqman primer-probe set (Applied Biosystems), and 5.0 μl of nuclease-free water were added to each well of a 384-well optical PCR plate (Applied Biosystems, catalog #4483315). 4.0 μl of the diluted cDNA was added to each well of the 384-well optical PCR plate. The plate was sealed using optically clear sealing tape and centrifuged for 1.0 min at 1000 x g at RT. 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, followed by 40 cycles of 95°C for 15 s and 60°C for 1.0 min.

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

human alpha-synuclein (catalog number Hs01103383ml) labeled with FAM;

human PROS1 (catalog number HS00165590_ml), labeled with FAM;

FAM-labeled cynomolgus monkey alpha-synuclein (catalog number Mf02793033_ml);

GAPDH of cynomolgus macaque (catalog number Mf04392546_gl), labeled with FAM;

Cynomolgus monkey GAPDH (catalog number Mf04392546_gl), VTC-labeled, with limited primer concentration;

rat alpha-synuclein (catalog number Rn01425141_ml) labeled with FAM;

rat GAPDH (catalog number Rn01775763-gl) labeled with FAM;

rat GAPDH (catalog number 4352338E), VTC-labeled, with limited primer concentration;

mouse GAPDH (catalog number Mm99999915-gl) labeled with FAM;

mouse GAPDH (catalog number 4352339E), labeled with VTC, with limited primer concentration.

Example 5. Analysis of in vivo activity and tolerability of ASO-005459 in mice.

ASO-005459 is a LNA-modified ASO specific for human SNCA. In vitro results (described above) demonstrate that ASO-005459 is active and selective in reducing SNCA mRNA levels in primary neurons. In vitro results also indicate that ASO-005459 is well tolerated.

Mice A53T-RAS.

To assess whether these results are also true 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 (above).

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As shown in Table 6 (below), ASO-005459 was well tolerated, with an overall mean tolerability score of 1. Additionally, ASO-005459 significantly reduced SNCA mRNA levels by >90% in the hippocampus 3 days after administration (Fig. 9).

Table 6

Tolerability of ASO-005459 in A53T-PAC Mice, 3-Day Study

Animal No. Hyperactivity Alertness Coordination of movements and strength Posture/breathing Tremors/convulsions Overall rating 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 value

SEM

1.00

1.00

To assess in vivo activity, A53T-PAC mice were administered 3.13 μg, 12.5 μg, 25 μg, or 50 μg of ASO-005459 via ICV injection. Tolerability was assessed by measuring body weights at 7 and 14 days post-dose. SNCA mRNA expression was assessed at 14 days post-dose, when animals were sacrificed and tissues were collected. SNCA mRNA knockdown was measured in three brain regions: the hippocampus, brainstem, and striatum. The brainstem and striatum are two of the regions that are most affected in the brain in patients with MSA and PD. In a separate study, C57BL/6 mice were dosed with 100 μg ASO-005459 and tolerability was assessed by measuring animal body weights at 7, 14, 21, and 28 days post-dose. A 100 μg dose of ASO-005459 was administered ICV to wild-type (WT) C57BL/6 mice and body weight and behavior were monitored over a 4-week period. Because ASO-005459 does not specifically target mouse SNCA, reduction in SNCA mRNA expression was not measured in these animals. As shown in Figs. 10A and 10B, there were no significant differences in body weight between mice (both A53T-PAC and C57BL/6 mice) treated with ASO005459 (at all concentrations) and vehicle control-treated mice. The animals (C57BL/6) did not exhibit any abnormal behavior during the experimental period. See Table 7 (below). These results demonstrate that ASO-005459 was well tolerated. In addition, mice treated with ASO-005459 showed a significant and dose-dependent reduction in SNCA mRNA expression in all three brain regions examined. See Figs. 11A-11C. 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 (Figs. 11A–11C): 75% and 46% knockdown were observed with 50 μg and 25 μg ASO-005459, respectively. However, there was no significant reduction in SNCA mRNA expression with 12.5 μg and 3.13 μg ASO-005459. Possible explanations for the lower activity observed in the striatum may be due to differences in ASO levels or the kinetics of SNCA mRNA knockdown.

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Table 7

Tolerability of ASO-005459 in a 28-day study

Animal No. Moment of time (day) Hyperactivity Alertness Coordination of movements and strength Posture/breathing Tremors/convulsions Overall rating 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 30 1 0 0 0 0 0 0 30 28 0 0 0 0 0 0

ASO-005459 levels were measured in all three brain regions of A53T-PAC mice. As shown in Fig. 12 and Table 8 (below), brain exposure magnitudes were approximately dose proportional and similar in all three regions, including the striatum. Exposure-response relationships for ASO-005459 for various brain regions are presented in Figs. 13A-13D. 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 high, suggesting slower kinetics of SNCA mRNA decline in this region. See Fig. 13C.

Table 8

Summary of the effects of ASO-005459 on the brain in a 14-day study in A53TPAC

Dose (mcg) Average value (nM) SD Regarding the hippocampus Hippocamp 3.13 214 43 12.5 411 140 25 600 223 50 1916 807 Trunk head 3.13 160 47 0,7 brain 12.5 266 47 0,6 25 440 102 0,7 50 707 202 0,4 Striped body 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 detailed dose-response/dynamic data, ASO-005459 was re-administered (0, 12.5, 25, or 50 μg) directly into the brain ventricles of A53T-PAC mice via manual ICV injection. Animals were sacrificed at 24 h, 3 days, 4, 8, 12, 16, and 20 weeks post-dose and SNCA mRNA expression in the brainstem and striatum was assessed.

As shown in Figs. 14A and 14B (and consistent with the data presented above), administration of ASO-005459 to A53T-PAC mice resulted in a significant reduction in SNCA mRNA expression levels (relative to vehicle control) in both the brainstem and striatum. The reduction was both time- and dose-dependent, with a peak reduction (~90%) observed in the brainstem at the 50 μg dose of ASO-005459 approximately 4 weeks post-dose (Fig. 14A). In the striatum, a peak reduction (~55%) was observed in the

- 52 046118 was observed at the 50 μg dose of ASO-005459 approximately 3 days post-dose (Fig. 14B). SNCA mRNA expression levels remained significantly reduced compared to vehicle controls at 4 weeks post-dose (Figs. 15A and 15B) and returned to baseline control levels at approximately 16 weeks post-dose (Figs. 14A and 14B).

As shown in Figs. 16A and 16B, administration of ASO-005459 to animals also resulted in a time- and dose-dependent decrease in SNCA protein levels in both brainstem and striatum tissues. The peak decrease (~75%) was observed in the brainstem at the 50 μg dose at 8 weeks post-dose (Fig. 16A). For striatum tissue, the peak decrease (~75%) was also observed at the 50 μg dose but at 4 weeks post-dose (Fig. 16B). Expression levels for individual mice at 8 weeks post-dose are presented in Figs. 17A (brainstem) and 17B (striatum). While SNCA protein levels returned close to baseline by approximately 12 weeks post-dose in the striatum, expression levels were still significantly reduced (~25%) in the brainstem up to 16 weeks post-dose.

Example 7. Analysis of the activity and tolerability of SNCA-targeting antisense oligonucleotides (ASOs) in vivo in cynomolgus monkeys.

To further evaluate the in vivo activity and tolerability of ASO, a cynomolgus macaque model with an intrathecal port (Cyno IT) was developed. This model allows for the evaluation of ASO-005459-mediated knockdown of SNCA, as well as knockdown of potential off-targets PROS1 and IKZF3 with a 1 base pair mismatch. The ASO-005459 target sites in SNCA, PROS1, and IKZF3 are fully conserved between humans and cynomolgus macaques.

As described above in Example 3, each animal was implanted with an intrathecal cerebrospinal fluid (CSF) catheter entering the L3 or L4 vertebrae. ASO (ASO-005459, ASO-005584, and ASO-005578) was dissolved in saline and administered to the animals as an infusion over 4.5 min using the IT port (2 animals per dose group). Each animal received one of the following: (i) ASO-005578 (4 mg total dose), (ii) ASO-005584 (4 or 8 mg total dose), and (iii) ASO-005459 (8 mg total dose). Animals were then sacrificed at various time points post-dose, at which time tissues were collected for analysis of ASO exposure and activity. The brain regions analyzed included the medulla oblongata (Med), pons (V-Pons), midbrain (V-MB), cerebellum (CBL), dorsal striatum (left and right) (CauF), pocampus (left and right) (Hip), frontal cortex (left and right) (FrC), temporal cortex (left and right) (TeC), parietal cortex (left and right) (PaC), occipital cortex (left and right) (Oce), and white matter (WM). In addition, spinal cord samples were collected from the cervical (CSC), thoracic (TSC), and lumbar (LSC) regions. Samples were also collected from the liver, kidney, heart, trigeminal nuclei, tibial nerve, and aorta to assess off-target pharmacological properties in these areas.

As observed in mice, the ASOs were well tolerated by cynomolgus monkeys with no observed adverse effects (data not shown). And as shown in Fig. 18A and Table 9 below, administration of ASO-005578 or ASO-005584 to animals resulted in significant reductions in SNCA mRNA levels in all brain tissues analyzed. At 2 weeks post-dose, ASO005578 induced reductions of 75%, 32%, 65%, 69%, 98%, and 87% in the medulla oblongata, dorsal striatum, pons, cerebellum, lumbar spinal cord, and frontal cortex, respectively. ASO-005584 induced reductions of 59%, 45%, 61%, 57%, 97%, and 88% in the medulla oblongata, dorsal striatum, pons, cerebellum, lumbar spinal cord, and frontal cortex, respectively. Similar reductions were observed at 2 weeks and 4 weeks post-dose with 8 mg of ASO-005584 (Fig. 18B). ASO-005459 also demonstrated robust knockdown of SNCA mRNA in various regions of the cynomolgus monkey brain (Table 9).

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Table 9

Effect of ASO on SNCA mRNA levels in the cynomolgus macaque brain

ASO No. Dose (mg) Moment in time (weeks) Med CBL FrC RaS CauP TeS Oss Hip Vmv VPons CSC TSC LSC WM ASO005578 4 2 25 32 13 27 68 24 38 45 27 35 15 7 2 64 ASO005584 ASO- 005459 4 8 8 8 2 2 4 24 hours 41 21 36 174 44 62 70 140 12 10 11 146 34 16 23 55 62 65 149 8 6 7 38 19 28 44 36 48 53 34 48 39 23 37 137 16 2 5 9 2 4 3 2 3 73 45 38 78 8 24 hours 129 108 138 133 145 90 8 3 days 162 69 69 112 62 19 8 3 days 127 67 72 114 87 31 2 2 133 140 109 161 104 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 30 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 30 10 8 8 121 85 40 98 78 5 8 8 93 52 14 61 36 11 8 13 30 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 11

To further characterize the above-described time-dependent decrease in SNCA mRNA levels, cynomolgus monkeys were dosed with ASO-005459 (8 mg total dose per animal) and sacrificed at 24 h, 3 days, 2, 4, 8, 13, or 20 weeks post-dose to assess SNCA mRNA expression levels in various tissues. As shown in Fig. 19A, peak decreases were observed at 2 weeks and 13 weeks post-dose. Peak decreases of 70, 65, 75, 35, 94, and 99% were observed in the medulla, cerebellum, pons, dorsal striatum, frontal cortex, and lumbar spinal cord, respectively. This decrease in SNCA mRNA expression levels also correlated with the time-dependent decrease in SNCA protein expression levels.

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Claims (28)

(Fig. 19B). Next, to further evaluate whether the reduction in expression levels in cynomolgus monkeys was also dose dependent, animals were given 2, 4, or 8 mg of ASO-005459 and then sacrificed 2 weeks post-dose. As shown in Figs. 20A and 20B, the reduction in both SNCA mRNA and SNCA protein expression levels was also dose dependent, with the greatest reduction observed with 8 mg of ASO-005459. The results presented herein demonstrate that ASO-005459 is potent and selective in reducing SNCA mRNA levels and that ASO-005459 is well tolerated in neurons and in preclinical animal studies in vivo. Furthermore, the results from A53T-PAC neurons confirm that ASO-005459-mediated reductions in mRNA levels result in reductions in SNCA protein levels in vitro and in vivo. Additionally, results in A53T-PAC mice and cynomolgus monkeys demonstrate that ASO005459 reduces SNCA mRNA and SNCA protein levels in the brain at well-tolerated doses. Collectively, these results support the continued development of ASO-005459 as a disease-modifying therapeutic for synucleopathies. This PCT application claims priority from U.S. Provisional Patent Application No. 62/616,994, filed January 12, 2018, which is hereby incorporated by reference in its entirety. CLAUSE OF THE INVENTION 1. An antisense oligonucleotide for the treatment of synucleopathy, comprising a continuous nucleotide sequence of 10-30 nucleotides in length and comprising a sequence as set forth in: (i) SEQ ID NO: 7; (ii) SEQ ID NO: 4; (iii) SEQ ID NO: 5; (iv) SEQ ID NO: 6; (v) SEQ ID NO: 8; (vi) SEQ ID NO: 9; (vii) SEQ ID NO: 10; (viii) SEQ ID NO: 11; (ix) SEQ ID NO: 12; (x) SEQ ID NO: 13; (xi) SEQ ID NO: 14; (xii) SEQ ID NO: 16; (xiii) SEQ ID NO: 17; (xiv) SEQ ID NO: 18; (xv) SEQ ID NO: 19; (xvi) SEQ ID NO: 20; (xvii) SEQ ID NO: 21; and the antisense oligonucleotide is a gapmer with alternating nucleotides in the flanking regions. 2. An antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide is capable of inhibiting the expression of a human SNCA transcript in a cell that expresses a human SNCA transcript. 3. The antisense oligonucleotide of claim 2, wherein the human SNCA transcript comprises the nucleotide sequence set forth in SEQ ID NO: 1. 4. An antisense oligonucleotide according to any one of claims 1 to 3, wherein the gapmer with alternating nucleotides in the flanking regions contains a nucleotide analogue which is a nucleoside with a modified sugar. 5. The antisense oligonucleotide of claim 4, wherein the sugar-modified nucleoside is an affinity-enhancing sugar-modified nucleoside. 6. An antisense oligonucleotide according to claim 4 or 5, which comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten nucleotide analogues. 7. An antisense oligonucleotide according to any one of claims 4 to 6, wherein (i) the sugar-modified nucleoside is selected from the group consisting of locked nucleic acid (LNA); 2'-O-alkyl RNA; 2'-amino DNA; 2'-fluoro DNA; arabino nucleic acid (ANA); 2'-Φτορ-ΑΝΑ, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleoside analogue (cEt), 2'-O-methyl nucleic acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), and any combination thereof; or - 55 046118 (ii) A nucleoside with a modified sugar contains a bicyclic sugar. 8. The antisense oligonucleotide of claim 7, wherein the bicyclic sugar comprises cEt, 2',4' sterically hindered 2'-O-methoxyethyl (cMOE), LNA, α-L-LNA, P-D-LNA, 2'-O,4'-C-ethylene bridged nucleic acids (ENA), amino-LNA, oxy-LNA or thio-LNA. 9. An antisense oligonucleotide according to any one of claims 1 to 8, wherein the continuous nucleotide sequence comprises one or more 5'-methylcytosine nucleotide bases. 10. An antisense oligonucleotide according to claim 7, which comprises: (i) two to five LNAs in the 5' region of the antisense oligonucleotide; or (ii) two to five LNAs in the 3' region of the antisense oligonucleotide. 11. An antisense oligonucleotide according to any one of claims 1 to 10, wherein the continuous nucleotide sequence comprises the sequence set out in: (i) SEQ ID NO: 7 with the scheme AttCctttacaccACaCT (ASO-005578), AttCctttacaccAcACT (ASO-005584), AttCctttacaccaCACT (ASO-005597) or AttCctttacaccacACT (ASO-005611); (ii) SEQ ID NO: 9 with the scheme AatTCctttacaccacACT (ASO-005717) or AAttCctttacaccaCACT (ASO005700); (iii) SEQ ID NO: 12 with the scheme ttccTttacaccacac (ASO-288906), ttCctttacaccAcAc (ASO-287957), TtcctttacaccaCaC (ASO-287959) or ttCctttacaccACac (ASO-287962); or (iv) SEQ ID NO: 14 with the scheme AAttcctttacACCAcAC (ASO-005650), AAttCctttacacCaCAC (ASO005496) or AAttCCtTtacaccacAC (ASO-005392); wherein the uppercase letter represents a nucleoside with a modified sugar and the lowercase letter represents DNA. 12. An antisense oligonucleotide according to any one of claims 1 to 11, which comprises an internucleoside bridge selected from: a phosphodiester bridge, a phosphotriester bridge, a methylphosphonate bridge, a phosphoramidate bridge, a phosphorothioate bridge, and combinations thereof. 13. An antisense oligonucleotide according to claim 12, wherein the internucleoside bridge comprises one or more modified phosphate bridges determined by the stereochemical structure. 14. An antisense oligonucleotide according to any one of claims 1 to 13, wherein the antisense oligonucleotide is covalently attached to at least one non-nucleotide moiety or moiety other than a polynucleotide. 15. The antisense oligonucleotide of claim 14, wherein the non-nucleotide fragment or the fragment other than a polynucleotide comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a protein, or any combination thereof. 16. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. 17. The composition according to claim 16, which additionally contains a therapeutic agent. 18. The composition of claim 17, wherein the therapeutic agent is an alphasynuclein antagonist. 19. The composition of claim 18, wherein the alpha-synuclein antagonist is an alpha-synuclein antibody or fragment thereof. 20. A kit for treating synucleopathy, comprising an antisense oligonucleotide according to any one of claims 1-15 or a composition according to any one of claims 16-19 and instructions for use. 21. A method for inhibiting or reducing the expression of SNCA mRNA or the expression of SNCA protein in a cell, wherein the method comprises contacting a cell expressing SNCA mRNA or SNCA protein with an antisense oligonucleotide according to any one of claims 1-15 or a composition according to any one of claims 16-19, wherein the expression of SNCA mRNA or the expression of SNCA protein in the cell is inhibited or reduced after such contact. 22. The method according to claim 21, wherein the cell is a neuron. 23. A method for treating synucleopathy in a subject in need thereof, comprising administering to the subject an effective amount of an antisense oligonucleotide according to any one of claims 1-15 or a composition according to any one of claims 16-19. 24. The method of claim 21, wherein the subject is a human, and wherein the synucleopathy is selected from the group consisting of Parkinson's disease, Parkinson's disease dementia (PDD), multiple system atrophy, Lewy body dementia, and any combinations thereof. 25. Use of an antisense oligonucleotide according to any one of claims 1 to 15 for treating synucleopathy in a subject in need thereof. 26. The use of claim 25, wherein the subject is a human, and wherein the synucleopathy 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. 27. Use of a composition according to any one of claims 16-19 for the treatment of synucleopathy in a subject in need thereof. 28. The use of claim 27, wherein the subject is a human, and wherein the synucleopathy 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. -