Stereopure phosphorothioate linkage in antisense oligonucleotides

Abstract

Compositions comprising antisense oligonucleotides (ASO) wherein certain phosphorothioate (PS) linkages are stereopure and methods for increasing the efficacy of the ASO by designing certain ASOs wherein certain PS linkages are stereopure.

Description

[0001] STEREOPURE PHOSPHOROTHIOATE LINKAGE IN ANTISENSE OLIGONUCLEOTIDES TECHNICAL FIELD

[0002] The present disclosure relates to compositions comprising antisense oligonucleotides (ASO) wherein certain phosphorothioate (PS) linkages are stereopure and methods for increasing the efficacy of the ASO by designing certain ASOs wherein certain PS linkages are stereopure.

[0003] SEQUENCE LISTING

[0004] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 300145-PCT01-NP_SL.xml (Mouse calcium / calmodulin-dependent protein kinase II type delta (CAMK2D) transcript ENSEMBL Accession No. ENST0000511664.6 (SEQ ID NO: 1)), (Rho guanine nucleotide exchange factor 6 (ARHGEF6) transcript ENSEMBL Accession No. ENST00000250617.7 (SEQ ID NO: 85)). The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’ -OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2’ -OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of an uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, unless otherwise stated, including, but not limited to such nucleic acids having modified nucleobases.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] Antisense oligonucleotides (ASOs) are short (12-25 nucleotides), synthetic, single stranded DNA or RNA molecules with a sequence that is complementary to a nucleotide sequence of a target nucleic acid. Classic single stranded antisense oligonucleotides (ASOs) can act in the nucleus to cleave mRNAs via an RNase H dependent mechanism. (Juliano, RL(2016) Nuc. Acids. Res. 44(14):6518-6548). Many therapeutic ASOs are ‘gapmers’, which have a central DNA region that supports RNase H activity that is flanked by chemically modified ends to increase affinity and reduce susceptibility to nucleases. (Bennett et al.

[0007] (2010) Annu. Rev. Pharmacol. Toxicol. 50:259-293).

[0008] However, despite the promising therapeutic potential, challenges related to delivery, half-life, and potency persist. To overcome this, chemical modification in the phosphodiester backbone, the ribose sugar moiety, and the nucleobase has been implemented (Collotta et al. Front Pharmacol. (2023) 14:1304342). One of the most significant modifications is the phosphorothioate (PS) linkage, which replaces one of the non-bridging oxygen atoms in the phosphodiester linkage with a sulphur atom (Shen et al. (2018). Nucleic Acids Res. 46(4): 1584-1600). This structural change not only improves the resistance to nucleases but also prolongs circulation in the body, facilitating better tissue uptake (Crooke et al. Nucleic Acids Res. 48(10): 5235-5253). However, a consequence of the PS modification is that it creates a stereocenter at the phosphorus atom, resulting in (Rp)- and (Sp)- PS diastereomers (Oka et al. Chemical Soc. Reviews (2011) 40(12): 5829-5843). Currently, the PS linkages are synthesized in a non-stereoselective manner, which is less than ideal since the properties, e.g., binding affinity, stability to nucleases, and enzyme activity, are affected by the stereochemistry (Poredos et al. Organic Process Research & Development (2024) 28(12): 4194-4214).

[0009] SUMMARY OF THE DISCLOSURE

[0010] Compositions comprising antisense oligonucleotides (ASO) wherein certain phosphorothioate (PS) linkages are stereopure and methods for increasing the efficacy of the ASO by designing certain ASOs wherein certain PS linkages are stereopure are provided.

[0011] In one aspect, there is provided an ASO that follow the chimeric gapmer design, where a central DNA segment is flanked by RNA-based wings, and where all the phospodiesters (PO) in the backbone are replaced by phosphorothioates (PS), and where at least three consecutive PS are stereopure.

[0012] In one aspect, the central DNA segment is 6-14 nucleotides long and the RNA-based wings are 2-4 nucleotides long.

[0013] In one aspect, the central DNA segment is 6-12 nucleotides long and the RNA-based wings are 2-4 nucleotides long.

[0014] In one aspect, the central DNA segment is 6-10 nucleotides long and the RNA-based wings are 2-4 nucleotides long.In one aspect, the central DNA segment is 10 nucleotides long and the RNA-based wings are 3 nucleotides long.

[0015] In one aspect, the RNA-based wings include one or more modified nucleotides.

[0016] In one aspect, in the RNA-based wings the one or more modified nucleotides comprise: 2’0-methyl (2’OMe), 2’ O-m ethoxy ethyl (MOE), peptide nucleic acid (PNA), phosphoroamidate morpholino (PMO), locked nucleic acid (LNA), 2’ -deoxy -2 ’-fluoro (2’-F) or any other 2’ modified oligonucleotide.

[0017] In one aspect, at least three consecutive stereopure PS comprise a 5’-SpSpSp-3’ (SSS), 5’-SpRpSp-3’ (SRS) or a 5’-SpSpRp-3’ (SSR) motif.

[0018] In one aspect, three consecutive stereopure PS are 5’-SpSpSp-3’ (SSS), 5’-SpRpSp-3’ SRS) or 5’-SpSpRp-3’ (SSR).

[0019] In one aspect, three consecutive stereopure PS are 5’-SpSpSp-3’ (SSS).

[0020] In one aspect, three consecutive stereopure PS are 5’-SpRpSp-3’ (SRS).

[0021] In one aspect, three consecutive stereopure PS are 5’-SpSpRp-3’ (SSR).

[0022] In one aspect, the at least three consecutive stereopure PS, 5’-SpSpSp-3’ (SSS), 5’-SpRpSp-3’ (SRS) or 5’-SpSpRp-3’ (SSR), are located in the RNase Hl binding site.

[0023] In one aspect, three consecutive stereopure PS, 5’-SpSpSp-3’ (SSS), 5’-SpRpSp-3’ (SRS) or 5’-SpSpRp-3’ (SSR), are located in the RNase Hl binding site.

[0024] In one aspect, three consecutive stereopure PS, 5’-SpSpSp-3’ (SSS), are located in the RNase Hl binding site.

[0025] In one aspect, three consecutive stereopure PS, 5’-SpRpSp-3’ (SRS), are located in the RNase Hl binding site.

[0026] In one aspect, three consecutive stereopure PS, 5’-SpSpRp-3’ (SSR), are located in the RNase Hl binding site.

[0027] The calcium / calmodulin-dependent protein kinase II type delta (CAMK2D) enzyme is a serine / threonine-specific protein kinase that is encoded by the CAMK2D gene. This enzyme plays a significant role in the cardiovascular system, particularly in cardiac muscle contraction and heart function, through the regulation of Ca2+signaling. CAMK2D is essential for maintaining heart Ca2+homeostasis, and dysregulation of the CAMK2D gene has been linked to cardiac decompensation and heart failure.

[0028] Rho guanine nucleotide exchange factor 6 (ARHGEF6) is a GEF protein encoded in humans by the ARHGEF6 gene. Dysregulation of the ARHGEF6 gene have potential utility in thetreatment of e.g. kidney conditions, including diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS), renal hypertension (HTN), IgA nephropathy / Berger’s disease (IgAN), rapidly progressive glomerulonephritis (RPGN) and systemic lupus erythematosus (SLE).

[0029] Throughout this specification ASO sequences targeting the ARHGEF6 and CAMK2D genes are being used for illustrative purposes.

[0030] Control Sequences CAMK2D

[0031] A set of control CAMK2D ASOs was synthesized to ensure comparative analysis, as summarized in Table 1. A series of five control ASOs were prepared. These included the full-length PO version of the parent sequence, the full-length PO with a non-methylated cytosine DNA gap, a full-length PS (stereorandom) of the parent sequence, a full-length PS (stereorandom) with a non-methylated cytosine DNA gap, and a full Rp-gap version of the parent sequence.

[0032] Table 1. All control sequences for the CAMK2D libraries.

[0033] SEQ ID Sequence (5’— >3’) Sugar composition Sugar Linker pattern

[0034] SEQ ID NO: 2 GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 PO SEQ ID NO: 3 GTTTGGTATTCTTTAG LNA-DNA-LNA 3-10-3 PO SEQ ID NO: 4 GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 PS SEQ ID NO: 5 GTTTGGTATTCTTTAG LNA-DNA-LNA 3-10-3 PS SEQ ID NO: 6 GTTTGGTATTCTTTAG LNA-DNA-LNA 3-10-3 A / ?-PS

[0035]

[0036] A: Adenine; C: Cytosine; mC: C-5-methyl cytosine; G: Guanine; T: Thymine; LNA: Lockec Nucleic Acid

[0037] A targeted library with a 5’-SpSpSp-3’ and 5’-SpSpRp-3’ triad was synthesized for all CAMK2D sequences to enhance efficacy and RNase Hl cleavage, as shown in Table 2 and Figure 1. A 4S,5S,6S,7R,SR,9R,10R ASO was synthesized for maximal stereocontrol.

[0038] Table 2. Design of stereopure triads sequences for the CAMK2D libraries.SEQ ID Sequence (5’— >3’) Sugar composition Sugar Linker pattern

[0039] SEQ ID NO: 7 GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 4,5,6 Sp- PS SEQ ID NO: 8 GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 5,6,7 Sp- PS SEQ ID NO: 9 GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 6,7,8 Sp- PS SEQ ID NO: GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 4,5 Sp- 10 PS; 6 Ap-PS SEQ ID NO: GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 5,6 Sp- 11 PS; 7 Ap-PS SEQ ID NO: GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 6,7 Sp- 12 PS; 8 Ap-PS SEQ ID NO: GTTTGGTATTmCTTTAG LNA-DNA-LNA 3-10-3 4,5,6 Sp- 13 PS;

[0040] 7,8,9,10 Ap-PS

[0041]

[0042] A: Adenine; C: Cytosine; mC: C-5-methyl cytosine; G: Guanine; T: Thymine; LNA: Locked Nucleic Acid

[0043] Binding Affinity and Hepatotoxicity

[0044] There is no significant difference in the binding affinity of the stereopure triads compared to the parent ASO. Overall, the SSS triads exhibits slightly lower binding affinity than the AS7 triads, likely due to the absence of the Rp linkage (Table 3 and Figure 2). The ASO with maximal stereocontrol had a slightly higher melting temperature than the parent ASO, but the values were very similar, which was anticipated given that there were four stereocontrolled Rp linkages and three stereocontrolled Sp linkages. When both Sp and Rp linkages are present, they tend to balance each other, resulting in a binding affinity similar to that of theparent ASO. This is expected since Rp isomer stabilize the duplex, while the Sp isomer tend to destabilize it.

[0045] Table 3. Melting temperature for the CAMK2D stereopure triads and references.

[0046] SEQ ID Tim (°C) Tm2(°C) Average SEQ ID NO: 2 77.77 77.72 77.75

[0047] SEQ ID NO: 4 69.87 70.53 70.20

[0048] SEQ ID NO: 6 70.93 71.40 71.17

[0049] SEQ ID NO: 7 68.53 68.54 68.54

[0050] SEQ ID NO: 8 67.88 67.57 67.73

[0051] SEQ ID NO: 9 68.27 68.13 68.20

[0052] SEQ ID NO: 10 68.17 68.55 68.36

[0053] SEQ ID NO: 11 69.04 68.94 68.99

[0054] SEQ ID NO: 12 68.63 68.32 68.48

[0055] SEQ ID NO: 13 68.84 68.84 68.84

[0056]

[0057] For the stereopure triads both the SSS and 5S7? triads significantly reduced caspase activation relative to the stereorandom parent as seen in Table 4 and Figure 3.

[0058] Table 4. Hepatotoxicity for the CAMK2D stereopure triads and references.

[0059] SEQ ID MEC (pM)

[0060] SEQ ID NO: 2 1.130

[0061] SEQ ID NO: 4 0.341

[0062] SEQ ID NO: 6 10.000

[0063] SEQ ID NO: 7 10.000

[0064] SEQ ID NO: 8 10.000

[0065] SEQ ID NO: 9 0.681

[0066] SEQ ID NO: 10 10.000

[0067] SEQ ID NO: 11 10.000

[0068] SEQ ID NO: 12 10.000

[0069] SEQ ID NO: 13 10.000

[0070]

[0071] Only the 65,75,85 ASO exhibit any caspase activation within the 555 triad. Insights from the approach can help identify specific positions that contribute to toxicity, which can assist in ASO design.

[0072] Potency and Enzymatic RNase Hl Cleavage

[0073] The ASOs from the 555 and 555 triad demonstrated higher potency than the parent ASO, but remained less potent than the full-length PS control ASO (Table 5 and Figure 4). In the 555 triad having the stereo controlled PS modifications in positions 55,65,75 was more favourable while having the 555 triad in position 45,55,65 was preferred. These ASOs showed more than a four-fold increase in potency and did not cause caspase activation according to the Hepatotoxicity assay. The shift in position preference may be attributed to RNase Hl's binding preference, as it is known that the presence of chemically modified nucleosides can influence this.

[0074] Table 5. IC50 for the CAMK2D stereopure triads and references.

[0075] SEQ ID IC50 pIC50 Fold Active or

[0076] (pM) Change Inactive

[0077] SEQ ID NO: 5 0.687 6.163 1.00 A

[0078] SEQ ID NO: 4 0.135 6.870 5.01 A

[0079] SEQ ID NO: 7 0.209 6.679 3.287 A

[0080] SEQ ID NO: 8 0.142 6.849 4.838 A

[0081] SEQ ID NO: 9 0.290 6.537 2.369 A

[0082] SEQ ID NO: 10 0.156 6.808 4.404 A

[0083] SEQ ID NO: 11 0.214 6.669 3.21 A

[0084] SEQ ID NO: 12 0.226 6.645 3.04 A

[0085] SEQ ID NO: 13 0.176 6.754 3.903 A

[0086]

[0087] Interestingly, the maximal stereocontrol ASO displayed comparable potency levels to that of the 55,65,75 and 45,55,65 ASOs. This suggest that the minimal number of linkages required to achieve a consistent enhancement in potency is represented by the triad structure.

[0088] Implementing a triad consistently enhances the potency of the stereocontrolled PS-modified ASO.

[0089] To understand the observed improvement in potency of the ASO, the cell-free enzymatic RNase Hl cleavage assay was performed. Unexpectedly, results indicated suboptimal RNaseHl cleavage for all ASOs tested, apart from the ASO with maximal stereocontrol which showed >50% increase in cleavage rate (Table 6 and Figure 5).

[0090] Table 6. RNase Hl cleavage of the CAMK2D stereopure triads and references.

[0091] RNase Hl activity Percentage Change Fold AZ ID vs Parent ASO (%) Change > 50% Change SEQ ID NO: 2 -45.36 No 0.55 SEQ ID NO: 3 -75.77 No 0.25 SEQ ID NO: 5 0.00 No 1.00 SEQ ID NO: 4 -29.43 No 0.71 SEQ ID NO: 6 -37.22 No 0.63 SEQ ID NO: 7 20.10 No 1.2 SEQ ID NO: 8 -12.10 No 0.88 SEQ ID NO: 9 21.9 No 1.22 SEQ ID NO: 10 17.10 No 1.17 SEQ ID NO: 11 38.80 No 1.39 SEQ ID NO: 12 -7.30 No 0.93 SEQ ID NO: 13 73.00 Yes 1.73

[0092]

[0093] There was an increase in cleavage rate for some of the ASOs, however, they were less than 50%. Interestingly, the 5S,6S,7R ASO was better preforming in this assay than the 5S,6S,7S ASO which was more potent based on the IC50 value. This outcome suggests a limitation within the assay, as differences were observed between cell-free RNase Hl cleavage and intracellular RNase Hl activity, making optimization in this assay necessary for future studies.

[0094] No correlation was established between RNase Hl cleavage efficiency and ASO potency. Overall, positioning the triad at the cleavage site of the ASOs seems to play a critical role in enhancing potency.

[0095] Control Sequences ARHGEF6

[0096] A set of control ARHGEF6 ASOs was synthesized to ensure comparative analysis, as summarized in Table 7. A series of four control ASOs were prepared.Table 7. All control sequences for the ARHGEF6 libraries.

[0097] SEQ Sequence (5’— >3’) Sugar composition Sugar Linker ID pattern

[0098] NO

[0099] 14 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 PS 15 TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 PS 16 GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 PS 17 AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 PS

[0100]

[0101] A: Adenine; C: Cytosine; mC: C-5-methyl cytosine; G: Guanine; T: Thymine; LNA: Lockec Nucleic Acid

[0102] A targeted library with 5’-SpSpSp-3’, 5’-SpSpRp-3’ and 5’-SpRpSp-3’ triads was synthesized for all ARHGEF6 sequences to enhance efficacy and RNase Hl cleavage, as shown in Table 8.

[0103] Table 8. Design of stereopure triads sequences for the ARHGEF6 libraries.

[0104] SEQ Sequence (5’— >3’) Sugar composition Sugar Linker ID pattern

[0105] NO

[0106] 18 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 1,2,3 Sp- PS

[0107] 19 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 4,5,6 Sp- PS

[0108] 20 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 5,6,7 Sp- PS

[0109] 21 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 6,7,8 Sp- PS

[0110] 22 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 8,9,10 Sp-PS 23 mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 1,25 / ?- PS; 3 Ap-PS

[0111]

[0112] mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 2,3 Sp- PS; 4 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 3,4 Sp- PS; 5 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 4,5 Sp- PS; 6 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 6,7 Sp- PS; 8 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 7,9 Sp- PS; 9 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 8,9 Sp- PS; 10 Ap-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 1 Sp-PS;

[0113] 2 A / ?-PS; 3 Sp-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 2 Sp-PS;

[0114] 3 A / ?-PS; 4 Sp-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 3 Sp-PS;

[0115] 4 A / ?-PS; 5 Sp-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 4 Sp-PS;

[0116] 5 A / ?-PS; 6 Sp-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 5 Sp-PS;

[0117] 6 A / ?-PS; 7 Sp-PS

[0118]

[0119] mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 75 / ?-PS;

[0120] 8 A / ?-PS; 9 Sp-PS mCmCATTTAATGTGmCmCTT LNA-DNA-LNA 3-10-3 8 Sp-PS;

[0121] 9 A / ?-PS; lOSp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 1,2,3 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 2,3,4 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 3,4,5 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 4,5,6 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 5,6,7 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 6,7,8 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 7,8,9 Sp- PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 8,9,10 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 1,25 / ?- PS; 3 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 3,4 Sp- PS; 5 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 4,5 Sp- PS; 6 Ap-PS

[0122]

[0123] TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 5,6 Sp- PS; 7 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 6,7 Sp- PS; 8 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 7,8 Sp- PS; 9 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 8,9 Sp- PS; 10 Ap-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 1 Sp-PS;

[0124] 2 A / ?-PS; 3 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 2 Sp-PS;

[0125] 3 A / ?-PS; 4 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 4 Sp-PS;

[0126] 5 A / ?-PS; 6 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 5 Sp-PS;

[0127] 6 A / ?-PS; 7 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 6 Sp-PS;

[0128] 7 A / ?-PS; 8 Sp-PS TTGATAGmCmCTTmCTAAA LNA-DNA-LNA 3-10-3 7 Sp-PS;

[0129] 8 A / ?-PS; 9 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 1,2,3 Sp- PS

[0130]

[0131] GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 2,3,4 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 3,4,5 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 4,5,6 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 5,6,7 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 6,7,8 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 7,8,9 Sp- PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 8,9,10 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 1,25 / ?- PS; 3 Ap-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 2,3 Sp- PS; 4 Ap-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 4,5 Sp- PS; 6 Ap-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 5,6 Sp- PS; 7 Ap-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 7,8 Sp- PS; 9 Ap-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 8,9 Sp- PS; 10 Ap-PS

[0132]

[0133] GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 1 Sp-PS;

[0134] 2 A / ?-PS; 3 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 2 Sp-PS;

[0135] 3 A / ?-PS; 4 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 3 Sp-PS;

[0136] 4 A / ?-PS; 5 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 5 Sp-PS;

[0137] 6 A / ?-PS; 7 Sp-PS GTATTTmCGTTGTmCTAG LNA-DNA-LNA 3-10-3 8 Sp-PS;

[0138] 9 A / ?-PS; lOSp-PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 1,2,3 Sp- PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 2,3,4 Sp- PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 4,5,6 Sp- PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 6,7,8 Sp- PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 7,8,9 Sp- PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 3,4 Sp- PS; 5 Ap-PS AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 5,6 Sp- PS; 7 Ap-PS

[0139]

[0140] 84 AGTTATGTGTAGGATT LNA-DNA-LNA 3-10-3 2 Sp-PS;

[0141] 3 A / ?-PS; 4 Sp-PS A

[0142]

[0143] : Adenine; C: Cytosine; mC: C-5-methyl cytosine; G: Guanine; T: Thymine; Lb A: Locked Nucleic Acid

[0144] Table 9. Hepatotoxicity for the ARHGEF6 stereopure triads and references.

[0145] SEQ ID MEC SEQ ID MEC SEQ ID MEC SEQ ID MEC NO (pM) NO (pM) NO (pM) NO (pM)

[0146] 14 0.118 15 0.025 56 0.063 75 10.000

[0147] 18 0.255 37 0.201 57 0.047 76 0.186

[0148] 19 0.064 38 0.071 16 10.000 17 10.000

[0149] 20 0.085 39 0.016 58 10.000 77 10.000

[0150] 21 0.148 40 0.048 59 10.000 78 10.000

[0151] 22 0.040 41 0.077 60 0.251 79 10.000

[0152] 23 0.076 42 0.078 61 0.175 80 10.000

[0153] 24 0.060 43 10.000 62 10.000 81 10.000

[0154] 25 0.027 44 0.031 63 0.205 82 10.000

[0155] 26 0.061 45 0.093 64 0.236 83 10.000

[0156] 27 0.032 46 0.016 65 10.000 84 10.000

[0157] 28 0.092 47 0.035 66 10.000

[0158] 29 0.019 48 0.217 67 10.000

[0159] 30 0.031 49 0.098 68 0.182

[0160] 31 0.085 50 0.101 69 0.152

[0161] 32 0.031 51 0.060 70 0.313

[0162] 33 0.027 52 0.013 71 0.197

[0163] 34 0.064 53 0.228 72 0.138

[0164] 35 0.339 54 0.027 73 0.252

[0165] 36 0.207 55 0.059 74 0.123

[0166]

[0167] Table 10. IC50 for the ARHGEF6 stereopure triads and references

[0168] SEQ ID IC50 SEQ ID IC50 SEQ ID IC50 SEQ ID IC50

[0169] NO (pM) NO (pM) NO (pM) NO (pM)

[0170] 14 0.443 15 0.631 56 1.073 75 1.349

[0171] 18 0.079 37 1.074 57 1.336 76 0.601

[0172] 19 0.153 38 0.90 16 0.589 17 1.558

[0173] 20 0.546 39 0.305 58 1.187 77 2.265

[0174] 21 0.267 40 0.34 59 0.646 78 2.666

[0175] 22 0.336 41 1.623 60 0.697 79 0.592

[0176] 23 0.299 42 0.888 61 0.855 80 1.363

[0177] 24 0.445 43 2.056 62 0.986 81 1.714

[0178] 25 0.495 44 0.464 63 1.054 82 1.956

[0179] 26 0.491 45 0.169 64 0.994 83 1.188

[0180] 27 0.143 46 0.175 65 1.51 84 1.366

[0181] 28 0.16 47 0.288 66 0.808

[0182] 29 0.191 48 1.055 67 1.162

[0183] 30 0.35 49 0.809 68 0.928

[0184] 31 0.244 50 1.388 69 0.497

[0185] 32 0.414 51 0.294 70 0.37

[0186] 33 1.085 52 0.173 71 0.845

[0187] 34 0.618 53 1.929 72 0.54

[0188] 35 0.17 54 0.365 73 1.284

[0189] 36 0.446 55 0.988 74 0.371

[0190]

[0191] Evaluation of Triad Design

[0192] The triad design library demonstrates that employing a minimum of three stereocontrolled PS linkages offers consistent improvement in ASO potency.

[0193] Despite primarily enhancing potency and impacting enzymatic cleavage, the triad design did not significantly alter other properties such as toxicity, indicating that modified ASOs tend to mimic the characteristics of the parent sequence. Given that a triad is essential for catalysis, combinations of 23or eight potential P-diastereomers, can be generated to optimize RNase Hl binding and recognition.The two triad designs, SSS and SSR, were systematically evaluated across all CAMK2D sequences. These demonstrated that stereochemical configurations play a pivotal role in RNase Hl interactions.

[0194] Overall, these findings demonstrate the potential of the SSS and SSR triad designs in improving ASO efficacy.

[0195] A Potential Approach for Improving Potency

[0196] The current strategy for optimizing ASOs using stereocontrolled PS modifications has primarily focused on implementing an SSR pattern throughout the DNA gap (Iwamoto, N., et al., Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides. Nat Biotechnol, 2017. 35(9): p. 845-851). While this method enhances ASO efficacy, it is not sustainable as the stereocontrolled PS linkages are difficult to synthesis and costly due to the starting materials not being commercially available.

[0197] Based on the insights gained from the triad library design, a potential approach has been proposed for optimizing ASO efficacy through strategic ASO design. This approach is based on implementing a stereocontrolled SSS or SSR triad in position contacted by the RNase Hl. Compared to the traditional method for optimization, it limits the amount of stereocontrolled PS linkages being utilized which decreases the manufacturing cost as well as difficulty associated with synthesis. Moreover, experimental data indicates that a minimum of three stereocontrolled PS linkages are required for a significant improvement in potency.

[0198] Incorporating additional stereocontrolled linkages may not improve potency.

[0199] The primary advantage of using this approach would be that the positions contacted by RNase Hl are easily identified through the RNA cleavage pattern. For RNase Hl to effectively bind and cleave its RNA target, a minimum of six nucleotides is required which corresponds to a cleavage site. This requirement is attributed to the enzyme’s need to interact with three linkages, equivalent to four nucleotides, and includes a cleavage site located two nucleotides downstream of the binding region. Since there are six well-established cleavage sites it is easy to utilize these to identify positions to implement the triad design (Ostergaard, M.E., et al., Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides. Nucleic Acids Res, 2020. 48(4): p. 1691-1700). Additionally, different ASO design would still be compatible with this approach. Asymmetric ASO designs, such as the 4-10-2 design as compared to the symmetric 3-10-3 design, would not impact the RNase Hl cleavage, as the overall DNA gap remains unchanged. The presence of longer ASOs may introduce additional cleavage siteswhile shorter ASOs would inherently possess fewer cleavage sites but still exhibit cleavage identical to the well-established cleavage sites.

[0200] However, this approach would be limited by the properties of the parent ASO, as the stereopure PS-modified ASO mimics these properties. Thus, having a balanced parent ASO is crucial when designing stereocontrolled PS-modified ASOs.

[0201] Brief description of the Figures

[0202] Figure 1: Design of stereopure triads sequences for the CAMK2D libraries.

[0203] Figure 2: Melting temperature with full-length PS ASO as reference for stereopure triads and ASO with maximal stereocontrol (CAMK2D).

[0204] Figure 3: Hepatotoxicity with full-length PS ASO as reference for stereopure triads and ASO with maximal stereocontrol (CAMK2D).

[0205] Figure 4: IC50 with full-length PS ASO as reference for stereopure triads and ASO with maximal stereocontrol (CAMK2D).

[0206] Figure 5: Enzymatic RNase Hl cleavage rate with full-length PS ASO as reference, of stereopure triads and ASO with maximal stereocontrol (CAMK2D).

[0207] DETAILED DESCRIPTION OF THE DISCLOSURE

[0208] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular, for example, “a” or “an,” include pluralities, e.g., “one or more” or “at least one” and the term “or” can mean “and / or” unless stated otherwise. The terms “including,” “includes” and “included” are not limiting. Ranges provided herein, of any type, include all values within a particular range described and values about an endpoint for a particular range.

[0209] An “antisense oligonucleotide” or “ASO” is an oligonucleotide that includes at least a portion of which is complementary to a target nucleic acid such that the ASO can hybridize to the target nucleic acid. An antisense oligonucleotide can increase or decrease expression of a target nucleic acid.

[0210] In this specification, “stereopure” or “stereocontrolled” phosphorothioate (PS) refers to PS where the Rp:Sp or Sp:Rp ratio is >90%, preferably >95%, most preferably >99%.Biophysical and Biological Assays

[0211] The post-synthetic evaluation of ASOs in vitro is critical in drug discovery for assessing their properties and eliminating unsuitable candidates before advancing to in vivo testing. Evaluations typically involve a variety of assays that investigate each compound's stability, efficacy, and toxicity. Conducting the in vitro studies is not only beneficial for finding suitable candidates but it also enhances understanding regarding their mechanisms of action and potential side effects, paving the way for more targeted therapies.

[0212] Melting Temperature

[0213] The antisense approach relies on ASOs to efficiently bind to the target sequence, making the assessment of ASOs affinity to target RNA crucial in initial investigations. The affinity is characterized by the melting temperature (Tm) of the ASO / RNA duplex, defined as the temperature where half of the ASO and RNA strands have dissociated into single strands. Tmis influenced by intrinsic factors including the length of the ASO, modifications, and the sequence composition, and extrinsic factors including pH, salt concentration, and solvent during measurement (Uhlmann, E. et al. Antisense oligonucleotides: a new therapeutic principle. Chemical Reviews, 1990. 90(4): p. 543-584; Mergny, J.L. et al., Analysis of thermal melting curves. Oligonucleotides, 2003. 13(6): p. 515-37.)

[0214] Tmmeasurement is conducted by gradually heating the duplex to break the hydrogen bonds between the strands. This process can be monitored using various techniques, each with its own set of advantages and limitations. UV / Vis-spectroscopy is one such technique, where the absorbance at 260 nm is monitored (Stulz, R., et al., An enhanced biophysical screening strategy to investigate the affinity of ASOs for their target RNA. RSC Chem Biol, 2023. 4(12): p. 1123-1130). At this wavelength, oligonucleotides exhibit higher absorbance in their singlestranded form compared to their duplex form — a phenomenon known as the hyperchromic shift (Sun, C., et al., SimpleDSFviewer : A tool to analyze and view differential scanning fluorimetry data for characterizing protein thermal stability and interactions. Protein Sci, 2020. 29(1): p.

[0215] 19-27). Although straightforward and a widely used technique for assessing nucleic acid stability, UV / Vis-spectroscopy may be impractical for a large number of samples due to instrument limitations.

[0216] Alternatively, fluorescence-based techniques offer a solution for high-throughput analysis either by using fluorescence-labelled nucleic acids or fluorescent dyes. In this study, a differential scanning fluorimetry (DSF) assay is utilized. This technique involves monitoring the fluorescence change of a dye that binds to the nucleic acids. Ribogreen® is commonly usedfor this purpose. In ASO / RNA duplexes, where strands are annealed, Ribogreen® exhibits high fluorescence. As temperature rises, the dye is progressively released from the duplex, decreasing fluorescence. This decrease continues until an inflection point is reached, corresponding to the Tmof the duplex. The fluorescence signal decreases to zero when the duplex is completely unfolded.

[0217] Caspase Activation Assay

[0218] Early toxicity assessments are crucial to identify the cytotoxic effects of ASOs or their delivery systems, thereby eliminating toxic compounds. To avoid testing compounds in vivo, various in vitro assays have been developed to measure toxicity through parameters such as viability, autophagy, or apoptosis. Apoptosis assays are designed to assess cell viability by quantitatively measuring the proportion of living and dead cells within a population (Kepp, O., et al., Cell death assays for drug discovery. Nature Reviews Drug Discovery, 2011. 10(3): p. 221-237). This is done by detecting the involvement of specific apoptotic markers, such as proteins and enzymes critical to the apoptosis process. Among these markers are caspases, a family of cysteine-dependent proteases. These are divided into two groups: (1) initiator caspases, which initiate apoptosis by activating effector caspases through proteolytic cleavage, and (2) effector caspases, which mediate cell death by cleaving vital cellular proteins, causing DNA degradation (Boatright, K.M. and G.S. Salvesen, Mechanisms of caspase activation. Current Opinion in Cell Biology, 2003. 15(6): p. 725-731).

[0219] Caspase activation assays can be performed using various methods, including colorimetric, immunological, fluorometric, and luminescent assays. In this study, the Caspase-Gio® 3 / 7 luminescent assay was employed. This assay is specifically designed to measure the activity of effector caspase 3 and caspase 7 in cell cultures. It utilizes a proluminescent caspase-3 / 7 DEVD-aminoluciferin substrate, which is cleaved by active caspase-3 / 7 to release aminoluciferin (Payne, A.M., et al., Caspase activation as a versatile assay platform for detection of cytotoxic bacterial toxins. J Clin Microbiol, 2013. 51(9): p. 2970-6; Caspase-Gio® 3 / 7 Assay Technical Bulletin (TB323). 2023: Promega Corporation). Upon binding the Ultra-Glo rLuciferase, it produces a luminescent signal. The luminescence intensity directly correlates with caspase 3 / 7 activity, with a higher luminescence indicating increased toxicity. Since the Caspase-Gio® 3 / 7 assay is designed as a single step, it is compatible with high-throughput screening applications and automation. However, while invaluable for evaluating compound toxicity, the assays measure the cleavage of synthetic substrates, which is an indirect measure of caspase activity. Consequently, any unspecific degradation occurs throughmechanisms that are not related to cell death, it cannot be distinguished with this assay. The notion that any caspase activation equals apoptosis does not reflect the complex regulation and interactions within the cell death cascades. Thus, it is often recommended to use a multiparametric approach for an accurate assessment of apoptosis (Kepp, O., et al., Cell death assays for drug discovery. Nature Reviews Drug Discovery, 2011. 10(3): p. 221-237).

[0220] RNase Hl Cleavage Assay

[0221] RNase Hl -dependent ASOs are designed to modulate gene expression through the recruitment of RNase Hl . Understanding how RNase Hl cleavage dynamics are influenced by ASO design and chemical modifications is crucial for enhancing the specificity and potency of ASOs. To determine the efficiency of RNase Hl cleavage, enzymatic RNase Hl cleavage assays are utilised.

[0222] Enzymatic assays are broadly employed to investigate enzyme kinetics and play a vital role in identifying and quantifying enzymes within biological samples. These assays measure the consumption of substrate or the generation of product over time and are categorised into continuous and discontinuous assays based on their sampling methodologies. Continuous assays provide a real-time reading of activity, determining reaction rates without additional processing. In contrast, discontinuous assays involve taking samples at intervals, stopping the reaction, and then measuring the concentration of substrates or products. Several factors influence the outcome of enzyme assays, including pH and buffer type, ionic strength, and temperature. Most enzymes have specific activity ranges for these parameters, with optimal conditions usually mirroring the physiological environment of the source organism. Deviations from these conditions can significantly impact enzyme activity.

[0223] Enzymatic RNase Hl cleavage assays can be performed using different methods to monitor the cleavage of RNA substrates. Among these, gel electrophoresis-based discontinuous assays are commonly utilised. Gel electrophoresis is a method used to separate DNA, RNA, or protein molecules according to their charge and size. By applying an electric field, molecules migrate through a gel matrix. In this study, the discontinuous gel electrophoresis assay was conducted using a 15% TBE-Urea gel (Novex®), with the fragments were detected by using Fluorescein Amidite (FAM) labelled mRNA. While this method is a powerful tool for identifying and quantifying cleavage fragments, it is not suitable for high-throughput applications in large- scale studies.

[0224] Quantitative PCRFor ASOs to be therapeutically beneficial, they must efficiently downregulate the levels of specific target mRNA and its corresponding protein. By measuring gene knockdown with quantitative PCR (qPCR), it is possible to directly determine how effectively the ASO reduces these levels.

[0225] qPCR, also known as real-time PCR or quantitative real-time PCR, is a technique that simultaneously amplifies and quantifies target DNA sequences (Dymond, J.S., Explanatory chapter: quantitative PCR. Methods Enzymol, 2013. 529: p. 279-89). For RNA-based targets, RNA is first reverse-transcribed to generate complementary DNA (cDNA), which is subsequently amplified. These amplifications occur in a thermal cycler instrument and involve repeated cycles of denaturation (94-98°C), annealing (50-70°C), and extension (68-72°C) phases. The quantification of DNA during this process is achieved by using fluorescent dyes that label double-stranded DNA (dsDNA) or specific DNA species of interest. The fluorescence signal, directly proportional to the quantity of DNA present, allows for the precise measurement of gene expression levels.

[0226] In this study, TaqMan probes were utilized for DNA quantification. The TaqMan probe are short oligonucleotides that hybridize with the target sequence and are labelled with a fluorophore at the 5 ’-end and a quencher at the 3 ’-ends. The proximity prevents the fluorescence signal due to Forster resonance energy transfer (FRET). During the PCR amplification, these probes are cleaved and degraded by the 5'— 3' exonuclease activity of Taq DNA polymerase, separating the fluorophore from the quencher. This process results in an irreversible increase in fluorescence signal, which can be measured in real time. The specificity of TaqMan probes ensures fluorescence occurs only if the probe hybridizes to the target. qPCR data analysis can be conducted using either absolute or relative quantification methods. Absolute quantification determines the exact copy number of the target DNA in the sample, providing a direct measure of gene expression. Relative quantification compares the levels of the target DNA to a reference, yielding a fold change in expression that is particularly useful for assessing the efficacy of gene knockdown by ASOs. Additionally, qPCR measurements at varied concentrations can create a dose-response curve to determine ASO IC50, the concentration at which the ASO is half-maximally effective.

[0227] To ensure accuracy, reactions are performed in triplicate and normalized against an internal control, given qPCR's extreme sensitivity to small differences in reaction composition, which can be magnified over the course of the amplifications. Other factors that have an impact on the qPCR assay include primer specificity for the target sequence, efficiency of reverse transcription for converting RNA to cDNA, PCR amplification efficiency, and sample qualityof the target, all of which must be carefully controlled and optimized to ensure reliability and accuracy.

[0228] Experimental Section

[0229] General. Automated oligonucleotide synthesis was performed on a MerMade 48X synthesizer (Biosearch Technologies) using controlled-pore glass (CPG) resin with a universal linker (Sigma-Aldrich). Standard dimethoxy trityl (DMT)-protected phosphoramidites and ancillary reagents were purchased from Wuxi or Sigma-Aldrich / Merck. Fully DMT-protected stereodefined bicyclic oxazaphospholidine (OAP) monomers were purchased from Wuxi. Stereocontrolled synthesis was prepared in a similar manner described by Wada et al. Solid-Phase Synthesis of Stereoregular Oligodeoxyribonucleoside Phosphorothioates Using Bicyclic Oxazaphospholidine Derivatives as Monomer Units. Journal of the American Chemical Society, 2008. 130(47): p. 16031-16037. The procedures were slightly modified to avoid purification.

[0230] Oligonucleotide solid-phase automated synthesis. Oligonucleotides were prepared on the MerMade 48X synthesiser at 1 pmol scale using CUTAG CPG solid support (1000 A, 35 mol / g). Phosphoramidites were incorporated using standard MerMade conditions as shown in Table 7, i.e., 3% DCA in toluene for deblocking, 0.25 M 5-[3,5-bis(trifhioromethyl)phenyl]-27 / -tetrazole (Activator 42®) in ACN as activator or 2.0 M CMPT in ACN (alternatively 1.4 M 1-Phenylimidazolium triflate, PhIMT, in ACN) as activator for stereocontrolled PS incorporations, 10% acetic anhydride in THF / Lutidine (8:1) (Cap A) and 16% NMI in THF (Cap B) for capping, 0.2 M Xanthane hydride in pyridine for thiolation, and 0.02 M iodine in THF / Pyridine / Water (90.6:0.4:9) for oxidation. DNA and LNA amidites were prepared at 0.1 M in ACN and incorporated using two applications of 1 min and 3 min coupling times, respectively. OAP monomers were prepared at 0.2 M in ACN / Toluene (1:1) + 2.5% pyridine and incorporated using three applications of 6 min coupling times. All oligonucleotides were synthesised in DMT-off mode, and the cyanoethyl group on the phosphate backbone was removed by treatment with 20% DEA in ACN on the synthesizer for 15 min. The column containing the oligonucleotides was then dried under vacuum before proceeding with the deprotection.

[0231] Table 7. Synthesis cycle for oligonucleotides

[0232] Step Operation Reagent / Sol vents Time1 Detritylation 3% DCA in toluene 4 min 2 Wash ACN 90 s 0.25 M Activator 42®2x 1 min3 Coupling (DNA)

[0233] 2x3 min (LNA) 2.0 M CMPT in ACN 3 x6 min (OAP) 4 Wash ACN 30 s Oxidation (PO) 0.02 M iodine in THF / PyridineAVater 1 min5(90.6:0.4:9)

[0234] Sulfurization (PS) 0.2 M Xanthane hydride in pyridine 6 min 6 Wash ACN 30 s 10% Acetic anhydride in

[0235] 7 Capping 1 min '&THF / Luti dine (8:1)

[0236] 16%NMI in THF

[0237] 8 Wash ACN 30 s

[0238] Gas deprotection. On-column gas deprotection was performed using a Heated Pressure Chamber with Digital Controller and Timer (Biolytic) according to the protocol in Table 8.

[0239] Columns with the oligonucleotides were precipitated with 0.1 M NaOAc in 85% EtOH (1 mL / well) followed by 85% EtOH solution (1 mL / well) and then dried in vacuum for 5 min. The oligonucleotides were then eluted with 600 pL of Milli-Q Water into a 96-well plate and concentrated by either lyophilization or by Speedvac.

[0240] Table 8. Gas deprotection protocols

[0241] Reagents Time (min) Temperature (°C) Pressure (psi) Methylamine 60 45 24 Ammonia190 65 40 Ammonia1300 65 401Columns were pre-wet with 200 pL Milli-Q Water prior to deprotection and dried in vacuum for 60 min after deprotection.Liquid deprotection. Liquid deprotection was performed by eluting with the selected deprotection reagent into a 96-well plate for 1 h, followed by heating in cleavage chuck (Bioresearch Technologies) according to the protocol in Table 9. After cooling to room temperature, the solutions were concentrated by using a Speedvac. The oligonucleotides in the plate were dissolved in Milli-Q water (225 pL / well) and precipitated by adding 3 M NaOAc (pH 5.2) (25 pL / well) and 95% EtOH (1 mL / well). The plate was sealed, shaken, and placed on dry ice for 4 h. It was the immediately centrifuged at 4°C at 3200 x g for at least 15 min, and the supernatant was removed. Next, 99.5% EtOH (750 pL / well) was added to the pellets. The plate was sealed, shaken, and placed on dry ice for 2 h. It was immediately centrifuged at 4°C at 3200 x g for at least 15 min, and the supernatant was removed again. The pellets were dried using a Speedvac and resuspended in Milli-Q water (600 pL / well). The plate was sealed, shaken at 800 rpm for 15 min, and then centrifuged at 3200 x g for 30 min. The supernatant was then transferred to a new plate, and the oligonucleotides were concentrated by either lyophilization or by Speedvac.

[0242] Table 9. Liquid deprotection protocols

[0243] Reagents Time Temperature (°C) Volume (pL / well) Methylamine 40wt% in H2O 10 min 65 600

[0244] 28-30% aq. Ammonia 16 h 55 600 AMA 1:1 15 min 65 600 25% aq. Ammonia / EtOH 5:1 (v:v) 48 h 55 1000

[0245] Oligonucleotide characterization. All oligonucleotides were characterized by LC-MS (ES-) on Waters ACQ-SQD2 with ACQUITY UPLC system using an analytic ACQUITY UPLC-BEH C18 column (2.1 mm x 50 mm) at 60 °C. Buffer A (10.3 mM TBuAA in Water) and Buffer B (ACN) was used as eluents with the gradient described in Table 10. UV absorbance was measured at 260 nm.

[0246] Table 10. Gradient for LC-MS characterization

[0247] Time Flow Rate Buffer A Buffer B

[0248] Curve (min) (mL / min) (%) (%)

[0249] 0.00 0.500 65.0 35.0 Initial 10.00 0.500 50.0 50.0 6 10.10 0.500 20.0 80.0 611.00 0.500 20.0 80.0 6 11.10 0.500 65.0 35.0 6 14.00 0.500 65.0 35.0 6

[0250] Oligonucleotide purification. If needed oligonucleotides were purified by Ion-pairing high performance liquid chromatography (HPLC) on Waters system with XBridge BEH Cl 8 OBD Prep column (10 mm x 250 mm). Buffer A (60 mM DBuAA in Water / ACN 95:5) and Buffer B (60 mM DBuAA in ACN) was used as eluents with the gradient described in Table 11. UV absorbance was measured at 260 nm. After purification oligonucleotides were concentrated by Speedvac and then precipitated two times with NaOAc (see Liquid deprotection) to remove the DBuAA.

[0251] Table 11. Gradient for HPLC purification

[0252] Time Flow Rate Buffer A Buffer B

[0253] Curve (min) (mL / min) (%) (%)

[0254] Initial 6.00 90.0 10.0 Initial 50.0 6.00 0.0 100.0 6 55.00 6.00 0.0 100.0 6 55.01 6.00 0.0 100.0 6

[0255] UV-monitored thermal denaturation temperatures. ASO, RNA, and buffer containing 0.01X Quant-iT™ Ribogreen® dye in IX PBS (137 mM NaCl, 2.7 mM KC1, 8 mM Na2HPO4, and 2 mM KH2PO4 at pH 7.4), supplemented with 10 mM MgCh, were prepared in 1:1:8 ratio (10 pM duplex) in a 384 cell-qPCR plate (SP-1384, Thermo Scientific). The plate was sealed with an optically clear adhesive seal (Thermo Scientific) and centrifuged at 1000 x g for 2 min. Thermal denaturation temperatures were performed using a LightCycler® 480 instrument (Roche), and the fluorescence (465-510 nm) was measured from 20 °C to 95 °C at a ramp rate of 0.01 °C / s with 35 acquisitions per °C.

[0256] Caspase-3 / 7 activation assay. A solution of 0.5% Lipofectamine RNAiMAX Transfection reagent in OptiMEM was dispensed (lOpL / well) into a 384 cell-assay plate containing ASO solutions (200nL / well) with selected concentrations (10-points serially diluted 3.162-fold to give concentration range lOpM-lmM). ASOs were incubated for at least 30 minutes prior to addition of the cell line. HepG2 / C3A cell line (ECL.3537) suspension was diluted to 2*105cells / mL in DMEM medium and dispensed (lOpL / well) to the ASOs. The plate was incubated at 37°C for 24 h. Caspase-Gio 3 / 7 (Promega, G8093) solution was prepared according to themanufacturer's instructions and added (lOpL / well) to the plate. The mixture was incubated at room temperature for 30 min. Luminescence was determined with a PHERAstar plate reader (BMG LABTECH).

[0257] Cell-free RNase Hl cleavage assay. ASO and human RNase Hl were combined in equal amounts in Eppendorf tubes and annealed for 15 minutes (50 nM complex). Following annealing, 5’-FAM labelled mRNA (24-mer) (3.125 nM) was added to reach the corresponding concentrations specified in Table 12. The reaction was incubated at room temperature for 90 min. The Eppendorf tubes were placed on ice, and the reaction was quenched by adding a stop buffer comprising of 90% formamide and 10% EDTA, equal in volume to the reaction mixture. A Ficoll solution (F5415, Sigma-Aldrich), equivalent to one-third of the reaction mixture volume, was added. The samples were denatured at 95°C for 2 min and then placed on ice. Gel electrophoresis was performed in IX TBE running buffer on 15% TBE-Urea gel (Novex®) at 180 V for 70 minutes. The cleaved mRNA was visualized using the ChemiDoc Touch Imaging System (Bio-Rad) on a Blue Sample Tray.

[0258] Table 12. Sample composition in RNase Hl cleavage assay

[0259] Reagent Concentration (nM) Ratio RNase Hl 10 1

[0260] ASO 10 1

[0261] 5 ’-FAM mRNA 250 25

[0262] Quantitative RT-qPCR assay. A solution of 1% Lipofectamine RNAiMAX Transfection reagent in OptiMEM was dispensed (lOpL / well) into a 384 cell-assay plate containing ASO solutions (400nL / well) with selected concentrations (Single Point qPCR: lOpM and Concentration Response qPCR: 10 points serially diluted 3.162-fold to give concentration range lOpM-lmM). The ASOs were incubated for 20 minutes prior to addition of the cell line. A HP1-L cell line suspension was diluted to 0.175 x io6cells / mL in Claycomb media, supplemented with 10% FBS, 0.1 mM Norepinephrine, and 2 mM Glutamine. The cell suspension was dispensed (30pL / well) into the wells containing the ASOs and incubated at 37°C for 48 h in a 5% CO2 and 95% humidity atmosphere. Post-transfection, the medium was removed using Bluewasher RegularSpin evacuation. Lysis buffer, composed of 96% in-house RLN buffer (50 mM Tris-Cl, 140 mM NaCl, 1.5 mM MgCh, 0.5% IGEPAL) and 4% RNA secure reagent was dispensed (20pL / well). The plate was then incubated by shaking at room temperature for 5 minutes. The mixture was transferred (2pL / well) to a new plate. A reversetranscription master mixture composed of IX Reverse Transcription buffer (Thermo Fisher Scientific, A4391852C), IX Reverse Transcription enzyme mix (Thermo Fisher Scientific, A4391852C) and RNase-free water was added (18pL / well). The reverse transcription reaction was performed in a C1000 Touch Thermal Cycler (Bio-Rad) according to the program in Table 13 to generate cDNA.

[0263] Table 13. Thermal Cycler program

[0264] Step Stage Cycles Temperature Time (min) (°C)

[0265] Reverse transcription 1 1 37 60 Reverse transcription 2 1 95 5 inactivation

[0266] Hold 3 1 4

[0267] The cDNA was then transferred (3pL / well) to a new plate, and a qPCR master mixture composed of 1.4X Fast Advance Taqman mastermix (Thermo Fisher Scientific, 4444558), 1.4X mCamK2d FAM-MGB assay (Applied Biosystems, 4331182, Mm00499266_ml), 1.4X mHPRT VIC-MGB assay (Applied Biosystems, 4448491, Mm03024075_ml) and RNase-free water was added (7pL / well). Analysis was done in a Quantstudio 7 Flex qPCR machine (Applied Biosystems) using the 42-minute program.

[0268] NMR. Amidite and precipitation was suspended in 600 pL of denatured DMF.JH NMR spectrum (499.80 MHz, DMF-d7) and31P NMR (202.32 MHz, DMF-d7) were acquired on an Oxford NMR 500 spectrometer equipped with BRUKER B-ACS-60 and were visualized using MestReNova x64. Chemical shifts were reported in parts per million (ppm).

[0269] Sp-GiBuOAP monomer unopened. 'H NMR (500 MHz, DMF) 5 8.24 (s, 1H), 7.48 - 7.37 (m, 5H), 7.36 - 7.21 (m, 11H), 6.93 - 6.81 (m, 4H), 6.37 (t, J = 6.6 Hz, 1H), 5.79 (d, J= 6.5 Hz, 1H), 5.09 - 4.99 (m, 1H), 4.18 (q, J = 4.3 Hz, 1H), 4.07 (q, J= 7.1 Hz, 1H), 4.03 - 3.97 (m, 1H), 3.79 (d, J = 1.1 Hz, 6H), 3.13 - 3.03 (m, 2H), 2.64 (ddd, J = 13.8, 6.4, 3.9 Hz, 1H), 2.02 (s, 1H), 1.70 - 1.46 (m, 2H), 1.29 - 1.15 (m, 9H), 0.90 (dq, J = 12.4, 8.3 Hz, 1H).31P NMR (202 MHz, DMF) 5 150.90.

[0270] Sp-GiBuOAP monomer resealed. 'HNMR (500 MHz, DMF) 58.24 (s, 1H), 7.46 - 7.38 (m, 5H), 7.35 - 7.21 (m, 11H), 6.87 (dd, J= 8.9, 6.6 Hz, 4H), 6.37 (t, J= 6.7 Hz, 1H), 5.79 (d, J= 6.6 Hz, 1H), 5.05 (td, J = 9.7, 3.6 Hz, 1H), 4.24 - 3.94 (m, 3H), 3.79 (s, 7H), 3.16 - 3.00 (m,2H), 2.02 (s, 1H), 1.65 - 1.46 (m, 2H), 1.30 - 1.15 (m, 9H), 0.98 - 0.84 (m, 1H).31P NMR (202 MHz, DMF) 5 150.90.

Claims

CLAIMS1. An antisense oligonucleotide comprising a central DNA segment 6-14 nucleotides long and RNA-based wings 2-4 nucleotides long, wherein all the phospodiesters (PO) in the backbone are replaced by phosphorothioates (PS), and where at least three consecutive PS are stereopure.

2. The antisense oligonucleotide of claim 1 where the three consecutive stereopure PS are 5’-SpSpSp-3’ (SSS), 5’-SpRpSp-3’ (SRS) or 5’-SpSpRp-3’ (SSR).

3. The antisense oligonucleotide of claim 2 where the central DNA segment is 6-12 nucleotides long and the RNA-based wings are 2-4 nucleotides long.

4. The antisense oligonucleotide of claim 2 where the central DNA segment is 6-12 nucleotides long and the RNA-based wings are 2-4 nucleotides long.

5. The antisense oligonucleotide of claim 2 where the central DNA segment is 10 nucleotides long and the RNA-based wings are 3 nucleotides long.

6. The antisense oligonucleotide of claim 2 where the RNA-based wings include one or more modified nucleotides.

7. The antisense oligonucleotide of claim 6 where the one or more modified nucleotides comprise 2’0-methyl (2’0Me), 2’0-methoxyethyl (MOE), peptide nucleic acid (PNA), phosphoroamidate morpholino (PMO), locked nucleic acid (LNA), 2’ -deoxy -2 ’-fluoro (2’-F) or any other 2’ modified oligonucleotide.

8. The antisense oligonucleotide of claim 2 where the three consecutive stereopure PS are located in the RNase Hl binding site.

9. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1-8 and a pharmaceutically acceptable diluent or carrier.

10. A method for the treatment and / or prevention of a disorder in a subject comprising administering to the subject a therapeutically effective amount of an antisense oligonucleotide according to any one of claims 1-8.

11. The method of claim 10 wherein the subject is a human.

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