Antisense oligonucleotides and their medical use

Abstract

The present invention provides antisense oligonucleotides having 2'-O-methoxyethyl sugar modifications at each position and at least one chiral internucleotide phosphorothioate bond having an Sp structure at the 3' end. The present invention further relates to the use of such antisense oligonucleotides in medicine. The present invention further provides compositions including pharmaceutical compositions containing such antisense oligonucleotides. Furthermore, methods for production are provided.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of antisense oligonucleotides, in particular splice-switching oligonucleotides, and their use in medicine, in particular in the treatment of spinal muscular atrophy. BACKGROUND ART

[0002] Antisense oligonucleotides: Antisense oligonucleotides (ASOs) are relatively short single-stranded nucleic acids, usually 10 to 30 nucleotides in length, capable of binding to complementary messenger RNA (mRNA, including pre-mRNA). Typically, in contrast to many conventional drugs that inhibit protein function, for example by binding to a specific pocket of an enzyme, ASOs alter the biosynthesis of a protein as the protein is being formed. General classification of ASOs may be made based on their respective mechanism of action, in particular based on whether they function via the so-called "target occupancy" mechanism or "occupancy-mediated degradation".

[0003] ASOs that function via "occupancy-mediated degradation" include in particular RNase H-dependent ASOs, which are typically used to downregulate disease-causing proteins via enzymatic degradation of the respective mRNA. One example of such an RNase H-dependent ASO is mipomersen, which binds to ApoB-100 mRNA and induces its degradation via RNase H, thereby inhibiting translation of the ApoB-100 protein.

[0004] In contrast, ASOs that act primarily through so-called "target occupation" mechanisms typically do not induce enzymatic RNA degradation. A notable example of so-called "target occupation" ASOs is splice-switching oligonucleotides (SSOs) that bind to specific regions of pre-mRNA involved in splicing, such as splice sites or regulatory elements, in order to regulate splicing when normal splicing is disrupted. In particular, SSOs can interfere with the endogenous RNA splicing mechanism by sterically blocking access to specific splice sites and / or regulatory elements.

[0005] The field of ASOs, particularly SSOs, has attracted attention in recent years due to advances made in the treatment of neurological disorders. For example, nusinersen, marketed as Spinraza, was the first drug approved for use in the treatment of spinal muscular atrophy (SMA), a rare neuromuscular disorder.

[0006] Treatment of spinal muscular atrophy: SMA is a rare neuromuscular disease caused by homozygous deletion of the SMN1 gene, which encodes the survival motor neuron (SMN) protein. In humans, SMN protein is also produced from the SMN2 gene. However, the production of SMN protein from the SMN2 gene is limited. Normally, SMN2 protein expression is suppressed by a splicing enhancer sequence that leads to the exclusion of exon 7, resulting in an unstable, cleaved form of SMN protein.

[0007] Nusinersen is an ASO that binds to SMN2 premRNA and alters its splicing, thereby restoring the loss of SMN proteins, including exon 7.

[0008] Furthermore, SMA can be treated with a gene therapy recently developed and marketed as Zolgensma. However, the high cost associated with this gene therapy method limits the application of Zolgensma.

[0009] Chemical oligonucleotide synthesis: General methods for chemical oligonucleotide synthesis are known in the art and typically involve the stepwise addition of nucleoside building blocks to the 5' end of an elongating oligonucleotide chain bound to a solid support. Each addition is often called a synthetic cycle and typically involves four chemical reactions: Deblocking: Removal of a protecting group (e.g., a 4,4'-dimethoxytrityl group) from the 5'-hydroxyl group of an oligonucleotide bound to a solid support, or, in the first synthetic cycle (i.e., when the nucleoside has not yet attached to the solid support, a general-purpose solid support), the hydroxyl protecting group present on the solid support. Coupling: Once the hydroxyl protecting group is removed, the free 5'-OH group of the oligonucleotide bound to the solid support or the free hydroxyl group of the solid support can react with the following nucleoside, which is usually added as a monomeric nucleoside building block, such as a phosphoramidite (PA) building block or an oxazaphosphoridine (OAP) building block. The coupling reaction results in the formation of a phosphite triester bond. Capping: Unreacted 5'-hydroxyl groups or free hydroxyl groups on a solid support are blocked, for example, by using acetic anhydride in the presence of a base. If not blocked, these hydroxyl groups react during the next cycle, thus leading to oligonucleotides containing internal deletions. Oxidation or sulfidation: The triester phosphite formed during coupling reactions is relatively unstable under the conditions used for oligonucleotide synthesis. Therefore, before the start of the next synthetic cycle, the triester phosphite must be converted to a more stable phosphorus species, particularly a phosphodiester bond by oxidation, usually using an aqueous iodine solution, or a phosphorothioate bond by sulfidation, usually using commercially available sulfiding reagents.

[0010] Each solution of the reagents used in oligonucleotide synthesis is prepared in advance and placed in reagent bottles (usually for laboratory-scale synthesis) or tanks (usually for commercial production) for the oligonucleotide synthesizer.

[0011] Modern oligonucleotide synthesis typically relies on the so-called phosphoramidite method, in which naturally protected nucleoside building blocks (PA or OAP) or chemically modified nucleosides are successfully added to an oligonucleotide chain bound to a support until the desired chain length is reached. Therefore, chemical synthesis allows for the precise synthesis of specified oligonucleotide sequences and enables the introduction of various sequence-specific chemical modifications. To allow for maximum freedom in sequence selection, oligonucleotides are often synthesized on commercially available general-purpose solid supports, i.e., solid supports configured for automated oligonucleotide synthesis but not yet bound to nucleosides. However, several solid supports with single nucleosides already pre-loaded are also available; these limit the user's freedom (as the 3'-terminal nucleoside is already determined by the solid support), but often result in better overall yields at the end of synthesis than general-purpose solid supports.

[0012] Automated synthesis of oligonucleotides with uniform chemical modifications, such as nucleosides entirely modified by the 2'-MOE moiety or nucleic acid bases entirely substituted with 5mC, is well-established in a cost-effective manner. However, the synthesis of oligonucleotides with heterogeneous modification patterns, such as oligonucleotides with specific stereochemistry at selected PS bonds combined with uncontrolled chiral PS bonds, requires the use (and maintenance) of many different reagents (various nucleoside construction blocks, activators, and reaction conditions for the synthesis of Rp, Sp, and sterically random PS bonds), making the synthesis process difficult and economically unattractive. Furthermore, the synthesis of oligonucleotides containing chirally controlled PS bonds requires the use of nucleoside construction blocks with chiral auxiliary groups, particularly oxazaphosphoridine construction blocks. However, the coupling yields of oxazaphosphoridine construction blocks are typically lower than those of commonly used nucleoside phosphoramidites (uncontrolled chirality).

[0013] Therefore, complete PS oligonucleotides that are not chiralized (sterically random, i.e., each PS bond is a mixture of Rp and Sp bonds) remain the absolute standard in oligonucleotide therapeutics to this day.

[0014] Chemical modification of ASO: The properties and functions of ASOs are primarily determined by the combination of their nucleotide sequences and chemical structures. Possible sites for chemical modification include the nucleic acid backbone, nucleic acid bases, sugar moieties, and the 5'- and 3' ends. Various chemical modifications are known to those skilled in the art, and their application to therapeutic oligonucleotides is described, for example, in Crooke et al. [JBC Reviews (2021) 296, 100416].

[0015] A prominent example of a chemically modified ASO is nusinersene (an 18-nucleotide ASO), in which the 2'-hydroxyl group of the ribofuranosyl ring is replaced with a 2'-O-2-methoxyethyl (2'-MOE) group at each position (i.e., at each nucleotide). Furthermore, all phosphate bonds (PO) are replaced with phosphorothioate (PS) bonds, and all cytosine nucleotide bases are replaced with 5-methylcytosine (5mC). Nusinersene is represented by Sequence ID 1:TCACTTTCATAATGCTGG, where T represents 2'-MOE-thymidine, C represents 2'-MOE-5-methylcytidine, A represents 2'-MOE-adenosine, and G represents 2'-MOE-guanosine.

[0016] While substitution of PO bonds with PS is known to increase metabolic stability, the substitution of one of the non-crosslinked oxygen atoms with sulfur converts the prochiral PO bond into a new stereocenter. In conventional oligonucleotide synthesis, Rp and Sp bonds typically occur in a ratio of approximately 1:1 [see, e.g., Oka et al., Nucleic Acids Symp. Ser. (Oxf) 2008, 335; and VT Ravikumar, DL Cole, Nucleosides, Nucleotides & Nucleic Acids (2003), 22, 1639]. Therefore, the 18-base oligonucleotide nusinersene contains a mixture of 2^17 = 131,072 potential diastereomers (2^n diastereomers, where n is the number of PS bonds), each with its own unique physical and biochemical properties.

[0017] However, the synthesis of chiral, sterically pure SSOs remains a challenge in this field. As a result, efforts to study the effects of stereochemistry on PS binding have also focused on modulating stereochemistry through the selection of activators used during RNA chemosynthesis [Jahns et al., Nat. Commun. 2015, 6, 6317], showing that higher proportions of Rp-PS binding lead to increased hybridization affinity and improved metabolic stability of small interfering RNAs (siRNAs). Furthermore, it has been reported that stereochemically pure, complete Rp 2'-O-(2-methoxyethyl)-phosphorothioate SSOs targeting ferrokeratase premRNA are more effective than their Sp controls but not as good as their sterically random parent SSOs [Li et al., Chem. Commun. 2017, 53, 541]. This is consistent with the report by Ostergaard et al., which found that no improvement in efficacy was observed with a series of chirally controlled ASOs compared to sterically random parent ASOs [Nucleic Acids Res. 2020;48(4):1691~1700].

[0018] While some literature reports have suggested the superiority of certain oligonucleotide drugs containing several chirally controlled phosphorothioate bonds, it is generally considered that "chiral purity in individual PS units does not result in a significant improvement in the therapeutic index and overall performance of PS ASOs" [Crooke et al., JBC Reviews (2021) 296, 100416]. Furthermore, clinical programs studying sterically pure ASOs for the treatment of Huntington's disease (rovanersen, lexanersen) and Duchenne muscular dystrophy (suvodirsen, see, e.g., https: / / ir.wavelifesciences.com / news-releases / news-release-details / wave-life-sciences-announces-discontinuation-suvodirsen) have been terminated due to a lack of efficiency. Given the current lack of approved oligonucleotide drugs with high steric purity, complete PS ASOs, which are not chirally controlled (sterically random), remain the absolute standard in antisense therapy to this day. [Prior art documents] [Patent Documents]

[0019] [Patent Document 1] US7202264 [Patent Document 2] WO2017 / 198775 [Non-patent literature]

[0020] [Non-Patent Document 1] Crooke et al. [JBC Reviews (2021) 296, 100416] [Non-Patent Document 2] Oka et al., Nucleic Acids Symp. Ser. (Oxf) 2008, 335 [Non-Patent Document 3] V. T. Ravikumar, D.L. Cole, Nucleosides, Nucleotides & Nucleic Acids (2003), 22, 1639

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Summary of the Invention

Problem to be Solved by the Invention

[0021] Description of the Invention

[0022] It is an object of the present invention to provide ASOs, particularly SSOs, with improved therapeutic properties, especially for the treatment of SMA.

[0023] In particular, an object of the present invention is to provide an ASO having increased metabolic stability compared to existing therapeutic ASOs, especially nusinersen. A further object of the present invention is to provide a method for producing such an improved ASO in an economically feasible manner. [Means for solving the problem]

[0024] The present invention provides novel ASOs comprising a 3'-terminal chiral nucleotide PS bond, also called a 3'-terminal PS bond, which includes a 2'-O-methoxyethyl sugar modification at each position, and further comprises the 3'-terminal PS bond having an Sp structure in its chiral-linked phosphorus. The present invention further relates to compositions comprising a plurality of such ASOs, and pharmaceutical compositions comprising such ASOs or a plurality of such ASOs. Furthermore, the present invention provides pharmaceutically acceptable carriers or diluents in lyophilized form, and kits comprising the ASOs or a plurality of such ASOs.

[0025] In a further embodiment, the present invention provides the use of the ASO, the plurality of ASOs, or the kit as a pharmaceutical, particularly in the treatment of SMA. Furthermore, the present invention provides a method for producing such ASOs.

[0026] It is understood that the various embodiments / characteristics, preferred ones and scopes provided / disclosed herein may be combined in any way, insofar as any particular combination of embodiments / characteristics is technically meaningful. In particular, it is understood that all of the definitions and characteristics of the ASO of the present invention provided in the first aspect are similarly applicable to the second, third, fourth, fifth and sixth aspects of the present invention.

[0027] Unless otherwise specified, the following definitions shall apply herein: When used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present invention (particularly in the context of the claims) should be construed to encompass both singular and plural forms unless otherwise indicated herein or unless explicitly stated otherwise.

[0028] The terms “including,” “containing,” and “comprising” are used herein in an open-ended and non-restrictive sense.

[0029] As used herein, the term "and / or" means that all or any one of the elements of the group may be present. For example, "A and / or B" means "A only, or B only, or both A and B." In the case of "A only," the term also covers the possibility that B is not present, i.e., "A only, not B."

[0030] The terms “including,” “containing,” and “comprising” are used herein in an open-ended and non-restrictive sense. It is understood that various embodiments, preferred and scopes may be combined as desired.

[0031] Treatment: As used herein, the terms “to treat,” “to treat,” and “treatment” include one or more of the following: (i) preventing the occurrence of a disease, pathological or medical condition (e.g., prevention); (ii) inhibiting or halting the progression of a disease, pathological or medical condition; (iii) alleviating a disease, pathological or medical condition; and (iv) reducing symptoms associated with a disease, pathological or medical condition. Thus, the terms “to treat,” “treatment,” and “to treat” include prevention and preventing, preventing, reducing, halting, or reversing the progression or severity of the condition or symptoms being treated. Therefore, the term “treatment” includes, as may be, medical, therapeutic, and / or prophylactic administration.

[0032] Gene: As used herein, “gene” means all DNA regions (including exons and introns) that encode a gene product, and furthermore, all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to the sequences that encode and / or transcribe them. Thus, a gene includes, but is not limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and intra-sequence ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix binding regions, and locus regulatory regions. As used herein, the term “gene” means, in particular, a human gene.

[0033] Nucleotides: The term “nucleotide” generally refers to deoxyribonucleotides, ribonucleotides, or derivatives thereof. A nucleotide may be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or a nucleotide analog. A nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base and / or a modified ribose moiety. A nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a nucleotide that does not exist in nature. Examples of nucleotides that do not exist in nature include nucleotides modified at the 2'-O position of a sugar, e.g., 2'-OMe, 2'-MOE, 2'-F modified nucleotides, especially 2'-MOE, as well as cross-linked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos.

[0034] Nucleic acid bases: The term "nucleic acid base" is known in the art and refers to the heterocyclic base portion of a nucleoside or nucleotide. Generally, a nucleic acid base is any group containing one or more atoms or groups of atoms that can hydrogen bond to a base of another nucleic acid. In addition to "unmodified" or "natural" nucleic acid bases, such as the purine nucleic acid bases adenine (A) and guanine (G), and the pyrimidine nucleic acid bases thymine (T), cytosine (C), and uracil (U), a wide range of modified nucleic acid bases are known and can be used in the context of this invention. The synthesis of modified nucleic acid bases is known in the art.

[0035] Oligonucleotides: The term "oligonucleotide" generally refers to deoxyribonucleotides or ribonucleotide polymers, or derivatives thereof. In oligonucleotides, adjacent nucleotides are covalently bonded by phosphate groups to form a sequence. The nucleotide bond is also called the oligonucleotide backbone. In most naturally occurring oligonucleotides, the backbone consists of 3'-5' phosphodiester bonds. However, synthetic oligonucleotides often involve chemical modifications, for example, to increase the binding affinity of ASOs to their target RNA, to increase nuclease resistance (metabolic stability), and / or to alter the pharmacokinetics of the oligonucleotide, such as its distribution in the body. As those skilled in the art will understand, the use of chemical modifications to increase the affinity of oligonucleotides to their target RNA allows for the use of relatively short oligonucleotides. Therefore, the term oligonucleotide includes modified oligonucleotides, which include nucleotides modified at the base (e.g., 5-methylcytosine), sugar (e.g., 2'-MOE), and / or phosphate moiety (e.g., PS bond).

[0036] Antisense oligonucleotides, or ASOs: This term is known in the art and relates to synthetic linear single-stranded nucleic acid polymers that can typically bind to complementary messenger RNA (including mRNA and premRNA) via Watson-Crick base pairing. Typically, ASOs have a length of 10 to 30 nucleotides and may include the modified and / or unmodified nucleotides described above. In particular, the ASOs of the present invention contain up to 20 nucleotides. Therefore, in the context of the present invention, ASOs have a length of 18 to 20 nucleotides.

[0037] Furthermore, in the ASO of the present invention, at each position (i.e., in each of its nucleotides), the 2'-hydroxyl group of the ribofuranosyl ring (i.e., sugar) is substituted with a 2'-MOE group. Furthermore, at least the 3'-terminal internucleotide phosphate bond is substituted with a PS bond having an Sp structure (i.e., at least the 3'-terminal PS bond is chiralized). As those skilled in the art will understand, the 3'-terminal PS bond is located between the 3' end and the second to last nucleotide, and the 3'-terminal nucleotide of the ASO contains a 3'-hydroxyl group instead of a further phosphate or phosphorothioate moiety. In the context of the present invention, the ASO preferably has a length of 18 to 20 nucleotides.

[0038] The term "ASO" specifically includes "SSO". SSO binds to premRNA and modulates splicing by, for example, blocking RNA-RNA base pairing or protein-RNA binding interactions that occur between components of the endogenous splicing mechanism and premRNA. In the context of this invention, the modulation of splicing specifically refers to altering the splicing of SMN2 premRNA so that exon 7 is included in SMN2 mRNA.

[0039] PO bond, PS bond: The terms "PO bond" and "PS bond" are well known in the art and refer to a phosphodiester bond between two nucleosides (nucleoside 1-O-PO2-O-nucleoside 2) or a phosphorothioate bond between two nucleosides (nucleoside 1-O-POS-O-nucleoside 2), respectively. Thus, these terms should not be confused with a single PO bond or a single PS bond.

[0040] Chiralized: This term is known in the art and, when used in the context of the present invention, particularly relates to chiralized internucleotide PS bonds in which one of the non-crosslinked oxygen atoms (present in the prochiral 3'-5' phosphodiester bond) is substituted with sulfur. Methods for determining the steric purity of PS bonds in phosphorus are known in the art, particularly RP-HPLC and 31This includes 1P NMR spectroscopy. As used herein, “chiral controlled” means that the steric purity of the chiral phosphorus of the PS bond is at least 90%, preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99%, as measured preferably by RP-HPLC. Preferably, the steric purity of each chiral controlled PS bond is at least 99%, as measured preferably by RP-HPLC.

[0041] Hybridization: The term "hybridization" is known in the art and relates to pairing, i.e., the binding of complementary nucleic acid strands, for example, the binding of an ASO to its target sequence. While not limited to specific mechanisms, the most common mechanisms of hybridization (binding) involve hydrogen bonding between the nucleic acid bases of complementary nucleotides, e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen base pairing, particularly Watson-Crick base pairing. The term "complementarity," or "complementary," as used herein, relates to the ability to form precise base pairs between two nucleic acid bases, for example, between an ASO and its target sequence. For example, a position in an ASO is considered a complementary position if a nucleic acid base at a particular position can pair with a nucleic acid base at a particular position in a target nucleic acid, particularly premRNA, e.g., SMN2 premRNA. Furthermore, an ASO and its target nucleic acid are considered complementary if a sufficient number of positions (nucleoside bases) are complementary so that stable and specific binding occurs between the ASO and the target nucleic acid, i.e., SMN2 premRNA. As is known in the art, the nucleic acid base adenine is complementary to the nucleic acid bases thymidine and uracil, and the nucleic acid base guanine is complementary to the nucleic acid bases cytosine and 5-methylcytosine. Those skilled in the art can determine, for example, whether an ASO is complementary to its target nucleic acid by determining the melting temperature (Tm) between the two strands. As is known in the art, Tm is a measure of hybridization affinity or binding affinity between the ASO and its target sequence. Methods for determining the Tm between an ASO and its target sequence are known in the art, and Tm can also be calculated by computation.

[0042] Polypeptide: The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues.

[0043] Throughout this specification, 2'-F 2'-Fluorine 2'-MOE 2'-methoxyethyl 2'-OMe 2'-O-methyl 5mC 5-methylcytosine ACN Acetonitrile AEX-HPLC Anion Exchange High-Performance Liquid Chromatography ASO Antisense Oligonucleotides BHQ-1 Black Hole Quencher 1 BTT 2-benzylthiotetrazol CPG porosity-controlled glass DCA (Dichloroacetic Acid) DCI 4,5-dicyanoimidazole DCM Dichloromethane DDTT 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thion DMTr 4,4'-Dimethoxytrityl ETT 5-ethylthio-1H-tetrazol FAM Carboxyfluorescein GalNAc N-acetyl-galactosamine GAPDH Glyceraldehyde 3-phosphate dehydrogenase HPLC (High-Performance Liquid Chromatography) mRNA messenger ribonucleic acid RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography) siRNA, small interfering RNA SMA (Spinal Muscular Atrophy) SMN2 Survival Motor Neuron 2 SPE solid phase extraction LC-MS Liquid Chromatography-Mass Spectrometry LNA cross-linked nucleic acid mAU (milli-absorbance unit, arbitrary unit) MMTr 4-Monomethoxytrityl mRNA messenger RNA NMR nuclear magnetic resonance N-PhIT N-phenylimidazolium triflate OAP Oxazaphosphoridine PA phosphoramidite Ph Phenyl PNA (Peptide Nucleic Acid) PO phosphodiester PS Phosphothioate R structure in Rp chirallin SSO Splice Switching Oligonucleotides S structure in sp chirallin THF (Tetrahydrofuran) Tm melting temperature Multiple abbreviations, including the one mentioned above, are used.

[0044] First aspect, antisense oligonucleotide: In a first embodiment, the present invention relates to an ASO comprising SEQ ID NO 1, or a pharmaceutically acceptable salt thereof, wherein each of the nucleotides has a 2'-O-methoxyethyl sugar modification and a chiral internucleotide phosphorothioate bond having an Sp structure at the 3' end, or a pharmaceutically acceptable salt thereof. As those skilled in the art will understand, the internucleotide phosphorothioate at the 3' end bond is located between the 3' end and the second to last nucleotide.

[0045] Sequence ID 1:TCACTTTCATAATGCTGG, where T represents 2'-MOE-thymidine, A represents 2'-MOE-adenosine, and G represents 2'-MOE-guanosine. C represents 2'-MOE-cytidine and / or 2'-MOE-5-methylcytidine; that is, in certain positions C may represent 2'-MOE-cytidine, and in other positions C may represent 2'-MOE-5-methylcytidine.

[0046] Preferably, the ASO contains at least one 5-methylcytosine. More preferably, C represents 2'-MOE-5-methylcytidine in SEQ ID NO: 1, i.e., all cytosine residues are 5-methylcytosine. Substituting cytosine with 5-methylcytosine has the advantage of increasing the hybridization affinity of the ASO to its target sequence. However, C may also represent 2'-MOE-cytidine at all positions.

[0047] At least the 3' terminal bond (i.e., the final internucleotide bond at the outermost 3' end of ASO) is a PS bond having an Sp structure in its chiral phosphate, but the remaining internucleotide bonds may be selected from PO bonds (prochiral) and / or PS bonds, where the PS bond may have an Sp structure, an Rp structure, and / or not be chiralized, i.e., it may contain a mixture of PS bonds as Sp and Rp structures.

[0048] Surprisingly, the ASOs of the present invention described herein were found to have improved metabolic stability compared to nusinersen (see Example 6; Figure 1A, ASO4, 5, 6 and 10; nusinersen corresponding to ASO1). As those skilled in the art will understand, metabolic stability is a crucial factor for the therapeutic activity of ASOs. It is particularly beneficial and surprising that a single PS bond with an Sp structure at the 3' terminus is already sufficient to achieve a significant increase in metabolic stability, i.e., increased stability against nuclease degradation (see Example 6; ASO1 vs ASO4).

[0049] As is commonly accepted in the art, a good alternative method for determining the in vivo metabolic stability of ASOs is incubation in whole tissue lysates, specifically mouse liver lysates. In preferred embodiments, at least 95% of the ASOs of the present invention described herein, i.e., 95% to 100%, preferably 98% to 100%, maintain their full length after incubation in mouse liver lysates for at least 5 days, as measured by LC-MS.

[0050] Preferably, as measured by LC-MS, there is an increase of at least 5 percentage points in the full length of the ASO of the present invention compared to nusinersen after incubation in mouse liver lysate for at least 5 days.

[0051] Preferably, the ASO of the present invention has PS bonds at all positions, which is also called a complete PS ASO (i.e., all internucleotide bonds are PS). Having PS bonds at all positions results in improved metabolic stability compared to an otherwise identical ASO containing at least one PO bond, e.g., 1 to 20 PO bonds, e.g., 1 to 5 PO bonds.

[0052] However, ASOs may contain PO bonds. In such embodiments, up to 50% of the PS bonds are replaced by PO bonds. In this embodiment as well, it is clear from the above that at least the 3' terminal bond is a PS bond. Including a limited number of PO bonds, e.g., 1 to 10 PO bonds, preferably 1 to 5 PO bonds, has the advantage of reducing side effects, for example, by reducing stimulation of the human immune system, particularly the innate immune system, while maintaining sufficient metabolic stability. Therefore, if present, the number of PO bonds is usually limited to 1 to 10 PO bonds, preferably 1 to 5 PO bonds, e.g., 1, 2, 3, 4, or 5 PO bonds, while the remaining internucleotide bonds are PS. In the typical scenario of ASOs described herein, namely, for a maximum length of 20 nucleotides, a total of 1 to 10 PO bonds corresponds to 5% to 50% of the bonds being PO bonds. The PO bonds may be located directly adjacent to each other, for example, ASO may have three PO bonds at positions 9, 10, and 11, or five PO bonds at positions 7, 8, 9, 10, and 11, or the PO bonds may be scattered, for example, ASO may have three PO bonds at positions 4, 8, and 11, or five PO bonds at positions 3, 5, 9, 13, and 15 (nucleotide positions are counted starting from the 5' end; the positions indicated for PO bonds are illustrative only).

[0053] Furthermore, the ASO of the present invention may include further chiralized internucleotide PS bonds, which are advantageously continuous with the 3'-terminal chiral internucleotide phosphorothioate bond. In an advantageous embodiment, the ASO further has at least one (see Example 6; Figure 1, ASO5) or at least two chiral internucleotide phosphorothioate bonds (see Example 6; Figure 1, ASO6) having an Sp structure, continuous with the 3'-terminal chiral internucleotide phosphorothioate bond (i.e., at least the last two or at least the last three internucleotide bonds at the outermost 3' end are chiralized PS bonds having an Sp structure).

[0054] The inclusion of a further chiral-controlled internucleotide PS bond having an Sp structure, adjacent to the aforementioned internucleotide PS bond having an Sp structure at the 3' end, is expected to result in even greater metabolic stability when administered in vivo compared to an ASO having a single chiral-controlled PS bond having an Sp structure at the 3' end. However, the ASO of the present invention may also include a chiral internucleotide PS bond having an Rp structure. For example, a chiral-controlled PS bond having an Rp structure may increase binding affinity to the target sequence and may further improve interaction with certain proteins in the endogenous splicing mechanism, thereby potentially leading to a higher proportion of exon 7 in the SMN2 mRNA.

[0055] In a favorable embodiment, the ASO contains one chiral internucleotide PS bond having an Sp structure at its 3' end, and all other internucleotide bonds are uncontrolled chiral PS bonds (see Example 6; Figure 1, ASO4), i.e., all other internucleotide bonds are a mixture of PS bonds having, for example, Rp and Sp structures with an Rp:Sp ratio of 0.1–0.9, e.g., 0.4–0.6. Such an ASO has the advantage of improved metabolic stability compared to the same ASO in which none of the PS bonds are chiralized, i.e., none of the PS bonds are synthesized under chiral control (also called a completely sterically random PS ASO, see Example 6, ASO1 vs ASO4), or in which the 3'-terminal PS bond has an Rp structure (see Example 6, ASO7, 8, and 9), while being able to be produced in a more cost-effective manner than ASOs containing multiple chiralized PS bonds.

[0056] In another advantageous embodiment, the ASO contains one chiral internucleotide PS bond having an Sp structure at its 3' end, while all other internucleotide bonds are chiralized PS bonds having an Rp structure (see Figure 1, ASO10). Such an ASO has the advantage of increased metabolic stability compared to the same ASO in which none of the PS bonds are chiralized (e.g., ASO1) or in which the 3'-terminal PS bond has an Rp structure (e.g., ASO2, 7, 8, and 9, see Figure 1), while also having the further advantage of improved target binding affinity, i.e., potency, compared to other ASOs in which none of the PS bonds are chiralized.

[0057] In other advantageous embodiments, the ASO contains two consecutive chiral nucleotide PS bonds having an Sp structure at the 3' end, with all other nucleotide bonds being uncontrolled chiral PS bonds (see Example 6; Figure 1, ASO5). In other advantageous embodiments, the ASO contains three consecutive chiral nucleotide PS bonds having an Sp structure at the 3' end, with all other nucleotide bonds being uncontrolled chiral PS bonds (see Example 6; Figure 1, ASO6). An ASO containing two or three consecutive chiralized nucleotide PS bonds having an Sp structure at the 3' end, with all other nucleotide bonds being uncontrolled chiral PS bonds, offers the advantage of further improved metabolic stability when administered in vivo. This is particularly relevant when the ASO is administered at long intervals, for example, once every four months, once every six months, or once every twelve months. ASOs containing two or three consecutive chiral-controlled internucleotide PS bonds having an Sp structure at the 3' end can still be produced in a cost-effective manner, i.e., with good yield and sufficient purity. In other embodiments, all internucleotide bonds are chiral-controlled PS bonds having an Sp structure. However, ASOs in which all internucleotide bonds are chiral-controlled PS bonds having an Sp structure are less desirable because their production is relatively extensive and the synthetic yield is usually lower compared to ASOs containing fewer chiral-controlled PS bonds.

[0058] Preferably, the ASO of the present invention is up to 20 nucleotides in length (i.e., contains up to 20 nucleotides). Therefore, the ASO preferably contains 18 to 20 nucleotides, for example, 18, 19, or 20 nucleotides. In one preferred embodiment, the ASO consists of 18 nucleotides, i.e., the ASO is represented by Sequence ID No. 1. In one advantageous embodiment, the ASO of the present invention is represented by ASO3 (see Figure 1). In another advantageous embodiment, the ASO of the present invention is represented by ASO4 (see Figure 1). In yet another advantageous embodiment, the ASO of the present invention is represented by ASO5. In yet another advantageous embodiment, the ASO of the present invention is represented by ASO6 (see Figure 1). In yet another advantageous embodiment, the ASO is represented by ASO3 (see Figure 1). In yet another advantageous embodiment, the ASO of the present invention is represented by ASO10 (see Figure 1).

[0059] Advantageously, all cytosine residues are 5-methylcytosine.

[0060] Furthermore, the ASO of the present invention may be conjugated to a non-nucleic acid moiety, for example, N-acetyl-galactosamine (GalNAc), for example, the previously described tripartite GalNAc linker [see, e.g., Nair et al., J Am Chem Soc, 2014, 136, pp. 16958-61], or a lipid moiety [see, e.g., Moroz et al., Mol. Ther.-Nucl. Acids (2016) 5, e364]. Such a non-nucleic acid moiety may be conjugated to either the 3' or 5' end of the ASO. Conjugation to a non-nucleic acid moiety improves tissue delivery and intracellular uptake in vivo. Conjugation to a non-nucleic acid moiety is within the realm of conventional art, and suitable construction blocks are commercially available, for example, from Glen Research (e.g., catalog numbers 10-1974 and 20-2974).

[0061] The ASOs of the present invention include esters in the internucleoside PS and / or PO bonds, such as S-acyl-2-thioethyl ester (see, e.g., Tosquellas et al., Nucleic Acids Res. 1998; 26(9): pp. 2069-2074), or any other functional chemotherapeutic equivalents (also called prooligonucleotides) that, when administered to animals or humans, particularly humans, result in the ASOs described herein. Sodium and potassium salts of ASOs, pharmaceutically acceptable salts including sodium salts, their esters, or other functional chemotherapeutic equivalents thereof are also claimed and described herein.

[0062] Second aspect; composition comprising multiple ASOs: In a second embodiment, the present invention relates to compositions comprising a plurality of ASOs and / or pharmaceutically acceptable salts thereof. Such plurality of ASOs include various variants of the ASOs described herein, e.g., ASOs whose stereochemistry at chiral PS bonds differs at specific positions (i.e., at specific internucleotide bonds). For example, such plurality of ASOs include ASOs comprising two or more chiralally controlled internucleotide PS bonds having an Sp structure at the 3' end, and / or ASO comprising a chiral-controlled internucleotide phosphorothioate bond having an Sp structure at its 3' end, and further comprising at least one chiral-controlled internucleotide phosphorothioate bond having an Rp structure. Combined with, ASO contains one chiralized internucleotide PS bond with an Sp structure at its 3' end, but all other internucleotide bonds are non-chiralized PS bonds. It may include.

[0063] Such multiple ASOs may also include combinations of ASOs of the present invention with different lengths, for example, the 18-base ASO and / or 19-base ASO and / or 20-base ASO described herein.

[0064] Compositions containing multiple ASOs may have several beneficial effects, such as a reduced risk of immune response due to lower amounts of each specific variant of ASO, and / or improved activity.

[0065] Third aspect, pharmaceutical composition: In a third embodiment, the present invention relates to a pharmaceutically acceptable carrier or diluent, and Antisense oligonucleotides and / or pharmaceutically acceptable salts thereof as described herein, Multiple antisense oligonucleotides and / or pharmaceutically acceptable salts thereof as described herein. This relates to a pharmaceutical composition containing any of the following.

[0066] As those skilled in the art will understand, such pharmaceutical compositions can be prepared by adding an effective amount of ASO as described herein to a suitable pharmaceutically acceptable diluent or carrier.

[0067] Pharmaceutically acceptable carriers and diluents are known in the art. As those skilled in the art will understand, the selection of a diluent or carrier is based on several factors, including the solubility of the ASO and the route of administration.

[0068] For example, the diluent or carrier may include an ionic strength adjustment medium containing a buffer and a salt, such as phosphate buffer, such as phosphate-buffered saline, isotonic NaCl solution, sugar solution, citrate buffer, isocitrate buffer, EDTA (including EDTA salts), and mixtures thereof.

[0069] Preferably, the pharmaceutical compositions described herein are provided as solutions for intrathecal administration.

[0070] Furthermore, the pharmaceutical compositions described herein are preferably provided in unit dosage forms.

[0071] The ASO of the present invention, or the plurality of ASOs, may be provided in a lyophilized form, separate from a pharmaceutically acceptable carrier or diluent. Therefore, the present invention provides a pharmaceutically acceptable carrier or diluent, and one of the following in a lyophilized form: ASO as described herein or a pharmaceutically acceptable salt thereof, A composition comprising several ASOs described herein and / or pharmaceutically acceptable salts thereof. Further information regarding the kit, which includes this.

[0072] Fourth aspect, therapy: A fourth aspect of the present invention relates to medical applications.

[0073] The ASOs of the present invention can be used to modulate SMN2 expression in animals or humans, particularly in human subjects, where such modification is required. In particular, the present invention provides a method for promoting the inclusion of exon 7 in an SMN2 transcript in a cell, tissue, or organ, comprising the step of contacting the cell, tissue, or organ in vitro with an ASO described herein or a pharmaceutically acceptable salt thereof. Furthermore, the present invention provides a method for promoting the inclusion of exon 7 in an SMN2 transcript in a cell, tissue, or organ, comprising the step of contacting the cell, tissue, or organ in vitro with a composition comprising a plurality of ASOs described herein and / or pharmaceutically acceptable salts thereof, or a pharmaceutical composition described herein or a kit described herein.

[0074] Therefore, the ASOs described herein or their pharmaceutically acceptable salts are useful as pharmaceuticals, particularly for the treatment of SMA. Furthermore, compositions comprising several ASOs and / or their pharmaceutically acceptable salts described herein, the pharmaceutical compositions described herein, and the kits described herein are available for use as pharmaceuticals, particularly for the treatment of SMA.

[0075] For example, pharmaceutical use includes a method comprising administering an effective amount of the ASO described herein, and / or a pharmaceutically acceptable salt thereof, to a human subject in need of therapy for a disease or condition associated with SMN2. As those skilled in the art will understand, diseases or conditions associated with SMN2 include, but are not limited to, spinal muscular atrophy. The ASO of the present invention can effectively modulate SMN2 splicing, resulting in increased exon 7 inclusion in SMN2 mRNA. Modulation of SMN2 expression can be measured, for example, in body fluids, such as serum. Such body fluids may or may not contain cells. Modulation of SMN2 expression can also be measured in tissue samples, usually obtained by biopsy. Methods for measuring the modulation of SMN2 expression are known in the art. As those skilled in the art will understand, the evaluation of safe and effective therapies typically involves measuring biomarkers, such as markers of renal and hepatic function, e.g., liver transaminases, albumin, blood urea nitrogen, bilirubin, creatine, and inflammatory markers, e.g., tumor necrosis factor, intracellular adhesion molecules, interleukins, C-reactive proteins, chemokines, and cytokines. Methods for measuring such biomarkers are known in the art.

[0076] Fifth aspect, method of manufacture: In a fifth aspect, the present invention provides a method for producing the ASOs described herein. From the viewpoint of this disclosure, those skilled in the art can synthesize the ASOs of the present invention using known methods of oligonucleotide synthesis. The following methods have proven particularly advantageous for the efficient synthesis of the ASOs described herein: Step a: Step a includes the step of preparing a functionalized solid support represented by Formula I.

[0077] [ka]

[0078] (In the formula, L is preferably:

[0079] [ka]

[0080] A linker selected from, SS represents a solid support, preferably a polystyrene solid support or a porosity-controlled glass (CPG) solid support. Ar1 is a phenyl (Ph, C6H5) optionally substituted with a halogen, C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C4 alkoxy. X is selected from sulfur and oxygen. R1 represents hydrogen, or a protecting group for a hydroxyl group, preferably selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl. R2 is selected from methyl and phenyl. R3 is selected from hydrogen, acetyl, and trifluoroacetyl.

[0081] Solid supports suitable for oligonucleotide synthesis, such as polystyrene and CPG solid supports, are known in the art and are commercially available. For example, a solid support configured for reaction with linker L, i.e., one that presents a functional group on its surface for covalent bonding to L, such as a long-chain aminoalkyl group, is commercially available, for example, from ChemGenes (catalog number: N-5100-05). Furthermore, a general-purpose solid support containing linker L and configured for oligonucleotide synthesis is described, for example, in US7202264 and is commercially available, for example, from ChemGenes (catalog numbers: N-4000-05 for CPG and N-4000-03 for polystyrene solid supports).

[0082] The functional solid supports described above can be obtained by several methods (see, for example, Example 1). For example, step a may further include step a-1), which includes the step of preparing a solid support represented by formula III.

[0083] [ka]

[0084] (In the formula, L is:

[0085] [ka]

[0086] Selected from, SS represents a solid support, preferably a polystyrene solid support or a CPG solid support. R1 represents hydrogen, or a protecting group for a hydroxyl group, preferably selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl. R2 is selected from methyl and phenyl).

[0087] If present, following step a-1), the nucleoside construction block of formula (IIa-G) is reacted with a solid support according to formula III to obtain nucleotides bound to the support, and thereafter Step a-2) includes reacting a nucleoside construction block of formula (IIb-G) with a nucleotide bound to a support, thereby obtaining a dinucleotide bound to a support, which corresponds to the functionalized solid support described above.

[0088] Step b): The method for generating ASO involves the following series of reactions: A step of reacting a functionalized solid support of formula I with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a trinucleotide oligonucleotide bound to the support, followed by A step of reacting a trinucleotide oligonucleotide bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), thereby obtaining a tetranucleotide oligonucleotide bound to a support, followed by The process involves reacting four bases bound to a support with a nucleoside construction block of formula (IIa-G), (IIb-G), or (IIc-G), preferably (IIc-G), thereby obtaining a five-base oligonucleotide bound to a support, followed by The process involves reacting five bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), thereby obtaining a six-base oligonucleotide bound to a support, followed by The steps include: reacting a 6-base compound bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), thereby obtaining a 7-base oligonucleotide bound to a support; followed by The process involves reacting seven bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), thereby obtaining an eight-base oligonucleotide bound to a support, followed by The process involves reacting eight bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a nine-base oligonucleotide bound to a support, followed by The process involves reacting nine bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), thereby obtaining a 10-base oligonucleotide bound to a support, followed by The process involves reacting 10 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), thereby obtaining an 11-base oligonucleotide bound to a support, followed by The process involves reacting 11 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 12-base oligonucleotide bound to a support, followed by The process involves reacting 12 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), thereby obtaining a 13-base oligonucleotide bound to a support, followed by The process involves reacting 13 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 14-base oligonucleotide bound to a support, followed by The process involves reacting 14 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), thereby obtaining a 15-base oligonucleotide bound to a support, followed by The process involves reacting 15 bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), thereby obtaining a 16-base oligonucleotide bound to a support, followed by The process involves reacting 16 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), thereby obtaining a 17-base oligonucleotide bound to a support, followed by The process involves reacting 17 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), thereby obtaining an 18-base oligonucleotide containing SEQ ID NO: 1 bound to the support. The process of carrying out the above is further included as process b).

[0089] The nucleoside construction blocks of formulas (IIa-G), (IIa-T), (IIa-C), (IIa-A), (IIb-G), (IIb-T), (IIb-C), (IIb-A), (IIc-G), (IIc-T), (IIc-C), and (IIc-A) are given by the following formulas, respectively:

[0090] [ka]

[0091] Represented by (In the formula, Ar2 is optionally substituted with a halogen, C1-C6 alkyl, C3-C6 cycloalkyl or C1-C4 alkoxy phenyl (Ph, C6H5), R4 represents a hydroxyl group protecting group selected from triphenylmethyl, 4-monomethoxytrityl (MMTr), 4,4'-dimethoxytrityl (DMTr), 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from DMTr and MMTr.

[0092] Methods for reacting nucleoside construction blocks with solid supports containing functionalized solid supports, and with oligonucleotides (i.e., extended or nascent oligonucleotide chains) bound to the supports, are known in the art (see, for example, WO2017 / 198775; Li et al., Chem. Commun. 2017, 53, 541; Jahns et al., Nat. Commun. 2015, 6, 6317). Exemplary synthesis of ASO is also described in Example 2.

[0093] Based on their chemical structure, the nucleoside construction blocks of formulas (IIa-G), (IIa-T), (IIa-C), and (IIa-A) are also called phosphoramidite (PA) construction blocks, particularly 2'-MOE PA, and the nucleoside construction blocks of formulas (IIb-G), (IIb-T), (IIb-C), (IIb-A), (IIc-G), (IIc-T), (IIc-C), and (IIc-A) are also called oxazaphosphoridine construction blocks (OAP), particularly 2'-MOE OAP.

[0094] Generally, the process of reacting a nucleoside construction block (whether a PA or OAP construction block is used) involves the following steps: b-1) Deblocking, b-2) Coupling, b-3) Capping, b-4) Oxidation or sulfidation This includes, and each is publicly known in the art.

[0095] Deblocking (also called "detritylation"): Each synthetic cycle, i.e., the step of reacting a nucleoside construction block with a solid support or a nascent oligonucleotide chain bound to a solid support, is initiated by the removal of the protecting group of the 5'-hydroxyl group of the nucleoside or nascent oligonucleotide chain bound to the solid support (see R1 in formulas I, IIa-G, IIa-T, IIa-C, IIa-A, IIb-G, IIb-T, IIb-C, IIb-A, IIc-G, IIc-T, IIc-C, IIc-A, and III). As those skilled in the art will understand, when starting with a general-purpose solid support, i.e., a solid support that is not pre-loaded with nucleosides, such as a solid support of general formula III, deblocking refers to the deprotection of the hydroxyl group of the solid support in the first synthetic cycle so that the incoming nucleoside construction block can be bound to the solid support. Typically, the protecting group for the hydroxyl group is selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl. Methods of deblocking are known in the art and include deblocking using a 3% (w / w) dichloroacetic acid (DCA) solution in dichloromethane (DCM) or toluene, preferably in DCM.

[0096] Coupling: When the protecting group of the 5'-hydroxyl group is removed, the free 5'-OH of the nucleoside or nascent oligonucleotide chain (or the free hydroxyl of the solid support) can react with the next nucleoside, which is added as a monomeric nucleoside building block (phosphoamidite or oxazaphosphoridine building block), thereby forming a phosphite triester bond. The coupling reaction is usually carried out in the presence of activating compounds, such as 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 2-benzylthiotetrazole (BTT), and 4,5-dicyanoimidazole (DCI), and N-phenylimidazolium triflate (N-PhIT). Such activating compounds are commercially available. In the context of the present invention, N-PhIT has been found to be a particularly suitable activating compound when nucleoside construction blocks containing chiral auxiliary groups (e.g., oxazaphosphoridine construction blocks of formulas IIb-G, IIb-T, IIb-C, IIb-A, IIc-G, IIc-T, IIc-C, and IIc-A) are attached to form chirally controlled PS bonds. BTT has been found to be a particularly suitable activating compound when PAs without chiral auxiliary groups (e.g., PAs of formulas IIa-G, IIa-T, IIa-C, and IIa-A) are attached to form chirally uncontrolled PS bonds (also called sterically random PS bonds, i.e., a mixture of Rp and Sp PS bonds) or PO bonds (prochiral). As those skilled in the art will understand, commonly used phosphoramidite construction blocks (e.g., PA of formulas IIa-G, IIa-T, IIa-C, and IIa-A) are also chiral at phosphorus, but they do not bind stereospecifically, and therefore the resulting internucleotide PS bonds are not synthesized under chiral control; in other words, the resulting internucleotide PS bonds are not chirally controlled.

[0097] Capping: In the capping step, unreacted 5'-OH groups are blocked, otherwise these hydroxyl groups would react in the next cycle, thus leading to oligonucleotides containing internal deletions (i.e., one or more deficient nucleosides). Capping is usually carried out using a mixture of two capping reagents, commonly referred to as capping reagents A and B, which are applied separately to an oligonucleotide synthesis column containing the nascent oligonucleotide chain. For example, capping reagent A may contain a mixture of tetrahydrofuran (THF), lutidine, and acetic anhydride in a ratio of, for example, 8:1:1 (w / w / w), and capping reagent B may contain 16% (w / w) N-imidazole in THF. As those skilled in the art will understand, other capping reagents may be used in the present invention. For example, in capping reagent A, acetic anhydride may be substituted with trifluoroacetic anhydride.

[0098] Oxidation / Sulfurization: The phosphite triester bond has limited stability and may be converted to either a 5'-3'-phosphodiester bond (called oxidation; PO bond) or a 5'-3'-phosphorothioate bond (called sulfurization; PS bond). Oxidation to form the PO bond is usually carried out using an aqueous iodine solution in the presence of a weak base, such as pyridine, lutidine, or colidine. For example, oxidation may be carried out using a 0.02 M iodine solution in THF / pyridine / H2O in a ratio of preferably 7:2:1 (w / v / v / v).

[0099] A wide range of sulfiding reagents are known and suitable for the present invention. For example, sulfidation to form PS bonds may be carried out using 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazol-5-thione (DDTT; commercially available as sulfiding reagent II; Glen Research), for example, preferably using a 0.1 M solution of DDTT in a mixture of dry pyridine / ACN in a ratio of 9:1 (v / v).

[0100] In the context of this invention, sulfidation (to form PS bonds) is preferred over oxidation (to form PO bonds). Therefore, synthesis of complete PS ASO is preferred. However, including PO bonds at specific positions may be advantageous in reducing stimulation of the innate immune system.

[0101] As those skilled in the art will understand, the order of steps b-1 (deblocking), b-2 (coupling), b-3 (capping), and b-4 (oxidation or sulfidation, preferably sulfidation) may vary to some extent, that is, steps b-1) to b-4) may be carried out in any of the following order: b-1), b-2), b-3), b-4), or b-1), b-2), b-4), b-3), or b-2), b-3), b-4), b-1), or b-2), b-4), b-3), b-1). In the context of the present invention, in particular, when sulfidation is carried out in step b-4) in a preferred embodiment, steps b-1 to b-4 are preferably carried out in the order b-1 (deblocking), b-2 (coupling), b-3 (capping), and b-4 (oxidation).

[0102] Once all synthesis cycles are complete, i.e., when the ASO reaches its desired chain length, R1 may optionally be cleaved from the oligonucleotide bound to the support, i.e., further deblocking steps may be performed while the oligonucleotide remains bound to the solid support. Cleaving R1 is advantageous, especially on a large scale, when the ASO is purified by anion exchange high-performance liquid chromatography (AEX-HPLC). In this case, a single HPLC purification is usually sufficient to obtain an oligonucleotide of sufficient purity for therapeutic application, which is measured, for example, by AEX-HPLC or reverse-phase HPLC (RP-HPLC).

[0103] However, at smaller scales, it is often advantageous for the oligonucleotide to remain bound to the solid support without cleaving R1. In this case, the first purification is usually performed by RP-HPLC, taking advantage of the presence of a lipophilic protecting group, such as a DMT-protecting group, on the 5'-OH of the ASO, which is absent in the cleaved oligonucleotide (because the unreacted 5'-OH group is capped; see step b-3), thereby facilitating the separation of the ASO of the desired length from the cleaved oligonucleotide (imperfect sequence). After R1 is cleaved from the ASO obtained after the first purification (because R1 is cleaved only after the ASO is released from the solid support and in the unbound form; see step d below), a second purification is then performed by either RP-HPLC or AEX-HPLC.

[0104] As those skilled in the art will understand, the purification of oligonucleotides by either RP-HPLC or AEX-HPLC is carried out after the oligonucleotides have been released from the solid support (thereby simultaneously cleaving the extracyclic amino group and the protecting groups of the phosphate skeleton, including both PO and PS bonds; see step d below). Thus, the method described herein includes step d), during which the oligonucleotide bound to the support (e.g., the 18-base oligonucleotide bound to the support obtained after step b) is cleaved from the solid support, thereby yielding the oligonucleotide. Cleavage of oligonucleotides from solid support is known in the art and is achieved under basic conditions. As those skilled in the art will understand, cleavage of oligonucleotides from solid support under basic conditions also results in the cleavage of the extracyclic amino group and the protecting groups of the phosphate group (PO and PS). For example, cleavage from the solid support and cleavage of the protecting groups may be carried out using aqueous ammonia, for example, at 55°C for 24 hours. Thus, the oligonucleotide is released into solution, thereby obtained in an unbound form. The ASO (oligonucleotide) thus obtained may be used without further purification, or it may be further purified using, for example, AEX-HPLC or RP-HPLC, as described earlier in the context of the optional step c).

[0105] Methods for purifying oligonucleotides, including AEX-HPLC and RP-HPLC, and analytical methods for determining the purity of oligonucleotides, including liquid chromatography-mass spectrometry (LC-MS), are known in the art.

[0106] A sixth aspect, ASO obtained by the production method described herein: In a sixth aspect, the present invention relates to an ASO represented by Sequence ID No. 1, obtained by the previously described method (fifth aspect). As is understood in the art, the coupling of chiral oxazaphosphoridine constructs occurs with stereochemical inversion at the chiral phosphorus, i.e., when a 2'-MOE oxazaphosphoridine (MOE OAP) construct having an Rp structure (see formulas IIb-A, IIb-C, IIb-G, IIb-A) is sulfurized, it produces a PS bond having an Sp structure. Consistent with the first aspect described earlier, the ASO obtained by the previously described method (fifth aspect) has improved metabolic stability compared to current therapeutic ASOs, particularly nusinersen. In particular, at least 95% of the ASOs obtained by the previously described method, i.e., 95% to 100%, preferably 98% to 100%, maintain their full length after incubation in mouse liver lysate for at least 5 days, as measured by LC-MS.

[0107] Preferably, as measured by LC-MS, there is an increase of at least 5 percentage points in full-length ASO compared to nusinersen after incubation in mouse liver lysate for at least 5 days.

[0108] It is understood that all definitions and characteristics of ASO described in the first embodiment are equally applicable to this sixth embodiment.

[0109] The present invention will be better understood by referring to the following figures. [Brief explanation of the drawing]

[0110] [Figure 1] This figure shows the structures of exemplary ASOs of the present invention (ASO3, 4, 5, 6, and 10) having at least one 3'-terminal PS bond with an Sp structure, and comparative ASOs (ASO2, 7, 8, and 9) having a 3'-terminal PS bond with an Rp structure. All ASOs are represented by the sequence number TCACTTTCATAATGCTGG (18 bases). All ASOs have 2'-O-MOE sugar modifications at each position, and all internucleotide bonds are PS. C is 5mC in all examples. ASO2 and 3 have homogeneous stereochemistry, all Rp PS and all Sp PS, respectively. ASO4, 5, and 6 have one, two, and three highly chiral Sp PS internucleotide bonds at the 3' end, respectively, and the rest of the sequence is sterically random (i.e., not chiralized, i.e., a mixture of Rp and Sp PS bonds). ASO7, 8, and 9 have one, two, and three RpPS internucleotide bonds at their 3' ends, respectively, with the rest of the sequence being sterically random. ASO10 is a perfectly sterically pure RpPS ASO with a single internucleotide SpPS bond at its 3' end. B. Figures of the analysis of ASO1-3. UV chromatograms of ASO1 (nusinersene, not chiralized), ASO2 (complete PS, total Rp), and ASO3 (complete PS, total Sp). Thus, in ASO2 and ASO3, all PS bonds are chiralized. C. UV chromatograms of ASO2 and ASO3 simultaneously injected in various ratios (1:2, 1:1, 2:1) confirming that PS stereochemistry has an effect on the overall lipophilicity of oligonucleotides and demonstrating the success of the synthesis of unique diastereomers. Synthesis, including precision analysis and purification of ASO, is described in Example 2. Tm measurement is described in Example 4. The simultaneous injection of ASO2 and ASO3 is described in Example 5. [Figure 2]A. This graph shows the time-dependent metabolic stability of ASO1 (nusinersen), ASO4-6 (invention), and ASO7-9 (comparison). The relative amount of ASO that maintains its full length (i.e., 18 nucleotides) is plotted as a function of incubation time. B. This graph plots the relative amount of remaining full-length ASO as a function of incubation time. The percentages of full-length ASO, as well as their n-1 and n-2 metabolites (i.e., 17 nucleotides and 16 nucleotides, respectively; cleaved at the 3' end) detected in the sample are plotted against the number of incubation days. ASO4, 5, and 6 (having one, two, and three sterically pure SpPS bonds at the 3' end, respectively) remained unchanged after 5 days of incubation in mouse liver homogenate, indicating that a single SpPS bond at the 3' end is sufficient to improve ASO stability. ASO7, 8, and 9 (each possessing one, two, and three sterically pure RpPS bonds at their 3' ends) are metabolized faster than sterically random ASO1 (i.e., less full-length ASO after 5 days of incubation). Samples were analyzed by LC-MS after sample preparation using a combination of proteinase K digestion and solid-phase extraction (SPE). Samples were prepared and analyzed blindly and then opened up at the end of the experiment. Error bars represent the standard deviation of three biological replicates. Further details of the experiment are shown in Example 6. [Figure 3] Images showing in vitro (cellular) evaluation of splice switch activity in SMA patient fibroblasts of ASO1 (comparison; complete PS, not chiralized; nusinersen), ASO2 (comparison; complete PS, total Rp), and ASO3 (invention; complete PS, total Rp). Scrape loading delivery (24 hours) of 0, 8, 40, or 200 nM ASO1, ASO2, and ASO3 to cells. Images of agarose gels quantifying the amounts of full-length SMN2 premRNA with exon 7 (SMN2 FL) and SMN2 premRNA without exon 7 (SMN2d7). [Figure 4]These are 31P NMR spectra of stereochemically randomized (left) and chirally controlled SpPS dinucleotides (right). The SpPS dinucleotide (5'-A(Sp)C-3') exhibits a single peak at 55.36 ppm, indicating that the PS bond was formed with over 99% stereoselectivity. [Figure 5] This figure shows the chemical structure of ASO3 (perfect PS; all PS bonds have an Sp structure (i.e., all PS bonds are chiral)). [Figure 6] This figure shows the chemical structure of ASO4 (complete PS; the 3'-terminal PS bond has an Sp structure, and the other PS bonds are not chiralized, i.e., a mixture of Rp and Sp PS bonds). [Modes for carrying out the invention] [Examples]

[0111] To further illustrate the present invention, the following embodiments are provided in Example 6. These embodiments are provided without intent to limit the scope of the present invention.

[0112] (Example 1) Synthesis of Functionalized CPG Solid Supports Functionalized CPG solid supports pre-loaded with sterically pure phosphorothioate dinucleotides (GG; PS-linked with an Sp structure) were prepared using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The second nucleotide was incorporated using Rp G MOE OAP (formula IIb-G), which provides a sterically pure Sp-PS linkage. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0113] After the synthesis was complete, the surface loading of the solid support was determined using quantitative trityl analysis according to the following method: Two mg of a dried functionalized solid support was placed in a 10 ml volumetric flask, and a detritylation (deblocking) reagent was added to the flask to the mark. The flask was mixed, and an orange 4,4'-dimethoxytrityl (DMTr) cation was immediately formed. The absorbance spectrum from 400 to 600 nm was measured using a UV / visible spectrophotometer. The absorbance at the peak value between 503 and 505 nm was recorded. The loading of the support was calculated using the following formula:

[0114]

number

[0115] In the formula, loading is micromoles per gram, solution volume is in milliliters, support mass is in milligrams, and E is the extinction coefficient of the 4,4'-dimethoxytrityl group (76 ml cm⁻¹ μmol⁻¹). The surface loading was found to be 9.9 μmol / g.

[0116] (Example 2) Synthesis of ASO according to the present invention An ASO having the sequence TCACTTTCATAATGCTGG (SEQ ID NO: 1) and 2'-MOE sugar modifications at each position was prepared using a MerMade12 synthesizer at Bio Automation (BioAutomation Corp., Irving, TX, USA) with a CPG 500Å unylinker support (81.2 μmol / g, ChemGenes, Wilmington, MA, USA). Chemicals for oligonucleotide synthesis were purchased from Sigma Aldrich (Steinheim, Germany), Fluorochem (Hadfield, UK), and TCI (Eschborn, Germany). The deblocking reagent was 3% dichloroacetic acid in dichloromethane (DCM). Conventional 2'-O-MOE-2-cyanoethyl-N,N-diisopropyl-phosphoramidite (MOE PA; see formulas IIa-G, IIa-T, IIa-C, IIa-A) was obtained from Thermo Fisher Scientific (Waltham, MA, USA) and used as a 0.1 M solution in dry acetonitrile (ACN). MOE PA was incorporated using 0.24 M 5-(benzylthio)-1H-tetrazole (BTT; Biosolve BV, Valkenswaard, Netherlands) in dry acetonitrile (ACN) as an activator. Fully protected, sterically pure 2'-O-MOE oxazaphosphoridines (MOE OAP; see formulas IIb-G, IIb-T, IIb-C, IIb-A, IIc-G, IIc-T, IIc-C, IIc-A) were synthesized according to the previously described procedure (WO 2017 / 198775) and used as a 0.2 M solution in dry ACN. MOE OAP was incorporated using 1.4 M N-phenylimidazolium triflate (N-PhIT) in dry ACN as an activator. The capping process was carried out using capping reagent A (THF / lutidine / acetic anhydride, 8:1:1) and capping reagent B (16% N-imidazole / THF).Sulfidation was performed using a 0.1 M solution of 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazol-5-thione (DDTT; Sulfidation Reagent II; Glen Research, Virginia) in dried pyridine / ACN (9:1). Oxidation was performed using a 0.02 M iodine solution in THF / pyridine / H2O (70:20:10, w / v / v / v).

[0117] ASO1 (comparison; complete PS, no chiral controlled PS bonding; sterically random): ASO1 was synthesized using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The remaining nucleotides were also incorporated using their respective MOE PAs. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0118] The following conditions were used for the synthesis of ASO-1: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling: MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Oxidation / Sulfidation: The oxidation conditions (used only for the first cycle) were as follows: oxidizing reagent injection volume 2000 μl; contact time 480 s. The sulfidation conditions (used throughout all subsequent cycles) were as follows: DDTT injection volume 2000 μl; contact time 600 s.

[0119] ASO2 (Comparison, Full PS, Full Rp): ASO2 was generated using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The remaining nucleotides were incorporated using Sp MOE OAP (formula IIc-G, IIc-T, IIc-C, IIc-A), which yielded sterically pure Rp-PS bonds. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0120] The following conditions were used for the synthesis of ASO2: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling (used only in the first cycle): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Coupling (used in all subsequent cycles): Sp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Oxidation / Sulfidation: The oxidation conditions (used only for the first cycle) were as follows: oxidizing reagent injection volume 2000 μl; contact time 480 s. The sulfidation conditions (used for all subsequent cycles) were as follows: DDTT injection volume 2000 μl; contact time 600 s.

[0121] ASO3 (Invention; Complete PS, All Sp): ASO3 was generated using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using their respective Rp MOE OAPs (formulas IIb-G, IIb-T, IIb-C, IIb-A) which provided sterically pure SpPS bonds. The synthesis cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0122] The following conditions were used for the synthesis of ASO3: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling: Rp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings with a contact time of 360 s for each. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Sulfidation: DDTT injection volume 2000μl; contact time 600s.

[0123] ASO4 (Invention; complete PS; the 3'-terminal PS bond has an Sp structure, and other PS bonds are not chiralized): ASO4 was generated using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using the respective MOE PAs (Formulas IIa-G, IIa-T, IIa-C, IIa-A) which resulted in sterically random bonding (i.e., not chiral). The synthesis cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0124] The following conditions were used for the synthesis of ASO4: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling: MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Sulfidation: DDTT injection volume 2000μl; contact time 600s.

[0125] ASO5 (Invention; complete PS; the last two 3' terminal PS bonds have an Sp structure, and the other PS bonds are not chiralized): ASO5 was generated using 250 mg of the functionalized solid support synthesized in Example 1. The third nucleotide at the 3' end of ASO5 was incorporated using Rp T MOE OAP (formula IIb-T), which provides a second sterically pure SpPS bond. The remaining nucleotides were incorporated using their respective MOE PAs (formulas IIa-G, IIa-T, IIa-C, IIa-A), which provide sterically random bonding. The synthesis cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0126] The following conditions were used for the synthesis of ASO5: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling (used only in the first cycle): Rp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Coupling: MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Sulfidation: DDTT injection volume 2000μl; contact time 600s.

[0127] ASO6 (Invention; complete PS; the last three 3' terminal PS bonds have an Sp structure, and the other PS bonds are not chiralized): ASO6 was generated using 250 mg of the functionalized solid support synthesized in Example 1. The second nucleotide at the 3' end was incorporated using Rp G MOE OAP (Formula IIb-G), which provides the first sterically pure SpPS bond. The third nucleotide at the 3' end was incorporated using Rp T MOE OAP (Formula IIb-T), which provides the second sterically pure SpPS bond. The fourth nucleotide at the 3' end was incorporated using Rp C MOE OAP (Formula IIb-C), which provides the third sterically pure SpPS bond. The remaining nucleotides were incorporated using their respective MOE PAs (Formulas IIa-G, IIa-T, IIa-C, IIa-A), which provide sterically random bonding. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0128] The following conditions were used for the synthesis of ASO6: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling (used only for the first and second cycles): Rp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Coupling: MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Sulfidation: DDTT injection volume 2000μl; contact time 600s.

[0129] ASO7 (comparison; complete PS, 3' terminal PS bond has an Rp structure, other PS bonds are not chiralized): ASO7 was synthesized using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The second nucleotide at the 3' end was incorporated using Sp G MOE OAP (formula IIc-G) to obtain the first sterically pure Rp-PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (formulas IIa-G, IIa-T, IIa-C, IIa-A) to obtain sterically random bonds. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0130] The following conditions were used for the synthesis of ASO7: Deblocking: Deblocking reagent injection volume 2500 μl; followed by three deblocking steps, each with a contact time of 60 s. Coupling (used only in the first cycle): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Coupling (used only in the second cycle): Sp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Coupling (used in all subsequent cycles): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Oxidation or sulfidation: The oxidation conditions (used only for the first cycle) were as follows: oxidizing reagent injection volume 2000 μl; contact time 480 s. The sulfidation conditions (used for all subsequent cycles) were as follows: DDTT injection volume 2000 μl; contact time 600 s.

[0131] ASO8 (comparison; complete PS, the last two 3' terminal PS bonds have an Rp structure, and the other PS bonds are not chirally controlled): ASO8 was synthesized using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (Formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The second nucleotide at the 3' end was incorporated using Sp G MOE OAP (Formula IIc-G), which provided the first sterically pure Rp-PS bond. The third nucleotide at the 3' end was incorporated using Sp T MOE OAP (Formula IIc-T), which provided the second sterically pure Rp-PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (Formula IIa-G, IIa-T, IIa-C, IIa-A), which provided sterically random bonding. The synthetic cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0132] The following conditions were used for the synthesis of ASO-8: Deblocking: Deblocking reagent injection volume 2500 μl; followed by three deblocking steps, each with a contact time of 60 s. Coupling (used only in the first cycle): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Coupling (used only for the second and third cycles): Sp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Coupling (used in all subsequent cycles): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Oxidation or sulfidation: The oxidation conditions (used only for the first cycle) were as follows: oxidizing reagent injection volume 2000 μl; contact time 480 s.

[0133] ASO9 (comparison; complete PS, the last three 3' terminal PS bonds have an Rp structure, and the other PS bonds are not chirally controlled): ASO9 was generated using 250 mg of UnyLinker CPG 500 Å support. The first nucleotide at the 3' end was incorporated using MOE G PA (formula IIa-G) in a cycle encompassing the following steps: deblocking, coupling, capping, and oxidation. The second nucleotide at the 3' end was incorporated using Sp G MOE OAP (formula IIc-G) to provide the first sterically pure Rp-PS bond. The third nucleotide at the 3' end was incorporated using Sp T MOE OAP (formula IIc-T) to provide the second sterically pure Rp-PS bond. The fourth nucleotide at the 3' end was incorporated using Sp C MOE OAP (formula IIc-C) to provide the third sterically pure Rp-PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (formulas IIa-G, IIa-T, IIa-C, IIa-A) to provide sterically random bonding. The synthesis cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0134] The following conditions were used for the synthesis of ASO-9: Deblocking: Deblocking reagent injection volume 2500 μl; followed by three deblocking steps, each with a contact time of 60 s. Coupling (used only in the first cycle): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Coupling (used only for the 2nd, 3rd, and 4th cycles): Sp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings, each with a contact time of 360 s. Coupling (used in all subsequent cycles): MOE PA injection volume 600 μl, BTT injection volume 1200 μl; contact time 360 ​​s. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Oxidation / Sulfidation: The oxidation conditions (used only for the first cycle) were as follows: oxidizing reagent injection volume 2000 μl; contact time 480 s. The sulfidation conditions (used for all subsequent cycles) were as follows: DDTT injection volume 2000 μl; contact time 600 s.

[0135] ASO10 (the present invention; complete PS, 3' terminal PS bonded Sp structure, remaining PS bonded Rp structure): ASO10 was generated using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using Sp MOE OAPs (formulas IIc-G, IIc-T, IIc-C, IIc-A) that provided sterically pure Rp-PS bonding. The synthesis cycle consisted of the following steps in this order: deblocking, coupling, sulfidation, and capping.

[0136] The following conditions were used for the synthesis of ASO10: Deblocking: Deblocking reagent injection volume 2500 μl; contact time 60 s. Coupling: Sp MOE OAP injection volume 500 μl, N-PhIT injection volume 1000 μl; two subsequent couplings with a contact time of 360 s for each. Capping: Cap A injection volume 1400 μl, Cap B injection volume 1400 μl; contact time 90 s. Sulfidation: DDTT injection volume 2000μl; contact time 600s.

[0137] Cleavage and deprotection from solid support: After the synthesis was complete, ASO was cleaved from the solid support, and the protecting group was removed using 300 μl of aqueous ammonia at 55°C for 24 hours. The reaction was cooled to room temperature, the solid support was filtered, and washed with 400 μl of ethanol (EtOH) and H2O 1:1 (v / v). The filtrate was concentrated to a dry state, and the residue was dissolved in 200 μl of water.

[0138] Purification: Oligonucleotides were purified at 65°C by RP-HPLC on an Agilent 1200 series HPLC equipped with a Waters Xbridge OST C-18 column, 10 × 50 mm, 2.5 μm. Electrophoresis buffer for HPLC purification: Buffer A - 0.1 M triethylammonium acetate, Buffer B - acetonitrile, pH 8.0. Gradient for DMT-ON purification (i.e., before cleavage of the lipophilic DMTr protecting group): 5–80% buffer B over 8 minutes. The fractions were pooled, dried in a SpeedVac, and treated with 40% acetic acid at room temperature for 15 minutes for DMT deprotection. After drying in a SpeedVac, the oligonucleotides were dissolved in H2O and subjected to a second DMT-OFF purification in RP-HPLC. Gradient for DMT-OFF purification: 5–35% buffer B over 6 minutes. The fractions containing the product were collected and dried in a SpeedVac. ASO was analyzed at 65°C by LC-MS (Agilent 1200 / 6130 system) using a Waters Acquity OST C-18 column, 2.1 × 50 mm, 1.7 μm. Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: 10-50% Buffer B over 15 minutes; Flow rate: 0.3 ml / min.

[0139] (Example 3) Stereochemical purity of chiral-controlled PS bonds To confirm the steric purity of the PS MOE dinucleotide, 31P NMR analysis was performed on dinucleotides having a single PS bond as the Sp structure and a sterically random variant (a PS bond not controlled by chirality) with the sequence 5'-AC-3'. The dinucleotides were synthesized on a commercially available CPG 500 Å unylinker support. Detritylation (removal of 5'-DMT) of the dinucleotide bound to the support was carried out as described in Example 1, and the "DMT-off" dimer was released from the solid support by treatment with concentrated ammonium hydroxide at 55°C for 24 hours. In the subsequent step, the obtained product was filtered, and the filtrate (containing the unbound dinucleotide) was evaporated under reduced pressure. The remaining residue was then dissolved in heavy water (0.6 mL) and transferred to an NMR tube for analysis. The 31P NMR spectrum of the sterically random dinucleotide was measured first. Clear separation of two phosphorothioate diastereomer signals was observed (see Figure 4). The RpPS and SpPS structures of the phosphorothioate bond were based on a literature report [VT Ravikumar, DL Cole, Nucleosides, Nucleotides & Nucleic Acids (2003), 22, 1639]. Therefore, the high-field peak in the 31P NMR spectrum was assigned to the SpPS structure, and the low-field peak was designated as the RpPS structure.

[0140] The 31P NMR spectrum of the sterically pure SpPS dinucleotide showed a single peak at 55.36 ppm, which was assigned to the SpPS structure. This result indicates that the PS bond was formed with more than 99% stereoselectivity.

[0141] Furthermore, the steric purity of couplings of various MOE OAPs (including dinucleotides containing G) at the 5' end of sterically pure PS ASOs targeting ferrochelatase premRNA was previously demonstrated by Li et al. [Chem. Commun. 2017, 53, 541].

[0142] (Example 4) Thermal denaturation research (hybridization affinity) The melting temperatures (Tm) of ASO1, 2, and 3 were measured along with complementary PO RNA strands using a CARY 300 (Agilent) thermocontroller. ASO1-3 and their respective counterparts (unmodified RNA, complete PO) were combined to obtain an equimolar concentration of 1 μM in phosphate-buffered saline (5 mM Na2HPO4, 5 mM NaH2PO4, 100 mM NaCl, 0.1 mM EDTA). Absorption at 260 nm was measured in a 160 μl quartz cuvette. The temperature gradient was set to 0.5 K / min in the range of 20°C to 95°C. Absorbance readings were taken every 30 seconds. Each flow was repeated three times. Holding times were set to 5 minutes at both 95°C and 20°C to ensure thermal equilibrium. The melting profiles were fitted to sigmoid curves. Tm was calculated from the maximum value of the first derivative of the three melting curves. The following results were obtained (see Figure 1): ASO1: 63.0℃ ASO2: 63.4℃ ASO3: 55.3℃

[0143] Therefore, the Tm measured for ASO2 (18 bases, complete PS, total Rp) was 8.1°C higher than the Tm measured for ASO3 (18 bases, complete PS, total Sp).

[0144] Furthermore, compared to ASO1 (18-base complete PS, not chiralized), ASO2 exhibited a Tm approximately 0.2°C higher per PS bond. Conversely, the Tm of ASO3 was approximately 0.3°C lower per PS bond than that of sterically random ASO1. This data demonstrates the advantages of having several PS bonds (other than the 3' terminal PS bond, as is clearer above) as an Rp structure (e.g., ASO10) or not chiralized (e.g., ASO4, 5, and 6), as shown in the preferred embodiment.

[0145] (Example 5) Simultaneous LC-MS injection analysis of ASO2 and ASO3 with completely high steric purity. ASO2 and ASO3 were prepared at 10 μM concentrations. Subsequently, three distinct solutions were prepared: 5 μL ASO2 + 10 μL ASO3, 5 μL ASO2 + 5 μL ASO3, and 10 μL ASO2 + 5 μL ASO3. The mixtures were then analyzed by LC-MS (Agilent 1200 / 6130 system) on a Waters Acquity OST C-18 column, 2.1 × 50 mM, 1.7 μm, at 65°C. Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: 10-50% Buffer B over 20 minutes; Flow rate: 0.3 ml / min, injection volume 5 μL. The resulting chromatogram showed two distinct peaks, which provided further evidence of the stereochemical purity of the synthesized ASO. The corresponding UV traces are shown in Figure 1C.

[0146] (Example 6) Metabolic stability in mouse whole tissue homogenates The homogenization buffer consisted of 100 mM Tris-HCl and 1 mM magnesium acetate (pH 8.0, adjusted with HCl). Postmortem liver samples were collected at 19 weeks after carbon dioxide euthanasia and cervical dislocation of wild-type black 6 mice (C57BL / 6J). 217 mg of untreated mouse liver was placed in an Eppendorf tube, and 434 μl of homogenization buffer was added. These were mechanically homogenized using a TissueLyser II (QIAGEN). The resulting homogenate was further diluted with homogenization buffer to prepare several 200 μL volumes of liver homogenate. Based on an optimized ratio, 5 μL of liver homogenate (2.5% of the total volume) was added to 195 μL of buffer solution. To determine the shortmer formation rate of nusinersen, liver homogenate (200 μL) and ASO (25 μL 180 μM) were incubated at 37°C with gentle shaking (400 rpm) for 0, 24, 72, and 120 h using an Eppendorf Thermomixer-R (Hauppauge, NY, USA).

[0147] The incubation was quenched by adding 225 μl of incubation mixture to 62 μl of MasterPure tissue lysis solution (Epicentre, WI, USA) and 6 μl of proteinase K (20 mg / ml, Roche). Proteinase K digestion was carried out at 55°C for 1 hour. The sample was then treated by solid-phase extraction (SPE).

[0148] Following proteinase K digestion, SPE was performed using the Phenomenex Clarity OTX kit (Phenomenex, Torrance, CA, USA). Tissue samples were mixed with 600 μL of Clarity OTX buffer, vortexed for 5 minutes, and incubated at room temperature for a further 5 minutes. The SPE cartridge was conditioned with 1 mL of methanol, followed by 1 mL of equilibration buffer. The tissue samples were loaded onto the column. The cartridge was washed with 3 × 1 mL of equilibration buffer and 3 × 1 mL of washing buffer, and the analyte was eluted with 2 × 0.5 mL of elution buffer. The collected solution was evaporated under vacuum for 1 hour and directly analyzed by LC-MS (Agilent 1200 / 6130 system) at 65°C using a Waters Acquity OST C-18 column, 2.1 × 50 mm, 1.7 μm. Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: 10-50% buffer B over 15 minutes; flow rate: 0.3 ml / min, injection volume: 10 μL. Mass spectrometry was performed using an Agilent 6130 single quadrupole mass spectrometer equipped with an API-ES ion source. The instrument was operated in negative ion mode, with a scan range of m / z 500-m / z 1000.

[0149] The data show that ASO4-6, i.e., ASOs with at least one 3' terminal PS having an Sp structure, exhibit greater metabolic stability than their completely sterically random control (ASO1) and ASOs with 3' terminal PS bonds having an Rp structure (ASO7-9). Metabolic stability in mouse whole tissue homogenates reflects metabolic stability in vivo. The data are shown in Table 1 and Figure 2 below.

[0150] [Table 1]

[0151] (Example 7) Splicing correction assay in SMA patient fibroblasts Oligonucleotides were resuspended in nuclease-free water. Fibroblasts derived from type II SMA patients (GM03813; Coriell Institute for Medical Research) were maintained in standard medium (DMEM + GlutaMax, 10% fetal bovine serum (FBS)). When the cells reached approximately 80% confluence in a T75 flask, they were washed once with PBS and trypsinized with 2 mL of trypsin-EDTA. After incubation at 37°C for 3 minutes, 3 mL of DMEM-10% FBS was added and the cells were counted. The cells were seeded into 6-well plates at 120,000 or 150,000 cells per well and incubated overnight at 37°C and 5% CO2.

[0152] For scrape delivery, ASO was added to 1 mL of fresh medium (at the concentration shown in the figure). Cells were scraped off with a cell scraper S (TPP), resuspended by pipetting, transferred to an Eppendorf tube, and centrifuged at 250 RCF for 5 minutes. The cells were resuspended in 1 mL of fresh medium, transferred to a new 6-well plate, and incubated for a further 48 hours.

[0153] After 48 hours, RNA was extracted using 500 μL of Trizole reagent. 100 μL of cold chloroform was added to the sample, vortexed completely, and centrifuged at 12,000xg for 15 minutes at 4°C. The aqueous phase was collected and an equal volume of anhydrous ethanol was added. The solution was mixed by pipetting up and down, transferred to a Zymo-Spin IC column, rotated, and the flow-through was discarded. 400 μL of RNA Prep buffer was added, rotated, and the flow-through was discarded. Finally, 700 μL of RNA wash buffer was added, rotated for 2 minutes, and the flow-through was discarded. The sample was eluted with 15 μL of nuclease-free solution, and the RNA concentration was measured by NanoDrop. Reverse transcription was performed using 1 μg of total RNA with the Lunascript RT SuperMix kit. The reverse transcription reaction was performed using a thermocycler. RT program: Step 1: 25°C (2 min), 55°C (10 min), 95°C (1 min). In the gel-based splice assay, 50 ng of cDNA was PCR-amplified using an SMN2 primer spanning exon 7 (see Table 2). PCR program: Step 1: 95°C (2 min); Step 2: 95°C (30 s); Step 3: 65°C (30 s); Step 4: 74°C (30 s); Step 5: Repeat steps 2-4 for 25 cycles; Step 6: 74°C (5 min). The PCR product was loaded onto a 2% agarose gel in TAE buffer and 1 / 10,000 GelRed nucleic acid stain (41003, Chemie Brunschwig) and electrophoresed at 100V for 1 hour.

[0154] [Table 2]

[0155] The results are shown in Figure 3. The data showed that ASO2 (invention; complete PS, total Rp) had superior activity in cells compared to ASO3 (comparison; complete PS, total Sp). As those skilled in the art will understand, the increased in vivo metabolic stability of the ASOs of the invention (e.g., ASO3, 4, 5, 6, 10; see Example 6; Figure 2) affects the potency of the ASOs in vivo, but in cell experiments, since the ASOs are not exposed to the harsh conditions encountered in tissues, it is not expected that this will provide further advantages in cell experiments.

Claims

1. An antisense oligonucleotide containing Sequence ID No. 1, or a pharmaceutically acceptable salt thereof, Each of these nucleotides has a 2'-O-methoxyethyl sugar modification and a chiral internucleotide phosphorothioate bond with an Sp structure at its 3' end. Antisense oligonucleotides, or pharmaceutically acceptable salts thereof.

2. An antisense oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising only a phosphorothioate bond.

3. An antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, further comprising at least one or at least two chiral nucleotide phosphorothioate bonds having an Sp structure, continuous with the 3'-terminal chiral nucleotide phosphorothioate bond.

4. All cytosine residues are 5-methylcytosine, Antisense oligonucleotides It contains one chiral internucleotide phosphorothioate bond with an Sp structure at its 3' end, but all other internucleotide bonds are either uncontrolled chiral phosphorothioate bonds or It contains two consecutive chiral internucleotide phosphorothioate bonds with an Sp structure at the 3' end, but all other internucleotide bonds are either uncontrolled chiral phosphorothioate bonds or It contains three consecutive chiral internucleotide phosphorothioate bonds with an Sp structure at the 3' end, but all other internucleotide bonds are either uncontrolled chiral phosphorothioate bonds or It contains one chiral internucleotide phosphorothioate bond with an Sp structure at its 3' end, but all other internucleotide bonds are chiral-controlled phosphorothioate bonds with an Rp structure. The antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1.

5. A composition comprising a plurality of antisense oligonucleotides and / or pharmaceutically acceptable salts thereof according to any one of claims 1 to 4.

6. Pharmacologically acceptable carriers or diluents, and An antisense oligonucleotide and / or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, Any of the plurality of antisense oligonucleotides as defined in claim 5 and / or any pharmaceutically acceptable salts thereof A pharmaceutical composition containing the following:

7. A pharmaceutically acceptable carrier or diluent, and one of the following in lyophilized form: An antisense oligonucleotide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, Composition according to claim 5 A kit that includes this.

8. For use as a medicine, An antisense oligonucleotide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, The composition according to claim 5, or The pharmaceutical composition according to claim 6, or The kit according to claim 7.

9. For use in the treatment of spinal muscular atrophy, An antisense oligonucleotide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, The composition according to claim 5, or The pharmaceutical composition according to claim 6, or The kit according to claim 7.

10. A method for promoting the inclusion of exon 7 in an SMN2 transcript in a cell, tissue, or organ, wherein the cell, tissue, or organ is subjected in vitro to an antisense oligonucleotide according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or the composition according to claim 5, Pharmaceutical composition according to claim 6 A method that includes the step of bringing into contact with.

11. The following steps: a) A step of preparing a functionalized solid support represented by formula I, 【Chemistry 1】 (In the formula, L is preferably: 【Chemistry 2】 A linker selected from, SS stands for solid support, Ar 1 Halogen and C are optional choices. 1 ~C 6 Alkyl, C 3 ~C 6 Cycloalkyl or C 1 ~C 4 It is a phenyl substituted with an alkoxy, X is selected from sulfur and oxygen. R 1 This represents hydrogen, or a protecting group for a hydroxyl group, selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl. R 2 is selected from methyl and phenyl, R 3 (Selected from hydrogen, acetyl, and trifluoroacetyl) b) The following series of reactions: The process involves reacting a functionalized solid support of formula I with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a trinucleotide oligonucleotide bound to the support, followed by A step of reacting a trinucleotide oligonucleotide bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a tetranucleotide bound to a support, followed by The process involves reacting four bases bound to a support with a nucleoside construction block of formula (IIa-G), (IIb-G), or (IIc-G) to obtain a five-base oligonucleotide bound to a support, followed by... The process involves reacting a 5-base oligonucleotide bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 6-base oligonucleotide bound to a support, followed by... The process involves reacting a 6-base molecule bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 7-base oligonucleotide bound to a support, followed by... The process involves reacting seven bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain an eight-base oligonucleotide bound to the support, followed by... The process involves reacting eight bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a nine-base oligonucleotide bound to the support, followed by... The process involves reacting nine bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 10-base oligonucleotide bound to a support, followed by... The process involves reacting 10 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain an 11-base oligonucleotide bound to a support, followed by... The process involves reacting 11 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 12-base oligonucleotide bound to a support, followed by... The process involves reacting 12 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 13-base oligonucleotide bound to the support, followed by... The process involves reacting 13 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 14-base oligonucleotide bound to the support, followed by... The process involves reacting 14 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a 15-base oligonucleotide bound to a support, followed by... The process involves reacting 15 bases bound to a support with a nucleoside construction block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 16-base oligonucleotide bound to a support, followed by... The process involves reacting 16 bases bound to a support with a nucleoside construction block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a 17-base oligonucleotide bound to a support, followed by... The process involves reacting 17 bases bound to a support with a nucleoside construction block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain an 18-base oligonucleotide containing SEQ ID NO: 1 bound to the support. The process of carrying out the following: The reaction process is as follows: The order is b-1), b-2), b-3), b-4); b-1), b-2), b-4), b-3); b-2), b-3), b-4), b-1); or b-2), b-4), b-3), b-1). b-1) Deblocking, b-2) Coupling, b-3) Capping, b-4) Oxidation or sulfidation, preferably sulfidation Processes including c) Optionally, R from an oligonucleotide bound to a support 1 The process of cutting, d) A step of cleaving an 18-base oligonucleotide bound to a solid support from the support to obtain an antisense oligonucleotide. Includes, The nucleoside construction blocks of equations (IIa-G), (IIa-T), (IIa-C), (IIa-A), (IIb-G), (IIb-T), (IIb-C), (IIb-A), (IIc-G), (IIc-T), (IIc-C), and (IIc-A) are respectively given by the following equations: 【Transformation 3】 Represented by, (In the formula, Ar 2 The following are optional choices: halogen, C 1 ~C 6 Alkyl, C 3 ~C 6 Cycloalkyl or C 1 ~C 4 It is a phenyl substituted with an alkoxy, R 4 (This represents a protecting group for a hydroxyl group, selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl.) A method for generating an antisense oligonucleotide according to any one of claims 1 to 4.

12. Step a) is, a-1) A step of preparing a solid support represented by Equation III, 【Chemistry 4】 (In the formula, L is: 【Transformation 5】 Selected from, SS stands for solid support SS, R 1 This represents hydrogen, or a protecting group for a hydroxyl group, selected from triphenylmethyl, 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, 9-phenylxanthin-9-yl, and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytrityl and 4-monomethoxytrityl. R 2 (is selected from methyl and phenyl), followed by a-2) A step of reacting a nucleoside construction block of formula (IIa-G) with a solid support according to formula III to obtain a nucleotide bound to the support, followed by The process involves reacting a nucleoside construction block of formula (IIb-G) with a nucleotide bound to a support, thereby obtaining a dinucleotide bound to the support. The method according to claim 11, including the method described in claim 11.

13. An antisense oligonucleotide represented by Sequence ID No. 1, obtained by the method described in claim 11 or 12.

14. The antisense oligonucleotide according to claim 13, wherein at least 95% of the antisense oligonucleotides maintain their full length after incubation in mouse liver lysate for at least 5 days, as measured by LC-MS.

15. The antisense oligonucleotide according to claim 13 or 14, wherein, as measured by LC-MS, there is an increase of at least 5 percentage points in the full-length antisense oligonucleotide compared to nusinersene after incubation in mouse liver lysate for at least 5 days.

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