Antisense oligonucleotides and medical uses thereof

Abstract

The present invention provides antisense oligonucleotides having a 2 '-O-methoxyethyl sugar modification at each position, and having at least one chiral inter-nucleotide thiophosphate bond having an Sp configuration at the 3'-terminus. The invention also relates to the use of such antisense oligonucleotides in medicine. The invention also provides compositions, including pharmaceutical compositions, comprising such antisense oligonucleotides. In addition, the invention also provides a manufacturing method.

Description

Technical Field

[0001] This invention relates to the field of antisense oligonucleotides, particularly splice switching oligonucleotides and their use in medicine, especially in the treatment of spinal muscular atrophy. Background Technology

[0002] Antisense oligonucleotides:

[0003] Antisense oligonucleotides (ASOs) are relatively short single-stranded nucleic acids, typically between 10 and 30 nucleotides in length, that bind to complementary messenger RNA (mRNA, including precursor mRNA). Unlike many traditional drugs that typically inhibit protein function by binding to specific pockets of enzymes, ASOs alter protein biosynthesis during protein formation. ASOs can be generally classified according to their respective mechanisms of action, particularly whether they function through a so-called "target occupancy" mechanism or an "occupancy-mediated degradation" mechanism.

[0004] ASOs that function through "occupation-mediated degradation" include RNase H-dependent ASOs, which are typically used to downregulate pathogenic proteins by enzymatically degrading their respective mRNAs. 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 the translation of the ApoB-100 protein.

[0005] In contrast, ASOs that primarily function through a so-called "target-occupying" mechanism typically do not induce enzymatic RNA degradation. A notable example of a so-called "target-occupying" ASO is the splice-conversion oligonucleotide (SSO), which binds to certain regions of precursor mRNA involved in splicing, such as splice sites or regulatory elements, to regulate splicing in the event of disruption of normal splicing. In particular, SSOs can interfere with endogenous RNA splicing mechanisms by spatially blocking the accessibility of certain splice sites and / or regulatory elements.

[0006] The field of ASO (Anaerobic Spondylitis) and, particularly, SSO (Spinal Muscular Spondylitis) has recently garnered attention due to advances in the treatment of neurological diseases. For example, nusinersen, marketed under the name Spinraza, became the first drug approved for the treatment of spinal muscular atrophy (SMA, a rare neuromuscular disease).

[0007] Treatment of spinal muscular atrophy:

[0008] SMA is a rare neuromuscular disease caused by homozygous deletion of the SMN1 gene, which encodes the survival motor neuron (SMN) protein. In humans, the SMN protein is additionally produced by the SMN2 gene. However, the production of SMN protein from the SMN2 gene is limited. Typically, SMN2 protein expression is suppressed by splicing enhancer sequences, leading to the exclusion of exon 7 and resulting in an unstable truncated SMN protein.

[0009] Nusinersen sodium is an ASO that binds to SMN2 precursor mRNA and alters its splicing, thereby including exon 7 and restoring the lost SMN protein.

[0010] In addition, SMA can be treated with a recently developed gene therapy called zolgensma. However, the application of zolgensma is limited due to the high cost associated with this gene therapy.

[0011] Chemical oligonucleotide synthesis:

[0012] General methods for the chemical synthesis of oligonucleotides are known in the art and typically involve the stepwise addition of nucleoside building blocks to the 5' end of a growing oligonucleotide chain bound to a solid support. Each addition is generally referred to as a synthetic cycle and typically involves four chemical reactions:

[0013] Deprotection: Removing the 5'-hydroxyl (e.g., 4,4'-dimethoxytriphenylmethyl) protecting group from the oligonucleotide linked to the solid support or the hydroxyl protecting group present on the solid support in the first synthesis cycle (i.e., when no nucleoside is attached to the solid support, or a general solid support).

[0014] Coupling: Once the hydroxyl protecting group is removed, the free 5'-OH of the oligonucleotide linked to the solid support, or the free hydroxyl group of the solid support, can react with the next nucleoside, which is typically added as a monomeric nucleoside building block, such as a phosphoramidite (PA) building block or an oxazaphospholidine (OAP) building block. The coupling reaction leads to the formation of a phosphite trimer bond.

[0015] End-capping: Unreacted 5'-hydroxyl groups (or free hydroxyl groups) of the solid support are capped, for example, using acetic anhydride in the presence of a base. If not capped, these hydroxyl groups will react in the next cycle, resulting in oligonucleotides containing internal deletions.

[0016] Oxidation or sulfidation: The phosphite triesters formed during the coupling reaction are relatively unstable under oligonucleotide synthesis conditions. Therefore, they must be converted to more stable phosphorus species before the start of the next synthetic cycle, typically by oxidation to phosphodiester bonds using aqueous iodine solution, or by sulfidation to thiophosphate bonds using commercially available sulfidation reagents.

[0017] Solutions of each reagent used for oligonucleotide synthesis are prepared in advance and loaded into reagent bottles (typically for laboratory-scale synthesis) or tanks (typically for commercial manufacturing) of the oligonucleotide synthesizer.

[0018] Modern oligonucleotide synthesis typically relies on the so-called phosphoramidite process, in which protected nucleoside building blocks (PAs or OAPs) of natural or chemically modified nucleosides are sequentially 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 defined oligonucleotide sequences and enables the introduction of various chemical modifications in a sequence-specific manner. To achieve maximum flexibility in sequence selection, oligonucleotides are often synthesized on commercially available universal solid supports—that is, solid supports configured for automated oligonucleotide synthesis but not yet coupled with nucleosides. However, in addition to these, there are solid supports pre-loaded with a single nucleoside, which limits user flexibility (because the 3'-terminal nucleoside is already determined by the solid support) but generally provides a better overall yield at the end of synthesis than universal solid supports.

[0019] While automated synthesis of oligonucleotides including homogeneous chemical modifications (e.g., all nucleosides partially modified with 2'-MOE or all nucleobases substituted with 5mC) has been established cost-effectively, the synthesis of oligonucleotides involving heterogeneous modification patterns—such as those with defined stereochemistry at selected PS bonds or those combining with chiral-controlled PS bonds—requires the use (and maintenance) of many different reagents (different nucleoside building blocks, activators, and reaction conditions for synthesizing Rp, Sp, and stereorandom PS bonds), making the synthesis laborious and unattractive. Furthermore, the synthesis of oligonucleotides containing chiral-controlled PS bonds requires the use of nucleoside building blocks carrying chiral auxiliaries, particularly oxo-nitro-phosphorus heterocyclic pentane building blocks. However, the coupling yield of oxo-nitro-phosphorus heterocyclic pentane building blocks is generally lower than that of commonly used nucleoside phosphoramids (which lack chiral control).

[0020] Therefore, chiral-free, fully PS oligonucleotides (stereo-random, i.e., each PS bond is a mixture of Rp and Sp bonds) remain the gold standard for oligonucleotide therapy to date.

[0021] Chemical modification of ASO:

[0022] The properties and functions of ASOs depend primarily on the combination of their nucleotide sequence and chemical structure. Potential sites for chemical modification include the nucleic acid backbone, nucleobases, sugar moieties, and 5' and 3' ends. Various chemical modifications are known to those skilled in the art, and their applications in therapeutic oligonucleotides are described, for example, in Crooke et al. [JBC Reviews (2021) 296, 100416].

[0023] A prominent example of a chemically modified ASO is nusinersen sodium (18-mer ASO), in which the 2'-hydroxyl group of the furanyl ribosyl ring at each position (i.e., on each nucleotide) is replaced by a 2'-O-2-methoxyethyl (2'-MOE) group (i.e., the furanyl ribosyl ring is replaced by 2-methoxyethyl (MOE) based on the 2'-hydroxyl group). Furthermore, all phosphate bonds (PO) are replaced by phosphate thiophosphate (PS) bonds, and all cytosine nucleobases are replaced by 5-methylcytosine (5mC). Nusinersen sodium is represented by SEQ ID NO:1:TCACTTTCATAATGCTGG, where T represents 2'-MOE thymidine, C represents 2'-MOE-5-methylcytosine, A represents 2'-MOE adenosine, and G represents 2'-MOE guanosine.

[0024] While it is known that replacing the PO bond with PS can improve metabolic stability, replacing one of the non-bridging oxygens with sulfur can transform the prochiral PO bond into a new stereocenter. In conventional oligonucleotide synthesis, Rp and Sp bonds typically occur in an approximately 1:1 ratio [see, for example, Oka et al., Nucleic Acids Symp. Ser. (Oxf) 2008, 335; and VTRavikumar, DL Cole, Nucleosides, Nucleotides & Nucleic Acids (2003), 22, 1639]. Therefore, the 18-mer oligonucleotide nusinersen contains 2 17 =131072 possible diastereomers (2 n A mixture of diastereomers (where n is the number of p- bonds), each with its own unique physical and biochemical properties.

[0025] However, synthesizing chiral-controlled stereoSSOs remains a challenge in this field. Therefore, efforts to investigate the effect of stereochemistry on the PS bond have focused on modulating stereochemistry by selecting activators used in RNA chemical synthesis [Jahns et al., Nat. Commun. 2015, 66317], which suggests that a higher proportion of Rp PS bonds results in higher hybridization affinity and better metabolic stability of small interfering RNA (siRNA). Furthermore, stereochemically pure all-Rp 2′-O-(2-methoxyethyl) thiophosphate SSOs targeting ferrochelate precursor mRNA have been reported to be more effective than their Sp counterparts, but not superior to their stereorandom parental SSOs [Li et al., Chem. Commun. 2017, 53541]. This aligns with a report by Ostergaard et al., which found no improvement in the potency of a series of chiral-controlled ASOs compared to stereorandom parental ASOs [NucleicAcids Res. 2020; 48(4):1691-1700]. While some literature reports suggest that certain oligonucleotide drugs containing several chiral-controlled phosphate-thioester bonds are superior, it is generally accepted that "the chiral purity of a single PS unit does not meaningfully improve the therapeutic index and overall performance of PSASO" [Crooke et al., JBC Reviews (2021) 296, 100416]. Furthermore, clinical programs for stereopure ASO (rovanersen, lexanersen) for Huntington's disease and stereopure ASO (suvodirsen, see, e.g., https: / / ir.wavelifesciences.com / news-releases / news-release-details / wave-life-sciences-announces-discontinuation-suvodirsen) for Duchenne muscular dystrophy have been terminated due to lack of efficacy. Given the absence of currently approved stereopure oligonucleotide drugs, chiral-controlled (stereorandomized) all-PS ASO remains the gold standard for antisense therapy. Summary of the Invention

[0026] The object of this invention is to provide an ASO with improved therapeutic properties, particularly an SSO, especially for the treatment of SMA.

[0027] In particular, an object of the present invention is to provide an ASO with higher metabolic stability compared to current therapeutic ASOs (especially nusinersen). Another object of the present invention is to provide a method for producing such an improved ASO in an economically feasible manner.

[0028] This invention provides a novel ASO comprising a 2'-O-methoxyethyl sugar modification at each position and further comprising a chiral internucleotide PS bond, also referred to as a 3'-terminal PS bond, at the 3'-terminus having an Sp configuration at its chiral phosphate junction. This invention also relates to compositions comprising multiple such ASOs, and pharmaceutical compositions comprising such ASOs or multiple such ASOs. Furthermore, this invention provides a kit comprising a pharmaceutically acceptable carrier or diluent, and the ASO or multiple such ASOs in lyophilized form.

[0029] In another aspect, the present invention provides the use of the ASO, the various ASOs, or the kit as a medicine, particularly in the treatment of SMA. Furthermore, the present invention provides a method for preparing such an ASO.

[0030] It should be understood that various embodiments / features, preferred options, and scopes provided / disclosed in this specification can be freely combined, provided that a specific combination of embodiments / features is technically meaningful. In particular, it should be understood that all definitions and features of the ASO of the present invention provided in the first aspect also apply to the second, third, fourth, fifth, and sixth aspects of the present invention.

[0031] Unless otherwise stated, the following definition This instruction manual applies to:

[0032] As used herein, unless otherwise stated herein or clearly contradicted by the context, the terms “a”, “an”, “the” and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to cover both the singular and the plural.

[0033] The terms “including,” “containing,” and “comprising” are used in an open, non-restrictive sense herein.

[0034] As used herein, the term "and / or" means that all elements of the group or only one element 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, but no B".

[0035] The terms “including,” “containing,” and “comprising” are used herein in an open and non-restrictive sense. It should be understood that various embodiments, preferred embodiments, and scopes can be combined freely.

[0036] Treatment: As used herein, the terms “treating,” “treat,” and “treatment” include one or more of the following: (i) preventing the occurrence of a disease, pathology, or medical condition (e.g., prevention); (ii) suppressing or halting the development of a disease, pathology, or medical condition; (iii) alleviating a disease, pathology, or medical condition; and (iv) reducing symptoms associated with a disease, pathology, or medical condition. Therefore, the terms “treat,” “treatment,” and “treating” extend to prevention, including prevention, prevention, deterrence, reduction, cessation, or reversal of the progression or severity of the treated condition or symptoms. Thus, the term “treatment” includes medical, therapeutic, and / or preventative administration (as applicable).

[0037] Gene: As used herein, “gene” refers to a region of DNA (including exons and introns) that encodes a gene product, as well as all DNA regions that regulate the production of gene products, regardless of whether these regulatory sequences are adjacent to coding and / or transcriptional sequences. Therefore, genes include, but are not limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions. As used herein, the term “gene” specifically refers to the human gene.

[0038] Nucleotides: The term "nucleotide" generally refers to deoxyribonucleotides, ribonucleotides, or derivatives thereof. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or nucleotide analogs. Nucleotide analogs are nucleotides having modified purine or pyrimidine bases and / or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (such as inosine) or non-naturally occurring nucleic acids. Examples of non-naturally occurring nucleotides include nucleotides modified at the 2'-O position of the sugar, such as 2'-OMe, 2'-MOE, 2'-F modified nucleotides, especially 2'-MOE, as well as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino oligonucleotides.

[0039] Nucleobase: The term "nucleobase" is known in the art to refer to the heterocyclic base portion of a nucleoside or nucleotide. Generally, a nucleobase refers to any group containing one or more atoms or groups of atoms capable of hydrogen bonding with another nucleic acid base. In addition to "unmodified" or "natural" nucleobases, such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U), a wide range of modified nucleobases are known and can be used in this invention. The synthesis of modified nucleobases is known in the art.

[0040] Oligonucleotide: The term "oligonucleotide" generally refers to deoxyribonucleotides or ribonucleotide polymers, or derivatives thereof. In an oligonucleotide, adjacent nucleotides are covalently linked by phosphate groups to form a sequence. The bonds between nucleotides are also referred to as the backbone of the oligonucleotide. In most naturally occurring oligonucleotides, the backbone consists of 3' to 5' phosphodiester bonds. However, synthetic oligonucleotides often involve chemical modifications, such as increasing the binding affinity of ASO to its target RNA, increasing nuclease resistance (metabolic stability), and / or altering the pharmacokinetics of the oligonucleotide, such as its biodistribution. As will be understood by those skilled in the art, the use of chemical modifications that increase the affinity of the oligonucleotide to its target RNA can allow the use of relatively short oligonucleotides. Therefore, the term oligonucleotide includes modified oligonucleotides that contain nucleotides modified in the base (e.g., 5-methylcytosine), sugar (e.g., 2'-MOE), and / or phosphate moiety (e.g., PS bond).

[0041] Antisense oligonucleotides (ASOs): This term is known in the art and refers to synthetic, linear, single-stranded nucleic acid polymers that typically bind to complementary messenger RNA (mRNA, including precursor mRNA) via Watson-Crick base pairing. Typically, ASOs are between 10 and 30 nucleotides in length and may include modified and / or unmodified nucleotides as described above. In particular, the ASOs of this invention contain up to 20 nucleotides. Therefore, in the context of this invention, the length of an ASO is 18 to 20 nucleotides.

[0042] Furthermore, in the ASO of the present invention, at each position (i.e., on each nucleotide), the 2'-hydroxyl group of the furanyl ribosyl ring (i.e., the sugar) is replaced by a 2'-MOE group. Additionally, at least the 3'-terminal internucleotide phosphate bonds are replaced by PS bonds having an Sp configuration (i.e., at least the 3'-terminal PS bonds are chiral controlled). Those skilled in the art will understand that the 3'-terminal PS bond is located between the 3'-terminus and the penultimate nucleotide, and that the 3'-terminal nucleotide of the ASO contains a 3'-hydroxyl group, rather than another phosphate or thiophosphate moiety. In the context of the present invention, the ASO preferably has a length of 18 to 20 nucleotides.

[0043] The term "ASO" specifically includes "SSO". SSO binds to precursor mRNA and regulates splicing, for example, by blocking RNA-RNA base pairing or protein-RNA binding interactions between components of the endogenous splicing mechanism and precursor mRNA. In the context of this invention, splicing regulation specifically refers to altering the splicing of SMN2 precursor mRNA to include exon 7 in SMN2 mRNA.

[0044] PO bond, PS bond: The terms “PO bond” and “PS bond” are known in the art to refer to a phosphodiester bond between two nucleosides (nucleoside 1-O-PO2-O-nucleoside 2) or a thiophosphate bond between two nucleosides (nucleoside 1-O-POS-O-nucleoside 2), respectively. Therefore, the terms should not be confused with a single PO bond or a single PS bond.

[0045] Chiral control: This term is known in the art and is used in the context of this invention, particularly relating to chiral-controlled internucleotide PS bonds, in which a non-bridging oxygen (such as that present in a pre-chiral 3'-5'-phosphodiester bond) is substituted with sulfur. Methods for determining the stereopurity of phosphorus atoms in PS bonds are known in the art, and particularly include RP-HPLC and... 31 P NMR spectroscopy. As used herein, "chiral control" means that the stereopurity of chiral phosphorus at the PS bond is at least 90%, preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, preferably measured by RP-HPLC. Preferably, the stereopurity at each chiral control PS bond is at least 99%, preferably measured by RP-HPLC.

[0046] Hybridization: The term “hybridization” is known in the art and refers to the pairing, or binding, of complementary nucleic acid strands, such as the binding of an ASO to its target sequence. While not limited to specific mechanisms, the most common hybridization (binding) mechanisms involve hydrogen bonds between the nucleobases of complementary nucleotides, such as Watson-Crick, Hoogsteen, or reverse Hoogsteen base pairing, particularly Watson-Crick base pairing. As used herein, the term “complementarity” or “complementarity” refers to the ability of two nucleobases to pair precisely, such as the ability of an ASO to pair with its target sequence. For example, a position is considered complementary if a nucleobase at a location in the ASO can pair with a nucleobase at a location in the target nucleic acid (particularly in precursor mRNA, such as SMN2 precursor mRNA). Furthermore, an ASO and its target nucleic acid are considered complementary if a sufficient number of positions (nucleobases) are complementary, allowing for stable and specific binding between the ASO and the target nucleic acid (i.e., SMN2 precursor mRNA). As is known in the art, adenine is complementary to thymine and uracil, while guanine is complementary to cytosine and 5-methylcytosine. Those skilled in the art can determine whether an ASO is complementary to its target nucleic acid, for example, 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 an ASO and its target sequence. Methods for determining Tm between an ASO and its target sequence are known in the art, and Tm can also be calculated.

[0047] Polypeptide: The terms “polypeptide” and “protein” are used interchangeably and refer to a polymer of amino acid residues.

[0048] This manual uses many abbreviations, including:

[0049] 2'-F 2'-fluorine

[0050] 2'-MOE 2'-Methoxyethyl

[0051] 2'-OMe 2'-O-methyl

[0052] 5mC 5-methylcytosine

[0053] ACN Acetonitrile

[0054] AEX-HPLC Anion Exchange High Performance Liquid Chromatography

[0055] ASO antisense oligonucleotides

[0056] BHQ-1 Black Hole Quenching Agent 1

[0057] BTT 2-benzylthiotetrazole

[0058] CPG controllable aperture glass

[0059] DCA dichloroacetic acid

[0060] DCI 4,5-Dicyanoimidazole

[0061] DCM dichloromethane

[0062] DDTT 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazolyl-5-thione

[0063] DMTr 4,4'-Dimethoxytriphenylmethyl

[0064] ETT 5-ethylthio-1H-tetrazole

[0065] FAM Carboxyfluorescein

[0066] GalNAc N-acetylgalactosamine

[0067] GAPDH glyceraldehyde-3-phosphate dehydrogenase

[0068] HPLC (High Performance Liquid Chromatography)

[0069] mRNA messenger ribonucleic acid

[0070] RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography)

[0071] siRNA (small interfering RNA)

[0072] Spinal muscular atrophy (SMA)

[0073] SMN2 motor neuron survival 2

[0074] SPE solid phase extraction

[0075] LC-MS (Liquid Chromatography-Mass Spectrometry)

[0076] LNA locked nucleic acid

[0077] mAU (milliosorbance units)

[0078] MMTr 4-Monomethoxytriphenylmethyl

[0079] mRNA messenger RNA

[0080] NMR (Nuclear Magnetic Resonance)

[0081] N- PhIT N-methylbenzimidazole trifluoro ester

[0082] OAP (oxygen, nitrogen, phosphorus, heterocyclic pentane)

[0083] PA phosphorus amide

[0084] Ph phenyl

[0085] PNA peptide nucleic acid

[0086] PO phosphate diester

[0087] PS Thiophosphate

[0088] R-configuration of chiral phosphorus (Rp)

[0089] SSO splicing conversion oligonucleotides

[0090] S configuration of chiral phosphorus (Sp)

[0091] THF Tetrahydrofuran

[0092] Tm melting temperature

[0093] Firstly, antisense oligonucleotides:

[0094] In a first aspect, the present invention relates to an ASO comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof, wherein the ASO has a 2'-O-methoxyethyl sugar modification on each of its nucleotides and a chiral internucleotide thiophosphate bond having an Sp configuration at its 3' end. As understood by those skilled in the art, the internucleotide thiophosphate at the 3'-terminal bond is located between the 3'-terminus and the penultimate nucleotide.

[0095] SEQ ID NO:1:TCACTTTCATAATGCTGG, where T represents 2'-MOE thymidine, A represents 2'-MOE adenosine, G represents 2'-MOE guanosine, and C represents 2'-MOE cytidine and / or 2'-MOE-5-methylcytidine. That is, C can represent 2'-MOE cytidine in some positions and 2'-MOE-5-methylcytidine in others.

[0096] Preferably, the ASO comprises at least one 5-methylcytosine. More preferably, C in SEQ ID NO:1 represents 2'-MOE-5-methylcytidine, i.e., all cytosine residues are 5-methylcytosine. Replacing cytosine with 5-methylcytosine has the advantage of increasing the hybridization affinity of the ASO with its target sequence. However, C may also represent 2'-MOE cytidine at each position.

[0097] Although at least the 3'-terminal bond (i.e. the final internucleotide bond at the very end of the ASO 3') is a PS bond with an Sp configuration on its chiral phosphorus atom, the remaining internucleotide bonds may be selected from PO bonds (prochiral) and / or PS bonds, wherein the PS bonds may have an Sp configuration, an Rp configuration and / or may be uncontrolled by chirality, i.e., a mixture of PS bonds with Sp and Rp configurations.

[0098] Surprisingly, it has been found that the ASO of the present invention described herein has better metabolic stability compared to nusinersen sodium (see Example 6); Figure 1 A, ASO 4, 5, 6, and 10; Nusinersen corresponds to ASO1). Those skilled in the art will understand that metabolic stability is a key factor in the therapeutic activity of ASOs. It is believed that a single PS bond with an Sp configuration at the 3'-terminus is sufficient to significantly improve metabolic stability, i.e., improve stability against nuclease degradation, which is particularly beneficial and surprising (see Example 6; ASO1 vs ASO4).

[0099] As is generally accepted in the art, a good alternative measure of the metabolic stability of ASO in vivo is incubation in whole tissue lysates, particularly in mouse liver lysates. In a preferred embodiment, as measured by LC-MS, at least 95%, i.e., 95%-100%, preferably 98%-100%, of the ASO of the present invention as described herein retains its full length after incubation in mouse liver lysates for at least 5 days.

[0100] Preferably, as measured by LC-MS, after incubation in mouse liver lysate for at least 5 days, the full-length ASO of the present invention increased by at least 5 percentage points compared to nusinersen sodium.

[0101] Preferably, the ASO of the present invention has a PS bond at every position, also known as a full PS ASO (i.e., all internucleotide bonds are PS). Compared with other identical ASOs containing at least one PO bond (e.g., 1-20 PO bonds, such as 1-5 PO bonds), having a PS bond at every position can improve metabolic stability.

[0102] However, ASOs may also include PO bonds. In these embodiments, up to 50% of the PS bonds are replaced by PO bonds. As can be clearly seen from the above, in this embodiment, at least the 3'-terminal bond is PS. Including a limited number of PO bonds, such as one to ten PO bonds, preferably one to five PO bonds, may have the advantage of reducing side effects, for example, by reducing stimulation of the human immune system, especially the innate immune system, while maintaining sufficient metabolic stability. Therefore, if PO bonds are present, the number of PO bonds is generally limited to one to ten PO bonds, preferably one to five PO bonds (e.g., one, two, three, four, or five PO bonds), while the remaining internucleotide bonds are PS. In a typical setting of the ASO described herein, i.e., up to 20 nucleotides in length, a total of 1 to 10 PO bonds corresponds to 5%-50% of the PO bonds. The PO bonds can be directly adjacent; for example, an ASO may have three PO bonds at positions 9, 10, and 11, or five PO bonds at positions 7, 8, 9, 10, and 11. Alternatively, the PO links can be interleaved. For example, an ASO may have three PO links 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 PO bond positions are indicated for illustrative purposes only).

[0103] Furthermore, the ASO of the present invention may comprise a further chiral-controlled internucleotide PS bond, advantageously, said further chiral-controlled internucleotide PS bond being sequential with a 3'-terminal chiral internucleotide thiophosphate bond. In an advantageous embodiment, the ASO also has at least one (see Example 6; Figure 1 (ASO5) or at least two chiral nucleotides with Sp configurations inter-thiophosphate bonds (see Example 6); Figure 2 These bonds (ASO6) are adjacent to the 3'-terminal chiral nucleotide thiophosphate bonds (i.e., at the 3' end, at least the last two or at least the last three nucleotide bonds are chiral controlled PS bonds with Sp conformation).

[0104] Compared to ASOs with a single chiral-controlled PS bond of Sp conformation at the 3' end, the inclusion of an additional chiral-controlled internucleotide PS bond of Sp conformation continuous with the 3'-terminal Sp-conformation internucleotide PS bond during in vivo administration is expected to further improve metabolic stability. However, the ASOs of the present invention may also contain chiral internucleotide PS bonds of Rp conformation. For example, chiral-controlled PS bonds of Rp conformation can increase binding affinity to target sequences and, moreover, improve interactions with certain proteins involved in endogenous splicing mechanisms, resulting in higher exon 7 inclusion in SMN2 mRNA.

[0105] In an advantageous embodiment, the ASO comprises a chiral internucleotide PS bond with an Sp configuration at the 3' end, while all other internucleotide bonds are chiral PS bonds (see Example 6). Figure 1 ASO1 (ASO4) is a mixture of all other nucleotide internal bonds consisting of PS bonds with Rp and Sp configurations, for example, an Rp:Sp ratio of 0.1 to 0.9, such as 0.4 to 0.6. Compared to the same ASO where no PS bond is chirally controlled, this ASO has the advantage of improved metabolic stability, i.e., synthesis without PS bonds and with chirality control (also known as a fully stereorandom PS ASO, see Example 6, ASO1 and ASO4), or where the 3'-terminal PS bond has an Rp configuration (see Example 6, ASO 7, 8 and 9), while being able to be manufactured in a more cost-effective manner than ASOs containing multiple chirally controlled PS bonds.

[0106] In another advantageous embodiment, the ASO comprises a chiral internucleotide PS bond with an Sp conformation at the 3' end, while all other internucleotide bonds are chiral controlled PS bonds with an Rp conformation (see [link to relevant documentation]). Figure 1 (ASO10). Similar to the same ASO without chiral control of the PS bond (such as ASO1) or ASOs with an Rp configuration of the 3'-terminal PS bond (e.g., ASO 2, 7, 8, and 9, see ASO 10). Figure 1 Compared to other ASOs without chiral control of PS bonds, this type of ASO has the advantage of improved metabolic stability, and also has the additional advantage of improved target binding affinity (i.e., potency) compared to other ASOs without chiral control of PS bonds.

[0107] In other advantageous embodiments, the ASO comprises two consecutive chiral internucleotide PS bonds with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral controlled PS bonds (see Example 6). Figure 1 ASO5). In other advantageous embodiments, ASO comprises three consecutive chiral internucleotide PS bonds with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral controlled PS bonds (see Example 6; Figure 1 ASOs (ASO6) contain two or three consecutive chiral-controlled internucleotide PS bonds with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral PS bonds. This provides the advantage of higher metabolic stability when administered in vivo. This is especially important when ASOs are administered at longer intervals, such as every four months, every six months, or every twelve months. ASOs containing two or three consecutive chiral-controlled internucleotide PS bonds with an Sp configuration at the 3' end can still be manufactured cost-effectively, i.e., in good yields and with sufficient purity. In other embodiments, all internucleotide bonds are chiral-controlled PS bonds with an Sp configuration. However, ASOs (where all internucleotide bonds are chiral-controlled PS bonds with an Sp configuration) are less desirable because they are relatively expensive to manufacture and generally have lower synthetic yields compared to ASOs containing fewer chiral-controlled PS bonds.

[0108] Preferably, the ASO of the present invention is at most 20 nucleotides in length (i.e., contains at most 20 nucleotides). Therefore, the ASO preferably contains 18 to 20 nucleotides, for example 18, 19, or 20 nucleotides. In a preferred embodiment, the ASO consists of 18 nucleotides, i.e., the ASO is represented by SEQ ID NO:1. In an 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 ASO4). Figure 1 In another advantageous embodiment, the ASO of the present invention is represented by ASO5. In another advantageous embodiment, the ASO of the present invention is represented by ASO6 (see ASO6). Figure 1 In another advantageous implementation, ASO is represented by ASO3 (see ASO3). Figure 1 In other advantageous embodiments, the ASO of the present invention is represented by ASO10 (see ASO10). Figure 1 ).

[0109] Advantageously, all cytosine residues are 5-methylcytosine.

[0110] Furthermore, the ASO of the present invention can bind to a non-nucleic acid moiety, such as N-acetylgalactosamine (GalNAc), as described previously with a tri-antennae GalNAc connector [see, for example, Nair et al., J Am Chem Soc, 2014, 136, 16958-61], or a lipid moiety [see, for example, Moroz et al., Mol. Ther. Nucl. Acids (2016) 5, e364]. This non-nucleic acid moiety can bind to either the 3' or 5' end of the ASO. Binding to the non-nucleic acid moiety improves tissue delivery and intracellular uptake in vivo. Within the scope of common technology, suitable building blocks for binding to non-nucleic acid moieties are commercially available, for example, from Glen Research (e.g., catalog numbers 10-1974 and 20-2974).

[0111] The ASOs of the present invention include esters, such as S-acyl-2-thioethyl esters at the internucleotide PS and / or PO bonds (see, for example, Tosquellas et al., Nucleic Acids Res. 1998; 26(9): 2069-2074), or any other functional chemical equivalents, which, when administered to animals or humans, particularly humans, provide the ASOs described herein (also known as pro-oligonucleotides). Pharmaceutically acceptable salts of ASOs, including sodium and potassium salts, particularly sodium salts, their esters, or other functional chemical equivalents, are also claimed and described herein.

[0112] The second aspect; compositions containing multiple ASOs:

[0113] In a second aspect, the present invention relates to a composition comprising a variety of ASOs and / or their pharmaceutically acceptable salts. This diversity includes different variants of the ASOs described herein, for example, ASOs that differ at certain positions (i.e., at certain internucleotide bonds) in the stereochemistry of chiral PS bonds. For example, such a variety may include… ASOs contain a chiral, controlled internucleotide PS bond with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral PS bonds. ASOs containing two or more chiral PS bonds between nucleotides with Sp conformation at the 3' end, and / or An ASO comprising a chiral, controlled internucleotide thiophosphate bond with a Sp configuration at the 3'-terminus, and further comprising at least one chiral, controlled internucleotide thiophosphate bond with an Rp configuration.

[0114] Such a variety may also include ASOs of different lengths as described herein, for example, combinations of 18mer ASOs, and / or 19mer ASOs and / or 20mer ASOs as described herein.

[0115] Compositions containing such multiple ASOs may have a variety of beneficial effects, such as reducing the risk of immune responses and / or increasing activity due to the lower amounts of each specific variant of ASO.

[0116] Thirdly, the pharmaceutical composition:

[0117] In a third aspect, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and

[0118] Antisense oligonucleotides and / or their pharmaceutically acceptable salts as described herein, or

[0119] As described herein, there are various antisense oligonucleotides and / or their pharmaceutically acceptable salts.

[0120] Those skilled in the art will understand that such pharmaceutical compositions can be prepared by adding an effective amount of ASO as described herein to a suitable pharmaceutically acceptable diluent or carrier.

[0121] Pharmaceutically acceptable carriers and diluents are known in the art. Those skilled in the art will understand that the selection of a diluent or carrier is based on many factors, including the solubility of ASO and the route of administration.

[0122] For example, diluents or carriers may include ionic strength regulating media, including buffers and salts, such as phosphate buffers, such as phosphate-buffered saline, isotonic NaCl solutions, sugar solutions, citrate buffers, isocitrate buffers, EDTA (including EDTA salts) and mixtures thereof.

[0123] Preferably, the pharmaceutical composition described herein is provided as a solution for intrathecal administration.

[0124] Furthermore, the pharmaceutical compositions described herein are preferably provided in unit dosage form.

[0125] The ASO or the various ASOs of the present invention can also be provided separately from a pharmaceutically acceptable carrier or diluent in a lyophilized form. Therefore, the present invention also relates to a kit comprising a pharmaceutically acceptable carrier or diluent and one of the following substances in a lyophilized form: As described herein, ASO or its pharmaceutically acceptable salt, or The composition comprises a variety of ASOs and / or their pharmaceutically acceptable salts, as described herein.

[0126] Fourthly, treatment:

[0127] The fourth aspect of this invention relates to medical applications.

[0128] The ASO of the present invention can be used to modulate SMN2 expression in animals or humans, particularly human subjects, where desired. Specifically, the present invention provides a method for promoting the inclusion of exon 7 into SMN2 transcripts in cells, tissues, or organs, comprising contacting said cells, tissues, or organs in vitro with the ASO described herein or a pharmaceutically acceptable salt thereof. Furthermore, the present invention provides a method for promoting the inclusion of exon 7 into SMN2 transcripts in cells, tissues, or organs, comprising contacting said cells, tissues, or organs in vitro with a composition as described herein comprising multiple ASOs and / or pharmaceutically acceptable salts thereof, or a pharmaceutical composition as described herein, or a kit as shown herein.

[0129] Therefore, the ASOs or pharmaceutically acceptable salts thereof described herein can be used as medicines, particularly for the treatment of SMA. Furthermore, the compositions comprising multiple ASOs and / or their pharmaceutically acceptable salts, the pharmaceutical compositions described herein, and the kits described herein can be used as medicines, particularly for the treatment of SMA.

[0130] For example, use as a medicine includes administering an effective amount of ASO and / or a pharmaceutically acceptable salt thereof to a human subject requiring treatment for a disease or condition related to SMN2, as described herein. Those skilled in the art will understand that diseases or conditions related to SMN2 include, but are not limited to, spinal muscular atrophy. The ASO of the present invention can effectively modulate SMN2 splicing, resulting in increased inclusion of exon 7 in SMN2 mRNA. For example, regulation of SMN2 expression can be measured in bodily fluids (such as serum). Such bodily fluids may or may not contain cells. Regulation of SMN2 expression can also be measured in tissue samples, which are typically obtained via biopsy. Methods for measuring regulation of SMN2 expression are known in the art. As those skilled in the art will understand, safe and effective treatment evaluation typically includes measuring biomarkers, such as markers of kidney and liver function, such as liver transaminases, albumin, blood urea nitrogen, bilirubin, creatine, and markers of inflammation, such as tumor necrosis factor, intracellular adhesion molecules, interleukins, C-reactive protein, chemokines, and cytokines. Methods for measuring such biomarkers are known in the art.

[0131] Fifthly, manufacturing method:

[0132] In a fifth aspect, the present invention provides a method for producing the ASO described herein. According to this disclosure, those skilled in the art can synthesize the ASO of the present invention using known oligonucleotide synthesis methods. The following methods have been found to be particularly advantageous for the efficient synthesis of the ASO described herein:

[0133] Step a:

[0134] Step a includes providing a functionalized solid support represented by formula I.

[0135] Formula I

[0136]

[0137] in

[0138] L is the connector, preferably selected from:

[0139] and ;

[0140] SS stands for solid support, preferably polystyrene solid support or controlled porosity glass (CPG) solid support;

[0141] Ar1 is a phenyl (Ph, C6H5), optionally substituted with a halogen, a C1-C6 alkyl, a C3-C6 cycloalkyl or a C1-C4 alkoxy.

[0142] X is selected from sulfur and oxygen;

[0143] R1 represents a protecting group for hydrogen or hydroxyl groups, preferably selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanthin-9-yl and 9-(p-methoxyphenyl)xanthin-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethoxytriphenylmethyl;

[0144] R2 is selected from methyl and phenyl;

[0145] R3 is selected from hydrogen, acetyl, and trifluoroacetyl.

[0146] Solid supports suitable for oligonucleotide synthesis, such as polystyrene and CPG solid supports, are known in the art and are commercially available. For example, solid supports configured for reaction with linker L, i.e., having functional groups, such as long-chain aminoalkyl groups, on their surface for covalent coupling with L, are commercially available from ChemGenes (catalog number: N-5100-05). Furthermore, general-purpose solid supports containing linker L and configured for oligonucleotide synthesis have been described, for example, in US7202264, and are commercially available from, for example, ChemGenes (catalog number: N-4000-05 for CPG, N-4000-03 for polystyrene solid supports).

[0147] The aforementioned functional solid support can be obtained by several methods (see, for example, Example 1). For instance, step a may also include step a-1, which includes providing a solid support represented by Formula III.

[0148] Formula III

[0149] ,

[0150] Where L is selected from:

[0151] and ;

[0152] SS stands for solid support, preferably polystyrene solid support or CPG solid support;

[0153] R1 represents a protecting group for hydrogen or hydroxyl groups, preferably selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanol-9-yl and 9-(p-methoxyphenyl)xanol-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethoxytriphenyl;

[0154] R2 is selected from methyl and phenyl.

[0155] If present, step a-1) is followed by step a-2), which involves reacting the nucleoside building blocks of formula (IIa-G) with the solid support of formula III to obtain nucleotides bound to the support, and then...

[0156] The nucleoside building block of formula (IIb-G) is reacted with a nucleotide bound to the support to obtain a 2-mer oligonucleotide bound to the support, corresponding to the above-described functionalized solid support.

[0157] Step b):

[0158] The method for producing ASO also includes carrying out the following series of reactions as step b): The functionalized solid support of formula I is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 3-mer oligonucleotide bound to the support. The support-bound 3-mer oligonucleotide is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), to obtain a support-bound 4-mer oligonucleotide. The 4-mer bound to the support is reacted with a nucleoside building block of formula (IIa-G), (IIb-G), or (IIc-G), preferably (IIc-G), to obtain a 5-mer oligonucleotide bound to the support. The 5-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 6-mer oligonucleotide bound to the support. The 6-mer bound to the support is reacted with a nucleoside building block of formula (IIa-a), (IIb-a), or (IIc-a), preferably (IIc-A), to obtain a 7-mer oligonucleotide bound to the support, subsequently... The 7-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), to obtain an 8-mer oligonucleotide bound to the support, subsequently... The 8-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 9-mer oligonucleotide bound to the support. The 9-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), to obtain a 10-mer oligonucleotide bound to the support, subsequently... The 10-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), to obtain an 11-mer oligonucleotide bound to the support. The 11-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 12-mer oligonucleotide bound to the support. The 12-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 13-mer oligonucleotide bound to the support. The 13-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T), preferably (IIc-T), to obtain a 14-mer oligonucleotide bound to the support, subsequently... The 14-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), to obtain a 15-mer oligonucleotide bound to the support. The 15-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A), preferably (IIc-A), to obtain a 16-mer oligonucleotide bound to the support, subsequently... The 16-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C), preferably (IIc-C), to obtain a 17-mer oligonucleotide bound to the support. The 17-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T) or (IIc-T), preferably (IIc-T), to obtain an 18-mer oligonucleotide bound to the support comprising SEQ ID NO:1.

[0159] The nucleoside building 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 shown in the following formulas. Formula (IIa-G) Formula (IIa-T) Formula (IIa-C) Formula (IIa-A)

[0160]

[0161] Formula (IIb-G) Formula (IIb-T) Formula (IIb-C) Formula (IIb-A)

[0162]

[0163] Formula (IIc-G) Formula (IIc-T) Formula (IIc-C) Formula (IIc-A)

[0164]

[0165] Wherein Ar2 is a phenyl (Ph, C6H5), optionally substituted with a halogen, C1-C6 alkyl, C3-C6 cycloalkyl, or C1-C4 alkoxy group, and

[0166] R4 represents a protecting group for a hydroxyl group, selected from triphenylmethyl, 4-monomethoxytriphenylmethyl (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr), 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanol-9-yl and 9-(p-methoxyphenyl)xanol-9-yl, preferably selected from DMTr and MMTr.

[0167] Methods for reacting nucleoside building blocks with a solid support (including a functionalized solid support) and oligonucleotides bound to the support (i.e., growing or newly formed oligonucleotide chains) are known in the art (see, for example, WO 2017 / 198775; Li et al., Chem. Commun. 2017, 53541; Jahns et al., Nat. Commun. 2015, 6, 6317). An illustrative synthesis of ASO is also described in Example 2.

[0168] Based on their chemical structures, the nucleoside building units of formulas (IIa-G), (IIa-T), (IIa-C), and (IIa-A) are also referred to as phosphoramidite (PA) building units, especially 2'-MOE PA, while the nucleoside building units of formulas (IIb-G), (IIb-T), (IIb-C), (IIb-A), (IIc-G), (IIc-T), (IIc-C), and (IIc-A) are also referred to as oxygen-nitrogen-phosphorus heterocyclic pentane building units (OAP), especially 2'-MOE OAP.

[0169] Generally, reacting nucleoside building blocks (whether using PA or OAP building blocks) involves the following steps, each of which is known in the art: b-1) Deprotection, b-2) Coupling, b-3) End capping, and b-4) Oxidation or sulfidation.

[0170] Deprotection (also known as "detriphenylmethylation"): Each synthetic cycle, i.e., the reaction of the nucleoside building block and the solid support or the nascent oligonucleotide chain bound to the solid support, is initiated by removing the 5'-hydroxyl protecting group of the nucleoside or nascent oligonucleotide chain linked 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 will be understood by those skilled in the art, when starting with a general solid support, i.e., a solid support without pre-loaded nucleosides, such as the solid support of Formula III, deprotection refers to the deprotection of the hydroxyl groups of the solid support in the first synthetic cycle so that the incoming nucleoside building block can be coupled to the solid support. Typically, the protecting group of the hydroxyl group is selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanol-9-yl, and 9-(p-methoxyphenyl)xanol-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethoxytriphenylmethyl. Deprotection methods are known in the art and include deprotection using a solution of dichloromethane (DCM) or toluene (preferably DCM) containing 3% (w / w) dichloroacetic acid (DCA).

[0171] Coupling: Once the protecting group of the 5'-hydroxyl group is removed, the free 5'-OH of the nucleoside bound to the solid support or the free hydroxyl group of the nascent oligonucleotide chain can react with the next nucleoside, which is added as a monomeric nucleoside building unit (phosphamide or oxonium-phosphorus heterocyclic pentane building unit), thereby forming a phosphite trimer bond. Coupling reactions are usually carried out in the presence of activating compounds such as 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 5-benzylthiotetrazole (BTT), 4,5-dicyanimidazole (DCI), and N-phenylimidazolium trifluoromethanesulfonate (BCI). N- PhIT). This activator compound is commercially available. In the context of this invention, when nucleoside building units containing chiral auxiliary groups (e.g., oxo-nitrogen-phosphorus heterocyclic pentane building units of formula IIb-G, IIb-T, IIb-C, IIb-A, IIc-G, IIc-T, IIc-C, and IIc-A) are coupled to form chiral controlled PS bonds, it has been found that… N-PhIT is a particularly suitable activator compound. BTT has been found to be a particularly suitable activator compound when coupled with a PA (e.g., PAs of formulas IIa-G, IIa-T, IIa-C, and IIa-A) that does not contain a chiral auxiliary group, thereby forming an achiral-controlled PS bond (also known as a stereorandom PS bond, i.e., a mixture of Rp and Sp forms of PS bonds) or a PO bond (prochiral). Those skilled in the art will understand that commonly used phosphoramide building blocks (e.g., PAs of formulas IIa-G, IIa-T, IIa-C, and IIa-A) are also chiral at the phosphorus atom, but do not undergo stereospecific coupling; therefore, the resulting internucleotide PS bonds are not synthesized under chiral control, i.e., the resulting intranucleotide PS bonds are not chiral-controlled.

[0172] Capping: In the capping step, unreacted 5'-OH groups are blocked, otherwise these hydroxyl groups would react in the next cycle, resulting in oligonucleotides containing internally missing segments (i.e., one or more missing nucleosides). Capping is typically performed using a mixture of two capping reagents, commonly referred to as capping reagents A and B, which are applied to the oligonucleotide synthesis column containing the nascent oligonucleotide chain. For example, capping reagent A may comprise a mixture of tetrahydrofuran (THF), dimethylpyridine, and acetic anhydride, for example, in a ratio of 8:1:1 (w / w / w), and capping reagent B may comprise THF containing 16% (w / w) N-imidazole. Those skilled in the art will understand that other capping reagents may be used in this invention. For example, in capping reagent A, acetic anhydride may be substituted with trifluoroacetic anhydride.

[0173] Oxidation / Sulfation: The stability of the phosphite trimer bond is limited, and it can be converted into a 5'-3'-phosphodiester bond (referred to as oxidation; PO bond) or a 5'-3'-thiophosphate bond (referred to as sulfation; PS bond). Oxidation, thus forming the PO bond, is typically carried out using an aqueous solution of iodine in the presence of a weak base such as pyridine, dimethylpyridine, or trimethylpyridine. For example, oxidation can be carried out using a THF / pyridine / H2O solution containing 0.02M iodine, preferably in a 7:2:1 (w / v / v / v) ratio.

[0174] Various sulfiding agents are known and suitable for use in this invention. For example, sulfidation to form PS bonds can be carried out using 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazol-5-thione (DDTT; commercially available as Sulfiding Agent II; Glen Research), for example using a 0.1 M DDTT solution, preferably in a dry pyridine / ACN mixture, for example at a 9:1 (v / v) ratio.

[0175] In the context of this invention, sulfidation (thus forming PS bonds) is preferred over oxidation (thus forming PO bonds). Therefore, the synthesis of all-PS ASO is preferred. However, incorporating PO bonds at certain positions may be beneficial in reducing stimulation of the innate immune system.

[0176] As those skilled in the art will understand, the order of steps b-1 (deprotection), b-2 (coupling), b-3 (end-capping), and b-4 (oxidation or sulfidation, preferably sulfidation) can vary to some extent. That is, steps b-1)-b-4) can be performed in the order b-1), b-2), b-3), b-4), or in the order b-1), b-2), b-4), b-3), or in the order b-2), b-3), b-4), b-1), or in the order b-2), b-4), b-3), b-1). In the context of this invention, particularly when sulfidation is performed in step b-4), as in the preferred embodiment, steps b-1 to b-4 are preferably performed in the order b-1 (deprotection), b-2 (coupling), b-3 (end-capping), and b-4 (oxidation).

[0177] Once all synthetic cycles are complete, i.e., the ASO reaches its desired chain length, R1 can optionally be cleaved from the oligonucleotide bound to the support, i.e., an additional deprotection step can be performed while the oligonucleotide is still bound to the solid support. Cleavage of R1 is advantageous when purifying ASO by anion exchange-high performance liquid chromatography (AEX-HPLC), especially for large-scale purification. In this case, a single HPLC purification is often sufficient to obtain oligonucleotides with adequate purity for therapeutic applications, measured, for example, by AEX-HPLC or reversed-phase HPLC (RP-HPLC).

[0178] However, at smaller scales, it is often advantageous not to cleave R1 while the oligonucleotide is still bound to the solid support. In this case, a first purification is typically performed on RP-HPLC, utilizing the lipophilic protecting group present on the 5'-OH of ASO, such as the DMT protecting group, which is absent on the truncated oligonucleotide (because the unreacted 5'-OH group is capped; see step b-3), thereby facilitating the separation of ASO with the desired length from the truncated oligonucleotide (failure sequence). Then, a second purification is performed on RP-HPLC or AEX-HPLC after R1 is cleaved from the ASO obtained after the first purification (the ASO is in an unbound form because R1 is only cleaved after the ASO is released from the solid support; see step d below).

[0179] As is known to those skilled in the art, oligonucleotides, after being released from a solid support, are purified by RP-HPLC or AEX-HPLC (thereby simultaneously cleaving the protecting groups of the exocyclic amino and phosphate backbones, including PO and PS bonds; see step d below). Therefore, the method described herein includes step d), during which the oligonucleotide bound to the support (e.g., the support-linked 18-mer oligonucleotide obtained after step b) is cleaved from the solid support to obtain the oligonucleotide. The cleavage of the oligonucleotide from the solid support is known in the art and is achieved under basic conditions. Those skilled in the art will understand that cleavage of the oligonucleotide from the solid support under alkaline conditions will also result in the cleavage of the protecting groups (PO and PS) of the exocyclic amino and phosphate groups. For example, cleavage from the solid support and the cleavage of the protecting groups can be performed using ammonia, for example, at 55°C for 24 hours. Thus, the oligonucleotide is released into solution, thereby obtained in an unbound form. The resulting ASO (oligonucleotide) can be used without further purification or can be further purified, for example using AEX-HPLC or RP-HPLC, as described above in the context of optional step c).

[0180] 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.

[0181] Sixthly, the ASO that can be obtained through the production method described in this article:

[0182] In a sixth aspect, the present invention relates to an ASO represented by SEQ ID NO:1, which can be obtained by the method described above (fifth aspect). As understood in the art, the coupling of the chiral oxynitrophosphoric heterocyclopentane building units occurs in the stereochemical inversion of their chiral phosphorus atoms, i.e., the 2'-MOE oxynitrophosphoric heterocyclopentane (MOE OAP) building units with the Rp configuration (see formulas IIb-A, IIb-C, IIb-G, IIb-A) generate a PS bond with the Sp configuration upon sulfidation. According to the first aspect described above, the ASO obtained by the method described above (fifth aspect) has improved metabolic stability compared to current therapeutic ASOs (particularly nusinersen). In particular, as measured by LC-MS, at least 95%, i.e., 95%-100%, preferably 98%-100%, of the ASO obtained by the method described above retains its full length after incubation in mouse liver lysate for at least 5 days.

[0183] Preferably, as measured by LC-MS, after incubation in mouse liver lysate for at least 5 days, the full-length ASO increased by at least 5 percentage points compared to nusinersen.

[0184] It should be understood that all the definitions and characteristics of ASO described in the first aspect also apply to the sixth aspect. Attached Figure Description

[0185] The invention will be better understood with reference to the following figures:

[0186] Figure 1 The structures of Nusinersen sodium (ASO1), exemplary inventive ASOs (ASO 3, 4, 5, 6, and 10) having at least one 3' terminal PS bond with an Sp configuration, and comparative ASOs (ASO 2, 7, 8, and 9) having a 3' terminal PS bond with an Rp configuration are described. All ASOs are represented by SEQ ID NO:TCACTTTCATAATGCTGG (18-mer). All ASOs have a 2'-O-MOE sugar modification at each position, and all internucleotide bonds are PS. In all cases, C is 5mC. ASO 2 and 3 have homogeneous stereochemistry, both being Rp PS and Sp PS, respectively. ASO 4, 5, and 6 have one, two, and three chiral pure Sp PS internucleotide bonds at the 3' end, respectively, while the remaining sequences are stereo-random (i.e., not chiral controlled, i.e., a mixture of Rp PS and Sp PS bonds). ASO 7, 8, and 9 have one, two, and three Rp PS nucleotide internucleotide bonds at the 3' end, respectively, while the rest of the sequence is stereo-random. ASO 10 is a completely stereo-based Rp PS ASO with one internucleotide sp PS bond at the 3' end.

[0187] B. Analysis of ASO 1-3. UV chromatograms of ASO1 (nusinersen sodium, non-chiral controlled), ASO2 (all PS, all Rp), and ASO3 (all PS, all Sp). Therefore, in ASO2 and ASO3, all PS bonds are chiral controlled.

[0188] C. UV chromatograms of ASO2 and ASO3 co-injected in different ratios (1:2, 1:1, 2:1) confirm that PS stereochemistry affects the overall lipophilicity of oligonucleotides and demonstrates the successful synthesis of different diastereomers.

[0189] Example 2 describes the synthesis of ASO, including post-processing and purification. Example 4 describes Tm measurement. Example 5 describes the co-injection of ASO 2 and 3.

[0190] Figure 2 Time-dependent metabolic stability of ASO1 (nusinersen), ASO 4-6 (this invention), and ASO 7-9 (comparative). The relative amounts of ASO retained in the full length (i.e., 18 nucleotides) were plotted as a function of incubation time.

[0191] B. The relative amount of residual full-length ASO was plotted as a function of incubation time. A graph showing the percentage of full-length ASO and its n-1 and n-2 metabolites (i.e., 17 and 16 nucleotides, respectively; truncated from the 3' end) detected in the sample versus incubation days is presented. After 5 days of incubation in mouse liver homogenate, ASO 4, 5, and 6 (with one, two, and three stereosp PS bonds, respectively, at the 3' end) remained intact, indicating that a single Sp PS bond at the 3' end was sufficient to enhance ASO stability. ASO 7, 8, and 9 (with one, two, and three stereorogue Rp PS bonds, respectively, at the 3' end) were metabolized faster than stereorogue ASO 1 (i.e., less full-length ASO after 5 days of incubation). Samples were analyzed by LC-MS after preparation using a combination of proteinase K digestion and solid-phase extraction (SPE). Samples were prepared and analyzed blinded, with unblinded analysis performed at the end of the experiment. Error bars represent the standard deviation of three biological replicates. Experimental details are shown in the examples.

[0192] Figure 3 In vitro (cellular) assessment of fibroblast splicing conversion activity in SMA patients using ASO1 (comparative; all PS, non-chiral control; nusinersen), ASO2 (comparative; all PS, all Rp), and ASO3 (creative; all PS, all Rp). Scrapes of 0, 8, 40, or 200 nM ASO1, ASO2, and ASO3 were loaded and delivered (24 hours) into cells. Agarose gel images were used to quantify the amounts of full-length SMN2 precursor mRNA with exon 7 (SMN2 FL) and SMN2 pre-messenger ribonucleic acid without exon 7 (SMN2 d7).

[0193] Figure 4 Stereo-random (left) and chiral-controlled sp PS dinucleotides (right) 31 P NMR. A singlet was observed at 55.36 ppm for the Sp PS dinucleotide (5'-A(Sp)C-3'), indicating that the PS bond was formed with >99% stereoselectivity.

[0194] Figure 5 Chemical structure of ASO3 (all PS; all PS bonds have Sp configuration (i.e., all PS bonds are chiral controlled)).

[0195] Figure 6 The chemical structure of ASO4 (all PS; with a 3'-terminal PS bond in the Sp configuration, the other PS bonds are not chiral controlled, i.e., a mixture of Rp PS bonds and Sp PS bonds). Detailed Implementation Example

[0196] To further illustrate the present invention, the following embodiments are provided in Embodiment 6. These embodiments are not intended to limit the scope of the invention.

[0197] Example 1; Synthesis of functionalized CPG solid support

[0198] Functionalized CPG solid supports preloaded with stereopure phosphate dinucleotides (GG; Sp-type PS bonds) were prepared using 250 mg UnyLinker CPG 500 Å support. The 3'-terminal initial nucleotide was incorporated into a MOE G PA (Formula IIa-G) via a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation. A second nucleotide was incorporated using Rp G MOE OAP (Formula IIb-G) to provide stereopure Sp-PS bonds. The synthetic cycle consisted of the following sequential steps: deprotection, coupling, sulfidation, and capping.

[0199] After synthesis, the surface loading of the solid support was determined using quantitative triphenylmethyl analysis according to the following method:

[0200] Place 2 mg of the dry, functionalized solid support into a 10 mL volumetric flask. Add the detrimethylation (deprotection) reagent to the mark. Mix the flasks; an orange 4,4'-dimethoxytriphenylmethyl (DMTr) cation will immediately form. Measure the absorption spectrum from 400 to 600 nm using a UV / Vis spectrophotometer. Record the absorbance of the maximum peak from 503 to 505 nm. Calculate the support loading using the following equation:

[0201]

[0202] The loading is expressed in μmol / g, the solution volume in mL, the support weight in mg, and E is the extinction coefficient of 4,4'-dimethoxytriphenylmethyl (76 mL cm⁻¹μmol⁻¹). The measured surface loading is 9.9 μmol / g.

[0203] Example 2; Synthesis of ASO of the present invention

[0204] ASOs with the sequence TCACTTTCATAATGCTGG (SEQ ID NO:1) and 2'-MOE sugar modification at each position were prepared using a CPG 500 Å unyllinker vector (81.2 µmol / g, ChemGenes, Wilmington, Massachusetts, USA) on a MerMade 12 synthesizer from Bio Automation (BioAutomation Corp., Irving, Texas, USA). Chemicals used in the oligonucleotide synthesis were purchased from Sigma Aldrich (Stanheim, Germany), Fluorochem (Hardfield, UK), and TCI (Eschborn, Germany). The deprotecting agent was a 3% dichloroacetic acid solution in dichloromethane (DCM). Conventional 2'-O-MOE-2-cyanoethyl-N,N-diisopropylphosphonamide (MOE-PA; see formulas IIa-G, IIa-T, IIa-C, IIa-A) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA) and used as a 0.1 M solution of dry acetonitrile (ACN). Anhydrous acetonitrile (ACN) containing 0.24 M 5-(benzylthio)-1H-tetrazole (BTT; Biosolve BV, Vankenswald, Netherlands) was incorporated into MOE PA as an activator. Fully protected stereopure 2'-O-MOE-oxynitrophosphoric cyclopentanes (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 of dry ACN. Using 1.4 M N -Phenylidene imidazolium trifluoromethanesulfonate (Phenylidene imidazolium trifluoromethanesulfonate) N Dry ACN (-PhIT) was incorporated with MOE OAP as an activator. Capping reagent A (cap A; THF / dimethylpyridine / acetic anhydride, 8:1:1) and capping reagent B (cap B; 16%) were used. N End-capping was performed using imidazole / THF. Sulfidation was carried out using a dry pyridine / ACN (9:1) solution containing 0.1 M of 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazol-5-thione (DDTT; Sulfidating Agent II; Glenn Institute, Virginia). Oxidation was carried out using THF / pyridine / H₂O (70:20:10, w / v / v / v) containing 0.02 M iodine solution.

[0205] ASO1 (Comparison; Full PS, no PS key manual control; 3D random):

[0206] ASO1 was produced using 250 mg UnyLinker CPG 500 Å carrier. The initial nucleotide was incorporated into the 3' end using MOE G PA (Formula IIa-G) via a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation. Remaining nucleotides were also incorporated using their respective MOE PAs. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and capping.

[0207] The synthesis of ASO-1 was performed under the following conditions:

[0208] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0209] Coupling: MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0210] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0211] Oxidation / Sulfation:

[0212] The oxidation conditions (for the initial cycle only) were as follows: oxidant injection volume 2000 µl; contact time 480 seconds.

[0213] The vulcanization conditions (for all subsequent cycles) were as follows: DDTT injection volume 2000 µl; contact time 600 seconds.

[0214] ASO2 (Comparison, Full PS, Full RP):

[0215] ASO2 was produced using 250 mg UnyLinker CPG 500 Å carrier. The initial nucleotide was incorporated into the 3' end using MOE G PA (formula IIa-G) via a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation. The remaining nucleotides were incorporated using their respective Sp MOE OAPs (formulas IIc-G, IIc-T, IIc-C, IIc-A) to provide a stereo RpPS bond. The synthetic cycle consisted of the following sequential steps: deprotection, coupling, sulfidation, and capping.

[0216] The synthesis of ASO2 uses the following conditions:

[0217] Deprotectant injection: 2500µl of deprotectant was injected; contact time was 60s.

[0218] Coupling (for initial cycle only): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0219] Coupling (for all subsequent cycles): Sp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0220] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0221] Oxidation / Sulfation:

[0222] The oxidation conditions (for the initial cycle only) were as follows: oxidant injection volume 2000 µl; contact time 480 seconds.

[0223] The vulcanization conditions (for all subsequent cycles) were as follows: DDTT injection volume 2000 µl; contact time 600 seconds.

[0224] ASO3 (This invention; all PS, all SP):

[0225] ASO3 was prepared using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using their respective RpMOE OAPs (formulas IIb-G, IIb-T, IIb-C, IIb-A) to provide stereosp-PS bonds. The synthetic cycle consisted of the following sequential steps: deprotection, coupling, sulfidation, and end-capping.

[0226] The synthesis of ASO3 uses the following conditions:

[0227] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0228] Coupling: Rp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0229] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0230] Vulcanization: DDTT injection volume 2000µl; contact time 600 seconds.

[0231] ASO4 (This invention; all PS; 3'-terminal PS bond has Sp configuration, other PS bonds have no chiral control):

[0232] ASO4 was prepared using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using their respective MOE-PAs (formulas IIa-G, IIa-T, IIa-C, IIa-A) to provide stereo random bonds (i.e., no chiral control). The synthetic cycle consisted of the following sequential steps: deprotection, coupling, sulfidation, and end-capping.

[0233] The synthesis of ASO4 uses the following conditions:

[0234] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0235] Coupling: MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0236] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0237] Vulcanization: DDTT injection volume 2000µl; contact time 600 seconds.

[0238] ASO5 (This invention; all PS; the last two 3' terminal PS bonds have an Sp configuration, and the other PS bonds have no chiral control):

[0239] ASO5 was prepared using 250 mg of the functionalized solid support synthesized in Example 1. A second stereospPS bond was provided by incorporating the third nucleotide at the 3' end of ASO5 using Rp T MOEOAP (formula IIb-T). The remaining nucleotides were incorporated using their respective MOE PAs (formulas IIa-G, IIa-T-IIa-C, IIa-A) to provide stereorandom bonds. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and end-capping.

[0240] The synthesis of ASO5 was performed under the following conditions:

[0241] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0242] Coupling (for the first cycle only): Rp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0243] Coupling: MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0244] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0245] Vulcanization: DDTT injection volume 2000µl; contact time 600 seconds.

[0246] ASO6 (This invention; all PS; the last three 3' PS bonds have an Sp configuration, and the other PS bonds have no chiral control):

[0247] ASO6 was prepared using 250 mg of the functionalized solid support synthesized in Example 1. The first stereospPS bond was provided by incorporating the second nucleotide at the 3' end using Rp G MOE OAP (Formula IIb-G). The second stereospPS bond was provided by incorporating the third nucleotide at the 3' end using Rp T MOE OAP (Formula IIb-T). The third stereospPS bond was provided by incorporating the fourth nucleotide at the 3' end using Rp C MOE OAP (Formula IIb-C). The remaining nucleotides were incorporated using their respective MOE-PAs (Formulas IIa-G, IIa-T-IIa-C, IIa-A) to provide stereorandom bonds. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and end-capping.

[0248] The synthesis of ASO6 was performed under the following conditions:

[0249] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0250] Coupling (for first and second cycles only): Rp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0251] Coupling: MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0252] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0253] Vulcanization: DDTT injection volume 2000µl; contact time 600 seconds.

[0254] ASO7 (Comparison; all PS, the 3' terminal PS bond has an Rp configuration, other PS bonds have no chiral control):

[0255] ASO7 was produced using 250 mg UnyLinker CPG 500 Å carrier. The initial nucleotide at the 3' end was incorporated into a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation using MOE G PA (formula IIa-G). A second nucleotide at the 3' end was incorporated using Sp G MOE OAP (formula IIc-G), providing the first stereo Rp PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (formulas IIa-G, IIa-T-IIa-C, IIa-A), providing stereo random bonds. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and capping.

[0256] The synthesis of ASO7 uses the following conditions:

[0257] Deprotection: 2500µl of deprotectant was injected; three subsequent deprotection steps, each with a contact time of 60 seconds.

[0258] Coupling (for initial cycle only): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0259] Coupling (for the second cycle only): Sp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0260] Coupling (for all subsequent cycles): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0261] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0262] Oxidation or sulfidation:

[0263] The oxidation conditions (for the initial cycle only) were as follows: oxidant injection volume 2000 µl; contact time 480 seconds.

[0264] The vulcanization conditions (for all subsequent cycles) were as follows: DDTT injection volume 2000 µl; contact time 600 seconds.

[0265] ASO8 (Comparison; all PS, the last two 3' terminal PS bonds have Rp configuration, the other PS bonds have no chiral control):

[0266] ASO8 was produced using 250 mg UnyLinker CPG 500 Å carrier. The initial nucleotide at the 3' end was incorporated into a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation. A second nucleotide at the 3' end was incorporated using Sp G MOE OAP (Formula IIa-G), providing the first stereo Rp PS bond. A third nucleotide at the 3' end was incorporated using Sp T MOE OAP (Formula IIc-T), providing the second stereo Rp PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (Formula IIa-G, IIa-T-IIa-C, IIa-A), providing stereo random bonds. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and capping.

[0267] The synthesis of ASO-5 was performed under the following conditions:

[0268] Deprotection: 2500µl of deprotectant was injected; three subsequent deprotection steps, each with a contact time of 60 seconds.

[0269] Coupling (for initial cycle only): MOE PA injection volume 600 μl, BTT injection volume 1200 µl; contact time 360 ​​seconds.

[0270] Coupling (for the second and third cycles only): Sp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0271] Coupling (for all subsequent cycles): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0272] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0273] Oxidation or sulfidation:

[0274] The oxidation conditions (for the initial cycle only) were as follows: oxidant injection volume 2000 µl; contact time 480 seconds.

[0275] ASO9 (Comparison; all PS, the last three 3' PS keys have Rp configuration, the other PS keys have no chiral control):

[0276] ASO9 was produced using 250 mg UnyLinker CPG 500 Å carrier. The initial nucleotide at the 3' end was incorporated into a cycle comprising subsequent steps: deprotection, coupling, capping, and oxidation using MOE G PA (Formula IIa-G). A second nucleotide at the 3' end was incorporated using Sp G MOE OAP (Formula IIc-G), providing the first stereo Rp PS bond. A third nucleotide at the 3' end was incorporated using Sp T MOE OAP (Formula IIc-T), providing the second stereo Rp PS bond. A fourth nucleotide at the 3' end was incorporated using Sp C MOE OAP (Formula IIc-C), providing the third stereo Rp PS bond. The remaining nucleotides were incorporated using their respective MOE PAs (Formula IIa-G, IIa-T-IIa-C, IIa-A), providing stereo random bonds. The synthetic cycle consisted of the following steps in sequence: deprotection, coupling, sulfidation, and capping.

[0277] The synthesis of ASO-6 was performed under the following conditions:

[0278] Deprotection: 2500µl of deprotectant was injected; three subsequent deprotection steps, each with a contact time of 60 seconds.

[0279] Coupling (for initial cycle only): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0280] Coupling (for the second, third, and fourth cycles only): Sp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0281] Coupling (for all subsequent cycles): MOE PA injection volume 600µl, BTT injection volume 1200µl; contact time 360 ​​seconds.

[0282] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0283] Oxidation / Sulfation:

[0284] The oxidation conditions (for the initial cycle only) were as follows: oxidant injection volume 2000 µl; contact time 480 seconds.

[0285] The vulcanization conditions (for all subsequent cycles) were as follows: DDTT injection volume 2000 µl; contact time 600 seconds.

[0286] ASO10 (This invention; all PS, 3'-terminal PS bond Sp configuration, the remaining PS bonds have Rp configuration):

[0287] ASO10 was prepared using 250 mg of the functionalized solid support synthesized in Example 1. The remaining nucleotides were incorporated using their respective Sp MOE OAPs (formulas IIc-G, IIc-T, IIc-C, IIc-A) to provide stereo Rp PS bonds. The synthetic cycle consisted of the following sequential steps: deprotection, coupling, sulfidation, and end-capping.

[0288] The synthesis of ASO10 was performed under the following conditions:

[0289] Deprotection: The deprotectant was injected in a volume of 2500 µl; the contact time was 60 s.

[0290] Coupling: Sp MOE OAP injection volume 500µl, N -PhIT injection volume 1000µl; two subsequent couplings, each contact time 360 ​​seconds.

[0291] End capping: Cap A injection volume 1400µl, Cap B injection volume 1400µl; contact time 90 seconds.

[0292] Vulcanization: DDTT injection volume 2000µl; contact time 600 seconds.

[0293] Splicing and Deprotection from the Solid Support: After synthesis, ASO was spliced ​​from the solid support, and the protecting group was removed at 55 °C with 300 µl of ammonia for 24 hours. The reaction was cooled to room temperature, the solid support was filtered, and the mixture was washed with 400 µl of ethanol (EtOH) and 1:1 (v / v) H₂O. The filtrate was concentrated to dryness, and the residue was dissolved in 200 µl of water.

[0294] Purification: The oligonucleotides were purified by RP-HPLC on an Agilent 1200 series HPLC system equipped with a WatersXbridge OST C-18 column (10 × 50 mm, 2.5 μm) at 65 °C. HPLC purification run buffer: Buffer A – 0.1 M triethylamine acetate, Buffer B – acetonitrile, pH 8.0. DMT-ON purification gradient (i.e., before splicing of the lipophilic DMT-protecting group): Add 5–80% Buffer B over 8 minutes. Combine the fractions, dry in a SpeedVac, and treat with 40% acetic acid for 15 minutes at room temperature for DMT deprotection. After drying in a SpeedVac, dissolve the oligonucleotides in H2O and perform a second DMT-OFF purification on RP-HPLC. DMT-OFF purification gradient: Use 5–35% Buffer B over 6 minutes. Collect the fraction containing the product and dry in a SpeedVac. ASO was analyzed by LC-MS (Agilent 1200 / 6130 system) on a Waters Acquity OST C-18 column (2.1 x 50 mm, 1.7 μm, 65 °C). Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: Buffer B 10-50% for 15 min; Flow rate: 0.3 mL / min.

[0295] Example 3; Chiral control of the stereopurity of PS bonds

[0296] To confirm the stereopurity of PS MOE dinucleotides, dinucleotides with the sequence 5'-AC-3' were subjected to... 31 PNMR analysis revealed that the dinucleotide possessed a single PS bond with an Sp configuration and its stereorandom variant (the PS bond was not chiral-controlled). The dinucleotide was synthesized on a commercially available CPG 500 Å unyllinker support. As described in Example 1, the dinucleotide bound to the support was detriphenylmethylated (removing 5'-DMT) and treated with concentrated ammonium hydroxide at 55°C for 24 hours to release the "DMT-off" dimer from the solid support. In a subsequent step, the obtained product was filtered, and the filtrate (containing the unbound form of the dinucleotide) was evaporated under reduced pressure. The remaining residues were then dissolved in deuterium oxide (0.6 mL) and transferred to an NMR tube for analysis. The stereorandom dinucleotide was first measured. 31 P NMR spectra. Note the clear separation of signals from the two diastereomers of thiophosphate (see P NMR spectra). Figure 4The RpPS and SpPS configurations of the thiophosphate ester bonds were determined based on literature reports [VT Ravikumar, DLCole, Nucleosides, Nucleotides & Nucleic Acids (2003), 22, 1639]. Therefore, 31 The high-field peaks in the p NMR spectrum are designated as the Sp PS configuration, and the low-field peaks are designated as the Rp PS configuration.

[0297] Stereo-pure SpPS dinucleotides 31 The p NMR spectrum showed a singlet at 55.36 ppm, which was accordingly designated as the SpPS configuration. This result indicates that the formation of PS bonds has a stereoselectivity of >99%.

[0298] In addition, Li et al. [Chem. Commun. 2017, 53, 541] previously demonstrated the stereopurity of various MOE OAPs (including G-containing dinucleotides) conjugated to the 5' end of stereopure PS ASO precursor mRNA.

[0299] Example 4; Thermal denaturation study (hybridization affinity)

[0300] The melting temperatures (Tm) of ASO 1, 2, and 3 with complementary PO RNA strands were measured on a CARY 300 (Agilent) equipped with a thermal controller. ASO 1–3 were bound with their corresponding reverse strands (unmodified RNA, full PO) to produce an equimolar concentration of 1 μM in phosphate-buffered saline (5 mM Na₂HPO₄, 5 mM NaH₂PO₄, 100 mM NaCl, 0.1 mM EDTA). Absorbance was measured at 260 nm in 160 µl quartz tubes. The temperature gradient was set at 0.5 K / min over a range of 20 °C to 95 °C. Absorbance readings were taken every 30 seconds. Each series was performed three times. Holding times were set to 5 minutes at 95 °C and 20 °C, respectively, to ensure thermal equilibrium. The melting curves conformed to an S-shaped curve. Tm was calculated from the maximum of the first derivatives of the three melting curves. The following results were obtained (see [link to results]). Figure 1 ):

[0301] ASO1: 63.0℃

[0302] ASO2: 63.4℃

[0303] ASO3: 55.3℃

[0304] Therefore, the Tm of ASO2 (18mer, all pS, all Rp) is 8.1℃ higher than that of ASO3 (18mer, all pS, all Sp).

[0305] Furthermore, compared to ASO1 (18mer all-PS, non-chiral control), ASO2 has a Tm per PS bond that is approximately 0.2 °C higher. Conversely, ASO3 has a Tm that is approximately 0.3 °C lower than that of stereo-random ASO1. This data suggests the advantages of having some PS bonds (other than the 3' terminal PS bonds mentioned above) or no chiral control (such as ASO4, 5, and 6) in Rp configurations (such as ASO10), as shown in the preferred embodiments.

[0306] Example 5; LC-MS co-injection analysis of fully stereochemical pure ASO2 and ASO3

[0307] ASO2 and ASO3 were prepared at a concentration of 10 µM. Subsequently, three different schemes were created: 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 x 50 mm, 1.7 μm, 65 °C). Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: Buffer B 10-50% 20 min; Flow rate: 0.3 ml / min, injection volume 5 µL. The resulting chromatogram showed two distinct peaks, further confirming the stereochemical purity of the synthesized ASO. The corresponding UV traces are shown below. Figure 1 As shown in C.

[0308] Example 6; Metabolic stability of mouse whole tissue homogenate

[0309] The homogenization buffer consisted of 100 mM Tris-HCl and 1 mM magnesium acetate (pH 8.0, adjusted with HCl). Postmortem liver samples from wild-type Black 6 mice (C57BL / 6J) were collected at 19 weeks of age after carbon dioxide euthanasia and cervical dislocation. 217 mg of untreated mouse liver was placed in Eppendorf tubes, and 434 μL of homogenization buffer was added. These were mechanically homogenized using TissueLyser II (QIAGEN). The resulting homogenates were further diluted with homogenization buffer to prepare several 200 μL aliquots of liver homogenate. Following an optimized ratio, 5 μL of liver homogenate (2.5% of the total volume) was added to 195 μL of buffer. The rate of formation of short chains of nusinersen sodium was determined by incubating liver homogenate (200 μL) with ASO (25 μL 180 μM) for 0, 24, 72, and 120 hours with gentle shaking (400 rpm) at 37 °C using an Eppendorf (Hauppauge, New York, USA) Thermomixer-R.

[0310] Incubation was quenched by adding the incubation mixture (225 μl) to 62 μl of Master-Pure tissue lysis solution (Epicenter, Wisconsin, USA) and 6 μl of proteinase K (20 mg / ml, Roche). Proteinase K digestion was performed at 55 °C for 1 hour. Samples were then treated with solid-phase extraction (SPE).

[0311] After proteinase K digestion, SPE was performed using the Phenomenex Clarity OTX kit (Phenomenez, Torrance, California, USA). Tissue samples were mixed with 600 μL of Clarity OTX buffer, vortexed for 5 min, and incubated at room temperature for another 5 min. The SPE column was conditioned with 1 mL methanol and 1 mL equilibration buffer. Tissue samples were loaded onto the column. The kit was washed with 3 x 1 mL equilibration buffer and 3 x 1 mL wash buffer, and the analytes were eluted with 2 x 0.5 mL elution buffer. The collected solution was evaporated under vacuum for 1 h and analyzed directly by LC-MS (Agilent 1200 / 6130 system) on a Waters Acquity OST C-18 column (2.1 x 50 mm, 1.7 μm, 65 °C). Buffer A: 0.4 M HLIP, 15 mM triethylamine; Buffer B: MeOH. Gradient: Buffer B 10-50% for 15 min; flow rate: 0.3 ml / min, injection volume 10 μL. Mass spectrometry analysis 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 to m / z 1000.

[0312] Data showed that ASO 4-6, i.e., ASOs with at least one 3' terminal PS bond in the Sp configuration, exhibited greater metabolic stability than their completely stereorandom counterparts (ASO1) and ASOs with a 3' terminal PS bond in the Rp configuration (ASO 7-9). Metabolic stability in mouse whole tissue homogenates indicates in vivo metabolic stability. Data are shown in Table 1 below. Figure 2 As shown.

[0313] Table 1. Time-dependent metabolic stability of ASO1 (comparative; all-PS, stereo-randomized; nusinersen) compared to ASO 4-6 (inventive; all-PS, with one, two, and three 3'-terminal PS bonds of the Sp configuration, respectively) and ASO 7-9 (comparative; all-PS, with one, two, and three 3'-terminal PS bonds of the Rp configuration, respectively) in mouse liver homogenates. Data are reported as the mean of three biological replicates.

[0314] Example 7; Fibroblast Splicing Correction Experiment in SMA Patients

[0315] The oligonucleotides were resuspended in nuclease-free water. Fibroblasts derived from type II SMA patients (GM03813; Coriell Institute of Medicine) were maintained in standard medium (DMEM + GlutaMax, 10% fetal bovine serum (FBS)). Once cells reached approximately 80% confluence in T75 flasks, they were washed once with PBS and digested with 2 mL of trypsin-EDTA. After incubation at 37°C for 3 minutes, 3 mL of DMEM-10%FBS was added, and cell counts were performed. Cells were seeded at 120,000 or 150,000 cells per well in 6-well plates and incubated overnight at 37°C and 5% CO2.

[0316] For scraping delivery, add ASO (concentration shown in the figure) to 1 mL of fresh culture medium. Scrape cells with a cell scraper S (TPP), resuspend them by pipetting, and transfer them to Eppendorf tubes for centrifugation at 250 RCF for 5 minutes. Resuspend the cells in 1 mL of fresh culture medium, transfer them to new 6-well plates, and incubate for another 48 hours.

[0317] Forty-eight hours later, RNA was extracted using 500 µL of Trizol reagent. 100 µL of cold chloroform was added to the sample, vortexed thoroughly, and centrifuged at 12,000 x g for 15 minutes at 4 °C. The aqueous phase was harvested, and an equal volume of anhydrous ethanol was added. The solution was mixed by pipetting and then transferred to a Zymo Spin IC column, vortexed, and the flow-through was discarded. 400 µL of RNA preparation buffer was added, vortexed, and the flow-through was discarded. Finally, 700 µL of RNA wash buffer was added, vortexed for 2 minutes, and the flow-through was discarded. The sample was eluted with 15 µL of nuclease-free water, and RNA concentration was measured using NanoDrop. Reverse transcription was performed using the Lunascript RT SuperMix kit with 1 µg of total RNA. The reverse transcription reaction was performed in a thermal cycler. RT program: Step 1: 25 °C (2 min), 55 °C (10 min), 95 °C (1 min). For gel-based splicing analysis, PCR amplification of 50 ng cDNA was performed using SMN2 primers spanning exon 7 (see Table 2). PCR program: Step 1: 95℃ (2 min); Step 2: 95℃ (30 s); Step 3: 65℃ (30 s); Step 4: 74℃ (30 s); Step 5: Repeat steps 2-4 25 times; Step 6: 74℃ (5 min). PCR products were loaded onto 2% agarose gels in TAE buffer and 1 / 10000 GelRed nucleic acid staining agent (41003, Chemie Brunschwig) and run at 100V for 1 hour.

[0318] Table 2. qPCR primers used for semi-quantitative measurement of SMN2 FL and Δ7 mRNA levels.

[0319] The results are as follows Figure 3 As shown. Data shows that ASO2 (in this invention; all PS, all Rp) has superior activity in cells compared to ASO3 (comparative; all PS, all Sp). As those skilled in the art will know, the ASOs of this invention (e.g., ASO 3, 4, 5, 6, 10; see Example 6;) Figure 2 Increased metabolic stability in vivo may affect the potency of the ASO in vivo, but it is not expected to provide any additional advantage in cell experiments, as the ASO is not exposed to the harsh conditions encountered in tissues in cell experiments.

Claims

1. An antisense oligonucleotide comprising SEQ ID NO:1 or a pharmaceutically acceptable salt thereof, The antisense oligonucleotides described therein have a 2'-O-methoxyethyl sugar modification on each of their nucleotides and have an inter-thiophosphate bond of the Sp configuration at the 3' end.

2. The antisense oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide contains only phosphate thioester bonds.

3. The antisense oligonucleotide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide further has at least one or at least two chiral internucleotide thiophosphate bonds having an Sp configuration, the Sp configuration being continuous with the chiral internucleotide thiophosphate bond at the 3'-end.

4. The antisense oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof. All of its cytosine residues are 5-methylcytosine, and The antisense oligonucleotides mentioned above include: A phosphate thioester bond between chiral nucleotides with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral-controlled phosphate thioester bonds; or A phosphate thioester bond between two consecutive chiral nucleotides with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral-controlled phosphate thioester bonds; or Phosphothioester bonds are formed between three consecutive chiral nucleotides with an Sp configuration at the 3' end, while all other internucleotide bonds are achiral-controlled phosphothioester bonds; or One internucleotide thiophosphate bond is a chiral nucleotide bond with the Sp configuration at the 3'-end, while all other internucleotide bonds are chiral controlled thiophosphate bonds with the Rp configuration.

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

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and The antisense oligonucleotide and / or its pharmaceutically acceptable salt according to any one of claims 1 to 4, or The various antisense oligonucleotides and / or their pharmaceutically acceptable salts as described in claim 5.

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

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

9. The antisense oligonucleotide according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or The composition according to claim 5, or The pharmaceutical composition according to claim 6, or The kit according to claim 7, Used to treat spinal muscular atrophy.

10. A method for promoting the inclusion of exon 7 in an SMN2 transcript in a cell, tissue, or organ, comprising contacting said cell, tissue, or organ in vitro with an antisense oligonucleotide of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a composition of claim 5, or a pharmaceutical composition of claim 6.

11. A method for preparing antisense oligonucleotides according to any one of claims 1 to 4, comprising the following steps: a) Provide a functionalized solid support represented by formula I, Formula I ， Where L is the connector, preferably selected from: and ; SS stands for solid support; Ar1 is a phenyl group, which may be optionally substituted with a halogen, a C1-C6 alkyl, a C3-C6 cycloalkyl or a C1-C4 alkoxy group; X is selected from sulfur and oxygen; R1 represents a protecting group for hydrogen or hydroxyl groups, selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanol-9-yl and 9-(p-methoxyphenyl)xanol-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethylthiotriphenylmethyl; R2 is selected from methyl and phenyl; R3 is selected from hydrogen, acetyl, and trifluoroacetyl; b) The following series of reactions will occur: The functionalized solid support of formula I is reacted with nucleoside building blocks of formula (IIa-T), (IIb-T), or (IIc-T) to obtain 3-mer oligonucleotides bound to the support, and then... The 3-mer oligonucleotide bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a 4-mer oligonucleotide bound to the support, subsequently... The 4-mer bound to the support is reacted with a nucleoside building block of formula (IIa-G), (IIb-G), or (IIc-G) to obtain a 5-mer oligonucleotide bound to the support, and then... The 5-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 6-mer oligonucleotide bound to the support, and then... The 6-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 7-mer oligonucleotide bound to the support, and then... The 7-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain an 8-mer oligonucleotide bound to the support, and then... The 8-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 9-mer oligonucleotide bound to the support, and then... The 9-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 10-mer oligonucleotide bound to the support, and then... The 10-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain an 11-mer oligonucleotide bound to the support, and then... The 11-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 12-mer oligonucleotide bound to the support, and then... The 12-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 13-mer oligonucleotide bound to the support, and then... The 13-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain a 14-mer oligonucleotide bound to the support, and then... The 14-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a 15-mer oligonucleotide bound to the support, and then... The 15-mer bound to the support is reacted with a nucleoside building block of formula (IIa-A), (IIb-A), or (IIc-A) to obtain a 16-mer oligonucleotide bound to the support, and then... The 16-mer bound to the support is reacted with a nucleoside building block of formula (IIa-C), (IIb-C), or (IIc-C) to obtain a 17-mer oligonucleotide bound to the support, and then... The 17-mer bound to the support is reacted with a nucleoside building block of formula (IIa-T), (IIb-T), or (IIc-T) to obtain an 18-mer oligonucleotide bound to the support containing SEQ ID NO:

1. The reaction includes the following steps: b-1) Deprotection, b-2) Coupling, b-3) End capping, and b-4) Oxidation or sulfidation, Proceed in the following order: 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); c) Optionally, R1 is cleaved from the oligonucleotide bound to the support; and d) Cutting the 18-mer oligonucleotides bound to the solid support from the solid support to obtain antisense oligonucleotides. The nucleic acid building 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 respectively represented by the following formulas: Formula (IIa-G) Formula (IIa-T) Formula (IIa-C) Formula (IIa-A) Formula (IIb-G) Formula (IIb-T) Formula (IIb-C) Formula (IIb-A) Formula (IIc-G) Formula (IIc-T) Formula (IIc-C) Formula (IIc-A) Wherein Ar2 is a phenyl group, optionally substituted with a halogen, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or a C1-C4 alkoxy group, and R4 represents a protecting group for a hydroxyl group, selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenyl-tonol-9-yl and 9-(p-methoxyphenyl)tonol-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethylthiotriphenylmethyl.

12. The method of claim 11, wherein step a) comprises a-1) Provide a solid support represented by Equation III Formula III , Where L is selected from: and ; SS stands for Solid Support; R1 represents a protecting group for hydrogen or hydroxyl groups, selected from triphenylmethyl, 4-monomethoxytriphenylmethyl, 4,4'-dimethoxytriphenylmethyl, 4,4',4''-trimethoxytriphenylmethyl, 9-phenylxanol-9-yl and 9-(p-methoxyphenyl)xanol-9-yl, preferably selected from 4,4'-dimethoxytriphenylmethyl and 4-monomethylthiotriphenylmethyl; R2 is selected from methyl and phenyl; then a-2) React the nucleoside building block of formula (IIa-G) with the solid support of formula III to obtain a nucleotide bound to the support, then The nucleoside building block of formula (IIb-G) is reacted with a nucleotide bound to the support to obtain a 2-mer oligonucleotide bound to the support.

13. An antisense oligonucleotide represented by SEQ ID NO:1, which can be obtained by the method according to claim 11 or 12.

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

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

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