Oligonucleotide and use thereof

Abstract

An oligonucleotide capable of reducing SCN10A expression and a use.

Description

Oligonucleotides and their applications Technical Field

[0001] This invention belongs to the field of oligonucleotide drugs, specifically relating to oligonucleotides that can reduce SCN10A expression and their uses. Background Technology

[0002] Ion channels are closed intrinsic membrane proteins containing water pores that regulate the voltage potential on the cell membrane by modulating the ion flow across the cell. Studies have shown that alterations in ion channels are the molecular basis for peripheral sensitization, central sensitization, and disinhibition following inflammation or neuropathological injury, and are also an important molecular mechanism in the development of pain. There are nine types of sodium ion channels in humans, Nav1.1–Nav1.9. Among them, Nav1.8, encoded by the SCN10A (Sodium Voltage-Gated Channel Alpha Subunit 10) gene, is mainly expressed in the trigeminal ganglion (TRG) and dorsal root ganglion (DRG), and is an important ion channel involved in chronic neuropathic and chronic inflammatory pain, making it a highly selective target for pain treatment.

[0003] More than one billion people worldwide suffer from pain, and this number is increasing by hundreds of millions each year. Pain has become the third leading health problem after cardiovascular disease and cancer. The pathogenesis of pain is relatively complex, resulting in unsatisfactory treatment effects from many approaches, such as the persistent pain caused by osteoarthritis and the severe chronic pain caused by cancer. Existing pain medications often suffer from problems such as low tolerability, poor long-term safety, and potential drug abuse. For example, opioids are commonly used to treat pain, and while they are very effective pain medications, the abuse of these addictive drugs can cause social problems. Some people do not respond to or cannot tolerate the side effects on the digestive system and kidney function caused by nonsteroidal anti-inflammatory drugs (NSAIDs). Other serious drawbacks of NSAID and opioid treatments include high mortality rates.

[0004] Therefore, there is an urgent need for highly effective and safe drugs to treat pain. It is anticipated that targeting the SCN10A gene with siRNA or ASO drugs to achieve specific and efficient inhibition of the target gene mRNA or pre-mRNA and protein could provide beneficial effects in the treatment and prevention of acute and chronic pain. Currently, there are no small nucleic acid drugs on the market targeting this site, and further development of such drugs with better efficacy, long-lasting effects, specific targeting, and / or safety is still needed. Summary of the Invention

[0005] This invention relates to oligonucleotides for the treatment or prevention of pain, wherein the oligonucleotides inhibit the expression of Nav1.8 in cells expressing Nav1.8. The oligonucleotides are capable of inhibiting the expression of the target gene SCN10A in mammalian cells, particularly inhibiting the expression of human SCN10A mRNA or precursor mRNA.

[0006] In a first aspect, the present invention relates to an oligonucleotide capable of inhibiting the expression of SCN10A (Nav1.8), wherein the oligonucleotide comprises at least a first nucleotide region comprising a region at least partially complementary to at least a portion of RNA transcribed from the SCN10A gene, and wherein the at least partially complementary region comprises at least 15 consecutive nucleotides differing from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3 nucleotides.

[0007] In some embodiments, the oligonucleotide further comprises a second nucleotide region that is at least partially complementary to the first nucleotide region.

[0008] In some embodiments, the oligonucleotide comprises a first nucleotide region and a second nucleotide region, the first nucleotide region comprising a region at least partially complementary to at least a portion of the RNA transcribed from the SCN10A gene, and wherein the at least partially complementary region comprises at least 15 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3 nucleotides.

[0009] In some implementations, the second nucleotide region is complementary or substantially complementary to at least a portion of the first nucleotide region to form a double strand.

[0010] In some embodiments, the second nucleotide region comprises at least 15 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 3 nucleotides.

[0011] In some embodiments, the first nucleotide region contains 18-35 nucleotides, preferably 18-30, 18-23, or 19-25 nucleotides, for example, 18, 19, 20, 21, 22, or 23 nucleotides.

[0012] In some embodiments, the first nucleotide region contains 18-35 nucleotides, preferably 18-23 nucleotides, for example, 18, 19, 20, 21, 22 or 23 nucleotides.

[0013] In some embodiments, the second nucleotide region contains 16-35 nucleotides, preferably 18-30, 16-23, or 19-25 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0014] In some embodiments, the second nucleotide region contains 16-35 nucleotides, preferably 16-23 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides.

[0015] In some embodiments, the oligonucleotide includes at least one complementary double-stranded region, the complementary double-stranded region including at least a portion of the first nucleotide region, the first nucleotide region being directly or indirectly linked to at least a portion of the second nucleotide region to form a double strand.

[0016] In some implementations, the double-stranded region is 10-25, 10-19, 12-19, 12-15, or 19-23 base pairs long.

[0017] In some implementations, the double-stranded region is 19, 20, 21, 22, or 23 base pairs long.

[0018] In some embodiments, the oligonucleotide includes at least one complementary double-stranded region, the complementary double-stranded region including at least a portion of the first nucleotide region, the first nucleotide region being directly or indirectly linked to at least a portion of the second nucleotide region, the double-stranded region being 10-19, 12-19, or 12-15 base pairs long, wherein optionally, there is a mismatch within the double-stranded region.

[0019] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 15 consecutive nucleotides selected from any one of SEQ ID NO: 1-502.

[0020] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 18, 19, 20, 21, 22, or 23 consecutive nucleotides selected from any one of SEQ ID NO: 1-502.

[0021] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 18 nucleotides selected from any one of SEQ ID NO: 1-502.

[0022] In some implementations, the first nucleotide region is 19-23 nucleotides in length, for example, 19, 20, 21, 22, or 23 nucleotides in length.

[0023] In some embodiments, the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3, 2, 1, or 0 nucleotides.

[0024] In some embodiments, the first nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 2, 1, or 0 nucleotides.

[0025] In some embodiments, the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3, 2, 1, or 0 nucleotides.

[0026] In some embodiments, the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 3, 2, 1, or 0 nucleotides.

[0027] In some embodiments, the second nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 2, 1, or 0 nucleotides.

[0028] In some embodiments, the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 3, 2, 1, or 0 nucleotides.

[0029] In some embodiments, the first nucleotide region comprises the sequence of any one of SEQ ID NO: 503-1004; and the second nucleotide region comprises the sequence of any one of SEQ ID NO: 1-502.

[0030] In some implementations, the first nucleotide region and the second nucleotide region form a double strand comprising any of the nucleic acid sequence pairs shown in Table A.

[0031] In some embodiments, the oligonucleotide has a nucleotide duplex as shown in any of Table A, having no more than 3 (preferably no more than 1 or 2, more preferably 1) nucleotide differences.

[0032] In some implementations, each of the first nucleotide region and the second nucleotide region has a 5' to 3' orientation, thereby defining a 5' region and a 3' region, respectively.

[0033] In some embodiments, the 5' region of the first nucleotide region is directly or indirectly connected to the 3' region of the second nucleotide region, for example, through complementary base pairing, wherein, preferably, the 5' terminal nucleotide of the first nucleotide region is paired with the 3' terminal nucleotide of the second nucleotide region.

[0034] In some embodiments, the 3' region of the first nucleotide region is directly or indirectly connected to the 5' region of the second nucleotide region, wherein, preferably, the first nucleotide region is directly or indirectly connected to the second nucleotide region via, for example, a phosphate ester, a thiophosphate ester, or a dithiophosphate ester.

[0035] In some embodiments, the first nucleotide region contains one or more modified nucleotides, and optionally, one or more nucleoside bonds are modified nucleoside bonds.

[0036] In some embodiments, the second nucleotide region contains one or more modified nucleotides, and optionally, one or more nucleoside bonds are modified nucleoside bonds.

[0037] In some implementations, all nucleotides in the first nucleotide region are modified nucleotides.

[0038] In some implementations, all nucleotides in the second nucleotide region are modified nucleotides.

[0039] In some embodiments, all nucleotides of the oligonucleotide are modified nucleotides.

[0040] In some embodiments, the modification includes at least one nucleotide selected from the group consisting of 2'-fluorine, hexitol nucleotides, lock nucleotides, cyclohexene nucleotides, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxy, and diol-modified nucleotides; and combinations thereof.

[0041] In some embodiments, the modified nucleotide is independently selected from at least one of the following: 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-hexadecyl-modified nucleotides, 2'-deoxynucleotides, debased nucleotides, reverse debased nucleotides, deoxynucleotides, locked nucleotides, 2'-aminonucleotides, 2'-hydroxynucleotides, 2'-O-allyl nucleotides, 2'-C 1-25 Alkyl nucleotides, 2'-OC1-25 Alkyl nucleotides, 2'-methoxyethyl nucleotides, configuration-restricted nucleotides, 2'-allyl nucleotides, morpholino nucleotides, tetrahydropyran-modified nucleotides, hexitol nucleotides, 1,5-dehydrated hexitol nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate nucleotides, 5'-thiophosphate nucleotides, 5'-methylphosphonate nucleotides, nucleotides containing vinyl phosphates, 5'-methylcytosine-modified nucleotides, diol-modified nucleotides, and nucleic acid analogs; and combinations thereof.

[0042] In some embodiments, the modified nucleotide includes at least one modified nucleotide selected from 2'-fluoromodified nucleotides, vinyl phosphate nucleotides, reverse debased nucleotides, locked nucleotides, 2'-methoxynucleotides, and 5'-thiophosphate nucleotides.

[0043] In some embodiments, the modified nucleotide includes at least one modified nucleotide selected from 2'-fluoro-modified nucleotides (2'-fluoronucleotides), 2'-methoxy-modified nucleotides (2'-methoxynucleotides), 2'-O-hexadecyl-modified nucleotides (2'-O-hexadecylnucleotides), 2'-deoxynucleotides, nucleotides containing vinyl phosphate, reverse debased nucleotides, locked nucleotides, and 5'-thiophosphate nucleotides.

[0044] In some implementations, the first nucleotide region comprises a 3' overhang of 2 nucleotides.

[0045] In some embodiments, the one or more modified nucleotides are independently 2'-fluoromodified nucleotides or 2'-methoxymodified nucleotides.

[0046] In some embodiments, the one or more modified nucleotides are independently 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, or 2'-O-hexadecyl-modified nucleotides.

[0047] In some embodiments, the one or more modified nucleotides are independently 2'-O-hexadecyluracil (Uhd), 2'-O-hexadecyladenine (Ahd), 2'-O-hexadecylcytosine (Chd), or 2'-O-hexadecylguanine (Ghd).

[0048] In some embodiments, the first nucleotide region and / or the second nucleotide region contains one or more modified nucleoside inter-bonds; the modified nucleoside inter-bonds are thiophosphate or dithiophosphate nucleoside inter-bonds.

[0049] In some embodiments, the modified nucleoside inter-bond is a thiophosphate nucleoside inter-bond.

[0050] In some embodiments, the first nucleotide region and / or the second nucleotide region contains one or more nucleotide internucleotide bonds modified with thiophosphate.

[0051] In some embodiments, the first nucleotide region and / or the second nucleotide region independently contain 0, 1, 2, 3, 4 or 5 phosphate thioester nucleoside bonds.

[0052] In some implementations, the first nucleotide region and / or the second nucleotide region independently contain 3-5 phosphate thioester nucleoside bonds.

[0053] In some implementations, the first nucleotide region and / or the second nucleotide region independently contain 4 or 5 phosphate thioester nucleoside bonds.

[0054] In some embodiments, the nucleotide at the 5' end of the first nucleotide region has a phosphate ester group or a phosphate ester analog.

[0055] In some embodiments, the nucleotide at the 5' end of the first nucleotide region has a 5' phosphonate, such as 5' methylene phosphonate (5'-MP) or 5'-(E)-vinylphosphonate (5'-VP).

[0056] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 15 consecutive nucleotides selected from any one of SEQ ID NO: 1-502.

[0057] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 18, 19, 20, 21, 22, or 23 consecutive nucleotides selected from any one of SEQ ID NO: 1-502.

[0058] In some embodiments, the first nucleotide region comprises a sequence complementary to at least 18 nucleotides selected from any one of SEQ ID NO: 1-502.

[0059] In some implementations, the first nucleotide region is 19-23 nucleotides in length, for example, 19, 20, 21, 22, or 23 nucleotides in length.

[0060] In some embodiments, the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 1499-1992 by no more than 3, 2, 1 or 0 nucleotides.

[0061] In some embodiments, the first nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1499-1992 by no more than 2, 1, or 0 nucleotides.

[0062] In some embodiments, the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1499-1992 by no more than 3, 2, 1, or 0 nucleotides.

[0063] In some embodiments, the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 1005-1498 by no more than 3, 2, 1 or 0 nucleotides.

[0064] In some embodiments, the second nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1005-1498 by no more than 2, 1, or 0 nucleotides.

[0065] In some embodiments, the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1005-1498 by no more than 3, 2, 1, or 0 nucleotides.

[0066] In some embodiments, the first nucleotide region comprises the sequence of any one of SEQ ID NO: 1499-1992; and the second nucleotide region comprises the sequence of any one of SEQ ID NO: 1005-1498.

[0067] In some embodiments, the first nucleotide region comprises the sequence of any one of SEQ ID NO: 1499-1992; and the second nucleotide region comprises the sequence of any one of SEQ ID NO: 1005-1498.

[0068] In some implementations, the first nucleotide region and the second nucleotide region form a double strand comprising any of the nucleic acid sequence pairs shown in Table B.

[0069] In some embodiments, the oligonucleotide has a nucleotide duplex as shown in any of Table B, having no more than 3 (preferably no more than 1 or 2, more preferably 1) nucleotide differences.

[0070] In some embodiments, the oligonucleotide is an RNAi agent, an RNA interference agent, or siRNA.

[0071] In some embodiments, the oligonucleotide is siRNA comprising a sense strand and an antisense strand.

[0072] In some implementations, the antisense strand is the first nucleotide region.

[0073] In some implementations, the positive strand is the second nucleotide region.

[0074] The present invention also relates to an siRNA conjugate. In some embodiments, the siRNA conjugate contains the oligonucleotide described in the first aspect and its conjugated delivery portion (ligand).

[0075] In some embodiments, the oligonucleotide further comprises one or more delivery moieties (ligands). The oligonucleotide conjugates with the delivery moieties to form siRNA conjugates.

[0076] In some embodiments, the delivery portion is a lipophilic delivery portion.

[0077] In some embodiments, the delivery portion is a lipid, cholesterol, carbohydrate, aptamer, vitamin, polypeptide, or antibody or an antigen-binding fragment thereof that binds to a specific target on the cell membrane or cell surface.

[0078] In some embodiments, the delivery portion is an anti-transferrin receptor antibody or its antigen-binding fragment.

[0079] In some implementations, the delivery portion is a nerve cell, nerve tissue, or nervous system targeted delivery portion.

[0080] In some embodiments, the delivery moiety is 2'-O-hexadecyl or

[0081] In some embodiments, the delivery moiety is 2'-O-hexadecyl.

[0082] In some implementations, the one or more delivery portions are coupled to the second nucleotide region and / or the first nucleotide region.

[0083] In some embodiments, the one or more delivery portions are coupled to the 3' region of the second nucleotide region and / or the first nucleotide region, preferably to the 3' end of the second nucleotide region and / or the first nucleotide region.

[0084] In a second aspect, the present invention relates to a pharmaceutical composition comprising the oligonucleotide of the first aspect and a pharmaceutically acceptable excipient, carrier, or diluent.

[0085] In some implementations, the oligonucleotide is a siRNA conjugate.

[0086] Thirdly, the present invention relates to the use of the oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect, said use including the following aspects:

[0087] Inhibit SCN10A expression; and / or,

[0088] Used in the preparation of medicaments for diseases and / or conditions associated with SCN10A expression; and / or,

[0089] Treatment or prevention of diseases and / or conditions associated with SCN10A expression.

[0090] In some implementations, the disease and / or symptom associated with SCN10A expression is pain.

[0091] The present invention also relates to the use of the oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect in the preparation of a medicament for treating or preventing pain. In some embodiments, the pain includes acute pain, chronic pain, inflammatory pain, cancer pain, neuropathic pain, musculoskeletal pain, primary pain, postoperative pain, visceral pain, and idiopathic pain.

[0092] In some implementations, the pain is chronic pain.

[0093] The present invention also relates to a method for treating diseases and / or conditions associated with SCN10A expression, the method comprising administering a therapeutically effective amount of the aforementioned oligonucleotide (such as siRNA or siRNA conjugate) or the pharmaceutical composition to a subject in need.

[0094] In some implementations, the diseases and / or conditions associated with SCN10A expression include pain.

[0095] In some implementations, the subjects mentioned above are human beings. Detailed Implementation Plan

[0096] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0097] definition

[0098] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0099] Unless the context clearly indicates otherwise, references without a specific quantity include their plural forms. For example, mentioning "one cell" includes multiple such cells and equivalents known to those skilled in the art, etc.

[0100] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0101] Pain can be classified in several ways, including acute pain and chronic pain. Acute pain signal transmission plays a crucial physiological role: it allows humans to sense and avoid dangerous environments, and it is highly conserved evolutionarily. Chronic pain, on the other hand, is often a pathological phenomenon caused by injury and disease, and is related to sensitization of the somatosensory nervous system. Primary sensory neurons are involved in both of these processes.

[0102] Voltage-gated sodium channels (VGSCS) are key determinants of the excitability of sensory neurons: they are crucial for the initial transduction of sensory stimuli, the generation of action potentials, and the release of neurotransmitters at the neuronal terminals. VGSCS are transmembrane glycoprotein complexes widely found on the cell membranes of excitable cells such as neurons and skeletal muscle cells, consisting of an α subunit and several β subunits. The α subunit is the functional carrier of the sodium ion channel, composed of 1700–2000 amino acids forming four domains (I-IV), each containing six transmembrane segments (S1–S6). S4, rich in basic amino acid residues, is considered the voltage-sensitive element of the sodium ion channel. The β subunits mainly play an auxiliary role, modifying the kinetics and voltage-gated dependence of the ion channel; four isotypes (β1–β4) have been identified. Sodium ion channels can be classified based on their α subunits. Currently, nine sodium ion channel subtypes have been identified in mammals. Because their amino acid sequence similarity is greater than 50%, they are considered to belong to the same family, namely Nav1 (Nav1.1–Nav1.9). Based on their ability to be effectively inhibited by tetrodotoxin (TTX), sodium ion channels are divided into TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) types. Nav1.1, Nav1.2, Nav1.3, and Nav1.7 are TTX-S type, with their encoding genes located on human chromosome 2q23-24, and they are highly expressed in neurons. Nav1.5, Nav1.8, and Nav1.9 are TTX-R type, with their encoding genes located on human chromosome 3p21-24. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all expressed in human sensory neurons, and they play a decisive role in the transmission of pain signals.

[0103] The terms "SCNxA" and "voltage-gated sodium channel α subunit" used in this article encompass all family members, mutants, alleles, isotypes, fragments, species, coding and non-coding sequences, and sense and antisense oligonucleotide chains. SCNxA refers to the SCNxA gene family, composed of 11 known members: SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A (also known as SCN6A), SCN8A, SCN9A, SCN10A, SCN11A, and SCN12A.

[0104] target genes

[0105] In this invention, oligonucleotide siRNA “targeting” a target gene (or a designated segment of the target gene) means that the antisense strand of the siRNA is at least partially complementary to at least a portion of the RNA transcribed from the target gene (or a designated segment thereof).

[0106] In some embodiments, the target gene of this invention is the SCN10A (Nav1.8) gene. In some embodiments, the siRNA provided by this invention targets the SCN10A (Nav1.8) gene. In some embodiments, the antisense strand of the siRNA provided by this invention is at least partially or completely complementary to the target fragment of the RNA transcribed from the SCN10A gene.

[0107] In some embodiments, the location of the target fragment of the SCN10A gene targeted by the antisense strand of the siRNA provided by the present invention can be based on its location in the SCN10A gene transcript.

[0108] Characteristics and properties of the SCN10A gene

[0109] The SCN10A (voltage-gated sodium channel alpha subunit 10) gene is the gene encoding the α subunit Nav1.8 of the voltage-gated sodium ion channel. It can also be called voltage-gated sodium channel α subunit 10, voltage-gated sodium channel X-type α polypeptide, voltage-gated sodium channel subunit αNav1.8, sodium channel protein X-type subunit α, peripheral nerve sodium channel 3, HPN3, PN3, Nav1.8, FEPS2, and SNS.

[0110] The SCN10A gene is located in the 3P21-22 region of human chromosome 3, adjacent to the SCN5A region. SCN10A has 6380 SNPs, 26 introns, and 27 exons.

[0111] SCN10A encodes Nav1.8, which is primarily found in trigeminal ganglion neurons and DRG neurons. It exhibits slow inactivation and rapid recovery electrophysiological characteristics and plays a crucial role in the mechanisms of neuropathic pain. The use of the oligonucleotides and pharmaceutical compositions described herein to act on the SCN10A gene can be used to treat, prevent, and / or alleviate the symptoms of the diseases and conditions described herein.

[0112] As used herein, the terms “nucleic acid,” “oligonucleotide,” and “polynucleotide” are used interchangeably. A nucleic acid molecule is a polymer of nucleotides consisting of at least two nucleotides covalently linked together. Nucleic acid molecules are DNA (deoxyribonucleotides), RNA (ribonucleotides), and recombinant RNA and DNA molecules or analogs of DNA or RNA generated using nucleotide analogs. Nucleic acids can be single-stranded or double-stranded, linear or circular. Within the scope of this document, fragments of nucleic acids are also included, such as naturally occurring RNA or DNA that can be recovered using the disclosed extraction methods, or artificially synthesized DNA or RNA molecules in vitro (i.e., synthetic polynucleotides). The molecular weight of nucleic acids is also not limited and can be selected from a few base pairs (bp) to from hundreds of base pairs, for example, from about 2 nucleotides to about 10,000 nucleotides, or from about 10 nucleotides to about 5,000 nucleotides, or from about 10 nucleotides to about 1,000 nucleotides. When the relevant nucleic acid molecule typically contains fewer than about 100 bases, it is often referred to as an “oligonucleotide.” When a nucleic acid molecule typically contains more than about 100 bases, it is often referred to as a "polynucleotide".

[0113] Oligonucleotides can be 10-200 nucleotides long, or 10-100 nucleotides long, or 10-50 nucleotides long, or 50-100 nucleotides long. In some embodiments, the oligonucleotide may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

[0114] RNA interference

[0115] RNA interference (RNAi) is an evolutionarily conserved process that inhibits the expression of a target gene by expressing or introducing RNA with the same or highly similar sequence as the target gene, leading to sequence-specific degradation of the messenger RNA (mRNA) transcribed from the target gene or specific post-transcriptional gene silencing (PTGS). This biological process is well known in the art and is mediated by short interfering nucleic acid molecules. As used herein, “RNAi agent, RNA interference agent, or siRNA” includes both sense and antisense strands and refers to any molecule that can downregulate, reduce, or inhibit RNA interference function or activity in cells or organisms. RNAi inhibitors can downregulate, reduce, or inhibit RNAi (e.g., RNAi-mediated cleavage of target polynucleotides, translational repression, or transcriptional silencing) by interacting with or interfering with the function of any component of the RNAi pathway, including protein components such as RISC, or nucleic acid components such as miRNA or siRNA. RNAi inhibitors can be siNA molecules, antisense molecules, aptamers, or small molecules that interact with or interfere with the function of RISC, miRNA, siRNA, or any other component of the RNAi pathway in a cell or organism. By inhibiting RNAi (e.g., RNAi-mediated cleavage of target polynucleotides, translational repression, or transcriptional silencing), the RNAi inhibitors of the present invention can be used to regulate (e.g., upregulate or downregulate) the expression of target genes. In some embodiments, the RNAi interfering agent typically consists of a nucleic acid sequence or a nucleic acid analog with target gene specificity (such as SCN10A).

[0116] As used herein, “short interfering nucleic acid,” “siRNA,” “short interfering RNA,” “siRNA,” “short interfering nucleic acid molecule,” “short interfering oligonucleotide molecule,” or “chemically modified short interfering nucleic acid molecule” refers to any nucleic acid molecule capable of inhibiting or downregulating gene expression or viral replication in a sequence-specific manner by mediating RNA interference (RNAi) or gene silencing. These terms may refer to a single nucleic acid molecule, multiple such nucleic acid molecules, or a library of such nucleic acid molecules. siRNA can be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a target nucleic acid molecule, and the sense strand comprises a nucleotide sequence corresponding to or partially complementary to the target nucleic acid sequence. siRNA can be a polynucleotide having a double strand, an asymmetric double strand, a hairpin or asymmetric hairpin secondary structure, and self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a separate target nucleic acid molecule, and the sense region comprises a nucleotide sequence corresponding to or partially complementary to the target nucleic acid sequence. siRNA may also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in the target nucleic acid molecule (e.g., where such siRNA molecules do not require the presence of a nucleotide sequence corresponding to or partially thereof within the siRNA molecule), wherein the single-stranded polynucleotide may further comprise a terminal phosphate group, such as 5'-phosphate or 5',3'-bisphosphate. In some embodiments, when said siRNA coexists or is expressed with the SCN10A gene in the same cell, the siRNA has the ability to reduce or inhibit the expression of the SCN10A gene.

[0117] The small interfering RNA (siRNA) used according to the present invention can be synthesized and used according to methods known in the art and familiar to those skilled in the art. The siRNA used in the methods of the present invention suitably comprises about 1 to about 50 nucleotides (nt). In examples of non-limiting embodiments, the siRNA may comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20 to about 25 nt.

[0118] Modified nucleotides

[0119] The "modified nucleotide" or "nucleotide modification" mentioned in this invention refers to a nucleotide that has been modified by introducing one or more modifications to the sugar moiety or (nucleo)base moiety compared to an unmodified DNA or RNA nucleotide.

[0120] In some embodiments, modification refers to any modification to the chemical structure of a nucleotide that differs from that of native siRNA or RNA. This includes additions, substitutions, or modifications of native siRNA or RNA at sugar, base, or nucleotide internucleotide bonds, as described herein or known elsewhere in the art. In specific embodiments, the term "modification" may refer to a specific form of RNA that is naturally present in a particular biological system, such as 2'-O-methyl modification or inosine modification. Nucleic acids can be modified in the base moiety, sugar moiety, or phosphate backbone. Modifications include, but are not limited to, 2'-position sugar modifications, 5'-position pyrimidine modifications, 8'-position purine modifications, modifications at the outer cyclic amine, substitution of 4-thiouridine, substitution of 5-bromopyrimidine or 5-iodo-uridine, backbone modifications, thiophosphate or alkyl phosphate modifications, methylation, and aberrant base pairing combinations, such as isobasic cytosine and isoguanidine. Modifications may also include 3' and 5' modifications, such as capping. Nucleic acid molecules can also be modified by conjugation to portions having desired biological properties. Such components may include, but are not limited to, compounds, peptides and proteins, carbohydrates, antibodies, enzymes, polymers, drugs, and fluorophores.

[0121] In some embodiments, the modified nucleotide comprises a modified sugar moiety.

[0122] In some embodiments, the nucleobase moiety is modified, preferably with a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-mercaptocytosine, 5-propynylcytosine, 5-propynyluracil, 5-bromouracil, 5-thiazouracil, 2-thiouracil, 2'-thiothymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0123] In some embodiments, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-hexadecyl-modified nucleotides, 2'-deoxynucleotides, debased nucleotides, reverse debased nucleotides, deoxynucleotides, locked nucleotides, 2'-aminonucleotides, 2'-hydroxynucleotides, 2'-O-allyl nucleotides, and 2'-C 1-25 Alkyl nucleotides, 2'-OC 1-25Alkyl nucleotides, 2'-methoxyethyl nucleotides, configuration-restricted nucleotides, 2'-allyl nucleotides, morpholino nucleotides, tetrahydropyran-modified nucleotides, hexitol nucleotides, 1,5-dehydrated hexitol nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate nucleotides, 5'-thiophosphate nucleotides, 5'-methylphosphonate nucleotides, nucleotides containing vinyl phosphates, 5'-methylcytosine-modified nucleotides, diol-modified nucleotides, and nucleic acid analogs.

[0124] In some embodiments, the oligonucleotide may further comprise one or more phosphate ester analogs. The term "phosphate ester analog" as used in this invention refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate ester group. In some embodiments, the phosphate ester analog is located at the 5' terminal nucleotide of the oligonucleotide, rather than the 5'-phosphate ester that is typically readily removable by enzymatic reactions. Examples of phosphate ester analogs include, but are not limited to, 5'-methylenephosphonates (5'-MP or MP), 5'-(E)-vinylphosphonates (5'-VP or VP), and 5'-cyclic phosphonates (e.g., 5'-cyclopropylphosphonate (CprP)).

[0125] According to embodiments of the present invention, the nucleotides modified with 5'-phosphate mimics are selected from EVpu, EVpa, cPrpu, and cPrpa; the structures are shown below:

[0126] Modified nucleotide bonds

[0127] In this document, "nucleotide inter-bond" and "nucleoside inter-bond" are used interchangeably. The "modified nucleotide inter-bond" described in this invention refers to a bond other than the phosphodiester (PO) bond that covalently links two nucleosides together. Therefore, the oligonucleotides described in this invention may contain modified nucleotide inter-bonds. For naturally occurring oligonucleotides, the nucleotide inter-bond includes a phosphate ester group that forms a phosphodiester bond between adjacent nucleosides.

[0128] In some implementations, the modified internucleotide bonds increase the nuclease resistance of the oligonucleotides compared to phosphodiester bonds. These modified internucleotide bonds can be used to stabilize the oligonucleotides.

[0129] In some embodiments, the oligonucleotide comprises one or more internucleotide bonds modified by natural phosphodiester.

[0130] In some embodiments, the nucleoside linking the oligonucleotide of the present invention to a non-nucleotide functional group such as a conjugate can be a phosphate diester.

[0131] In some embodiments, all internucleotide bonds of the oligonucleotide or its continuous nucleotide sequence are nuclease-resistant internucleotide bonds.

[0132] In some embodiments, the oligonucleotide comprises one or more modified internucleotide bonds. In some embodiments, the modified internucleotide bonds include modified phosphate groups, such as thiophosphates, selenophosphates, boron phosphates, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and dithiophosphates.

[0133] In some implementations, the oligonucleotide uses a phosphate thioester nucleotide inter-bond.

[0134] In some embodiments, one or more inverted abasic residues (invAbs) are added to the 3' end of the sense and / or antisense strands. In some embodiments, one or more inverted abasic residues (invAbs) are added to the 5' end of the sense and / or antisense strands. In some embodiments, one or more inverted abasic residues are inserted between the ligand and the nucleotide sequence of the sense and / or antisense strands. In some embodiments, the inverted abasic residues may be linked via phosphate ester, thiophosphate, or other nucleoside internucleotide bonds.

[0135] In some embodiments, the positive strand may include one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capped residue.” As used herein, a “capped residue” is a nonnucleotide compound or other portion that may be incorporated at one or more ends of the nucleotide sequence of the RNAi reagent disclosed herein. In some cases, capping residues may provide certain beneficial properties to the RNAi reagent, such as protection against exonuclease degradation. In some embodiments, inverse debasement residues (invAbs) (also referred to in the art as “inverse debasement sites”) are added as capping residues. Capping residues are generally known in the art and include, for example, inverse debasement residues and carbon chains, such as terminal C3H7 (propyl), C6H… 13 (Hexyl) or C 12 H 25(Dodecyl) group. In some embodiments, the capping residue is present at the 5' end, 3' end, or both 5' and 3' ends of the sense strand. In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 3' end of the sense strand and / or antisense strand. In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 5' end of the sense strand and / or antisense strand. In some embodiments, one or more inverted abasic residues or inverted abasic nucleotides are inserted between the target ligand and the nucleotide sequence of the sense strand. In some alternative embodiments of the invention, one or more inverted abasic nucleotides are inserted between the ligand and the nucleotide sequence of the sense strand and / or antisense strand. In some alternative embodiments of the invention, the inverted abasic nucleotides may be linked via phosphate esters, thiophosphate esters, or other nucleoside internucleotide bonds. In some embodiments, the structural formulas of the "inverted abasic modification" and the "inverted abasic nucleotide" are shown below:

[0136] Here, 'a' is the 5' end of the chain pointing toward the justice chain or the antisense chain, and 'b' is the 3' end of the chain pointing toward the justice chain or the antisense chain.

[0137] In some alternative embodiments, when invAb is located at the 5' end:

[0138] When invAb is in the middle of the sequence:

[0139] When invAb is at the 3' end:

[0140] In this article, "phosphoramidite" refers to the starting material in oligonucleotide solid-phase synthesis by linking modified or unmodified nucleotides, linkers, or conjugation groups to oligonucleotides to form phosphate esters or thiophosphate esters.

[0141] As used herein, "double helix" refers to a structure (nucleic acid sequence pair) formed by hydrogen bonds between two antiparallel nucleotide sequences that are complementary base pairing, under appropriate conditions. In some implementations, double helixes (double strands) can also be formed even without perfect complementarity (basic complementarity).

[0142] The term "identity" as used in this article refers to the matching between two nucleic acid sequences. The percentage identity of two nucleic acid sequences is determined by aligning the sequences for optimal comparison (e.g., introducing a gap in the first nucleic acid sequence for best alignment with the second). Nucleotides at corresponding nucleotide positions are then compared. The molecules are considered identical at that position when a position in the first sequence is occupied by the same nucleotide as its corresponding position in the second sequence. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%).

[0143] As used herein, “complementarity” or “complementarity” has the general meaning in the art. In some embodiments, complementarity refers to the formation or presence of one or more hydrogen bonds between one nucleotide sequence and another nucleotide sequence via conventional Watson-Crick or other non-conventional types of bonding as described herein. The binding free energy of the sense and antisense strands in the oligonucleotides of the present invention is sufficient to enable the oligonucleotide to perform activities such as RNAi activity. Complete complementarity means that all adjacent residues of the nucleic acid sequence will hydrogen bond with the same number of adjacent residues in a second nucleic acid sequence. Partial complementarity may include various mismatched or non-base-paired nucleotides within a nucleic acid molecule (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatched, non-nucleotide linkers or non-base-paired nucleotides) that can result in protrusions, loops or overhangs between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and the corresponding target nucleic acid molecule. Such partial complementarity can be expressed as a percentage complementarity determined by the number of non-base-paired nucleotides, i.e., approximately 50%, 60%, 70%, 80%, or 90%, depending on the total number of nucleotides involved. This type of partial complementarity allows nucleic acid molecules (e.g., siNA) to maintain their function to a certain extent, such as their ability to mediate sequence-specific RNAi.

[0144] In some implementations, the basic complementarity means that the sense strand and the antisense strand mismatch no more than 3 nucleotides in the double-stranded region; preferably, there are 2 or 1 mismatches in the double-stranded region. In this document, "mismatch" means that the bases at corresponding positions are not paired in a complementary manner.

[0145] conjugates and conjugates

[0146] Unless otherwise stated, “conjugation” as used herein means the connection between two or more chemical parts, each having a specific function, in a covalent or non-covalent manner; correspondingly, “conjugate” as used herein refers to two or more parts connected by direct or indirect covalent or non-covalent interactions.

[0147] In some embodiments, the terms "targeting ligand," "delivery portion," or "ligand" are used interchangeably and refer to a siRNA comprising a pharmaceutically acceptable scaffold group and at least one targeting group, wherein the siRNA is sequentially linked to the scaffold group and the targeting group, i.e., "siRNA-scaffold group-target group." In some embodiments, there are 2, 3, or 4 targeting groups. In some embodiments, the siRNA conjugation site is at the 3' or 5' end of the positive strand. In some embodiments, the conjugation may be the conjugation of one or more targeting ligands.

[0148] In some embodiments, the "targeting group" refers to a targeting agent for cells or tissues in an animal or human body, such as binding to specific cells. In some embodiments, the targeting group may be N-acetamide-galactosamine (GalNAc), lipids, cholesterol, steroids, transferrin, polylactose, polygalactose, glycosylated polyamino acids, RGD peptides or their analogues, folic acid, glycoproteins, etc.

[0149] In some implementations, the interactions are covalent. In some implementations, the covalent interactions are mediated by the connector portion.

[0150] In some implementations, the interactions are non-covalent (e.g., charge interactions, affinity interactions, metal coordination, physical adsorption, hydrophobic interactions, stacking interactions, hydrogen bonding interactions (e.g. with “viscous sequences”), van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.).

[0151] As used in this article, "linker" refers to a structure used to bind a nucleotide (e.g., an oligonucleotide) to a target ligand or delivery moiety.

[0152] In some embodiments, the linker may be "cleavable" or "cuttable". In some embodiments, the linker is biocleavable, for example, cleavable by endogenous proteases (e.g., present in target tissues and / or cells). In some embodiments, the linker includes a protease-cleavable site. In some embodiments, the linker includes a pH-sensitive site, such as a site sensitive to acidic pH, for example, a site hydrolyzed under acidic conditions.

[0153] In some embodiments, the linker is stable under physiological conditions. In some embodiments, the selected linker is cleavable in vivo. Cleavable linkers may include chemically or enzymatically unstable or cleavable links. Cleavable linkers typically rely on intracellular processes to release the drug, such as cytoplasmic reduction, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. Cleavable linkers typically incorporate one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker is non-cleavable.

[0154] In some embodiments, the cleavable linker may include cleavable chemical moieties, such as disulfides that can be cleaved by reduction, diols or glycols that can be cleaved by periodate, diazonium bonds that can be cleaved by dithionite, esters that can be cleaved by hydroxylamine, sulfones that can be cleaved by base, etc.

[0155] In some implementations, the cleavable linker is a linker containing a disulfide bond, which can be cleaved by reducing the disulfide bond. As used herein, “treatment” or “prevention” refers to any treatment or prevention of a disease or symptom in a patient, including: preventing the development of the disease in a susceptible but not yet diagnosed individual; suppressing the disease, i.e., preventing its further development; suppressing the symptoms of the disease; and alleviating the disease, i.e., causing the disease to subside or reducing the symptoms of the disease.

[0156] As used herein, the terms "individual," "subject," or "patient" are used interchangeably and refer to any subject requiring diagnosis, treatment, or therapy. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0157] As used herein, "therapeutic effective amount" refers to the amount of the composition of the present invention that is sufficient to administer treatment to a subject requiring such treatment, according to the following definition. Therapeutic effective amount will vary depending on the specific activity of the therapeutic agent used, as well as the patient's age, physical condition, presence of other disease states, and nutritional status. Furthermore, other medications the patient may be receiving will affect the determination of the therapeutic effective amount of the administered therapeutic agent.

[0158] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc. The use of media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is conceivable unless any conventional media or reagent is incompatible with the active ingredient. Additional active ingredients can also be introduced into the composition.

[0159] As used herein, “administration” means a method or route by which the siRNAs and pharmaceutical compositions of the present invention are at least partially located at a desired site in order to deliver the composition to a subject. The siRNAs and pharmaceutical compositions of the present invention can be administered via any suitable route that produces an effective treatment in the subject.

[0160] The siRNAs and pharmaceutical compositions according to the invention can be administered to a subject alone or in combination with one or more other therapeutic agents, such as anticancer agents or anti-inflammatory agents.

[0161] Carrier / Delivery System

[0162] The siRNA molecules of the present invention can be added directly, or can be complexed with cationic lipids, packaged in liposomes, or used as recombinant plasmids or viral vectors for expressing siRNA molecules, or delivered to target cells or tissues in other ways.

[0163] In some implementations, the carrier system is a lipid-based carrier system, cationic lipids, or liposomal nucleic acid complexes, liposomes, microclusters, virions, lipid nanoparticles, or mixtures thereof.

[0164] In other embodiments, the carrier system is a polymer-based carrier system, such as a cationic polymer-nucleic acid complex. In further embodiments, the carrier system is a cyclodextrin-based carrier system, such as a cyclodextrin polymer-nucleic acid complex. In a more advanced embodiment, the carrier system is a protein-based carrier system, such as a cationic peptide-nucleic acid complex. Preferably, the carrier system is a lipid nanoparticle (LNP) formulation. In other embodiments, conjugates of siRNA molecules are provided. Such conjugates can be used to facilitate the delivery of siRNA molecules into biological systems, such as cells. The conjugates provided by the present invention can confer therapeutic activity by transferring therapeutic compounds across cell membranes, altering drug metabolism kinetics, and / or modulating the localization of the nucleic acid molecules of the present invention.

[0165] Table A:

[0166] Table B:

[0167] 1) The lowercase letters c, g, u, a, and t are all nucleotides modified with 2'-methoxy groups (that is, c, g, u, and a respectively represent that the ribosyl 2'-OH of the nucleotide represented by the corresponding uppercase letters is replaced by a methoxy group);

[0168] 2) f indicates that the nucleotide represented by the letter before f is a 2'-fluorinated nucleotide (that is, the 2'-OH of the nucleotide represented by the letter before f is replaced by a fluorine atom);

[0169] 3) s indicates that the two adjacent nucleotide residues to the left and right of s are thiophosphate esters (that is, the 5'-phosphate ester group of the nucleotide represented by the letter before s is replaced by a 5'-thiophosphate ester group).

[0170] 4) EV represents (E)-vinylphosphonate: Where B represents a base;

[0171] 5) Uhd represents 2'-O-hexadecyluracil, Ahd represents 2'-O-hexadecyladenine, Chd represents 2'-O-hexadecylcytosine, and Ghd represents 2'-O-hexadecylguanine.

[0172] Example 1: Synthesis of Oligonucleotides (siRNA)

[0173] The specific steps are as follows:

[0174] 1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramide solid-phase synthesis technology. This was achieved using a universally controllable porous glass CPG... The synthesis was carried out on the above. All phosphoramidite monomers (purchased from Tangzhi Pharmaceutical & Shanghai Zhaowei) were dissolved in anhydrous acetonitrile (Suzhou Kelema) and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT) as an activator (Suzhou Kelama) was 8–12 minutes. Phosphate ester bonds were constructed using a 0.05 M iodine solution (dissolved in pyridine / water = 9:1, Suzhou Kelama); thiophosphate ester bonds were generated using a 0.2 M hydroflavin (Suzhou Kelama) solution in anhydrous acetonitrile / pyridine (v / v = 1 / 1), with a reaction time of 5 minutes. The synthesis was complete after the final removal of the DMT group from all sequences.

[0175] 2) Cleavage and deprotection of oligonucleotides bound to CPG: After termination of solid-phase synthesis, the protecting groups were removed by treating with an acetonitrile solution containing 20% ​​diethylamine (Sinopharm) for 10 minutes. The obtained CPG carrier was then subjected to ammoniation with concentrated ammonia (Sinopharm) to remove the protecting groups on the carrier and bases. Some oligonucleotides were deprotected by treating with a mixture of DMSO / triethylamine hydrogen fluoride (Beijing Bailingwei Technology Co., Ltd.) (5:1) at 25 degrees Celsius for 4 hours. After filtration, a solution containing the product was obtained.

[0176] 3) Purification of single-stranded oligonucleotides: Oligomers were obtained by HPLC purification using NanoQ anion exchange. Buffer A was a 20 mM sodium hydroxide solution; and buffer B contained a 20 mM sodium hydroxide solution and 3 M sodium chloride. The target product was then separated. The obtained target product was further desalted by gel electrophoresis.

[0177] 4) The sense and antisense strands obtained by chemical synthesis were subjected to base complementation in a molar ratio of 1:1 under the reaction conditions of 70℃ for 10 min and then slowly restored to room temperature to finally obtain oligonucleotides (siRNA).

[0178] The nucleotide sequences of the sense and antisense strands of the oligonucleotides (siRNA) obtained in this invention are shown in Tables A and B.

[0179] Test Example 1: In vitro screening of HEK293 cells using dual-luciferase psiCHECK-2 vector

[0180] In a 96-well cell plate, siRNA was prepared and added to each well in duplicate. Then, a plasmid carrying the SCN10A gene from the psiCHECK-2 vector (hereinafter referred to as plasmid) was added to each well. Opti-MEM medium (Gibco, catalog number 31985-070) was added to each well to obtain a mixture containing siRNA and plasmid. Lipofectamine 2000 (Invitrogen, catalog number 11668-019) was added to the Opti-MEM medium and incubated for 5 minutes. Then, it was added to the mixture containing siRNA and plasmid in each well. After incubation at room temperature for 20 minutes, 100 μL of Dulbecco's Modified Eagle Medium (Gibco, catalog number C11995500BT) containing 5 × 10⁴ HEK293 cells (ATCC) was added to the mixture containing siRNA, plasmid, and Lipofectamine 2000 transfection reagent in each well. After incubating the cells for 24 hours, firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter gene assay kit (Novizan, catalog number DD1205-02). Different concentrations of siRNA were used for the assay.

[0181] The test results showed that the siRNAs of the present invention all had good silencing effects, indicating that the nucleotides of the present invention have the ability to reduce or inhibit the expression of the SCN10A gene. Specifically, the measurement results of some exemplary siRNAs (corresponding to Tables A and B) at certain concentrations in this test example are shown in Table 1.

[0182] Table 1:

[0183] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An oligonucleotide capable of inhibiting the expression of Nav1.8, wherein the oligonucleotide comprises at least a first nucleotide region, the first nucleotide region comprising a region at least partially complementary to RNA transcribed from the SCN10A gene, and wherein the at least partially complementary region comprises at least 15 consecutive nucleotides differing from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3 nucleotides.

2. The oligonucleotide of claim 1, further comprising a second nucleotide region, the second nucleotide region being at least partially complementary to the first nucleotide region; Preferably, the second nucleotide region comprises at least 15 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 3 nucleotides.

3. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region comprises 18-35 nucleotides, preferably 18-30, 18-23, or 19-25 nucleotides, for example, comprising 18, 19, 20, 21, 22, or 23 nucleotides.

4. The oligonucleotide according to claim 1 or 2, wherein the second nucleotide region comprises 16-35 nucleotides, preferably 18-30, 16-23, or 19-25 nucleotides, for example, comprising 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

5. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 3, 2, 1, or 0 nucleotides; Preferably, the first nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 503-1004 by no more than 2, 1, or 0 nucleotides.

6. The oligonucleotide according to claim 1 or 2, wherein the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 3, 2, 1, or 0 nucleotides; Preferably, the second nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1-502 by no more than 2, 1, or 0 nucleotides.

7. The oligonucleotide according to claim 1 or 2, comprising at least one complementary double-stranded region, the complementary double-stranded region comprising at least a portion of the first nucleotide region, the first nucleotide region being directly or indirectly connected to at least a portion of the second nucleotide region to form a double strand; Preferably, the double-stranded region is 10-25, 10-19, 12-19, 12-15, or 19-23 base pairs long; Preferably, the double-stranded region is 19, 20, 21, 22, or 23 base pairs long.

8. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region comprises the sequence of any one of SEQ ID NO: 503-1004; and the second nucleotide region comprises the sequence of any one of SEQ ID NO: 1-502.

9. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region and the second nucleotide region form a duplex comprising any one of the nucleic acid sequence pairs shown in Table A.

10. The oligonucleotide according to claim 1 or 2, wherein the 5' region of the first nucleotide region is directly or indirectly connected to the 3' region of the second nucleotide region, for example, through complementary base pairing; preferably, the 5' terminal nucleotide of the first nucleotide region is base-paired with the 3' terminal nucleotide of the second nucleotide region.

11. The oligonucleotide according to claim 1 or 2, wherein the 3' region of the first nucleotide region is directly or indirectly connected to the 5' region of the second nucleotide region; Preferably, the first nucleotide region is directly or indirectly connected to the second nucleotide region via, for example, a phosphate ester, a thiophosphate ester, or a dithiophosphate ester.

12. The oligonucleotide of claim 1, wherein the first nucleotide region comprises one or more modified nucleotides, and optionally, one or more internucleotide bonds are modified internucleotide bonds.

13. The oligonucleotide according to claim 1 or 2, wherein the second nucleotide region comprises one or more modified nucleotides, and optionally, one or more internucleotide bonds are modified internucleotide bonds; Preferably, all nucleotides of the oligonucleotide are modified nucleotides.

14. The oligonucleotide according to claim 12 or 13, wherein the modified nucleotide is independently selected from at least one of the following: 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-hexadecyl-modified nucleotides, 2'-deoxynucleotides, debased nucleotides, reverse debased nucleotides, deoxynucleotides, locked nucleotides, 2'-aminonucleotides, 2'-hydroxynucleotides, 2'-O-allyl nucleotides, 2'-C 1-25 Alkyl nucleotides, 2'-OC 1-25 Alkyl nucleotides, 2'-methoxyethyl nucleotides, configuration-restricted nucleotides, 2'-allyl nucleotides, morpholino nucleotides, tetrahydropyran-modified nucleotides, hexitol nucleotides, 1,5-dehydrated hexitol nucleotides, cyclohexenyl-modified nucleotides, PEG-modified nucleotides, 5'-aminophosphate nucleotides, 5'-thiophosphate nucleotides, 5'-methylphosphonate nucleotides, nucleotides containing vinyl phosphates, 5'-methylcytosine-modified nucleotides, diol-modified nucleotides, and nucleic acid analogs; Preferably, the modified nucleotide includes at least one modified nucleotide selected from 2'-fluoromodified nucleotides, 2'-methoxymodified nucleotides, 2'-O-hexadecylmodified nucleotides, 2'-deoxynucleotides, nucleotides containing vinyl phosphate, reverse debased nucleotides, locked nucleotides, and 5'-thiophosphate nucleotides.

15. The oligonucleotide according to claim 12 or 13, wherein the one or more modified nucleotides are independently 2'-fluoromodified nucleotides, 2'-methoxymodified nucleotides, or 2'-O-hexadecylmodified nucleotides.

16. The oligonucleotide according to claim 12 or 13, wherein the first nucleotide region and / or the second nucleotide region comprises one or more modified nucleoside internucleotide bonds; the modified nucleoside internucleotide bond is a thiophosphate or dithiophosphate nucleoside internucleotide bond; preferably, the modified nucleoside internucleotide bond is a thiophosphate nucleoside internucleotide bond.

17. The oligonucleotide according to claim 12 or 13, wherein the first nucleotide region and / or the second nucleotide region independently comprise 0, 1, 2, 3, 4 or 5 phosphate thioester nucleoside bonds; Preferably, the first nucleotide region contains 4 or 5 phosphate thioester nucleoside bonds; Preferably, the second nucleotide region contains 4 or 5 thiophosphate nucleoside bonds.

18. The oligonucleotide according to claim 1 or 10, wherein the nucleotide at the 5' end of the first nucleotide region has a phosphate ester group or a phosphate ester analog.

19. The oligonucleotide according to claim 1 or 10, wherein the nucleotide at the 5' end of the first nucleotide region has a 5'-phosphonate, preferably 5'-methylenephosphonate (5'-MP) or 5'-(E)-vinylphosphonate (5'-VP).

20. The oligonucleotide of claim 1, wherein the first nucleotide region comprises a sequence complementary to at least 15 consecutive nucleotides selected from any one of SEQ ID NO: 1-502; preferably, the first nucleotide region comprises a sequence complementary to at least 18, 19, 20, 21, 22, 23 consecutive nucleotides selected from any one of SEQ ID NO: 1-502.

21. The oligonucleotide of claim 1, wherein the first nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 1499-1992 by no more than 3, 2, 1 or 0 nucleotides; Preferably, the first nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1499-1992 by no more than 2, 1, or 0 nucleotides.

22. The oligonucleotide of claim 1 or 2, wherein the second nucleotide region comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides that differ from the sequence of any one of SEQ ID NO: 1005-1498 by no more than 3, 2, 1 or 0 nucleotides; Preferably, the second nucleotide region comprises at least 21, 22, or 23 consecutive nucleotides that differ from the sequence of any one of SEQ ID NO: 1005-1498 by no more than 2, 1, or 0 nucleotides.

23. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region comprises the sequence of any one of SEQ ID NO: 1499-1992; and the second nucleotide region comprises the sequence of any one of SEQ ID NO: 1005-1498.

24. The oligonucleotide according to claim 1 or 2, wherein the first nucleotide region and the second nucleotide region form a duplex comprising any one of the nucleic acid sequence pairs shown in Table B.

25. The oligonucleotide according to claim 1 or 2, further comprising one or more conjugated delivery moieties; Preferably, the one or more delivery portions are coupled to the second nucleotide region and / or the first nucleotide region.

26. The oligonucleotide of claim 25, wherein the delivery portion is a lipophilic delivery portion; Alternatively, the delivery portion may be a lipid, cholesterol, carbohydrate, aptamer, vitamin, polypeptide, or antibody or its antigen-binding fragment that binds to a specific target on the cell membrane or cell surface; preferably, the delivery portion may be an anti-transferrin receptor antibody or its antigen-binding fragment. Preferably, the delivery portion is 2'-O-hexadecyl.

27. The oligonucleotide of claim 25, wherein the delivery portion is a nerve cell, nerve tissue, or nervous system targeted delivery portion.

28. A pharmaceutical composition comprising an oligonucleotide of any one of claims 1-27 and a pharmaceutically acceptable excipient, carrier, or diluent.

29. Use of the oligonucleotide according to any one of claims 1-27 or the pharmaceutical composition according to claim 28, comprising: Inhibit SCN10A expression; and / or, Used in the preparation of medicaments for diseases and / or conditions associated with SCN10A expression; and / or, Treatment or prevention of diseases and / or conditions associated with SCN10A expression; Preferably, the disease and / or symptom associated with SCN10A expression is pain.

30. The use of the oligonucleotide according to any one of claims 1-27 or the pharmaceutical composition according to claim 28 in the preparation of a medicament for treating pain; Preferably, the pain includes acute pain, chronic pain, inflammatory pain, cancer pain, neuropathic pain, musculoskeletal pain, primary pain, postoperative pain, visceral pain, and idiopathic pain.

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