Compositions and methods for modulating Na V 1.8

Abstract

The present disclosure relates to oligonucleotides for inhibiting Na V 1.8 Oligonucleotides, antibody-siRNA conjugates, or pharmaceutical compositions thereof for gene expression, and methods of using such oligonucleotides, antibody-siRNA conjugates, or pharmaceutical compositions thereof for reducing / treating pain or diseases / disorders associated with Na V 1.8.

Description

Technical Field

[0001] This disclosure relates to oligonucleotides and their role in inhibiting / regulating Na+. V 1.8 Use in the expression of the gene (SCNA10A gene / transcript). This disclosure also relates to conjugates comprising oligonucleotides and anti-TfR antibodies or their antigen-binding fragments, and their use in the treatment or relief of pain and / or with Na V 1.8 Uses in diseases associated with the channel.

[0002] sequence list

[0003] This application is submitted together with a sequence list in ST.26 XML format. The sequence list is provided as a file with the title "30152_WO", created on November 20, 2024, and with a size of 1132 kilobytes. The full text of the ST.26 XML format sequence list information is incorporated herein by reference. Background Technology

[0004] The treatment of chronic pain is a significant unmet medical need; more than a quarter of the U.S. population suffers from chronic pain (Nahin RL, “Estimates of Pain Prevalence and Severity in Adults: United States 2012,” J. Pain.16(8):769-80 (2015)). Furthermore, pain is one of the most common chief complaints in outpatient clinics. Pain has multiple categories and types, including neuropathic, nociceptive, musculoskeletal, inflammatory, psychogenic, and mechanical.

[0005] Chronic pain can be categorized into three types: neuropathic, inflammatory, and mixed pain, which combines both neuropathic and inflammatory aspects. Neuropathic pain can be further divided into peripheral neuropathic pain and central neuropathic pain. Peripheral neuropathic pain is caused by damage or infection of peripheral sensory nerves, while central neuropathic pain is caused by damage to the CNS and / or spinal cord. Both peripheral and central neuropathic pain can occur without apparent initial nerve damage. Pain caused by diabetic peripheral neuropathy (DPN) is a typical example of peripheral neuropathic pain. The International Association for the Study of Pain (IASP, Seattle, Washington, USA) provides a similar definition: peripheral neuropathic pain is pain caused or induced by primary lesions or dysfunctions in the peripheral nervous system. Central neuropathic pain is pain caused or induced by primary lesions or dysfunctions in the central nervous system. Inflammatory pain refers to increased sensitivity caused by an inflammatory response associated with tissue damage. Inflammatory pain results from increased excitability of peripheral nociceptive sensory fibers due to the action of inflammatory mediators. This excitatory effect is a result of altered ion channel activity within the affected sensory fibers. Conditions exhibiting characteristics of both nociceptive and neuropathic pain, such as chronic low back pain (CLBP), are classified as mixed pain.

[0006] Sodium channels play a central role in the generation of action potentials in all excitable cells, such as neurons and muscle cells, and also in disease states such as pain (Waxman et al., “Sodium Channels and Pain” Proc. Natl. Acad. Sci. USA 96(14): 7635-9 (1999) and Waxman et al., (2000) “Voltage-gated Sodium Channels and the Molecular Pathogenesis of Pain: A Review,” J. Rehabil. Res. Dev. 37(5): 517-28 (2000)). Voltage-gated sodium channels play an important role in regulating neuronal excitability in both normal and pathological pain states. However, sodium channels (such as Na+) V 1.7 and Na V 1.8) Its function in the pathophysiology of chronic pain is not fully elucidated. V Non-selective antagonists of the pain pathway can reduce pain signals and can be used to treat a variety of pain conditions; however, these analgesics (such as painkillers) can cause a range of adverse reactions and are generally not very effective in completely relieving pain.

[0007] Common analgesics such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) cannot adequately improve pathological pain, such as peripheral and central nervous system pathological pain, due to insufficient efficacy and / or dose-limiting side effects. Side effects also limit the use of nonselective sodium... V The practicality of antagonists is limited by side effects including dizziness, drowsiness, nausea, and vomiting (Tremont-Lukats et al., “Anticonvulsants for Neuropathic Pain Syndromes: Mechanisms of Action and Place in Therapy,” Drugs 60:1029-1052 (2000)), thus restricting the use of Na+. V The practicality of antagonists in treating pain. These side effects are believed to be at least partly due to the blockage of multiple Na+ receptors. V This is due to the subtype.

[0008] The ability to selectively and effectively regulate Na is still needed. V 1.8 Channel activity for use in medications to treat or relieve pain. Selective inhibition of Na+. V The 1.8-channel reagent compared to non-selective Na V Antagonists can provide beneficial treatment and reduce adverse reactions. Additionally, there is a need for analgesics that combine high biological activity and in vivo stability for treating or relieving pain. Summary of the Invention

[0009] This disclosure provides oligonucleotides, conjugates comprising oligonucleotides and anti-transferrin receptor (anti-TfR) antibodies or their antigen-binding fragments, pharmaceutical compositions, and methods for inhibiting Na+ in cells or mammals. V 1.8 Methods for gene expression. This invention also provides methods for treating chronic pain and [the use of Na+]. V 1.8 Drug compositions and methods for other diseases caused by gene expression.

[0010] Na V 1.8 is a voltage-gated sodium channel, expressed almost exclusively in the nociceptor population of primary afferent dorsal root ganglion neurons in humans. It is responsible for the terminal phase of the rising limb of the action potential in primary afferent fibers. Several sodium channels have been identified in the human population. V 1.8 Gain-of-function mutations, which lead to primary afferent fiber overexcitation and multiple chronic pain phenotypes (Xiao et al., "Increased Resurgent Sodium Currents in Na...). V1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies,” J Neuroscience 39, Vol. 8: 1539-1550 (2019)). Furthermore, targeting Na… V Selective small molecules of 1.8 have shown efficacy in both acute and chronic pain indications (Qin, H. et al., “Discovery of Selective Na+”). V 1.8 Inhibitors Based on 5-chloro-2-(4, 4-difluoroazepan-1-yl)-6-methyl Nicotinamide Scaffold for the Treatment of Pain," European Journal of Medicinal Chemistry, 254, p. 115371 (2023); Jones et al., "Selective Inhibition of Na V 1.8 with VX-548 for Acute Pain, “New England Journal of Medicine 389, Vol. 5: 393-405 (2023)”, and several small molecule projects are currently in various stages of clinical trials. However, targeting peripheral sodium channels with small molecules has historically been challenging (Waxman, Stephen G., “Targeting a Peripheral Sodium Channel to Treat Pain,” New England Journal of Medicine 389: 466-469 (2023)). This is likely due to several factors, including molecular selectivity, variability of the blood-nerve barrier, and inconsistent target binding in multiple compartments of primary afferent neurons. Direct delivery of therapeutic oligonucleotides to primary afferent neurons could provide precise molecular selectivity and consistent inhibition of Na+ in multiple compartments of primary afferent neurons (cutaneous terminals, nerve axons, neuronal cell bodies, and proximal dorsal horn synapses). V 1.8 Opportunities for protein expression.

[0011] In some embodiments, the conjugates described herein have the superior benefit of delivering oligonucleotides to dorsal root ganglion (DRG) neurons. In some aspects, this disclosure provides conjugates with the superior benefit of delivering oligonucleotides to DRG neurons with a reduced risk of immunogenicity compared to other conjugates containing anti-TfR antibodies. In some embodiments, the conjugates described herein induce pain relief and can be used to treat chronic pain in human subjects.

[0012] In some embodiments, the TfR antibody disclosed herein is a bivalent antibody against human / cynomolgus monkey reactive transferrin receptor, which binds to Na+ via a linker. V 1.8 (SCN10A gene / transcription) siRNA linking forms an antibody-oligonucleotide conjugate (AOC), which is engineered for uptake into peripheral tissues via the transferrin receptor. In some embodiments, upon administration, the AOC binds to the apical domain of the transferrin receptor expressed on peripheral tissues, including the dorsal root ganglion, thereby undergoing receptor-mediated internalization and entering the lysosomal degradation compartment. Then, targeting Na... V 1.8% of the siRNA is presumed to be released into the cytoplasm to trigger the RNAi targeting mechanism.

[0013] One aspect of this disclosure relates to the use of human Na+ in cells for inhibiting Na+ V 1.8 Oligonucleotides expressed by the gene (SCN10A), which include single-stranded or double-stranded oligonucleotides containing Na+. V 1.8 Regions complementary to mRNA.

[0014] Another aspect of this disclosure relates to a method for inhibiting human Na+ in cells. V 1.8 Oligonucleotides expressed by gene (SCN10A), comprising: a sense strand and an antisense strand, wherein the sense strand and / or antisense strand form a double-stranded region, and the antisense strand contains Na+. V 1.8 The complementary region of the mRNA (i.e., SCN10A mRNA), wherein the length of the complementary region is less than 30 nucleotides.

[0015] Another aspect of this disclosure relates to cells containing one of the oligonucleotides disclosed herein. The cell is preferably a mammalian cell, such as a human cell.

[0016] In some embodiments, the oligonucleotides disclosed herein are single-stranded (ss) oligonucleotides comprising an antisense strand containing Na V1.8 A region complementary to the mRNA (i.e., SCN10A mRNA), wherein the length of the complementary region is less than 30 nucleotides. In some embodiments, the antisense strand is 15 to 30 nucleotides, 18 to 25 nucleotides, 19 nucleotides, or 21 nucleotides in length. In some embodiments, the antisense strand includes a region complementary to the target sequence of any of SEQ ID NO: 1 to 141. In some embodiments, the complementary region of the ss oligonucleotide is at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides in length. In some embodiments, the antisense strand includes a 3' sequence of one or more nucleotides in length. In some embodiments, the antisense strand includes at least one modified nucleotide. In some embodiments, all nucleotides of the antisense strand are modified. In some embodiments, the antisense strand has modified nucleotides, and the modified nucleotides include a 2'-modification on the sugar. In some embodiments, the 2'-modification is 2'-fluorine, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the modification is 2'-fluorine, vinylphosphonic uridine (VpUq), or 2'-O-methyl. In some embodiments, the 5' end of the antisense strand contains at least one vinylphosphonic uridine (VpUq). In some embodiments, one or more nucleotides at positions 1-21 of the antisense strand are modified with 2'-fluorine. In some embodiments, one or more nucleotides at positions 1-21 of the antisense strand are modified with 2'-O-methyl. In some embodiments, the antisense strand has at least one modified internucleotide linker. In some embodiments, at least one modified internucleotide linker is a thiophosphate linker or a dithiophosphate linker. In some embodiments, at least two terminal nucleotides at the 5' or 3' end of the antisense strand have a thiophosphate linker or a dithiophosphate linker. In some embodiments, at least three terminal nucleotides at the 5' or 3' end of the antisense strand have phosphate thioester or phosphate dithioester linkages. In some embodiments, a linker portion is attached to one or more ends of the antisense strand. In some embodiments, the linker is 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC). In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to cholesterol, lipids, peptides, or antibodies or their antigen-binding fragments. In some embodiments, the antisense strand has the following sequence: i) any odd number from SEQ ID NO: 143 to 423, or ii) any one from SEQ ID NO: 424 to 564. In some embodiments, (i) the antisense strand has the sequence shown in Table 2, or (ii) the antisense strand has the sequence shown in Table 3.

[0017] In another aspect, this disclosure provides a method for inhibiting Na+ in organisms. V 1.8 A gene-expressing pharmaceutical composition comprising one or more oligonucleotides of the present invention and comprising a pharmaceutically acceptable carrier.

[0018] In another aspect, this disclosure provides a cell comprising one of the oligonucleotides of the present invention. The cell is preferably a mammalian cell, such as a human cell.

[0019] In another aspect, this disclosure provides a method for inhibiting Na+ in cells. V 1.8 Cells containing a gene expression vector. The vector contains a regulatory sequence operatively linked to a nucleotide sequence encoding at least one strand of one of the oligonucleotides of the present invention.

[0020] Another aspect of this disclosure relates to a conjugate comprising the oligonucleotide of this disclosure and conjugated to a target Na+. V 1.8 nucleotide sense strand or targeting Na V An anti-transferrin receptor antibody or an antigen-binding fragment thereof comprising the antisense strand of an 1.8-nucleotide chain. One embodiment of this disclosure relates to a pharmaceutical composition comprising a conjugate of this disclosure and comprising a pharmaceutically acceptable carrier.

[0021] Another aspect of this disclosure relates to a method for preventing, treating, or alleviating pain in a patient in need, the method comprising administering to the patient an effective amount of the oligonucleotide, conjugate, or pharmaceutical composition of this disclosure.

[0022] In another aspect, the present invention provides a method for inhibiting Na+ in cells. V 1.8 A method for gene expression, comprising the steps of: a) introducing an oligonucleotide or conjugate of the present disclosure into a cell; and b) maintaining the cells produced in step a) for a sufficient time to obtain Na+. V Degradation of the mRNA transcript of gene 1.8, thereby inhibiting Na+ in cells. V 1.8 Gene expression. Attached Figure Description

[0023] Figure 1A and Figure 1B Oligonucleotides that can be used in this disclosure (e.g., those targeting Na) are shown. V Modification patterns in siRNA (1.8). In the attached figure, VpUq is vinylphosphonic uridine, 2'-OMe is 2'-O-methyl modification, 2'F is 2'-fluorine, and PS is thiophosphate. Figure 1B The markings shown at the bottom are applicable Figure 1A and Figure 1B Both.

[0024] Figure 2 A comparison is shown between modified siRNA 410 without VpUq (i.e., siRNA number 285 in Table 4) and modified siRNA 410 with VpUq (i.e., siRNA number 305 in Table 4).

[0025] Figure 3 A comparison is shown between modified siRNA 535 without VpUq (i.e., siRNA number 291 in Table 4) and modified siRNA 535 with VpUq (i.e., siRNA number 311 in Table 4).

[0026] Figure 4 Various Na are shown V 1.8 Potency of siRNA sequences in primary human dorsal root ganglion neurons (DRG). The most potent siRNAs showed equal to or greater than 70% Na+ in DRG neurons. V 1.8 mRNA reduction. This figure identifies siRNAs by the start position of the target region on the human SCN10A transcript.

[0027] Figure 5 Some Na were shown V 1.8 siRNA concentration-response relationship. This figure identifies siRNAs by the starting position of the target region on the human SCN10A transcript.

[0028] Figures 6A to 6C The RT-qPCR analysis and ELISA analysis of SCN10A expression of the two siRNAs are shown.

[0029] Figure 7 Yes V 1.8 A representative structure of one embodiment of the antibody-oligonucleotide conjugate, also referred to herein as “AOC” or “conjugate”. The antibody is shown in the figure as a “Y” shape, linked to the siRNA molecule via an SMCC linker. In some embodiments, two siRNA molecules may be linked to the antibody molecule (e.g., using eCys or nCys to link each siRNA molecule to each heavy chain of the antibody via a linker).

[0030] Figure 8 Na using SAX method 1 is shown V 1.8 Representative analytical strong anion exchange column (SAX) chromatogram of the AOC reaction mixture.

[0031] Figure 9 Na using FPLC method 1 is shown V 1.8 Representative rapid protein liquid chromatography (FPLC) chromatograms of the AOC reaction mixture.

[0032] Figure 10 A representative size exclusion chromatography (SEC) spectrum using SEC method 1 is shown.

[0033] Figure 11 A representative strong anion exchange (SAX) spectrum using SAX method 1 is shown.

[0034] Figure 12 The SDS-PAGE analysis of AOC is shown. MWM represents the molecular weight marker, and the molecular weight (MW) of each band of this marker is labeled. The gel on the left shows TBP1-410, and the gel on the right shows TBP2-410.

[0035] Figure 13 This demonstrates that TBP1-si410 AOC reduces Na in human DRG in vitro. V 1.8 mRNA expression. TBP1-si410AOC contains TBP1 antibody and modified 410 siRNA linked via SMCC adapter.

[0036] Figure 14 This shows the effect of humanized Na V 1.8 Reversal of cold-induced noxious responses in rats following intravenous administration of AOC (6 mg / kg single intravenous dose, based on oligonucleotide weight, for 13 days of exposure) and subsequent administration of OX26-410 and OX26-535 AOC. Compared to the PBS control (white bar), rats treated with OX26-410 AOC (grey bar) showed a statistically significant reduction in cold-induced noxious behavior at both tested temperatures (2°C and -1°C) (p<0.05, one-way ANOVA, Dunnet post-hoc test). Rats treated with OX26-535 AOC (black bar) showed a reduced response to cold, but this effect did not reach statistical significance.

[0037] Figure 15 The effects of applying OX26-410 and OX26-535 AOC on humanized Na were demonstrated. V 1.8 Na in rat DRG V 1.8 protein reduction. Administration of targeted Na... V 1.8% AOC (OX26-410 and OX26-535, 6 mg / kg, intravenous, exposure for 13 days) resulted in Na+ levels measured in the dorsal root ganglion (DRG). V 1.8 protein was significantly reduced, and it was associated with humanized Na+. V 1.8 Cold-induced reduction of noxious behavior in rats. Rats treated with AOC OX26-535 (black squares) showed a reduction in DRG Na compared to PBS controls (white circles).V A significant reduction in 1.8% protein was observed, but this did not lead to a significant reduction in cold behavior. Humanized Na+ treated with AOC OX26-410 (grey triangle) V 1.8 Rats showed Na V 1.8 Both DRG protein levels and cold-induced noxious behavior were statistically significantly reduced (p<0.05, one-way ANOVA, Dunnet post-hoc test).

[0038] Figure 16 The study showed that Na+ levels in hairless skin of rats after administration of OX26-410 and OX26-535 AOC were [not specified]. V 1.8 protein reduction. Administration of targeted Na... V An AOC of 1.8 (OX26-410 and OX26-535, 6 mg / kg, intravenously, for 13 days of exposure) resulted in Na levels measured in hairless claw skin. V 1.8 protein was significantly reduced, and it was associated with humanized Na+. V 1.8 Reduction of cold-induced noxious behavior in rats. Rats treated with AOC OX26-535 (black squares) showed reduced Na+ in hairless paw skin compared to PBS controls (white circles). V A significant reduction in 1.8% protein was observed, but this did not lead to a significant reduction in cold behavior. Humanized Na+ treated with AOC OX26-410 (grey triangle) V 1.8 Rats showed Na V 1.8 Both hairless claw skin protein levels and cold-induced noxious behavior were statistically significantly reduced (one-way ANOVA, Dunnet post-hoc test, p<0.05).

[0039] Figure 17 The study illustrates the effects of single-dose anti-Tfr-410 nCys mAb (dark gray bar; TBP1-siRNA410 AOC), anti-Tfr-410 eCys mAb (light gray bar; TBP2-siRNA410 AOC), anti-Tfr-410 Fab (shaded gray bar; TBP3-siRNA410 AOC), two-dose anti-Tfr-410 nCys mAb, or PBS (white bar) on Na+ levels in the hairless paw skin of cynomolgus monkeys. V 1.8 Protein reduction. Single-dose groups received 1.0 mg / kg or 6.0 mg / kg (by oligonucleotide weight) of AOC. Two-dose groups of anti-Tfr-410nCys mAb received AOC twice, on days 1 and 8, at a dose of 3.0 mg / kg (by oligonucleotide weight), which resulted in a decrease in Na+ in hairless claw skin. V 1.8 A significant decrease in protein levels (p<0.05, one-way ANOVA). All exposures lasted 29 days.

[0040] Figure 18 This demonstrates the effect of TBP2-410 eCys AOC on Na in the hairless paw skin of cynomolgus monkeys (NHP). V 1.8 Effect of protein knockdown. After treatment with TBP2-410 eCys AOC, Na in hairless claw skin... V 1.8 Protein Decrease. Groups received PBS (control), 0.3 mg / kg, 3.0 mg / kg, or 30.0 mg / kg (by weight of oligonucleotides) of TBP2-410 eCys AOC (N=4 / group, except for the 30.0 mg / kg group, where N=2). AOC was administered intravenously once weekly for four weeks. Administration of 3.0 mg / kg and 30.0 mg / kg (by weight of oligonucleotides) of AOC resulted in decreased sodium levels in hairless claw skin. V 1.8 Significant reduction in protein levels (52.3% and 42.2% of the PBS control group, respectively, *p<0.05, one-way ANOVA). All exposures occurred 27 days after the last dose (total study duration 49 days). Detailed Implementation

[0041] One aspect of this disclosure relates to oligonucleotides, conjugates comprising such oligonucleotides, pharmaceutical compositions, and the use of oligonucleotides, conjugates, or compositions to inhibit Na+ in cells or mammals. V 1.8 Methods for gene expression. This invention also provides methods for treating Na+-related gene expression using oligonucleotides, conjugates, or combinations thereof. V 1.8 Compositions and methods for mammalian pathological conditions and diseases caused by genes. In some embodiments, oligonucleotides guide Na through a process called RNA interference (RNAi). V 1.8 Sequence-specific degradation of mRNA (i.e. SCN10A mRNA).

[0042] Using these oligonucleotides and conjugates, it is possible to achieve Na+ related to mammalian pain responses. V 1.8 Targeted degradation of mRNA. This disclosure has demonstrated that these oligonucleotides or conjugates can specifically and effectively mediate RNAi, leading to Na+ degradation. V 1.8 Significant inhibition of gene expression. This disclosure also demonstrates that oligonucleotides and conjugates (such as those with anti-TfR antibodies) can be efficiently delivered to neurons (such as dorsal root ganglion neurons), leading to Na+ inhibition. V 1.8 Significant inhibition of gene expression and reduction / treatment of pain. Therefore, the methods and compositions of the present invention, comprising these oligonucleotides and conjugates, can be used to relieve or treat pain.

[0043] The following detailed description discloses how to prepare and use oligonucleotides, conjugates, and related compositions to inhibit target Na+. V 1.8 Gene expression and its use in treating Na+ V 1.8 Compositions and methods for expressing diseases and symptoms (such as pain).

[0044] The conjugates disclosed herein comprise oligonucleotides having an antisense strand containing Na V 1.8 At least a complementary region of the RNA transcript of the gene and anti-TfR antibody (anti-transferrin receptor antibody) or its antigen-binding fragment.

[0045] The pharmaceutical compositions disclosed herein comprise oligonucleotides (having an antisense strand, the antisense strand comprising Na+, ... V 1.8 The RNA transcript of the gene (at least a portion of the complementary region), and a pharmaceutically acceptable carrier. Another embodiment of the pharmaceutical composition comprises a conjugate of the present disclosure and a pharmaceutically acceptable carrier. Thus, certain aspects of the present disclosure provide pharmaceutical compositions comprising the oligonucleotide or conjugate of the present disclosure and a pharmaceutically acceptable carrier, and the use of such pharmaceutical compositions to inhibit Na+. V 1.8 Methods for gene expression and the use of pharmaceutical compositions to treat Na+ V 1.8 Methods for treating or alleviating pain caused by gene expression.

[0046] One aspect of this disclosure relates to a method for inhibiting human Na+ in cells. V 1.8 Oligonucleotides for gene expression (e.g., siRNA). These oligonucleotides comprise: a sense strand and / or an antisense strand, wherein the sense strand and / or antisense strand form a double-stranded region, and the antisense strand contains a Na+ / Na ... V 1.8 A complementary region of mRNA, wherein the length of the complementary region is less than 30 nucleotides.

[0047] In some embodiments, the oligonucleotides of this disclosure cause the antisense strand to bind with Na. V 1.8 The complementary region of the mRNA target sequence is at least 15 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, or at least 21 consecutive nucleotides in length. In some embodiments, the antisense strand is related to Na... V 1.8 The complementary region of the mRNA target sequence is at least 16, 17, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

[0048] In some embodiments, the sense strand of the oligonucleotide is 15 to 30 nucleotides, 18 to 25 nucleotides, or 19 nucleotides in length. In some embodiments, the sense strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length.

[0049] In some embodiments, the antisense strand of the oligonucleotide is 15 to 30 nucleotides, 18 to 25 nucleotides, 19 nucleotides, or 21 nucleotides in length. In some embodiments, the antisense strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the antisense strand is 19 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length, and the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length, and the antisense strand is 19 nucleotides in length.

[0050] In some embodiments, the oligonucleotide includes an antisense strand containing a region complementary to the target sequence of any of SEQ ID NO: 1 to 141. Table 1 shows Na V 1.8 Target sequences present in mRNA.

[0051] Table 1: Target sequences (18-mer) of SCNA10A mRNA transcripts .

[0052]

[0053]

[0054]

[0055] In some implementations, the oligonucleotide contains an antisense strand that contains Na+. V 1.8 A region complementary to the mRNA target sequence, wherein the length of the complementary region of the antisense strand to the target sequence (e.g., as shown in Table 1) is at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides.

[0056] In some embodiments, the oligonucleotide has the sense strand having a sequence numbered as an even number among SEQ ID NO: 142 to 422. In some embodiments, the antisense strand of the oligonucleotide has a sequence numbered as an odd number among SEQ ID NO: 143 to 423. In some embodiments, the antisense strand of the oligonucleotide has a sequence numbered as an any one of SEQ ID NO: 424 to 564.

[0057] Tables 2 and 3 below illustrate some exemplary oligonucleotides of this disclosure.

[0058] Table 2: 19-mer Na V 1.8 Nucleic acid sequence of siRNA

[0059]

[0060]

[0061]

[0062] Table 3: 19 / 21 polymer Na V 1.8 Nucleic acid sequence of siRNA

[0063]

[0064]

[0065]

[0066] In some embodiments, the oligonucleotides of this disclosure cause the sense strand and antisense strand to comprise nucleotide sequences selected from the group consisting of:

[0067] (a) are SEQ ID NO: 565 and 566, respectively;

[0068] (b) are SEQ ID NO: 567 and 568, respectively;

[0069] (c) are SEQ ID NO: 569 and 570, respectively;

[0070] (d) are SEQ ID NO: 571 and 572, respectively;

[0071] (e) are SEQ ID NO: 573 and 574, respectively;

[0072] (f) are SEQ ID NO: 575 and 576, respectively;

[0073] (g) are SEQ ID NO: 577 and 578, respectively;

[0074] (h) are SEQ ID NO: 579 and 580, respectively;

[0075] (i) are SEQ ID NO: 581 and 582, respectively;

[0076] (j) are SEQ ID NO: 583 and 584, respectively;

[0077] (k) are SEQ ID NO: 585 and 586, respectively;

[0078] (l) are SEQ ID NO: 587 and 588, respectively;

[0079] (m) are SEQ ID NO: 589 and 590, respectively;

[0080] (n) are SEQ ID NO: 591 and 592, respectively;

[0081] (o) are SEQ ID NO: 593 and 594, respectively;

[0082] (p) are SEQ ID NO: 595 and 596, respectively;

[0083] (q) are SEQ ID NO: 597 and 598, respectively;

[0084] (r) are SEQ ID NO: 599 and 600, respectively;

[0085] (s) are SEQ ID NO: 601 and 602, respectively; and

[0086] (t) are SEQ ID NO: 603 and 604, respectively.

[0087] In some embodiments, the oligonucleotides of this disclosure cause the sense strand and antisense strand to comprise nucleotide sequences selected from the group consisting of:

[0088] (a) are SEQ ID NO: 565 and 605, respectively;

[0089] (b) are SEQ ID NO: 567 and 606, respectively;

[0090] (c) are SEQ ID NO: 569 and 607 respectively;

[0091] (d) are SEQ ID NO: 571 and 608, respectively;

[0092] (e) are SEQ ID NO: 573 and 609, respectively;

[0093] (f) are SEQ ID NO: 575 and 610, respectively;

[0094] (g) are SEQ ID NO: 577 and 611, respectively;

[0095] (h) are SEQ ID NO: 579 and 612, respectively;

[0096] (i) are SEQ ID NO: 581 and 613, respectively;

[0097] (j) are SEQ ID NO: 583 and 614, respectively;

[0098] (k) are SEQ ID NO: 585 and 615, respectively;

[0099] (l) are SEQ ID NO: 587 and 616, respectively;

[0100] (m) are SEQ ID NO: 589 and 617 respectively;

[0101] (n) are SEQ ID NO: 591 and 618, respectively;

[0102] (o) are SEQ ID NO: 593 and 619 respectively;

[0103] (p) are SEQ ID NO: 595 and 620, respectively;

[0104] (q) are SEQ ID NO: 597 and 621, respectively;

[0105] (r) are SEQ ID NO: 599 and 622, respectively;

[0106] (s) are SEQ ID NO: 601 and 623 respectively; and

[0107] (t) are SEQ ID NO: 603 and 624, respectively.

[0108] In some embodiments, the oligonucleotides of this disclosure are such that: i) the sense strand has the sequence shown in Table 2, and the antisense strand has the sequence shown in Table 2; ii) the sense strand has the sequence shown in Table 3, and the antisense strand has the sequence shown in Table 3; iii) the oligonucleotide has the sequence shown by any of siRNA numbers 1 to 141; iv) the oligonucleotide has the sequence shown by any of siRNA numbers 142 to 282; or v) the oligonucleotide has the sequence shown by any of siRNA numbers 283 to 322.

[0109] RNA modification

[0110] Various oligonucleotide types and / or structures can be used to target SCN10A mRNA, including but not limited to oligonucleotides, antisense oligonucleotides (ASOs), miRNAs, etc. Any oligonucleotide type described herein or elsewhere is intended to be used as a framework to be incorporated into the target sequences herein for the purpose of inhibiting SCN10A activity. In some embodiments, the oligonucleotides herein inhibit SCN10A activity by acting on upstream or downstream RNAi pathways involved by Dicer. For example, oligonucleotides of about 19 to 25 nucleotides per strand have been developed and have at least one to five nucleotide 3' overhangs (see, for example, U.S. Patent No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, for example, U.S. Patent No. 8,883,996). Further work has yielded extended RNAi oligonucleotides in which at least one end of at least one strand extends beyond the double-stranded targeting region, including structures in which one strand comprises a thermodynamically stable four-membered ring (see, for example, U.S. Patent Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010 / 033225). Such structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0111] In some embodiments, the oligonucleotides described herein act on downstream RNAi pathways involving Dicer (e.g., Dicer cleavage). In some embodiments, the oligonucleotide has a 3' overhang (e.g., one, two, or three nucleotides in length) at the 3' end of the sense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a 21-nucleotide antisense strand antisense to the target mRNA (e.g., SCN10AmRNA) and a complementary sense strand, wherein the two strands anneal to form a 19-bp duplex and have a two-nucleotide overhang at either one or both 3' ends. Longer oligonucleotide designs are also anticipated, including oligonucleotides with a 23-nucleotide antisense strand and a 21-nucleotide sense strand, wherein there is a blunt end on the right side of the molecule (3' end of the sense strand / 5' end of the antisense strand) and a two-nucleotide antisense 3' overhang on the left side of the molecule (5' end of the sense strand / 3' end of the antisense strand). In such molecules, a 21-bp duplex region is present. See, for example, U.S. Patent Nos. 9,012,138, 9,012,621 and 9,193,753.

[0112] In some embodiments, the oligonucleotides of this disclosure have the following characteristics: Figure 1A The modification pattern shown optionally does not contain an amine or cholesterol handle. In some embodiments, the oligonucleotide has, for example, Figure 1B The modified pattern shown optionally does not contain amine or cholesterol connecting stems.

[0113] In some embodiments, the oligonucleotide of this disclosure has an antisense strand comprising a 3' overhang sequence of one or more nucleotides in length. In some embodiments, the 3' overhang sequence is two nucleotides in length. In some embodiments, the 3' overhang sequence is UU. In some embodiments, the 3' overhang sequence is AA, GG, or CC.

[0114] In some embodiments, the oligonucleotide of this disclosure comprises at least one modified nucleotide. In some embodiments, all nucleotides of the oligonucleotide are modified.

[0115] Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, wherein, for example, one or more modifications occur at the 2', 3', 4' and / or 5' carbon positions of the sugar. Modified sugars also include non-natural, alternative carbon structures, such as those present in locked nucleic acids (“LNA”; see, for example, Koshkin et al., “LNA (Locked Nucleic Acids): Synthesis of the Adenine, Cytosine, Guanine, 5-methylcytosine, Thymine and Uracil Bicyclonucleoside Monomers, Oligomerisation, and Unprecedented Nucleic Acid Recognition,” Tetrahedron 54, Vol. 14 (1998): 3607-3630), unlocking nucleic acids (“UNA”; see, for example, Snead et al., “5' Unlocked Nucleic Acid Modification Improves siRNA Targeting,” Molecular Therapy-Nucleic Acids 2 (2013)), and bridging nucleic acids (“BNA”; see, for example, Imanishi, Takeshi, and Satoshi Obika, “BNAs: Novel Nucleic Acid Analogs with a Those mentioned in "Bridged Sugar Moiety," Chemical Communications 16 (2002): 1653-1659.

[0116] In some embodiments, the nucleotide modification in the sugar is a 2'-modification, such as 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-F (2'-fluoro), 2'-aminoethyl (EA), 2'-OMe (2'-O-methyl), 2'-MOE (2'-methoxyethyl), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-FANA (2'-fluoro-arabinonucleotide). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In other embodiments, the modification in the sugar is a modification of the sugar ring, which includes modification of one or more carbons of the sugar ring. For example, the modification in the sugar is the attachment of the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or the attachment of the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In other embodiments, the modification is an acyclic sugar lacking the 2'-carbon and 3'-carbon bonds. In other embodiments, the modification is a thiol group, such as, for example, at the 4' position of the sugar.

[0117] The oligonucleotides described herein comprise at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 or more). In some embodiments, the sense strand comprises at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or more). In some embodiments, the antisense strand comprises at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20 or more).

[0118] In some embodiments, all nucleotides in the sense strand except for tetraL (also referred to herein as a four-membered ring) are modified. Similarly, all nucleotides in the antisense strand are modified. In some embodiments, all nucleotides of the oligonucleotide (i.e., the paired nucleotides of the sense and antisense strands) are modified. As described above, and in some embodiments, the modified nucleotides are 2'-modified (e.g., 2'-F, 2'-OMe, 2'-MOE, and / or 2'-FANA). In some embodiments, the modified nucleotides are 2'-modified, such as, for example, 2'-F or 2'-OMe.

[0119] In some embodiments, the modified nucleotide contains a 2'-modification. In some embodiments, the 2'-modification can be, for example, 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, or 2'-deoxy-2'-fluoro-β-d-arabinonucleotide. In some embodiments, the oligonucleotide is modified and contains a 2'-fluoro, vinylphosphonic uridine (VpUq), or 2'-O-methyl modification.

[0120] In some embodiments, the oligonucleotide of this disclosure comprises at least one vinylphosphonate uridine (VpUq) at the 5' end of the antisense strand. In some embodiments, the oligonucleotide of this disclosure comprises at least two or more vinylphosphonate uridines (VpUq) at the 5' end of the antisense strand. In some embodiments, the oligonucleotide is modified and contains a vinylphosphonate ester (Vp).

[0121] In some embodiments, the oligonucleotide comprises a sense strand, and about 50% to 90% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand, and about 50% to 60% of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand, and about 60% to 70% of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand, and about 70% to 80% of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand, and about 80% to 90% of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises a sense strand, and about 90% to 100% of the nucleotides of the sense strand are modified with 2'-OMe. In some embodiments, approximately 78% of the nucleotides in the sense strand contain a 2'-OMe modification. In some embodiments, approximately 79% of the nucleotides in the sense strand contain a 2'-OMe modification. In some embodiments, approximately 80% of the nucleotides in the sense strand contain a 2'-OMe modification. In some embodiments, approximately 81% of the nucleotides in the sense strand contain a 2'-OMe modification. In some embodiments, approximately 84% of the nucleotides in the sense strand contain a 2'-OMe modification.

[0122] In some embodiments, the oligonucleotide comprises an antisense strand, and about 50% to 90% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises an antisense strand, and about 50% to 60% of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises an antisense strand, and about 60% to 70% of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises an antisense strand, and about 70% to 80% of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises an antisense strand, and about 80% to 90% of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, the oligonucleotide comprises an antisense strand, and about 90% to 100% of the nucleotides of the antisense strand are modified with 2'-OMe. In some embodiments, approximately 78% of the nucleotides of the antisense strand contain a 2'-OMe modification. In some embodiments, approximately 79% of the nucleotides of the antisense strand contain a 2'-OMe modification. In some embodiments, approximately 80% of the nucleotides of the antisense strand contain a 2'-OMe modification. In some embodiments, approximately 81% of the nucleotides of the antisense strand contain a 2'-OMe modification.

[0123] In some embodiments, about 15% to about 25% (e.g., 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, about 35% to 45% (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%) of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, about 45% to 85% (e.g., 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%) of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, about 70% of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, about 75% of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, about 80% of the nucleotides of the oligonucleotide contain a 2'-OMe modification. In some embodiments, approximately 85% of the nucleotides in the oligonucleotide contain a 2'-OMe modification. In some embodiments, approximately 90% of the nucleotides in the oligonucleotide contain a 2'-OMe modification. In some embodiments, approximately 95% of the nucleotides in the oligonucleotide contain a 2'-OMe modification.

[0124] In some embodiments, the oligonucleotide comprises a sense strand, and about 10% to 20% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) of the nucleotides of this sense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, and about 21% to 30% (e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%) of the nucleotides of this sense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, and about 38% to 43% (e.g., 38%, 39%, 40%, 41%, 42%, or 43%) of the nucleotides of this sense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 38% to 43% (e.g., 38%, 39%, 40%, 41%, 42%, or 43%) of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 15% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 16% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 17% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 18% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 19% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand in which about 20% of the nucleotides contain a 2'-F modification.

[0125] In some embodiments, the oligonucleotide comprises an antisense strand, and about 10% to 20% (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) of the nucleotides of the antisense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, and about 21% to 30% (e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%) of the nucleotides of the antisense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, and about 38% to 43% (e.g., 38%, 39%, 40%, 41%, 42%, or 43%) of the nucleotides of the antisense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, and about 15% of the nucleotides of the antisense strand contains a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, of which approximately 16% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, of which approximately 17% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, of which approximately 18% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, of which approximately 19% of the nucleotides contain a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, of which approximately 20% of the nucleotides contain a 2'-F modification.

[0126] In some embodiments, about 14% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 15% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 16% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 17% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 18% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 20% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 21% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 22% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 23% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 24% of the nucleotides in the oligonucleotide contain a 2'-F modification. In some embodiments, about 25% of the nucleotides in the oligonucleotide contain a 2'-F modification.

[0127] Furthermore, the oligonucleotides described herein can have different modification patterns. In some embodiments, the modified oligonucleotides contain the following as shown in Table 4 (and...). Figure 1A or Figure 1BThe meaningful chain sequence of the modification pattern shown in Table 4 and the antisense chain with the modification pattern shown in Table 4 are shown in Table 4.

[0128] In some embodiments, the oligonucleotide of this disclosure comprises a 2'-fluorine (2'-F) modification at one or more nucleotides at positions 7, 8, or 9 of the sense strand. In some embodiments, the oligonucleotide causes one or more nucleotides at positions 2, 6, 14, or 16 of the antisense strand to be modified with 2'-fluorine. In some embodiments, the oligonucleotide causes one or more nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the sense strand to be modified with 2'-O-methyl (2'-OMe). In some embodiments, the oligonucleotide causes one or more nucleotides at positions 1, 3, 4, 5, 7 to 13, 15, 17, 18, 19, 20, or 21 of the antisense strand to be modified with 2'-O-methyl.

[0129] In some embodiments, the oligonucleotides of this disclosure comprise at least one modified internucleotide linker. The internucleotide linker may be a phosphate thioester linker or a phosphate dithioester linker. In some embodiments, at least two terminal nucleotides at the 5' or 3' end of the antisense or sense strand have phosphate thioester linkers or phosphate dithioester linkers. In some embodiments, at least three terminal nucleotides at the 5' or 3' end of the antisense or sense strand have phosphate thioester linkers or phosphate dithioester linkers.

[0130] In some implementations, the oligonucleotide makes the sense strand have 19 nucleotides, and the internucleotide linkages between the nucleotides at positions 1 and 2, 2 and 3, 17 and 18, or 18 and 19 of the sense strand are modified with phosphate thioester linkages.

[0131] In some implementations, the oligonucleotide makes the antisense strand have 21 nucleotides, and the internucleotide linkages between one or more nucleotides of the antisense strand between positions 1 and 2, 2 and 3, 19 and 20, or 20 and 21 are modified with phosphate thioester linkages.

[0132] In some embodiments, the oligonucleotides of this disclosure are selected from Table 4. Table 4 shows some embodiments of the modified siRNA sequences of this disclosure.

[0133] Table 4: Modified Na V 1.8 siRNA

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Legend: (N) = 2'-OH (ribose; e.g., A, G, C, or U); m(N) = 2'-O-methylribose; p = phosphate; [fl2r] (N) = 2'-fluororibose; [ps] = thiophosphate skeleton modification; 5vp_una2moe = vinylphosphonate-2'-O-methoxyethylribose-UNA-U; "S" indicates sense chain; "AS" indicates antisense chain. At the 5' end of the sense chain, there may be free C6-NH2 or cholesterol conjugated to an amino linker.

[0140] The sense and antisense strands of the oligonucleotides disclosed herein can be synthesized using any nucleic acid polymerization method known in the art, such as solid-phase synthesis using phosphoramide chemistry (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonates, phosphate triesters, or enzymatic synthesis. Automated commercial synthesizers, such as the MerMade from LGC Biosearch Technologies, can be used. ™ 12, or other synthesizers from BioAutomation or AppliedBiosystems. The thiophosphate linkage can be introduced using sulfiding agents such as phenylacetyl disulfide or DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-diathiazoline-3-thione). The synthesis of modified or conjugated oligonucleotides using similar techniques and commercially available modified phosphoramides and controlled-porosity glass (CPG) products is well-known.

[0141] Unwanted impurities can be removed from the final oligonucleotide product using purification methods. Common purification techniques for single-stranded oligonucleotides include reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC), capillary gel electrophoresis (CGE), anion-exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, the oligonucleotides can be analyzed by mass spectrometry and quantified spectrophotometrically at a wavelength of 260 nm. The sense and antisense strands can then be annealed to form dsRNA.

[0142] In addition to the modifications described above, the oligonucleotides herein also contain one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as a base analog) is linked at the 1' position of the nucleotide sugar moiety. In some embodiments, the modified nucleobase is a nitrogenous base. In other embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 2008 / 0274462. In some other embodiments, the modified nucleotide is a universal base. However, in some embodiments, the modified nucleotide does not contain a nucleobase (base-free).

[0143] Regarding universal bases, they contain a heterocyclic moiety located at the 1' position of the nucleotide sugar moiety in the modified nucleotide or at an equivalent position in the substitution of the nucleotide sugar moiety. When present in a duplex, this heterocyclic moiety is located opposite more than one type of base without significantly altering the structure of the duplex. Furthermore, duplexes formed by ss nucleic acids with universal bases and target nucleic acids have a lower melting temperature (T0) compared to duplexes formed with complementary nucleic acids, compared to reference single-stranded (ss) nucleic acids (e.g., oligonucleotides) that are perfectly complementary to the target nucleic acid. m However, when compared to a reference ss nucleic acid in which universal bases have been replaced to produce a single mismatch, the duplex formed by the ss nucleic acid with universal bases and the target nucleic acid has a higher T than the duplex formed with the nucleic acid with mismatched bases. m .

[0144] The oligonucleotides described herein, or their pharmaceutically acceptable salts (e.g., trifluoroacetate, acetate, or hydrochloride), are incorporated into formulations or pharmaceutical compositions. Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to the individual or cellular environment using formulations that minimize degradation, promote delivery and / or uptake, or provide another beneficial property to the oligonucleotides in the formulation. In some embodiments, the oligonucleotides are formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micelle structures, and capsids.

[0145] To improve in vivo compatibility and efficacy, oligonucleotides can react with any of a variety of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Common methods for preparing pharmaceutically acceptable salts are well known in the art.

[0146] Antibodies (TfR-binding proteins) and antibody-oligonucleotide conjugates

[0147] One aspect of this disclosure is based, at least in part, on the development of transferrin receptor-binding proteins (also referred to herein as anti-TfR antibodies or antigen-binding fragments thereof) that bind to transferrin receptors, have a low risk of immunogenicity, and can be used to deliver molecular payloads (such as therapeutic agents, single-stranded RNA (ssRNA), ASO, dsRNA, siRNA, or drugs) to different tissues in a subject's body, such as muscles, pancreas, brain, central nervous system (CNS), peripheral nervous system (PNS), or to different cell types, such as cancer cells or dorsal root ganglion (DRG) neurons.

[0148] In another aspect, this document provides conjugates comprising the human TfR-binding protein described herein and oligonucleotides. In some embodiments, the oligonucleotide is a single-stranded RNA (ssRNA). In some embodiments, the oligonucleotide is a double-stranded RNA (dsRNA). In some embodiments, the oligonucleotide comprises a sense strand and / or an antisense strand, wherein the antisense strand is complementary to at least a portion of the SCN10A mRNA. In some embodiments, the oligonucleotide comprises a sense strand and / or an antisense strand, wherein the antisense strand is complementary to a portion of the SNC10A mRNA.

[0149] In some aspects, this disclosure provides data demonstrating that certain conjugates containing transferrin-binding protein (TBP; also described herein as anti-TfR antibodies) have superior benefits in that they have a reduced risk of immunogenicity compared to other conjugates containing anti-TfR antibodies. In some aspects, this disclosure provides conjugates containing TBP that have the superior benefit of delivering molecular payloads to dorsal root ganglion (DRG) neurons. In some aspects, this disclosure provides conjugates containing TBP that have the superior benefit of delivering molecular payloads to dorsal root ganglion (DRG) neurons with a reduced risk of immunogenicity compared to other anti-TfR antibodies.

[0150] In some embodiments, the transferrin receptor-binding protein (TBP) described herein binds to the human transferrin receptor with high specificity, high affinity, and reduced immunogenicity risk. In some embodiments, TBP provides a range of affinities for TfR.

[0151] In some embodiments, the TBP described herein can be used to target tissues and / or cells expressing TfR. In some embodiments, the TBP described herein can be used to deliver molecular payloads to target cells or tissues (e.g., cells or tissues expressing transferrin receptors).

[0152] In some aspects, this disclosure relates to a complex (or conjugate) comprising at least one of the disclosed TBPs conjugated (e.g., covalently linked) to at least one molecular payload (e.g., a therapeutic agent, siRNA). In some embodiments, the TBP of this disclosure can be used to deliver a conjugate (which comprises TBP and a molecular payload) to cells or tissues expressing TfR1 (e.g., DRG or brain) for the treatment or relief of pain; and / or for the treatment of diseases associated with SCN10A.

[0153] One aspect of this disclosure relates to a conjugate comprising an oligonucleotide of the present disclosure and an anti-transferrin receptor antibody (anti-TfR antibody) or a fragment thereof conjugated to the sense strand or antisense strand of the oligonucleotide. In some embodiments, the anti-transferrin receptor antibody (anti-TfR antibody) or a fragment thereof is conjugated to at least one sense strand of the oligonucleotide. In some embodiments, the anti-transferrin receptor antibody or an antigen-binding fragment thereof is conjugated to at least two sense strands of at least two oligonucleotides. In some embodiments, the anti-transferrin receptor antibody (anti-TfR antibody) or a fragment thereof is conjugated to at least one antisense strand of the oligonucleotide. In some embodiments, the anti-transferrin receptor antibody or an antigen-binding fragment thereof is conjugated to at least two antisense strands of at least two oligonucleotides.

[0154] In some implementations, this document provides a conjugate of formula (I): P-(LR) n Wherein R is an oligonucleotide comprising sense and antisense strands as disclosed herein; where P is an anti-TfR antibody or its antigen-binding fragment as described herein; and where L is a linker, which may optionally be absent; and where "n" may be 1, 2 or more. In some embodiments, n is 1. In one embodiment, n is 2.

[0155] In some embodiments, R is selected from Table 2, Table 3, or Table 4. In some embodiments, R is selected from siRNA numbers 1 to 322 disclosed in Table 2, Table 3, or Table 4. In some embodiments, the antisense strand of R has 90% sequence similarity to any antisense strand disclosed in Table 2, Table 3, or Table 4. In some embodiments, the antisense strand of R has 95% sequence similarity to any antisense strand disclosed in Table 2, Table 3, or Table 4. In some embodiments, the sense strand of R has 90% sequence similarity to any sense strand disclosed in Table 2, Table 3, or Table 4. In some embodiments, the sense strand of R has 95% sequence similarity to any sense strand disclosed in Table 2, Table 3, or Table 4. In some embodiments, P is selected from Table 8.

[0156] In some implementations, the oligonucleotides of the conjugate mediate targeting of human Na+. V1.8 RNA interference with mRNA. In some embodiments, the conjugated oligonucleotide preferentially mediates targeting of human Na+ in the DRG cells of human subjects. V 1.8 RNA interference with mRNA. In some embodiments, conjugates of this disclosure containing oligonucleotides mediate RNA interference against human Na+. V 1.8 RNA interference with mRNA and modulation of pain in subjects. In some embodiments, conjugates of this disclosure containing oligonucleotides mediate targeting of human Na+. V 1.8 RNA interference with mRNA and treatment of pain in subjects. In some embodiments, conjugates of this disclosure containing oligonucleotides mediate targeting of human Na+. V 1.8 RNA interference with mRNA and reduction of pain in subjects.

[0157] In some embodiments, the anti-TfR antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: HCDR1 comprises SEQ ID NO: 629, HCDR2 comprises SEQ ID NO: 630, HCDR3 comprises SEQ ID NO: 631, LCDR1 comprises SEQ ID NO: 632, LCDR2 comprises SEQ ID NO: 633, and LCDR3 comprises SEQ ID NO: 634. Some examples of HCDRs of the present disclosure TBP are provided in Table 5.

[0158] Table 5: Examples of heavy chain complementarity-determining regions (HCDRs) of transferrin receptor-binding protein (TBP)

[0159]

[0160] Some examples of LCDRs for the TBP disclosed herein are provided in Table 6.

[0161] Table 6: Examples of the light chain complementarity-determining region (LCDR) of transferrin receptor-binding protein (TBP)

[0162]

[0163] Some examples of HCVR and LCVR of the TBP disclosed herein are provided in Table 7.

[0164] Table 7: Heavy chain variable region (HCVR) and light chain variable region (LCVR) of transferrin receptor-binding protein (TBP)

[0165]

[0166] Some examples of the HC and LC of TBP disclosed herein are provided in Table 8. TBP2 is a heterologous monoclonal antibody and therefore has two heavy chains (i.e., HCA and HCB). Such heterologous monoclonal antibodies are described, for example, in Example 1 of U.S. Patent Application Publication No. 2021 / 0054103, which is incorporated herein by reference in its entirety. Heterodimeric antibodies, such as heterologous monoclonal antibodies, orthogonal monoclonal antibodies, or bimeric antibodies, have been described in WO2014150973, WO2016118742, WO2018118616, and WO2011131746, which are incorporated herein by reference in their entirety. The nucleic acid sequence of TBP is provided in Table 9 below.

[0167] Table 8: Transferrin receptor-binding protein heavy chain (HC) and light chain (LC)

[0168]

[0169] In Table 8 and this disclosure, the term "EN" refers to an effector null mutation. In some embodiments, the term "EN" refers to the mutation L234A / L235E / G237A / A330S / P331S in the Fc region of the antibody or its derivative.

[0170] In Table 8 and this disclosure, the term “AAS” refers to the L234A / L235A / D265S mutation. This is an example of an effector null mutation in the Fc region. For more information, see, for example, Pejchal et al., “Profiling the BiophysicalDevelopability Properties of Common IgG1 Fc Effector Silencing Variants,” Antibodies, 12, 54 (2023), which is incorporated herein by reference in its entirety.

[0171] In Table 8 and this disclosure, the term "211" refers to an antibody or a derivative thereof that binds only to the top domain of TfR.

[0172] In Table 8 and this disclosure, “Com29 B09” (e.g., TBP1) refers to an antibody or a derivative thereof engineered to reduce affinity and immunogenicity risk. In Table 8 and this disclosure, “TBP2” specifically has an asymmetric / single eCys site. “TBP3” is a monovalent Fab fragment of TBP2, which specifically has an engineered eCys site. In some embodiments, the conjugates (or AOCs) of this disclosure comprise an antibody or an antigen-binding fragment thereof containing at least one heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index number). In some embodiments, the conjugates (or AOCs) of this disclosure comprise an antibody or an antigen-binding fragment thereof containing two heavy chain constant regions containing a cysteine ​​residue at residue 124 (according to EU index number).

[0173] Table 9: Nucleic acid sequences of the heavy chain (HC) and light chain (LC) of transferrin receptor-binding protein (TBP)

[0174]

[0175] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure comprises HCVR and LCVR, wherein HCVR comprises a sequence having at least 95% sequence identity with SEQ ID NO: 627, and LCVR comprises a sequence having at least 95% sequence identity with SEQ ID NO: 628.

[0176] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure comprises HCVR and LCVR, wherein HCVR comprises a sequence having at least 90% sequence identity with SEQ ID NO: 627, and LCVR comprises a sequence having at least 90% sequence identity with SEQ ID NO: 628.

[0177] In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof disclosed herein is a human IgG1 antibody having an effector null (EN) mutation comprising HC and LC, wherein HC comprises the sequence of SEQ ID NO: 625 and LC comprises the sequence of SEQ ID NO: 626.

[0178] In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof disclosed herein is a human IgG1 antibody having an effector null (EN) mutation, comprising HC and LC, wherein HC comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 625, and LC comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 626.

[0179] In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof disclosed herein is a human IgG1 antibody having an effector null (EN) mutation, comprising HC and LC, wherein HC comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 625, and LC comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 626.

[0180] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is a human IgG1 heterologous monoclonal antibody with an AAS effector null mutation, and the TBP comprises two HC variants (HCA and HCB) and an LC, wherein HCA comprises the sequence comprising SEQ ID NO: 635 including 124S, HCB comprises the sequence comprising SEQ ID NO: 636 including 124C, and the LC comprises the sequence comprising SEQ ID NO: 626.

[0181] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is a human IgG1 heterologous monoclonal antibody with an AAS effector null mutation, and the TBP comprises two HC variants (HCA and HCB) and an LC, wherein HCA comprises a sequence having at least 95% sequence identity with the sequence comprising SEQ ID NO: 635 including 124S, HCB comprises a sequence having at least 95% sequence identity with the sequence comprising SEQ ID NO: 636 including 124C, and the LC comprises a sequence having at least 95% sequence identity with the sequence comprising SEQ ID NO: 626.

[0182] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is a human IgG1 heterologous monoclonal antibody with an AAS effector null mutation, and the TBP comprises two HC variants (HCA and HCB) and an LC, wherein HCA comprises a sequence having at least 90% sequence identity with the sequence comprising SEQ ID NO: 635 including 124S, HCB comprises a sequence having at least 90% sequence identity with the sequence comprising SEQ ID NO: 636 including 124C, and the LC comprises a sequence having at least 90% sequence identity with the sequence comprising SEQ ID NO: 626.

[0183] In some embodiments, the anti-TfR antibody or antigen-binding fragment thereof disclosed herein is a human IgG1 Fab having a cysteine ​​residue at position 124 of HC, and the Fab comprises HC and LC, wherein HC comprises the sequence of SEQ ID NO: 637 and LC comprises the sequence of SEQ ID NO: 638.

[0184] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is a human IgG1 Fab with a cysteine ​​residue at position 124 of HC, and the Fab comprises HC and LC, wherein HC comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 637, and LC comprises a sequence having at least 90% sequence identity with the sequence of SEQ ID NO: 638.

[0185] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is a human IgG1 Fab with a cysteine ​​residue at position 124 of HC, and the Fab comprises HC and LC, wherein HC comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 637, and LC comprises a sequence having at least 95% sequence identity with the sequence of SEQ ID NO: 638.

[0186] In some embodiments, the conjugates disclosed herein are selected from Table 10 below. In some embodiments, the conjugate has an SMCC linker that links an antibody to an oligonucleotide.

[0187] Table 10: Some embodiments of the conjugates disclosed herein .

[0188]

[0189]

[0190] *In some implementations, the “R” oligonucleotide has at least 90% or 95% sequence identity with the listed sequence.

[0191] The TfR-binding proteins described herein can be recombinantly generated in host cells, for example, using expression vectors. For instance, the expression vector may contain a sequence encoding one or more signal peptides that promote the secretion of polypeptides from host cells. Expression vectors containing polynucleotides of interest (e.g., polynucleotides encoding the heavy or light chain of a TfR-binding protein) can be transferred into host cells using well-known methods. Furthermore, the expression vector may contain one or more selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired polynucleotide sequence.

[0192] Host cells (e.g., mammalian cells) include cells stably or transiently transfected, transformed, transduced, or infected with one or more expression vectors expressing all or part of the TfR-binding proteins described herein. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing HC peptides of the TfR-binding proteins described herein and LC peptides of the TfR-binding proteins described herein. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced, or infected with expression vectors expressing both HC and LC peptides of the TfR-binding proteins described herein. TfR-binding proteins can be produced in mammalian cells such as CHO, NSO, HEK293, or COS cells according to techniques well known in the art.

[0193] In some implementations, cell growth media in which TfR-binding proteins have been secreted can be purified using a hybrid approach combining conventional techniques such as ion exchange and hydrophobic interaction chromatography. For example, cell growth media can be loaded onto a protein A or G column and eluted using conventional methods; a hybrid approach combining ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and polymers can be effectively removed using common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. A variety of protein purification methods can be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

[0194] Embodiments of this disclosure also include antibody fragments or antigen-binding fragments, as used herein, comprising at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFv-Fc, biantibodies, scFab, scFv-CH3, Fv, scFa, disulfide-linked Fv (sdFv) and Fd fragments.

[0195] In some embodiments, the TfR-binding protein is scFv. In some embodiments, the TfR-binding protein is Fab. In some embodiments, the TfR-binding protein is Fab and VHH linked together, wherein VHH binds human serum albumin (HSA). In some embodiments, the TfR-binding protein also includes a heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index numbering). The EU numbering system is used in this disclosure to number the residues of antibodies or fragments thereof.

[0196] In some embodiments, the antibody or its antigen-binding fragment is conjugated to an oligonucleotide using the “nCys” method. “nCys” refers to a conjugation method in which an antibody or its antigen-binding fragment is conjugated to a molecular payload (e.g., ASO or siRNA) using one or more native cysteine ​​residues. In some embodiments, the human TfR-binding protein described herein comprises one or more native cysteine ​​residues that can be used for conjugation. For example, in some embodiments, the human TfR-binding protein described herein comprises a native cysteine ​​residue at position 214 of the light chain and / or native cysteine ​​residues at positions 220, 226, and / or 229 of the heavy chain, which can be used for conjugation (all residues are numbered according to EU index).

[0197] In some embodiments, the antibody or its antigen-binding fragment is conjugated to an oligonucleotide using the “eCys” method. This is a method of artificially incorporating one or more engineered cysteine ​​residues into an antibody or its antigen-binding fragment and conjugating the antibody or its antigen-binding fragment to a molecular payload (e.g., siRNA). Methods incorporating engineered cysteine ​​residues as a conjugation means are described in WO2018232088, which is incorporated herein by reference in its entirety. In some embodiments, the human TfR-binding protein described herein comprises a heavy chain containing one or more cysteine ​​residues at the following residues: 124, 157, 162, 262, 373, 375, 397, 415 (all residues are according to EU index numbers). In some embodiments, the human TfR-binding protein described herein comprises a light chain (e.g., a κ light chain) containing one or more cysteine ​​residues at the following residues: 156, 171, 191, 193, 202, 208 (all residues are according to EU index numbers). In some embodiments, the human TfR-binding protein described herein comprises a heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index number). In some embodiments, the human TfR-binding protein described herein comprises a light chain constant region containing a cysteine ​​residue at residue 156 (according to EU index number). In some embodiments, the human TfR-binding protein described herein comprises an immunoglobulin Fc region containing a cysteine ​​residue at residue 378 (according to EU index number).

[0198] In some embodiments, the antibody or its antigen-binding fragment is conjugated to one payload (e.g., an oligonucleotide), and its drug-antibody ratio (DAR) is 1. In some embodiments, the antibody or its antigen-binding fragment is conjugated to two payloads, and its drug-antibody ratio (DAR) is 2. In some embodiments, two variants of the heavy chain (HCA and HCB) are such that one variant contains an engineered cysteine ​​(eCys) for site-specific conjugation (e.g., with the payload). For example, such conjugation with a variant of the heavy or light chain can produce a highly homogeneous DAR1 conjugate or product. In some embodiments, HCA has an eCys site for site-specific conjugation. In some embodiments, HCB has an eCys site for site-specific conjugation.

[0199] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein further comprise a half-life extender, such as an immunoglobulin Fc region or a VHH binding to human serum albumin (see, for example, WO2022169766, which is incorporated herein by reference in its entirety). In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise an immunoglobulin Fc region, such as a modified human IgG4 Fc region or a modified human IgG1 Fc region. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise a modified human IgG4 Fc region containing proline at residue 228 and alanine at residues 234 and 235 (all residues are numbered according to EU index numbers, also referred to as the hIgG4PAA Fc region). In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein contains a modified human IgG1 Fc region containing alanine at residues 234, 235 and 329, serine at position 265 and aspartic acid at position 436 (all residues are numbered according to EU index numbers and are collectively referred to as hIgG1 effector null or hIgG1EN Fc region).

[0200] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a VHH that binds to HSA. In some embodiments, the VHH also binds to mouse, rat, and / or cynomolgus monkey albumin.

[0201] connector

[0202] In some embodiments, the oligonucleotides of this disclosure include a linker portion attached to one or more ends of a sense strand or antisense strand. Such linkers can be used to attach antibodies to the oligonucleotide. In some embodiments, the linker is an SMCC and optionally attached to the 5' end of the sense strand. In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to cholesterol, lipids, peptides, or antibodies or fragments thereof. In some embodiments, the linker is used to conjugate cholesterol, lipids, peptides, or antibodies or fragments thereof to the oligonucleotide.

[0203] In some embodiments, the anti-TfR antibody or its antigen-binding fragment of the present disclosure is linked to the sense or antisense strand of the oligonucleotide via a linker. In some embodiments, this is an SMCC linker. In some embodiments, the linker is an SMCC linker that is attached to: i) the 5' end of the sense strand, ii) the 5' end of the antisense strand, iii) the 3' end of the sense strand, or iv) the 3' end of the antisense strand.

[0204] In some embodiments, the connector described herein is either a cuttable connector or a non-cuttable connector. In some cases, the connector is a cuttable connector. In other cases, the connector is a non-cuttable connector.

[0205] In some cases, the joint is a nonpolymer joint. A nonpolymer joint is a joint that does not contain repeating monomer units generated through the polymerization process. Exemplary nonpolymer joints include, but are not limited to, C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homo-bifunctional crosslinkers, hetero-bifunctional crosslinkers, peptide joints, traceless joints, self-eliminating joints, maleimide-based joints, or combinations thereof. In some cases, a nonpolymer joint contains C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homo-bifunctional crosslinkers, hetero-bifunctional crosslinkers, peptide joints, traceless joints, self-eliminating joints, maleimide-based joints, or combinations thereof.

[0206] In some embodiments, the connector comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer group. In some cases, the maleimide group also comprises hexanoic acid, forming a maleimide hexanoyl (mc). In some cases, the connector comprises a maleimide hexanoyl (mc). In some cases, the connector is a maleimide hexanoyl (mc). In other cases, the maleimide group includes a maleimide methyl group, such as 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide ester (sMCC) or 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester (sulfo-sMCC) as described above.

[0207] In some embodiments, the maleimide group is a self-stabilized maleimide. In some cases, the self-stabilized maleimide utilizes diaminopropionic acid (DPR) to introduce a basic amino group near the maleimide to provide intramolecular catalysis for the ring hydrolysis of thiosuccinimide, thereby preventing the maleimide from undergoing elimination via the reverse Michael reaction. In some cases, the self-stabilized maleimide is the maleimide group described by Lyon et al., “Self-hydrolyzingmaleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some cases, the linker contains a self-stabilized maleimide. In some cases, the linker is a self-stabilized maleimide.

[0208] In some embodiments, the linker comprises a peptide moiety. In some cases, the peptide moiety comprises at least 2, 3, 4, 5, or 6 more amino acid residues. In some cases, the peptide moiety comprises up to 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some cases, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some cases, the peptide moiety is a cleavable peptide moiety (e.g., by enzymatic or chemical means). In some cases, the peptide moiety is an incleavable peptide moiety.

[0209] In some embodiments, the connector comprises a benzoic acid group or a derivative thereof. In some cases, the benzoic acid group or a derivative thereof comprises p-aminobenzoic acid (PABA). In some cases, the benzoic acid group or a derivative thereof comprises γ-aminobutyric acid (GABA).

[0210] In some embodiments, the connector comprises any combination of one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the connector comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some cases, the maleimide group is maleimide hexanoyl (mc). In some cases, the peptide group is val-cit. In some cases, the benzoic acid group is PABA. In some cases, the connector comprises an mc-val-cit group. In some cases, the connector comprises a val-cit-PABA group. In other cases, the connector comprises an mc-val-cit-PABA group.

[0211] In some embodiments, the connector is a self-eliminating connector or a self-degrading connector. In some cases, the connector is a self-eliminating connector. In other cases, the connector is a self-degrading connector (e.g., a cyclized self-degrading connector). In some cases, the connector comprises the connector described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0212] In some cases, the connector includes a bifunctional connector. Exemplary bifunctional connectors include, but are not limited to, Lomant reagents dithiobis(succinimide propionate) DSP, 3'3'-dithiobis(succinimide sulfonylpropionate) (DTSSP), disuccinimide octanoate (DSS), bis(sulfonylsuccinimide) octanoate (BS), disuccinimide tartrate (DST), and disulfonylsuccinimide tartrate (sulfo DST), ethylene glycol bis(succinimide succinate) (EGS), disuccinimide glutarate (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl hexamethyleneimine (DMA), dimethyl heptamethine (DMP), dimethyl octylimine (DMS), dimethyl-3,3'-dithiobispropionylimine ester (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimide hexane (BMH), compounds containing aryl halides (DFDNB), such as... Examples include 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylic acid)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide).

[0213] In some implementations, the adapter is a dendritic adapter. In some cases, the dendritic adapter includes branched, multifunctional adapter portions. In some cases, the dendritic adapter is used to increase the molar ratio of siRNA to antibody. In some cases, the dendritic adapter comprises a polyamidoamine (PAMAM) dendrite.

[0214] In some cases, the connector is the connector described in the following documents: U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239, 2013 / 028919, 2014 / 286970, 2013 / 0309256, 2015 / 037360, or 2014 / 0294851; or PCT Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0215] Expression vectors and cells

[0216] In some embodiments, the present invention provides methods for inhibiting Na+ in cells. V 1.8 A gene expression vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding at least one strand of one of the oligonucleotides of the present invention.

[0217] In another embodiment, the present invention provides a method for inhibiting Na+ in cells. V 1.8 Cells containing a gene expression vector. The vector contains a regulatory sequence operatively linked to a nucleotide sequence encoding at least one strand of one of the oligonucleotides of the present invention.

[0218] In another aspect of the invention, adjusting Na V 1.8 Na for gene expression activity V 1.8 Specific oligonucleotide molecules are expressed from transcriptional units inserted into DNA or RNA vectors known in the art. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and can be incorporated and inherited as transgenes integrated into the host genome. Transgenes can also be constructed to allow their inheritance as extrachromosomal plasmids (Gassmann et al., PNAS (1995) 92:1292).

[0219] Each strand of the oligonucleotide can be transcribed from promoters on two separate expression vectors and co-transfected into target cells. Alternatively, each individual strand of the oligonucleotide can be transcribed from promoters located on the same expression plasmid. The recombinant oligonucleotide expression vector is preferably a DNA plasmid or a viral vector. Viral vectors expressing oligonucleotides can be constructed based on, but are not limited to, the following viruses: adeno-associated virus (for review, see Muzyczka et al., Curr. Topics Micro. Immunol. (1992) 158:97-129); adenovirus (see, for example, Berkner et al., BioTechniques (1998) 6:616; Rosenfeld et al., (1991, Science 252:431-434); and Rosenfeld et al., (1992), Cell 68:143-155); or alphavirus, and other viruses known in the art. Oligonucleotide molecules can also be inserted into vectors and used as gene therapy vectors for human patients.

[0220] Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the oligonucleotide transgene. Furthermore, the expression of the transgene can be precisely regulated, for example, by using inducible regulatory sequences and expression systems, such as regulatory sequences sensitive to certain physiological regulators (e.g., circulating glucose levels or hormones) (Docherty et al., 1994, FASEB J. 8:20-24).

[0221] Pharmaceutical Composition

[0222] In one embodiment, this disclosure provides a pharmaceutical composition comprising an oligonucleotide as described herein and a pharmaceutically acceptable carrier. In another embodiment, this disclosure provides a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising oligonucleotides or conjugates can be used to treat conditions related to sodium... V 1.8 Diseases or conditions associated with gene expression or activity, such as chronic pain, including neuropathic pain, inflammatory pain, or mixed pain.

[0223] In another embodiment, this disclosure provides a mixture containing at least two substances designed to target Na. V 1.8 Oligonucleotides from different regions of the gene and comprising a pharmaceutically acceptable carrier. In yet another embodiment, this disclosure provides a pharmaceutical composition comprising at least two oligonucleotides designed to target Na+. V 1.8 Pharmaceutical compositions of conjugates for different regions of a gene and pharmaceutically acceptable carriers. In this embodiment, an oligonucleotide may have a conjugate with Na+. V1.8 The gene has at least a substantially complementary nucleotide sequence, while another oligonucleotide has a sequence similar to Na. V 1.8 The nucleotide sequences of different parts of the gene are substantially complementary. Multiple oligonucleotides or conjugates can be combined in the same pharmaceutical composition or formulated individually. If formulated individually, compositions containing individual oligonucleotides or conjugates can contain the same or different carriers and can be administered using the same or different routes of administration. Furthermore, pharmaceutical compositions containing individual oligonucleotides or conjugates can be administered substantially simultaneously, sequentially, or at predetermined intervals within a day or treatment period.

[0224] The pharmaceutical composition disclosed herein is sufficient to inhibit Na V 1.8 Dosage administration of gene expression. Generally, an appropriate dose of oligonucleotide will be in the range of 0.01 mg to 10.0 mg per kilogram of recipient body weight per day. Those skilled in the art will understand that certain factors may affect the dose and duration required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or a series of treatments. Estimates of the effective dose and in vivo half-life of the individual oligonucleotides covered by this disclosure can be made using conventional methods or based on in vivo testing using appropriate animal models.

[0225] The pharmaceutical compositions covered by this disclosure may be administered in any manner known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, epidural, intrathecal, intraventricular, intraparenchymal (in the peripheral or central nervous system), subcutaneous, transdermal, intranasal, airway (aerosol), rectal, vaginal, and local (including buccal and sublingual) administration.

[0226] For intrathecal, intraventricular, intramuscular, intraparenchymal (peripheral or central nervous system), subcutaneous, and intravenous use, the pharmaceutical compositions of this disclosure will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous solvents include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists only of an aqueous buffer solution. In this context, "only" means the absence of substances that could affect or mediate the expression of oligonucleotides in Na+. V 1.8 Cellular uptake of cellular adjuvants or encapsulating substances. Such substances include, for example, micelle structures, such as liposomes or capsids as described below.

[0227] The pharmaceutical compositions useful according to this disclosure also include encapsulation formulations to protect oligonucleotides from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions (including liposomes targeting cells infected with antiviral antigen monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811, PCT Publication WO 91 / 06309, and European Patent Publication EP-A-43075, which are incorporated herein by reference. Lipid nanoparticles (LNPs) can also be used to deliver the oligonucleotides or conjugates of this disclosure.

[0228] In addition to being administered alone or in combination as discussed above, the oligonucleotides or conjugates of this disclosure can also be administered in combination with other known effective agents for the treatment of pain. In any case, the administering physician may adjust the dosage and timing of administration of the oligonucleotides or conjugates disclosed herein based on results observed using standard efficacy measurements known in the art or described herein.

[0229] In some embodiments, the compositions herein comprise a carrier that can confer improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the carrier is a buffer (e.g., sodium citrate, sodium phosphate, trimethylolaminomethane base, or sodium hydroxide) or a solvent (e.g., buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides herein are lyophilized to extend shelf life and then formulated into a solution prior to use (e.g., administration to an individual). Thus, in pharmaceutical compositions comprising one or more oligonucleotides, the carrier is a lyophilization protectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature regulator (e.g., dextran, Ficoll). ™ Or gelatin).

[0230] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0231] Methods to reduce SCN10A activity in cells, tissues, organs and organisms

[0232] The oligonucleotides, conjugates, or pharmaceutical compositions disclosed herein are used to reduce SCN10A mRNA and Na+ in cells, tissues, organs, or individuals. V 1.8 Protein and / or NaV 1.8 Activity. These methods include the steps described herein, and these steps may, but must, be performed in the order described. However, other orders are also contemplated. Furthermore, single or multiple steps may be performed in parallel and / or overlap in time and / or individually or as multiple repetitions. In addition, these methods include additional, unspecified steps.

[0233] These methods include contacting or delivering to cells, cell populations, tissues, organs, or individuals an effective amount of any oligonucleotide, conjugate, or pharmaceutical composition disclosed herein for reducing SCN10A expression. In some embodiments, the reduced SCN10A activity is measured by measuring SCN10A mRNA and Na+ in cells. V 1.8 Protein and / or Na V 1.8 The decrease in the amount or level of protein activity is used to determine this.

[0234] Regarding the appropriate cell type, this cell type is any cell expressing SCN10A mRNA (e.g., DRG neurons). In some embodiments, the cells are primary cells obtained from an individual. In some embodiments, the cells are primary cells obtained from an individual's nervous system (CNS or PNS). In some embodiments, the primary cells have undergone a limited number of passages, such that the cells substantially retain their native phenotypic characteristics. In some embodiments, the cells are ex vivo, in vivo, or in vitro cells (i.e., such that one or more oligonucleotides or conjugates described herein can be delivered to cells in culture or the organism in which the cells reside).

[0235] In some embodiments, the oligonucleotides herein are delivered to cells or cell populations using nucleic acid delivery methods known in the art, including but not limited to injecting a solution containing the oligonucleotide, bombarding with particles coated with the oligonucleotide, exposing cells or cell populations to a solution containing the oligonucleotide, or electroporating cell membranes in the presence of the oligonucleotide. Other methods known in the art for delivering oligonucleotides to cells are also used, such as, for example, lipid-mediated carrier transport, chemically mediated transport, and cationic liposome transfection of calcium phosphate, etc. In some embodiments, the oligonucleotides are conjugated to an anti-TfR antibody or an antigen-binding fragment thereof for transport into cells, cell populations, tissues, or cells of a subject.

[0236] Decreased SCN10A activity is determined by an assay or technique (e.g., using SCN10A expression biomarkers) evaluating one or more molecules, properties, or characteristics of cells or cell populations associated with SCN10A gene expression, or by evaluating molecules that directly indicate SCN10A activity in cells or cell populations (e.g., SCN10A mRNA, Na+). V 1.8 Protein and / or NaV 1.8 The activity is determined by an assay or technique. In some embodiments, the extent to which the oligonucleotide reduces SCN10A activity is evaluated by comparing the SCN10A activity in cells or cell populations exposed to the oligonucleotide or conjugate with that in control cells or cell populations (e.g., cells or cell populations not exposed to the oligonucleotide or exposed to the control oligonucleotide). In some embodiments, a control amount or level of SCN10A activity in control cells or cell populations is predetermined, so that it is not necessary to measure the control amount or level each time an assay or technique is performed. The predetermined level or value takes many forms, including but not limited to a single cutoff value, such as a median or average.

[0237] Contacting or delivering the oligonucleotides, conjugates, or pharmaceutical compositions disclosed herein to cells or cell populations results in a reduction in SCN10A activity. In some embodiments, the reduced SCN10A activity is relative to a control amount or level of SCN10A activity in cells or cell populations that are not contacted, for example, with the oligonucleotide or are contacted with a control oligonucleotide. In some embodiments, the reduced SCN10A activity relative to a control amount or level of SCN10A activity is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less. In some embodiments, the control amount or level of SCN10A activity is SCN10A mRNA, Na+, or Na+ in cells or cell populations that have not been contacted with the oligonucleotides, conjugates, or pharmaceutical compositions disclosed herein. V 1.8 Protein and / or Na V1.8 Amount or level of activity. In some embodiments, the effect of delivering the oligonucleotide, conjugate, or pharmaceutical composition disclosed herein to cells or cell populations according to the methods herein is evaluated after any limited time period or time duration (e.g., minutes, hours, days, weeks, and / or months). For example, SCN10A activity in cells or cell populations is determined after at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, or about 24 hours. Alternatively, SCN10A activity in cells or cell populations may be determined at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or longer after contacting or delivering the oligonucleotide, conjugate, or pharmaceutical composition disclosed herein to cells or cell populations. In other embodiments, SCN10A activity in cells or cell populations may be determined at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or longer after contacting or delivering the oligonucleotide, conjugate, or pharmaceutical composition disclosed herein to cells or cell populations.

[0238] Methods of prevention, relief or treatment :

[0239] One aspect of this disclosure relates to a method of preventing, treating, or alleviating pain in a patient in need, the method comprising administering to the patient an effective amount of the oligonucleotide of this disclosure; an effective amount of the conjugate of this disclosure; or a pharmaceutical composition of this disclosure. Another aspect of this disclosure relates to the use of the oligonucleotide of this disclosure; the conjugate of this disclosure; or a pharmaceutical composition of this disclosure in the preparation of a medicament for preventing, treating, or alleviating pain. In yet another aspect, the oligonucleotide of this disclosure; the conjugate of this disclosure; or a pharmaceutical composition of this disclosure is used in the prevention, treatment, or alleviation of pain in a patient in need. In some embodiments, a method of treating an individual who has, is suspected of having, or is at risk of developing a disease, condition, or ailment associated with SCN10A activity comprises administering to that individual at least one or more of the oligonucleotides or conjugates described herein.

[0240] In some embodiments, the individual suffers from chronic pain, including: i) inflammatory pain, ii) neuropathic pain, or iii) mixed pain. In one embodiment, the oligonucleotide, conjugate, or pharmaceutical composition of this disclosure is administered intravenously according to at least one method described herein.

[0241] Furthermore, methods for treating or reducing the onset or progression of a disease, condition, or illness associated with SCN10A activity in an individual include using one or more oligonucleotides, pharmaceutical compositions, or conjugates described herein. Additionally, methods for achieving one or more therapeutic benefits in an individual suffering from a disease, condition, or illness associated with SCN10A activity include providing one or more oligonucleotides, pharmaceutical compositions, or conjugates disclosed herein. In some embodiments, an individual may be treated by administering a therapeutically effective amount of any one or more oligonucleotides, pharmaceutical compositions, or conjugates described herein. In some embodiments, treatment includes reducing SCN10A activity. In some embodiments, the individual receives therapeutic treatment. In some embodiments, the individual receives preventative treatment.

[0242] In some embodiments, one or more oligonucleotides, one or more conjugates, or pharmaceutical compositions comprising them are administered to an individual suffering from a disease, condition, or ailment associated with SCN10A activity, thereby treating the individual by reducing SCN10A activity in the individual. In some embodiments, the amount or level of SCN10A mRNA in the individual is reduced. In other embodiments, SCN10A (or Na+) in the individual is reduced. V 1.8) Decreased amount or level of protein. In other embodiments, the amount or level of SCN10A activity in the individual is decreased.

[0243] In some embodiments, when compared with the SCN10A activity prior to administration of one or more oligonucleotides, conjugates or pharmaceutical compositions thereof, the SCN10A activity in an individual is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater. In other embodiments, when compared with the SCN10A activity in an individual who has not received one or more oligonucleotides, conjugates, or pharmaceutical compositions, or who has received control oligonucleotides, conjugates, pharmaceutical compositions, or treatments (e.g., a reference or control individual), the SCN10A activity in the individual is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

[0244] In some embodiments, the amount or level of SCN10A mRNA in an individual is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of SCN10A mRNA prior to administration of one or more oligonucleotides, conjugates, or pharmaceutical compositions thereof. In some embodiments, when compared with the amount or level of SCN10A mRNA in an individual who has not been given one or more oligonucleotides, conjugates, or pharmaceutical compositions, or who has been given a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual), the amount or level of SCN10A mRNA in the individual is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

[0245] In some embodiments, when compared to the amount or level of SCN10A protein in an individual prior to administration of one or more oligonucleotides, conjugates, or pharmaceutical compositions thereof, the level of SCN10A (or Na+) in the individual is [not specified]. V 1.8) The amount or level of protein is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In other embodiments, when compared with the amount or level of SCN10A protein in an individual who has not been given one or more oligonucleotides, conjugates, or pharmaceutical compositions, or who has been given a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual), the amount or level of SCN10A protein in the individual is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%.

[0246] In some embodiments, when combined with Na prior to the administration of one or more oligonucleotides, conjugates, or pharmaceutical compositions thereof V 1.8 When comparing the amount or level of activity, the amount of Na in an individual V1.8 The amount or level of activity is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99%. In some embodiments, when compared with Na in individuals who have not been given one or more oligonucleotides or pharmaceutical compositions or who have been given control oligonucleotides, pharmaceutical compositions, or treatments (e.g., reference or control individuals). V 1.8 When comparing the amount or level of activity, the amount of Na in an individual V 1.8 The amount or level of activity is reduced by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99%.

[0247] Here, SCN10A activity, the amount or level of SCN10A mRNA, and Na... V 1.8 protein, Na V 1.8 Activity or any combination thereof is reduced in cells (e.g., DRG neurons), cell populations or cell groups (e.g., nerves), tissues, samples, organs, blood or fractions thereof, or any other biological material obtained or isolated from an individual. In some embodiments, SCN10A activity, the amount or level of SCN10A mRNA, Na V 1.8 protein, Na V 1.8 Activity or any combination thereof is reduced in more than one type of cell, more than one cell group, more than one type of tissue, more than one type of sample, more than one organ, or more than one fraction of blood obtained or isolated from an individual.

[0248] Examples of diseases, conditions, or illnesses associated with SCN10A activity include, but are not limited to, chronic pain, including neuropathic pain, inflammatory pain, and mixed pain. Examples of diseases that may be associated with SCN10A activity include inflammatory CNS diseases such as multiple sclerosis, myelitis, or syphilis; ischemia; hemorrhage; or arteriovenous malformations located in the thalamus, spinothalamic pathway, or thalamic cortical projections (e.g., post-stroke neuropathic pain); and syringomyelia (Koltzenburg, Pain 2002—An Updated Review: Refresher Course Syllabus; IASP Press, Seattle, 2002). Diseases or illnesses associated with SCN10A activity also include “pain and related conditions,” the term “related conditions” referring to conditions that cause or are associated with pain or have been shown to have mechanisms similar to pain. These conditions include addiction, seizures, stroke, ischemia, neurodegenerative diseases, anxiety, depression, headache, asthma, rheumatism, osteoarthritis, retinopathy, inflammatory eye disease, pruritus, ulcers, gastric disorders, urinary incontinence, inflammatory or unstable bladder disease, inflammatory bowel disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), gastroesophageal reflux disease (GERD), functional dyspepsia, functional chest pain presumed to be esophageal, functional dysphagia, non-cardiac chest pain, symptomatic gastroesophageal disorders, gastritis, aerophagia, functional constipation, functional diarrhea, bloating, chronic functional abdominal pain, recurrent abdominal pain (RAP), functional bloating, functional biliary pain, functional incontinence, functional anorectal pain, chronic pelvic pain, pelvic floor dyssynergia, unclassified functional anorectal disorders, interstitial cystitis, dysmenorrhea, and dyspareunia.

[0249] Due to their high specificity, the oligonucleotides or conjugates described herein specifically target the mRNA of target genes in cells, tissues, or organs. In disease prevention, the target gene is a gene required for the occurrence or maintenance of the disease, or a gene identified as being associated with a higher risk of developing the disease. In disease treatment, one or more oligonucleotides or conjugates described herein are brought into contact with cells, tissues, or organs that express or are responsible for mediating the disease. For example, with wild-type (i.e., natural) or mutant genes (that is, genes associated with SCN10A (or Na...). V 1.8) Oligonucleotides that are wholly or partially complementary to an activity-related disease, symptom or condition are contacted with or introduced into a cell or tissue type of interest, such as a DRG neuron.

[0250] In some implementations, the target gene is derived from any mammal, such as a human. According to the methods described herein, any gene can be silenced. Furthermore, the methods described herein generally involve administering to an individual a therapeutically effective amount of one or more oligonucleotides described herein, i.e., an amount capable of producing the desired therapeutic outcome. A therapeutically acceptable amount is an amount that therapeutically treats a disease, symptom, or condition. The appropriate dose for any individual will depend on several factors, including the individual's size, body surface area, age, the composition to be administered, the active ingredient in the composition, the time and route of administration, overall health, and any other therapeutic agents administered concurrently.

[0251] In these methods, any of the oligonucleotides, conjugates, or compositions described herein are administered to an individual via enteral (e.g., oral, via gastric tube, via duodenal tube, via gastrostomy, or rectal), parenteral (e.g., subcutaneous, intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intraventricular, or intrathecal), topical (e.g., transdermal, inhaled, via eye drops, or via mucous membranes), or by direct injection into a target organ (e.g., the individual's liver). Typically, the oligonucleotides or compositions are administered intravenously or subcutaneously.

[0252] As a non-limiting example group, the oligonucleotides or compositions described herein are typically administered quarterly (every three months), every two months (every two months), monthly, or weekly. For example, the oligonucleotides or compositions are administered weekly, every two weeks, or every three weeks. In some embodiments, the oligonucleotides or compositions are administered daily. In some embodiments, an individual is given one or more loading doses of the oligonucleotides or compositions, followed by one or more maintenance doses of the oligonucleotides or compositions.

[0253] In some implementations, the individual is a human, an NHP, or other mammal. In other implementations, the individual is a domestic animal, such as a dog or cat; livestock, such as a horse, cow, pig, sheep, goat, or chicken; and an animal, such as a mouse, rat, guinea pig, or hamster.

[0254] The oligonucleotides, conjugates, or compositions disclosed herein can be used, or modified, for the treatment of individuals who will benefit from reduced SCN10A activity (e.g., those with conditions related to Na+). V 1.8 Individuals with SCN10A-associated diseases, conditions, or illnesses. In some embodiments, the oligonucleotide is provided for, or modified for, treating individuals suffering from, a disease, condition, or illness associated with SCN10A activity. Additionally, the oligonucleotide is provided for, or can be modified for, preparing a medicament or pharmaceutical composition for treating, a disease, condition, or illness associated with SCN10A activity. In other embodiments, the oligonucleotide or conjugate is provided for, or can be modified for, targeting SCN10A mRNA and reducing Na+. V1.8 Activity (e.g., via the RNAi pathway). In other embodiments, oligonucleotides or conjugates are provided for, or can be tuned for, targeting SCN10A mRNA and reducing SCN10A mRNA, Na+. V 1.8 Protein and / or Na V 1.8 The amount or level of activity.

[0255] The oligonucleotide, conjugate, or pharmaceutical compositions disclosed herein can be integrated into a kit containing one or more oligonucleotide, conjugate, or pharmaceutical compositions, along with instructions for use. In some embodiments, the kit contains one or more oligonucleotide, conjugate, or pharmaceutical compositions, along with a packaging insert containing instructions for use of the kit and / or any of its components. In other embodiments, the kit contains a suitable container, one or more oligonucleotide, conjugate, or pharmaceutical compositions, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art.

[0256] In some embodiments, the container may be at least one vial, well, test tube, flask, bottle, syringe, or other container device to hold one or more oligonucleotides, conjugates, or pharmaceutical compositions, and in some embodiments, appropriately aliquoted. In other embodiments, when additional components are provided, the kit includes an additional container for holding that component. The kit also includes means for tightly sealing one or more oligonucleotides, conjugates, or pharmaceutical compositions, along with any other reagents, for commercial sale. Such containers include injection-molded or blow-molded plastic containers in which the desired vial is retained. The container and / or kit include labels with instructions for use and / or warnings.

[0257] In some implementations, the kit contains one or more oligonucleotides, conjugates described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition containing one or more oligonucleotides or conjugates, along with instructions for treating or delaying the progression of a disease, condition, or illness associated with SCN10A activity in an individual in need.

[0258] Abbreviations and Definitions

[0259] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, unless the context explicitly requires the presence of one and only one element, reference to an element with the indefinite articles “a” or “an” does not preclude the possibility of the presence of more than one element. Therefore, the indefinite articles “a” or “an” generally mean “at least one”. Moreover, the use of “including” and other forms such as “include,” “includes,” and “included” does not constitute limitation.

[0260] Some of the definitions used in this article are as follows:

[0261] As used herein, “about” means within a statistically significant range of one or more numerical values, such as, for example, a specified concentration, length, molecular weight, pH, sequence similarity, time range, temperature, volume, etc. Such values ​​or ranges can be on the order of typically 20%, more typically 10%, and even more typically 5% of a given value or range. The permissible variation covered by “about” will depend on the specific system or subject being studied and will be readily understood by those skilled in the art.

[0262] As used herein, “application”, “administration”, etc., refer to the provision of a substance (e.g., oligonucleotide, conjugate, AOC, or composition herein) to an individual in a pharmacologically useful manner (e.g., to treat a disease, symptom or condition in an individual or patient).

[0263] As used in this article, "target sequence" refers to the sequence in Na. V 1.8 The continuous portion of the nucleotide sequence of the mRNA molecule formed during transcription of the gene (SCN10A), including the mRNA of the RNA processing product as a primary transcription product.

[0264] As used herein, “antisense strand” refers to an oligonucleotide of this article that is complementary to a region of the target sequence. Similarly, and as used herein, “sense strand” refers to an oligonucleotide of this article that is complementary to a region of the antisense strand.

[0265] As used herein, “reduction”, “alleviation”, etc., refer to a reduction or effective cessation. As a non-limiting example, one or more treatments described herein can reduce or effectively stop the onset or progression of pain and related diseases, conditions, and ailments (such as, for example, chronic pain) in an individual. Such reduction can be illustrated by, for example, a reduction in one or more aspects of an individual (e.g., symptoms, tissue characteristics, and cellular, inflammatory, or immune activity, etc.) and related diseases, conditions, and ailments.

[0266] As used herein, and unless otherwise specified, the term "complementary" when used to describe the relationship between a first nucleotide sequence and a second nucleotide sequence means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure, as will be understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PPIES pH 6.4, 1 mM EDTA, at 50 or 70°C for 12 to 16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may be encountered in vivo, may also be applied. Those skilled in the art will be able to determine the most suitable set of conditions for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides. This includes base pairing of an oligonucleotide or polynucleotide containing the first nucleotide sequence with an oligonucleotide or polynucleotide containing the second nucleotide sequence along the entire length of the first and second nucleotide sequences. Such sequences may be referred to herein as "perfectly complementary" relative to each other. However, when the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be perfectly complementary, or they may form one or more, but preferably no more than four, three, or two mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their final application. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches when determining complementarity. For example, a dsRNA comprising one 21-nucleotide oligonucleotide and another 23-nucleotide oligonucleotide, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as “perfectly complementary” for the purposes of this invention.

[0267] As used herein, “complementary” sequences may also include or consist entirely of base pairs formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding their hybridization ability are met.

[0268] The terms “complementary,” “fully complementary,” and “substantially complementary” in this article can be understood, depending on the context in which they are used, as the base matching between the antisense strand of ssRNA and the target sequence, between the sense strand of dsRNA and the antisense strand, or between the antisense strand of dsRNA and the target sequence.

[0269] As used herein, a polynucleotide that is substantially complementary to at least a portion of messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to the mRNA of interest (e.g., encoding Na+). V 1.8) are polynucleotides whose continuous portions are substantially complementary. For example, if the polynucleotide sequence is the same as that encoding Na VThe uninterrupted portion of the 1.8 mRNA is essentially complementary, so this polynucleotide is compatible with Na. V 1.8 At least a portion of the mRNA is complementary.

[0270] As used herein, "contact" refers to the direct or indirect introduction or delivery of oligonucleotides (such as oligonucleotides or AOCs) into cells, for example, by promoting or enabling uptake or absorption into cells.

[0271] When referring to ssRNA or dsRNA, “introducing into cells” means promoting uptake or absorption into cells, as understood by those skilled in the art. The uptake or absorption of ssRNA or dsRNA can occur through unassisted diffusion or active cellular processes, or through an adjuvant or device. The meaning of this term is not limited to in vitro cells; ssRNA or dsRNA can also be “introduced into cells” where the cells are part of a living organism. In this case, introducing into cells will include delivery to the organism. For example, for in vivo delivery, ssRNA or dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection.

[0272] As used herein, “deoxyribonucleotide” means a nucleotide in which a hydrogen hydroxyl group is replaced at the 2' position of the pentose sugar compared to a ribonucleotide. Modified deoxyribonucleotides have one or more modifications or substitutions on atoms other than the 2' position, including modifications or substitutions in or above the nucleobase, sugar, or phosphate group.

[0273] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or ribonucleic acid complex with a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands as defined above. The two strands forming the double-stranded structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are thus linked by an unbroken nucleotide chain between the 3' end of one strand and the 5' end of the corresponding strand forming the double-stranded structure, the linking RNA strand is called a "hairpin loop." When the two strands are covalently linked by a means other than an unbroken nucleotide chain between the 3' end of one strand and the 5' end of the corresponding strand forming the double-stranded structure, the linking structure is called a "connector." The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. In addition to the double-stranded structure, dsRNA may also contain one or more nucleotide overhangs.

[0274] As used in this article, a "nucleotide overhang" refers to an unpaired nucleotide protruding from the double-stranded structure of a dsRNA when the 3' end of one strand extends beyond the 5' end of the other, and vice versa. "Flat-ended" or "blunt-ended" means that there is no unpaired nucleotide at one end of the dsRNA; that is, there is no nucleotide overhang. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its entire length, meaning that there is no nucleotide overhang at either end of the molecule.

[0275] As used in this article, the term "double helix" in relation to nucleic acids (e.g., oligonucleotides) refers to a structure formed by complementary base pairing of two antiparallel nucleotide sequences.

[0276] As used herein, the term "antibody" refers to a molecule that binds to an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, heterodimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the antibody disclosed herein is an IgG1 antibody.

[0277] Immunoglobulin G (IgG) antibodies consist of four polypeptide chains: two heavy chains (HC) and two light chains (LC), which are cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of approximately 100 to 125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further subdivided into subtypes (e.g., IgG1, IgG2, IgG3, and IgG4).

[0278] The VH (also referred to as HCVR in this paper) and VL (also referred to as LCVR in this paper) regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). CDRs are exposed on the protein surface and are important regions for antigen-binding specificity of antibodies. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In this paper, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." These CDRs contain most of the residues that specifically interact with the antigen. The allocation of amino acid residues to CDRs can be performed according to well-known schemes, including those described in the following literature: Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. (1991), which is incorporated herein by reference in its entirety); Chothia (Chothia et al., “Canonical Structures for the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol., 196, 901-917 (1987); Al-Lazikani et al., “Standard Conformations for the Canonical Structures of Immunoglobulins,” J. Mol. Biol., 273, 927-948 (1997), which are incorporated herein by reference in their entirety); North (North et al., “A New Clustering of Antibody CDR Loop Conformations,” J. Mol. Biol., 406, 228-256). (2011)); or IMGT ® (The International Immunogenetics Database is available at imgt.org; see Lefranc et al., Nucleic Acids Res. 27:209-212 (1999), which are incorporated herein by reference in their entirety.)

[0279] As used herein, the term "antigen-binding fragment" refers to the portion of an antibody that binds to an antigen or antigenic epitope. For example, "TfR-binding protein" refers to an antibody or a portion of an antibody fragment that binds to TfR or a TfR epitope.

[0280] As mentioned herein, the term "epitope" refers to an amino acid residue in an antigen that is bound by an antibody. An epitope can be a linear epitope, a conformational epitope, or a heterozygous epitope. The term "epitope" can also be used to refer to a structural epitope. According to some embodiments, a structural epitope can be used to describe an antigenic region covered by an antibody or antigen-binding protein. In some embodiments, a structural epitope can describe amino acid residues of an antigen that are within a specified proximity (e.g., within a specified number of angstroms) to the amino acid residues of an antibody or antigen-binding protein. The term "epitope" can also be used to refer to a functional epitope. According to some embodiments, a functional epitope can be used to describe amino acid residues of an antigen that interact with the amino acid residues of an antibody or antigen-binding protein in a manner that contributes to the binding energy between the antigen and the antibody or antigen-binding protein.

[0281] Epitopes can be determined using various experimental techniques (also known as “eptope mapping techniques”). It should be understood that epitope determination may vary depending on the specific epitope mapping technique used, as well as the experimental conditions employed, such as antigen conformational changes or cleavage induced by particular experimental conditions. Epitope mapping techniques are known in the art (e.g., Rockberg and Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd edition, 2018), and include, but are not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species exchange mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays.

[0282] As used herein, the term "Fc region" refers to a polypeptide containing the CH2 and CH3 domains of a constant region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). Optionally, the Fc region may contain a portion or the entire hinge region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is a human IgG Fc region, such as the human IgG1 Fc region, human IgG2 Fc region, human IgG3 Fc region, or human IgG4 Fc region. In some embodiments, the Fc region is a modified IgG Fc region having reduced or eliminated effector function compared to the corresponding wild-type IgG Fc region. The residues in the Fc region are numbered based on the EU index as described in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Bethesda, MD: US Dept. of Health and Human Services, Public Health Service, National Institutes of Health, 1991). The boundaries of the Fc region of the immunoglobulin heavy chain may vary, and the human IgG heavy chain Fc region is generally defined as a segment from the N-terminus of the CH2 domain (e.g., amino acid residue 231 according to the EU index number) to the C-terminus of the CH3 domain (or the C-terminus of the immunoglobulin).

[0283] The term "knockdown" or "expression knockdown" refers to the reduction of gene mRNA or protein expression after treatment with reagents.

[0284] Unless otherwise stated, the term “binding” as used herein refers to the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in the two proteins or molecules coming into close proximity, as determined by commonly used methods known in the art.

[0285] The term "% sequence identity" or "percentage sequence identity" for reference nucleic acid sequences is defined as the percentage of nucleotides, nucleosides, or nucleosides in a candidate sequence that are identical to those in the reference nucleic acid sequence, after optimal alignment and, if necessary, the introduction of gaps or overhangs to achieve maximum percentage sequence identity. Alignments used to determine the percentage of nucleic acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software programs such as those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 1987, Supplement Vol. 30, Section 7.7.18, Table 7.7.1), including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0, or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. The "sequence identity" percentage is determined by comparing two best-aligned sequences in a comparison window, where the nucleic acid sequence fragments in the comparison window may contain additions or deletions (e.g., gaps or protrusions) to achieve optimal alignment of the two sequences compared to a reference sequence (which contains no additions or deletions). The percentage is calculated by determining the number of positions in both sequences where the same nucleotide, nucleoside, or nucleotide appears to generate a number of matching positions, dividing that number by the total number of positions in the comparison window, and multiplying the result by 100. The output is the identity percentage of the test sequence relative to the query sequence.

[0286] As used herein, “TfR” refers to the transferrin receptor protein or polypeptide, such as the human transferrin receptor protein or polypeptide. The amino acid sequence of the human transferrin receptor protein (hTfR) can be found in the NCBI reference sequence: NP_001121620.1, which is incorporated herein by reference in its entirety.

[0287] The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. Cell culture media are explicitly excluded. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginate are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0288] As used in this article, “individual” means any mammal, including cats, dogs, mice, rats, and primates, especially humans. Furthermore, “subject” or “patient” may be used interchangeably with “individual.”

[0289] As used herein, "instable joint" means a joint that can be cut (e.g., by acidic pH). Similarly, "relatively stable joint" means a joint that cannot be cut.

[0290] As used herein, "modified internucleotide linker" refers to an internucleotide linker that has one or more chemical modifications compared to a reference internucleotide linker having a phosphodiester bond. The modified nucleotides may be non-naturally occurring links. Typically, modified internucleotide linkers impart one or more desired properties to the nucleic acid in which the modified linker is located. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, and reduce immunogenicity.

[0291] As used herein, a "modified nucleotide" means a nucleotide that has one or more chemical modifications compared to a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymine deoxyribonucleotide. Modified nucleotides may be non-naturally occurring nucleotides. Modified nucleotides may, for example, have one or more chemical modifications in their sugar, nucleotide, and / or phosphate groups. Additionally or alternatively, modified nucleotides may have one or more chemical moieties conjugated to the corresponding reference nucleotide. Generally, modified nucleotides impart one or more desired properties to nucleic acids in which the modified nucleotide is present. For example, modified nucleotides may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0292] As used herein, “nucleoside” refers to a nucleobase-sugar combination, where the nucleobase portion is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. The sugar is typically a pentose, such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0293] As used herein, “nucleotide” means an organic molecule having a nucleoside (nucleobases such as, for example, adenine, cytosine, guanine, thymine, or uracil; and a pentose, such as, for example, ribose or 2'-deoxyribose) and a phosphate group, which can be used as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0294] As used herein, “oligonucleotide” means a short nucleic acid molecule (e.g., less than about 100 nucleotides in length). Oligonucleotides can be single-stranded (ss) or double-stranded (ds). Oligonucleotides may or may not have double-stranded regions. As a set of non-limiting examples, oligonucleotides can be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (DsiRNA), antisense oligonucleotides (ASO), short siRNA, or ss siRNA. In some cases, the oligonucleotide is a phosphorodiamide morpholine oligomer (PMO), which is a short single-stranded oligonucleotide analogue built on a morpholine ring backbone linked by phosphorodiamide linkages. The oligonucleotide is a single-stranded (ss) oligonucleotide (e.g., ASO) or a double-stranded (ds) oligonucleotide (e.g., siRNA).

[0295] As used herein, “phosphate ester analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate ester analog is located at the 5' terminal nucleotide of the oligonucleotide in place of the 5'-phosphate, which is generally readily removable by enzymatic reactions. The 5' phosphate ester analog may contain an antiphosphatase linker. Examples of phosphate ester analogs include, but are not limited to, 5' phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). The oligonucleotide may have a phosphate ester analog at the 4'-carbon position of the sugar at the 5'-terminal nucleotide (referred to as a “4'-phosphate ester analog”). An example of a 4'-phosphate ester analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed for the 5' end of oligonucleotides (see, for example, International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al., (2015) Nucleic Acids Res. 43:2993-3011).

[0296] As used herein, “reduced expression” or “reduced activity” of a gene (e.g., SCN10A) means, when compared with a suitable reference (e.g., reference cells, cell populations, samples, or individuals), a decrease in the expression of the RNA transcript (e.g., SCN10A mRNA) or the activity of the protein (e.g., Na+) encoded by that gene. V A decrease in the amount or level of 1.8 protein, and / or a decrease in the amount or level of activity of the gene or protein in cells, cell populations, samples or subjects.

[0297] Involving Na V When dealing with 1.8 genes, the terms "silencing" and "repressed expression" in this paper refer to at least partial suppression of Na+ expression. V 1.8 Gene expression, which is manifested in the expression of Na+ from Na+ compared to the second cell group or cell group (control cells that are substantially the same as the first cell group or cell group but have not undergone such treatment). V 1.8 The gene was transcribed and has been treated to make Na V 1.8 Na isolated from the first cell or cell group in which gene expression was suppressed. V 1.8 The amount of mRNA transcribed from genes is reduced.

[0298] Alternatively, the degree of inhibition can be determined based on the relationship with Na. V 1.8 The decrease in functionally related parameters of gene transcription, such as the reduction in Na+ secreted by cells. V1.8 The amount of protein encoded by a gene, or the number of cells exhibiting a certain phenotype (e.g., apoptosis). In principle, Na... V 1.8 Gene silencing can be determined by any appropriate assay in any cell that expresses the target (constitutively or through genome engineering). However, when a reference is needed to determine whether a given siRNA inhibits Na+ to a certain extent... V 1.8 When the expression of genes is included and is therefore covered by this invention.

[0299] As used herein, "complementary region" refers to a nucleotide sequence of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel nucleotide sequence to allow the two nucleotide sequences to hybridize under appropriate hybridization conditions (e.g., in phosphate-buffered saline, in cells, etc.). Oligonucleotides in this article include targeting sequences having regions complementary to the mRNA target sequence.

[0300] As used herein, "ribonucleotide" means a nucleotide having ribose as its pentose sugar and a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides having one or more modifications or substitutions on atoms other than the 2' position, including modifications or substitutions on or above nucleobases, sugars, or phosphate groups.

[0301] As used herein, siRNA comprises a double-stranded oligonucleotide having a sense strand and an antisense strand, wherein the antisense strand or a portion thereof is used by the Argonaute 2 (Ago2) endonuclease of the RNA-induced silencing complex (RISC) to cleave the target mRNA.

[0302] As used herein, the term "single-stranded oligonucleotide" includes an antisense strand (or a portion thereof) that, in combination with a ribonuclease H (RNase H) endonuclease, mediates the cleavage of target mRNA.

[0303] As used herein, a “chain” refers to a single, continuous sequence of nucleotides linked together by internucleotide linkages (e.g., phosphodiester linkages or thiophosphate linkages). A chain may have two free ends (e.g., a 5' end and a 3' end).

[0304] As used herein, “synthetic” means nucleic acid or other molecules that are artificially synthesized (e.g., using a machine such as a solid-state nucleic acid synthesizer) or otherwise not derived from the natural source (e.g., a cell or organism) that normally produces the nucleic acid or other molecule.

[0305] As used herein, “treatment” means the act of providing care to an individual in need, for example by administering a therapeutic agent (e.g., an oligonucleotide as described herein) to an individual with the aim of improving the individual’s health and / or well-being with respect to an existing disease, condition, or illness, or preventing or reducing the likelihood of the occurrence of the disease, condition, or illness. Treatment may also involve reducing the frequency or severity of at least one sign, symptom, or contributing factor of a disease, condition, or illness experienced by an individual. The term “treatment” also includes reducing or alleviating pain perception, including reducing or alleviating the intensity and / or duration of pain (e.g., burning, tingling, electric shock-like sensation, etc.) experienced by a subject in response to a given stimulus (e.g., pressure, tissue damage, cold temperature, etc.). Reduction or alleviation of pain perception can be any detectable reduction in the intensity or duration of pain. Treatment may occur in subjects (e.g., humans or companion animals) suffering from a pain condition or having one or more symptoms of a pain-related condition that can be treated according to this disclosure, or in animal models of pain.

[0306] As used in this article, "Na" V 1.8” refers to any Na+ associated with the development or maintenance of ion channels. V 1.8 Proteins, peptides, or polypeptides. The term "Na" is used in this context. V 1.8” also refers to encoding any Na V 1.8 Nucleic acid sequence of a protein, peptide, or polypeptide. Encoding Na V The gene at 1.8 is called SCN10A.

[0307] As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an oligonucleotide or conjugate, and a pharmaceutically acceptable carrier. As used herein, a “pharmacologically effective amount,” a “therapeuticly effective amount,” or simply an “effective amount,” refers to the amount of RNA that is effective in producing the expected pharmacological, therapeutic, or preventative outcome. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 25%, then a therapeutically effective amount of a drug used to treat that disease or condition is the amount necessary to achieve that at least 25% reduction in the parameter.

[0308] As used herein, the phrases “therapeutic effective amount” and “preventive effective amount” refer to the amount that provides therapeutic benefit in the treatment, prevention, or management of pain or obvious symptoms of pain. The specific amount that is therapeutically effective can be readily determined by a general medical practitioner and can vary based on factors known in the art, such as, for example, the type of pain, the patient’s medical history and age, the stage of pain, and the administration of other analgesics.

[0309] As used in this article, "transformed cell" refers to a cell in which a vector has been introduced, from which dsRNA molecules can be expressed.

[0310] While similar or equivalent methods and materials described herein may be used to practice or test the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0311] Example

[0312] The following non-limiting examples are provided for illustrative purposes and not for limitation.

[0313] Example 1: Bioinformatics-based siRNA testing / selection .

[0314] Design and computer simulation selection of siRNAs targeting human SCN10A messenger RNA

[0315] Computer simulations were performed to classify and select targets for SCN10A (sodium voltage-gated channel α subunit 10, gene ID: 6336, alias: NA). V 1.8) siRNA sequences were used for in vitro testing. All 5856 possible 19-mer siRNA sequences for SCN10A human messenger RNA (mRNA) were created in a computer using the SCN10A reference sequence NM_006514.3 (SEQ ID NO: 648), which are incorporated herein by reference in their entirety. For bioinformatics analysis, all transcript RNA sequences were obtained from NCBI RefSeq DB version 95 (July 2019) and Ensembl DB version 97 (July 2019). Table 11 shows the reference sequences used for computer simulation screening and selection. The selection criteria used for the initial round of siRNAs were:

[0316] a. A complete sequence match (19-mer) with human SCN10A and cynomolgus / rhesus monkey SCN10A is required, and cross-reactivity with mice or rats has been analyzed, but this is not mandatory.

[0317] b. The sequence within positions 2-18 (5'-3') of the antisense strand does not completely match the non-SCN10A gene or has a mismatch.

[0318] c. For a different set of sodium channel sequences, including SCN4A (Na V 1.4, Gene ID 6329); SCN5A (Na V 1.5, Gene ID 6331); SCN9A (Na V 1.7, gene ID 6335) and SCN11A (Na V1.9 (gene ID 11280) requires three or more mismatches in the antisense strand.

[0319] d. Based on miRBase R22 (human, rhesus monkey, mouse, rat, March 2018), sequences with the same seed region as known miRNAs were excluded.

[0320] e. Based on the human SNP database (NCBI dbSNP Build 151, October 2017), siRNA sequences targeting regions of SNPs with a known suballele frequency (MAF) of >1% were excluded.

[0321] Table 11: Used for computer simulation of Na V 1.8 Transcript sequences for siRNA design and classification .

[0322]

[0323] Using Na selection V 1.8 Exclusion criteria for siRNAs: Computer simulation screening identified 141 siRNA sequences (see Table 2 above), representing approximately 2.4% of all possible 19-mer sequences. Each selected siRNA met the required selection criteria for predicted cross-reactivity between humans and cynomolgus / rhesus macaques (Table 12) and exhibited low predicted off-target effects and miRNA activity.

[0324] Table 12: Cross-reactivity of 19-mer siRNAs from computer simulation screening ("X" indicates the dominant siRNA species) (This shows the cross-reactivity predicted by computer simulation) .

[0325]

[0326]

[0327]

[0328] In target human Na V Of the 5856 possible 19-mer siRNA sequences for the 1.8 transcript (reference sequence NM_006514.3), 141 sequences met the selected criteria, such as low potential off-target toxicity to other NAV channels and genes, and perfect 19-mer match with known cynomolgus monkey and rhesus monkey SCN10A mRNA transcripts. These 141 siRNAs were then selected for synthesis and in vitro screening.

[0329] Example 2: Preparation method of siRNA molecules and description of RNA modification

[0330] Na was synthesized V 1.8 21-mer double strands, which have 19 complementary bases and a guide strand targeting human SCN10AmRNA (also known as Na) V1.8 mRNA) 3' dinucleotide overhang (see Table 3). RNA modification was used to optimize the potency of the siRNA duplex and reduce immunogenicity (e.g. Figure 1A or Figure 1B As shown; the 5' end of the transit chain may or may not have an amine or cholesterol linker.

[0331] All siRNA single strands were fully assembled on the solid phase using standard phosphoramide chemistry and purified by HPLC. The purified single strands were annealed to obtain double-stranded 21-mer / 19-mer siRNAs (Malecova et al., “Targeted Tissue Delivery of RNA Therapeutics using Antibody–oligonucleotide Conjugates (AOCs),” Nucleic Acids Res. Vol. 51, No. 12, pp. 5901–5910 (2023)).

[0332] A non-targeted control siRNA sequence (siNTC) was synthesized. This is a publicly available non-targeted control sequence with a 21-mer double strand and 19 complementary bases and a 3' dinucleotide overhang on the guide strand (Burke et al., "siRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds," Pharm. Res. 31, 3445-3460 (2014)). The sequence of siNTC is shown in Table 13.

[0333] Table 13: Non-targeted control siRNA sequences .

[0334]

[0335] In some implementations, the siRNA contains a vinylphosphonate-modified nucleotide (VpUq) in the antisense strand, such as Figure 1B As shown in the diagram and Table 4 (5vp_una2moe), VpUq is a vinylphosphonate-2'-O-methoxyethyl ribose unlocking nucleic acid with a uridine base. It was synthesized according to the procedure described in US Patent No. 11,110,180, which is incorporated herein by reference in its entirety. The chemical structure of VpUq is shown below:

[0336]

[0337] Example 3: VpUq guiding chain modification in Na V 1.8 Effects of siRNA .

[0338] siRNAs with the VpUq modification pattern are described in Table 4 and Figure 1B The text describes the process of evaluating the activity and potency of VpUq modification in siRNA, testing Na+ with and without VpUq modification in primary human neurons. V 1.8 siRNA sequences. Modified siRNA 410 (i.e., siRNA number 285 in Table 4) without VpUq and modified siRNA 410 (i.e., siRNA number 305 in Table 4) containing VpUq were tested, as were modified siRNA 535 (i.e., siRNA number 291 in Table 4) without VpUq and modified siRNA 535 (i.e., siRNA number 311 in Table 4) containing VpUq. All tested siRNAs were encapsulated in lipid nanoparticles as described below.

[0339] Using human dorsal root ganglion neurons (DRG) as the expression of Na V 1.8 Primary cell source of mRNA. Fresh primary human DRG was purchased from AnaBios (San Diego, CA) and maintained in serum-free NbActive4 maintenance medium (Axol, Easter Brush, United Kingdom) containing 25 ng / mL recombinant human nerve growth factor (NGF, Axol). Primary DRG was treated with siRNA encapsulated in lipid nanoparticles (LNP, Precision Nanosystems, Vancouver, Canada) as recommended by the manufacturer. LNP-siRNA (with VpUq) was introduced into the growth medium at concentrations ranging from 150 nM to 0.008 nM and maintained for 3 days without transfection reagents.

[0340] The treated human DRG was kept at 37°C and 5% CO2 for 3 days, and then in phosphate-buffered saline (PBS, ThermoFisher). ™ Wash in TRIzol and store in 300 µL of TRIzol. ™ (ThermoFisher) ™The mRNA was stored at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA). RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_ml, STMN2: Hs00975900_m1, and SNAP25: Hs00938957_m1). V 1.8 The relative reduction in transcripts was determined by normalizing the expression of siRNA-treated DRG relative to the simulated transfection group using the DDCt method, using the following formula: [%hNa V 1.8 mRNA = 100 * 2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and housekeeping genes STMN2 and SNAP25 {avg DCt=[((2*Ct(SCN10A))-Ct(STMN2)-Ct(SNAP25)) / 2]} (Green MR, Sambrook J. “Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR),” Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD, “Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,”Methods, 25(4), 402–8 (2001)).

[0341] The siRNA concentration-response data were fitted to a three-parameter nonlinear regression model [log(siRNA concentration) versus %hNa] using GraphPad Prism software (GraphPad Software, LLC). V IC50 was calculated using 1.8 mRNA.

[0342] The dose response curve is in Figure 2 and Figure 3 As shown in Table 14, adding the modified nucleotide VpUq to siRNA had minimal effect on the maximum reduction in mRNA levels. However, the presence of VpUq at the 5' end of the guide strand affected the siRNA IC50 value in a sequence-specific manner. For siRNA number 305 (start position 410), the calculated IC50 values ​​showed little difference; while for siRNA number 311 (start position 535), adding VpUq reduced the siRNA IC50 value from 23.4 nM to 6.9 nM, a reduction of more than 3-fold, as shown in Table 14.

[0343] Table 14: Effects of VpUq Modification Mode

[0344]

[0345] In primary human DRG, the incorporation of VpUq-modified nucleotides produces beneficial effects in a sequence-specific manner. Data shows that Na... V 1.8 siRNA Sequence: The IC50 value of siRNA number 311 was increased by more than approximately 3-fold. However, in Na... V 1.8 Minor differences were observed in siRNA sequence number 305. A slight change was observed in the maximum mRNA reduction after incorporating the modified nucleotide VpUq into the siRNA.

[0346] Example 4: Na V 1.8 Activity of siRNA in HEK293 cells in vitro .

[0347] Using the expression Na V 1.8 HEK293 cells containing transcripts (HEK293-hNa) V 1.8) To determine the in vitro activity of the siRNA sequence, cells were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, Billings, MT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning, Corning, NY), 1% Pen / Strep (Gibco), and 0.1 mg / mL Zeocin. ™ (Gibco) and 0.5 μg / mL puromycin (ThermoFisher, Waltham, MA). For transfection, HEK293-hNa V 1.8 Cells were seeded at 15,000 cells per well in 96-well tissue culture plates, with 100 μL of culture medium added to each well. Transfection was performed within 24 hours post-seeding. siRNA was then mixed with Lipofectamine. ™Gently mix the RNAiMAX transfection reagent (ThermoFisher) and add it to the wells according to the manufacturer's recommendations, with a final concentration of 10 nM or 1 nM.

[0348] The treated cells were kept at 37°C and 5% CO2 for 72 hours, then washed in phosphate-buffered saline (PBS, ThermoFisher) and stored in 300 μL TRIzol. ™ In ThermoFisher, store at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).

[0349] RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1 and PPIB: Hs00168719_m1). V 1.8 Relative reduction of transcripts by using the DDCt method to treat HEK-hNa with siRNA V 1.8 Cell expression was determined by normalization relative to the simulated transfection group using the following formula: [%hNa] V 1.8 mRNA = 100*2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and the housekeeping gene PPIB [DCt=Ct(SCN10A)-Ct(PPIB)] (Green MR, Sambrook J. “Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR),” Cold SpringHarb.Protoc., 2018(10): pdb.prot095042; Livak KJ, Schmittgen TD, “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,” Methods, 25(4), 402–8 (2001)).

[0350] Table 15 summarizes the screening results, showing the in vitro activity ranking of Na-targeting RNA based on 10 nM siRNA concentration. V 1.8 siRNA sequence. %hNa V 1.8 mRNA represents the amount of human NA remaining after siRNA treatment compared to simulated transfection control cells. V 1.8 Relative expression of mRNA. Next to the siRNA number in the table is the start position of the siRNA sequence based on the SCN10A reference transcript sequence (NM_006514.3).

[0351] Non-targeting human siRNA sequences (siNTC, Table 13) were used as transfection controls to account for non-specific mRNA reductions. Control results are also shown in Table 15. Significant mRNA reduction cutoffs were determined by subtracting three standard deviations (std.dev) from the combined mean of the siNTC groups (10 nM and 1 nM siRNA concentrations) (siNTC mean = 95.6%; std.dev = 14.8%).

[0352] Table 15: In HEK-293 hNa V 1.8 In vitro activity of two siRNAs at different concentrations in cells. siRNAs such as... Figure 1A As shown Modified, but without amino / cholesterol linker .

[0353]

[0354]

[0355]

[0356] *: Indicates a non-targeted control siRNA (siNTC) sequence.

[0357] 141 siRNA sequences selected from computer-simulated screening were synthesized and expressed in human Na+. V Further in vitro evaluation was performed in HEK293 cells at 1.8 μm. Na+ was removed using lipofectamine. V 1.8 siRNA sequence transfected into HEK293-hNa V 1.8 cells. In HEK293-hNa V Of the 141 siRNA sequences tested in cells, 52 siRNA sequences induced more than 50% Na+ at 10 nM or 1 nM. V 1.8 Decreased mRNA levels (%hNa) V 1.8 mRNA <51.2% (see Table 15). siRNA sequence: siRNA number 142 (target region at start position 407 on human SCN10A transcript) in reducing Na V1.8 It exhibits optimal activity at the mRNA level, with Na+ at 10 nM. V 1.8 mRNA levels decreased by more than 85% (%hNa) V 1.8 mRNA = 17.8%.

[0358] In HEK293-hNa V In cell line 1.8, Na was observed. V 1.8 mRNA levels can be affected by Na+. V 1.8 siRNA sequence downregulation. Although computer simulation screening identified 141 19-mer siRNA sequences, the tested Na... V 1.8 52 siRNA sequences showed greater than 50% inhibitory activity at 10 nM or 1 nM (% hNa). V 1.8 mRNA <51.2%.

[0359] Example 5: Na V 1.8 siRNA in the expression of human Na V Concentration-dependent activity of HEK293 at 1.8 .

[0360] Using the expression Na V 1.8 HEK293 cells containing transcripts (HEK293-hNa) V 1.8) Determine in vitro hNa V 1.8 mRNA reduction and the half-maximal inhibitory concentration (IC50) of the selected lead siRNA sequence. Cells were cultured in DMEM (Gibco, Billings, MT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning, Corning, NY), 1% Pen / Strep (Gibco), and 0.1 mg / mL Zeocin. ™ (Gibco) and 0.5 μg / mL puromycin (ThermoFisher, Waltham, MA). For transfection, HEK293-hNa V 1.8 Cells were seeded at 15,000 cells per well in 96-well tissue culture plates, with 100 μL of culture medium added to each well. Transfection was performed within 24 hours post-seeding. siRNA was then mixed with Lipofectamine. ™ Gently mix the RNAiMAX transfection reagent (ThermoFisher) and add it to the wells according to the manufacturer's recommendations, with a final concentration range of 67 nM to 0.27 nM.

[0361] The treated cells were kept at 37°C and 5% CO2 for 72 hours, washed in phosphate-buffered saline (PBS, ThermoFisher), and stored in 300 μL TRIzol. ™In ThermoFisher, store at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).

[0362] RNA expression levels were determined by RT-qPCR using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1 and PPIB: Hs00168719_m1). V 1.8 Relative reduction of transcripts by using the DDCt method to treat HEK-hNa with siRNA V 1.8 Cell expression was determined by normalization relative to the simulated transfection group using the following formula: [%hNa] V 1.8 mRNA = 100*2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and the housekeeping gene PPIB [DCt=Ct(SCN10A)-Ct(PPIB)] (Green MR, Sambrook J. "Quantification of RNA by Real-Time Reverse Transcription-Polymerase ChainReaction (RT-PCR)," Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD. "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,"Methods, 25(4), 402–8 (2001)).

[0363] Table 16 summarizes the screening results, showing the target Na based on the siRNA gene start position sequencing according to the SCN10A reference transcript sequence (NM_006514.3). VThe siRNA sequence was 1.8. The siRNA concentration-response data were fitted to a three-parameter nonlinear regression model [log(siRNA concentration) versus %hNa] using GraphPad Prism software (GraphPad Software, LLC). V 1.8 mRNA] to calculate IC50 and maximum hNa V 1.8 Knockdown percentage (%maxKD) of target mRNA. Maximum knockdown (maxKD) is calculated using the following formula: %maxKD = 100 - hNa V 1.8 eMax, where eMax represents the predicted lowest value of the fitted concentration-response curve (the bottom value of the three-parameter nonlinear regression curve). Based on computer simulation predictive analysis, the expected effect on Na+ in mice and / or rats... V 1.8 Computer simulation predictions of active siRNAs from mRNA were also included along with in vitro results.

[0364] Table 16: HEK293-hNa V 1.8 Na in cells V 1.8 Summary of siRNA concentration-dependent response assay. (siRNA figure shown) The modification shown in 1A is performed, but without the amino / cholesterol linker. .

[0365]

[0366] Legend: ND indicates undetermined; X indicates expected effect on Na+ in mice and / or rats based on computer simulation analysis. V 1.8 mRNA activity.

[0367] Based on the siRNA sequence activity screened in vitro from HEK cells (as shown in Table 15), the highest inhibitory activity (lowest % hNa) was observed at 10 nM or 1 nM. V 31 siRNA sequences (1.8 mRNA) in HEK293-hNa V 1.8 Further concentration-dependent response assays were performed in the cell lines (the first 30 rows of Table 15 plus the siRNA sequence number 237 (start position 860)) to determine the maximum Na+ concentration. V 1.8 Transcription reduction and siRNA half-maximal inhibition concentration.

[0368] As shown in Table 16, most siRNA sequences induced more than 50% of Na+. V 1.8 mRNA levels decreased. Na V 1.8 The siRNA sequence number 145 (start position 410) has the highest maxKD, at 80%; while Na V 1.8 The siRNA sequence number 142 (start position 407) had the lowest IC50 of 0.13. Overall, 31 Na... V1.8 The mean maxKD of the siRNA sequence was 63.8% (std.dev=7.3%), and the mean IC50 was 0.54 nM (std.dev=0.37 nM). As expected, the effect of the siNTC negative control was negligible, and the nonlinear regression R-squared was poor (R... 2 <0.1). A summary of the three-parameter nonlinear regression is also shown in Table 16.

[0369] The selected siRNA sequence is located in HEK293-hNa V 1.8 The cell line showed in vitro activity and potency, with 7 siRNA sequences delivering Na+. V 1.8 Transcript expression levels decreased by more than 70%. Regarding IC50, 31 Na... V 1.8 Of the siRNAs, 26 had activity in the sub-nanomolar range (<1 nM), while the remaining siRNA sequences had activity in the low single-digit nanomolar range (<2 nM).

[0370] Example 6: Na V 1.8 Evaluation of off-target effects of siRNA .

[0371] Batch RNA-seq analysis was performed on human skeletal muscle myotubes (Institute of Myology, France), using appropriate culture media from Promocell (Heidelberg, Germany) for growth and differentiation. After differentiation, a 50 nM LNP-siRNA negative control (siNTC) or Na+ was used. V 1.8 siRNA treatment of cells. The siRNAs tested were: siRNA No. 283 (start position 407), siRNA No. 285 (start position 410), siRNA No. 291 (start position 535), and siRNA No. 301 (start position 1172) in Table 4, and were treated for 24 hours (n=4, each LNP-siRNA). The modification patterns of the siRNA sequences are described in Table 4.

[0372] Using TRIzol ™ (ThermoFisher) ™ Total RNA was isolated, followed by chloroform extraction and isopropanol precipitation, and then processed using QIAGEN RNeasy according to the manufacturer's recommendations. ®Purification was performed using a small kit (QIAGEN, Hilden, Germany). The isolated RNA was treated with DNAseI (QIAGEN) on a column, resuspended, and quantified using a Nanodrop (ThermoFisher) at a 260 / 280 nm ratio. cDNA libraries were prepared using Poly(A)+ selection. Sequencing reads were normalized to kilobase million transcripts per mille (TPM) for downstream analysis.

[0373] Off-target analysis was performed on human skeletal muscle myotube cells using siRNA sequences siRNA 283, siRNA 285, siRNA 291, and siRNA 301. Batch RNA-seq analysis was performed, and differentially expressed (DE) genes were identified using the DESeq2 package with R and an adjusted p-value <0.05 (Love MI et al., “Moderated Estimation of Fold Change and Dispersion for RNA-seq Data with DESeq2,” Genome Biol. 15(12):550(2014)). Cells treated with siNTC were used as baseline comparison conditions. The number of DE genes with a log2 fold change (FC) greater than 2 is reported in Table 17.

[0374] Four types of Na V 1.8 The siRNA sequences were delivered in vitro to human skeletal muscle myotubes via lipid nanoparticles, showing negligible to slight downregulation of SCN10A-unrelated genes. siRNA number 291 (start position 535) showed the highest unintended off-target profile (14 DE genes, with a log2 FC greater than 2 relative to siNTC), while siRNA number 285 (start position 410) showed the lowest off-target effect (no DE genes with a log2 FC greater than 2 relative to siNTC).

[0375] Table 17: Using Na V 1.8 Off-target analysis of siRNA-treated human skeletal muscle myotubes .

[0376]

[0377] Overall, all leading Na V 1.8 All siRNAs showed little to negligible off-target profiles, with siRNA sequence number 285 (start position 410) showing the best overall performance in vitro, with negligible off-target profiles after 24 hours of LNP-siRNA treatment.

[0378] Example 7: Leader Na V 1.8-siRNA activity in DRG neurons .

[0379] Using functional NaV 1.8 mRNA from human neurons to confirm the leader Na V 1.8 siRNA activity. The top 14 Na+ molecules with the highest %maxKD and / or lowest IC50 were selected. V 1.8 The siRNA sequence was further evaluated. The effect on mouse Na+ was predicted based on computer simulations. V 1.8 Species cross-reactivity of mRNA was assessed, and five additional siRNA sequences were selected. Based on their high sequence similarity to siRNA number 142 (start position 407) and siRNA number 145 (start position 410) in Table 3, another siRNA sequence, siRNA number 205 (start position 409) in Table 3, was selected. Therefore, a total of 20 siRNA sequences were preferentially synthesized, using... Figure 1A The modified pattern shown features a cholesterol linker at the 5' end of the transient chain, enabling efficient transfection in primary human neurons without the need for lipofectamine reagent.

[0380] Using human dorsal root ganglion neurons (DRG) as the expression of functional Na+ V 1.8 Primary cell sources of mRNA and protein. Fresh primary human DRG was purchased from AnaBios (San Diego, CA) and maintained in serum-free NbActive4 maintenance medium (Axol, Easter Brush, United Kingdom) containing 25 ng / mL recombinant human NGF (Axol). Cholesterol-conjugated siRNA (Chol-siRNA) was introduced into the growth medium twice as is (day 0 and day 3, for a total of 5 days) at a concentration of 1 µM, without the use of transfection reagents.

[0381] The treated human DRG was kept at 37°C and 5% CO2 for 5 days, then washed in phosphate-buffered saline (PBS, ThermoFisher) and stored in 300 µL TRIzol. ™ In ThermoFisher, store at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).

[0382] RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1, STMN2: Hs00975900_m1, and SNAP25: Hs00938957_m1). V 1.8 The relative reduction in transcripts was determined by normalizing the expression of siRNA-treated DRG relative to the simulated transfection group using the DDCt method, using the following formula: [%hNA] V 1.8 mRNA = 100*2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and housekeeping genes STMN2 and SNAP25 {avg DCt=[((2*Ct(SCN10A))-Ct(STMN2)-Ct(SNAP25)) / 2]} (Green MR, Sambrook J.“Quantification of RNA by Real-Time Reverse Transcription-Polymerase ChainReaction (RT-PCR),” Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD, “Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,”Methods, 25(4), 402–8 (2001)).

[0383] Figure 4 Results in human DRG were summarized, demonstrating the targeting of Na+ based on in vitro activity ranking at 1 µM siRNA concentration. V 1.8 Cholesterol-modified siRNA sequence. %hNa V 1.8 mRNA represents the residual human Na+ after siRNA treatment compared to simulated transfection control cells. V 1.8 Relative mRNA expression. The number below each bar in the bar chart indicates the siRNA sequence start position based on the SCN10A reference transcript sequence (GenBank: NM_006514.3). A non-targeted cholesterol-modified siRNA sequence (siNTC) was used as a transfection control to explain the non-specific mRNA decrease. Commercially available Accell cholesterol-modified Na+ was used.V 1.8 The siRNA pool served as a positive control (Horizon Discovery Ltd, Waterbeach, UK). Adjusted p-values ​​were determined using ordinary one-way ANOVA with Dunnett's multiple comparison test, employing the software tool GraphPad Prism (GraphPad Software, LLC).

[0384] Based on the use of HEK293-hNa V 1.8 In vitro cell assays further selected 14 Na+ cells with the highest %maxKD and / or lowest IC50 values. V 1.8 The siRNA sequence and six additional siRNA sequences selected through computer simulation were synthesized and cholesterol-modified. These cholesterol-modified siRNA sequences were then used to express functional Na+. V 1.8 mRNA in human neurons using cholesterol-modified Na V 1.8 siRNA sequence transfection was performed, followed by in vitro assays. For example... Figure 4 As shown, the selected Na V 1.8 Cholesterol-modified siRNA sequences reduced Na in human DRG at a concentration of 1 µM siRNA. V 1.8 mRNA level. Of the 20 siRNA sequences tested in primary human DRG, 10 siRNA sequences showed significantly lower levels of NA compared to siNTC sequences in primary human DRG. V 1.8 Transcript levels were reduced by at least 50% (adjusted P < 0.05). siRNA sequence number 285 (start position 410) reduced Na+ in human primary DRG. V It exhibits optimal activity at the 1.8 mRNA level.

[0385] In HEK293-hNa V Of the 18 siRNA sequences identified in the initial screening of cell lines (Table 14), 10 showed significant Na+ expression in primary human DRG cell cultures using cholesterol-modified siRNA. V 1.8 Transcriptional degradation. siRNA sequences 285 (start position 410), 291 (start position 535), and 301 (start position 1172) showed consistently high repressive activity in vitro. V 1.8 Overall mRNA reduction was equal to or greater than 70%.

[0386] Example 8: Selected Leader Na V 1.8 siRNA concentration-response in human DRG: IC50 at sub-nanomolar levels. Within range .

[0387] To further characterize the first three Na... V 1.8 The activity and potency of siRNA in human DRG were determined in vitro using concentration-response assays. Three siRNA sequences exhibiting the highest activity in primary neurons were tested. The siRNAs tested were: siRNA No. 285 (start position 410), siRNA No. 291 (start position 535), siRNA No. 301 (start position 1172), and siRNA No. 283 (start position 407) in Table 4. The siRNAs were encapsulated in lipid nanoparticles.

[0388] Using human dorsal root ganglion neurons (DRG) as the expression of Na V 1.8 Primary cell source of mRNA. Fresh primary human DRG was obtained from AnaBios (San Diego, CA) and maintained in serum-free NbActive4 maintenance medium (Axol, Easter Brush, United Kingdom) containing 25 ng / mL recombinant human NGF (Axol). Primary DRG was treated with siRNA encapsulated in lipid nanoparticles (LNP, Precision Nanosystems, Vancouver, Canada) as recommended by the manufacturer. LNP-siRNA was introduced into the growth medium at concentrations ranging from 150 nM to 0.008 nM and maintained for 3 days without transfection. The modification patterns of the siRNA sequence are described in Table 4.

[0389] The treated human DRG was kept at 37°C and 5% CO2 for 3 days, then washed in phosphate-buffered saline (PBS, ThermoFisher) and stored in 300 µL TRIzol. ™ In ThermoFisher, store at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).

[0390] RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1, STMN2: Hs00975900_m1, and SNAP25: Hs00938957_m1). V1.8 The relative reduction in transcripts was determined by normalizing the expression of siRNA-treated DRG relative to the simulated transfection group using the DDCt method, using the following formula: [%hNa V 1.8 mRNA = 100*2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and housekeeping genes STMN2 and SNAP25 {avg DCt=[((2*Ct(SCN10A))-Ct(STMN2)-Ct(SNAP25)) / 2]} (Green MR, Sambrook J.“Quantification of RNA by Real-Time Reverse Transcription-Polymerase ChainReaction (RT-PCR),” Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD, “Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,”Methods, 25(4), 402–8 (2001)).

[0391] The siRNA concentration-response data were fitted to a three-parameter nonlinear regression model [log(siRNA concentration) versus %hNa] using GraphPad Prism software (GraphPad Software, LLC). V 1.8 mRNA] to calculate IC50 and maximum hNa V 1.8 Knockdown percentage (%maxKD) of target mRNA. Maximum knockdown (maxKD) is calculated using the following formula: %maxKD = 100 - hNa V 1.8 eMax, where eMax represents the predicted minimum value of the fitted concentration-response curve (the bottom value of the three-parameter nonlinear regression curve).

[0392] like Figure 5 As shown, the concentration-response curves of the first three lead siRNA sequences and siRNA number 283 exhibit similar maximum Na+ levels in human DRG. V 1.8 mRNA reduction: All tested sequences achieved greater than 75% reduction in target transcripts in LNP form. Na V1.8 The IC50 of siRNA was in the sub-nanomolar (<1 nM) range, with 3 out of the 4 sequences tested (siRNA numbers 285, 291, and 283), and siRNA number 301 in the single-digit nanomolar range (<2 nM) (see Table 18).

[0393] Table 18: Summary Results of Concentration-Response Measurement in Primary Human DRG .

[0394]

[0395] Na in all tests V 1.8 siRNA sequences all induce Na+ in human primary DRG. V 1.8 mRNA levels were significantly reduced by more than 70%. siRNA number 285 showed the highest activity, with transcript reduction greater than 90% and an IC50 of 71 pM.

[0396] Example 9: Downregulation of SCN10A transcript using siRNA-LNP leads to Na+ reduction in human primary neurons V 1.8 Protein Expression reduce .

[0397] The test siRNA sequences (i.e., siRNA number 283 (start position 407) and siRNA number 285 (start position 410) in Table 4) were used to reduce Na+ in human primary neurons. V 1.8 Protein expression capacity. The modification patterns of the siRNA sequence are described in Table 4.

[0398] Using human dorsal root ganglion neurons (DRG) as the expression of Na V 1.8 Primary cell source of mRNA. Fresh primary human DRG was purchased from AnaBios (San Diego, CA) and maintained in serum-free NbActive4 maintenance medium (Axol, Easter Brush, United Kingdom) containing 25 ng / mL recombinant human NGF (Axol). Primary DRG was treated with test siRNA encapsulated in lipid nanoparticles (LNP, Precision Nanosystems, Vancouver, Canada) as recommended by the manufacturer. LNP-siRNA was introduced into the growth medium twice at a concentration of 0.5 µg / mL for 6 days, without the use of transfection reagents.

[0399] Human DRG was treated and kept at 37°C and 5% CO2 for 6 days, then washed in phosphate-buffered saline (PBS, ThermoFisher) and stored in 300 µL TRIzol (ThermoFisher) at -80°C until RNA extraction. Total mRNA was isolated using the ZYMO 96-well RNA Kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA).

[0400] RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1, STMN2: Hs00975900_m1, and SNAP25: Hs00938957_m1). V 1.8 The relative reduction in transcripts was determined by normalizing the expression of siRNA-treated DRG relative to the simulated transfection group using the DDCt method, using the following formula: [%hNa V 1.8 mRNA = 100*2 -DCt (siRNA处理细胞)-DCt (模拟转染细胞) ], where DCt is determined by the difference in Ct values ​​between SCN10A and housekeeping genes STMN2 and SNAP25 {avg DCt=((2*Ct(SCN10A))-Ct(STMN2)-Ct(SNAP25)) / 2} (Green MR, Sambrook J.“Quantification of RNA by Real-Time Reverse Transcription-Polymerase ChainReaction (RT-PCR),” Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD, “Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,”Methods, 25(4), 402–8 (2001)).

[0401] Na+ in protein lysates of cultured human DRG neurons was measured using ELISA. V1.8 Protein Concentration. The growth medium was aspirated, and the cultured human DRG neurons were washed once in Dulbecco phosphate-buffered saline (DPBS; Gibco, catalog 14190-136). Whole-cell lysates were prepared by resuspending the cultured human DRG neurons in 100 µL of 1X lysis buffer (CST, catalog 9803) per well, along with Halt protease and phosphatase inhibitors (TFS, catalog 78440) diluted to 2X, and PMSF (Sigma, #78830-25G, 17.4 mg dissolved in 1 mL isopropanol, diluted to 1X). The lysates were incubated on ice for 5 min, then centrifuged at 800 × g for 20 min at 4 °C. The supernatant was collected and frozen at -80 °C for analysis the next day. Pierce was used. ™ BCA Protein Assay Kit (catalog number 23227) was used to measure protein concentrations according to the “Microplate Procedure” described in the manufacturer’s manual. Samples were further diluted 1:3 with cell lysis buffer before plating. Na+ in cultured human DRG neuron protein lysates was detected using the MesoScale Discovery (MSD) S-Plex platform. V 1.8 Protein Levels. The custom-developed assay was performed using the S-PLEX Development Kit B, SECTOR (25 plates) (catalog number K15601S-4). Capture and detection antibodies were labeled with MSD. The capture antibody (NeuroMab, catalog number 75-166) was diluted to 0.5 µg / mL, and the detection antibody was diluted to 0.2 µg / mL. The calibrator was human sodium. V 1.8 recombinant C-terminal fragment. Sample lysate was diluted 4-fold in assay buffer. The assay buffer matrix was Diluent 39 (MSD, catalog number R5ABB2), with added MSD. ® Each component from the inhibitor package (catalog number R70AA-1) was added in 100 µL. The washing step used MSD Tris wash buffer (MSD, catalog number R61TX-1) diluted to 1X in double-distilled water (ddH2O). MSD plates were read using a MESO QuickPlex SQ 120MM plate reader with MSD Methodical Mind software. Data were analyzed using Discovery Workbench v4. The Na+ of each lysate sample was measured. V 1.8 The data were normalized by dividing the protein concentration by the total protein concentration. Statistical significance was determined using one-way ANOVA.

[0402] Results: siRNA sequences siRNA 283 and siRNA 285, possessing minimal off-target spectrum, were transfected into human DRG as lipid nanoparticle (LNP) siRNA sequences, and further in vitro assays were performed. Na+ was measured by qPCR. V 1.8 mRNA level ( Figure 6A ), and Na was measured by ELISA. V 1.8 protein levels ( Figure 6B Negligible transcriptional regulation was observed using LNP-siNTC negative control sequences. When the assay was normalized to total protein levels, Na… V 1.8 Protein expression decreased by approximately 30%. When corrected for Na by CGRP protein expression (a human DRG-specific protein)... V 1.8 No difference was observed in protein expression between the untreated group and the siNTC group. Na V 1.8 LNP-siRNA downregulated SCN10A transcript by over 90%. More importantly, Na... V 1.8 Decreased mRNA levels were associated with inhibition of protein expression levels exceeding 80%, regardless of the normalization method (total protein loading or human DRG normalization).

[0403] In human primary DRG cultured in vitro, Na V 1.8 mRNA and protein expression are highly correlated. The lead siRNA sequences, siRNA number 283 and siRNA number 285, are used with LNP-Na. V 1.8 siRNA treatment resulted in a significant reduction in mRNA and protein levels in vitro. Na V 1.8 Na in the siRNA-treated group V 1.8 The decrease in protein was independent of the protein normalization method. This confirmed that in cultured human DRG, Na... V 1.8 There is a correlation between mRNA regulation and protein translation.

[0404] Example 10: Expression and purification of transferrin receptor antibody (TfR) .

[0405] The exemplary anti-TfR antibodies and antibody fragments of this disclosure are expressed and purified as described below. Antibodies TBP1, TBP2, and TBP3 (as shown in Table 8 above) are expressed in appropriate host cells (such as CHO cells) using an optimal predetermined HC:LC vector ratio or a single-vector system encoding both HC and LC, via transient or stable transfection expression systems to secrete TBP1 or TBP3 antibodies.

[0406] For TBP2 antibodies, use the optimal predetermined HCA:HCB:LC vector ratio or a single-vector system encoding HCA, HCB, and LC. The expression plasmid contains cDNA versions of the LC and HC genes for antibodies TBP1, TBP2, or TBP3; and is expressed by a commonly used and suitable construct for this purpose, such as a construct based on the human cytomegalovirus major immediate early promoter.

[0407] The culture medium in which antibodies are secreted can be purified using conventional techniques such as mixed-mode methods involving ion exchange and hydrophobic interaction chromatography. For example, culture medium containing TBP1 or TBP2 antibodies can be loaded onto a Protein A column (Cytiva) and eluted using conventional methods. Culture medium containing TBP3 antibodies can be loaded onto a CaptureSelect column using conventional methods. ™ CH1-XL column (Thermo Scientific) ™ And wash it off.

[0408] Soluble aggregates, polymers, and fragments can be produced using POROS. ™ HS 50 column (Thermo Scientific) ™ Cation exchange chromatography effectively removes the antibodies using conventional methods. The product can be immediately frozen (e.g., at -70°C), refrigerated, or lyophilized. Various protein purification methods can be employed, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd edition, Springer, NY (1994). Antibodies TBP1, TBP2, or TBP3 can be immediately frozen at -70°C or stored at 2 to 8°C for several months, or lyophilized, or stored at 4°C for immediate use. The amino acid sequences of the antibodies disclosed herein are shown in Tables 5 to 8. The nucleic acid sequences of the antibodies are provided in Table 9.

[0409] Example 11: Method for preparing antibody oligonucleotide conjugates (AOC) conjugated with natural cysteine ​​(nCys)

[0410] Na V 1.8-AOC ( Figure 7 ) is an antibody-siRNA conjugate (also referred to herein as an antibody oligonucleotide conjugate (AOC)), which is formed by combining a human IgG1 antibody specific for human transferrin receptor 1 (anti-TFR1 antibody) with a target Na+ nucleotide conjugate. V 1.8 mRNA double-stranded siRNA oligonucleotide (Na) V1.8 Formed by conjugation with siRNA. Any antibody provided in Tables 5 through 8 can be used in the AOC and conjugated with any of the siRNAs provided in Tables 2 through 4. Conjugation can be performed via a linker (e.g., an SMCC linker).

[0411] 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) has a maleimide linker located at the 5' end of the transchain and is conjugated to the antibody via a cysteine ​​residue in the antibody's amino acid sequence. This conjugate binds to the human transferrin receptor on the cell surface, internalizes into the cell, and delivers the siRNA oligonucleotide to the intracellular compartment. After being taken up by the cell, the siRNA is loaded into the RNA-induced silencing complex (RISC) and hydrolyzes intracellular Na+. V 1.8 mRNA.

[0412] Step 1: Reduce interchain disulfide bonds of the antibody using TCEP

[0413] The antibody (anti-human transferrin receptor 1, TBP1) was prepared in phosphate-buffered saline (PBS) at pH 7.4 and adjusted to 2 mM ethylenediaminetetraacetic acid (EDTA). Two equivalents (EQ) of tris(2-carboxyethyl)phosphine (TCEP) dissolved in water were added to this solution, and the mixture was incubated at room temperature (RT) for 4 hours. A solution of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid (MCC)-siRNA (0.9 EQ) dissolved in 10 mM sodium acetate at pH 6 was added to the resulting reaction mixture, and the mixture was incubated at room temperature for 1 hour. Analysis of the reaction mixture by analytical strong anion exchange (SAX) column chromatography revealed the antibody-siRNA conjugate as well as unreacted antibody and siRNA. Figure 8 The reaction mixture was treated with 10 equivalents of N-ethylmaleimide (dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL) to block any remaining free cysteine ​​residues.

[0414] Step 2: Purification

[0415] The crude reaction mixture was purified by anion exchange chromatography using ÄKTA explorer rapid purification liquid chromatography (FPLC). Figure 9 Fractions containing DAR1 siRNA conjugates were isolated, concentrated, and prepared with PBS at pH 7.4.

[0416] FPLC Method 1 Details

[0417] Column: HiScale ™ 50 / 20 column, TSKgel ® SuperQ-5PW resin

[0418] Solvent A: 20mM phosphate buffer, pH 7.2

[0419] Solvent B: 20mM phosphate buffer, 1.5M NaCl, pH 7.2

[0420] Gradients used (%A / %B): 90:10; 81:19; 68:32

[0421] Used volumes: 0.5, 0.2, 6

[0422] Step 3: Analysis of the purified conjugate

[0423] The separated conjugates were characterized by size exclusion (SEC) and SAX chromatography. The purity of the conjugates was assessed by analytical high-performance liquid chromatography (HPLC), and the purity of the separated DAR1 conjugates was greater than 90%. Figure 10 and Figure 11 ).

[0424] SAX Method 1 Details

[0425] Column: Thermo Scientific, PropPac ™ SAX-10, Bio LC, 4×250mm

[0426] Solvent A: 80% 10mM Tris, pH 8, 20% ethanol

[0427] Solvent B: 80% 10mM Tris pH 8, 20% ethanol, 1.5M NaCl

[0428] Flow rate: 0.9 mL / min

[0429] Gradient (%A / %B): 84:16; 60:40; 46.5:53.5; 40:60; 84:16; 84:16

[0430] Time intervals related to the gradient: 0, 2, 4, 4.5, 5, 6.5

[0431] SEC Method 1 Details

[0432] Chromatographic column: Waters ™ Bioresolve SEC mAb, 2.5µm, 4.6×150mm

[0433] Solvent A: 55mM KH₂PO₄, 62mM Na₂HPO₄·7H₂O, 100mM Na₂SO₄, 0.05% w / v NaN₃, pH 6.7

[0434] Flow rate: 0.4 mL / min

[0435] Isogradation (%A): 100

[0436] Time related to isotropy: 0, 5.5

[0437] Example 12: Method for producing engineered cysteine ​​(eCys) conjugated AOC .

[0438] dsRNA (siRNA) is conjugated using site-specific natural or engineered cysteine ​​amino acid residues from antibody molecules. Cysteine ​​residues can be engineered into the primary amino acid sequence of the TfR-binding protein (TBP) disclosed herein. Methods for introducing cysteine ​​as a conjugation method are described in WO 2018 / 232088, which is incorporated herein by reference in its entirety, including the specific details concerning conjugation via cysteine ​​residues. For engineered cysteine ​​conjugation, the TfR-binding protein is first reduced at room temperature for two hours with 10 molar equivalents of the reducing agent tris(2-carboxyethyl)phosphine (TCEP), followed by buffer exchange, for example, tangential flow filtration (TFF), to remove the reducing agent. For TBP2, the TfR-binding protein is subsequently incubated at room temperature for two hours with 20 molar equivalents of dehydroascorbic acid (DHAA) to re-oxidize the TfR-binding protein and reform interchain disulfide bonds. Buffer exchange is performed again to remove the oxidizing agent. For TBP3, since the scaffold does not contain interchain disulfide bonds, the re-oxidation step is omitted.

[0439] dsRNA was conjugated to TfR-binding proteins using SMCC-functionalized dsRNA. The prepared TBP was incubated with 0.9 to 1.0 molar equivalents of SMCC-dsRNA at room temperature for 1 to 2 hours to allow conjugation. Conjugation was monitored using analytical anion exchange chromatography (AEX). ProPac was used to monitor the conjugation. ™ SAX-10 HPLC column, 10µm particles, 4mm diameter, 50mm length (Thermo Scientific) ™ The following method was used: flow rate 1 mL / min, buffer A: 20 mM TRIS pH 7.0, buffer B: 20 ​​mM TRIS pH 7.0 + 1 M NaCl, temperature 30 °C. The mobile phase gradient is shown in Table 19. The drug antibody / protein ratio (DAR) was calculated based on the peak area % from the analytical anion exchange (aAEX) chromatogram.

[0440] Table 19: HPLC gradient used to determine dsRNA conjugation to TfR-binding protein

[0441]

[0442] Example 13: In vitro stability of antibody oligonucleotide conjugates (AOC) .

[0443] The stability of the native cysteine-conjugated divalent mAb (TBP1), engineered cysteine-conjugated divalent mAb (TBP2), and engineered cysteine-conjugated monovalent Fab (TBP3) DAR1 AOC was evaluated in vitro. The siRNA portion of the AOC is as follows... Figure 1B The modification shown is to include an amine linker.

[0444] Samples were prepared at a protein concentration of 1 mg / mL in phosphate-buffered saline (PBS) at pH 7.2 and then incubated at 4°C and 40°C for 2 weeks. The samples were analyzed by analytical size exclusion chromatography (SEC), AEX (as described in Example 12), and reduced and non-reduced CE-SDS. SEC was performed using an Acquity UPLC Protein BEH SEC 200 Å column with a particle size of 1.7 μM, a diameter of 4.6 mm, and a length of 150 mm (Waters). ™ The mobile phase consisted of 50 mM sodium phosphate, 0.3 M NaCl, and 0.005% sodium azide, at pH 6.8. Isocratic elution was performed at a flow rate of 0.3 mL / min for 8 minutes. Reduced CE-SDS was performed using Maurice (BioTechne) according to the manufacturer's protocol. Non-reduced CE-SDS was performed using Labchip. ® GXII (PerkinElmer) is manufactured according to the manufacturer's specifications.

[0445] The results are shown in Table 20. In all assays, the native cysteine-conjugated TBP1-410 AOC showed reduced stability compared to the engineered cysteine-conjugated TBP2-410 AOC. The monovalent Fab-engineered cysteine ​​AOC (TBP3-410 AOC) showed comparable stability compared to the divalent mAb-engineered cysteine ​​AOC.

[0446] AOC was analyzed by non-reducing and reducing SDS-PAGE to assess covalent assembly. Figure 12 Using NuPAGE ™ 4-12% Bis-Tris gel, LDS sample buffer, and MES SDS run buffer (ThermoFisher Scientific). Use Mark 12. ™ Molecular weight markers (MWM) were used as standards. Non-reduced samples were analyzed with and without iodoacetamide (IAM) alkylation to confirm that the apparent fragmentation under denaturing conditions was not the result of artificial reduction during SDS-PAGE sample preparation.

[0447] For TBP1-410 AOC, multiple fragments were observed with and without IAM alkylation, corresponding to light chain, heavy chain, half antibody, heavy chain dimer, heavy chain dimer + light chain, and intact antibody. These fragments were observed in TBP1-410, while in TBP2-410 AOC, mainly intact antibody was observed, with only a few fragment types. In reduction analysis, the samples were comparable, showing roughly equal band intensities for heavy chain and heavy chain + RNA, with very few or no light chain + RNA observed. Fragmentation observed in TBP1-410 AOC but not in TBP2-410 AOC is an expected result of the conjugation method and may contribute to the observed stability differences. In summary, these results indicate that engineered cysteine ​​conjugation methods provide more homogeneous and stable AOCs compared to native cysteine ​​conjugation methods.

[0448] Table 20: In vitro stability results show the change of the main peak at 40℃ relative to 4℃ after 2 weeks of incubation. .

[0449]

[0450] a Due to significant peak heterogeneity, non-reducing CE-SDS analysis of TBP1-410 was not possible.

[0451] b No percentage change in peak area was observed, but the main peak shifted due to maleimide ring hydrolysis.

[0452] Example 14: TfR1-Na V 1.8 In vitro evaluation of siRNA AOC in human DRG .

[0453] To confirm that the anti-transferrin antibody (anti-TfR1) of this disclosure can deliver the siRNA sequence to human DRG, an anti-TfR1 antibody (TBP1-si410 AOC) conjugated to siRNA 305 in Table 4 was synthesized and evaluated in an in vitro assay using primary human DRG. The siRNA sequence used in the AOC is siRNA 305 as shown in Table 4, and this siRNA is conjugated to the antibody via an SMCC linker, as shown below. Figure 7 As shown in Table 8, the antibody used in AOC is TBP1.

[0454] Using human dorsal root ganglion neurons (DRG) as the expression of Na V1.8 Primary cell source of mRNA. Fresh primary human DRG was purchased from AnaBios (San Diego, CA) and maintained in serum-free NbActive4 maintenance medium (Axol, Easter Brush, United Kingdom) containing 25 ng / mL recombinant human NGF (Axol). Primary DRG was treated twice with 100 nM AOC for 5 days.

[0455] Human DRG was treated and kept at 37°C and 5% CO2 for 5 days, then washed in phosphate-buffered saline (PBS, ThermoFisher) and stored in 300 µL TRIzol (ThermoFisher) at -80°C until RNA extraction. Total mRNA was isolated using a ZYMO 96-well RNA kit (Zymo Research, Irvine, CA), and cDNA was generated from 100 ng to 250 ng of purified mRNA using an iScript cDNA synthesis kit (BioRad, Hercules, CA).

[0456] RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using commercially available TaqMan probes (Life Technologies; SCN10A: Hs01045150_m1, STMN2: Hs00975900_m1, and SNAP25: Hs00938957_m1). V 1.8 The relative reduction of transcripts was determined by normalizing the expression of DRG after TBP1-si410 AOC treatment relative to the siNTC group using the DDCt method, using the following formula: [%hNa V 1.8 mRNA = 100 * 2 -DCt (si410处理细胞)-DCt (siNTC处理细胞)], where DCt is determined by the difference in Ct values ​​between SCN10A and housekeeping genes STMN2 and SNAP25 {avg DCt=((2*Ct(SCN10A))-Ct(STMN2)-Ct(SNAP25)) / 2} (Green MR, Sambrook J., “Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR),” Cold Spring Harb Protoc. Oct 2018, 2018(10): pdb.prot095042; and Livak KJ, Schmittgen TD, “Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method,”Methods, 25(4), 402–8 (2001)). Parametric unpaired t-tests were performed using the software tool GraphPad Prism (GraphPad Software, LLC).

[0457] like Figure 13 As demonstrated, when compared with the negative control cells (TFR1-siNTC AOC) in primary human DRG, the tested AOC significantly reduced Na... V 1.8 mRNA level (approximately 30%). In primary human DRG, it was demonstrated that AOC can deliver Na+. V 1.8 Specific siRNA. AOC treatment leads to receptor-mediated Na+... V 1.8 siRNA uptake, because human DRG expresses TfR1 on its surface. AOC uptake leads to siRNA-mediated Na+ uptake. V 1.8 mRNA knockdown.

[0458] Example 15: OX26-410 and OX26-535 tools AOC in humanized Na V 1.8 In vivo activity in rats .

[0459] A humanized Nav 1.8 rat was developed in which the rat SCN10A gene was knocked out and replaced with the human SCN10A gene to allow testing of oligonucleotide therapeutics that have predictive activity against human SCN10A transcripts only.

[0460] Test siRNAs 410 and 535 were conjugated with a commercial rat transferrin antibody (OX26 clone, BioXcell) using natural cysteine ​​conjugation and purified to a drug-to-antibody ratio of 1.0. Both test siRNAs exhibited the following properties: Figure 1B The modifications shown are not amine or cholesterol-containing linkers.

[0461] Table 21: Description of AOC

[0462]

[0463] Female rats approximately 4 weeks old received intravenous (IV) administration of either a solvent (PBS), OX26-siRNA410 (6 mg / kg oligonucleotide), or OX26-siRNA535 (6 mg / kg oligonucleotide). Thirteen days post-administration, the rats were tested three times in a cold plate system. Rats were first tested for 5 minutes at room temperature (23°C), followed by 10 minutes of rest in their cages. Then, half of the rats were tested for 4 minutes at 2°C, and the other half at -1°C. Approximately 2 hours later, the rats were tested at another temperature. No effect was observed from the testing order. Noxious behavior was defined as the time spent licking the hind paw or guarding the hind paw (raising the paw towards the body). This time was accumulated to form a noxious behavior score, which was reported on [date missing]. Figure 14 , Figure 15 and Figure 16 On the y-axis. Statistical analysis was performed using ANOVA via each temperature test using JMP version 15, and in the case of knockdown studies, for ANOVA with a p-value less than 0.05, a Dunnet post-hoc analysis was subsequently performed using a PBS control as a comparison. In these studies, an α level of 0.05 was used to determine statistical significance. After collecting behavioral data, rats were humanely euthanized, and samples of dorsal root ganglia and hairless skin were collected, and Na+ levels were measured. V 1.8 protein level.

[0464] Na in hairless skin V 1.8 The expression is entirely attributed to Na+ in sensory nerve endings. V 1.8, therefore, measurements of both DRG cell bodies and hairless skin can confirm the concentration of Na in neurons throughout the dorsal root ganglion. V 1.8 protein knockdown. To determine Na... V1.8 Protein concentration: Tissue lysates were prepared by suspending tissues in 200 µL of 1X lysis buffer (CST, catalog 9803) per sample, along with Halt protease and phosphatase inhibitors (TFS, catalog 78440) diluted to 2X, and benzyl sulfonyl fluoride (PMSF; Sigma, #78830-25G, 17.4 mg dissolved in 1 mL isopropanol, diluted to 1X), and two (2) 5 mm tissue homogenizer beads (QIAGEN). Tissue tubes were run in a tissue homogenizer (QIAGEN) at 30 1 / s for 3 minutes, followed by centrifugation at 5000 × g for 20 minutes at 4°C. The supernatant was collected and frozen at -80°C for analysis the following day. Pierce was used. ™ BCA Protein Assay Kit (Catalogue No. 23227), measure protein concentrations according to the “Microplate Procedure” described in the manufacturer’s manual. Before plating, further dilute the sample 1:3 with cell lysis buffer.

[0465] Na was detected using the Meso Scale Discovery (MSD) S-plex platform. V 1.8 Protein Levels. A custom-developed assay was performed using the S-PLEX Development Kit B, SECTOR (25 plates) (catalog number K15601S-4). Capture and detection antibodies were labeled with MSD. The capture antibody (NeuroMab, catalog number 75-166) was diluted to 0.5 µg / mL, and the detection antibody (EliLilly) was diluted to 0.2 µg / mL. The calibrator was an internally generated human Na+ molecule. V 1.8 recombinant C-terminal fragment. Sample lysate was diluted 4-fold in assay buffer. The assay buffer matrix was Diluent 39 (MSD, catalog number R5ABB2), with added MSD. ® Each component from the inhibitor package (catalog number R70AA-1) was added in 100 µL. The washing step used MSD Tris wash buffer (MSD, catalog number R61TX-1) diluted to 1X in double-distilled water (ddH2O). The plates were read using a MESO QuickPlex SQ 120 plate reader with MSD Methodical Mind software. Data were analyzed using Discovery Workbench v4. The Na+ of each lysate sample was measured. V 1.8 The protein concentration was divided by the total protein concentration to normalize the data. Statistical significance was determined by one-way ANOVA, p < 0.05.

[0466] Selective Na V1.8 The effects of small molecules on cold-press testing in early clinical trials have been reported (Hijma et al., "A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Crossover Study to Evaluate the Pharmacodynamic Effects of VX-150, a Highly Selective Na...). V 1.8 Inhibitor, in Healthy Male Adults, “Pain Medicine, 22 (8), pp. 1814-26, (2021)”. Intravenous administration of targeted Na V Similar effects were observed in rodent cold behavior following AOC administration of 1.8%. Administration of AOC OX26-siRNA410 and OX26-siRNA535 resulted in a reduction in cold-induced noxious behavior; however, only animals receiving OX26-siRNA410 showed a statistically significant effect (p<0.05, one-way ANOVA, Dunnet post-hoc test). This finding was consistent at both temperatures measured (2℃ and -1℃). Figure 14 Administration of OX26-siRNA410 and OX26-siRNA535 AOC also resulted in increased Na levels in DRG cell bodies and hairless claws in both treatment groups. V 1.8 Protein was significantly reduced (a later finding confirmed that Na+ in sensory nerve endings was significantly reduced). V 1.8 protein knockdown (p<0.05, one-way ANOVA).

[0467] In addition, these data indicate that Na V 1.8 The degree of protein knockdown was well correlated with the reversal of cold-induced behavior. These AOCs used the same OX26 delivery antibody, differing only in the conjugated siRNA. Compared to the AOC containing siRNA535 and the PBS control, the AOC containing siRNA410 showed superior in vivo efficacy, reflected in greater reversal of noxious cold behavior and a greater degree of Na+ reduction. V 1.8 protein knockdown.

[0468] Example 16: In vivo activity of TfR-410 siRNA AOC in non-human primates

[0469] The goal of this study was to determine the pharmacodynamic effects of intravenous bolus administration of the anti-TfR antibody-siRNA410 AOC (TBP1 anti-TfR antibody conjugated to 410 siRNA via an SMCC linker) in cynomolgus monkeys. Groups included PBS control, TBP1-410 nCys mAb, TBP2-410 eCys mAb, and TBP3-410 eCys Fab AOC, with N=4 in each group. The siRNA molecule is as follows: Figure 1B The product is modified as shown, but does not contain amine or cholesterol connecting stems.

[0470] Table 22: Description of AOC Testing

[0471]

[0472] Female cynomolgus macaques (aged 2.4 to 4.0 years, weighing 2.4 kg to 4.0 kg) received a single intravenous dose of PBS (control), or 1.0 mg / kg or 6.0 mg / kg (based on oligonucleotide weight) of one of the three tested AOCs. Additionally, a separate group received TBP1-siRNA410 nCys mAb twice on days 1 and 8 at a dose of 3.0 mg / kg (based on oligonucleotide weight). Humane euthanasia was performed on day 29, and hairless paw skin was collected for Na+ determination. V 1.8 protein levels. Na in hairless claw skin V 1.8 Protein Indicators of Protein Expression in Sensory Nerve Endings. NHP skin lysates were prepared by suspending tissue in 150 µL of 1X lysis buffer (CST, catalog 9803) and Halt protease and phosphatase inhibitors (TFS, catalog 78440) diluted to 2X, and PMSF (Sigma, #78830-25G, 17.4 mg dissolved in 1 mL isopropanol, diluted to 1X) and two (2) 5 mm tissue homogenizer beads (QIAGEN). Tissue tubes were run in a tissue homogenizer (QIAGEN) at 30 1 / s for 3 min, followed by centrifugation at 5000 × g for 20 min at 4 °C. The supernatant was collected and frozen at -80 °C for analysis the next day. Pierce was used. ™ BCA Protein Assay Kit (catalog number 23227) was used to measure protein concentrations according to the “Microplate Procedure” described in the manufacturer’s manual. Samples were further diluted 1:3 with cell lysis buffer before plating. Na+ in cultured human DRG neuron protein lysates was detected using the MesoScale Discovery (MSD) S-Plex platform. V1.8 Protein Levels. The custom-developed assay was performed using the S-PLEX Development Kit B, SECTOR (25 plates) (catalog number K15601S-4). Capture and detection antibodies were labeled with MSD. The capture antibody (NeuroMab, catalog number 75-166) was diluted to 0.5 µg / mL, and the detection antibody was diluted to 0.2 µg / mL. The calibrator was human sodium. V 1.8 recombinant C-terminal fragment. Sample lysate was diluted 4-fold in assay buffer. The assay buffer matrix was Diluent 39 (MSD, catalog number R5ABB2), with added MSD. ® Each component from the inhibitor package (catalog number R70AA-1) was added in 100 µL. The washing step used MSD Tris wash buffer (MSD, catalog number R61TX-1) diluted to 1X in ddH2O. The plates were read using an MSD plate reader (MESOQuickPlex SQ 120) with MSD Methodical Mind software. Data were analyzed using Discovery Workbench v4. The Na+ of each lysate sample was measured. V 1.8 The data were normalized by dividing the protein concentration by the total protein concentration. Statistical significance was determined using one-way ANOVA.

[0473] When compared with the PBS control, all groups that received a single dose of the tested AOC showed Na+. V 1.8 Protein levels remained consistent but moderately decreased. Figure 17 However, none of these reductions reached statistical significance. Nevertheless, the treatment group receiving two doses of 3.0 mg / kg (by oligonucleotide weight) TBP1-siRNA410 nCys mAb showed Na... V A significant decrease in 1.8 protein was observed (47.3% of the PBS control group, p < 0.05, one-way ANOVA). Although the effect of cold-induced behavior was not tested in these animals, it has been previously observed in humanized Na+... V Tests in rats 1.8 showed that this level of Na in the skin V 1.8 protein knockdown is expected to produce significant effects, and this indicates that it is related to Na+. V Physiologically relevant decreases in 1.8 protein.

[0474] Example 17: Dose-response study of TBP2-410 eCys in non-human primates .

[0475] The objective of this study was to determine the pharmacodynamic effects of intravenous bolus administration of TBP2-410 eCys mAb DAR1 (hereinafter referred to as TBP2-410 eCys) in cynomolgus monkeys.

[0476] Table 23: Description of AOC

[0477]

[0478] Female cynomolgus macaques received four weekly intravenous doses of either PBS control or three dose groups of TBP2-410 eCys at doses of 0.3 mg / kg, 3.0 mg / kg, and 30.0 mg / kg (based on oligonucleotide weight). The PBS control and two low-dose groups had N=4 per group, while the high-dose groups had N=2 per group. Humane euthanasia was performed on day 49 (27 days after the last administration), and hairless paw skin was collected for Na+ determination. V 1.8 protein levels. Na in hairless claw skin V 1.8 Protein indicators of protein expression in sensory nerve endings.

[0479] NHP skin lysates were prepared by suspending tissue in 150 µl of 1X lysis buffer (CST, catalog 9803) diluted to 2X with Halt protease and phosphatase inhibitors (TFS, catalog 78440) and PMSF (Sigma, #78830-25G, 17.4 mg dissolved in 1 mL isopropanol, diluted to 1X) and two (2) 5 mm tissue homogenizer beads (QIAGEN). Tissue tubes were run in the QIAGEN at 30 1 / s for 3 min, then centrifuged at 5000 × g for 20 min at 4 °C. The supernatant was collected and frozen at -80 °C for analysis the following day. Pierce was used. ™ BCA Protein Assay Kit (Catalogue No. 23227) measures protein concentration according to the “Microplate Procedure” described in the manufacturer’s manual.

[0480] Before plating, the samples were further diluted 1:3 with cell lysis buffer. Na+ in cultured human DRG neuron protein lysates was detected using the Meso Scale Discovery (MSD) S-Plex platform. V 1.8 Protein Levels. Custom-developed assays were performed using the S-PLEX Development Kit B, SECTOR (25 plates) (catalog number K15601S-4). Capture and detection antibodies were labeled with MSD. The capture antibody (NeuroMab, catalog number 75-166) was diluted to 0.5 µg / mL, and the detection antibody (6B2, field-manufactured by Lilly Research Laboratories) was diluted to 0.2 µg / mL. Calibrators were human Na+ produced by Lilly Research Laboratories. V1.8 recombinant C-terminal fragment. Sample lysate was diluted 4-fold in assay buffer. The assay buffer matrix was Diluent 39 (MSD, catalog number R5ABB2), with added MSD. ® Each component from the inhibitor package (catalog number R70AA-1) was added in 100 µL. The washing step used MSD Tris wash buffer (MSD, catalog number R61TX-1) diluted to 1X in ddH2O. The plates were read using an MSD plate reader (MESOQuickPlex SQ 120) with MSD Methodical Mind software. Data were analyzed using Discovery Workbench v4. The Na+ of each lysate sample was measured. V 1.8 The data were normalized by dividing the protein concentration by the total protein concentration. Statistical significance was determined using one-way ANOVA.

[0481] like Figure 18 As shown, administration of medium and high doses of TBP2-410 eCys (3.0 mg / kg and 30.0 mg / kg, based on oligonucleotide weight) resulted in increased Na+ levels in the skin of hairless claws. V 1.8 protein was significantly reduced (52.3% and 42.2% of the PBS control group, respectively; *p<0.05, one-way ANOVA). Although the effect of cold-induced behavior was not tested in these animals, previous studies in humanized Na+... V Tests in rats 1.8 showed that this level of Na in the skin V 1.8 protein knockdown is expected to produce significant effects, and this indicates that it is related to Na+. V Physiologically relevant decreases in 1.8 protein.

[0482] sequence

[0483] The following nucleic acid and / or amino acid sequences of the antibodies disclosed herein are provided for reference.

[0484] TBP1;211 Com29 B09 hIgG1-EN mAb:

[0485] Heavy chain (HC) (SEQ ID NO: 625)

[0486] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0487] Light chain (LC) (SEQ ID NO: 626)

[0488] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0489] Heavy chain variable region (HCVR) (SEQ ID NO: 627)

[0490] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSS

[0491] Light chain variable region (LCVR) (SEQ ID NO: 628)

[0492] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK

[0493] HCDR1 (SEQ ID NO: 629)

[0494] SYSMN

[0495] HCDR2 (SEQ ID NO: 630)

[0496] SISSSSSYIYYADSVKG

[0497] HCDR3 (SEQ ID NO: 631)

[0498] RHGYSNSDAFDT

[0499] LCDR1 (SEQ ID NO: 632)

[0500] RASQGISHYLV

[0501] LCDR2 (SEQ ID NO: 633)

[0502] AASSLQS

[0503] LCDR3 (SEQ ID NO: 634)

[0504] LQHNSYPWT

[0505] TBP2; 211 Com29 B09 hIgG1-AAS heterologous monoclonal antibody 124C (for eCys DAR1 conjugation):

[0506] Heavy chain A (HCA) (SEQ ID NO: 635)

[0507] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPRVYTLPPSREEMTKNQVSLVCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0508] Heavy chain B (HCB) (SEQ ID NO: 636)<00015^45>[

[0509] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTDNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLMSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0510] Light chain (LC) (SEQ ID NO: 626)

[0511] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0512] HCVR (SEQ ID NO: 627)

[0513] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSS

[0514] LCVR (SEQ ID NO: 628)

[0515] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK

[0516] HCDR1 (SEQ ID NO: 629)

[0517] SYSMN

[0518] HCDR2 (SEQ ID NO: 630)

[0519] SISSSSSYIYYADSVKG

[0520] HCDR3 (SEQ ID NO: 631)

[0521] RHGYSNSDAFDT

[0522] LCDR (SEQ ID NO: 632)

[0523] RASQGISHYLV

[0524] LCDR2 (SEQ ID NO: 633)

[0525] AASSLQS

[0526] LCDR3 (SEQ ID NO: 634)

[0527] LQHNSYPWT

[0528] TBP3; 211 Com29 B09 hIgG1 124C Fab:

[0529] Heavy chain (HC) (SEQ ID NO: 637)

[0530] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0531] Light chain (LC) (SEQ ID NO: 638)

[0532] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

[0533] HCVR (SEQ ID NO: 627)

[0534] EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDTWGQGTLVTVSS

[0535] LCVR (SEQ ID NO: 628)

[0536] DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIK

[0537] [[ID=,7]]HCDR1 (SEQ ID NO: 629)

[0538] SYSMN

[0539] HCDR2 (SEQ ID NO: 630)

[0540] SISSSSSYIYYADSVKG

[0541] HCDR3 (SEQ ID NO: 631)

[0542] RHGYSNSDAFDT

[0543] LCDR1 (SEQ ID NO: 632)

[0544] RASQGISHYLV

[0545] LCDR2 (SEQ ID NO: 633)

[0546] AASSLQS

[0547] LCDR3 (SEQ ID NO: 634)

[0548] LQHNSYPWT

[0549] TBP1; Heavy chain (SEQ ID NO: 639)

[0550]

[0551] TBP2; Heavy chain A (SEQ ID NO: 640)

[0552]

[0553] TBP3; heavy chain (SEQ ID NO: 641)

[0554] GAAGTGCAGCTGGTCGAGTCAGGAGGCGGCCTCGTGAAACCGGGTGGTAGCCTGAGGCTGTCCTGCGTGGCATCAGGGTTCACCTTCTCGTCCTACTCCATGAACTGGGTCAGACAGGCCCCCGGAAAGGGACTTGAATGGGTGTCCAGCATCAGCAGCTCCTCCTCGTACATCTACTACGCCGATTCCGTGAAGGGCCGGTTCACCATTAGCCGCGACAATGCCAAGAACTCGCTGTATTTGCAAATGAACTCTCTGCGCGCCGAGGACACTGCTGTGTACTACTGTGCGCGGAGACACGGGTACTCCAACTCCGATGCCTTTGACACCTGGGGCCAGGGAACTCTCGTGACCGTGTCGTCCGCCAGCACCAAGGGCCCATGCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCACTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT

[0555] TBP2; heavy chain B (SEQ ID NO: 642)

[0556]

[0557] TBP1; light chain (SEQ ID NO: 643)

[0558] GACATCCAGATGACCCAGTCTCCATCTGCCATGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCCACTATTTAGTGTGGTTTCAGCAGAAACCAGGGAAAGTCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0559] TBP2; light chain (SEQ ID NO: 644)

[0560] GACATCCAGATGACCCAGTCTCCATCTGCCATGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCCACTATTTAGTGTGGTTTCAGCAGAAACCAGGGAAAGTCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

[0561] TBP3; light chain (SEQ ID NO: 645)

[0562] GACATCCAGATGACCCAGTCTCCATCTGCCATGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCCACTATTTAGTGTGGTTTCAGCAGAAACCAGGGAAAGTCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG

[0563] siNTC (SEQ ID NO: 646)

[0564] CUAGCUGGACACGUCGAUA

[0565] siNTC (SEQ ID NO: 647)

[0566] UAUCGACGUGUCCAGCUAGUU

[0567] Homo sapiens sodium voltage-gated channel alpha subunit 10 (SCN10A), transcript variant 1, mRNA (SEQ ID NO: 648)

[0568]

Claims

1. A method for inhibiting human sodium in cells V 1.8 Oligonucleotides expressed by the gene (SCN10A), said oligonucleotides comprising single-stranded or double-stranded oligonucleotides, said single-stranded or double-stranded oligonucleotides containing Na V 1.8 Regions complementary to mRNA.

2. The oligonucleotide of claim 1, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and the antisense strand comprises a Na+ region. V 1.8 A complementary region of mRNA, wherein the length of the complementary region is less than 30 nucleotides.

3. The oligonucleotide according to claim 2, wherein the antisense strand is conjugated with Na. V 1.8 The length of the complementary region of the mRNA target sequence is at least 15 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides, or at least 21 consecutive nucleotides.

4. The oligonucleotide of claim 2, wherein the length of the sense strand is 15 to 30 nucleotides, 18 to 25 nucleotides, or 19 nucleotides.

5. The oligonucleotide of claim 2, wherein the antisense strand is 15 to 30 nucleotides, 18 to 25 nucleotides, 19 nucleotides, or 21 nucleotides in length.

6. The oligonucleotide of claim 2, wherein the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

7. The oligonucleotide of claim 2, wherein the antisense strand comprises a region complementary to the target sequence of any one of SEQ ID NO: 1 to 141.

8. The oligonucleotide of claim 2, wherein the length of the complementary region is at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides.

9. The oligonucleotide according to any one of claims 2 to 8, wherein the antisense strand comprises a 3' overhang sequence of one or more nucleotides in length.

10. The oligonucleotide of claim 9, wherein the 3' overhang sequence is 2 nucleotides in length, and optionally wherein the 3' overhang sequence is UU.

11. The oligonucleotide according to any one of claims 2 to 10, wherein the oligonucleotide comprises at least one modified nucleotide.

12. The oligonucleotide of claim 11, wherein all nucleotides of the oligonucleotide are modified.

13. The oligonucleotide of claim 11 or 12, wherein the modified nucleotide comprises a 2'-modification on the sugar.

14. The oligonucleotide of claim 13, wherein the 2'-modification is 2'-fluorine, 2'-O-methyl, or 2'-O-methoxyethyl.

15. The oligonucleotide of claim 12, wherein the modification is 2'-fluoro, vinylphosphonic uridine (VpUq), or 2'-O-methyl.

16. The oligonucleotide according to any one of claims 11 to 15, wherein the 5' end of the antisense strand comprises at least one vinylphosphonic uridine (VpUq).

17. The oligonucleotide according to any one of claims 11 to 16, wherein one or more nucleotides at position 7, 8 or 9 of the sense strand are modified with 2'-fluorine.

18. The oligonucleotide according to any one of claims 11 to 17, wherein one or more nucleotides at positions 2, 6, 14 or 16 of the antisense strand are modified with 2'-fluorine.

19. The oligonucleotide according to any one of claims 11 to 18, wherein one or more nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the sense strand are modified with 2'-O-methyl.

20. The oligonucleotide according to any one of claims 11 to 19, wherein one or more nucleotides at positions 1, 3, 4, 5, 7 to 13, 15, 17, 18, 19, 20 or 21 of the antisense strand are modified with 2'-O-methyl.

21. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleotide linker.

22. The oligonucleotide of claim 21, wherein at least one modified internucleotide linking bond is a phosphate thioester linking bond or a phosphate dithioester linking bond.

23. The oligonucleotide of claim 22, wherein at least two terminal nucleotides at the 5' or 3' end of the antisense strand or the sense strand have phosphate thioester linkages or phosphate dithioester linkages.

24. The oligonucleotide of claim 22, wherein at least three terminal nucleotides at the 5' or 3' end of the antisense strand or the sense strand have phosphate thioester linkages or phosphate dithioester linkages.

25. The oligonucleotide according to any one of claims 21 to 24, wherein the sense strand has 19 nucleotides, and the internucleotide linkages between the nucleotides at positions 1 and 2, 2 and 3, 17 and 18, or 18 and 19 of the sense strand are modified with the phosphate thioester linkage.

26. The oligonucleotide according to any one of claims 21 to 25, wherein the antisense strand has 21 nucleotides, and the internucleotide linking bond of one or more nucleotides between positions 1 and 2, between positions 2 and 3, between positions 19 and 20, or between positions 20 and 21 of the antisense strand is modified with the phosphate thioester linking bond.

27. The oligonucleotide according to any one of claims 2 to 26, wherein the linker portion is attached to one or more ends of the sense strand or the antisense strand.

28. The oligonucleotide of claim 26, wherein the linker is SMCC, optionally attached to the 5' end of the sense strand.

29. The oligonucleotide according to any one of claims 2 to 28, wherein the oligonucleotide has the modification pattern shown in FIG1A, and optionally does not contain an amine or cholesterol linker.

30. The oligonucleotide according to any one of claims 2 to 28, wherein the oligonucleotide has the modification pattern shown in FIG1B, and optionally does not contain an amine or cholesterol linker.

31. The oligonucleotide according to any one of the preceding claims, wherein at least one nucleotide of the oligonucleotide is conjugated to cholesterol, lipid, polypeptide, or antibody or antigen-binding fragment thereof.

32. The oligonucleotide according to any one of claims 2 to 30, wherein the sense strand has a sequence numbered by any even number from SEQ ID NO: 1 to 422.

33. The oligonucleotide according to any one of claims 2 to 30, wherein the antisense strand has i) a sequence with an odd number from SEQ ID NO: 143 to 423, or ii) a sequence from SEQ ID NO: 424 to 564.

34. The oligonucleotide according to any one of claims 2 to 30, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 565 and 566, respectively; (b) are SEQ ID NO: 567 and 568, respectively; (c) are SEQ ID NO: 569 and 570, respectively; (d) are SEQ ID NO: 571 and 572, respectively; (e) are SEQ ID NO: 573 and 574, respectively; (f) are SEQ ID NO: 575 and 576, respectively; (g) are SEQ ID NO: 577 and 578, respectively; (h) are SEQ ID NO: 579 and 580, respectively; (i) are SEQ ID NO: 581 and 582, respectively; (j) are SEQ ID NO: 583 and 584, respectively; (k) are SEQ ID NO: 585 and 586, respectively; (l) are SEQ ID NO: 587 and 588, respectively; (m) are SEQ ID NO: 589 and 590, respectively; (n) are SEQ ID NO: 591 and 592, respectively; (o) are SEQ ID NO: 593 and 594, respectively; (p) are SEQ ID NO: 595 and 596, respectively; (q) are SEQ ID NO: 597 and 598, respectively; (r) are SEQ ID NO: 599 and 600, respectively; (s) are SEQ ID NO: 601 and 602, respectively; and (t) are SEQ ID NO: 603 and 604, respectively.

35. The oligonucleotide according to any one of claims 2 to 30, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 565 and 605, respectively; (b) are SEQ ID NO: 567 and 606, respectively; (c) are SEQ ID NO: 569 and 607 respectively; (d) are SEQ ID NO: 571 and 608, respectively; (e) are SEQ ID NO: 573 and 609, respectively; (f) are SEQ ID NO: 575 and 610, respectively; (g) are SEQ ID NO: 577 and 611, respectively; (h) are SEQ ID NO: 579 and 612, respectively; (i) are SEQ ID NO: 581 and 613, respectively; (j) are SEQ ID NO: 583 and 614, respectively; (k) are SEQ ID NO: 585 and 615, respectively; (l) are SEQ ID NO: 587 and 616, respectively; (m) are SEQ ID NO: 589 and 617 respectively; (n) are SEQ ID NO: 591 and 618, respectively; (o) are SEQ ID NO: 593 and 619 respectively; (p) are SEQ ID NO: 595 and 620, respectively; (q) are SEQ ID NO: 597 and 621, respectively; (r) are SEQ ID NO: 599 and 622, respectively; (s) are SEQ ID NO: 601 and 623 respectively; and (t) are SEQ ID NO: 603 and 624, respectively.

36. The oligonucleotide according to any one of claims 2 to 30, wherein i) The sense chain has a sequence as shown in Table 2, and the antisense chain has a sequence as shown in Table 2; ii) The sense chain has the sequence shown in Table 3, and the antisense chain has the sequence shown in Table 3; iii) The oligonucleotide has a sequence shown by any of the siRNAs numbered 1 to 141; iv) The oligonucleotide has a sequence shown by any of siRNA numbers 142 to 282; or v) The oligonucleotide has a sequence shown by any of the siRNA numbers 283 to 322.

37. The oligonucleotide of claim 1, wherein the single-stranded oligonucleotide comprises an antisense strand, the antisense strand comprising Na V 1.8 A complementary region of mRNA, wherein the length of the complementary region is less than 30 nucleotides.

38. The oligonucleotide of claim 37, wherein the antisense strand is 15 to 30 nucleotides, 18 to 25 nucleotides, 19 nucleotides, or 21 nucleotides in length.

39. The oligonucleotide of claim 37, wherein the antisense strand comprises a region complementary to the target sequence of any one of SEQ ID NO: 1 to 141.

40. The oligonucleotide of claim 37, wherein the length of the complementary region is at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides.

41. The oligonucleotide according to any one of claims 37 to 40, wherein the antisense strand comprises a 3' sequence of one or more nucleotides in length.

42. The oligonucleotide according to any one of claims 37 to 41, wherein the oligonucleotide comprises at least one modified nucleotide.

43. The oligonucleotide of claim 42, wherein all nucleotides of the oligonucleotide are modified.

44. The oligonucleotide of claim 42 or 43, wherein the modified nucleotide comprises a 2'-modification on the sugar.

45. The oligonucleotide of claim 44, wherein the 2'-modification is 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl.

46. ​​The oligonucleotide of claim 45, wherein the modification is 2'-fluoro, vinylphosphonic uridine (VpUq), or 2'-O-methyl.

47. The oligonucleotide according to any one of claims 41 to 46, wherein the 5' end of the antisense strand comprises at least one vinylphosphonic uridine (VpUq).

48. The oligonucleotide according to any one of claims 42 to 47, wherein one or more nucleotides at positions 1 to 21 of the antisense strand are modified with 2'-fluorine.

49. The oligonucleotide according to any one of claims 42 to 48, wherein one or more nucleotides at positions 1 to 21 of the antisense strand are modified with a 2'-O-methyl group.

50. The oligonucleotide according to any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleotide linker.

51. The oligonucleotide of claim 50, wherein the at least one modified internucleotide linker is a phosphate thioester linker or a phosphate dithioester linker.

52. The oligonucleotide of claim 51, wherein at least two terminal nucleotides at the 5' or 3' end of the antisense strand have phosphate thioester linkages or phosphate dithioester linkages.

53. The oligonucleotide of claim 51, wherein at least three terminal nucleotides at the 5' or 3' end of the antisense strand have phosphate thioester linkages or phosphate dithioester linkages.

54. The oligonucleotide according to any one of claims 2 to 53, wherein the linker portion is attached to one or more ends of the antisense strand.

55. The oligonucleotide of claim 54, wherein the linker is SMCC.

56. The oligonucleotide according to any one of the preceding claims, wherein at least one nucleotide of the oligonucleotide is conjugated to cholesterol, lipid, polypeptide, or antibody or antigen-binding fragment thereof.

57. The oligonucleotide according to any one of claims 37 to 56, wherein the antisense strand has i) a sequence with an odd number from SEQ ID NO: 143 to 423, or ii) a sequence from SEQ ID NO: 424 to 564.

58. The oligonucleotide according to any one of claims 37 to 56, wherein (i) the antisense strand has a sequence as shown in Table 2, or (ii) the antisense strand has a sequence as shown in Table 3.

59. A cell comprising an oligonucleotide according to any one of claims 1 to 58.

60. A vector comprising a nucleotide sequence encoding an oligonucleotide according to any one of claims 1 to 58.

61. The vector of claim 60, wherein the nucleotide sequence encoding the oligonucleotide is operatively linked to a regulatory sequence.

62. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 58 and comprising a pharmaceutically acceptable carrier.

63. A conjugate comprising an oligonucleotide according to any one of claims 1 to 58 and an anti-transferrin receptor antibody or an antigen-binding fragment thereof conjugated to the sense strand or the antisense strand of the oligonucleotide.

64. The conjugate of claim 63, wherein the antibody or its antigen-binding fragment is connected to the sense strand or the antisense strand via a adapter.

65. The conjugate according to claim 64, wherein the connector is an SMCC connector.

66. The conjugate according to claim 65, wherein the connector is an SMCC connector connected to: i) the 5' end of the sense chain, ii) the 5' end of the antisense chain, iii) the 3' end of the sense chain, or iv) the 3' end of the antisense chain.

67. The conjugate according to any one of claims 63 to 66, wherein the oligonucleotide preferentially mediates targeting of human Na+ in the DRG cells of a human subject. V 1.8 RNA interference with mRNA.

68. The conjugate of claim 67, wherein the conjugate is effective against the human Na V 1.8 RNA interference of mRNA mediates pain in subjects.

69. The conjugate according to claim 67, wherein the conjugate is effective against the human Na V 1.8 RNA interference mediated by mRNA reduced pain in subjects.

70. The conjugate according to any one of claims 63 to 67, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the LCVR comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following sequences: HCDR1 comprises SEQ ID NO: 629, HCDR2 comprises SEQ ID NO: 630, HCDR3 comprises SEQ ID NO: 631, LCDR1 comprises SEQ ID NO: 632, LCDR2 comprises SEQ ID NO: 633, and LCDR3 comprises SEQ ID NO:

634.

71. The conjugate according to any one of claims 63 to 67, wherein the antibody or antigen-binding fragment thereof comprises HCVR of SEQ ID NO: 627 and LCVR of SEQ ID NO:

628.

72. The conjugate according to any one of claims 63 to 67, wherein the antibody comprises a heavy chain (HC) and a light chain (LC) of the following sequence: (a) HC of SEQ ID NO: 625 and LC of SEQ ID NO: 626; (b) HCA (first heavy chain) of SEQ ID NO: 635, HCB (second heavy chain) of SEQ ID NO: 636, and LC of SEQ ID NO: 626; or (c) HC of SEQ ID NO: 637 and LC of SEQ ID NO:

638.

73. The conjugate according to any one of claims 63 to 72, wherein the antibody fragment is Fab, scFv, Fv, or scFab.

74. The conjugate according to any one of claims 63 to 73, wherein the antibody or its antigen-binding fragment comprises at least one heavy chain constant region containing a cysteine ​​residue at residue 124 (according to EU index number).

75. The conjugate according to any one of claims 63 to 73, wherein the antibody or its antigen-binding fragment comprises two heavy chain constant regions containing cysteine ​​residues at residue 124 (according to EU index number).

76. The conjugate according to any one of claims 63 to 73, wherein the antibody or its antigen-binding fragment further comprises a half-life extender.

77. The conjugate according to claim 76, wherein the half-life extender is a VHH bound to human serum albumin (HSA).

78. A pharmaceutical composition comprising a conjugate according to any one of claims 63 to 77 and comprising a pharmaceutically acceptable carrier.

79. A method for preventing, treating, or alleviating pain in a patient in need, the method comprising administering to the patient an effective amount of an oligonucleotide according to any one of claims 1 to 58; an effective amount of a conjugate according to any one of claims 63 to 77; a pharmaceutical composition according to claim 62; or a pharmaceutical composition according to claim 78.

80. The method of claim 79, wherein the pain is chronic pain.

81. The method of claim 79, wherein the pain is: i) inflammatory pain, ii) neuropathic pain, or iii) mixed pain.

82. The method according to any one of claims 79 to 81, wherein the oligonucleotide, the conjugate, or the pharmaceutical composition is administered intravenously.

83. The method according to any one of claims 79 to 81, wherein the oligonucleotide, the conjugate, or the pharmaceutical composition is administered subcutaneously.

84. The oligonucleotide according to any one of claims 1 to 58; the conjugate according to any one of claims 63 to 77; the pharmaceutical composition according to claim 62; or the use of the pharmaceutical composition according to claim 78 in the preparation of a medicament for the prevention, treatment or relief of pain.

85. The use according to claim 84, wherein the pain is chronic pain.

86. The use according to claim 84, wherein the pain is: i) inflammatory pain, ii) neuropathic pain, or iii) mixed pain.

87. The use according to any one of claims 84 to 86, wherein the oligonucleotide, the conjugate, or the pharmaceutical composition is administered intravenously.

88. The oligonucleotide according to any one of claims 1 to 58; the conjugate according to any one of claims 63 to 77; the pharmaceutical composition according to claim 62; or the pharmaceutical composition according to claim 78, for use in the prevention, treatment or relief of pain in patients in need.

89. The oligonucleotide, conjugate, or pharmaceutical composition according to claim 88, wherein the pain is chronic pain.

90. The oligonucleotide, conjugate, or pharmaceutical composition according to claim 89, wherein the pain is: i) inflammatory pain, ii) neuropathic pain, or iii) mixed pain.

91. The oligonucleotide, conjugate, or pharmaceutical composition according to any one of claims 88 to 90, wherein the conjugate is administered intravenously.

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