Antisense oligonucleotides for the treatment of chronic pain

Abstract

The disclosure relates to the field of chronic pain and the treatment thereof. The disclosure involves antisense oligonucleotides and the use thereof in pre-mRNA and mRNA modulation, in one aspect related to RNA editing using endogenous ADAR enzymes, in targeting an adenosine in a (pre-) mRNA for human Nav1.7, preferably to change from a lysine residue to an arginine residue in the protein at position 1406 (K1406R), thereby impairing the ability of the Nav1.7 protein to act as a sodium ion channel. In another aspect the disclosure relates to exon skipping wherein antisense oligonucleotides are used to induce skipping of exon 23 in the generation of human SCN9A mRNA thereby generating a shortened Nav1.7 protein with an impaired ability to act as a sodium ion channel. The disclosure relates to methods and means to alter the Nav1.7 protein in cells of the peripheral nervous system, preferably large nociceptor cells (type Aα / Aβ), to provide a treatment of chronic pain.

Description

ANTISENSE OLIGONUCLEOTIDES FOR THE TREATMENT OF CHRONIC PAIN CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This PCT application claims the priority benefit of U.S. Provisional Applications 63 / 706,367, filed on October 11, 2024, and 63 / 841,889, filed on July 10, 2025, which are herein incorporated by reference in their entireties. TECHNICAL FIELD

[0002] This disclosure relates to the field of medicine, to the field of neurological disorders, and more in particular to the field of chronic pain. The disclosure describes antisense oligonucleotides that cause the modulation of the human Sodium voltage-gated Channel Alpha subunit 9 (SCN9A) gene transcript such that it influences the sodium ion channel activity of the encoded protein, Nav1.7, thereby providing a treatment for certain chronic pain disorders. BACKGROUND

[0003] Pain is an unpleasant sensory and emotional experience associated or resembling to be associated with actual or potential tissue damage. Pain is important from a survival point-of- view of an organism; it teaches caution in dangerous situations and helps with acting carefully to promote wound healing. However, when the sensation shifts from acute pain to chronic pain then pain may be classed as a disease. Indeed, the major reason why individuals seek medical care is due to pain.

[0004] Chronic pain is a debilitating condition that affects up to 30% of individuals worldwide. Unlike acute pain, chronic pain has no evolutionary benefit. Pain is defined as chronic if it persists for more than three months after onset. Moreover, chronic pain can be classified as follows: i) nociceptive (i.e., caused by injury to tissue, e.g., by burns, a bone fracture, etc.); ii) neuropathic (i.e., caused by injury to nerves themselves, e.g., by a spinal cord injury); or iii) nociplastic (i.e., caused by dysregulation of the nervous system with no obvious sign of trauma or nerve damage, e.g., fibromyalgia). Nociceptive pain is the most common form of chronic pain, while neuropathic pain accounts for 15-20% of chronic pain. Chronic pain has a negative impacton quality of life, causing depression, anxiety, and isolation among other adverse social conditions, due to conflation of multiple biological, social, and psychological factors. It has been suggested that currently chronic pain costs up to $635 billion per year, including direct healthcare expenses and lost days of work. Current treatment of chronic pain is complicated by the long-term nature of the disorder and expectations of the individuals of their pain management because there is no “quick fix” and there are limitations to current pain medications.

[0005] In humans, pain is perceived in the cortex of the brain, but of course, painful incidents often occur on the skin or in deeper tissues, such as bone and / or muscle. The detection and propagation of intense painful stimuli is signalled from the source to the cortex via the spinal cord through action potentials propagated by specialized peripheral afferent neurons called “nociceptors”. Nociceptors are polymodal in the sense that they respond to any painful stimulus including thermal, chemical, or mechanical stimuli by constantly surveilling the environment that is local to their axonal terminals. A key feature of nociception is ‘sensitization’, which is the increasing response to stimuli instead of ‘adaptation’. Sensitization of nociceptors often leads to chronic pain, as injury results in maladaptation plasticity of nociceptors.

[0006] Nociceptors have a high degree of functional specificity, which is controlled by their highly heterogenous nature. Nociceptors are most often classified according to their size: large (myelinated Aα / Aβ), intermediate (Aδ), and small (unmyelinated C). This in general corresponds to their stimulus specificity. To facilitate pain signalling, nociceptors express a myriad of different receptors at their cell surface, such as G protein-coupled receptors and ion channels. Amongst these, Voltage-Gated Sodium Channels (VGSC), also known as Nav-channels, are highly expressed in nociceptors and are responsible for the initiation and propagation of action potentials towards the brain. An individual channel comprising a pore-forming α-subunit plus one or two smaller β-subunits allows the conduction of sodium ions across the membrane to generate action potentials. The α-subunit is made up of four domains each composed of six transmembrane regions, four of which act as voltage sensor while the remaining two form the pore. Nav channels specifically conduct sodium ions via the presence of a selectivity filter made up of a locus of four highly conserved single amino acids from each domain, the so-called “DEKA” locus, in which D is for aspartic acid, E is for glutamic acid, K is for lysine, and A is for alanine. Each amino acid from this locus is important for sodium selectivity; the aspartic acid from the first domain and glutamic acid from the second domain electrically coordinate sodium ion conductance to the channel, while the lysine from the third domain narrows the channel, while providing energetically favourable conditions for sodium conductance.

[0007] The Nav-channel α-subunit family consists of nine highly conserved transmembrane channels (Nav1.1 to Nav1.9) with tissue specific expressions. In human adults, Nav1.2 and 1.3 are mainly expressed in the Central Nervous System (CNS), Nav1.4 is expressed in skeletal muscles, and Nav1.5 is expressed in cardiomyocytes. Nav1.7, Nav1.8, and Nav1.9 are generally expressed in the Peripheral Nervous System (PNS), while Nav1.1 and Nav1.6 are expressed in both CNS and PNS. In the PNS, the expression pattern of Nav channels is dependent on the signalling profile; that is, large nociceptors (type Aα / Aβ, see above) express Nav1.1, Nav1.6 and Nav1.7 to amplify rising phase of action potential formation, while small nociceptors express Nav1.8 and Nav1.9 as major contributors to the rising phase.

[0008] As the main sodium channel found in the PNS, Nav1.7 is expressed in vagal sensory neurons and Dorsal Root Ganglion (DRG) sensory neurons. Interestingly, Nav1.7 is also expressed in olfactory sensory neurons in the CNS. Nav1.7 has been shown to have a role in pain signalling through multiple genetic and behavioural studies, whereby loss of Nav1.7 results in deficiencies in pain perception. Overall, loss of Nav1.7 expression causes failure of action potential initiation and propagation of signal to second order spinal cord neurons, changes in pre-synapse neurotransmitter release, and upregulation of endogenous opioid signalling. Furthermore, Nav1.7 is heavily implicated in pain disorders, both through loss-of-function and gain-of-function mutations. Congenital Insensitivity to Pain (CIP) is a disorder resulting in individuals being unable to feel pain, regardless of stimulus. Often, this leads to multiple small injuries combining to cause major debility in individuals suffering from CIP. Numerous loss-of-function mutations in Nav1.7 cause CIP, although the complete mechanism by which loss-of-function of Nav1.7 results in individuals suffering from CIP and that are not being able to feel pain has not been fully elucidated. Studies in rodents have shown that the opioid antagonist naloxone can restore pain sensation.

[0009] Inherited Erythromelalgia (IEM) is a severe painful condition in which patients experience acute pain in the extremities, particularly the feet, which is exacerbated by warmth. Indeed, IEM pain is often relieved by local cooling. Multiple gain-of-function mutations in Nav1.7 have been described in relation to IEM. These mutations result in lower threshold for nociceptor firing and increased repetitive firing. Several attempts have been made to target Nav1.7 sodium ion channels by using sodium channel blockers to decrease pain perception in patients with chronic pain, such as IEM. Three phase II clinical studies have been conducted with vixotrigine, a drug that preferably blocks Nav1.7 channels of neurons with a high firing frequency. Even though some reduction of pain was observed in these studies, two of them failed to achieve the primary outcome by lack of statistical significance.

[0010] A small proof of principal study and two phase II clinical studies have been executed with a drug referred to as PF-05089771. This drug blocks the Nav1.7 channel by interacting with its voltage-sensor and has a 100-fold selectivity for Nav1.7 over cardiac Nav- channels. None of these studies were able to reach the primary outcome which were focused on the reduction of pain within a certain timeframe or its superiority over ibuprofen.

[0011] Funapide is an inhibitor of both Nav1.7 and Nav1.8 and has been used in multiple studies. In a small proof of principal and one phase IIa clinical trial, outcomes regarding a reduction of pain after administration of the drug were found to be significant. However, a larger proof of principal and two phase II clinical studies did not find a significant difference in pain scores of the treated group compared to the placebo-controlled group.

[0012] Lucosamide is another drug that not only inhibits Nav1.7 channels but also inhibits Nav1.3 and Nav1.8 channels. This drug is in a later stage as three phase III clinical studies and one phase II clinical trial have been reported. In three of the studies the primary outcomes – which were defined as a reduction in pain score – were achieved. Even though statistical significance was reached, there was not enough evidence that this drug could be used for diabetic neuropathy because of the variability of responses over time and comparing the effects to established drugs.

[0013] In addition to these phase II and III clinical trials, multiple phase I clinical trials with other drugs blocking Nav1.7 channels were reported in which safety, tolerability and pharmacokinetics were important endpoints. Of these drugs, GDC-0276 and CC8464 were well tolerated, and the results of the latter one has led to the initiation of a phase II trial. The safety and tolerability of DSP-2230 and GDC-0310 have been tested in phase I clinical studies but results are not reported yet.

[0014] To summarize, clinical trials have investigated the possibility to block Nav1.7 channels with drugs to decrease pain perception in individuals with pain disorders. However, most of the trials did not achieve statistical significance. Blocking of Nav channels also has a counter side since sodium channel blockers are often not specific enough for a subtype of any of the Nav channels. In other words, such blockers might, as a side-effect, inhibit multiple Navchannels each with their important roles in other tissues. Therefore, there is a high need of other types of drugs that decrease the functionality of the Nav1.7 channels, and preferably only the Nav1.7 channels, to inhibit the extensive action potential firing.

[0015] US 2016 / 024208 discloses human antibodies against Nav1.7 to downregulate its activity. US 8,183,221 (see also Intl. Patent Application Publication No. WO2009 / 033027) discloses the use of siRNA oligonucleotides targeting a sequence in exon 8 of the SCN9A mRNAto suppress the expression and / or function of Nav1.7. Intl. Patent Application Publication Nos. WO2018 / 051175 and WO2018 / 138585 disclose SCN9A antisense peptide nucleic acid oligonucleotides to skip exon 4 from the SCN9A pre-mRNA to inhibit the expression of a functional Nav1.7. Antisense oligonucleotides (ASOs) targeting Nav1.7 and characterization of the pharmacodynamics of ASOs in spinal cord and DRG in rodents were also reported (Mohan A et al. Pain 2018, 159(1):139-149).

[0016] Intl. Patent Application Publication No. WO2019 / 243430 discloses Locked Nucleic Acid (LNA)-containing ASOs (RNAi agents) to inhibit the expression of Nav1.7. Intl. Patent Application Publication No. WO2022 / 061108 discloses gapmers to degrade the target SCN9A mRNA and thereby lower the expression of Nav1.7. Intl. Patent Application Publication No. WO2022 / 147541 discloses chemically modified ASOs and Spherical Nucleic Acids (SNAs) with such oligonucleotides and liposomes as a core compartment that act as RNAi agents to lower the expression of Nav1.7.

[0017] As described above, Navchannels are dependent upon the sodium selectivity filter motif DEKA, made up of four single amino acids from each domain of the protein. For human Nav1.7, the DEKA motif is generated by the aspartic acid (D) at position 361, the glutamic acid (E) at position 927, the lysine (K) at position 1406, and the alanine (A) at position 1698. It has been shown via site-directed mutagenesis studies that altering the lysine (K) residue of the DEKA motif to an arginine (R) residue decreases the selectivity of the filter from sodium ions only, but also allows potassium ions to pass through but not calcium ions (Favre I et al. Biophys J. 1996, 71(6):3110-3125). The upshot of this change in selectivity is that a Nav1.7 channel with this change retains its activation biophysics but has a greater outward conductance at positive potentials (i.e., action potential generation is reduced), and it fires at a slower rate than an unmutated Navchannel. This altered function of Nav1.7 has applicability for individuals with chronic pain, whereby slowing Nav1.7 signalling could relieve pain sensation.

[0018] ASO approaches with a complete knockdown of Nav1.7 expression and / or functionality, either by exon skippers causing an early termination of the translation product (by out-of-frame exon skips), gapmers, siRNA or other RNAi agents (discussed above) cause a complete absence of Nav1.7 that is not always needed or required, or could have undesirable effects. The present disclosure, in contrast, relates to new and revolutionary methods and means that specifically target human SCN9A mRNA to alter it and to provide a loss-of-function Nav1.7 variant without completely abolishing its expression, thereby providing an alternative approach in the treatment of chronic pain.SUMMARY

[0019] Disclosed herein is an antisense oligonucleotide (herein generally abbreviated to AON) capable of modulating a human SCN9A transcript molecule in a cell, wherein the AON forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, and wherein the modulation of the transcript molecule results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel.

[0020] In one aspect, the modulation is the deamination of a target adenosine by an endogenous ADAR enzyme that is naturally present in the cell, wherein the region of the SCN9A transcript molecule comprises the target adenosine, wherein the nucleotide in the AON that is directly opposite the target adenosine is the orphan nucleotide, and wherein the double-stranded complex can recruit the ADAR enzyme to deaminate the target adenosine into an inosine, thereby editing the SCN9A transcript molecule. In one embodiment, the deamination of the target adenosine into an inosine results in an amino acid change from lysine to arginine at position 1406 (K1406R) in the encoded Nav1.7 protein.

[0021] In one aspect, the AON is directed at deamination of the target adenosine into an inosine that results in an amino acid change from lysine to arginine at position 1406 (K1406R) in the encoded Nav1.7 protein, wherein the AON is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80. In a preferred aspect, the AON is directed at deamination of the target adenosine into an inosine that results in an amino acid change from lysine to arginine at position 1406 (K1406R) in the encoded Nav1.7 protein, wherein the AON is selected from the group consisting of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79.

[0022] In one aspect, the modulation is the skip of exon 23 from the SCN9A transcript molecule, and wherein the AON is partly or fully complementary to a sequence within exon 23 of the human consensus SCN9A pre-mRNA sequence corresponding to NCBI Ref No. NG_012798.

[0023] In one aspect, the AON is directed at the skip of exon 23 from the SCN9A transcript molecule, wherein the AON is selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, and 109. In a preferred aspect, the AON is directed at the skip of exon 23 from the SCN9A transcript molecule, wherein the AON is selected from the group consisting of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, and 66.

[0024] In one aspect, the AON comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety.

[0025] In one aspect, the AON comprises one or more modifications in the linkage moiety, which is each independently selected from the group consisting of: phosphorothioate, phosphonoacetate, phosphorodithioate, methylphosphonate, sulfonylphosphoramidate, (1,3- dimethylimidazolidin-2-ylidene) phosphoramidate, and mesyl phosphoramidate.

[0026] In one aspect, the AON comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and / or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; - O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O- alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and - dimethylaminoethoxyethoxy.

[0027] In one aspect, the disclosure relates to a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding an AON as disclosed herein.

[0028] In one aspect, the disclosure relates to an AON, or a vector, as disclosed herein, for use in the treatment of pain, preferably chronic pain.

[0029] In one aspect, the disclosure relates to a use of an AON as disclosed herein in the manufacture of a medicament for the treatment of pain, preferably chronic pain.

[0030] In one aspect, the disclosure relates to a method of treating pain in an individual in need thereof, the method comprising contacting a SCN9A transcript molecule in a cell of the individual with an AON or a vector as disclosed herein, thereby treating the individual.

[0031] In one aspect, the disclosure relates to an in vitro, ex vivo or in vivo method of impairing the ability of human Nav1.7 to act as a sodium ion channel in a cell, the method comprising administering to the cell an AON or a vector as disclosed herein. In a preferred embodiment, the cell is a neuron, preferably a cell of the PNS, more preferably a large nociceptor cell (= type Aα / Aβ, as discussed above).

[0032] In one aspect, the disclosure relates to an in vitro, ex vivo or in vivo method of impairing the ability of human Nav1.7 to act as a sodium ion channel in a cell, the method comprising administering to the cell an AON as disclosed herein.

[0033] In one aspect, the disclosure relates to an in vitro, ex vivo, or in vivo method of modulating a human SCN9A transcript molecule in a cell, wherein the transcript molecule is a pre- mRNA or an mRNA molecule, wherein the modulation is: i) the deamination of a target adenosine into an inosine in the transcript molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, wherein the AON forms a double-stranded complex with a region of the transcript molecule, wherein the double-stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine; and / or ii) the skip of exon 23 from the transcript molecule, wherein the skip of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23; wherein the modulation results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel.

[0034] In one aspect, the disclosure relates to a method as disclosed herein, wherein administering of the AON is to the central nervous system, optionally via an intrathecal delivery.

[0035] In one aspect, the disclosure relates to an in vitro, ex vivo, or in vivo method of deaminating a target adenosine into an inosine in a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the double- stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine.

[0036] In one aspect, the disclosure relates to an in vitro, ex vivo, or in vivo method of skipping exon 23 from a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre-mRNA molecule, wherein the skipping of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] One or more embodiments will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0038] FIG.1 shows on top part of the human SCN9A mRNA target transcript sequence (5’ to 3’; SEQ ID NO:1). The AAG codon encoding lysine (K) at position 1406 in human Nav1.7 is underlined. The target adenosine is the middle nucleotide in this codon (provided in bold). The sequence of exon 23 is grey-boxed. Below the target sequence, the sequences (also 5’ to 3’) are given of an initial set of 21 antisense oligonucleotides (AONs RM116707 to RM116727; SEQ ID NO: 2 to 22, respectively) that were designed to bring about editing of the target adenosine. The chemical modifications in the AONs are as follows: Gm, Am, Um, and Cm are 2’-O-methyl (2’- OMe)-modified guanosine, adenosine, uridine, and cytidine, respectively; Ae is 2’-O-methoxy- ethyl (2’-MOE)-modified adenosine; m5Ce is 2’-MOE modified 5-methylcytidine; m5Ue is 2’- MOE modified 5-methyluridine (also sometimes named “Te”; 2’-MOE modified thymidine); Af, Uf, Gf, and Cf are 2’-F modified adenosine, uridine, guanosine, and cytosine, respectively; Cd is deoxycytidine; Zd is a cytidine analog that is also referred to as a nucleoside carrying a Benner’s base (as further outlined herein), with a deoxy moiety (= DNA) at the 2’ ribose position; m5Ud is a deoxynucleotide with a 5-methyluridine; * refers to a phosphorothioate (PS) linkage; ! refers to a (1,3-dimethylimidazolidin-2-ylidene) phosphoramidate (PNdmi) linkage; ^ refers to a methylphosphonate (MP) linkage; and ^ refers to a PO linkage.

[0039] FIG. 2 shows editing percentage of the target adenosine, which is the middle adenosine in the AAG codon for lysine at position 1406 in human Nav1.7, in the (pre-) mRNA of human SCN9A in iPSC-derived sensory neurons after gymnotic uptake of the indicated AONs, upon two weeks of treatment. A non-treated sample (NT) was taken as a negative control.

[0040] FIG. 3 shows the percentage exon 23 skipping from the pre-mRNA of human SCN9A in the same samples as FIG.2.

[0041] FIG.4 shows a set of 30 AONs (with their RM names and SEQ ID NO’s indicated) that are directed at causing the deamination of the second adenosine in the AAG codon for lysine at position 1406 in human Nav1.7. The AONs are complementary to the 5’ part of exon 23 and to the 3’ part of exon 22. The chemical modifications are as outlined in FIG.1, with Ge representing 2’-MOE modified guanosine, # referring to a PNms linkage, and with X representing an a-basic nucleotide (= a nucleotide lacking a nucleobase). The underlined nucleotides (including the orphanZd) mismatch with their opposite counterparts in the target sequence (a-basic, C-C, G-G, U-U, U- C, A-G, and C-U).

[0042] FIG. 5 shows editing percentage of the target adenosine, which is the middle adenosine in the AAG codon for lysine at position 1406 in human Nav1.7, in the (pre-) mRNA of human SCN9A in iPSC-derived sensory neurons after transfection of the AONs shown in FIG.4, upon three days of treatment. A mock and a non-treated sample (NT) were taken as negative controls.

[0043] FIG. 6 shows the percentage exon 23 skipping from the pre-mRNA of human SCN9A in the same samples as FIG.5.

[0044] FIG. 7 shows an additional set of 20 AONs (with their RM names and SEQ ID NO’s indicated) that are directed at causing the deamination of the second adenosine in the AAG codon for lysine at position 1406 in human Nav1.7. The chemical modifications are as outlined in FIG.1 and FIG.4, with m5Ud referring to a deoxynucleotide with a 5-methyluridine.

[0045] FIG. 8 shows editing percentage of the target adenosine, which is the middle adenosine in the AAG codon for lysine at position 1406 in human Nav1.7, in the (pre-) mRNA of human SCN9A in iPSC-derived sensory neurons after transfection uptake of the AONs shown in FIG.7, upon three days of treatment.

[0046] FIG. 9 shows the percentage exon 23 skipping from the pre-mRNA of human SCN9A in the same samples as FIG.8.

[0047] FIG. 10 shows an additional set of 12 AONs (with their RM names and SEQ ID NOs indicated) that are specifically directed at skipping of exon 23 from the human SCN9A pre- mRNA, but that should not cause the deamination of the second adenosine in the AAG codon for lysine at position 1406 in human Nav1.7. The chemical modifications are as provided in FIG. 1, FIG.4 and FIG.7. Below the 12 AONs a further 17 AONs (SEQ ID NO:93 to 109) are provided without any chemical modifications. Preferred modifications for these ‘plain’ exon skipping- giving AONs are provided in the accompanying examples, and relate to PS linkages, 2’-OMe and / or 2’-MOE modifications, and PNms linkages at the terminal linkage positions.

[0048] FIG. 11 shows editing percentage of the target adenosine, which is the middle adenosine in the AAG codon for lysine at position 1406 in human Nav1.7, in the (pre-) mRNA of human SCN9A in iPSC-derived sensory neurons after gymnotic uptake of the AONs shown in FIG. 10, upon one week of treatment. As expected, none of the 12 AONs provided the deamination effect, although the positive controls for deamination (RM116707, RM116709, RM116711, and RM116713) did show editing. A non-treated sample (NT) was taken as negative control.

[0049] FIG. 12 shows the percentage exon 23 skipping from the pre-mRNA of human SCN9A in the same samples as FIG.11.

[0050] FIG.13 shows a set of 37 AONs (RM122072 to RM122871; SEQ ID NO:110 to 146, respectively) that were designed to lower exon skip and to increase the editing percentage of the target adenosine as disclosed herein. The chemical modifications are as provided in FIG. 1, FIG.4, and FIG.7. Also shown here is a further set of 17 AONs (RM126178 to RM126194; SEQ ID NO: 152 to 168, respectively) with a variety of PNdmi internucleoside linkage positions. RM126194 is a scrambled (negative) control AON.

[0051] FIG. 14 shows the percentage exon 23 skipping from the pre-mRNA of human SCN9A in iPSC-derived sensory neurons after transfection with the first set of 37 AONs (RM122072 to RM122871; SEQ ID NO:110 to 146, respectively) provided in FIG.13.

[0052] FIG.15 shows the chemical structure (formula (VI) of the RM122635 AON (SEQ ID NO: 136).

[0053] FIG.16 shows the chemical structure (formula (VII) of the RM122637 AON (SEQ ID NO: 138).

[0054] FIG.17 shows the chemical structure (formula (VIII) of the RM122870 AON (SEQ ID NO: 145).

[0055] FIG.18 shows the chemical structure (formula (IX) of the RM122871 AON (SEQ ID NO: 146) DETAILED DESCRIPTION

[0056] It is an object of the present disclosure to create a human Nav1.7 with a partial loss- of-function without downregulating the expression of the protein completely, and without knocking out the functionality of the protein completely. It is an object of the present disclosure to provide methods and means to impair the sodium ion channel functionality of human Nav1.7 in the PNS, preferably large nociceptor cells (type Aα / Aβ) in individuals that suffer from chronic pain. Hence, it is not preferred to knockout Nav1.7 completely. The functionality of the sodium ion channel in Nav1.7 lies in the DEKA motif (discussed above). Taking out the lysine (K) from this motif impairs the ability of Nav1.7 to function properly as a sodium ion channel. This can be achieved for instance by trying to alter the amino acid sequence of Nav1.7 such that the lysine is no longer there or such that it is being replaced by an alternative amino acid. Both aspects are a subject of the present disclosure.

[0057] The disclosure is related to AONs that can cause the modulation of a transcript of the human SCN9A gene, encoding Nav1.7. Modulation in this sense means that the (preferably unmodulated version, generally referred to by NCBI Ref No. NG_012798; also referred to as wild type) transcript molecules encoded by the human SCN9A gene are altered in part such that the encoded protein no longer has the same function as a unmodulated protein, preferably no longer functions, or functions less efficiently as a sodium ion channel.

[0058] The transcript modulation can in one aspect be the modulation of splicing within the SCN9A pre-mRNA molecule, preferably such that exon 23 is skipped from the transcript in the generation of the mRNA. This results in a shortened Nav1.7 that has lost the ability to act as a sodium channel protein. Exon 23 (grey box in FIG. 1) contains 54 nucleotides and because it is ‘in-frame’, the skipping will not result in early termination during translation.

[0059] The transcript modulation can in another aspect be bringing about the specific deamination of a single adenosine in the human SCN9A pre-mRNA and / or mRNA transcript molecule, using a technology generally referred to as “RNA editing” leading to an inosine at the position of the adenosine, which is subsequently read by the translation machinery as a guanosine. The RNA editing is aimed at changing a single amino acid residue in the translated protein, preferably wherein the lysine of the DEKA motif at position 1406 of Nav1.7 is changed to an arginine, thereby generating a K1406R variant.

[0060] Disclosed herein are AONs that, when delivered to a cell under suitable conditions, result in exon skipping in a human (preferably wildtype) transcript molecule and / or RNA editing of a target adenosine in a target transcript molecule, such as pre-mRNA and / or mRNA. The target transcript molecule as disclosed herein (i.e., human SCN9A (pre-) mRNA) comprises a sequence encoding the DEKA motif and the editing results in a transcript that encodes a protein with a loss- of-function, which in this case means that the mutant Nav1.7 variant (K1406R) is no longer able, or less active, or has a lowered or diminished ability to act as a sodium ion channel. The skipping is preferably of exon 23, which in turn also results in the disappearance of the lysine at position 1406, since one of the 18 amino acids encoded by exon 23 (54 nucleotides in length) is the lysine at position 1406. The loss-of-function subsequently results in a lowered, diminished, or most preferably even absent Nav1.7 signalling that subsequently should relieve pain sensation. Exon skipping

[0061] AONs for exon skipping are small polynucleotide molecules (e.g., 19- to 35-mers) that may modulate splicing as their sequence is complementary to that of their target pre-mRNAmolecules. The complementarity may be solely in an exon, thereby influencing the attachment or involvement of splice factors to certain recognition sites in the pre-mRNA, but the complementarity may also be partly in an exon and partly in an upstream and / or downstream intron, for the same purpose of influencing the attachment or involvement of splice factors. In any case, it is part of the present disclosure that the an AON is provided that modulates the correct splicing of the introns surrounding exon 23, such that exon 23 is co-spliced from the pre-mRNA and no longer ends up in the mature mRNA. It is then skipped. In short, the envisioned mechanism is such that upon binding of an AON to a target sequence, with which it is complementary, the targeted region within the pre-mRNA is no longer available for splicing factors, which in turn, results in skipping of the targeted exon.

[0062] Therapeutically, this methodology can be used in a variety of ways: a) to redirect normal splicing of genes in which mutations activate cryptic splice sites; b) to skip exons that carry mutations such that the reading frame of the mRNA remains intact and a (partially or fully) functional protein is made; and c) to induce exon skipping of an exon normally present to generate a complete knock-out of the protein when the exon is out-of-frame, or to generate a protein with a loss-of-function or a gain-of-function because the exon is in-frame and the resulting protein is shorter. The present disclosure relates to the third option: to provide the methods and means to skip exon 23 of the (preferably wildtype) human SCN9A pre-mRNA, thereby generating a shortened Nav1.7 that has not completely lost its functionality, albeit that the function as a sodium ion channel is impaired. This loss-of-function of Nav1.7 provides a potential treatment for chronic pain. RNA editing

[0063] The site-specific deamination of a target adenosine in a transcript molecule is often and herein referred to as ‘RNA editing’. The change in the human SCN9A mRNA transcript molecule leads to a change from a lysine (K) amino acid at position 1406 in the encoded human Nav1.7 to an arginine (R) amino acid at that position, providing a Nav1.7 variant that is herein referred to as K1406R. According to a preferred aspect, targeted RNA editing is mediated by Adenosine Deaminase Acting on RNA (ADAR) that is endogenous (i.e., naturally present) to the cell. According to yet another aspect, the ADAR endogenous to the cell is being recruited to the target RNA in conjunction with a guide RNA (i.e., an AON as disclosed herein) that is at least partially complementary to the target RNA in the region of the proposed change. According to yet another aspect, the AON is designed to bind the target RNA in the region of the intended edit to form a (partially) double-stranded (ds) RNA sequence allowing the endogenous ADAR to bindand exerts an enzymatic deamination of a target adenosine in the target RNA, changing the adenosine into inosine. Preferably, the target adenosine is the adenosine in the triplet coding for lysine at position 1406 in human Nav1.7, effectively changing the triplet so that – upon canonical translation – the triplet codes for arginine. Further aspects of the disclosure and how to put it into practice are set forth in the detailed description herein.

[0064] The RNA editing AONs as disclosed herein can recruit deaminating enzymes, such as ADAR1 and / or ADAR2 that are endogenously present in a cell. An RNA editing AON as disclosed herein can mediate RNA editing of a target adenosine present in a target RNA molecule after it is bound to the target RNA molecule, because the deaminating enzymes are recruited to the double-stranded AON / target RNA molecule complex and which subsequently deaminate the target adenosine into an inosine.

[0065] ADARs are multidomain proteins with N-terminal ds RNA binding domains (dsRBDs) and C-terminal deaminase domains. Two ADAR genes encode catalytically active ADARs in humans (ADAR encoding ADAR1 proteins and ADARB1 encoding the ADAR2 protein). ADAR1 is expressed in two protein isoforms (p110 and p150) that differ in their N- terminal structures. Since the substrate for ADARs is an RNA duplex, the enzymes access the reactive adenosine using a base flipping mechanism (Stephens OM et al. Biochemistry. 2000. 39(40): 12243-12251). Also, because ADARs require duplex RNA for activity, their reaction can be directed to specific adenosines in different transcripts using complementary guide strands for duplex formation at the target sites. This approach is currently being pursued to develop therapeutic guide strands that recruit ADARs to correct disease-causing mutations in RNA (Qu L. et al. Nat. Biotechnol. 2019. 37(9):1059-1069; Merkle T. et al. Nat. Biotechnol. 2019. 37(2):133-138; Katrekar D. et al. Nat. Methods 2019. 16(3):239-242; Monian P. et al. Nat. Biotechnol. 2022. 40(7):1093-1102). While this approach is promising, ADARs have sequence preferences that make certain adenosines disfavored for reaction, limiting the current scope of this approach. For instance, the nearest neighbor nucleotide preferences for ADARs show a strong bias against reaction at adenosines in 5’-GA sites (Eggington JM et al. Nat. Commun.2011.2(319):1-9). This preference is explained by structural studies of ADAR2 bound to transition state analog-containing RNA that suggest a clash between the 2-amino group of the 5’-G and G489 of the ADAR2 loop involved in stabilizing the flipped-out conformation required for the adenosine deamination reaction (Matthews et al. Nat Struct Mol Biol 2016. 23(5):426-433). Earlier work with fusion proteins bearing ADAR deaminase domains indicated that editing efficiency at 5’-GA sites could be improved with a G-A or G-G pair at the 5’ nearest neighbor (Schneider MF et al. Nucleic AcidsRes.2014.42(10):p.e87). However, the basis for this effect has not been reported nor has this effect been established for full length ADARs bearing native dsRBD RNA binding domains. In Intl. Patent Application Publication No. WO2024 / 013361, it is shown that G-A and G-G pairs on the 5’ side of an editing site improve editing efficiency compared to a 5’ G-C pair for full-length ADAR2 and ADAR1 p110. Using X-ray crystallography, the structure of an active fragment of human ADAR2 bound to duplex RNA bearing a G:G pair adjacent to an editing site was determined. Intl. Patent Application Publication No. WO2024 / 013361 discloses that the ADAR enzyme prefers the Gsyn:Gantipairing since the 2-amino group of the 5’-G in the syn conformation does not clash with the minor groove of the enzyme. It was shown that the use of nucleosides capable of stable pairing with the 5’-G in the syn conformation enables more efficient editing within 5’-GA target sites, providing a solution to the problem in deaminating these unfavored editing sites. In the present disclosure related to editing of the second adenosine in the AAG codon for lysine at position 1406 no 5’-G is present next to the target adenosine. There is an adenosine in that position.

[0066] The antisense oligonucleotides are herein abbreviated to “AONs”, or exon skipping AONs when they induce exon skipping in the target transcript molecule, but sometimes the AONs are referred to as ‘RNA editing oligonucleotides’, ‘editing oligonucleotides’ or ‘EONs’, even though the RNA editing event itself is performed by the deamination enzyme and the action of the oligonucleotide only triggers the RNA editing to take place. AONs as disclosed herein may cause both modulations in the transcript molecule, one being the skip of exon 23 from the SCN9A pre- mRNA, the other being the deamination of the target adenosine when the target adenosine is still present in the transcript molecule. Clearly, when the exon is skipped during splicing, the deamination of the target adenosine is no longer useful. Depending on the need, skipping may be preferred as the outcome, but specific target adenosine deamination may be preferred as the outcome in another setting, for instance when it is not desired that the encoded protein is shortened.

[0067] There is a constant need for improving the pharmacokinetic properties of the AONs without: i) negatively affecting its skipping induction ability, or ii) negatively affecting the efficiency in which the target adenosine is edited in the target RNA, and / or without negatively affecting the stability of the AON itself, which is constantly prone to breakdown because of nucleases present in a natural cell. Many chemical modifications are available for the generation of AONs (and many have been applied in the art). Although some are very suitable for generating AONs that mediate exon skipping (for instance AONs that are fully modified by 2’-OMe and / or 2’-MOE, many of these properties are not always compatible with the desire of achieving efficientRNA editing. In the search for better pharmacokinetic properties, it was found earlier that a 2’- MOE modification of the ribose of some, but not all, nucleotides surprisingly appeared compatible with efficient ADAR engagement and editing (Intl. Patent Application Publication No. WO2019 / 158475), while a fully 2’-OMe-modified AON cannot provide RNA editing, when the nucleobase complementarity to the target sequence (except for the orphan position) is 100%. In a similar fashion, it was found earlier that a PS linkage at some, but not all, internucleoside linkages surprisingly appeared compatible with efficient ADAR engagement and editing (Intl. Patent Application Publication No. WO2019 / 219581). Also, it was found earlier that phosphonoacetate (PA) linkage modifications and / or Unlocked Nucleic Acid (UNA) ribose modifications of some, but not all, positions in the AON appeared compatible with efficient engagement of an enzyme with nucleotide deamination activity and with subsequent deamination (Intl. Patent Application Publication No. WO2020 / 165077). Whereas the properties of PA and UNA modifications were known as such, the compatibility thereof with engagement of enzymes with nucleotide deamination activity and with the deamination reaction was not known. Also, it was found earlier that methyl phosphonate (MP) linkage modifications of some, but not all, positions in the AON appeared compatible with efficient engagement of an enzyme with nucleotide deamination activity and with subsequent deamination (Intl. Patent Application Publication No. WO2020 / 201406). Also, it was found earlier that mesyl phosphoramidate linkages (PNms) at some positions in the AON appeared compatible with efficient engagement of an enzyme with nucleotide deamination activity and with subsequent deamination (Intl. Patent Application Publication No. WO2024 / 200278).

[0068] RNA editing is often applied to correct G>A mutations that cause a disease, such as a TGG (encoding tryptophan; W) to TAG or TGA mutation, each being a premature translation stop codon. This is not the case in the present disclosure. Here, RNA editing is applied to introduce a mutation that causes the resulting protein to act less effectively, or in another aspect with a complete loss in functionality in respect of pain signalling. Particularly, the variant that is disclosed herein (K1406R) may keep its potential to allow certain ions to pass. It is preferred that some sodium ions can still pass through the channel, albeit at a lower rate than in the unmodified situation.

[0069] The present disclosure relates to AONs that mediate RNA editing, using endogenous (naturally present) ADAR enzymes in the host cell (preferably nociceptive neurons at the DRG and trigeminal ganglion, and sympathetic ganglion neurons of the autonomic nervous system) of an adenosine present in the transcript of the human SCN9A gene. An AON as disclosed herein aims to lower the activity of Nav1.7.

[0070] The present disclosure also relates to AONs that not only mediate RNA editing as discussed above but also mediate skipping of exon 23 from the human SCN9A pre-mRNA transcript, such that the 18 amino acids encoded by exon 23 are left out of the resulting Nav1.7.

[0071] An AON as disclosed herein can (in some aspects of the disclosure) cause both modulations: i) cause RNA editing as discussed herein on the one hand, and / or ii) induce exon 23 skipping on the other hand. Both modulations will result in an impaired functionality of Nav1.7.

[0072] In another aspect, the present disclosure relates to AONs that do not mediate RNA editing as discussed herein but are solely designed and generated for the purpose of exon 23 skipping. Such can for instance be achieved by chemically modifying the orphan nucleotide by introducing a 2’-OMe modification in the ribose, or by having a 2’-OMe modification of the orphan nucleotide and both of its neighbour nucleotides (the first 5’ and the first 3’ from the orphan nucleotide, representing nucleotide +1 and -1, respectively), and / or having a uridine opposite the adenosine at the RNA editing target site, where it then should not cause editing (see the accompanying examples). The disclosure relates to AONs that that can be fine-tuned such that the level of skipping can be controlled to a certain level, and the RNA editing can be controlled to a certain level. Without wishing to be bound by theory, it may be that the skipping of exon 23, and the deletion of the 18 amino acids encoded thereby, influences the functionality and / or the structure of Nav1.7 in a more significant manner than only the very specific change of the lysine residue to an arginine residue at position 1406 in the DEKA motif. Definitions

[0073] Whenever reference is made to an oligonucleotide, oligo, ON, ASO, oligonucleotide composition, AON, (RNA) editing oligonucleotide, EON, and RNA (antisense) oligonucleotide, both oligoribonucleotides and deoxyoligoribonucleotides are meant unless the context dictates otherwise. Potentially the oligonucleotide may completely lack RNA and DNA nucleotides (as they appear in nature) and may consist completely of modified nucleotides. Whenever reference is made to an ‘oligoribonucleotide’ it may comprise the bases A, G, C, U, or I. Whenever reference is made to a ‘deoxyoligoribonucleotide’ it may comprise the bases A, G, C, T, or I. However, an oligonucleotide as disclosed herein may comprise a mix of ribonucleotides and deoxyribonucleotides. When a deoxyribonucleotide is used, hence without a modification at the 2’ position of the sugar, the nucleotide is often abbreviated to dA (or Ad), dC (or Cd), dG (or Gd), dI (or Id), m5Ud (or T) in which the ‘d’ represents the deoxy nature of the nucleoside, whilea ribonucleoside that is either normal RNA or modified at the 2’ position is often abbreviated without the ‘d’, and often abbreviated with their respective modifications and as explained herein.

[0074] The term ‘nucleoside’ refers to the nucleobase linked to the (deoxy)ribosyl sugar, without phosphate groups. A ‘nucleotide’ is composed of a nucleoside and one or more phosphate groups. The term ‘nucleotide’ thus refers to the respective nucleobase-(deoxy)ribosyl- phospholinker, as well as any chemical modifications of the ribose moiety or the phospho group. Thus, the term would include a nucleotide including a locked ribosyl moiety (comprising a 2’-4’ bridge, comprising a methylene group or any other group), an UNA, a Threose Nucleic Acid (TNA), a nucleotide including a linker comprising a phosphodiester, PA, phosphotriester, PS, phosphoro(di)thioate, MP (or sometimes abbreviated to MeP), methyl thiophosphonate, phosphoramidate linkages, PNdmi according to the structure of formula (IV) as described herein, and a linkage according to the structure of formula (I) as described herein, preferably PNms. Sometimes the terms nucleobase, nucleoside and nucleotide are used interchangeably, unless the context clearly requires differently, for instance when a nucleoside is linked to a neighbouring nucleoside and the linkage between these nucleosides is modified. As stated herein, a nucleotide is a nucleoside plus one or more phosphate groups. The terms ‘ribonucleoside’ and ‘deoxyribonucleoside’, or ‘ribose’ and ‘deoxyribose’ are as used in the art.

[0075] Sometimes the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, inosine, and hypoxanthine, are used interchangeably to refer to the corresponding nucleobase on the one hand, and the nucleoside or nucleotide on the other. The nucleobase thymine (T) is also known as 5-methyluracil (sometimes abbreviated to m5U) and is a uracil (U) derivative; thymine and 5-methyluracil can be interchanged throughout the document text. Likewise, the nucleotide thymidine is also known as 5- methyluridine and is a uridine derivative; thymidine and 5-methyluridine can be interchanged throughout the document text.

[0076] Whenever reference is made to nucleotides in the oligonucleotide, such as cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5- hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine are included. Whenever reference is made to adenine, N6-methyladenine, 8-oxo-adenine, 2,6-diaminopurine and 7-methyladenine are included. Whenever reference is made to uracil, dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudouracil, 4-thiouracil and 5-hydroxymethyluracil are included. Whenever reference is made to guanine, 1-methylguanine, 7-methylguanosine, N2,N2- dimethylguanosine, N2,N2,7-trimethylguanosine and N2,7-dimethylguanosine are included.Whenever reference is made to nucleosides or nucleotides, ribofuranose derivatives, such as 2’- deoxy, 2’-hydroxy, and 2’-O–substituted variants, such as 2’-OMe, are included, as well as other modifications, including 2’-4’ bridged variants. Whenever reference is made to oligonucleotides, one or more linkages may be a naturally occurring PO linkage, whereas the remaining linkages between two mononucleotides may be a modified linkage. Examples of such modified linkages are PA, phosphotriester, PS, phosphoro(di)thioate, MP, phosphoramidate linkages, phosphoryl guanidine, thiophosphoryl guanidine, sulfono phosphoramidate, PNdmi according to the formula (IV) as further outlined below, and the linkage structure according to formula (I) as further outlined in detail below, preferably PNms.

[0077] The term ‘comprising’ encompasses ‘including’ as well as ‘consisting of’, e.g., a composition ‘comprising X’ may consist exclusively of X or may include something additional, e.g., X + Y. The term ‘about’ in relation to a numerical value x is optional and means, e.g., x+10%.

[0078] The word ‘substantially’ does not exclude ‘completely’, e.g., a composition which is ‘substantially free from Y’ may be completely free from Y. Where relevant, the word ‘substantially’ may be omitted from the definition of the invention.

[0079] The term ‘conducive to’ or ‘mediate’ can be used interchangeably with ‘capable of facilitating’. When used in the context of an AON that is conducive to ADAR editing (or can mediate ADAR editing), this means that the AON, after entry into the cell, interacts with the target RNA sequence, thereby forming a ds structure which is recognized by the ADAR enzyme, which can then deaminate the target adenosine into an inosine. Hence, the AON itself does not have the enzymatic function (the ADAR enzyme has), but it can trigger, induce, cause, organize, mediate, provide, give, produce, facilitate, result in RNA editing after binding to the target RNA molecule. When used in the context of an AON that is conducive to exon skipping (or can mediate the skip of exon 23 from the pre-mRNA), this means that the AON, after entry into the cell, interacts with the target RNA sequence, thereby forming a ds structure that influences the splicing machinery such that exon 23 is no longer recognized as such and is spliced out of the pre-mRNA together with its upstream and downstream intron, resulting in a direct connection between exon 22 and exon 24. Hence, the AON itself does not have the splicing function, but it can prevent incorporating exon 23 in the mature mRNA after binding to the target RNA molecule.

[0080] The term ‘mismatch’ is used herein to refer to opposing nucleotides in a double- stranded RNA complex that do not form perfect base pairs according to the Watson-Crick base pairing rules. In the historical sense, mismatched nucleotides are G-A, C-A, U-C, A-A, G-G, C-C, U-U pairs. A nucleotide mismatches also with the opposing nucleotide when that oppositenucleotide is a-basic (lacks a nucleobase). In some embodiments AONs as disclosed herein comprise fewer than four mismatches with the target sequence, for example 0, 1 or 2 mismatches. ‘Wobble’ base pairs are G-U, I-U, I-A, and I-C base pairs. When a U is placed opposite the target A, there is no mismatch, and the AON may be 100% complementary, although an iso-uridine (iso- U) opposite the target adenosine in an RNA editing AON qualifies as a mismatch, since it does not pair according to the Watson-Crick rules of base pairing. When a C is placed opposite the target A, there is at least 1 mismatch between the AON and the target sequence. Although a G:G pairing would be considered a mismatch, that does not necessarily mean that the interaction is unstable, which means that the term ‘mismatch’ may be somewhat outdated based on the current disclosure where a Hoogsteen base-pairing may be seen as a mismatch based on the origin of the nucleotide but still be relatively stable. An isolated G:G pairing in duplex RNA can for instance be quite stable but will still be defined as a mismatch. Analysis of natural targets of ADAR enzymes has indicated that these generally include mismatches between the two strands that form the RNA helix edited by ADAR1 or 2. It has been suggested that these mismatches enhance the specificity of the editing reaction (Stefl et al. Structure 2006,14(2):345-355; Tian et al. Nucleic Acids Res 2011, 39(13):5669-5681). Characterization of optimal patterns of paired / mismatched nucleotides between the AONs and the target RNA also appears important to the development of efficient ADAR-based AON therapy.

[0081] The term ‘complementary’ as used herein refers to the fact that the AON hybridizes under physiological conditions to a second nucleic acid strand. Examples are (i) when the AON as a first nucleic acid strand (= guide oligonucleotide) forms a heteroduplex RNA editing oligonucleotide complex with second complementary nucleic acid strand (in vitro), or (ii) when it forms a double-stranded complex with the target RNA molecule. The term does not necessarily mean that each nucleotide in a nucleic acid strand has a perfect pairing with its opposite nucleotide in the opposite sequence. In other words, while an AON may be complementary to a target sequence, there may be mismatches, wobbles and / or bulges between the AON and the target sequence, while under physiological conditions that AON still hybridizes to the target sequence such that the cellular RNA editing enzymes can deaminate the target adenosine to an inosine. The term ‘substantially complementary’ therefore also means that despite the presence of the mismatches, wobbles, and / or bulges, the AON has enough matching nucleotides with the target sequence that under physiological conditions the AON hybridizes to the target RNA molecule. As shown herein, an AON may be complementary, but may also comprise one or more mismatches, wobbles and / or bulges with the target sequence, if under physiological conditions the AON is ableto hybridize to its target. In one aspect, when the AON is aimed at providing exon skipping as efficient as possible, the AON is fully complementary to the target sequence. There is no need for mismatches such as for instance when the orphan nucleotide mismatches with the target adenosine when RNA editing is desired. Notably, when the orphan nucleotide is uridine, it also does not mismatch and the RNA editing oligonucleotide may also be 100% complementary to the target sequence.

[0082] The term ‘orphan nucleotide’ relates to the nucleotide in the AON that is directly opposite the target adenosine, which is the adenosine that is deaminated by the deaminating enzyme. The orphan nucleotide may be a natural cytidine, a deoxycytidine, a uridine, or a deoxyuridine. It may also be a chemically modified nucleotide, as further described in detail below, or a known or chemically modified analog of a natural (deoxy)cytidine, such as a nucleotide carrying a Benner’s base, or a known or chemically modified analog of a natural (deoxy)uridine, such as iso-uridine, as further outlined in detail below. When the sole aim is to skip exon 23 from the human SCN9A pre-mRNA, there is no need for an orphan nucleotide. Hence, the term orphan nucleotide is only used in the context of RNA editing of a specific target adenosine, which needs an orphan nucleotide directly opposite in the annealing AON, such that endogenously present ADAR enzyme recognizes it as a target adenosine to be deaminated to an inosine.

[0083] A ‘nucleotide analog’ refers to an analog of a nucleic acid nucleotide. The nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine or deoxyuridine.

[0084] The term ‘downstream’ in relation to a nucleic acid sequence means further along the sequence in the 3' direction; the term ‘upstream’ means the converse. Thus, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand but is downstream of the stop codon in the antisense strand. The same holds true for the AONs as disclosed herein. Nucleotides that are upstream of the orphan nucleotide in the antisense oligonucleotide are located towards the 5’ terminus, and nucleotides that are downstream of the orphan nucleotide are located towards the 3’ terminus. Also, exon 23 is upstream of exon 23 in the SCN9A gene and pre-mRNA, whereas exon 25 is downstream of exon 23 in the SCN9A gene and pre-mRNA.

[0085] The nucleotide ‘numbering’ in an AON as disclosed herein is such that the orphan nucleotide is number 0 and the nucleotide 5’ from the orphan nucleotide is number +1. Counting is further positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end, wherein the first nucleotide 3’ from the orphan nucleotide is number -1.

[0086] The internucleoside linkage numbering in the AON is such that linkage number 0 is the linkage 5’ from the orphan nucleotide, and the linkage positions in the oligonucleotide are positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end.

[0087] References to ‘hybridisation’ typically refer to specific hybridisation and exclude non-specific hybridisation. Specific hybridisation can occur under experimental conditions chosen, using techniques well known in the art, to ensure that most stable interactions between probe and target are where the probe and target have at least 70%, preferably at least 80%, more preferably at least 90% sequence identity.

[0088] In all embodiments as disclosed herein, the terms ‘modulating splicing’ and ‘exon skipping’ are synonymous. In respect of human SCN9A, ‘splice switching’, ‘modulating splicing’ or ‘exon skipping’ are to be construed as the exclusion of exon 23 from the resulting human SCN9A mRNA. The term ‘exon skipping’ is herein defined as inducing, producing, or increasing production within a cell of a mature mRNA that does not contain a particular exon (in the current case, exon 23 of the human SCN9A gene) that would be present in the mature mRNA without exon skipping. Exon skipping is achieved by providing a cell expressing the pre-mRNA of said mature mRNA with a molecule capable of interfering with sequences such as, for example, the splice donor or splice acceptor sequence required for allowing the enzymatic process of splicing, or with a molecule that is capable of interfering with an exon inclusion signal required for recognition of a stretch of nucleotides as an exon to be included in the mature mRNA; such molecules are herein referred to as ‘exon skipping molecules’, as ‘exon 23 skipping molecules’, as ‘AONs capable of skipping exon 23 from human SCN9A pre-mRNA’, or as ‘exon skipping AONs’, and varieties thereof. The term ‘pre-mRNA’ refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template of a cell by transcription, such as in the nucleus.

[0089] Whenever a ‘naked’ form in relation to the AON as disclosed herein is referred to, it means that the AON is manufactured in a laboratory or manufacturing facility, through which it is generally chemically modified to prevent it from rapid degradation after it enters the mammalian body or a tissue, or cell, upon administration. A naked form of an AON is therefore different from a form in which the AON is encoded (and delivered) by a viral genome or from a plasmid vector. When such viral vectors or plasmid vectors are administered, the encoded AON is expressed from the viral vector genome or from the plasmid in the cell to which the viral vector or plasmid vector is delivered. Consequently, the AON is then not chemically modified, and comprises solely naturally occurring RNA nucleotides.

[0090] The length of the AON as disclosed herein, and when delivered in a naked form is preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length. However, when the AON as disclosed herein is to be delivered through the expression of a viral vector, then the AON may be longer, such as 70, 80, 90, 100, 150, or 200 or more nucleotides in length. An exon skipping AON is preferably 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length and may be fully complementary to a sequence within exon 23, which means that all consecutive nucleotides in the AON are complementary to consecutive nucleotides that are all in exon 23. In one aspect, the exon skipping AON may comprise a stretch of consecutive nucleotides that is partly complementary to a stretch of consecutive nucleotides in exon 23, and that is further complementary to a stretch of consecutive nucleotides in the intron that is upstream of exon 23. In other words, the exon skipping AON may be complementary to a stretch of consecutive nucleotides in the human wildtype SCN9A transcript that includes the boundary between exon 23 and its upstream intron. The skilled person understands that the stretch that is complementary in the upstream intron may be zero and may be as short as a single nucleotide. Similarly, the exon skipping AON may comprise a stretch of consecutive nucleotides that is partly complementary to a stretch of consecutive nucleotides in exon 23, and that is further complementary to a stretch of consecutive nucleotides in the intron that is upstream or downstream of exon 23. In other words, the exon skipping AON may be complementary to a stretch of consecutive nucleotides in the human wildtype SCN9A transcript that includes the boundary between exon 23 and its downstream or upstream intron. The skilled person understands that the stretch that is complementary in the upstream or downstream intron may be zero and may be as short as a single nucleotide.

[0091] The term ‘HEON’ refers to a heteroduplex ds complex molecule wherein an AON as disclosed herein is hybridized to a partially or fully complementary, partially or fully overlapping sense oligonucleotide. Because the AON as disclosed herein often has specified chemical modifications that are different from the chemical modifications in the sense strand, the two strands form such a heteroduplex RNA editing oligonucleotide complex. The sense strand may be chemically modified almost in its entirety, similar or different to what is performed in the AON as disclosed herein, for example by providing nucleotides with a ribose sugar moiety carrying a 2’- OMe substitution, a 2’-F substitution, or a 2’-MOE substitution. The sense strand in an HEON is preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotidesin length. The HEON is often generated in vitro and used as a delivery tool to protect the AON from degradation when administered to the cell. In other words, the HEON is preferably formed before the AON is administered to the cell. Preferred aspects of HEONs that may be used for AONs as disclosed herein are discussed in Intl. Patent Application Publication No. WO2024 / 084048. Embodiments

[0092] The present disclosure relates to an AON capable of modulating a human SCN9A transcript molecule in a cell, wherein the AON forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, and wherein the modulation of the transcript molecule results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel. In one aspect the human SCN9A transcript molecule is a transcript molecule encoded by a SCN9A gene according to NCBI Ref No. NG_012798.

[0093] In one aspect, the modulation is the deamination of a target adenosine by an endogenous ADAR enzyme that is naturally present in the cell, wherein the region of the SCN9A transcript molecule comprises the target adenosine, wherein the nucleotide in the AON that is directly opposite the target adenosine is the orphan nucleotide, and wherein the double-stranded complex can recruit the ADAR enzyme to deaminate the target adenosine into an inosine, thereby editing the SCN9A transcript molecule. In a preferred aspect, the orphan nucleotide comprises a 6- amino-5-nitro-3-yl-2(1H)-pyridone nucleobase. In a preferred aspect, the deamination of the target adenosine into an inosine results in an amino acid change from lysine to arginine at position 1406 in the encoded human Nav1.7 protein (see NCBI Ref No. NG_012798 and UniProt Q15858). The skilled person appreciates that position 1406 in isoform 2 of Nav1.7 is the same amino acid as position 1395 in isoform 1 of Nav1.7, which only differs from isoform 2 because of an alternative splicing site in exon 12. Hence, where K1406R is referred to herein, the change in isoform 2 is meant. Where K1395R is referred to, the change in isoform 1 is meant. Both isoforms are targeted using the AONs as disclosed herein.

[0094] In one aspect, the AON as disclosed herein comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety. In one aspect, the AON comprises one or more modifications in the linkage moiety, which is each independently selected from the group consisting of: phosphorothioate (PS), phosphonoacetate (PA), phosphorodithioate, methylphosphonate (MP), sulfonylphosphoramidate, (1,3-dimethylimidazolidin-2-ylidene) phosphoramidate (PNdmi), and mesyl phosphoramidate (PNms).

[0095] In one aspect, the internucleoside linkage numbering in the AON is such that linkage number 0 is the linkage 5’ from the orphan nucleotide, and the linkage positions in the oligonucleotide are positively (+) incremented towards the 5’ end and negatively (-) incremented towards the 3’ end, and in a preferred aspect, linkage position -2 is an MP linkage or a PNms linkage.

[0096] In one preferred aspect, the linkage between the most terminal two nucleotides on the 5’ and / or 3’ terminus of the AON is a PNdmi linkage or a PNms linkage.

[0097] In one aspect, the AON comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and / or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; - O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O- alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and - dimethylaminoethoxyethoxy.

[0098] In one aspect, the AON as disclosed herein is covalently or non-covalently, directly or through a linker, bound to an endosomal release agant, such as a triterpene glycoside, for example AG1856. AG1856 is also referred to herein and elsewhere as a ‘saponin’ or as a ‘triterpene saponin’ (see Int. Patent Application Publication Nos. WO2021 / 122998 and WO2024 / 153801). Without wishing to be bound by theory, the saponin is thought to cause improved endosomal release of the AON once taken up by the target cell and may provide more efficient editing during treatment. The saponin may be co-administered (or administered before / after administration of the AON), but is preferably conjugated to the AON, either directly or indirectly through one or more linking moieties. The skilled person is capable of to find the best format of such conjugates to serve the purpose of targeting the SCN9A pre-mRNA and / or mRNA and to direct the AON to the target cell of choice.

[0099] The present disclosure also relates to a vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding an AON as disclosed herein. The skilled person appreciates that when an AON is encoded by a nucleic acid in a viral vector, the AON is not non-naturally chemically modified.

[0100] In one aspect, the AON or a vector as disclosed herein is for use in the treatment of pain, preferably chronic pain.

[0101] In one aspect, the disclosure relates to a use of an AON as disclosed herein in the manufacture of a medicament for the treatment of pain, preferably chronic pain.

[0102] In one aspect, the modulation is the skip of exon 23 from the SCN9A transcript molecule, and wherein the AON is partly or fully complementary to a sequence within exon 23 of the human wildtype SCN9A pre-mRNA. The AON for exon skipping may be completely complementary to the target sequence, including the position of the target adenosine that is targeted for RNA editing as disclosed herein. The AON as disclosed herein may be completely complementary to the target transcript molecule but may, in-part, be complementary to the exon and in-part complementary the sequence of the upstream or downstream intron sequence. Also, when the AON is predominantly applied for exon skipping, it is preferred that the AON is as stable as possible and may therefore comprise a 2’-OMe or a 2’-MOE modification (or any other suitable stability increasing modification) at the nucleotide position that is opposite the second adenosine in the AAG codon for lysine at position 1406. Such modifications are generally not suitable when RNA editing is required.

[0103] In yet another aspect, RNA editing or exon skipping are achieved by a single AON, which means that exon skipping is achieved by the fact that the AON influences splicing on the one hand, and causes RNA editing of the target adenosine on the other. For RNA editing, the orphan nucleotide is preferably modified as disclosed herein. In one aspect, when the AON is aimed at causing exon skipping, the AON comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety. Preferably, the AON comprises one or more modifications in the linkage moiety, which is each independently selected from the group consisting of: phosphorothioate, phosphonoacetate, phosphorodithioate, methylphosphonate, sulfonylphosphoramidate, (1,3-dimethylimidazolidin-2-ylidene) phosphoramidate, and mesyl phosphoramidate.

[0104] In one aspect, the AON comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and / or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; - O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O-, S-, or N-allyl; -O-alkyl-O- alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and - dimethylaminoethoxyethoxy.

[0105] In one aspect, the present disclosure also relates to a method of treating pain in an individual in need thereof, the method comprising contacting a SCN9A transcript molecule in a cell of the individual with an AON as disclosed herein, or a vector as disclosed herein, thereby treating the individual.

[0106] In one aspect, the present disclosure relates to an in vitro, ex vivo or in vivo method of impairing the ability of human Nav1.7 to act as a sodium ion channel in a cell, the method comprising administering to the cell an AON as disclosed herein, or a vector as disclosed herein.

[0107] In one preferred aspect, the cell to which the AON is administered, is a neuron, preferably a cell of the PNS, more preferably a large nociceptor cell, type Aα / Aβ.

[0108] Disclosed herein is also a pharmaceutical composition comprising an AON, or a vector as disclosed herein, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art.

[0109] In one aspect, an AON as disclosed herein is in a naked form. In one aspect, an AON as disclosed herein is in a circular format. In one aspect, an AON as disclosed herein is not in a naked form but is expressed from the genome of a viral vector. In one aspect, an AON as disclosed herein is not in a naked form but is expressed from an expression vector such as a plasmid. In one aspect, when the AON as disclosed herein is not in a naked form, the AON is 15 to 60 nucleotides in length as indicated above, or in another embodiment, from 61 to 300 nucleotides in length.

[0110] In one aspect, the present disclosure relates to a method of deaminating a target adenosine in a human SCN9A pre-mRNA or mRNA molecule in a cell, the method comprising contacting the cell with an AON as disclosed herein under conditions suitable for: i) allowing uptake by the cell of the AON; ii) allowing annealing of the AON to the SCN9A pre-mRNA or mRNA molecule; iii) allowing an endogenous ADAR enzyme to deaminate the target adenosine in the target RNA molecule to an inosine. Optionally, the presence of the inosine in the target RNA molecule is assessed.

[0111] In one aspect, the present disclosure relates to a method of skipping an exon from the SCN9A pre-mRNA coding for human Nav1.7 in a cell, comprising administering an AON as disclosed herein to the cell and allowing the hybridization of the AON to the target pre-mRNA and allowing the splicing machinery to exclude the exon from incorporation into the mature mRNA. In one aspect, the skipped exon is exon 23. Optionally, the absence of the exon in the target RNA molecule is assessed.

[0112] In one aspect, the present disclosure relates to An in vitro, ex vivo or in vivo method of impairing the ability of human Nav1.7 to act as a sodium ion channel in a cell, the method comprising administering to the cell an AON as disclosed herein.

[0113] In one aspect, the present disclosure relates to an in vitro, ex vivo, or in vivo method of modulating a human SCN9A transcript molecule in a cell, wherein the transcript molecule is apre-mRNA or an mRNA molecule, wherein the modulation is: i) the deamination of a target adenosine into an inosine in the transcript molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, wherein the AON forms a double-stranded complex with a region of the transcript molecule, wherein the double-stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine; and / or ii) the skip of exon 23 from the transcript molecule, wherein the skip of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23, wherein the modulation results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel.

[0114] In one aspect, the present disclosure relates to an in vitro, ex vivo, or in vivo method of deaminating a target adenosine into an inosine in a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the double- stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine.

[0115] In a preferred embodiment, the AON is selected from the group consisting of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79.

[0116] In one aspect, the present disclosure relates to an in vitro, ex vivo, or in vivo method of skipping exon 23 from a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre-mRNA molecule, wherein the skipping of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule. In a preferred embodiment, the AON is selected from the group consisting of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, and 66.

[0117] In one aspect, the disclosure relates to a method as disclosed herein, wherein the step of administering the AON is to the central nervous system, optionally via an intrathecal delivery.Chemical modifications

[0118] Various chemistries and modifications are known in the field of oligonucleotides that can be readily used in accordance with the disclosure. All chemical modifications listed herein that may be used in the AON as disclosed herein may also be used for a sense strand that is complementary to the AON, when the AON and the complementary strand form a HEON complex, such as described in Intl. Patent Application Publication No. WO2024 / 084048 and as disclosed above, except that the opposite sense strand does not have an orphan nucleotide. Hence, the modification related to the orphan nucleotide relate only to the AON as disclosed herein, but all other modifications relate to the AON as disclosed herein and any (protecting) sense oligonucleotide that may be used together with the AON in a pharmaceutical product. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands, that have also been described herein, and in detail in Intl. Patent Application Publication No. WO2024 / 084048, which may either be bound to the AON or its opposite strand, or both.

[0119] The skilled person knows that an oligonucleotide, such as an AON as outlined herein, generally consists of repeating monomers. Such a monomer is most often a nucleotide or a chemically modified nucleotide. The most common naturally occurring nucleotides in RNA are adenosine monophosphate (A), cytidine monophosphate (C), guanosine monophosphate (G), and uridine monophosphate (U). These consist of a pentose sugar, a ribose, a 5’-linked phosphate group that is linked via a phosphate ester, and a 1’-linked base. The sugar connects the base and the phosphate and is therefore often referred to as the “scaffold” of the nucleotide. A modification in the pentose sugar is therefore often referred to as a ‘scaffold modification’. The original pentose sugar may be replaced in its entirety by another moiety that similarly connects the base and the phosphate. It is therefore understood that while a pentose sugar is often a scaffold, a scaffold is not necessarily a pentose sugar. Examples of scaffold modifications that may be applied in the monomers of the AON as disclosed herein are disclosed in Intl. Patent Application Publication Nos. WO2020 / 154342, WO2020 / 154343, and WO2020 / 154344.

[0120] A nucleoside in the AON as disclosed herein may be a natural nucleoside (deoxyribonucleoside or ribonucleoside) or a non-natural nucleoside. It is noted that for RNA editing, in which ds RNA is generally the substrate for enzymes with deamination activity (such as ADARs), ribonucleosides are considered ‘natural’, while deoxyribonucleosides may then be, for the sake of argument, considered as non-natural, or considered as modified, simply because DNA is not present in the RNA-RNA ds (natural) substrate configurations. The skilled personappreciates that when the nucleotide has a natural ribose moiety, it may still be non-naturally modified in the base and / or the linkage.

[0121] It is recognized in the art that common limiting factors in oligonucleotide-based therapies are the oligonucleotide’s ability to be taken up by the cell, when delivered per se, or ‘naked’ (= without applying a viral vector or plasmid), the biodistribution and the resistance to nuclease-mediated breakdown. The skilled person is aware, and it has been described in detail in the art, that a variety of chemical modifications can assist in overcoming such limitations. Examples of such now commonly used chemical modifications are the 2’-OMe, 2’-F, 2’,2’-diF, and 2’-MOE modifications of the sugar and the use of PS linkages between nucleosides, as described herein. Scaffold modifications (ribose)

[0122] The ribose 2’ groups in all nucleotides of the AON as disclosed herein, except for the ribose sugar moiety of the orphan nucleotide that has certain limitations in respect of compatibility with RNA editing, can be independently selected from 2’-H (i.e., DNA), 2’-OH (i.e., RNA), 2’-OMe, 2’-MOE, 2’-F, or 2’-4’-linked (for instance a LNA), or other ribosyl 1’- substitutions, 2’ substitutions, 3’ substitutions, 4’ substitutions or 5’ substitutions. The orphan nucleotide in the AON that comprises no other chemical modifications to the ribose sugar, the base, or the linkage preferably does not carry a 2’-OMe or 2’-MOE substitution when the nucleobase is a naturally occurring cytosine, but may carry a 2’-F, a 2’,2’-difluoro (diF), or 2’-ara- F (FANA) substitution or may be DNA. Intl. Patent Application Publication No. WO2024 / 013360 discloses the modification of the 2’ position of the ribose sugar moiety of the orphan nucleotide by a 2’,2’-disubstituted substitution such as diF, which is also applicable to what is disclosed herein. The 2’-4’ linkage can be selected from many linkers known in the art such as a methylene linker, amide linker, or constrained ethyl linker (cEt). Clearly, as outlined herein and as known to the person skilled in the art, 2’-OMe modifications as well as 2’-MOE modifications and combinations thereof can be used throughout the AON when the sole purpose is to skip an exon, preferably exon 23 from human SCN9A transcript molecules, preferably human wildtype SCN9A pre-mRNA molecules.

[0123] An AON as disclosed herein may comprise one or more nucleotides carrying a 2’- MOE ribose modification. Also, an AON as disclosed herein may comprise one or more nucleotides not carrying a 2’-MOE ribose modification, or wherein the 2’-MOE ribose modifications are at positions that do not prevent the ADAR enzyme from deaminating the targetadenosine. An AON as disclosed herein may comprise a 2’-OMe ribose modification at a position that does not comprise a 2’-MOE ribose modification. An AON as disclosed herein may comprise deoxynucleotides at positions that do not comprise a 2’-MOE or a 2’-OMe ribose modification, or other 2’ ribose substitution. An AON as disclosed herein may comprise one or more nucleotides comprising a 2’ substitution comprising a 2’-MOE, 2’-OMe, 2’-OH, 2’-deoxy, TNA, 2’-F, diF modification, 2’-fluoro-2’-C-methyl modification, or a 2’-4’-linkage (i.e., a bridged nucleic acid such as a LNA or examples mentioned in e.g., Intl. Patent Application Publication No. WO2018 / 007475). Other nucleic acid monomers that may be used in an AON as disclosed herein are arabinonucleic acids and FANA, for instance for improved affinity purposes. The 2’-4’ linkage can be selected from linkers known in the art, such as a methylene linker or constrained ethyl linker. A wide variety of 2’ modifications that may present in an AON as disclosed herein are known in the art, including but not limited to the modifications outlined in detail in Intl. Patent Application Publication Nos. WO2016 / 097212, WO2017 / 220751, WO2018 / 041973, WO2018 / 134301, WO2019 / 219581, WO2019 / 158475, and WO2022 / 099159. In all cases, the modifications should be compatible with RNA editing such that the AON fulfils its role as an oligonucleotide that can form a ds nucleic acid complex with the target RNA and by generating this ds nucleic acid complex, recruit a deaminating enzyme, which can subsequently deaminate the target adenosine. Where a monomer in an AON as disclosed herein comprises an UNA ribose modification, that monomer can have a 2’ position comprising the same modifications discussed above, such as a 2’-MOE, a 2’-OMe, a 2’-OH, a 2’-deoxy, a 2’-F, a diF, a 2’-fluoro-2’-C-methyl, an arabinonucleic acid, a FANA, or a 2’-4’-linkage (i.e., a bridged nucleic acids such as an LNA. In one aspect, the AON as disclosed herein comprises at least one nucleotide comprising a TNA ribose modification. In one aspect, the AON as disclosed herein comprises at least one nucleotide with a sugar moiety that comprises a 2’-F modification. A preferred position for the nucleotide that carries a 2’-F modification is position -3 in an RNA editing AON. Base modifications

[0124] A base, sometimes called a nucleobase, is generally adenine, cytosine, guanine, thymine or uracil, or a derivative thereof. A nucleobase is defined as a moiety that can bond to another nucleobase through H-bonds, polarized bonds (such as through CF moieties) or aromatic electronic interactions. Cytosine, thymine, and uracil are pyrimidine bases, and are generally linked to the scaffold through their 1-nitrogen. Adenine and guanine are purine bases and are generally linked to the scaffold through their 9-nitrogen. The terms ‘adenine’, ‘guanine’, ‘cytosine’,‘thymine’, ‘uracil’ and ‘hypoxanthine’ as used herein refer to the nucleobases as such. The terms ‘adenosine’, ‘guanosine’, ‘cytidine’, ‘thymidine’, ‘uridine’ and ‘inosine’ refer to the nucleobases linked to the (deoxy)ribosyl sugar. The nucleobases in an AON as disclosed herein can be adenine, cytosine, guanine, thymine, or uracil or any other moiety able to interact with another nucleobase through H-bonds, polarized bonds (such as CF) or aromatic electronic interactions. The nucleobases at any position in the AON as disclosed herein can be a modified form of adenine, cytosine, guanine, or uracil, such as hypoxanthine (the nucleobase in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseudouracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2-thiothymine, 5-substituted pyrimidine (e.g., 5-halouracil, 5- halomethyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5-propynylcytosine, 5- aminomethyluracil, 5-hydroxymethyluracil, 5-formyluracil, 5-aminomethylcytosine, 5- formylcytosine), 5-hydroxymethylcytosine, 7-deazaguanine, 7-deazaadenine, 7-deaza-2,6- diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, 8-oxo-adenine, 3-deazapurine (such as a 3-deaza-adenosine), pseudoisocytosine, N4- ethylcytosine, N2-cyclopentylguanine, N2-cyclopentyl-2-aminopurine, N2-propyl-2-aminopurine, 2,6-diaminopurine, 2-aminopurine, G-clamp and its derivatives, Super A, Super T, Super G, amino-modified nucleobases or derivatives thereof; and degenerate or universal bases, like 2,6- difluorotoluene, or absent like abasic sites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O- methylribose, azaribose). Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, - alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art. Purine nucleobases and / or pyrimidine nucleobases may be modified to alter their properties, for example by amination or deamination of the heterocyclic rings. The exact chemistries and formats may vary from oligonucleotide construct to oligonucleotide construct and from application to application, and may be worked out in accordance with the wishes and preferences of those of skill in the art.

[0125] A scaffold modification indicates the presence of a modified version of the ribosyl moiety as naturally occurring in RNA (i.e., the pentose moiety), such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2’-modified sugars, 4’-modified sugar, 5’-modified sugars and 4’-substituted sugars. Examples of suitable modifications include, but are not limited to 2’-O-modified RNA monomers, such as 2’-O-alkyl or 2’-O-(substituted)alkyl such as 2’-OMe, 2’-O-(2-cyanoethyl), 2’-MOE, 2’-O-(2-thiomethyl)ethyl, 2’-O-butyryl, 2’-O-propargyl, 2’-O- allyl, 2’-O-(2-aminopropyl), 2’-O-(2-(dimethylamino)propyl), 2’-O-(2-amino)ethyl, 2’-O-(2-(dimethylamino)ethyl); 2’-deoxy (DNA); 2’-O-(haloalkyl)methyl such as 2’-O-(2- chloroethoxy)methyl (MCEM), 2’-O-(2,2-dichloroethoxy)methyl (DCEM); 2’-O-alkoxycarbonyl such as 2’-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2’-O-[2-N-methylcarbamoyl)ethyl] (MCE), 2’-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2’-halo e.g. 2’-F, FANA; 2'-O-[2- (methylamino)-2-oxoethyl] (NMA); a bicyclic or bridged nucleic acid (BNA) scaffold modification such as a conformationally restricted nucleotide (CRN) monomer, an LNA monomer, a xylo-LNA monomer, an α-LNA monomer, an α-l-LNA monomer, a β-d-LNA monomer, a 2’- amino-LNA monomer, a 2’-(alkylamino)-LNA monomer, a 2’-(acylamino)-LNA monomer, a 2’- N-substituted 2’-amino-LNA monomer, a 2’-thio-LNA monomer, a (2’-O,4’-C) constrained ethyl (cEt) BNA monomer, a (2’-O,4’-C) constrained methoxyethyl (cMOE) BNA monomer, a 2’,4’- BNANC(NH) monomer, a 2’,4’-BNANC(NMe) monomer, a 2’,4’-BNANC(NBn) monomer, an ethylene-bridged nucleic acid (ENA) monomer, a carba-LNA (cLNA) monomer, a 3,4-dihydro- 2H-pyran nucleic acid (DpNA) monomer, a 2’-C-bridged bicyclic nucleotide (CBBN) monomer, an oxo-CBBN monomer, a heterocyclic-bridged BNA monomer (such as triazolyl or tetrazolyl- linked), an amido-bridged BNA monomer (such as AmNA), an urea-bridged BNA monomer, a sulfonamide-bridged BNA monomer, a bicyclic carbocyclic nucleotide monomer, a TriNA monomer, an α-l-TriNA monomer, a bicyclo DNA (bcDNA) monomer, an F-bcDNA monomer, a tricyclo DNA (tcDNA) monomer, an F-tcDNA monomer, an alpha anomeric bicyclo DNA (abcDNA) monomer, an oxetane nucleotide monomer, a locked PMO monomer derived from 2’- amino LNA, a guanidine-bridged nucleic acid (GuNA) monomer, a spirocyclopropylene-bridged nucleic acid (scpBNA) monomer, and derivatives thereof; cyclohexenyl nucleic acid (CeNA) monomer, altriol nucleic acid (ANA) monomer, hexitol nucleic acid (HNA) monomer, fluorinated HNA (F-HNA) monomer, pyranosyl-RNA (p-RNA) monomer, 3’-deoxypyranosyl DNA (p- DNA), UNA; an inverted version of any of the monomers above. All these modifications are known to the person skilled in the art. The orphan nucleotide in RNA editing AONs

[0126] Mutagenesis studies of human ADAR2 revealed that a single mutation at residue 488 from glutamate to glutamine (E488Q), gave an increase in the rate constant of deamination by 60-fold when compared to the unmutated enzyme (Kuttan & Bass. Proc Natl Acad Sci USA 2012. 109(48):3295-3304). During the deamination reaction, ADAR flips the edited base out of its RNA duplex, and into the enzyme active site (Matthews et al. 2016, supra). When ADAR2 edits adenosines in the preferred context (an A:C mismatch) the nucleotide opposite the target adenosineis often referred to as the ‘orphan nucleotide’ (or ‘orphan cytidine’ as the case may be), as indicated above. The crystal structure of ADAR2 E488Q bound to ds RNA (dsRNA) revealed that the glutamine (Gln; Q) side chain at position 488 can donate an H-bond to the N3 position of the orphan cytidine, which leads to the increased catalytic rate of ADAR2 E488Q. In the unmutated enzyme, wherein a glutamate (or glutamic acid; Glu; E) is present at position 488 instead of a glutamine (Gln; Q) the amide group of the glutamine is absent and is instead a carboxylic acid. To obtain the same contact of the orphan cytidine with the E488Q mutant would then, for the unmutated situation, require protonation for this contact to occur. To make use of endogenously expressed ADAR2 to correct disease relevant mutations, it is essential to maximize the editing efficiency of the ADAR2 enzyme present in the cell. Intl. Patent Application Publication No. WO2020 / 252376 discloses the use of AONs with modified RNA bases, especially at the position of the orphan cytidine to mimic the hydrogen-bonding pattern observed by the E488Q ADAR2 mutant. By replacing the nucleotide opposite the target adenosine in the AON with cytidine analogs that serve as H-bond donors at N3, it was envisioned that it would be possible to stabilize the same contact that is believed to provide the increase in catalytic rate for the mutant enzyme. Two cytidine analogs were of particular interest: pseudoisocytidine (also referred to as ‘piC’; Lu et al. J Org Chem 2009, 74(21):8021-8030; Burchenal et al. Cancer Res 1976, 36:1520-1523) and Benner’s base Z (also referred to as ‘dZ’ or ‘Zd’ when the ribose comprises a 2’-H group (DNA); Yang et al. Nucl Acid Res 2006, 34(21):6095-6101) that were initially selected because they offer hydrogen-bond donation at N3 with minimal perturbation to the shape of the nucleobase. Benner’s base is also referred to with its chemical name “6-amino-5-nitro-3-yl-2(1H)-pyridone” and sometimes also as “6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone”. The presence of the cytidine analog in the AON may exist in addition to modifications to the ribose 2’ group. The ribose 2’ groups in the orphan nucleotide can be independently selected from 2’-H (i.e., DNA), 2’-OH (i.e., RNA), 2’-OMe, 2’-MOE, 2’-F, or 2’-4’-linked (i.e., a bridged nucleic acid such as LNA), or other 2’ substitutions. The 2’-4’ linkage can be selected from linkers known in the art, such as a methylene linker or constrained ethyl linker.

[0127] The orphan nucleotide in the AON as disclosed herein is preferably a cytidine or analog thereof (such as a nucleotide carrying a Benner’s base) or a uridine or analog thereof (such as iso-uridine). The orphan nucleotide, whether it is a cytidine or analog thereof, or a uridine or analog thereof, preferably comprises a deoxyribose (2’-H; = DNA) but may also comprise a diF modification at the 2’ position of the sugar. In one aspect at least one and in another aspect both the neighbouring (directly adjacent) nucleotides flanking the orphan nucleotide do not comprise a2’-OMe modification. Complete modification wherein all nucleotides of the oligonucleotide hold a 2’-OMe modification (including the orphan nucleotide), with natural bases, results in a non- functional oligonucleotide as far as RNA editing goes (known in the art), presumably because it hinders the ADAR activity at the targeted position. In general, an adenosine in a target RNA can be protected from editing by providing an opposing nucleotide with a 2'-OMe group (at least when there are no other chemical substitutions or modifications within the nucleotide), or by providing a guanine or adenine as opposing base, as these two nucleobases are also able to reduce editing of the opposing adenosine. Linkage modifications

[0128] A nucleoside is generally connected to neighboring nucleosides through condensation of its 5’-phosphate moiety to the 3’-hydroxyl moiety of the neighboring nucleotide monomer. Similarly, its 3’-hydroxyl moiety is generally connected to the 5’-phosphate of a neighboring nucleotide monomer. This forms phosphodiester (PO) bonds. The PO’s and the scaffold form an alternating copolymer. The bases are grafted on this copolymer, namely to the scaffold moieties. Because of this characteristic, the alternating copolymer formed by linked scaffolds of an oligonucleotide is often called the ‘backbone’ of the oligonucleotide. Because PO bonds connect neighboring monomers together, they are often referred to as “backbone linkages”. It is understood that when a phosphate group is modified so that it is instead an analogous moiety such as a PS, such a moiety is still referred to as the backbone linkage of the monomer. This is referred to as a “backbone linkage modification”. In general terms, the backbone of an oligonucleotide comprises alternating scaffolds and backbone linkages.

[0129] As outlined in detail herein, naked AONs as disclosed herein comprise at least one, preferably multiple linkage modifications. It is generally more preferred that the AON as disclosed herein comprises linkage modifications at most, and potentially all positions if the AON is capable of mediating RNA editing through the deamination enzyme when the AON is bound to the target RNA nucleic acid molecule. A linkage modification can be, but is not limited to, a modified version of the PO present in RNA, such as PS, chirally pure PS, (R)-PS, (S)-PS, MP (also referred to as MeP), chirally pure MP, (R)-MP, (S)-MP, phosphoryl guanidine (such as PNdmi), chirally pure phosphoryl guanidine, (R)-phosphoryl guanidine, (S)-phosphoryl guanidine, phosphorodithioate (PS2), phosphonacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methyl phosphorohioate, methyl thiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borano PS, metylboranophosphate, methyl borano PS, methyl boranophosphonate, methyl boranophosphothioate, phosphate, phosphotriester, aminoalkylphosphotriester, and their derivatives. Another modification includes phosphoramidite, phosphoramidate, N3’^P5’ phosphoramidate, phosphorodiamidate, phosphorothiodiamidate, sulfamate, diethylenesulfoxide, amide, sulfonate, siloxane, sulfide, sulfone, formacetyl, alkenyl, methylenehydrazino, sulfonamide, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamide nucleic acid (TANA); and their derivatives. Various salts, mixed salts, deprotonated, protonated, tautomeric, and free acid forms are also included, as well as 3’^3’ and 2’^5’ linkages.

[0130] An AON as disclosed herein may also comprise one or more linkage modifications according to the structure of formula (I):wherein: X = O or S; and R = an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a C1-C6alkoxy, a substituted C1-C6alkoxy, a C1-C20 alkyl, a substituted C1-C20 alkyl, a C1-C6 alkenyl, a C1-C6 substituted alkenyl, a C1-C6alkynyl, a substituted C1-C6alkynyl, or a conjugate group. In a preferred embodiment, X = O and R = methyl and the linkage modification is referred to as “mesyl phosphoramidate” (abbreviated to MsPA or PNms).

[0131] In other preferred aspects, R equals one of the following structures (a), (b), (c), (d), (e), (f), (g), (h), or (i):

[0132] Disclosed herein is also an AON that is able to mediate adenosine deamination by recruitment of a deaminating enzyme in a cell after the AON has formed a ds complex with a region of a target RNA nucleic acid molecule in a cell, wherein the region comprises a target adenosine, wherein the deaminating enzyme can deaminate the target adenosine into an inosine, and wherein the AON comprises a moiety with a structure according to formula (II):wherein: X = O or S; Y = O- or S-; and R = an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a C1-C6alkoxy, a substituted C1-C6 alkoxy, a C1-C20 alkyl, a substituted C1-C20 alkyl, a C1-C6 alkenyl, a C1-C6 substituted alkenyl, a C1-C6alkynyl, a substituted C1-C6alkynyl, or a conjugate group. In a preferred embodiment, X = O and R = methyl.

[0133] An AON as disclosed herein may comprise a substitution of one of the non-bridging oxygens in the PO linkage. This modification slightly destabilizes base pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises PS, phosphonoacetate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H- phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'-alkylenephosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate. Particularly preferred are internucleoside linkages that are modified to contain a PS. Particularly preferred are internucleoside linkages that are modified to contain a PNms. Particularly preferred are internucleoside linkages that are modified to contain a PNdmi. The regular internucleoside linkages between the nucleosides may be altered by mono- or di-thioation of the PO bonds to yield PS esters or phosphorodithioate esters, respectively. Generally, all linkages in an exon skipping AON are modified to increase stability, for instance by having all PS linkages and no PO linkages. In RNA editing AON this may be different in the sense that not all linkages are preferably PS, but some linkages may be PNdmi, PNms, MP, or even, when needed, PO. Clearly, as outlined herein, an RNA editing AON with such varieties of linkages, base, and ribose modifications may still be regarded as an exon skipping AON when it provides RNA editing of a specific target adenosine, but in the meantime has beneficial properties in exon skipping.

[0134] Other modifications of the internucleoside linkages are possible, including amidation and peptide linkers. The skilled person can determine for what target RNA nucleic acid molecule the AON comprises a certain linkage modification at each linkage position of the AON as disclosed herein to generate the most effective and most stable oligonucleotide compound.

[0135] Many of the non-naturally occurring modifications of the linkage, such as PS, are chiral. This means that there are Rp and Sp configurations, known to the person skilled in the art. In one embodiment, the chirality of the PS linkages is controlled, which means that each of the linkages is either in the Rp or in the Sp configuration, whichever is preferred. The choice of an Rp or Sp configuration at a specified linkage position may depend on the target sequence and the efficiency of binding and induction of causing RNA editing of the target adenosine. However, if such is not specifically desired, a composition may comprise AONs as active compounds with both Rp and Sp configurations at a certain specified linkage position. Mixtures of such AONs are also feasible, wherein certain positions preferably have either one of the configurations, while for other positions such does not matter. In one aspect, the AON as disclosed herein comprises one or more (chirally pure or chirally mixed) PS linkages. In one aspect, the AON as disclosed herein comprises one of more (chirally pure or chirally mixed) phosphoramidate (PN) linkages. In one aspect, the AON as disclosed herein comprises one or more (chirally pure or chirally mixed) PNms linkages.

[0136] In one aspect, a PN linkage connects the terminal two nucleotides on each end of the AON. AONs as disclosed herein may also comprise linkage modifications at all positions thatare not chirally controlled. The AON as disclosed herein may also comprise one or more naturally occurring internucleoside linkages. The choice and number of modified linkages may depend on the specific target, the sequence, the length, and the stability of the AON observed in a particular cell type of interest, which can be assessed by methods known to the person skilled in the art.

[0137] In one aspect, at least one, at least two, at least three, or at least four internucleoside linkages between the 5’ and / or the 3’ terminal two, three, four, or five nucleosides respectively of the AON as disclosed herein are modified internucleoside linkages. In one aspect, the AON as disclosed herein comprises at least one MP internucleoside linkage according to the structure of formula (III): (III)

[0138] As was noted in the art, a preferred position for an MP linkage in an RNA editing AON is linkage position -2, thereby connecting the nucleoside at position -1 with the nucleoside at position -2. In one aspect, this position, in an AON as disclosed herein, comprises a linkage modification according to the structure of formula (I), preferably PNms, instead of an MP linkage. Intl. Patent Application Publication No. WO2020 / 201406 discloses the use of MP linkage modifications at certain positions surrounding the orphan nucleotide in the first nucleic acid strand. Although the presence of MP linkages is compatible with RNA editing by human ADAR enzymes, introducing MP linkages during the manufacturing of oligonucleotides is challenging in view of additional manufacturing (purification) steps in the coupling and decoupling process. In one aspect, the AON does not comprise an MP linkage.

[0139] In one aspect, the AON as disclosed herein comprises at least one PNdmi linkage, preferably linking the most terminal two nucleosides at the 5’ and / or 3’ end of the AON. A PNdmi linkage as preferably used in the AONs as disclosed herein has the structure of formula (IV):PNdmi (IV)linkage

[0140] Other internucleoside linkages that may be used in the AONs as disclosed herein are those that are disclosed in Intl. Patent Application Publication No. WO2023 / 278589. In one aspect, the AON as disclosed herein comprises at least one phosphonoacetate and / or at least one phosphonoacetamide internucleoside linkage. Conjugate chemistries

[0141] In one aspect, the AON as disclosed herein, or the sense strand to which it may be annealed before entering a target cell (in an HEON as disclosed herein), is bound to a hydrophobic moiety, such as palmityl or an analog thereof, cholesterol or analog thereof, or tocopherol or analog thereof. It is preferably bound to the 5’ terminus. In case a hydrophobic moiety is bound to the 5’ terminus as well as to the 3’ terminus, such hydrophobic moieties may the same or different. The hydrophobic moiety bound to the oligonucleotide may be bound directly, or indirectly mediated by another substance. When the hydrophobic moiety is bound directly, it is sufficient if the moiety is bound via a covalent bond, an ionic bond, a hydrogen bond, or the like. When the hydrophobic moiety is bound indirectly, it may be bound via a linking group (a linker). The linker may be a cleavable or an uncleavable linker. A cleavable linker refers to a linker that can be cleaved under physiological conditions, for example, in a cell or an animal body (e.g., a human body). A cleavable linker is selectively cleaved by an endogenous enzyme such as a nuclease, or by physiological circumstances specific to parts of the body or cell, such as pH or reducing environment (such as glutathione concentrations). Examples of a cleavable linker comprise, but are not limited to, an amide, an ester, one or both esters of a phosphodiester, a phosphoester, a carbamate, and a disulfide bond, as well as a natural DNA linker. Cleavable linkers also include self-immolative linkers. An uncleavable linker refers to a linker that is not cleaved under physiological conditions, or very slowly compared to a cleavable linker, for example, in a PS linkage, modified or unmodified deoxyribonucleosides linked by a PS linkage, a spacer connected through a PS bond and a linker consisting of modified or unmodified ribonucleosides. There is no restriction on the chain length,when a linker is a nucleic acid such as DNA, or an oligonucleotide. However, it may be usually from 2 to 20 bases in length, from 3 to 10 bases in length, or from 4 to 6 bases in length. There is no restriction on the length or composition of a spacer that is connects the ligand and the oligonucleotide, and may include for example ethylene glycol, triethylene glycol (TEG), HEG, alkyl chains, propyl, 6-aminohexyl, or dodecyl. One or more other types of molecules may be bound to the AON through one or more linkers, including peptides, sugars, vitamins, polymers, aptamers, (fragments of) antibodies, small molecules, and the like. General

[0142] In addition to the specific preferred chemical modifications at certain positions in compounds as disclosed herein, AONs as disclosed herein may comprise one or more (additional) modifications to the nucleobase, scaffold and / or backbone linkage, which may or may not be present in the same monomer, for instance at the 3’ and / or 5’ position. In one aspect, the AON as disclosed herein comprises at least one internucleoside linkage according to the structure of formula (I), (II), (III), and / or (IV), and / or the AON further comprises at least one nucleotide with a sugar moiety that comprises a 2’-OMe modification, and / or the AON comprises at least one nucleotide with a sugar moiety that comprises a 2’-MOE modification, and / or the AON comprises at least one nucleotide with a sugar moiety that comprises a 2’-F modification, and / or the AON comprises an orphan nucleotide that carries a 2’-H in the sugar moiety and is therefore referred to as a DNA nucleotide, even though additional modifications may exist in its base and / or linkage to its neighbouring nucleosides. In one aspect, the orphan nucleotide carries a 2’-F in the sugar moiety. In one aspect, the orphan nucleotide carries a diF substitution in the sugar moiety. In one aspect, the orphan nucleotide carries a 2’-F and a 2’-C-methyl in the sugar moiety. In one aspect, the orphan nucleotide comprises a FANA in the sugar moiety.

[0143] In one aspect, the AON is an antisense oligonucleotide that can form a ds nucleic acid complex with a target RNA molecule, wherein the ds nucleic acid complex can recruit an adenosine deaminating enzyme for deamination of a target adenosine in the target RNA molecule, wherein the nucleotide in the AON that is opposite the target adenosine is the orphan nucleotide, and wherein the orphan nucleotide has the structure of formula (V):wherein: X is O, NH, OCH2, CH2, Se, or S; B is a nitrogenous base selected from the group consisting of: cytosine, uracil, isouracil, N3-glycosylated uracil, pseudoisocytosine, 8-oxo- adenine, and 6-amino-5-nitro-3-yl-2(1H)-pyridone; R1 and R2 are both selected, independently, from H, OH, F or CH3; R3is the part of the AON that is 5’ of the orphan nucleotide, consisting of 7 to 30 nucleotides; and R4 is the part of the AON that is 3’ of the orphan nucleotide, consisting of 4 to 25 nucleotides. The nucleotide 3’ and / or 5’ from the orphan nucleotide may be DNA, more preferably the nucleotide at the 3’ (position -1).

[0144] Other chemical modifications of the AON as disclosed herein include the substitution of one or more than one of any of the hydrogen atoms with deuterium or tritium, examples of which can be found in e.g., Intl. Patent Application Publication Nos. WO2014 / 022566 or WO2015 / 011694. Again, in all cases, the modifications should be compatible with editing such that the AON fulfils its role as an oligonucleotide that can, after binding to its target sequence, recruit an enzyme with adenosine deaminase activity because of the ds nucleic acid entity that arises. In all aspects of the disclosure, the enzyme with adenosine deaminase activity is preferably ADAR1, ADAR2, or ADAT.

[0145] AONs as disclosed herein preferably do not include a 5’-terminal O6- benzylguanosine (O6-BG) or a 5’-terminal amino modification and preferably are not covalently linked to a SNAP-tag domain (an engineered O6-alkylguanosine-DNA-alkyl transferase). An AON as disclosed herein preferably does not comprise a boxB RNA hairpin sequence. In one aspect, an AON as disclosed herein comprises 0, 1, 2 or 3 wobble base pairs with the target sequence, and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatching base pairs with the target RNA sequence. No mismatch exists when the orphan nucleotide is uridine, which may be defined differently when the orphan nucleotide is a uridine analog or derivative. One alternative for uridine is positioning an iso-uridine opposite the target adenosine, which likely does not pair like G pairs with U. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the AON that is directly opposite the target adenosine.

[0146] As outlined above, an AON as disclosed herein makes use of specific nucleotide modifications at predefined spots to ensure stability as well as proper ADAR binding and activity.These changes may vary and may include modifications in the backbone of the AON, in the sugar moiety of the nucleotides as well as in the nucleobases or the phosphodiester linkages, as outlined in detail herein. They may also be variably distributed throughout the sequence of the AON. Specific modifications may be needed to support interactions of different amino acid residues within the RNA-binding domains of ADAR enzymes, as well as those in the deaminase domain. For example, PS linkages between nucleotides or 2’-OMe or 2’-MOE modifications may be tolerated in some parts of the AON, while in other parts they should be avoided so as not to disrupt crucial interactions of the enzyme with the phosphate and 2’-OH groups. Specific nucleotide modifications may also be necessary to enhance the editing activity on substrate RNAs where the target sequence is not optimal for ADAR editing. Previous work has established that certain sequence contexts are more amenable to editing. For example, a target sequence 5’-UAG-3’ (with the target A in the middle) contains the most preferred nearest-neighbor nucleotides for ADAR2, whereas a 5’-CAA-3’ target sequence is disfavored (Schneider et al. Nucleic Acids Res 2014, 42(10):e87). The structural analysis of ADAR2 deaminase domain hints at the possibility of enhancing editing by careful selection of the nucleotides that are opposite to the target trinucleotide. For example, the 5’-CAA-3’ target sequence, paired to a 3’-GCU-5’ sequence on the opposing strand (with the A-C mismatch formed in the middle), is disfavored because the guanosine base sterically clashes with an amino acid side chain of ADAR2. The guanosine opposite the C in such circumstances (= at the -1 position within the AON) is preferably replaced by an inosine, more preferably a deoxyinosine.

[0147] The AON as disclosed herein, in contrast to what has been described for siRNA, or gapmers and their relation towards RNase breakdown and the use of such gapmers in ds complexes (see, e.g., EP Patent Application No. EP 3954395 A1), does not comprise a stretch of DNA nucleotides that would make a target sequence (or a sense nucleic acid strand) a target for RNase- mediated breakdown. It is not desired that the target transcript molecule is degraded through the binding of the AON to the transcript molecule. In one embodiment, the AON does not comprise four or more consecutive DNA nucleotides anywhere within its sequence. In an embodiment, the AON is composed of as much (chemically) modified nucleotides as possible to enhance the resistance towards RNase-mediated breakdown, while at the same time being as efficient as possible in producing an RNA editing effect. This means that the orphan nucleotide and several other nucleotides within the AON may be DNA, but also that there is no stretch of four or more consecutive DNA nucleotides within the AON. Hence, the AON as disclosed herein is not a gapmer. A gapmer reduces the expression of a target transcript but does not produce RNA editingof a specified adenosine within the target transcript and in general does not induce exon skipping. A gapmer is in principle a single-stranded nucleic acid consisting of a central region (DNA gap region with at least four consecutive deoxyribonucleotides) and wing regions positioned directly at the 5’ end (5’ wing region) and the 3’ end (3’ wing region) thereof. In contrast, the AON as disclosed herein may be any oligonucleotide that produces exon skipping by interfering with the splicing machinery when bound to the target transcript sequence, and / or an RNA editing effect in which a target adenosine in a target RNA molecule is deaminated to an inosine, and accordingly is resistant to RNase-mediated breakdown as much as possible to yield this effect and to allow the resulting mRNA transcript being translated into a protein. Hence, the AON as disclosed herein causes the modulation of the target transcript molecule but is not aimed at the degradation of the target transcript molecule.

[0148] The AONs as disclosed herein may also be administered in the context of aids that will increase the entry of the AON into the target cell and / or its endosomal escape as soon as it is in the cell. Moieties that can be applied for such applications are for example a set of chemical compounds (generally purified from nature) referred to as ‘saponins’, see above. A saponin that can be used in the methods as disclosed herein is AG1856, disclosed in Intl. Patent Application Publication No. WO2021 / 122998 and further described for use in the context of RNA editing producing oligonucleotides, in Intl. Patent Application Publication No. WO2024 / 153801. In one aspect, the saponin may be conjugated to an AON as disclosed herein, providing a 1:1 ratio of saponin versus AON for optimal endosomal release of the AON, once it has reached the target cell in which it should act.

[0149] Disclosed herein is also a pharmaceutical composition comprising the AON as disclosed herein, and further comprising a pharmaceutically acceptable carrier, solvent, diluent, and / or other additive and may be dissolved in a pharmaceutically acceptable organic solvent, or the like. Dosage forms in which the AON or the pharmaceutical composition are administered may depend on the disorder to be treated and the tissue that needs to be targeted and can be selected according to common procedures in the art. The pharmaceutical compositions may be administered by a single-dose administration or by multiple dose administration. It may be administered daily or at appropriate time intervals, which may be determined using common general knowledge in the field and may be adjusted based on the disorder, the severity thereof, and the efficacy of the active ingredient.

[0150] Although in a preferred embodiment, the AON as disclosed herein is a single- stranded oligonucleotide comprising an orphan nucleotide opposite the target adenosine, whereinthe orphan nucleotide is chemically modified as disclosed herein, and wherein the remainder of the oligonucleotide is chemically modified to prevent it from nuclease breakdown also as disclosed herein, in another embodiment, disclosed is any kind of oligonucleotide or heteroduplex oligonucleotide complex, that may or may not be bound to hairpin structures (internally or at the terminal end(s)), that may be bound to ADAR or catalytic domains thereof, or wherein the oligonucleotide is in a circular format. In a preferred aspect, the AON as disclosed herein is a ‘naked’ oligonucleotide, comprising a variety of chemical modifications in the ribose sugar and / or the base of one or more of the nucleotides within the sequence, that preferably comprises at least one linkage according to the structure of formula (I) as disclosed herein, that can hybridize to the target transcript or a part thereof that includes the target adenosine, and can recruit endogenous (naturally present) ADAR in the target cell for the deamination of the target adenosine. In another aspect, the AON as disclosed herein, that is delivered in a ‘naked’ form, does not comprise a stem- loop structure for recruitment of the deaminating enzyme, which allows for a shorter AON and improved cellular delivery and trafficking.

[0151] Notably, when the AON comprises chemical modifications, as detailed herein, it may still be delivered through the means of a delivery vehicle. Suitable delivery vehicles are nanoparticle delivery vehicles such as polymeric nanoparticles, dendrimers, inorganic nanoparticles and nanocrystals, organic nanocrystals, and liposomes. Preferred nanoparticles are Lipid Nanoparticles (LNP’s) that are nano-sized lipid vesicles that carry the AON of the present invention and aid to the delivery of target cells. If an LNP is applied or any other similar type of carrier, the AON is still considered naked because it is not transcribed from an encoding polynucleotide (such as in the case of a plasmid or a vector, in which the AON is not regarded as ‘naked’). So, even though a chemically modified AON is encapsulated by a carrier, preferably an LNP, it is still seen as naked, as it has been manufactured as such in a laboratory setting and encapsulated thereafter in the carrier using methods known to the person skilled in the art. The disclosure also relates to a delivery vehicle, preferably an LNP, which comprises a ‘naked’ and chemically modified AON as disclosed herein, even more preferably as disclosed in any one of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, and 146.

[0152] The person skilled in the art understands that when a delivery moiety, or attachment to the AON is used that the AON is still seen as naked as well, when encapsulated in a delivery vehicle such as an LNP. In other words, a variety of non-limiting administration methods is feasible: i) a naked AON as is; ii) a naked AON encapsulated in a delivery vehicle, preferably an LNP; iii) a naked AON administered together or separately from (but not bound to) an endosomal release agent; iv) a naked AON conjugated to an endosomal release agent; v) a naked AON conjugated to an endosomal release agent, and wherein the conjugate is encapsulated in a delivery vehicle, preferably an LNP; or vi) through an encoding vector, such as a plasmid or a viral vector from which the AON is transcribed. Depending on the disease target and the cells that need to be targeted an administration method is being selected, although such is preferably an administration in which the AON is in a naked form, either or not conjugated to a delivery moiety (or endosomal release agent), or either or not encapsulated in a delivery vehicle such as an LNP. Preferred moieties are those that allow the passage across the blood brain barrier, known to the person skilled in the art.

[0153] It is known in the art that RNA editing entities (such as human ADAR enzymes) edit dsRNA structures with varying specificity, depending on several factors. One important factor is the degree of complementarity of the two strands making up the dsRNA sequence. Perfect complementarity of the two strands usually causes the catalytic domain of human ADAR to deaminate adenosines in a non-discriminative manner, reacting with any adenosine it encounters. The specificity of hADAR1 and 2 can be increased by introducing chemical modifications and / or ensuring several mismatches in the dsRNA, which presumably helps to position the dsRNA binding domains in a way that has not been clearly defined yet. Additionally, the deamination reaction itself can be enhanced by providing an oligonucleotide that comprises a mismatch opposite the adenosine to be edited. Following the instructions in the present application, those skilled in the art will be capable of designing the complementary portion of the oligonucleotide according to their needs.

[0154] It will be understood by a person having ordinary skill in the art that the extent to which the editing enzymes inside the cell are redirected to other target sites may be regulated by varying the affinity of the first nucleic acid strand for the recognition domain of the editing enzyme. The exact modification may be determined through some trial and error and / or through computational methods based on structural interactions between the AON and the recognition domain of the editing enzyme. In addition, or alternatively, the degree of recruiting and redirecting the editing enzyme resident in the cell may be regulated by the dosing and the dosing regimen ofthe AON. This is something to be determined by the experimenter (in vitro) or the clinician, usually in phase I and / or II clinical trials.

[0155] Disclosed herein is the site-specific editing of target adenosines in RNA sequences in eukaryotic, preferably metazoan, more preferably mammalian, even more preferably human cells, most preferably a cell of the PNS, such as large nociceptor cells (type Aα / Aβ) involved in the existence of chronic pain. The target cell can be located in vitro, ex vivo or in vivo. One advantage of the AON as disclosed herein is that it can be used with cells in situ in a living organism, but it can also be used with cells in culture. The AON as disclosed herein can also be used to edit target RNA sequences in cells from a transplant or within a so-called organoid, e.g., a brain tissue organoid. Organoids can be thought of as three-dimensional in vitro–derived tissues but are driven using specific conditions to generate individual, isolated tissues.

[0156] Without wishing to be bound by theory, the RNA editing through human ADAR2 for example is thought to take place on primary transcripts in the nucleus, during transcription or splicing, or in the cytoplasm, where e.g., mature mRNA, miRNA or ncRNA can be edited. Generally spoken, RNA editing may be used to create RNA sequences with different properties. Such properties may be coding properties (creating proteins with different sequences or length, leading to altered protein properties or functions), or binding properties (causing inhibition or over- expression of the RNA itself or a target or binding partner; entire expression pathways may be altered by recoding miRNAs or their cognate sequences on target RNAs). Protein function or localization may be changed at will, by functional domains or recognition motifs, including but not limited to signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co- or post-translational modification, catalytic sites of enzymes, binding sites for binding partners, signals for degradation or activation and so on. These and other forms of RNA and protein “engineering”, whether to prevent, delay or treat disease or for any other purpose, in medicine or biotechnology, as diagnostic, prophylactic, therapeutic, research tool or otherwise, are encompassed by the present disclosure.

[0157] A therapeutically amount of AON to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, age, weight, gender, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity, but these can and should be assessed by trial and error during in vitro research, in pre-clinical and clinical trials. The trials are particularly straightforward when the modified sequence leads to an easily detected phenotypic change, or a change in (the level of, or activity of) a specified biomarker. It is possible that higher doses of AONs couldcompete for binding to an ADAR enzyme within a cell, thereby depleting the amount of the enzyme, which is free to take part in RNA editing, but routine dosing trials will reveal any such effects for a given AON and a given target.

[0158] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of an AON may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

[0159] A “therapeutically effective amount” is an amount of a pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen.

[0160] One suitable trial technique involves delivering the AON to cell lines, or a test organism and then taking biopsy samples at various time points thereafter. The sequence of the target RNA can be assessed in the biopsy sample and the proportion of cells having the modification can easily be followed. A method as disclosed herein can include a step of identifying the presence of the desired change in the cell’s target RNA sequence, thereby verifying that the target RNA sequence has been modified. This step will typically involve sequencing of the relevant part of the target RNA, or a cDNA copy thereof (or a cDNA copy of a splicing product thereof, in case the target RNA is a pre-mRNA), as discussed above, and the sequence change can thus be easily verified. Alternatively, as indicated above, the change may be assessed on the function of the protein before, during, and / or after treatment or assessing any other potential marker, which measurements are preferably performed in vitro on samples obtained from the treated subject.

[0161] After RNA editing has occurred in a cell, the modified RNA can become diluted over time, for example due to cell division, limited half-life of the edited RNAs, etc. Thus, in practical therapeutic terms a method as disclosed herein may involve repeated delivery of an AON until enough target RNAs have been modified to provide a tangible benefit to the patient and / or to maintain the benefits over time.

[0162] AONs as disclosed herein are particularly suitable for therapeutic use, and so disclosed is also a pharmaceutical composition comprising an AON as disclosed herein and a pharmaceutically acceptable carrier, solvent, or diluent. In some embodiments the pharmaceutically acceptable carrier can simply be a saline solution. This can usefully be isotonic or hypotonic, particularly for pulmonary delivery. The AON as disclosed herein is suitably administrated in aqueous solution, e.g., saline, or in suspension, optionally comprising additives,excipients and other ingredients, compatible with pharmaceutical use, at concentrations ranging from 1 ng / ml to 1 g / ml, preferably from 10 ng / ml to 500 mg / ml, more preferably from 100 ng / ml to 100 mg / ml. Dosage may suitably range from between about 1 µg / kg to about 100 mg / kg, preferably from about 10 µg / kg to about 10 mg / kg, more preferably from about 100 µg / kg to about 1 mg / kg. As outlined above, the AONs of the present disclosure may also be delivered through a delivery vehicle such as an LNP. Amounts of LNP carrying AONs as disclosed herein can and will also be determined in (pre-) clinical phases. Administration may be by injection or infusion, intracranially, intrathecally, intranasally, orally, intravenously, subcutaneously, intradermally, intramuscularly, intra-tracheally, intra-peritoneally, intrarectally, intra-cisterna magna, parenterally, and the like. Administration may be in solid form, in the form of a powder, a pill, a gel, a solution, a slow-release formulation, or in any other form compatible with pharmaceutical use in humans.

[0163] In one embodiment, depending on the ultimate deamination effect of A to I conversion, the identification step of whether the editing has taken place, comprises the following steps: sequencing the target RNA; assessing the presence or absence of a non-, or less-functional protein; assessing whether splicing of the pre-mRNA was altered by the deamination; assessing the concentration and / or presence / absence of a biomarker; or using a functional read-out. A functional assessment will generally be according to methods known to the skilled person. A suitable manner to identify the presence of an inosine after deamination of the target adenosine is of course dPCR or even sequencing, using methods that are well-known to the person skilled in the art. However, the person skilled in the art of diseases such as chronic pain will preferably apply tests to monitor whether the chronic pain is less, lowered, diminished, or absent in the subject after treatment with the AON as disclosed herein.

[0164] In one embodiment, a method as disclosed herein comprises the steps of administering to the subject an AON, a nanoparticle delivery vehicle formulation as disclosed herein, or a pharmaceutical composition as disclosed herein, allowing the formation of a ds nucleic acid complex of the AON with its specific complementary target nucleic acid molecule in a cell in the subject; allowing the engagement of an endogenous present adenosine deaminating enzyme, such as ADAR 1 or ADAR2; and allowing the enzyme to deaminate the target adenosine in the target nucleic target molecule to an inosine, thereby alleviating, treating, ameliorating, or taking away chronic pain.

[0165] RNA editing molecules present in the cell will usually be proteinaceous in nature, such as the ADAR enzymes found in metazoans, including mammals. The ones of most interestare the human ADARs, hADAR1 and hADAR2, including any isoforms thereof. RNA editing enzymes known in the art, for which oligonucleotide constructs as disclosed herein may conveniently be designed, include the adenosine deaminases acting on RNA (ADARs), such as hADAR1 and hADAR2 in humans or human cells and cytidine deaminases. It is known that hADAR1 exists in two isoforms; a long 150 kDa interferon inducible version and a shorter, 110 kDa version, that is produced through alternative splicing from a common pre-mRNA. Consequently, the level of the 150 kDa isoform available in the cell may be influenced by interferon, particularly interferon-gamma (IFN-γ). hADAR1 is also inducible by TNF-α. This provides an opportunity to develop combination therapy, whereby IFN-γ or TNF-α and AONs as disclosed herein are administered to a patient either as a combination product, or as separate products, either simultaneously or subsequently, in any order. Certain disease conditions may already coincide with increased IFN-γ or TNF-α levels in certain tissues of a patient, creating further opportunities to make editing more specific for diseased tissues. It will be understood by a person having ordinary skill in the art that the extent to which the editing entities inside the cell are redirected to other target sites may be regulated by varying the affinity of the first nucleic acid strand for the recognition domain of the editing molecule.

[0166] An AON as disclosed herein can utilise endogenous cellular pathways and naturally available ADAR enzymes to specifically edit a target adenosine in the target RNA sequence. An AON as disclosed herein is capable of recruiting ADAR and complex with it and then facilitates the deamination of a (single) specific target adenosine nucleotide in a target RNA sequence to which it is bound. Ideally, only one adenosine is deaminated. An AON as disclosed herein, when complexed to ADAR, preferably brings about the deamination of a single target adenosine.

[0167] An AON as disclosed herein, especially when it is in a naked form, is normally equal to, or longer than 13 nucleotides, preferably longer than 14, 15, 16 nucleotides, still more preferably longer than than 17 nucleotides. In one aspect the AON as disclosed herein is longer than 20 nucleotides. The AON as disclosed herein is preferably shorter than 100 nucleotides, still more preferably shorter than 60 nucleotides, still more preferably shorter than 50 nucleotides. In a preferred aspect, the AON as disclosed herein comprises 18 to 70 nucleotides, more preferably comprises 18 to 60 nucleotides, and even more preferably comprises 18 to 50 nucleotides. Hence, in a particularly preferred aspect, the AON as disclosed herein comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.EXAMPLES Example 1. RNA editing of the human SCN9A transcript using a variety of antisense oligonucleotides.

[0168] A set of 21 AONs was designed to target the middle adenosine (underlined and bolded) in the AAG codon encoding lysine at position 1406 in the human Nav1.7 protein, to allow an endogenous ADAR enzyme to deaminate the adenosine to an inosine, thereby rendering a change to arginine at this position (K1406R). The design and chemical modifications of these AONs are provided in FIG. 1. The sequences and chemical modifications of these AONs are provided as SEQ ID NO:2 to 22, respectively. FIG.1 also shows part of the (pre-) mRNA of human SCN9A, which is SEQ ID NO:1.

[0169] For the initial screen of the AONs, the following was performed. For gymnotic uptake (without any transfection or cell-delivery enhancing means), on day 7, iPSC-derived sensory neurons (approximately 2.47x105cells / well) were incubated with 10 µM AONs to allow for uptake during a total of 14 days; in triplicates. The plates were held at 37°C, 5% CO2, and the medium was half-refreshed every 2 days and a complete medium refreshment was done after 7 days. On day 21 (14 days after the start of the gymnotic treatment) the supernatants were discarded, and subsequent analysis was performed as outlined below.

[0170] For a transfection experiment, iPSC-derived sensory neurons (approximately 2.47x105cells / well) were incubated with 200 nM AONs and RNAiMax transfectant reagent, 1:2 ratio, for 72 hrs, according to the instructions from the manufacturer. On day 14 (72 hrs post transfection / plating) the supernatants were discarded, and subsequent analysis was performed as outlined below.

[0171] Cells were collected and used for RNA isolation using a ReliaPrep™ RNA Cell Miniprep System (use of products Z6010, Z6011 and Z6012 were all according to the manufacturer’s instructions). The total RNAs were then reverse-transcribed using the Maxima Reverse Transcriptase (Thermo-EP0742) kit with oligo-dT primer, random Hexamer Primer, and dNTP Mix (10 mM each). Prior to the RT reaction, RNA was incubated with only the dNTP mix, oligo-dT primer and random Hexamer Primer and incubated for 5 min at 75°C, followed by 1 min at 4°C. Then the 5 x RT buffer and the Maxima Reverse Transcriptase were added, and reaction was run according to the manufacturer’s instructions.

[0172] A quantitative PCR was then performed with the Digital PCR System (QIAGEN, QIAcuity Four) in 12 μl aliquots of reaction mixtures containing cDNA, appropriate pairs of primers and dPCR Supermix. The primers given in Table 1 were used with a PCR program that was as follows: Nanoplate 8.5K 96-well plate; Specific priming for QIAcuity Probe based (RT-) PCR Kits; 2 min at 95°C; 40 cycles for 15 sec at 95°C and 30 sec at 60°C, followed by an imaging step. The editing percentage was calculated by pooling the three replicates for each gymnosis / transfection for all A and G counts, together with the Ctrl region of the SCN9A gene, and then scored according to the formula: score = SUM(G) / CTRL * 100.

[0173] Table 1. Primers and probes for the ddPCR of K1406R editing in human SCN9A transcripts. The “+” symbol represents an LNA at the 3’ side of the symbol.

[0174] The RNA editing results from the gymnotic uptake experiment are given in FIG.2 and show that the efficiency of RNA editing is relatively low, with RM116712 (SEQ ID NO:7) and RM116727 (SEQ ID NO:22) performing best with around 3% editing. The editing was significant since the non-treated control (NT) did not show any editing. It is noted that even though the editing is relatively low, the experiment was conducted with gymnotic uptake, which is in vitro a rather inefficient manner of AON administration. Nevertheless, it shows that editing can be achieved even in the absence of transfection force or by the co-administration of an endosomal release agent.

[0175] Since editing appeared low, and it was known that some of the AONs (for instance RM116707 to RM116715 and RM116725 to RM116727) were designed with their 3’ end being exactly at the first nucleotide of exon 23, it was investigated whether the gymnotic uptake of these AONs would have any effect on skipping of exon 23. This was performed on the same extracted RNA samples as used for the RNA editing ddPCT experiment. The experiment was identical to tracing editing, only with different primers / probes used in the dPCR (included in Table 1).

[0176] The exon skipping results are shown in FIG. 3 and quite surprisingly showed an unprecedented high level of almost 100% skip of exon 23 in the case of RM116707 (SEQ ID NO:2), RM116708 (SEQ ID NO:3), RM116721 (SEQ ID NO:16), and RM116722 (SEQ ID NO:17). These skipping levels were very surprising also because only gymnotic uptake was applied. Apparently, these AONs are hugely efficient in providing exon skip. The NT did not reveal any skip of exon 23, indicating that exon 23 skipping does not appear to be a natural event, leading to a different isoform of the Nav1.7 protein.

[0177] These results together show that it was possible to obtain RNA editing of the target adenosine in exon 23 of the human SCN9A transcript, which would lead to a change from lysine to arginine at position 1406 in the human Nav1.7 protein. Quite surprisingly, the results also show that it was possible to almost take out exon 23 completely from the human SCN9A transcript molecules, which in turn would give rise to a human Nav1.7 protein lacking 18 amino acids, including the lysine at position 1406. This should render a protein that is as non-functional as a sodium ion channel as a Nav1.7 protein lacking the lysine at position 1406.

[0178] These experiments have now paved the way for obtaining and using antisense oligonucleotides in the treatment of chronic pain, by either generating a K1406R variant, or by generating a previously unknown isoform, in which the amino acids encoded by exon 23 in the SCN9A gene are lacking. Example 2. RNA editing of the human SCN9A transcript with exon-exon overlapping AONs.

[0179] To investigate whether exon 23 skipping could be inhibited (in contrast to the results shown in Example 1), while the editing effect could be increased, a further set of AONs was designed that are shown in FIG. 4. These AONs are asymmetric in the sense that the orphan nucleotide is in the 5’ portion of the AON (and is complementary to the 5’ part of exon 23), while part of the 3’ portion of the AON is complementary to the ultimate 3’ part of exon 22 (see FIG.1 for the mRNA target sequence). This means that the AONs are partly complementary to exon 23 (and overlap with the position of the target adenosine, as outlined herein) and are partly complementary to upstream exon 22. This means that the AONs are likely to hybridize to the mature mRNA, and not to any intron sequences within the pre-mRNA, before splicing. These AONs are only partly complementary to the pre-mRNA. Some AONs shown in FIG.4 comprise abasic nucleotides (i.e., no nucleobase; noted with an X), which means that they mismatch with the opposite nucleotide in the target sequence. At these positions of the abasic nucleotides, infurther AONs, nucleotides with mismatching nucleobases were designed. These AONs were used in a similar gymnotic uptake / transfection experiment, followed by RNA analysis through dPCR as described above.

[0180] The RNA editing results are given in FIG.5 and show that the efficiency of RNA editing remained relatively low, with RM119143 (SEQ ID NO:46) and RM119148 (SEQ ID NO:51) performing best with around 6% editing. Strikingly, the fully complementary AONs (i.e., without abasic or mismatched nucleotides) appeared to be the best performers in this screen. The addition of abasic or mismatched nucleotides did not increase editing efficiency.

[0181] The exon-skipping results are given in FIG. 6 and show that skip of exon 23 of SCN9A is vastly reduced in comparison to the results shown in FIG.3, from around 90% down to less than 10%, when using a variety of the new designed AONs. Interestingly, AONs with abasic or mismatched nucleotides showed even less exon 23 skipping, with exon skipping levels between 1-2%. In general, the best performing AONs for editing appeared also to yield the most exon skip, see for instance RM119143, RM119128, RM119133, RM119148, and RM120247.

[0182] Since editing percentages observed on the target adenosine was still relatively low, a further set of AONs was designed, which do not overlap with the intron preceding exon 23, but instead are partly hybridizing to exon 23 and partly to exon 22, such that risk of skipping exon 23 (in the processing of pre-mRNA to mRNA) is lowered. These AONs are provided in FIG. 7. A range of chemical modifications was applied in combination with mismatched nucleotides. These AONs were used in a similar gymnotic uptake / transfection experiment as outlined above, followed by RNA analysis through dPCR as described above.

[0183] The RNA editing results are given in FIG. 8. It appeared possible to reach significant high levels of RNA editing that were much higher than shown in FIG.2 and FIG. 5. RM120309 (SEQ ID NO:69) and RM120319 (SEQ ID NO:79) provided editing up to 35% to 40%. Very strikingly, and completely unexpectedly, these best performers (as far as RNA editing goes) gave extremely low levels of exon skipping, as shown in FIG.9. For both AONs, RM120309 (SEQ ID NO:69) and RM120319 (SEQ ID NO:79) exon skipping was less than 0.5%.

[0184] These results show that by carefully designing the nucleotide sequences, mismatches and chemical modifications of the targeting guide oligonucleotides, skipping of exon 23 could be prevented, while RNA editing of the target adenosine, as disclosed herein, increased in efficiency at the same time.Example 3. Exon 23 skipping in the wildtype human SCN9A transcript with AONs that are fully complementary to the exon 23 sequence.

[0185] Since it may be that removal of exon 23 from the human SCN9A mRNA is similarly beneficial as introducing a K1406R mutation, since it means complete removal of the lysine at position 1406 with its directly surrounding amino acids, it was investigated whether exon 23 skipping could be further induced (on top of the results shown above). For this, a further set of AONs was designed that are shown in FIG. 10 (SEQ ID NO:81 to 92), with their respective chemical modifications. These AONs do not comprise an orphan nucleotide opposite the target adenosine, or a Central Triplet for editing with the orphan nucleotide in the middle, because they are not intended to cause RNA editing. Instead, opposite the second adenosine in the AAG codon for lysine at position 1406 a 2’-OMe or a 2’-MOE modified uridine (Um or m5Ue, respectively) is positioned, which should prevent deamination completely. The chemical modifications of these exon-skipping AONs may be much simpler, with solely 2’-MOE modifications throughout the sequence, or other useful modifications as disclosed herein. The basic nucleotide sequence of the AONs comprising 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, without chemical modifications are also depicted in FIG. 10 (SEQ ID NO:109 to 93, [hence, in reverse order], respectively). The AONs of FIG. 10 are only meant to provide exon 23 skipping from the human SCN9A pre-mRNA. SEQ ID NO:81 shows an AON that comprises nucleotides that are all substituted with 2’-OMe in the ribose. Nevertheless, in a preferred aspect, any one of the AONs as shown in SEQ ID NO:93 to 109 comprises one or more, preferably all nucleotides with a substitution at the 2’ position in the ribose with 2’-MOE. In another preferred aspect, one or more linkages are PS linkages. In another preferred aspect, the ultimate linkage at the 5’ terminus and / or the ultimate linkage at the 3’ terminus is not a PS, but a PNdmi or PNms linkage, preferably a PNms linkage.

[0186] AONs RM119378 to RM120108 were tested for editing and exon skipping efficiencies in comparison to RM116707 (SEQ ID NO:2), RM116709 (SEQ ID NO:4), RM116711 (SEQ ID NO:6), and RM116713 (SEQ ID NO:8), after gymnotic uptake in cells, following the procedures as outlined in Example 1.

[0187] The RNA editing results are given in FIG. 11 and show, not unexpectedly, that AONs designed without an orphan nucleotide opposite the target adenosine do not edit the target transcript, as they have the same editing value as non-treated cells (NT control).

[0188] The exon skipping results are given in FIG. 12 and show that AONs designed without an orphan nucleotide opposite the target adenosine maintain exon skipping. The exon skipping levels were lower than observed and shown in FIG. 3, which may be explained by inefficient entry of the AONs into the cells during this gymnotic uptake experiment (which also shows low editing percentages in the positive controls (see FIG.11). AONs containing the same modification as AONs shown in FIG. 1 but without the orphan nucleotide (for instance see the comparison between RM116707 and RM119379) show skipping to the same level, when the results of FIG.3 are compared to the results shown here, albeit at a lower ultimate level, likely due to inefficient cell entry. In any case RM119379 (SEQ ID NO:82, with basic design of SEQ ID NO:93) showed an even higher potency than RM116707 as far as exon skipping is concerned.

[0189] Together, these results show that it is possible to generate a human SCN9A mRNA in which exon 23 is efficiently skipped, to generate a Nav1.7 protein lacking the amino acids encoded by exon 23, including the lysine at position 1406. Example 4. RNA editing of the human SCN9A transcript with AONs.

[0190] An additional set of AONs was designed such that exon skipping could be lowered significantly, while RNA editing would still be significant. The sequences and the chemical modifications of these 37 AONs (RM122072 to RM122871; SEQ ID NO:110 to 146, respectively) are provided in FIG. 13. These AONs display a shift in the position of the orphan nucleotide towards the 5’ terminus.

[0191] The AONs were tested in iPSC-derived sensory neurons after transfection as outlined in Example 1, and editing was determined using ddPCR as outlined above. The editing (with each triplicate) for each AON is provided in Table 2.

[0192] Table 2. Editing percentages after transfection of the indicated AONs in iPSC- derived sensory neurons determined by ddPCR. Percentages for each of the triplicates (n=1, 2, and 3) is provided. The average of the triplicates is provided in the right comlumn.

[0193] Table 2 shows that a number of AONs demonstrate, such as RM122635 (SEQ ID NO:136), RM122637 (SEQ ID NO:138), RM122870 (SEQ ID NO:145), and RM122871 (SEQ ID NO:146), with average editing values shown in the last column. The exon skip percentages caused by these AONs were also assessed, generally as described above. The results are provided in FIG. 14 that clearly shows that (except for RM122638) all exon skip levels were in the range of 0.5% which is significantly less that observed with the earlier AONs designs, as disclosed above. RM122871 is an AON showing high levels of editing, and low levels of exon 23 skipping.

[0194] Subsequently, a further set of AONs was designed. The sequences and the chemical modifications of these 17 AONs (RM126178 to RM126194; SEQ ID NO: 152 to 168, respectively)are also provided in FIG.13. RM126194 is a scrambled negative control AON that should not give editing.

[0195] These AONs were also tested in iPSC-derived sensory neurons after transfection as outlined in Example 1 and in this example, and editing was determined using ddPCR as outlined above. The editing (with each triplicate) for each AON is provided in Table 3.

[0196] Table 3. Editing percentages (column 2 to 5) determined by ddPCR and skip levels (columns 6 to 9) after transfection of the indicated AONs (column 1) in iPSC-derived sensory neurons. Results for each of the triplicates (n=1, 2, and 3) are provided. The average of the triplicates is provided in column 5 (editing) and in column 9 (skip).

[0197] The data in Tables 2 and 3 shows that some AONs achieved relatively high deamination levels of the target adenosine to generate an mRNA transcript molecule, that encodes a Nav1.7 protein with a K1406R change in the DEKA motif, while maintaining skip of exon 23 relatively low. Especially RM122635 (SEQ ID NO: 136), RM122637 (SEQ ID NO: 138), RM122870 (SEQ ID NO: 145), RM122871 (SEQ ID NO: 146), RM126180 (SEQ ID NO: 154), RM126181 (SEQ ID NO: 155), RM126182 (SEQ ID NO: 156), RM126183 (SEQ ID NO: 157), RM126186 (SEQ ID NO: 160), and RM126189 (SEQ ID NO: 163) performed particularly well. The use of the negative control (scrambled) RM126194 (SEQ ID NO: 168) resulted in very low editing levels, as expected. Column 9 shows that some of the very potent editing oligonucleotides,such as RM126183, scored above 42% editing (with one of the triplicates providing >55% editing) with less than background levels of skip.

[0198] In a further functional assay, RM122635, RM122637, and RM122871 were tested for Nav1.7 function after RNA editing in human HEK cells using patch clamp electrophysiology on single cells. This displayed a distinct effect in Na reversal potential and increase in reduction of hyperexcitability in all three cases (data not shown), which is indicative that the editing of the SCN9A transcript resulting in the K1406R change in the Nav1.7 protein, impairs its functionality. Example 5. Intrathecal delivery of an editing oligonucleotide.

[0199] This example describes a non-human primate (NHP) study delivering an editing guide oligonucleotide via intrathecal (IT) route in cynomolgus monkeys. The animals were administered with an editing guide oligonucleotide (“test article”) according to the study design shown in Table 4. In this example the test article is a guide oligonucleotide according to SEQ ID NO:147, targeting Beta-actin transcripts. The study shows editing efficiency and exposure after IT delivery. SEQ ID NO:147 represents: 5’- Gm!Am*Am*Am*Gm*Cf*Am*Af*Um*Gf*m5Ce*Zd*Ad^Um*Cf*Ae*Cf*Cm*Uf*Cm*Cf*C m*Cm*Um!Gm-3’ wherein the modifications are as provided in FIG.1. Methods

[0200] 1. Animals. Twenty drug-naïve female cynomolgus monkeys had an IT catheter implanted in the lumbar region to facilitate test article administration.

[0201] 2. Reagents. Test article consisted of a single-stranded editing oligonucleotide according to SEQ ID NO:147.

[0202] 3. In-Life Study Design. Dose groups and terminal necropsy time points are depicted in Table 4. Test article was administered via IT catheter at total doses, volumes, and concentrations depicted in Table 4. Assessment of toxicity was based on mortality, clinical observations, body weights, qualitative food consumption, neurobehavioral observations, and clinical and anatomic pathology.

[0203] 4. Tissue Collection. Serial plasma and cerebrospinal fluid (CSF) collection was performed on dosing days for up to 360 hours post-dose. Terminal tissue collection was performedon the necropsy day indicated in Table 4. Tissues (when present) from each animal were preserved, as indicated in Table 5. Histology.

[0204] All tissues denoted by “E” in Table 5 (Necropsy, Organ Weights, and Macroscopic Observations section) from each monkey were incubated in 10% neutral buffered formalin (NBF) for at least 24 hrs, but no more than 28 hrs, at RT. Upon removal from NBF, tissues were rinsed three times with 1x PBS and stored in PBS at 4ºC for up to 72 hrs until processed to paraffin block. Once processed to paraffin block, tissues were sectioned at a nominal 5 micron, and slides were prepared and stained with haematoxylin and eosin. For spinal cord (mid-thoracic and mid-lumbar), one transverse and one transverse oblique section (including the injection site) were taken. For spinal cord (mid cervical) one transverse and one parasagittal longitudinal section was taken. Microscopic Observations.

[0205] All tissues denoted by “E” in Table 5 (Necropsy, Organ Weights, and Macroscopic Observations section) from all animals were examined microscopically. Following completion of the primary microscopic evaluation, an independent peer review evaluation was performed. Frozen Tissue Collection for Exploratory Analysis.

[0206] After the collection of any of the following tissue samples for microscopic evaluation, the following samples were collected from each monkey: Brain, left frontal cortex; Brain left, temporal cortex; Brain, left caudate; Brain, left putamen; Brain, left hippocampus; Brain, left thalamus; Brain, left medulla; Brain left, pons; Brain left, substantia nigra; Brain left, amygdala; Brain left, parietal cortex; Brain left, cerebellum; Dorsal Root Ganglion, lumbar (to target L5); Dorsal Root Ganglion, thoracic; Dorsal Root Ganglion, cervical; Heart, apex; Kidney, left cortex; Liver, left lateral lobe; Sciatic, left; Spinal cord, cervical; Spinal cord, thoracic; Spinal cord, lumbar; Spleen; Superior cervical ganglion, left.

[0207] For the left brain hemisphere for each brain region (frontal cortex, temporal cortex, caudate, putamen, hippocampus, thalamus, medulla, pons, substantia nigra, amygdala, parietal cortex, and cerebellum), up to four samples (25 to 100 mg each) were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain -60 to -80°C. All sample weights were recorded.

[0208] For the spinal cord region (cervical, thoracic, and lumbar), up to four samples (25 to 100 mg each) were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain -60 to -80°C. All sample weights were recorded.

[0209] For the dorsal root ganglions (DRG), four DRG (two bilateral pairs) from each region (cervical, thoracic, and lumbar) were collected, weighed, and had nerve roots trimmed out. Each bilateral pair was placed in a pre-chilled, 2 mL, RNase-free tube. The left superior cervical ganglion was sampled whole, weighed, and placed into a pre-chilled, 2 mL, RNase-free tube. Each sample was snap frozen in liquid nitrogen and placed on dry ice until transferred to a freezer, set to maintain -60 to -80°C. All sample weights were recorded.

[0210] For the liver (left lateral lobe), spleen, heart (apex), left kidney (cortex), and left sciatic nerve sample, up to four samples (25 to 100 mg each) of each tissue were collected and placed in a pre-chilled, 2 mL, RNase-free tube; snap frozen in liquid nitrogen; and placed on dry ice until transferred to a freezer, set to maintain -60 to -80°C. All sample weights were recorded. RNA isolation.

[0211] RNA was extracted from exploratory tissue lysates for exploratory pharmacodynamic endpoints (e.g. RNA editing efficiency). Frozen tissue chunks were transferred to 1 ml of cold Trizol (Life Technologies) and immediately homogenized using a Qiagen Ruptor or OMNI Soft Tissue Disruptor on ice (high speed for 10-30 sec, depending on the tissue). Homogenates equivalent to up to 35 mg of tissue weight (25 mg for liver, kidney, and spleen) were diluted with Trizol to a volume of 1 ml at RT. RNA was extracted by adding 200 µl of chloroform and shaking vigorously. After centrifugation, 450-500 µl of the upper aqueous phase was mixed with an equal volume of 70% ethanol. The mixture was subjected to RNA isolation using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. DNase on-column treatment was applied. RNA was eluted with 30 µl of RNase-free water. RNA concentration and quality were determined using an Agilent Bioanalyzer 4200 with RNA ScreenTape (Agilent), and RNA samples were stored at -80°C. cDNA synthesis.

[0212] A 0.5 µg RNA sample was mixed with 0.5 µl of 100 mM Oligo(dT)18 (Thermo Scientific), 0.5 µl of 100 µM Random Hexamer (Thermo Scientific), 1.0 µl of dNTP mix (Thermo Scientific), and RNase- and DNase-free water to make a final volume of 15.5 µl. The solution wasincubated at 65°C for 5 min and cooled at 4°C for 1 min. After mixing with 4 µl of 5x RT buffer and 0.5 µl of Maxima Reverse Transcriptase (Thermo Scientific), the reaction solution was placed in a thermocycler and run under the following conditions: 25°C for 10 min, 60°C for 30 min, 85°C for 5 min, and 4°C indefinitely. After the reaction, cDNA samples were diluted with RNase- and DNase-free water at a 1:80 ratio. Digital droplet PCR.

[0213] 1 µl of cDNA sample was used to perform digital droplet PCR (ddPCR) with ddPCR Multiplex Supermix (4x) (BioRad) in a reaction volume of 20 µl. ddPCR probes and primers were custom-made by IDT.

[0214] The sequences of probes are as follows: / 56-FAM / AGGTGA+T+G+GCATTGCTTTCGT / 3IABkFQ / (SEQ ID NO:148) and / 5HEX / AG+GTGA+T+A+GCATTGCTTTCGTGT / 3IABkFQ / (SEQ ID NO:149).

[0215] The sequences of primers are as follows: 5’-AGTCCTCTCCCGAGTCCACA-3’ (SEQ ID NO:150) and 5’-GGGGCATGAAGGCTCATTATTCAA-3’ (SEQ ID NO:151).

[0216] A final concentration of 250 nM (probes) was used. A final concentration of 500 nM (primers) was used. Droplets were generated on a Bio-Rad Automated Droplet Generator (BioRad). PCR was performed in a C1000 Touch™ Thermal Cycler (BioRad) using the following conditions: 95°C for 10 min for 1 cycle; 95°C for 30 sec and 64°C for 1 min for 40 cycles; 98°C for 10 min for 1 cycle; and 4°C indefinitely. After the reaction was completed, droplets were analysed on a QX600 Droplet Reader (BioRad). Data was acquired and analysed using BioRad QX manager Standard Edition v.2.20 software. FAM-positive, HEX-positive, and FAM / HEX- positive populations were gated in the 2D plot of channel 1 and channel 2 amplitude. Editing efficiency percentage was calculated using the equation: G(cp / µl) / (G(cp / µl) + A(cp / µl)) x 100%.

[0217] Table 4. NHP study design of different doses, timepoints and repeat dosing. Group 1 did not receive the test article, but was treated in line with the other animals, and served as negative control.

[0218] Table 5. Organ / tissue collection.Results

[0219] The editing efficiencies measured across various tissues are provided in Table 6.

[0220] Table 6. Editing efficiency measured across tissues.Exposure analyses. Tissue Materials, Methods, and Data from HPLC / HRMS Analysis.

[0221] To analyse test article delivery to tissues and understand exposure response relationships, tissue samples were collected at timepoints corresponding to the editing measurements, and the exposure of the total guide oligonucleotide in tissue was quantified by HP- LC / MS. Briefly, tissue standards were prepared in control tissue homogenate. To control assay variability, an internal standard was added to all standards and samples.

[0222] Tissue samples were homogenized in cell lysis buffer. For total guide oligonucleotide measurements, tissue standards and samples were digested with proteinase K priorto being loaded onto an Oasis Wax micro-elution solid phase extraction (SPE) plate (Waters Inc, Milford, MA) for isolation. The SPE plate was washed with wash buffers and then analytes were eluted with elution buffer. Eluants from the SPE plates were dried, reconstituted, and injected onto an LC / MS system.

[0223] The total guide oligonucleotide concentrations were measured using a Thermo Orbitrap Exploris 240 (Thermo Scientific, San Jose, CA) mass spectrometer using the guide oligonucleotide peak for quantification. The mass spectrometer was operated in negative ion detection mode. All data were processed using Xcalibur version 4.4 (Thermo Scientific, San Jose, CA).

[0224] Table 7 shows that animals dosed intrathecally with test article had measurable exposure in multiple central nervous system regions. The variability in brain exposure in some groups is likely indicative of incomplete IT dosing. Peripheral tissues (liver and kidney) were also exposed to test article, indicating a significant amount of IT dosed compound reaching the systemic circulation. Kidney concentrations were consistently higher than liver.

[0225] Table 7. Summary of exposure data.

[0226] The IT study demonstrates that direct administration into the brain is a feasible way of getting an RNA editing ASO at the cell of interest, especially brain cells.

Claims

CLAIMS An antisense oligonucleotide (AON) capable of modulating a human SCN9A transcript molecule in a cell, wherein the AON forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, and wherein the modulation of the transcript molecule results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel. The AON according to claim 1, wherein the modulation is the deamination of a target adenosine by an endogenous ADAR enzyme that is naturally present in the cell, wherein the region of the SCN9A transcript molecule comprises the target adenosine, wherein the nucleotide in the AON that is directly opposite the target adenosine is the orphan nucleotide, and wherein the double-stranded complex can recruit the ADAR enzyme to deaminate the target adenosine into an inosine, thereby editing the SCN9A transcript molecule. The AON according to claim 2, wherein the deamination of the target adenosine into an inosine that results in an amino acid change from lysine to arginine at position 1406 in the Nav1.7 protein encoded by the nucleic acid sequence of NCBI Ref No. NG_012798. The AON according to claim 2 or 3, wherein the AON comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety. The AON according to claim 4, wherein the AON comprises one or more modifications in the linkage moiety, which is each independently selected from the group consisting of: phosphorothioate, phosphonoacetate, phosphorodithioate, methylphosphonate, sulfonylphosphoramidate, (1,3-dimethylimidazolidin-2-ylidene) phosphoramidate, and mesyl phosphoramidate. The AON according to claim 4 or 5, wherein the AON comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and / or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O- , S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy.The AON according to any one of claims 1 to 6, wherein the AON is selected from the group consisting of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79. An AON comprising a nucleic acid sequence of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79. An AON consisting of a nucleic acid sequence of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79. A vector, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, comprising a nucleic acid molecule encoding an AON according to any one of claims 1 to 3. A pharmaceutical composition comprising the AON according to any one of claims 1 to 9, or a vector according to claim 10, and a pharmaceutically acceptable carrier. An AON according to any one of claims 1 to 9, a vector according to claim 10, or a pharmaceutical composition according to claim 11, for use as a medicament or for use in therapy. An AON according to any one of claims 1 to 9, a vector according to claim 10, or a pharmaceutical composition according to claim 11, for use in the treatment of pain, preferably chronic pain. Use of an AON according to any one of claims 1 to 9, or a vector according to claim 10, in the manufacture of a medicament for the treatment of pain, preferably chronic pain. The AON according to claim 1, wherein the modulation is the skip of exon 23 from the SCN9A transcript molecule, and wherein the AON is partly or fully complementary to a sequence within exon 23 of the human wildtype SCN9A pre-mRNA. The AON according to claim 15, wherein the AON comprises one or more non-naturally occurring chemical modifications in the ribose, linkage, or base moiety. The AON according to claim 16, wherein the AON comprises one or more modifications in the linkage moiety, which is each independently selected from the group consisting of: phosphorothioate, phosphonoacetate, phosphorodithioate, methylphosphonate, sulfonylphosphoramidate, (1,3-dimethylimidazolidin-2-ylidene) phosphoramidate, and mesyl phosphoramidate.The AON according to claim 16 or 17, wherein the AON comprises one or more nucleotides comprising a mono- or di-substitution at the 2', 3' and / or 5' position of the ribose, each independently selected from the group consisting of: -OH; -F; substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; -O-, S-, or N-alkyl; -O-, S-, or N-alkenyl; -O-, S-, or N-alkynyl; -O- , S-, or N-allyl; -O-alkyl-O-alkyl; -methoxy; -aminopropoxy; -methoxyethoxy; -dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy. The AON according to any one of claims 15 to 18, wherein the AON is selected from the group consisting of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, and 66. An AON comprising a nucleic acid sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, and 66. An AON consisting of a nucleic acid sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, and 66. A vector, preferably a viral vector, more preferably an AAV vector, comprising a nucleic acid molecule encoding an AON according to claim 15. A pharmaceutical composition comprising the AON according to any one of claims 15 to 21, or a vector according to claim 22, and a pharmaceutically acceptable carrier. An AON according to any one of claims 15 to 21, a vector according to claim 22, or a pharmaceutical composition according to claim 23, for use as a medicament or for use in therapy. An AON according to any one of claims 15 to 21, a vector according to claim 22, or a pharmaceutical composition according to claim 23, for use in the treatment of pain, preferably chronic pain. Use of an AON according to any one of claims 15 to 21, or a vector according to claim 22, in the manufacture of a medicament for the treatment of pain, preferably chronic pain. A method of treating a Nav1.7 associated disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an AON according to any one of claims 1 to 9, 15 to 21, a vector according to claim 10 or 22, or apharmaceutical composition according to claim 11 or 23, preferably wherein the administering is to the central nervous system, optionally via an intrathecal delivery. The method of claim 27, wherein the disease or disorder is pain, optionally chronic pain. A method of treating a chronic pain disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an AON according to any one of claims 1 to 9, 15 to 21, a vector according to claim 10 or 22, or a pharmaceutical composition according to claim 11 or 23, preferably wherein the administering is to the central nervous system, optionally via an intrathecal delivery. A method of treating pain in an individual in need thereof, the method comprising contacting a SCN9A transcript molecule in a cell of the individual with an AON according to anyone of claims 1 to 9, 15 to 21, or a vector according to claim 10 or 22, thereby treating the individual, preferably wherein the AON is administered to the central nervous system, optionally via an intrathecal delivery. An in vitro, ex vivo or in vivo method of impairing the ability of human Nav1.7 to act as a sodium ion channel in a cell, the method comprising administering to the cell an AON according to any one of claims 1 to 7, 18 to 22, a vector according to claim 10 or 22, or a pharmaceutical composition according to claim 11 or 23. An in vitro, ex vivo, or in vivo method of modulating a human SCN9A transcript molecule in a cell, wherein the transcript molecule is a pre-mRNA or an mRNA molecule, comprising administering to the cell an AON, wherein the modulation is: i) the deamination of a target adenosine into an inosine in the transcript molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, wherein the AON forms a double-stranded complex with a region of the transcript molecule, wherein the double-stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine; and / or ii) the skip of exon 23 from the transcript molecule, wherein the skip of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23, wherein the modulation results in an encoded Nav1.7 protein that is impaired in its function as a sodium ion channel.An in vitro, ex vivo, or in vivo method of deaminating a target adenosine into an inosine in a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre- mRNA or an mRNA molecule, wherein the deamination results in an amino acid change from lysine to arginine at position 1406 in the encoded Nav1.7 protein, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule, wherein the double-stranded complex can recruit an ADAR enzyme that is naturally present in the cell to deaminate the target adenosine into an inosine. A method according to claim 32 or 33, wherein the AON comprises a sequence of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79; or wherein the AON consists of a sequence of SEQ ID NO: 146, 145, 138, 136, 154, 155, 156, 157, 160, 163, 7, 6, 10, 11, 5, 12, 17, 22, 46, 51, 31, 36, 56, 69, and 79. An in vitro, ex vivo, or in vivo method of skipping exon 23 from a transcript molecule of the human SCN9A gene in a cell, wherein the transcript molecule is a pre-mRNA molecule, wherein the skipping of exon 23 results in an encoded Nav1.7 protein lacking the amino acids encoded by exon 23, said method comprising the step of administering to the cell an AON that forms a double-stranded complex with a region of the SCN9A transcript molecule. A method according to claim 32 or 35, wherein the AON comprises a sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, or 66; or wherein the AON consists of a sequence of SEQ ID NO: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 82, 2, 16, 17, 3, 18, 19, 4, 5, 6, 10, 22, 46, 51, 31, 36, 56, 65, or 66. The method according to any one of claims 31 to 36, wherein the method is an in vivo method and the AON is administered to the central nervous system, optionally via an intrathecal delivery.

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