Antisense oligonucleotides for the treatment of neurological disorders

Abstract

This disclosure relates to the field of diseases caused by reduced synaptic inhibition, preferably diseases caused by reduced activity of the potassium (K) / chloride (Cl) cotransporter (KCC2). This disclosure relates to oligonucleotides in RNA editing methods and their use in targeting adenosine in a codon encoding a phosphorylation site in the SLC12A5 mRNA precursor or mRNA encoding KCC2, preferably adenosine in a codon encoding threonine at position 1007 of the KCC2b isoform. Through editing, threonine is replaced by alanine, thereby removing the phosphorylation site and thereby increasing the activity of the KCC2 protein in a process that restores its GABAergic inhibitory tendency. This disclosure further relates to oligonucleotides for use in the treatment of chronic pain and epilepsy.

Description

[Technical Field]

[0001] Cross-referencing and incorporation by reference of related applications This PCT application claims priority to U.S. Provisional Application No. 63 / 492,019, filed on 24 March 2023, which is incorporated in its entirety by reference herein.

[0002] Sequence listing reference Electronic submission The contents of the electronically submitted XML-format ST.26 sequence listing (name: 0058WO01ORD_20240315xmL.xml, size: 893,403 bytes, creation date: March 18, 2023) filed together with this application are incorporated in their entirety by reference herein.

[0003] This disclosure relates to the field of medicine, more specifically to the field of neurological disorders. This disclosure describes antisense oligonucleotides that mediate nucleotide-specific RNA editing in the human SLC12A5 gene transcript, resulting in amino acid changes of the encoded KCC2 protein that affect its activity. [Background technology]

[0004] Fast synaptic transmission depends on ion flux through ligand-opening channels. Therefore, maintaining a transmembrane ion gradient is essential for maintaining synaptic efficacy. In the central nervous system (CNS) of adult vertebrates, potassium (K) / chloride (Cl) cotransporters (KCC2) are strongly enriched in neurons, and Cl ions (Cl) are present. - ) continuously expels, thereby enabling the Cl necessary for inhibitory γ-aminobutyric acid (GABA) agonist and glycinergic neurotransmission. - Ensure that intracellular levels of Cl are kept low. -The gradient determines whether GABA receptors and glycine receptors (GlyRs) generate inhibitory or excitatory signals in the brain. In developed CNS, maintaining the inhibitory tone of GABA receptors and GlyRs is crucial for normal neurological function. Disruption of inhibitory signaling is associated with a wide range of neurological and psychiatric disorders. A major mechanism that has emerged in understanding the underlying mechanisms of disinhibition is the loss of KCC2 activity resulting from Cl - It involves the disruption of homeostasis. This mechanism is associated with spinal cord injury (Coull JA et al. 2003. Nature 424(6951):938-942), inflammation (Lu Y et al. 2008. J Physiol (Lond.) 586(Pt 23):5701-5715), and painful diabetic neuropathy (Jolivalt CG et al. 2008. Pain 140(1):48-57), trigeminal pain (Wei B et al. 2013. Neuroscience 228:334-348), morphine-induced hyperalgesia (Ferrini F et al. 2013. Nat Neurosci. 16(2):183-192), and epilepsy (Cohen I et al. 2002. Science 298:1418-1421, Huberfeld G. It appears across several pathological pain syndromes with diverse etiologies, including (et al. 2007. J Neurosci. 27(37):9866-9873). KCC2 is Cl - Because it is responsible for elimination, the disruption of its function leads to transmembrane Cl - Slope collapse and GABA A Reverse potential (E GABA) results in depolarizing shifts, which in turn lead to a decrease in inhibitory efficacy. In chronic pain, GABAergic transmission is impaired, causing circuit dysfunction and disrupting inhibitory neural circuitry. Furthermore, in chronic pathological pain, KCC2 expression has been found to be attenuated in primary sensory gates in neurons of the spinal cord dorsal horn (SCDH). Thus, it is well established that this major pathophysiological mechanism contributes to excitation / inhibition imbalance by disrupting inhibitory neurotransmission and causing dysfunction of inhibitory circuits. Notably, there is no "backup" protein that can rescue KCC2 expression deficiency.

[0005] There are two isoforms of KCC2, namely KCC2a and KCC2b, which arise from alternative transcriptional start sites within the human SLC12A5 gene. These transcripts are translated into two protein isoforms with different N-termini, and the KCC2a form constitutes the larger of the two splice variants. While KCC2a levels are maintained relatively constant during prenatal and postnatal development, KCC2b is barely present during prenatal development and is strongly upregulated during postnatal development. The upregulation of KCC2b expression is thought to mediate the "developmental shift" from depolarizing postsynaptic effects of inhibitory synapses in early neural circuitry to hyperpolarizing effects in mature neural circuitry, which is observed in mammals. Cl in neurons - In addition to its function in regulating homeostasis, the activity of KCC2 has been established to be also associated with transmembrane water fluxes that compensate for solute fluxes related to synaptic activity. Furthermore, the interaction between KCC2 and the actin cytoskeleton is thought to be important for both dendritic spine morphogenesis and the maintenance of glutamatergic synapses (Chamma I et al. 2012. Front Cell Neurosci. 6:5). KCC2b knockout mice can survive until day 17 postnatal due to the presence of only functional KCC2a, but these show low body weight, motor impairment, and generalized seizures. Complete KCC2 knockout in which neither KCC2a nor KCC2b is present results in postnatal death due to respiratory failure.

[0006] The lower inhibitory tendency suggests that enhancing KCC2 activity could potentially be used as a treatment for a wide variety of neurological disorders in which disease exacerbation or underlying causes occur. For example, increasing KCC2 function has been proposed for the treatment of pathogenic pain (Doyon N et al. 2013. Expert Rev Neurother. 13(5):469-471). Here, enhancing inhibitory signaling through increasing KCC2 function is observed in GABAergic pain. A It reduces the deficiency of and glycine inhibitory signaling (Lorenzo LE et al. 2020. Nature Communications 11:869). In epilepsy in which seizures are induced by increased excitatory neuronal activity or decreased inhibitory tendency, increasing KCC2 has been proposed as a therapeutic strategy (Moore YE et al. 2017. Trends Neurosci. 40(9):555-571). For example, genetic removal of phosphorylation sites that impair KCC2 activity has been shown to be sufficient to limit the onset and severity of seizures (Moore YE et al. 2018. Proc Natl Acad Sci USA. 115(40):10166-10171). Furthermore, impaired GABAergic inhibitory function has been observed in neurodevelopmental disorders such as autism spectrum disorder (ASD) and Rett syndrome (a severe form of ASD) (Tyzio R et al. 2014. Science 343(6171):675-679, Tang X et al. 2016. Proc Natl Acad Sci USA. 113(3):751-756). Significantly lower KCC2 expression was observed in postmortem brain samples from patients with Rett syndrome (Hinz L et al. 2019. Acta Neuropathol Commun. 7(1):196), and it has been shown that restoring KCC2 function in neurons derived from patients with Rett syndrome restores the functional deficits observed in these neurons (Tang et al. 2016). [Overview of the project]

[0007] Despite the use of the small molecule KCC2 "activator" CLP257, which does not appear to act on KCC2 itself, and despite numerous attempts to increase KCC2 activity in individuals suffering from neurological disorders such as chronic pain and epilepsy, the need for alternative methods to treat these severe quality-of-life impairments remains. This disclosure aims to provide such alternative and / or improved compounds and compositions for use in the treatment of neurological disorders in which increased KCC2 activity is beneficial.

[0008] Brief overview Disclosed herein are RNA-editing oligonucleotides (EONs) capable of forming a double-strand complex with a region of an endogenous human SLC12A5 transcript molecule in a cell, wherein the region of the SLC12A5 transcript molecule contains a target adenosine, and the double-strand complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine (A) to inosine (I), thereby editing the SLC12A5 transcript molecule. Preferably, the SLC12A5 transcript molecule is an mRNA precursor or mRNA molecule, and preferably, the SLC12A5 transcript molecule has a wild-type sequence. In a preferred embodiment, target A is in a codon encoding a phosphorylated amino acid, and more preferably, target A is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5, according to the sequence referenced by NCBI reference sequence number NP_065759.1. In another embodiment, target A is the first nucleotide of the codon encoding the threonine at position 1030 of the KCC2a isoform, which is encoded by SLC12A5, referenced by NCBI reference sequence number NP_001128243.1.

[0009] Also disclosed herein are EONs for use in treating disorders caused by reduced GABAergic inhibition, preferably disorders caused by reduced KCC2 activity, preferably EONs in which the disorder is chronic pain or epilepsy.

[0010] Also disclosed herein is a method for editing SLC12A5 polynucleotides, comprising contacting SLC12A5 polynucleotides with EON, which can induce ADAR-mediated editing of target A in a codon encoding an amino acid related to the phosphorylation of the protein KCC2 encoded by SLC12A5, from A to I, thereby editing the SLC12A5 polynucleotides. Also disclosed herein is a method for treating impairment caused by reduced GABAergic inhibition, preferably by reduced KCC2 activity, in an individual in need thereof, comprising contacting SLC12A5 polynucleotides in cells of interest with EON, which can induce ADAR-mediated editing of target A in a codon encoding an amino acid related to the phosphorylation of the protein KCC2 encoded by SLC12A5, thereby treating the individual. Also disclosed is a method for deaminating target A in an SLC12A5 mRNA precursor or mRNA molecule in a cell, comprising the steps of (i) providing the cell with the EON disclosed herein, (ii) causing the cell to take up the EON, (iii) annealing the EON with the SLC12A5 mRNA precursor or mRNA molecule, (iv) having an endogenous ADAR enzyme deaminate target A in the target RNA molecule to I, and optionally, (v) identifying the presence of I in the target RNA molecule.

[0011] One or more embodiments are described below, for illustrative purposes only, with reference to the accompanying drawings. [Brief explanation of the drawing]

[0012] [Figure 1A-1] The top of the figure shows the sequence (5' to 3', SEQ ID NO: 105) of the human SLC12A5 mRNA (precursor) target transcript, with target A shown in bold and the codon encoding threonine underlined. In Figure 1A, below the target sequence, the sequences (likewise from 5' to 3') of the initial 78 edited oligonucleotides (EON B1-B78) designed to result in editing of target A are shown. The SEQ ID NOs of each modified EON are shown in parentheses. The chemical modifications in EON are as follows: m5Ce is 2'-MOE modified 5-methylcytidine, m5Ue is 2'-MOE modified 5-methyluridine (Te is 2'-MOE modified thymidine), Ge is 2'-MOE modified guanosine, Ae is 2'-MOE modified adenosine, Gm, Am, Um, and Cm are 2'-OMe modified guanosine, adenosine, uridine, and cytidine, respectively, Af, Uf, Gf, Cf, and If are 2'-F modified adenosine, uridine, guanosine, cytosine, and inosine, respectively, and Zd is Benner's base Also called nucleosides (further outlined herein), these are cytidine analogs having a deoxy moiety (=DNA) at the 2'-ribose position, where Id is deoxyinosine, * indicates a phosphorothioate (PS) linkage, ! indicates a (1,3-dimethylimidazolidined-2-ylidene)phosphoamidate (PNdmi) linkage, and ^ indicates a methylsulfonate (MP) linkage. All other linkages are phosphate diester (PO) linkages. [Figure 1A-2] (Continuation of Figure 1A) [Figure 1A-3] (Continuation of Figure 1A) [Figure 1B]The top of the figure shows the sequence (5' to 3', SEQ ID NO: 105) of the human SLC12A5 mRNA (precursor) target transcript, with target A shown in bold and codons encoding threonine underlined. Figure 1B shows the nucleotide sequences (SEQ ID NOs: 79-104) of the edited oligonucleotides from Figure 1A, which have no chemical modifications other than at the Zd(Z) and Id(I) positions. SEQ ID NO: 82 is also the sequence of EONs from SEQ ID NOs: 141-147, which have no chemical modifications other than at the Zd(Z) and Id(I) positions. SEQ ID NO: 184 is also shown, which is the sequence of EONs from SEQ ID NOs: 154, 155, and 156, which have no chemical modifications other than at the Zd(Z) and Id(I) positions. [Figure 2A] Figure 1A shows the percentage of editing over time in an in vitro biochemical editing assay using EON B1-B20 and SLC12A5 transcript RNA generated in vitro, with the addition of purified ADAR enzyme. Figure 2A shows the percentage of editing for EON B1, B2, B3, B4, and B5. [Figure 2B] Figure 1A shows the percentage of editing over time in an in vitro biochemical editing assay using EON B1-B20 and SLC12A5 transcript RNA generated in vitro, with the addition of purified ADAR enzyme. Figure 2B shows the percentage of editing for EON B6, B7, B8, B9, and B10. [Figure 2C] Figure 1A shows the percentage of editing over time in an in vitro biochemical editing assay using EON B1-B20 and SLC12A5 transcript RNA generated in vitro, with the addition of purified ADAR enzyme. Figure 2C shows the percentage of editing for EON B6, B11, B12, and B13. [Figure 2D] Figure 1A shows the percentage of editing over time in an in vitro biochemical editing assay using EON B1-B20 and SLC12A5 transcript RNA generated in vitro, with the addition of purified ADAR enzyme. Figure 2D shows the percentage of editing for EON B6, B14, B15, B16, and B17. [Figure 2E] This is a graph showing the percentage of editing over time in an in vitro biochemical editing assay using EON B1 - B20 shown in FIG. 1A and SLC12A5 transcript RNA generated in vitro, with the addition of purified ADAR enzyme applied. FIG. 2E shows the percentage of editing of EON B18, B19, and B20. [Figure 3A] This is a graph showing the percentage of editing measured 14 days after the first screening using gymnotic uptake of EON from the culture medium during EON treatment in human retinal organoids cultured for 200 days. The EONs tested are as mentioned in each figure. FIG. 3A shows the percentage of editing of EON B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, and B13 shown in FIG. 1A. [Figure 3B] This is a graph showing the percentage of editing measured 14 days after the first screening using gymnotic uptake of EON from the culture medium during EON treatment in human retinal organoids cultured for 200 days. The EONs tested are as mentioned in each figure. FIG. 3B shows the percentage of editing of EON B6, B15, B16, B17, B18, B19, and B20 shown in FIG. 1A. [Figure 4] This is a graph showing the percentage of editing measured 14 days after the second screening using gymnotic uptake of EON from the culture medium during EON treatment in human retinal organoids cultured for 200 days. The EONs tested are B3, B14, B21, B22, B23, B24, B25, B26, B27, B28, B29, B30, B31, B32, B33, B34, B35, B36, B37, B38, B39, and B40 shown in FIG. 1A, and B3 was also used in the screening shown in FIG. 3A. [Figure 5]Figure showing the percentage of editing in HEK cells stably overexpressing human KCC2, 24 hours after transfection of the EONs into the cells, using the 78 EONs shown in Figure 1A. Figure 5A shows the results using B1 - B40, and Figure 5B shows the results using B41 - B78, both using mock transfection as the negative control. [Figure 6] Figure showing the percentage of editing 48 hours after transfection in HEK - KCC2 cells transfected with the indicated EONs. Using the same transfected cell samples, the effect on the amount of phosphorylated KCC2 during transfection with the specified EONs was determined. Figure 6B shows the normalized phosphorylated KCC2 (herein referred to as target B), divided by total KCC2, compared to mock - transfected cells, which is set to 100 here. The normalized values are shown within each bar. [Figure 7] Figure showing the percentage of editing in human iPSC neurons after 2 - week exposure to the indicated EONs using a wash - out procedure, using the 78 EONs shown in Figure 1A. Figure 7A shows the results using B1 - B40, and Figure 7B shows the results using B41 - B78, both using non - treated (NT) samples as the negative control. EON B51 was not available at the time of the experiment, and the percentage of editing for this EON is not provided. [Figure 8] Figure showing a set of EONs (B122 - B137, B140, and B141, their respective sequence numbers are shown within square brackets) based on EON B4 shown at the top (see Figure 1A). The EONs have various 2′ - F modifications throughout the design. The 2′ - F - modified nucleotides are given in gray boxes. The chemical modifications are as provided in Figure 1A. [Figure 9] Figure showing the percentage of editing obtained in human iPSC neurons treated with dimmunox using the EONs provided in Figure 8, using a 2 - week wash - out treatment. Non - treated (NT) samples were included as the negative control. [Figure 10]This figure shows a set of EONs (B1030-144 to B1030-172, also known as B144 to B172 respectively, with their respective sequence numbers shown in square brackets) largely based on the design of B137 (see Figure 8). The EONs have various 2'-F modifications, mismatches / fluctuations, PNdmi linkages, and 2'-deoxy modifications at the various positions shown. The chemical modifications are as provided in Figure 1A. [Figure 11] This figure shows the percentage of editing obtained in human iPSC neurons treated with gymnosis using the EON provided in Figure 10, as shown, using a two-week washout experiment. Untreated samples and samples with an equivalent rat Slc12a5 target sequence (rB1030-144) specific EON were included. B1030-152 was not initially manufactured and therefore was not tested. [Figure 12] This figure shows a set of EON sequences (each sequence number shown in square brackets) designed to target an equivalent A in the rat Slc12a15 transcript (compared to a human transcript) in vivo, similar to the change from a threonine codon to an alanine codon at position 1007. The EON names are similar to those of their equivalent EONs used to target human transcript molecules. For example, rB1030-4 has the same chemical modification as B4 in Figure 1A, but at position +14, it contains 2'-MOE modified adenosine (Ae, underlined) instead of 2'-MOE modified guanosine (Ge). [Figure 13] As shown, this figure shows the percentage of editing in the lumbar spinal cord of rats two weeks after single-dose intrathecal administration (directly into the spinal cord) of 300 μg of EON. As described in the examples, HD indicates a higher dose. All EONs provided in Figure 12 were tested together with three EONs (B-70, B-74, and B-145) complementary to the human SLC12A15 target sequence. Many injections were off-site and incidentally outside the spinal cord, which gives 0 editing. These misinjections were not included in the editing calculation. Artificial cerebrospinal fluid (aCSF), which was also the buffer in which the EONs were dissolved, served as a negative control. [Modes for carrying out the invention]

[0013] The KCC2 protein is extensively post-transcriptionally modified, and the functional properties of KCC2 are mutually regulated by serine / threonine phosphorylation. One site that is post-translationally phosphorylated is the threonine residue at position 1007 in the human KCC2b isoform (see NCBI reference sequence number NP_065759.1), which is equivalent to the threonine at position 1030 in the human KCC2a isoform (see NCBI reference sequence number NP_001128243.1). Since the targeting discussed herein is primarily for transcripts present in mature neurons, the target threonine is generally referred to as being at position 1007 (in KCC2b), but it should be understood that the equivalent threonine at position 1030 in KCC2a can also be altered using the compounds and compositions disclosed herein, and that when this disclosure refers to targeting threonine (or adenosine in a codon encoding threonine), both isoforms are included. Phosphorylation at this site has been shown to lead to decreased KCC2 channel activity and reduced inhibitory tendency (Pisella LI et al. 2019. Sci Signal. 12(603):eaay0300). Conversely, disruption of this phosphorylation site has been shown to enhance KCC2 function and subsequently enhance inhibitory signaling (Weber M et al. 2014. J Biol Chem 289(27):18668-18679, Moore et al. 2018., cited above). Together, these led us to recognize that post-transcriptional alteration of the threonine at this position to a different residue (one that cannot be phosphorylated) would, in principle, make the KCC2 protein more active in its inhibitory effect, and thereby allow for the potential treatment of disorders in which KCC2 activity is reduced through any of the post-translational modification processes, such as decreased expression, loss-of-function mutations, or increased phosphorylation of the threonine at position 1007.As mentioned above, due to the numerous impairments caused by reduced KCC2 activity, if KCC2 activity can be (transiently) upregulated to achieve a greater inhibitory effect, even if the human SLC12A5 gene encoding KCC2 is wild-type, any of these impairments can potentially be treated.

[0014] The technology envisioned by the inventors is generally called "RNA editing," in which a specific adenosine present in a transcript molecule, such as an mRNA precursor or mRNA molecule, is deaminated to inosine, which is then observed as guanosine by the translation mechanism. Therefore, by editing the ACC codon of the threonine residue at position 1007 in KCC2b (or position 1030 in KCC2a) to an ICC codon (=GCC) in the transcript, the resulting protein contains an alanine residue instead of threonine at this position, and the protein can no longer be phosphorylated at this site. It should be noted that the change from threonine to alanine is not a mutation identified in nature to date, but the inventors recognized that this change would reflect a gain-of-function modification of a protein that cannot be technically induced in human subjects through the knock-in procedure outlined above, as described by Moore et al. (2018), or by gene therapy. RNA editing technology offers a unique transient method for modifying the KCC2 protein in the CNS of human individuals in need, without altering the individual's genome, preferably in the treatment of (chronic) pain and / or epileptic seizure (epilepsy) disorders. This also makes it possible to treat CNS disorders in which the KCC2 protein is attenuated in its expression or mutates toward loss-of-function mutants, such as those identified by Stoedberg and colleagues (2015. Nat Commun 6:8038) and Saitsu and colleagues (2016. Sci Rep 6:30072).

[0015] In one embodiment, the disclosure relates to an EON used to specifically induce in vivo deamination of a particular target adenosine in the transcript of a (human) mutant SLC12A5 transcript (mRNA precursor and / or mRNA) using an endogenous deaminationase (see below) to produce a KCC2 protein that is not phosphorylated at the codon encoding where adenosine was previously located. As a result, the resulting KCC2 protein (whether KCC2a and / or KCC2b isoforms) has enhanced inhibitory signaling function.

[0016] RNA editing is a natural process by which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise manner, thereby increasing the repertoire of RNA encoded by the genome by several orders of magnitude. RNA editing enzymes have been described for eukaryotes across the animal and plant kingdoms, and these processes play a crucial role in maintaining cellular homeostasis in metazoans, from the simplest organisms (such as nematodes (Caenorhabditis elegans)) to humans. Examples of RNA editing include the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which occur via enzymes called adenosine deaminases acting on RNA (ADAR) and APOBEC / AID (cytidine deaminases acting on RNA), respectively.

[0017] ADAR is a multi-domain protein containing one catalytic domain and, depending on the enzyme, two to three double-stranded (ds)RNA recognition domains. Each recognition domain recognizes a specific dsRNA sequence and / or conformation. While the catalytic domain also plays a role in recognizing and binding to portions of the dsRNA helix, its primary function is to convert A to I at predefined positions near the target RNA by deamination of nucleic acid bases. As mentioned above, inosine is read as guanosine by the cell's translation mechanism, meaning that if edited adenosine is present in the coding region of mRNA or mRNA precursor, this can recode the protein sequence. The A-to-I conversion can also occur in the 5' non-coding sequence of the target mRNA, sometimes creating a new translation start site upstream of the original start site, resulting in an N-terminally extended protein or 3'UTR or other non-coding portion of the transcript, which can affect RNA processing and / or stability. Furthermore, the conversion from A to I occurs in the splicing elements of introns or exons in the mRNA precursor, thereby altering the splicing pattern. As a result, exons may be included or skipped. Enzymes that catalyze adenosine deamination are located within the ADAR enzyme family, which includes the human deaminationases hADAR1 and hADAR2, as well as hADAR3. However, deamination activity of hADAR3 has not been demonstrated.

[0018] The use of antisense oligonucleotides to edit target RNA by applying adenosine deaminose has been described (e.g., Woolf et al. 1995. PNAS 92:8298-8302, Montiel-Gonzalez et al. PNAS 2013, 110(45):18285-18290, Vogel et al. 2014. Angewandte Chemie Int Ed 53:267-271). A disadvantage of the method described by Montiel-Gonzalez et al. (2013) is that it requires a fusion protein consisting of the box B recognition domain of bacteriophage lambda N-protein genetically fused with the adenosine deaminose domain of the cleaved native ADAR protein. This requires either transducing the target cells using a fusion protein (which is a major obstacle) or transposing the target cells using a nucleic acid construct encoding a modified adenosine deaminose fusion protein for expression. The system described by Vogel et al. (2014) suffers from a similar drawback in that it is unclear how to apply this to a system that does not require first genetically modifying ADAR and then transposing or transforming cells with the target RNA in order to provide cells with the genetically modified protein. U.S. Patent No. 9,650,627 describes a similar system. The oligonucleotides of Woolf et al. (1995), which are 100% complementary to the target RNA sequence, suffer from a severe lack of specificity, as almost all of the adenosine in the target RNA chain complementary to the oligonucleotide is edited out.

[0019] ADARs are known to act on any dsRNA. Through a process sometimes called "promiscuous editing," the enzyme edits multiple adenosines in the dsRNA. Therefore, there has been a need for methods and means to avoid such promiscuous editing and target only specific adenosines in the target RNA molecule to be therapeutically available. Vogel et al. (2014) showed that such off-target editing can be suppressed by using a 2'-O-methyl (2'-OMe) modified nucleoside in the oligonucleotide at a position opposite the adenosine that should not be edited, and an unmodified nucleoside directly opposite the specific targeted adenosine on the target RNA. However, it has not been shown that a specific editing effect at the target nucleotide occurs without the use of a recombinant ADAR enzyme covalently bonded to the oligonucleotide.

[0020] Currently, several publications have shown that the recruitment of endogenous ADARs (and therefore without the need for exogenous and / or recombinant sources) is possible while maintaining specificity that allows for the targeting and deamination of a single adenosine within a target RNA molecule to inosine. International Patent Application Publication No. 2016 / 097212, which is incorporated in its entirety by reference herein, discloses oligonucleotides for targeted editing of RNA, characterized by the presence of a sequence complementary to the target RNA sequence (referred to therein as the “targeting moiety”) and, preferably, a stem-loop (or hairpin) structure (referred to therein as the “recruiting moiety”) that is non-complementary to the target RNA. Such oligonucleotides are called “self-loop-forming oligonucleotides”. The recruiting moiety acts to recruit a native ADAR enzyme present in the cell to a dsRNA formed by the hybridization of the target sequence and the targeting moiety. Thanks to the recruiting moiety, neither a conjugated entity nor the presence of a modified recombinant ADAR enzyme is required. International Patent Application Publication No. 2016 / 097212, which is incorporated in its entirety by reference herein, describes the recruiting portion as a Z-DNA structure known to be recognized by the native substrate of ADAR enzymes (e.g., the GluB receptor) or a dsRNA-binding domain, or a stem-loop structure that mimics either the Z-DNA-binding domain or the native substrate of the ADAR enzyme. The stem-loop structure may be an intermolecular stem-loop structure formed by two separate nucleic acid strands, or an intramolecular stem-loop structure formed within a single nucleic acid strand. The stem-loop structure of the recruiting portion described is an intramolecular stem-loop structure formed within the oligonucleotide itself and is thought to attract (endogenous) ADAR. Similar stem-loop structures for RNA editing are described in International Patent Applications Nos. 2017 / 050306, 2020 / 001793, 2017 / 010556, 2020 / 246560, and 2022 / 078995, all of which are incorporated herein in their entirety by reference.

[0021] International Patent Applications Nos. 2017 / 220751 and 2018 / 041973, which are incorporated by reference in their entirety, describe next-generation oligonucleotides that do not contain such stem-loop structures and are (almost completely) complementary to the target region. In one embodiment, one or more mismatched nucleotides, fluctuations, or bulges are present between the oligonucleotide and the target sequence. A single mismatch may be at the nucleoside site opposite the target adenosine, while in other embodiments, the oligonucleotide (often called "RNA editing oligonucleotides" and abbreviated as "EONs," which themselves do not possess enzymatic deamination or editing activity) is described to have multiple bulges and / or fluctuations when attached to the target sequence region. It was considered possible to achieve in vitro, ex vivo, and in vivo RNA editing using EONs lacking stem-loop structures, and using endogenous ADAR enzymes when the sequence of the EON is carefully selected to attract / mobilize ADARs. An "orphan nucleoside," defined as a nucleoside in the EON located directly opposite the target adenosine in the target RNA molecule, did not have a 2'-OMe modification. The orphan nucleoside could be a deoxyribonucleoside (DNA) with no substitutions at the 2' position of the ribose sugar moiety, while the rest of the EON could still have a 2'-O-alkyl modification (such as 2'-OMe) to its ribose sugar. The nucleotides directly surrounding the orphan nucleoside contained chemical modifications (including being DNA and not RNA) that further improved RNA editing efficiency and / or increased resistance to nucleases. Such effects could be further enhanced by using sense oligonucleotides (SONs) that protect the EON from degradation (as described in International Patent Application Publication No. 2018 / 134301). The use of chemical modifications and specific structures in oligonucleotides that can be used in ADAR-mediated editing of specific adenosines in target RNA has been the subject of numerous publications in this field, for example,All of these are incorporated herein by reference in their entirety by international patent applications Nos. 2019 / 111957, 2019 / 158475, 2020 / 165077, 2020 / 201406, 2020 / 211780, 2021 / 008447, 2021 / 020550, 2021 / 060527, and 2021 / 1177. These include specifications No. 29, No. 2021 / 136408, No. 2021 / 182474, No. 2021 / 216853, No. 2021 / 242778, No. 2021 / 242870, No. 2021 / 242889, No. 2022 / 007803, No. 2022 / 018207, No. 2022 / 026928, and No. 2022 / 124345, among others. The use of specific sugar moieties is disclosed, for example, in International Patent Applications Nos. 2020 / 154342, 2020 / 154343, 2020 / 154344, 2022 / 103839, and 2022 / 103852, which are incorporated by reference in their entirety herein, while the use of sterically defined linker moieties (generally, for example, for exon skipping related to a wide variety of target sequences, in gapmers, in siRNA, or especially for RNA editing oligonucleotides) is disclosed in International Patent Application No. 2011 / 0057, which is incorporated by reference in their entirety herein. Specification No. 61, Specification No. 2014 / 010250, Specification No. 2014 / 012081, Specification No. 2015 / 107425, Specification No. 2017 / 015575 (HTT ), Specification No. 2017 / 062862, Specification No. 2017 / 160741, Specification No. 2017 / 192664, Specification No. 2017 / 192679 (DMD), No. 20 Specification No. 17 / 198775, Specification No. 2017 / 210647, Specification No. 2018 / 067973, Specification No. 2018 / 098264, Specification No. 2018 / 223056 Specification (PNPLA3), Specification No. 2018 / 223073 (APOC3), Specification No. 2018 / 223081 (PNPLA3), Specification No. 2018 / 237194,Specification No. 2019 / 032607 (C9orf72), Specification No. 2019 / 055951, Specification No. 2019 / 075357 (SMA / ALS), Specification No. 2019 / 200185 (DM1), No. 2019 / 21778 Specification No. 4 (DM1), Specification No. 2019 / 219581, Specification No. 2020 / 118246 (DM1), Specification No. 2020 / 160336 (HTT), Specification No. 2020 / 191252, Specification No. 2020 / 196662 This is described in the specifications, specifications 2020 / 219981 (USH2A), 2020 / 219983 (RHO), 2020 / 227691 (C9orf72), 2021 / 071788 (C9orf72), 2021 / 071858, 2021 / 178237 (MAPT), 2021 / 234459, 2021 / 237223, and 2022 / 099159. Following these disclosures, a great number of publications relate to the targeting of specific RNA target molecules, or specific adenosines within such RNA target molecules, to repair mutations that result in immature stop codons or other disease-causing mutations. Examples of disclosures in which adenosine is specifically targeted within a target RNA molecule are International Patent Applications Nos. 2020 / 157008 and 2021 / 136404 (USH2A), 2021 / 113270 (APP), 2021 / 113390 (CMT1A), 2021 / 209010 (IDUA, Hurler syndrome), 2021 / 231673 and 2021 / 242903, which are incorporated herein by whole reference. Specification (LRRK2), Specification No. 2021 / 231675 (ASS1), Specification No. 2021 / 231679 (GJB2), Specification No. 2019 / 071274 and Specification No. 2021 / 231680 (MECP2), Specification No. 2021 / 231685 and Specification No. 2021 / 231692 (OTOF, Autosomal Recessive Non-Symptomatic Hearing Loss), Specification No. 2021 / 231691 (XLRS), Specification No. 2021 / 231698 (Argininosuccinate Desorption),These are specification No. 2021 / 130313 and No. 2021 / 231830 (ABCA4), and No. 2021 / 243023 (Serpin A1).

[0022] Disclosed herein are RNA-editing oligonucleotides (EONs) capable of forming a double-stranded complex with a region of an endogenous human SLC12A5 transcript molecule in a cell, wherein the region of the SLC12A5 transcript molecule contains target adenosine, the nucleotide in the EON directly opposite the target adenosine is an orphan nucleotide, the nucleotide counting in the EON is such that the orphan nucleotide is number 0, and the nucleotides 5' to the orphan nucleotide are positively (+) incremented toward the 5' end and negatively (-) incremented toward the 3' end, and the double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the SLC12A5 transcript molecule. Preferably, the SLC12A5 transcript molecule is an mRNA precursor or an mRNA molecule. In one embodiment, the SLC12A5 transcript molecule has a wild-type sequence. In one embodiment, the target adenosine is located in a codon encoding an amino acid that can be phosphorylated. Preferably, the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5. In an alternative embodiment, the target adenosine is the first nucleotide of the codon encoding threonine at position 1030 of the KCC2a isoform encoded by SLC12A5. In one embodiment, deamination of the target adenosine results in the KCC2 protein encoded by SLC12A5 having increased activity. Preferably, the increased activity results in higher GABAergic inhibition. In one embodiment, the cells in which editing of the SLC12A5 transcript occurs are (human) neurons, preferably (human) brain cells. In one embodiment, EON is selected from the group consisting of SEQ ID NOs: 1-104 and 116-164.In a preferred embodiment, EON is selected from the group consisting of Sequence IDs 3, 4, 14, 15, 22, 23, 28, 29, 33, 34, 35, 36, 40, 55, 63, 65, 69, 70, 73, 74, 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, 116, 119, 124, 127, 128, 129, 130, 131, 136, 137, 141, 142, 143, 145, 146, 147, 153, 154, 155, 156, and 184. In a preferred embodiment, the orphan nucleotide disclosed herein is an EON that is not cytidine containing a 2'-OMe ribose substitution, having at least one non-naturally occurring chemical modification and / or containing one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion. In a preferred embodiment, when the EON disclosed herein is produced in a manufacturing facility or laboratory, the orphan nucleotide is a deoxynucleotide containing a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base (also called Benner's base or Z), and the nucleotide at position -1 in the EON is deoxyinosine (Id). In a preferred embodiment, one or more additional modifications in the linkage are independently selected from internucleotide linkages of phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylsulfonate (MP or MeP), sulfonyl phosphoramidate, mesyl phosphoramidate (PNms), or (1,3-dimethylimidazolidined-2-ylidene)phosphoamidate (PNdmi). In a preferred embodiment, one or more additional modifications in the ribose moiety are independently selected from substituted or unsubstituted linear or branched lower (C1-C) molecules, which may be interrupted by -OH, -F, or one or more heteroatoms. 10) One or two substitutions at the 2', 3', and / or 5' positions of ribose, selected from the group consisting of alkyl, alkenyl, alkynyl, alkaryl, -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, -dimethylaminooxyethoxy, and -dimethylaminoethoxyethoxy. Also disclosed herein are vectors comprising nucleic acid molecules encoding an EON containing a sequence of any one of SEQ ID NOs: 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, and 184, preferably viral vectors, more preferably adeno-associated virus (AAV) vectors, wherein the orphan nucleotide is cytidine or uridine and the nucleotide at position -1 of the EON is guanosine. Also disclosed herein are pharmaceutical compositions comprising the EON or vector disclosed herein and a pharmaceutically acceptable carrier. This disclosure also relates to the EON or vector disclosed herein for use in treating disorders caused by reduced GABAergic inhibition, preferably caused by reduced KCC2 activity. In a preferred embodiment, the disorder is chronic pain or epilepsy. The disclosure also relates to the use of EON or vectors disclosed herein in the manufacture of pharmaceuticals for the treatment of disorders caused by reduced GABAergic inhibition, preferably by reduced KCC2 activity. In a preferred embodiment, the disorder is chronic pain or epilepsy.

[0023] The disclosure also relates to a method for editing an SLC12A5 polynucleotide, comprising contacting the SLC12A5 polynucleotide with an EON capable of resulting in ADAR-mediated editing of a target adenosine in a codon encoding an amino acid associated with the phosphorylation of the protein KCC2 encoded by SLC12A5, thereby editing the SLC12A5 polynucleotide. The disclosure also relates to a method for treating a disorder caused by reduced GABAergic inhibition, preferably caused by reduced KCC2 activity, in an individual in need thereof, comprising contacting an SLC12A5 polynucleotide in a cell of interest with an EON capable of resulting in ADAR-mediated editing of a target adenosine in a codon encoding an amino acid associated with the phosphorylation of the protein KCC2 encoded by SLC12A5, thereby treating the individual. In a preferred embodiment, the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5, or the target adenosine is the first nucleotide of the codon encoding threonine at position 1030 of the KCC2a isoform encoded by SLC12A5. The disclosure also relates to a method for treating a disorder caused by reduced GABAergic inhibition, preferably a disorder caused by reduced KCC2 activity, comprising administering a therapeutically effective amount of the EON, vector, or pharmaceutical composition disclosed herein to an individual in need thereof. In a preferred embodiment, the disorder is chronic pain or epilepsy.

[0024] The disclosure also relates to a method for deaminating a target adenosine in an SLC12A5 mRNA precursor or mRNA molecule in a cell, comprising the steps of (i) providing the cell with the EON disclosed herein, (ii) allowing the cell to take up the EON, (iii) annealing the EON with the SLC12A5 mRNA precursor or mRNA molecule, (iv) having an endogenous ADAR enzyme (such as ADAR1 or ADAR2) deaminate the target adenosine in the target RNA molecule to inosine, and optionally, (v) identifying the presence of inosine in the target RNA molecule. In a preferred embodiment, the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5, and in an alternative embodiment, the target adenosine is the first nucleotide of the codon encoding threonine at position 1030 of the KCC2a isoform encoded by SLC12A5. In a preferred embodiment, step (v) includes a) determining the sequence of an SLC12A5 mRNA precursor or mRNA molecule; b) evaluating the presence of a KCC2 protein encoded by SLC12A5 having a lower phosphorylation rate, preferably the presence of a KCC2 protein lacking phosphorylation at position 1007 in the KCC2b isoform (or position 1030 in the KCC2a isoform); or c) evaluating the level of GABAergic inhibition in cells, preferably using a functional readout.

[0025] This disclosure also relates to a nucleic acid molecule for editing a target adenosine in a human SLC12A5 mRNA precursor or mRNA molecule, wherein the target region is Sequence ID No. 105, and the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 (or position 1030 in the KCC2a isoform) of the KCC2b isoform encoded by SLC12A5. In a preferred embodiment, the nucleic acid molecule is preferably selected from the group consisting of SEQ ID NOs: 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, and 184, wherein the orphan nucleotide, which is a nucleotide in the nucleic acid directly opposite the target adenosine in the target region, is not a cytidine containing a 2'-OMe ribose substitution. In a preferred embodiment, and as outlined above, one or more additional modifications in the ligation region are each independently selected from PS, phosphonoacetate, phosphorodithioate, MP, sulfonyl phosphoramidate, PNms, or PNdmi nucleotide ligations. This disclosure also relates to a vector comprising a nucleotide sequence encoding a nucleic acid molecule disclosed herein, wherein the orphan nucleotide is cytidine or uridine, and the nucleotide at position -1 in the EON is guanosine.

[0026] An EON capable of mediating RNA editing of a target adenosine in human SLC12A5 transcript (mRNA precursor and / or mRNA) is disclosed herein, through which the resulting KCC2 protein is mutated at a specific phosphorylation site. The absence of phosphorylation at this site increases the activity of the resulting (mutant) KCC2 protein. In a preferred embodiment, the EON induces deamination of adenosine at position 3149 in wild-type SLC12A5 mRNA encoding the KCC2b isoform (see NCBI reference sequence NM_020708.5), thereby producing inosine. Similarly, adenosine at position 3168 in wild-type SLC12A5 mRNA encoding the KCC2a isoform can be targeted (see NCBI reference sequence NM_001134771.2). In detail, the ACC codon encoding threonine (wild-type form) at amino acid position 1007 (KCC2b) is converted to an ICC codon, which is read as GCC encoding alanine (mutant form). In another embodiment, EON as herein causes the deamination of another adenosine present in the SLC12A5 transcript, which may be any adenosine that, when deaminated to inosine, results in a gain-of-function KCC2 protein. In another embodiment, EON as herein causes the mutation to result in the resulting KCC2 protein having a loss-of-function form, or one or more of its functional properties, preferably Cl - In the case of a G>A mutation that inactivates it in relation to leakage, it causes deamination of adenosine present in the mutant SLC12A5 transcript. Other mutations that can be targeted via RNA editing, thereby restoring normal KCC2 function, may be present in the SLC12A5 gene (and transcript).

[0027] In preferred embodiments, EON as herein is a single-stranded oligonucleotide comprising the orphan nucleotide as defined above, wherein the orphan nucleotide is chemically modified as disclosed herein, and the remainder of the oligonucleotide is also chemically modified to prevent nuclease degradation, also as disclosed herein. In other embodiments, this disclosure relates to any type of oligonucleotide or heterodouble-stranded oligonucleotide complex, which may or may not be bound to a hairpin structure (internal or terminal), may be bound to ADAR or its catalytic domain, or the oligonucleotide is expressed through a vector such as adeno-associated virus (AAV), or the oligonucleotide is in a cyclic form. It should be understood that RNA editing based on any type of oligonucleotide is encompassed by this disclosure when relating to the deamination of adenosine in the SLC12A5 transcript, preferably adenosine at position 1 in the codon encoding threonine at position 1007 in KCC2b. In a preferred embodiment, the EON herein is a “naked” oligonucleotide that includes various chemical modifications in one or more ribose sugars, bases, and / or internucleoside linkages among the nucleotides in the sequence, and can hybridize with an SLC12A5 transcript or a portion thereof containing a target adenosine, and can mobilize endogenous ADAR for deamination of the target adenosine. The endogenous ADAR enzyme is preferably human ADAR1 or ADAR2. The cell is preferably a human neuronal cell. The SLC12A5 transcript molecule is preferably an mRNA precursor or mRNA molecule. The EON herein preferably targets adenosine for deamination, which causes gain-of-function of the KCC2 protein. Although several mutations causing dysfunction of the KCC2 protein are known, the preferred adenosine targeted through the EON disclosed herein is the adenosine in the codon encoding the phosphorylation site in KCC2, and the resulting codon (after deamination of adenosine) is no longer the phosphorylation site.Loss of phosphorylation at this site in KCC2 increases its GABAergic inhibitory activity, thereby reducing the synchronization underlying abnormal neuronal activation (e.g., that causing chronic neurological pain) and epileptic seizures. The preferred threonine modified through editing of the SLC12A5 transcript is the threonine at position 1007 in the KCC2b isoform, which is modified to alanine, which can no longer be phosphorylated. The EONs herein can result in the deamination of adenosine in the ACC codon encoding threonine, thereby producing an ICC codon, which is read as GCC and translated to alanine. In one embodiment, the EONs herein include or consist of a sequence of any of the EON sequences (SEQ ID NOs. 1-78) shown in Figure 1A. In one embodiment, the EONs herein include or consist entirely of nucleotides, each having the chemical modifications mentioned in Figure 1A.

[0028] In one embodiment, the orphan nucleotide is cytidine, deoxycytidine, a cytidine analog (such as a nucleoside containing Benner's base), uridine, deoxyuridine, or a uridine analog (such as isouridine). In one embodiment, the EON contains at least one mismatch with the (duplicate) sequence of the target transcript molecule. If the orphan nucleotide is uridine, the EON does not necessarily contain a mismatch. A mismatch may be introduced into other parts of the EON if necessary, as long as the EON can hybridize with the target transcript under natural conditions.

[0029] In one embodiment, the EON as herein comprises at least one nucleotide containing one or more non-naturally occurring chemical modifications, or containing one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion, provided that the orphan nucleotide is not a cytidine containing a 2'-OMe-ribose substitution. In one embodiment, the EON as herein comprises one or more mismatches, fluctuations, or bulges, and a single mismatch may be present if the target adenosine has an opposite cytidine or uridine analog (not a perfect match compared to uridine) in the EON. If the orphan nucleotide is a cytidine, as described above, that cytidine does not contain a 2'-OMe-ribose substitution. Preferably, if the orphan nucleotide is not a cytidine, it does not contain a 2'-OMe-ribose substitution if it interferes with deamination by the ADAR enzyme.

[0030] A vector containing a nucleic acid molecule encoding EON as described herein, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, is disclosed herein. When EON is delivered by a viral vector or plasmid vector, the generated EON in cells is chemically unmodified. A pharmaceutical composition comprising the EON disclosed herein, or the viral vector or plasmid vector disclosed herein, and a pharmaceutically acceptable carrier is further disclosed herein.

[0031] In one embodiment, an EON, vector, or pharmaceutical composition for use in the treatment of a subject in need thereof is disclosed herein, which suffers from a disorder in which KCC2 inhibitory activity is reduced or absent, through any of the following: loss-of-function mutation, reduced expression of transcripts and / or proteins, or post-translational modifications such as inhibitory phosphorylation of a specific site in the (wild-type) protein (at a potentially increased rate). In one embodiment, an EON or vector for the manufacture of a pharmaceutical for the treatment of a disorder in a subject in which KCC2 inhibitory activity in neurons is reduced or absent, through any of the following: loss-of-function mutation, reduced expression of transcripts and / or proteins, or post-translational modifications such as inhibitory phosphorylation of a specific site in the (wild-type) protein (at a potentially increased rate).

[0032] In one embodiment, a method for editing an SLC12A5 polynucleotide is disclosed, comprising contacting the SLC12A5 polynucleotide with EON, which can result in an ADAR-mediated change from adenosine to inosine of adenosine in a codon encoding threonine, which is related to the phosphorylation of the resulting protein KCC2, thereby editing the SLC12A5 polynucleotide. The SLC12A5 polynucleotide is preferably an mRNA precursor or mRNA nucleic acid molecule. In one embodiment, reduced or diminished KCC2 activity (in its GABAergic inhibitory effect, particularly Cl from nerve cells) is disclosed. -A method for treating a disorder caused by activity at too low a level of leakage, or a disorder caused by a loss-of-function mutant of KCC2, is disclosed, comprising contacting an SLC12A5 polynucleotide in a cell of interest with an EON capable of causing an ADAR-mediated change from adenosine to inosine of adenosine in threonine at position 1007 in the codon encoding the phosphorylation site, preferably in KCC2b, and thereby treating the patient. In one embodiment, a method for treating epilepsy or pathological neurological (chronic) pain in a human subject in need is disclosed, comprising administering to the subject a therapeutically effective amount of the EON, vector, or pharmaceutical composition disclosed herein.

[0033] definition The term "nucleoside" refers to a nucleic acid base linked to a (deoxy)ribosyl sugar, without a phosphate group. A "nucleotide" consists of a nucleoside and one or more phosphate groups. Therefore, the term "nucleotide" refers to each nucleic acid base-(deoxy)ribosyl-phospholinker, as well as any chemical modifications of the ribose moiety or phospho group. Thus, this term includes nucleotides containing linkers such as locked ribosyl moieties (including 2'-4' crosslinks containing a methylene group or any other group), unlocked nucleic acids (UNAs), threose nucleic acids (TNAs), phosphate diesters, phosphonoacetates, phosphate triesters, PS, phosphoro(di)thioates, MPs, methylthiophosphonates, and phosphoramidate linkages. In some cases, the terms adenosine and adenine, guanosine and guanine, cytidine and cytosine, uracil and uridine, thymine and thymidine / uridine, inosine and hypoxanthine are used interchangeably, with one referring to the corresponding nucleic acid base and the other to the nucleoside or nucleotide. Thymine (T) is derived from 5-methyluracil (m 5Thymine, 5-methyluracil, and uracil, also known as 5-methyluridine, are uracil (U) derivatives and can be interchanged throughout this document. Similarly, thymidine, also known as 5-methyluridine, is a uridine derivative and can be interchanged throughout this document. In some cases, the terms nucleic acid base, nucleoside, and nucleotide are used interchangeably, except when it is clearly required by the context to be otherwise, for example, when a nucleoside is linked to an adjacent nucleoside and the linkage between these nucleosides is modified. As described herein, a nucleotide is a nucleoside + one or more phosphate groups. The terms “ribonucleoside” and “deoxyribonucleoside,” or “ribose” and “deoxyribose,” are used as they are used in the art.

[0034] Whenever oligonucleotides, oligos, ONs, ASOs, oligonucleotide compositions, antisense oligonucleotides, AONs, (RNA)-edited oligonucleotides, EONs, and RNA (antisense) oligonucleotides are referred to, unless otherwise indicated by the content, both oligoribonucleotides and deoxyoligoribonucleotides. Potentially, oligonucleotides may be completely devoid of RNA or DNA nucleotides (as they are found in nature) and may be complete from modified nucleotides. Whenever "oligoribonucleotide" is referred to, it may contain the bases A, G, C, U, or I. Whenever "deoxyoligoribonucleotide" is referred to, it may contain the bases A, G, C, T, or I. However, EON as used herein may contain mixtures of ribonucleosides and deoxyribonucleosides. When using deoxyribonucleotides, and therefore without modification at the 2' position of the sugar, the nucleotide is often abbreviated as dA, dC, dG, or T, where "d" represents the deoxy nature of the nucleoside, while ribonucleosides that are either normal RNA or modified at the 2' position are often abbreviated without "d", and are often abbreviated using their respective modifications, as described herein.

[0035] Whenever referring to nucleotides in oligonucleotides such as cytosine, this includes 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-acetylcytosine, 5-hydroxycytosine, and β-D-glucosyl-5-hydroxymethylcytosine. Whenever referring to adenine, this includes N6-methyladenine, 8-oxoadenine, 2,6-diaminopurine, and 7-methyladenine. Whenever referring to uracil, this includes dihydrouracil, isouracil, N3-glycosylated uracil, pseudouracil, 5-methyluracil, N1-methylpseudracil, 4-thiouracil, and 5-hydroxymethyluracil. Whenever referring to guanine, this includes 1-methylguanine, 7-methylguanosine, N2,N2-dimethylguanosine, N2,N2,7-trimethylguanosine, and N2,7-dimethylguanosine. Whenever nucleosides or nucleotides are mentioned, other modifications include ribofuranose derivatives, e.g., 2'-deoxy, 2'-hydroxy, and 2'-O-substituted variants, e.g., 2'-OMe, and 2'-4' crosslinking variants. Whenever oligonucleotides are mentioned, the linkage between two mononucleotides may be phosphate diester linkages or modifications thereof, including phosphonoacetates, phosphate triesters, PS, phosphoro(di)thioates, MP, phosphoramide linkers, phosphorylguanidine, thiophosphorylguanidine, sulfonophosphoramides, and the like.

[0036] The term "comprising" encompasses "including" and "consisting of." For example, a composition "comprising X" may be exclusive to X or may include something additional, such as X + Y. The term "approximately" in relation to a numerical value x is optional and means, for example, x ± 10%.

[0037] The term "substantially" does not exclude "completely"; for example, a composition "substantially free of Y" may be completely free of Y. Where relevant, the term "substantially" may be omitted from the definition of this invention.

[0038] As used herein, the term “complementary” means that an EON hybridizes with a second nucleic acid chain under physiological conditions (for example, when an oligonucleotide as the first nucleic acid chain (=guide oligonucleotide) forms a heteroduplex RNA editing oligonucleotide complex (HEON) with another complementary nucleic acid chain) or forms a ds complex with a target RNA sequence. This term does not necessarily mean that each nucleotide in the nucleic acid chain has a perfect match with its counterpart nucleotide in the counterpart sequence. In other words, while an EON may be complementary to a target sequence, there may be mismatches, fluctuations, and / or bulges between the oligonucleotide and the target sequence, on the other hand, under physiological conditions, the EON may still hybridize with the target sequence so that the cellular RNA editing enzyme can edit the target adenosine. Thus, the term “substantially complementary” also means that, despite the presence of mismatches, fluctuations, and / or bulges, the EON has nucleotides that match well with the target sequence and, under physiological conditions, the EON hybridizes with the target RNA. As described herein, EONs may be complementary to the target sequence, but may also include one or more mismatches, fluctuations, and / or bulges if the EON can hybridize with its target under physiological conditions.

[0039] In relation to nucleic acid sequences, the term "downstream" means further along the sequence in the 3' direction, while the term "upstream" means the opposite. Therefore, in any sequence encoding a polypeptide, the start codon is upstream of the stop codon in the sense strand, but downstream of the stop codon in the antisense strand.

[0040] The term "hybridization" typically refers to specific hybridization, excluding nonspecific hybridization. Specific hybridization can occur under selected experimental conditions, using techniques well known in the art, to ensure that the most stable interaction between the probe and target occurs when the probe and target have at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity.

[0041] The term “mismatch” is used herein to refer to opposing nucleotides in a double-stranded RNA complex that do not form a perfect base pair according to the Watson-Crick base pairing rules. Historically, mismatched nucleotides are GA, CA, UC, AA, GG, CC, and UU pairs. In some embodiments, the EONs disclosed herein contain fewer than four mismatches in the target sequence, e.g., 0, 1, or 2 mismatches. “Fluctuating” base pairs are GU, IU, IA, and IC base pairs. While a G:G pairing would be considered a mismatch, this does not necessarily mean the interaction is unstable, meaning that a Hoogsteen base pairing, which may appear as a mismatch based on the origin of the nucleotides, is still relatively stable, and therefore, based on the current disclosure, the term “mismatch” may be somewhat outdated. For example, isolated G:G pairs in double-stranded RNA are very stable, but may still be defined as mismatches.

[0042] The term "splicing mutation" refers to a mutation in a gene encoding an mRNA precursor that causes dysfunction in the splicing mechanism, meaning that the splicing of introns from exons is disrupted due to abnormal splicing, subsequent translation is out of frame, and the encoded protein terminates prematurely. Such shortened proteins are often rapidly degraded and have no functional activity.

[0043] The EONs disclosed herein (and complementary nucleic acid chains if two oligonucleotides form a HEON) can be chemically modified substantially in their entirety by providing nucleotides having a ribose sugar moiety having, for example, a 2'-OMe substitution, a 2'-F substitution, or a 2'-O-methoxyethyl (2'-MOE) substitution. The orphan nucleotide in the EON is preferably cytidine or an analogue thereof (such as a nucleotide having Benner's base), or uridine or an analogue thereof (such as isouridine), and / or, in one embodiment, it includes a diF modification at the 2' position of the sugar, in another embodiment, it includes deoxyribose (2'-H, DNA), and in a further embodiment, at least one, in another embodiment both, of the adjacent nucleotides to the side of the orphan nucleotide does not include a 2'-OMe modification. Complete modification of the oligonucleotide, in which all nucleotides have a 2'-OMe modification with a native base, results in a non-functional oligonucleotide as far as RNA editing (as known in the art) is concerned, presumably because it interferes with ADAR activity at the targeted site. Generally, adenosine in target RNA can be protected from editing by providing an opposing nucleotide with a 2'-OMe group (at least, if no other chemical substitutions or modifications exist within the nucleotide), or by providing guanine or adenine as the opposing base (because these two nucleic acid bases can also reduce editing of the opposing adenosine).

[0044] Various chemical and modification methods readily available in accordance with this disclosure are known in the field of oligonucleotides. The usual nucleoside linkages between nucleotides can be altered by phosphate diester bond mono- or dithiolation to yield PS esters or phosphorodithioate esters, respectively. Other modifications of nucleoside linkages, including amidation and peptide linkers, are possible.

[0045] In one embodiment, the EONs described herein contain 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. Notably, when the EON is delivered via a (viral) vector, its length can increase to be longer than 60 nucleotides. However, when the EON is delivered on its own without a vector, also called "naked form," the length of the EON is 15 to 60 nucleotides to reduce the risk of degradation. Furthermore, in its naked form, EON is preferably chemically modified as outlined herein to reduce the risk of degradation.

[0046] RNA editing entities (such as human ADAR enzymes) are known in the art to edit dsRNA structures with varying degrees of specificity, depending on several factors. One important factor is the degree of complementarity between the two strands constituting the dsRNA sequence. Perfect complementarity between the two strands usually causes the catalytic domain of human ADAR to react with any adenosine it encounters, deaminating the adenosine in an indiscriminate manner. The specificity of hADAR1 and hADAR2 can be increased by introducing chemical modifications and / or ensuring some mismatches in the dsRNA, which may help to position the dsRNA-binding domain in ways that have not yet been clearly defined. Furthermore, the deamination reaction itself can be enhanced by providing oligonucleotides containing mismatches opposite the adenosine to be edited. Following the instructions herein, those skilled in the art will be able to design complementary portions of oligonucleotides according to their requirements.

[0047] The most interesting RNA editing proteins present in cells for use with the EON disclosed herein are human ADAR1 and ADAR2. Those skilled in the art will understand that the degree to which intracellular editing entities are redirected to other target sites can be regulated by varying the affinity of the first nucleic acid strand to the recognition domain of the editing molecule. Precise modification can be determined through some trial and error and / or by computational methods based on the structural interaction between the EON and the recognition domain of the editing molecule. In addition, or alternatively, the degree of recruitment and redirection of intracellular editing entities can be regulated by the EON dosage and dosage regimen, which is determined by experimenters (in vitro) or clinicians, typically in Phase I and / or Phase II clinical trials.

[0048] This disclosure relates to the modification of target RNA sequences in eukaryotes, preferably metazoans, more preferably mammals, even more preferably human cells, most preferably human nerve cells. The EONs, vectors, and pharmaceutical compositions herein are particularly suitable for modifying RNA sequences in cells and tissues in which KCC2 is expressed and its protein acts. Because KCC2 is exclusively produced in neurons and plays a vital role in chloride efflux, preferred target cells for the EONs herein are nerve cells. Target cells can be located in vitro, ex vivo, or in vivo. One advantage of the EONs herein is that they can be used in situ with cells in living organisms, but they can also be used with cells in culture. In some embodiments, cells are treated ex vivo and then introduced into living organisms (e.g., reintroduced into the organism from which they originally originated). The EONs herein can also be used to edit target RNA sequences in cells from grafts or in so-called organoids, such as brain tissue organoids. Organoids are three-dimensional in vitro derived tissues, but can be thought of as being driven using specific conditions to generate individual isolated tissues. In therapeutic settings, they are useful because they can be induced in vitro from the patient's cells, and then the organoids can be reintroduced into the patient as autologous material that is less likely to be rejected than conventional grafts.

[0049] Without being constrained by theory, it is thought that RNA editing via human ADARs occurs in the nucleus during transcription or splicing of primary transcripts, or in the cytoplasm (where, for example, mature mRNA, miRNA, or ncRNA can be edited).

[0050] If adenosine deamination results in increased or restored KCC2 protein function, it will be apparent that the targeted editing described herein can be applied to any adenosine in the SLC12A5 transcript. However, as outlined herein, it is preferable to target the first adenosine present in the threonine-encoding codon at position 1007 of the mature KCC2 splice mutant.

[0051] Generally speaking, RNA editing can be used to create RNA sequences with a variety of properties. Such properties may be coding properties (creating proteins with various sequences or lengths, resulting in altered protein properties or functions) or binding properties (causing inhibition or overexpression of the RNA itself or its target or binding partner, or altering the entire expression pathway by recoding a miRNA or its related sequence on the target RNA). Protein function or localization can be freely altered by functional domains or recognition motifs, including, but not limited to, signal sequences, targeting or localization signals, recognition sites for proteolytic cleavage or co-modification or post-translational modification, enzyme catalytic sites, binding sites for binding partners, or degradation or activation signals. These, along with other forms of RNA and protein "manipulation," are encompassed in this disclosure as diagnostic, prophylactic, therapeutic, research tools, or otherwise in medicine or biotechnology, whether for the purpose of preventing, delaying, or treating disease, or for any other purpose. Therefore, the EON disclosed herein may mediate RNA editing of any target adenosine in the SLC12A5 transcript, resulting in improvement or restoration of KCC2 protein function. This disclosure opens up an entirely new field of treating pathological pain (such as chronic pain) and epilepsy using gene editing techniques.

[0052] The amount, dosage, and administration regimen of EON administered may vary between cell types, the disease being treated, the target population, the mode of administration (e.g., systemic vs. topical), the severity of the disease, and the level of acceptable side effects. These can and should be evaluated through trial and error during in vitro studies, preclinical studies, and clinical trials. Trials are particularly straightforward when they result in phenotypic changes in which the modified sequence is readily detectable, or changes in the level or activity of a specified biomarker. Higher doses of EON may compete for binding to ADARs within cells, potentially depleting the amount of entities free to participate in RNA editing; however, routine drug trials will reveal any such effects for a given EON and a given target.

[0053] One suitable investigational technique involves delivering EON to a cell line or test organism and then collecting biopsy samples at various points in time thereafter. The target RNA sequence can be evaluated in the biopsy samples, and the percentage of cells with modification can be easily tracked. Also suitable biomarkers that can be used in accordance with this disclosure include detecting threonine phosphorylation at position 1007, as well as evaluating the function / activity of the KCC2 protein in a particular subject before and after treatment, or with or without treatment of the subject with the EON or vector disclosed herein. After this trial is performed once, the knowledge can be retained, and future deliveries can be made without the need to collect biopsy samples. Thus, the methods disclosed herein may include a step of identifying the presence of a desired change in the target RNA sequence of cells and thereby confirming that the target RNA sequence is modified. This step typically involves sequencing the relevant portion of the target RNA, or its cDNA copy (or, if the target RNA is an mRNA precursor, the cDNA copy of its splicing product), as described above, and thus the sequence change can be easily confirmed. Alternatively, as described above, the change can be evaluated for protein function, for example, by measuring the thallium transport capacity of KCC2. The transport of thallium, a potassium substitute, is directly proportional to the number of active KCC2 potassium transporters. Thallium transport can then be detected by introducing a highly sensitive thallium indicator dye before, during, and / or after treatment, or by evaluating any other potential markers, and the measurement is preferably performed in vitro on a sample obtained from the treated subject.

[0054] After RNA editing occurs in a cell, the modified RNA may be diluted over time due to factors such as cell division and the limited half-life of the edited RNA. Therefore, from a practical therapeutic standpoint, the methods disclosed herein may include repeated delivery of EON until sufficient target RNA has been modified to provide a visible benefit to the patient and / or maintain that benefit over time.

[0055] Since EONs as herein are particularly suitable for therapeutic use, this disclosure also relates to pharmaceutical compositions comprising an EON as herein, or a vector or plasmid encoding an EON as herein, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier may be simply saline solution, which may be usefully isotonic or hypotonic, particularly for pulmonary delivery. This disclosure also provides delivery devices (e.g., syringes) comprising the pharmaceutical compositions as herein.

[0056] This disclosure also provides, as described herein, EONs for use in methods for introducing phosphorylated mutations into a target SLC12A5 RNA sequence in mammalian, preferably human, neurons. Similarly, this disclosure provides, as described herein, the use of EONs in the manufacture of pharmaceuticals to alter a target SLC12A5 RNA sequence in mammalian, preferably human, neurons, thereby treating, preventing, or alleviating diseases associated with reduced GABAergic inhibition, such as those resulting from reduced KCC2 activity.

[0057] EON as used herein is administered appropriately in an aqueous solution, such as physiological saline, artificial cerebrospinal fluid, or suspension, at a concentration in the range of 1 ng / ml to 1 g / ml, preferably 10 ng / ml to 500 mg / ml, more preferably 100 ng / ml to 100 mg / ml, and optionally containing pharmaceutically suitable additives, excipients, and other components. The dosage may appropriately be in the range of about 1 μg / kg to about 100 mg / kg, preferably about 10 μg / kg to about 10 mg / kg, and more preferably about 100 μg / kg to about 1 mg / kg. Administration may be by inhalation (e.g., by spraying), intranasal, oral, injection or infusion, intravenous, subcutaneous, intradermal, intramuscular, intratracheal, intraperitoneal, intrarectal, subarachnoid, cisterna magna, parenteral, etc. Administration may be in solid form, powder form, pill, gel, liquid form, sustained-release formulation, or any other form suitable for pharmaceutically acceptable use in humans.

[0058] In one embodiment, the method herein comprises the steps of: administering EON or a pharmaceutical composition herein to a subject, enabling the formation of a ds nucleic acid complex between EON and a specific complementary target nucleic acid molecule in the cells of the subject; enabling engagement of an endogenous adenosine deaminationase such as ADAR2; and enabling the enzyme to deaminate target adenosine in the target nuclear target molecule to inosine, thereby mitigating, preventing, or alleviating diseases associated with reduced GABAergic inhibition. Diseases that can be treated according to this method are preferably, but not limited to, the CNS diseases listed herein, and any other diseases in which deamination of adenosine in the SLC12A5 transcript would restore the function of the KCC2 protein in the individual in need.

[0059] RNA editing molecules present in cells are typically of a proteinaceous nature, such as ADAR enzymes found in metazoans, including mammals. Preferably, the cellular editing entity is an enzyme, more preferably an adenosine deaminosection or cytidine deaminosection, and even more preferably an adenosine deaminosection. These are enzymes that possess ADAR activity. Of particular interest are human ADAR, hADAR1, and hADAR2, including any of their isoforms. RNA editing enzymes known in the art that may be conveniently used to design oligonucleotide constructs according to this disclosure include RNA-acting adenosine deaminosections (ADARs), such as hADAR1 and hADAR2 in human or human cells, as well as cytidine deaminosections. hADAR1 is known to exist in two isoforms, namely a longer 150 kDa interferon-inducible version and a shorter 110 kDa version, which are generated from a common mRNA precursor through alternative splicing. As a result, the level of 150 kDa isoforms available in cells can be influenced by interferons, particularly interferon-gamma (IFN-γ). hADAR1 is also induceable by TNF-α. This provides an opportunity to develop combination therapies in which IFN-γ or TNF-α and the EON disclosed herein are administered to the patient either as a combined product or as separate products simultaneously or in any subsequent order. Certain medical conditions may already coincide with increased IFN-γ or TNF-α levels in specific tissues of the patient, creating further opportunities to make editing more tissue-specific. Those skilled in the art will understand that the extent to which intracellular editing entities are redirected to other target sites can be regulated by varying the affinity of the first nucleic acid chain to the recognition domain of the editing molecule.

[0060] chemical modification All of the following chemical modifications that may be used in EON as described herein may also be used on a sense strand complementary to EON if EON and its complementary strand form a so-called HEON complex, as described in UK Patent Application Publication No. 2215614.5 (unpublished), except that the opposing sense strand does not contain an orphan nucleotide. Therefore, modifications related to orphan nucleotides are related only to EON as described herein, but all other modifications relate to any (protective) sense oligonucleotides that may be used with EON as described herein and with EON in pharmaceutical products. This includes the use of hydrophobic moieties (such as tocopherol and cholesterol) and cell-specific ligands (such as GalNAc moieties), which are described herein and detailed in UK Patent Application Publication No. 2215614.5 (unpublished), and which may be bound to EON or its opposing strand, or both.

[0061] The nucleoside linkages in oligonucleotides as used herein may include one or more naturally occurring nucleoside linkages and / or modified nucleoside linkages. However, at least one, at least two, or at least three nucleoside linkages from the 5' and / or 3' ends of an EON are preferably modified nucleoside linkages. A preferred modified nucleoside linkage is a PS linkage. In one embodiment, all nucleoside linkages in an EON are modified nucleoside linkages. In one embodiment, the EON includes a PNdmi linkage that links the outermost nucleoside at the 5' and / or 3' ends to the second-to-last nucleoside at each of these ends, respectively. A PNdmi linkage preferred for use in an EON as used herein has the structure of formula (I):

[0062] [ka] It holds.

[0063] Common limiting factors in oligonucleotide-based therapies are the ability of oligonucleotides to be taken up by cells (either by themselves or when delivered "naked" without the application of a delivery vehicle), their distribution in the body, and their resistance to nuclease-mediated degradation. Those skilled in the art recognize that various chemical modifications can help overcome such limitations, and this has been detailed in the art. Examples of chemical modifications currently in common use include 2'-O-methyl (often abbreviated as 2'-OMe or 2'-O-Me), 2'-F, and 2'-O-methoxyethyl (often also called 2'-methoxyethoxy or 2'-MOE) modifications of sugars, as well as the use of PS linkages between nucleosides. International Patent Application No. 2020 / 201406 discloses the use of MP linkage modifications at specific positions surrounding orphan nucleotides in a first nucleic acid chain. Apart from the ribose sugar moiety of orphan nucleotides, which have certain limitations in terms of compatibility with RNA editing, the ribose 2' group in all nucleotides of EON can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' ligatures (e.g., locked nucleic acid (LNA)), or other ribosyl 1'-substitutions, 2'-substitutions, 3'-substitutions, 4'-substitutions, or 5'-substitutions. Orphan nucleotides in EON that do not contain any ribose sugar, base, or other chemical modifications to the ligatures preferably do not have 2'-OMe or 2'-MOE substitutions, but may have 2'-F, 2',2'-difluoro (diF), or 2'-ala-F (FANA) substitutions, or may be DNA. The unpublished UK Patent Application Publication No. 2214347.3 describes modifications of the 2' position of the ribose sugar moiety of orphan nucleotides by 2',2'-disubstituted, such as diF, which are applicable herein. The 2'-4' linkage can be selected from a number of linkers known in the art, such as methylene linkers, amide linkers, or restricted ethyl linkers (cEt).

[0064] This disclosure relates to an EON for use in the deamination of a target nucleotide (preferably adenosine) in a target RNA, wherein the EON is complementary to the stretch of nucleotides in the target RNA containing the target adenosine, and the nucleotide in the first nucleic acid strand directly opposite the target nucleotide is an orphan nucleotide, and when the target nucleotide is adenosine, the orphan nucleotide preferably includes a base or a modified base or a base analog (e.g., Benner's base Z) having an NH moiety at a position similar to the ring nitrogen. The numbering of nucleotides in the EON is such that the orphan nucleotide is numbered 0, and the nucleotide 5' from the orphan nucleotide is numbered +1. The numbering further involves positive (+) incrementing toward the 5' end and negative (-) incrementing toward the 3' end, such that the first nucleotide 3' from the orphan nucleotide is numbered -1. The numbering of nucleoside junctions in EON is such that junction number 0 is the junction on the 5' side from the orphan nucleotide, and the junction positions in the oligonucleotide increase positively (+) toward the 5' end and negatively (-) toward the 3' end.

[0065] Preferably, the EON may contain one or more (chiral pure or chiral mixed) PS linkages. In one embodiment, the PS linkage connects to the terminal 3, 4, 5, 6, 7, or 8 nucleotides at each end of the first nucleic acid chain. In one embodiment, the EON may contain one or more phosphoramidate (PN) linkages. In one embodiment, the PN linkage connects to the terminal 2 nucleotides at each end of the EON.

[0066] Nucleosides in EON can be either natural nucleosides (deoxyribonucleosides or ribonucleosides) or unnatural nucleosides. It should be noted that in RNA editing, where double-stranded RNA is generally the substrate of an enzyme with deamination activity (such as ADAR), ribonucleosides are considered "natural," while deoxyribonucleosides, for the sake of argument, may be considered unnatural or modified simply because the DNA is not present in the RNA-RNA double-stranded substrate configuration. Those skilled in the art will understand that if a nucleotide has a natural ribose moiety, this can still be unnaturally modified in the base and / or ligation.

[0067] In addition to specific preferred chemical modifications at particular positions in the compounds herein, which may or may not be present in the same monomer, for example, at the 3' and / or 5' positions, scaffold modifications indicate the presence of modified versions of naturally occurring ribosyl moieties in RNA (i.e., pentose moieties), such as bicyclic sugars, tetrahydropyrans, hexoses, morpholinos, 2'-modified sugars, 4'-modified sugars, 5'-modified sugars, and 4'-substituted sugars.Examples of appropriate modifications, though not limited to these, include 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), and 2'-O-(2-(dimethylaminopropyl). )propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl), 2'-deoxy(DNA), 2'-O-(haloalkyl)methyl, e.g., 2'-O-(2-chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM), 2'-O-alkoxycarbonyl, e.g., 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), bicyclic or cross-linked nucleic acid (BNA) scaffold modifications, e.g., conformationally restricted nucleotide (CRN) monomers, locked nucleic acid (LNA) monomers, xylo-LNA monomers BNA monomer, α-LNA monomer, α-l-LNA monomer, β-d-LNA monomer, 2'-amino-LNA monomer, 2'-(alkylamino)-LNA monomer, 2'-(acylamino)-LNA monomer, 2'-N-substituted 2'-amino-LNA monomer, 2'-thio-LNA monomer, (2'-O,4'-C)-restricted ethyl (cEt)BNA monomer, (2'-O,4'-C)-restricted methoxyethyl (cMOE)BNA monomer, 2',4'-BNA. NC (NH) monomer, 2',4'-BNA NC (NMe) monomer, 2',4'-BNA NC(NBn) monomers, ethylene-bridged nucleic acid (ENA) monomers, carba-LNA (cLNA) monomers, 3,4-dihydro-2H-pyran nucleic acid (DpNA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, oxo-CBBN monomers, heterocyclic-bridged BNA monomers (triazolyl or tetrazolyl-linked, etc.), amide-bridged BNA monomers (AmNA, etc.), urea-bridged BNA monomers, sulfonamide-bridged BNA monomers, bicyclic carbocyclic nucleotide monomers, TriNA monomers, α-l-TriNA monomers, bicycloDNA (bcDNA) monomers, F-bcDNA monomers, tricycloDNA (tcDNA) monomers, F-tcDNA monomers Examples include alpha-anomeric bicycloDNA (abcDNA) monomers, oxetane nucleotide monomers, locked PMO monomers derived from 2'-aminoLNA, guanidine-bridged nucleic acid (GuNA) monomers, spirocyclopropylene-bridged nucleic acid (scpBNA) monomers and their derivatives, cyclohexenyl nucleic acid (CeNA) monomers, altriol nucleic acid (ANA) monomers, hexitol nucleic acid (HNA) monomers, fluorinated HNA (F-HNA) monomers, pyranosyl RNA (p-RNA) monomers, 3'-deoxypyranosyl DNA (p-DNA), unlocked nucleic acid (UNA), and inverted versions of any of the above monomers. All of these modifications are known to those skilled in the art.

[0068] The EON nucleotide sequence described herein is complementary to at least a portion of the nucleotide sequence of the target SLC12A5 transcript, which contains the target adenosine that is deaminated to inosine, and is therefore capable of annealing (or hybridizing) with the target transcript. Sequential complementarity can be determined by using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can hybridize, taking into account the inter-strand complementarity.

[0069] In contrast to what is described regarding gapmers and their relationship to RNase degradation and the use of such gapmers in double-stranded complexes (see, for example, European Patent Application Publication No. 3954395A1), the EONs herein do not contain stretches of DNA nucleotides that target a target sequence (or sense nucleic acid strand) for RNase-mediated degradation. In one embodiment, the EON does not contain four or more consecutive DNA nucleotides anywhere in its sequence. In one embodiment, the EON is composed of as many (chemically) modified nucleotides as possible to enhance resistance to RNase-mediated degradation, while at the same time being as efficient as possible in producing an RNA editing effect. This means that the orphan nucleotides and some other nucleotides in the EON can be DNA, but also that there are no stretches of four or more consecutive DNA nucleotides in the EON. Therefore, the EONs herein are not gapmers. Gapmers reduce the expression of a target transcript but do not produce RNA editing of a specified adenosine within the target transcript. A gapmer is, in principle, an ss nucleic acid consisting of a central region (a DNA gap region having at least four consecutive deoxyribonucleotides) and wing regions directly located at its 5' end (5' wing region) and 3' end (3' wing region). In contrast, EONs as used herein may be any oligonucleotide that produces an RNA editing effect in which the target adenosine in the target RNA molecule is deaminated to inosine, and is therefore as resistant to RNase-mediated degradation as possible to produce this effect. The purpose of EONs as used herein is to increase KCC2 activity, not to decrease it, for example, by causing degradation of the transcript molecule encoding KCC2.

[0070] In one embodiment, the EON, or the sense chain to which it can anneal before entering the target cell, is bound to or associated with a blood-brain-barrier shuttle or hydrophobic moiety, such as palmityl or its analogues, cholesterol or its analogues, or tocopherol or its analogues. This is preferably bound to the 5' end. If the hydrophobic moiety is bound to both the 5' and 3' ends, such hydrophobic moieties may be the same or different. The hydrophobic moiety bound to the oligonucleotide may be bound directly or indirectly mediated by another substance. If the hydrophobic moiety is directly bound, it is sufficient that the moiety is bound via covalent, ionic, hydrogen, etc. If the hydrophobic moiety is indirectly bound, this may be via a linker. The linker may be cleavable or incleavable. A cleavable linker is one that can be cleaved under physiological conditions, for example, within a cell or animal body (e.g., the human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases, or by physiological conditions specific to a body part or cell, such as pH or a reducing environment (e.g., glutathione concentration). Examples of cleavable linkers, but not limited to them, include amides, esters, esters of one or both of phosphate diesters, phosphoesters, carbamates, and disulfide bonds, as well as natural DNA linkers. Cleavable linkers also include self-sacrificing linkers. Non-cleavable linkers are those that are not cleaved under physiological conditions, or are cleaved very slowly compared to cleavable linkers, for example, in linkers consisting of PS links, modified or unmodified deoxyribonucleosides linked by PS links, spacers connected via PS links, and modified or unmodified ribonucleosides. When the linker is a nucleic acid such as DNA or an oligonucleotide, there is no limit to the length of the chain. However, this is usually a length of 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.There are no restrictions on the length or composition of the spacer connecting the ligand and oligonucleotide; examples include ethylene glycol, TEG, HEG, alkyl chains, propyl, 6-aminohexyl, or dodecyl.

[0071] This disclosure also relates to pharmaceutical compositions comprising EON as herein, further comprising pharmaceutically acceptable carriers and / or other additives, and soluble in pharmaceutically acceptable organic solvents, etc. The dosage form in which the EON or pharmaceutical composition is administered may depend on the disorder to be treated and the tissue to be targeted, and may be selected according to general procedures in the art. The pharmaceutical composition may be administered by single-dose or multi-dose administration. This may be administered daily or at appropriate time intervals, which may be determined using general knowledge in the art and may be adjusted based on the disorder and the potency of the active ingredient.

[0072] In one embodiment, EON comprises at least one nucleotide having a sugar moiety containing a 2'-OMe modification. In one embodiment, EON comprises at least one nucleotide having a sugar moiety containing a 2'-MOE modification. In one embodiment, EON comprises at least one nucleotide having a sugar moiety containing a 2'-F modification. In one embodiment, an orphan nucleotide is called a DNA nucleotide even though it has 2'-H in its sugar moiety and therefore additional modifications may be present in its base and / or in its linkage with adjacent nucleosides. In one embodiment, an orphan nucleotide has 2'-F in its sugar moiety. In one embodiment, an orphan nucleotide has a diF substitution in its sugar moiety. In one embodiment, an orphan nucleotide has 2'-F and 2'-C-methyl in its sugar moiety. In one embodiment, an orphan nucleotide contains 2'-F in the arabinose configuration (FANA) of its sugar moiety. In one embodiment, EON is an antisense oligonucleotide (sometimes commonly abbreviated as "ASO") capable of forming a double-stranded nucleic acid complex with a target RNA molecule, the double-stranded nucleic acid complex capable of recruiting adenosine deaminationase for deamination of target adenosine in the target SLC12A5 RNA molecule, the nucleotide in EON facing the target adenosine being an orphan nucleotide, the orphan nucleotide having the structure of formula (II):

[0073] [ka] [wherein X is O, NH, OCH2, CH2, Se, or S, B is a nitrogen base selected from the group consisting of cytosine, uracil, isouracil, N3-glycosylated uracil, pseudoisocytosine, 8-oxo-adenine, and 6-amino-5-nitro-2(1H)-pyridone, R1 and R2 are both independently selected from H, OH, F, or CH3, R3 is the 5' portion of the orphan nucleotide, consisting of 7 to 30 nucleotides in the EON, and R4 is the 3' portion of the orphan nucleotide, consisting of 4 to 25 nucleotides.] The nucleotides on the 3' and / or 5' side of the orphan nucleotide may be DNA, more preferably nucleotides at 3'(position-1).

[0074] In one embodiment, the structure of the first nucleic acid chain is given by formula (III):

[0075] [ka] It includes at least one MP nucleoside linkage by [a specific factor].

[0076] The preferred location for MP linkage in EONs disclosed herein is linkage position-2 (for example, shown for all EONs shown in Figure 1A, where Zd is an orphan nucleotide and Id is the nucleotide at position-1), thereby linking the nucleoside at position-1 to the nucleoside at position-2, but does not explicitly exclude other locations for MP linkage.

[0077] EON disclosed herein also has the structure of the following formula (IV):

[0078] [ka] [In the formula, X = O or S R = aryl, substituted aryl, heterocycle, substituted heterocycle, aromatic heterocycle, substituted aromatic heterocycle, C1-C6 alkoxy, substituted C1-C6 alkoxy, C1-C 20Alkyl, substituted C1-C 20 The modifications may also include one or more linkage modifications by alkyl, C1-C6 alkenyl, C1-C6 substituted alkenyl, C1-C6 alkynyl, substituted C1-C6 alkynyl, or conjugate groups. In preferred embodiments, X=O and R=methyl, and the linkage modification is referred to as “mesylphosphoramidate,” “MsPA,” or “PNms.” In one embodiment, PNms linkages are used instead of MP and / or PNdmi linkages.

[0079] In one embodiment, the EON disclosed herein comprises an internucleoside linkage of the structure of formula (IV) [wherein X=O and R=CH3], which is generally referred herein to as a PNms linkage (mesylphosphoramidate). In other preferred embodiments, R is the following structure (a), (b), (c), (d), (e), (f), (g), (h), or (i)

[0080] [ka] It is equal to one of the following.

[0081] Other nucleoside linkages that may be used in the EON of this disclosure are disclosed in International Publication No. 2023 / 278589.

[0082] In one embodiment, the EON comprises at least one nucleotide having a sugar moiety containing a 2'-fluoro(2'-F) modification. A preferred position for the nucleotide having the 2'-F modification is position-3 in the EON, which may be present in coordination with the same 2' modification in the orphan nucleotide as described above.

[0083] In one embodiment, EON comprises at least one phosphonoacetate or phosphonoacetamide nucleoside linkage.

[0084] In one embodiment, EON comprises at least one nucleotide comprising locked nucleic acid (LNA) ribose modification or unlocked nucleic acid (UNA) ribose modification. In one embodiment, EON comprises at least one nucleotide comprising threose nucleic acid (TNA) ribose modification.

[0085] Those skilled in the art will know that oligonucleotides such as EONs outlined herein generally consist of repeating monomers. Such monomers are most frequently nucleotides or chemically modified nucleotides. 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 sugar, a 5'-linked phosphate group linked via a phosphate ester, and a 1'-linked base. The sugar links the base and phosphate, and is therefore often called the “scaffold” of the nucleotide.

[0086] Therefore, modifications in pentoses are often called “scaffold modifications.” The original pentose may be replaced in its entirety by another part that connects, as well as a base and a phosphate. Thus, it will be understood that while a pentose is often the scaffold, the scaffold is not necessarily a pentose. Examples of scaffold modifications applicable to monomers of EON disclosed herein are shown in International Patent Application Publications 2020 / 154342, 2020 / 154343, and 2020 / 154344, which are incorporated herein in their entirety by reference.

[0087] In one embodiment, the EON as described herein may comprise one or more nucleotides having a 2'-MOE ribose modification. In another embodiment, the EON comprises one or more nucleotides that do not have a 2'-MOE ribose modification, and the 2'-MOE ribose modification is located in a position that does not prevent an enzyme having adenosine deaminose activity from deaminating the target adenosine. In yet another embodiment, the EON comprises a 2'-OMe ribose modification at a position that does not have a 2'-MOE ribose modification, and / or the oligonucleotide comprises a deoxynucleotide at a position that does not have a 2'-MOE ribose modification. In one embodiment, EON comprises one or more nucleotides including a 2' position with modifications including 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, TNA, 2'-fluoro(2'-F), 2',2'-difluoro(diF) modification, 2'-fluoro-2'-C-methyl modification, or 2'-4'-linking (i.e., crosslinked nucleic acids such as locked nucleic acids (LNA, or examples mentioned in, for example, International Patent Application Publication No. 2018 / 007475)). In another embodiment, other nucleic acid monomers to be applied are, for example, arabino nucleic acids and 2'-deoxy-2'-fluoroarabino nucleic acids (FANA) for improved affinity purposes. The 2'-4' linking can be selected from linkers known in the art, such as methylene linkers or restricted ethyl linkers. A wide variety of 2' modifications are known in the art. Further examples are disclosed, for example, in International Patent Application Publications No. 2016 / 097212, 2017 / 220751, 2018 / 041973, 2018 / 134301, 2019 / 219581, 2019 / 158475, and 2022 / 099159, which are incorporated in their entirety by reference herein. In all cases, the modification should be editable and compatible so that the EON fulfills its role as an oligonucleotide capable of editing a double-stranded complex with the target RNA, recruiting a deaminationase, and subsequently deaminating the target adenosine.If the unlocked nucleic acid (UNA) contains ribose modifications, the monomer may have a 2' position containing the same modifications as described above, such as 2'-MOE, 2'-OMe, 2'-OH, 2'-deoxy, 2'-F, 2',2'-diF, 2'-fluoro-2'-C-methyl, arabino nucleic acid, FANA, or 2'-4'-linking (i.e., cross-linked nucleic acids such as LNA).

[0088] Bases, sometimes called nucleic acid bases, are generally adenine, cytosine, guanine, thymine, or uracil, or their derivatives. A base is defined as a moiety that can bind to another nucleic acid base through H-bonds, polar bonds (such as through the CF moiety), or aromatic electron interactions. Cytosine, thymine, and uracil are pyrimidine bases and are generally linked to a scaffold via their 1-nitrogen. Adenine and guanine are purine bases and are generally linked to a scaffold via their 9-nitrogen. As used herein, the terms “adenine,” “guanine,” “cytosine,” “thymine,” “uracil,” and “hypoxanthine” refer to the nucleic acid base itself. The terms “adenosine,” “guanosine,” “cytidine,” “thymidine,” “uridine,” and “inosine” refer to the nucleic acid base linked to a (deoxy)ribosyl sugar.

[0089] The nucleic acid bases in EONs as used herein may be adenine, cytosine, guanine, thymine, or uracil, or any other part, which can interact with other nucleic acid bases through H-bonds, polar bonds (such as CF), or aromatic electron interactions. Any nucleic acid base at any position in the nucleic acid chain may be a modified form of adenine, cytosine, guanine, or uracil, e.g., hypoxanthine (the nucleic acid base in inosine), pseudouracil, pseudocytosine, isouracil, N3-glycosylated uracil, 1-methylpseudracil, orotic acid, agmatidine, lysidine, 2-thiouracil, 2-thiothymine, 5-substituted pyrimidines (e.g., 5-halouryl (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 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 nucleic acid bases or their derivatives, and 2,6-difluorotoluene may be degenerate or universal bases, or they may be absent, such as debase sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose, azaribose).

[0090] In one embodiment, the nucleotide analog is an analog of a nucleic acid nucleotide. In one embodiment, the nucleotide analog is an analog of adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In one embodiment, the nucleotide analog is not guanosine or deoxyguanosine. In one embodiment, the nucleotide analog is not a nucleic acid nucleotide. In one embodiment, the nucleotide analog is not adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, or deoxyuridine. In one embodiment, the uridine analog that may be an orphan nucleotide in EON is isouridine.

[0091] Nucleotides are generally linked to neighboring nucleotides through the condensation of their 5'-phosphate moiety with the 3'-hydroxyl moiety of a neighboring nucleotide monomer. Similarly, their 3'-hydroxyl moiety is generally linked to the 5'-phosphate of a neighboring nucleotide monomer. This forms a phosphate diester bond. The phosphate diester and scaffold form an alternating copolymer. Bases are transferred onto this copolymer, i.e., to the scaffold portion. For this reason, the alternating copolymer formed by the linked scaffold of an oligonucleotide is often called the "backbone" of the oligonucleotide. Because the phosphate diester bond links neighboring monomers together, these are often called "backbone links." It should be understood that if the phosphate group is modified to form an analogous portion such as PS instead, such a portion is still called the monomer backbone link. This is called a "backbone link modification." In general, the backbone of an oligonucleotide contains both the alternating scaffold and the backbone links.

[0092] EON as used herein may include ligation modifications. Ligation modifications may include, but are not limited to, phosphate diesterified versions present in RNA, such as PS, chiral pure PS, (R)-PS, (S)-PS, methyl phosphonate (MP), chiral pure methyl phosphonate, (R)-methyl phosphonate, (S)-methyl phosphonate, phosphoryl guanidine (PNdmi, etc.), chiral pure phosphoryl guanidine, (R)-phosphoyl guanidine, (S)-phosphoyl guanidine, phosphorodithioate (PS2), phosphonoacetate (PAC E) may be phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, methylphosphorothioate, methylthiophosphonate, PS prodrug, alkylated PS, H-phosphonate, ethyl phosphate, ethyl PS, boranophosphate, borano PS, methylboranophosphate, methylborano PS, methylboranophosphonate, methylboranophosphothioate, phosphate, phosphate triesters, aminoalkyl phosphotryesters, and derivatives thereof. Other modifications include phosphoramidites, phosphoramidates, N3'→P5' phosphoramidates, phosphorodiamidates, phosphorothiodiamidates, sulfamates, diethylene sulfoxides, amides, sulfonates, siloxanes, sulfides, sulfones, formacetyl, alkenyls, methylenehydrazinos, sulfonamides, triazoles, oxalyls, carbamates, methyleneiminos (MMIs), and thioacetamide nucleic acids (TANAs), as well as their derivatives. Various salts, mixed salts, and free acid forms are also included, as well as 3'→3' and 2'→5' linkages.

[0093] In one embodiment, EON includes the substitution of one of the non-crosslinked oxygens in the phosphate diester linkage. This modification slightly destabilizes the base pairing but adds resistance to nuclease degradation. Preferred nucleotide analogs or equivalents include PS, phosphonoacetates, phosphorodithioates, phosphate triesters, aminoalkyl phosphotryesters, H-phosphonates, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, selenophosphates, or boranophosphates. Particularly preferred are internucleoside linkages modified to contain PS. Many of these non-naturally occurring modifications of linkages, such as PS, are chiral, meaning that Rp and Sp configurations known to those skilled in the art are present. In one embodiment, the chirality of the PS linkages is controlled, meaning that each linkage is either the Rp or Sp configuration, whichever is preferred. The choice of Rp or Sp configuration at a given linkage site may depend on the target sequence and the introduction that provides binding efficiency and RNA editing. However, if such is not specifically desired, the composition may contain oligonucleotides as active compounds having both Rp and Sp configurations at a particular designated linkage site. A mixture of such EONs is also feasible, where a particular site preferably has one of the configurations and it is not important at other sites.

[0094] Here again, in all cases, the modification should be edit-compatible so as to fulfill its role as an editing oligonucleotide that can mobilize adenosine deaminosease due to the nature of the dsRNA produced when the EON attaches to its target sequence. In all embodiments of the present invention, the enzyme having adenosine deaminosease activity is preferably ADAR1, ADAR2, or ADAT. In a very preferred embodiment, the EON is an RNA editing oligonucleotide that targets an mRNA precursor or mRNA, the target nucleotide is adenosine in the target RNA, and the adenosine is deaminated to inosine, which is read as guanosine by the translation mechanism. The disclosure also relates to pharmaceutical compositions comprising the EON characterized herein and a pharmaceutically acceptable carrier.

[0095] Other chemical modifications of EON as described herein include substituting any one or more hydrogen atoms with deuterium or tritium, examples of which can be found, for example, in International Patent Application Publication No. 2014 / 022566 or No. 2015 / 011694, which are incorporated herein in their entirety by reference.

[0096] This disclosure further relates to EON as herein, or pharmaceutical compositions comprising EON as herein, for use in the treatment or prevention of CNS disorders caused by reduced KCC2 functionality, preferably related to reduced GABAergic inhibition. In one embodiment, this disclosure relates to EON as herein, or pharmaceutical compositions comprising EON as herein, for use in the treatment or prevention of CNS disorders caused by reduced KCC2 functionality, preferably related to reduced GABAergic inhibition. In one embodiment, this disclosure relates to EON as herein, or pharmaceutical compositions comprising EON as herein, for use in the treatment or prevention of pain or epilepsy, preferably caused by reduced KCC2 functionality.

[0097] The EONs described herein preferably do not contain a 5'-terminal O6-benzylguanosine or a 5'-terminal amino modification, and preferably are not covalently bonded to a SNAP tag domain (a modified O6-alkylguanosine-DNA-alkyltransferase). Similarly, the EONs described herein preferably do not contain a boxB RNA hairpin sequence. In one embodiment, the EONs described herein contain 0, 1, 2, or 3 fluctuation base pairs with the target sequence, and / or 0, 1, 2, 3, 4, 5, 6, 7, or 8 mismatch base pairs with the target RNA sequence. If the orphan nucleotide is uridine, no mismatches are present. One alternative to uridine is to position isouridine opposite the target adenosine, which may not pair in the same way that G pairs with U. Preferably, the target adenosine in the target sequence forms a mismatch base pair with the nucleoside in the EON directly opposite the target adenosine.

[0098] It should be noted that when EONs are delivered via a vector, such as an AAV vector, the chemical modifications that act on the target RNA molecule are not present in the EONs. While it is preferable to use “naked” EONs with the chemical modifications outlined herein, EONs delivered by other means, such as AAV vector expression, or via cyclic or hairpin-structured editing molecules (e.g., mobilized portions disclosed in International Patent Application Publications 2016 / 097212, 2017 / 050306, 2020 / 001793, 2017 / 010556, 2020 / 246560, and 2022 / 078995, whose entirety is incorporated herein by reference), are also covered by this disclosure, as they can also be applied to edit adenosine in a target SLC12A5 RNA molecule to produce a KCC2 protein with increased GABAergic inhibitory activity.

[0099] The EONs described herein can utilize endogenous cellular pathways and naturally available ADAR enzymes to specifically edit target adenosine in a target RNA sequence. The EONs described herein can recruit ADAR and a complex with it to facilitate the deamination of a (single) specific target adenosine nucleotide in the target RNA sequence. Ideally, only one adenosine is deaminated. When the EONs described herein are complexed with ADAR, they preferably result in the deamination of a single target adenosine.

[0100] Analysis of native targets of ADAR enzymes has shown that they generally contain mismatches between the two strands forming the RNA helix edited by ADAR1 or 2. These mismatches have been suggested to enhance the specificity of the editing reaction (Stefl et al. 2006. Structure 14(2):345-355, Tian et al. 2011. Nucleic Acids Res 39(13):5669-5681). Characterizing the optimal nucleotide pairing / mismatch pattern between EON and target RNA is also considered important in the development of efficient ADAR-based EON therapies.

[0101] As outlined above, the EONs described herein utilize specific nucleotide modifications at predetermined sites to ensure stability and proper ADAR binding and activity. These modifications can vary and include modifications in the EON backbone, in the sugar portions of nucleotides, and in nucleic acid base or phosphate diester linkages, as outlined in detail herein. They may also be variably distributed throughout the EON sequence. Specific modifications may be required to support the interactions of various amino acid residues within the RNA-binding domain and deaminoenzyme domain of the ADAR enzyme. For example, PS linkages or 2'-OMe or 2'-MOE modifications between nucleotides may be tolerable in some parts of the EON, while in others they should be avoided to avoid disrupting critical interactions between the enzyme and phosphate and 2'-OH groups. Additionally, if the target sequence is not optimal for ADAR editing, specific nucleotide modifications may be required to enhance editing activity toward the substrate RNA. Previous studies have established that certain sequence configurations are more receptive to editing. For example, the target sequence 5'-UAG-3' (with target A in the center) contains the most preferred nearest neighbor nucleotide of ADAR2, while the 5'-CAA-3' target sequence is unfavorable (Schneider et al. 2014. Nucleic Acids Res 42(10):e87), with 5'-guanosine (G) being the least preferred surrounding nucleotide. Structural analysis of the ADAR2 deaminoenzyme domain suggests the possibility of enhancing editing by carefully selecting the nucleotide opposite the target trinucleotide. For example, the pair of the 5'-CAA-3' target sequence with the 3'-GCU-5' sequence on the opposite strand (forming an AC mismatch in the center) is unfavorable because the guanosine base sterically collides with the amino acid side chain of ADAR2.In one preferred embodiment, targeting of the 5'-CAC-3' target sequence (see Figure 1A, as in the case of the human SLC12A5 transcript), where central A is the target adenosine for deamination, is performed using an EON containing a central tribase (sometimes called, even though it is not necessarily the "central" position in the oligonucleotide) opposite these three nucleotides, with deoxyinosine (dI) on the 3' side of the orphan nucleotide. In one preferred embodiment, the orphan nucleotide is dZ, which is a nucleotide having a Benner's base and a 2'-H (=DNA) in the ribose sugar. In another embodiment, the orphan nucleotide is a cytidine containing a 2'-F substitution or a 2',2'-difluoro substitution in the ribose sugar. In yet another embodiment, the orphan nucleotide is a deoxyuridine or nucleotide having a modified uracil nucleic acid base (such as isouridine), with a 2'-H (=DNA) at the 2' position of the ribose sugar. In a preferred embodiment, three nucleotides opposite the 5'-CAC-3' tribase in the target molecule form a 5'-moeG-dZ-di-3' tribase in EON, where "moeG" is a guanosine containing a 2'-MOE substitution in the ribose sugar moiety.

[0102] This disclosure relates to RNA editing oligonucleotides, commonly referred to herein as "EONs," that can result in the deamination of adenosine in the SLC12A5 transcript, wherein the resulting KCC2 protein has increased functionality, preferably due to a reduced phosphorylation state. This means that this disclosure is not strictly limited to the deamination of adenosine in wild-type SLC12A5, but may target other (single or more) adenosines, which may also result in increased KCC2 protein function. Other adenosines that may be important (or more important) to KCC2 function and may be targeted through RNA editing may be identified, for example, by genetic screening in a population or in silico, according to the teachings of this disclosure. All oligonucleotides that can be used for such RNA events and such targeting, regardless of the exact nuclear molecule or what the EON may look like, are encompassed by this disclosure.

[0103] Mutagenesis studies of human ADAR2 have revealed that a single mutation from glutamic acid to glutamine at residue 488 (E488Q) resulted in a 60-fold increase in the deamination rate constant compared to the wild-type enzyme (Kuttan & Bass. Proc Natl Acad Sci USA 2012. 109(48):3295-3304). During the deamination reaction, ADAR ejects the edited base from its RNA double helix and places it within the enzyme's active site (Matthews et al. 2016). When ADAR2 edits adenosine in a preferred context (A:C mismatch), the nucleotide opposite the target adenosine is "orphancytidine". Crystal structure analysis of ADAR2 E488Q bound to double-stranded RNA (dsRNA) revealed that the glutamine (Gln) side chain at position 488 can donate an H-bond to the N3 position of orphancytidine, resulting in an increased catalytic rate of ADAR2 E488Q. In the wild-type enzyme, where glutamic acid (Glu) is present at position 488 instead of glutamine (Gln), the amide group of glutamine is absent, replaced by a carboxylic acid. To achieve the same contact between orphancytidine and the E488Q mutant, protonation would likely be required in the wild-type environment for this contact to occur. To utilize endogenously expressed ADAR2 to correct disease-related mutations, it is essential to maximize the editing efficiency of the wild-type ADAR2 enzyme present in cells. International Patent Application Publication No. 2020 / 252376, which is incorporated in its entirety herein by reference, discloses the use of an EON having a modified RNA base at the orphancytidine position to mimic the hydrogen bonding pattern observed, in particular, by the E488Q ADAR2 mutant. It was hypothesized that by replacing the nucleotide opposite the target adenosine in the EON with a cytidine analog that acts as an H-bonding donor at N3, it would be possible to stabilize the same contact that is thought to provide an increase in the catalytic rate of the mutagenesis.Two cytidine analogs, namely pseudoisocytidine (also known as "piC", Lu et al. J Org Chem 2009. 74(21):8021-8030, Burchenal et al. (1976) Cancer Res 36:1520-1523) and Benner's base Z (also known as "dZ", Yang et al. Nucl Acid Res 2006. 34(21):6095-6101), are of interest and were initially selected because they provide hydrogen bonding at N3 with minimal disturbance to the shape of the nucleic acid base. Benner's base is also chemically known as the 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base. In addition to modifications to the ribose 2' group, the presence of cytidine analogs in EON may also exist. The ribose 2' group in EON can be independently selected from 2'-H (i.e., DNA), 2'-OH (i.e., RNA), 2'-OMe, 2'-MOE, 2'-F, or 2'-4' linkages (i.e., crosslinked nucleic acids such as locked nucleic acids (LNA)), or other 2' substitutions. The 2'-4' linkage can be selected from linkers known in the art, such as methylene linkers or restricted ethyl linkers.

[0104] In one embodiment, nucleotide analogs or equivalents within an EON include modifications or substitutions of one or more bases. Modified bases include synthetic and natural bases known or to be known in the art, such as inosine, xanthine, hypoxanthine, and other pyrimidine and purine bases, including -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, and thioalkyl derivatives. Purine and / or pyrimidine nucleic acid bases may be modified, for example, by heterocyclic amination or deamination, to alter their properties. The exact chemistry and form may vary between oligonucleotide constructs and applications and may be shaped according to the wishes and preferences of those skilled in the art.

[0105] In this specification, EONs are typically longer than 10 nucleotides, preferably longer than 11, 12, 13, 14, 15, or 16 nucleotides, and more preferably longer than 17 nucleotides. In one embodiment, EONs in this specification are longer than 20 nucleotides. In this specification, EONs are preferably shorter than 100 nucleotides, more preferably shorter than 60 nucleotides, and even more preferably shorter than 50 nucleotides. In a preferred embodiment, EONs in this specification contain 18 to 70 nucleotides, more preferably 18 to 60 nucleotides, and even more preferably 18 to 50 nucleotides. Therefore, in a particularly preferred embodiment, the EONs in this specification comprise 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 one embodiment, the EON has a length of 27, 28, 29, or 30 nucleotides.

[0106] In one embodiment, an inverted deoxy-T or dideoxy-T nucleotide is incorporated into one or both ends of the EONs in this specification.

[0107] As described above, in some embodiments, this disclosure provides EONs for forming double-stranded complexes with human SLC12A5 target RNA molecules in human neuronal cells, for example, in the brain. Therefore, the therapeutic effect is preferably on human nerve cells in vivo. Of course, this method can also be carried out in vitro or ex vivo.

[0108] This disclosure provides EON or the pharmaceutical compositions herein for use in the treatment of a disease. This disclosure also provides the use of EON or the pharmaceutical compositions herein in the manufacture of a pharmaceutical for the treatment of a disease. This disclosure also provides a method for treating a disease in a patient, comprising administering a therapeutically effective amount of EON or the pharmaceutical compositions herein. Preferably, the disease is caused by reduced GABAergic inhibition, which is generally caused by decreased activity of the major chloride efflux factor KCC2, but due to increased chloride concentrations in nerve cells. EON is administered therapeutically or prophylactically, as both types of treatment may be beneficial.

[0109] After RNA editing occurs in a cell, the modified RNA may be diluted over time due to factors such as cell division and the limited half-life of the edited RNA. Therefore, from a practical therapeutic standpoint, the methods described herein may include repeatedly delivering EONs described herein until sufficient target RNA has been modified to provide a visible benefit to the patient and / or to maintain that benefit over time.

[0110] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. Database entries and electronic publications disclosed in this disclosure are incorporated in their entirety by reference. The versions of database entries or electronic publications incorporated by reference in this application are the most recent versions of the database entries or electronic publications that were publicly available at the time this application was filed. Database entries disclosed in this application that correspond to gene or protein identifiers (for example, genes or proteins identified by accession numbers or database identifiers in public databases such as Genbank, Refseq, or Uniprot) are incorporated in their entirety by reference. The incorporated information relating to genes or proteins is not limited to the sequence data contained in the database entries. The incorporated information includes the entire content of the database entries in the most recent versions of the databases that were publicly available at the time this application was filed. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to limit the scope of this application. [Examples]

[0111] [Example 1] Editing of targeted adenosine in human SLC12A5 target RNA molecules using an in vitro biochemical editing assay. First, we designed an initial set of 78 SLC12A5-targeted EONs (shown in Figure 1A), and then tested several of them to work on editing human SLC12A5 target mRNA (precursor) in an in vitro biochemical editing assay (BEA). To obtain SLC12A5 target RNA, we used an SLC12A5 G-block (IDT) containing the T7 promoter sequence and (part of) the SLC12A5 sequence as a template, and performed PCR using forward primer 5'-CTC GAC GCA AGC CAT AAC AC-3' (SEQ ID NO: 106) and reverse primer 5'-TGG ACC GAC TGG AAA CGT AG-3' (SEQ ID NO: 107). The 5' to 3' G-BLOCK sequence (SEQ ID NO: 108) is shown below, with the target adenosine underlined in bold and the primer position underlined.

[0112] [ka]

[0113] Subsequently, the PCR product was used as a template for in vitro transcription. The MEGAscript T7 transcription kit was used for this reaction. RNA was purified on a urea gel, then extracted with 50 mM Tris-Cl, pH 7.4, 10 mM EDTA, 0.1% SDS, and 0.3 M NaCl buffer, followed by purification with phenol-chloroform. The purified RNA was used as a target in BEA.

[0114] First, EONs B1-B20 (see Figure 1A) were annealed with SLC12A5 target RNA in a buffer (5 mM Tris-Cl, pH 7.4, 0.5 mM EDTA, and 10 mM NaCl) at a target RNA to oligonucleotide ratio of 1:3 (600 nM oligonucleotide and 200 nM target). In this disclosure, references to EONs B-1-B-78 (with dashes) are equivalent to and interchangeable with B1-B78 (without dashes). The samples were heated at 95°C for 3 minutes and then slowly cooled to RT. Next, the editing reaction was performed. Annealed oligonucleotides / target RNA were mixed with protease inhibitors (cOmplete®, Mini, EDTA-free protease I, Sigma-Aldrich), RNase inhibitors (RNasin, Promega), poly(A) (Qiagen), tRNA (Invitrogen), and editing reaction buffer (15 mM Tris-Cl, pH 7.4, 1.5 mM EDTA, 3% glycerol, 60 mM KCl, 0.003% NP-40, 0.5 mM DTT, 40 mM K-glutamate, and 3 mM MgSO4) to a final concentration of 6 nM oligonucleotide and 2 nM target RNA. The reaction was initiated by adding purified ADAR2 (GenScript) to the mixture to a final concentration of 9 nM, and the mixture was incubated at 37°C for a predetermined time. Each reaction was stopped by adding 95 μl of 3 mM EDTA solution at 95°C. Subsequently, 6 μl aliquots of the stopped reaction mixture were used as templates for cDNA synthesis using a Maxima reverse transcriptase kit (Thermo Fisher) with random hexamer primers (ThermoFisher Scientific). Initial RNA denaturation was performed at 95°C for 5 minutes in the presence of primers and dNTPs, followed by slow cooling to 10°C. Then, according to the manufacturer's instructions, the first strand was synthesized in a total volume of 20 μl using an extension temperature of 62°C.For pyrosequencing analysis by PCR, the product was amplified using the Amplitaq gold 360 DNA polymerase kit (Applied Biosystems) according to the manufacturer's instructions, with 1 μl of cDNA as a template, using the forward primer 5'-AGGAGCCTGAGGGGGAAG-3' (SEQ ID NO: 109) and the biotin-labeled reverse primer 5'-GGGGCCCTTATTCTTCTCTGC-biotin-3' (SEQ ID NO: 110).

[0115] Subsequently, PCR was performed using the following thermal cycling protocol: initial denaturation at 95°C for 5 minutes, followed by 45 cycles of 30 seconds at 95°C, 30 seconds at 62°C, and 30 seconds at 72°C, as well as final extension at 72°C for 7 minutes.

[0116] During cDNA synthesis in the reverse transcription reaction, inosine base pairs with cytidine, so the nucleotide incorporated at the edited position during PCR becomes guanosine. The percentage of guanosine (edited) versus adenosine (unedited) was determined by pyrosequencing. Pyrosequencing and data analysis of the PCR product were performed using a PyroMark Q48 Autoprep instrument (QIAGEN) according to the manufacturer's instructions, with 10 μl of PCR product input and 4 μM sequencing primer: 5'-GGGAAGGGGAGACAG-3' (SEQ ID NO: 111).

[0117] The instrumental analysis provided results for selected nucleotides as percentages of adenosine and guanosine detected at that position; therefore, the degree of A-to-I editing at the selected position was measured by the percentage of guanosine at that position.

[0118] The results are shown in Figures 2A-2E, and since EON B6 (29 nts) gave the best and fastest editing percentages, the results using them are shown in Figures 2B, 2C, and 2D. The editing percentages observed using these initial 20 EONs in BEA clearly demonstrate that deamination of target adenosine representing adenosine in the ACC codon encoding threonine at position 1007 in the human KCC2b isoform is feasible.

[0119] [Example 2] Editing of targeted adenosine in human SLC12A5 target RNA molecules using retinal organoids. To explore the possibility of editing the human SLC12A5 transcript molecule in an environment more similar to that of in vivo, KCC2 expression was determined in human retinal organoids generated as described in International Patent Application Publication No. 2022 / 090256. KCC2 transcripts were detectable (approximately 1200 per cell), and the KCC2 protein could be observed using various anti-KCC2 antibodies in immunohistochemistry and Western blotting (data not shown), indicating that retinal organoids, although not representative of brain tissue, do indeed contain neurons and are useful for determining whether the SLC12A5 transcript can be edited in a cellular environment.

[0120] In the initial screening, EON B1-B13 and B15-B20 were tested for RNA editing. For this purpose, 10 μM EON was added to the culture medium of human wild-type retinal organoids approximately 200 days old and incubated for 14 days. Two days after EON addition, the organoids were washed with fresh medium (this removes EON from the medium), and the medium was subsequently changed every two days thereafter. RNA was extracted from the organoids, and the percentage of editing was then determined using ddPCR.

[0121] RNA was isolated from the sample using the Direct-zol RNA Microprep kit (Zymo) and eluted with 15 μL of RNAse-free water. Subsequently, 500 ng of RNA was used as a template for cDNA synthesis using the Maxima 1-strand cDNA synthesis kit (Thermo Scientific) with random hexamers and OligoDt, according to the manufacturer's instructions. The total reaction volume was 20 μL.

[0122] During cDNA synthesis in reverse transcription, inosine pairs with cytidine, so the nucleotide incorporated at the edited position during PCR becomes guanosine. The percentage of guanosine (edited) versus adenosine (unedited) was determined by ddPCR at exon 23 of the SLC12A5 transcript.

[0123] Each ddPCR sample contained 1× probe ddPCR supermix (without dUTP) (Biorad), 0.9 μM forward primer 5'-GTGCAGCTGATCCACGAT-3' (SEQ ID NO: 112), 0.9 μM reverse primer 5'-GCCCTTATTCTTCTCTGCCA-3' (SEQ ID NO: 113), 0.6 μM each of the double quench WT unedited transcription probes 5'-Hexa-TGCATCT+C+A+CCTGGA-3' (SEQ ID NO: 114) and the mutant edited transcription probe 5'-Fam-TGCATCT+C+G+CCTGG-3' (SEQ ID NO: 115) [+ indicates locked nucleic acid (LNA) at the 3' end], and template cDNA in a total volume of 21 μL. Droplets were prepared from the PCR mix using a QX200 droplet generator (Biorad). Next, droplet PCR was performed in a T100 thermal cycler (Biorad) using a heated lid at 105°C and a temperature gradient of 2°C / second. Polymerase was thermally activated at 95°C for 10 minutes. In each cycle, denaturation was performed at 95°C for 30 seconds, followed by annealing / extension at 59°C for 60 seconds. This was repeated for a total of 40 cycles. The enzyme was inactivated at 98°C for 10 minutes, and the reaction was maintained at 8°C. Fluorescence signals from the droplets were measured using a QX200 droplet reader (Biorad). The number of fluorescently positive droplets and subsequent absolute quantification were performed using QuantaSoft software (Bio-Rad). As an additional quality control, samples that significantly deviated from the rest in terms of absolute copy number were removed. This was determined by calculating the average number of SLC12A5 (A+G transcript) copies for each condition, and samples were removed if they had one-fifth or five times the number of SLC12A5 copies compared to the average.

[0124] Each sample was measured in pairs, and the average value was used to determine the percentage of editing. The percentage of editing was calculated by dividing the number of edited (G)SLC12A5 transcript copies per well by the total number of SLC12A5 copies per well (A+G transcript).

[0125] The results are shown in Figures 3A and 3B, clearly demonstrating that in certain EONs (e.g., B3, B4, and B15), editing of the SLC12A5 target transcript reached nearly 15%.

[0126] In the next screening, 20 other EONs (B21-B40, see Figure 1A) were used for RNA editing in human retinal organoids without any translocation, and were compared with EON B3 and EON B14 used in the first screening. The setup and ddPCR procedure were as described above. The results of the second screening are shown in Figure 4, and these results show that the percentage of editing can be further increased compared to B3, and that EON B22 and B23 provide the highest percentage of RNA editing. These results show that we can deaminate the target adenosine shown in Figure 1A in the SLC12A5 target transcript to a significant level, making it possible to achieve a lack of phosphorylation site at position 1007 (in the KCC2b isoform) in the KCC2 protein encoded by the transcript, thereby increasing KCC2 activity.

[0127] [Example 3] Editing of human SLC12A5 transcript in HEK cells overexpressing KCC2. To evaluate adenosine editing in the codon ACC encoding threonine at position 1007 of the KCC2b isoform, we first investigated which available cell lines or cell systems could detect sufficient KCC2 expression. Of the more than 10 available cell lines, none appeared to express KCC2 to a useful level. However, retinal organoids expressed KCC2 at levels that allowed them to be used for initial in vitro screening (see Example 2). Because organoids are not readily cultured and generated, requiring complex culture conditions and growth factors, we obtained a stable expression construct and generated a novel cell line based on human embryonic kidney 293 (HEK293) cells that stably overexpresses the human SLC12A5 transcript (and KCC2 protein). The expression construct was based on pcDNA3.1Neo with an open reading frame located downstream of the CMV promoter, using a general method well known to those skilled in the art. The resulting cell line was named "HEK-KCC2," and it overexpressed KCC2 to a very significant level (expression data not shown), allowing it to be used in subsequent RNA editing experiments.

[0128] In the initial in vitro editing experiment, all 78 EONs shown in Figure 1A were transfused into HEK-KCC2 cells. This required 79,000 cells / cm³. 2The cells were seeded and maintained in DMEM containing 10% FBS and 750 μg / ml Geneticin. Immediately after seeding, cells were transfused (also known as reverse transfusion) using Lipofectamine® 3000 (Thermo Fisher) with 200 nM EON, using a 1:3 ratio of EON to lipofection reagent, according to the manufacturer's protocol. Editing was determined 24 hours after the start of transfusion, and RNA was isolated using a Direct-zol RNA isolation kit (Zymo Research) according to the manufacturer's instructions. cDNA generation and dPCR analysis were performed as described above for the retinal organoids. The results are shown in Figures 5A and 5B. Certain EONs, particularly B33, B34, B35, B50, B55, B63, B65, B77, and B78, performed relatively well, reaching an editing level of 15%. These results suggest that, in the context of modifications introduced into EONs (see Figure 1A), having one or more 2'-F modified nucleotides in an EON is beneficial. However, no definitive conclusions regarding the location of modifications or specific types and locations of modifications could be drawn. All good-performing EONs have multiple 2'-F modified nucleotides in their sequence, some with contiguous stretches. Furthermore, PNdmi ligations do not specifically inhibit or induce RNA editing based on the length of the EON and the content of the selected sequence.

[0129] [Example 4] Detection of phosphorylated human KCC2 protein versus non-phosphorylated human KCC2 protein using antibodies. The question was whether the effect of editing, which should result in the emergence of KCC2 mutants that cannot be phosphorylated at position 1007 (in the KCC2b isoform), could also be determined by addressing the phosphorylation state of the protein. In principle, the amount of phosphorylated KCC2 should decrease with the RNA disclosed herein. Note that the KCC2 protein can exist in both phosphorylated and unphosphorylated states, even in the wild type which contains a threonine residue at position 1007. Because the peptide surrounding the target residue is conserved across various proteins, the entire protein needs to be immunoprecipitated to remove it from other proteins before evaluating the phosphorylation state of the naturally expressed KCC2 protein at position 1007. However, this is not necessary in the HEK-KCC2 cell line. Using competitive experiments with 22-amino acid-length peptides containing either phosphorylated Thr at position 12, unphosphorylated Thr at position 12, or unphosphorylated Ala at position 12 (a mutant representing the edited protein), we determined which could interfere with the binding of the antibody to the phosphorylated KCC2 protein. Ultimately, the Thr1007 phospho-KCC2 antibody (1:1000 dilution, Thermo Fisher, PA5-95677, polyclonal) specifically recognized phosphorylated KCC2 protein but not unphosphorylated KCC2 protein (data not shown), making it possible to distinguish between edited and unedited events.

[0130] To test whether RNA editing can reduce the amount of phosphorylated KCC2 in cells, HEK-KCC2 cells were transfused with EONs B33, B34, B35, B50, B55, B63, B65, B77, and B78 using the same transfusion method as described above. Cells were harvested 48 hours after the start of transfusion, and the percentage of editing was determined generally as described above. In addition, anti-KCC2 antibodies were used to determine the levels of non-phosphorylated and phosphorylated proteins, and β-tubulin background expression was taken as a control. The editing results are shown in Figure 6A. The editing observed for all nine EONs also reached 15% in the best cases, which was considered somewhat comparable. The results of determining the amount of phosphorylated KCC2 in these same cell samples are provided in Figure 6B. Transfusion of EONs, as shown, reduced the amount of phosphorylated KCC2 in almost all cases compared to the standard set (set to 100%) due to mock transfusion, indicating that the reductions caused by EON B65 and B78 are indeed significant. This suggests that RNA editing of target adenosine in the SLC12A5 transcript, which induces a gain-of-function mutation by introducing alanine instead of threonine at position 1007, can reduce the amount of phosphorylated KCC2, thereby providing a potential means to activate KCC2 to higher levels.

[0131] [Example 5] Editing of the SLC12A5 transcript in human neurons (iPSC neurons) induced from pluripotent stem cells. As mentioned above, KCC2 expression was very low in most cell lines other than retinal organoids. This finding was the main driving force behind the creation of HEK cell lines that overexpress human KCC2. However, it was noted that transcript expression varied significantly over time and across different cultures. Subsequently, neurons cultured from induced pluripotent human stem cells (further referred to herein as "iPSC neurons") were found to express useful levels of human KCC2, enabling the application of RNA editing and potentially downstream effects.

[0132] In the initial experiment, 78 EONs shown in Figure 1A were used in a gymnosis exposure experiment (adding only EONs to the culture medium without gymnosis uptake = translocation). Here, 5 μM EONs were administered to the culture medium, the cells were kept for 48 hours, and then the EONs were slowly washed away by replacing 50% of the culture medium with fresh medium every two days. Editing was determined 14 days after the start of the experiment. RNA was isolated using the miRVana RNA isolation kit (Thermo Fisher), and EONs were removed from the RNA sample using several modifications according to the manufacturer's protocol. After cell disruption, the supplied miRNA homogenate additive was added at 1 / 10 of the total volume. After phenol:chloroform (equal volume) extraction, the upper aqueous phase was heated at 60°C for 1 minute. 100% ethanol was added to the aqueous phase at 1 / 3 of the total volume. The sample was added to the supplied filter column, and after centrifugation, the flow solution was discarded. The mRNA-containing filter was washed according to the protocol and eluted using 30 μl of 95°C nuclease-free water. Subsequently, the purified RNA was treated with TURBO DNase (Thermo Fisher) according to the supplied protocol. cDNA generation and dPCR analysis were performed as described above. The results are shown in Figures 7A and 7B. Interestingly, using this gymnosis incorporation procedure instead of the active translocation assay, other EONs appeared to work more efficiently than those found in HEK-KCC2 cells. In particular, EONs B26, B39, B66, B68, B69, B70, B72, B73, B74, and B78 performed especially well, with B78 being the EON that also performed well in HEK-KCC2 cells.

[0133] [Example 6] Editing of SLC12A5 transcripts in iPSC neurons using EONs with various 2'-ribose modifications. Subsequently, to further investigate the potential role of 2'-F modification of ribose in the nucleotides of SLC12A5-targeted EONs, and to see if altering these could improve the level of editing in iPSC neurons, we designed a new set of EONs in which one or more positions contained 2'-F substitutions compared to B4 (see Figure 1A). These EONs and their specific modifications are shown in Figure 8. B138 (SEQ ID NO: 132) and B139 (SEQ ID NO: 133) could not be generated initially and were not tested in the first experiment. The SEQ ID NOs for these EONs are provided in square brackets. B122 is identical to B4 except for the PS ligation at ligation position 0 (B4 contains a PO ligation). 2'-F modified nucleotides are shown in gray boxes, and B122 shows that it has only a single 2'-F modified nucleotide (position -3), which was also present in all EONs B123-B137 and B140-B141.

[0134] 5 μM EON was administered to iPSC neurons for the gymnastic uptake described above. The same washout procedure was applied by changing 50% of the culture medium every two days. Culture was continued for 14 days, and dPCR analysis of RNA isolation, cDNA generation, and editing using the mirVana RNA isolation kit was performed as described above.

[0135] The results are shown in Figure 9, indicating that several EONs, particularly those containing 3, 4, or 5 2'-F modified nucleotides on the 3' side of the orphan nucleotide and 2 or fewer (e.g., 0) 2'-F modified nucleotides on the 5' side, performed particularly well. Examples of the best-performing EONs include B134, B135, B136, and B137. In contrast, having multiple 2'-F modified nucleotides on both sides of the orphan nucleotide, as in B140, does not appear to contribute to a high percentage of editing. Having a series of 2'-F modified nucleotides on the 3' side of the orphan nucleotide, while having a low number of 2'-F modified nucleotides on the 5' side of the orphan nucleotide, appears to be beneficial for obtaining a higher percentage of editing with respect to the SLC12A5 target transcript.

[0136] Based on these results, in which B137 performed best, B137 was used as the base design to see if further 2'-F modifications could further increase the percentage of editing. In addition to 2'-F and specific PNdmi ligation sites, specific mismatches and asymmetric designs with the human target sequence were also introduced. For this purpose, a new set of EONs was designed, shown in Figure 10 (B144-172). In all cases, the Zd position is an orphan nucleotide. Here again, all 2'-F modified nucleotides are shown in gray boxes. Nucleotides that are not complementary to the human target sequence are underlined. B155, B161, and B163 contain the central three bases (orphan nucleotide and the nucleotides immediately adjacent to its 3' and 5') which are 3×DNA. B156-B160 have an asymmetric design where the 5' portion as seen from the orphan nucleotide is shorter than the 3' portion as seen from the orphan nucleotide. B161-B164 have the opposite asymmetric design. Sequences B161-B164 also have a high number of 2'-F modified nucleotides in the 5' portion relative to the orphan nucleotide. Sequences B165-B172 have numerous nucleotides that do not match the human target sequence.

[0137] B144–B172 (except for B152, which was not initially manufactured for unknown reasons) were tested in human iPSC neurons using 5 μM EON in a 2-week washout setting, as described above. A rat Slc12a5 sequence-specific version of B144 was used (referred to as rB1030-144 in the figure). The percentage of edits obtained in this initial experiment is shown in Figure 11, from which it is immediately apparent that asymmetric designs with a lower number of nucleotides on the 5' side of the orphan nucleotide compared to the 3' side (B156–B160) performed significantly lower than B144, which is an EON similar to B137 (see above, except that PNdmi linkages are introduced at three positions). In general, asymmetric designs with a higher number of nucleotides on the 5' side of the orphan nucleotide compared to the 3' side (B161–B164) provided a higher percentage of edits than B144, indicating that this is the preferred design. The best-performing EON, called B162, has only six nucleotides 3' from the orphan nucleotide. Notably, B151 and B155 also perform significantly better, and in these, the orphan nucleotide is located slightly centrally within the EON. The presence of 3×DNA in the central three bases of B155 does not appear to hinder proper editing. B151 has a 2'-F pattern similar to the 2'-F pattern used in B137 and B144.

[0138] [Example 7] Editing of the Slc12a5 transcript in rat spinal cord. In the following in vivo experiments, we investigated whether administering EONs into cerebrospinal fluid resulted in editing of endogenous rat Slc12a5 transcripts in the lumbar spinal cord and cortex. First, based on the results with human SLC12A5 transcript-specific EONs (see above), we designed a set of rat-specific EONs with chemical modifications. These rat-specific EONs are provided in Figure 12. Numbering refers to their equivalents used in human iPSC neurons (see above), and the only difference between the human counterpart and the rat-specific EON is underlined, which is A at position +14 in the EON, opposite U in the rat target sequence. EONs targeting rat App transcripts and cerebrospinal fluid from untreated animals were used as negative controls. Sprague-Dolly rats were used as study subjects, and 300 μg of EON was administered intraarachnoidally using a single dose. The following EONs were tested: rB-4 (rB1030-4, SEQ ID NO: 165), rB-26 (rB1030-26, SEQ ID NO: 166), rB-39 (rB1030-39, SEQ ID NO: 167), rB-50 (rB1030-50, SEQ ID NO: 168), rB-65 (rB1030-65, SEQ ID NO: 169), rB-66 (rB1030-66, SEQ ID NO: 170), rB-67 (rB1030-67, SEQ ID NO: 171), rB-68 (rB1030-68, SEQ ID NO: 172), rB-69 (rB1030-69, SEQ ID NO: 173), rB-70 ( rB1030;70 (sequence number 174), rB-72 (rB1030-72, sequence number 175), rB-73 (rB1030-73, sequence number 176), rB-74 (rB1030-74, sequence number 177), rB-77 (rB1030-77, sequence number 178), rB-78 (rB1030-78, sequence number 179), rB-144 (rB1030-144, sequence number 180), rB-145 (rB1030-145, sequence number 181), B-70 (sequence number 70), B-74 (sequence number 74), and B-145 (sequence number 137). Some EONs were tested at higher doses: rB-4 (770 μg), rB-68 (575 μg), rB-70 (490 μg), rB-72 (605 μg), rB-73 (460 μg), and rB-74 (450 μg). After two weeks, the rats were sacrificed and cerebrospinal fluid was collected to determine equivalent A editing in the rat Slc12a5 transcript.Furthermore, rat brains were dissected and the cortex isolated. RNA was isolated using the mirVana RNA isolation kit (Thermo Fisher) as described above. cDNA synthesis and dPCR were performed using rat-specific forward primers 5'-GTGCAGCTGATCCATGAC-3' (SEQ ID NO: 182) and reverse primers 5'-GCCTTTGTTCTTCTGAGCCG-3' (SEQ ID NO: 183), as generally described above, and the same detection probes as for human KCC2 were used. It should be noted that administering the therapeutic agent into the spinal cord is a laborious and delicate procedure. In humans and monkeys, it is difficult to locate and target the spinal cord with a single injection. In mice, locating the spinal cord is nearly impossible due to its size, which is why rats were chosen for this study. Multiple injections appeared to be unsuccessful (based on in situ imaging of injection sites after sacrifice, data not shown), which is visualized in the edit results, which appeared close to zero for misinjected doses. Nevertheless, in several cases, administration was successful and edits could be detected. These results are presented in Figure 13, demonstrating that a high editing level of 8% can be achieved in rat spinal cord, with rB-73 exhibiting the best performance. Doses higher than 300 μg (indicated as "HD" in the figure) did not result in significantly higher editing levels. Here again, some rats were misinjected, resulting in an editing level of 0. Therefore, samples in which EON was not detected in situ (from spinal cord tissue extracted near the injection site) were not included in the calculations.

Claims

1. An RNA editing oligonucleotide (EON) capable of forming a double-stranded complex with a region of an endogenous human SLC12A5 transcript molecule in a cell, wherein the region of the SLC12A5 transcript molecule contains target adenosine, the nucleotide in the EON directly opposite the target adenosine is an orphan nucleotide, the nucleotides in the EON are numbered such that the orphan nucleotide is number 0, the nucleotides 5' to the orphan nucleotide are positively (+) incremented toward the 5' end and negatively (-) incremented toward the 3' end, and the double-stranded complex can recruit an endogenous ADAR enzyme to deaminate the target adenosine to inosine, thereby editing the SLC12A5 transcript molecule.

2. The EON according to claim 1, wherein the SLC12A5 transcript molecule is an mRNA precursor or an mRNA molecule.

3. The EON according to claim 1 or 2, wherein the SLC12A5 transcript molecule has a wild-type sequence.

4. The EON according to any one of claims 1 to 3, wherein the target adenosine is located in a codon encoding an amino acid that can be phosphorylated.

5. The EON according to claim 4, wherein the target adenosine is the first nucleotide of the codon encoding the threonine at position 1007 of the KCC2b isoform encoded by SLC12A5.

6. The EON according to claim 4, wherein the target adenosine is the first nucleotide of the codon encoding the threonine at position 1030 of the KCC2a isoform encoded by SLC12A5.

7. The EON according to any one of claims 1 to 6, wherein the deamination of the target adenosine results in a KCC2 protein encoded by SLC12A5 having increased activity.

8. The EON according to claim 7, wherein the increased activity results in higher GABAergic inhibition.

9. The EON according to any one of claims 1 to 8, wherein the cells are neurons, preferably brain cells.

10. The EON is selected from the group consisting of sequence numbers 1 to 104 and 116 to 164, preferably sequence numbers 3, 4, 14, 15, 22, 23, 28, 29, 33, 34, 35, 36, 40, 55, 63, 65, 69, 70, 73, 74, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 EON according to any one of claims 1 to 9, selected from the group consisting of 4, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 116, 119, 124, 127, 128, 129, 130, 131, 136, 137, 141, 142, 143, 145, 146, 147, 153, 154, 155, 156, and 184.

11. An EON according to any one of claims 1 to 10, comprising at least one non-naturally occurring chemical modification and / or comprising one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion, wherein the orphan nucleotide is not a cytidine comprising a 2'-OMe ribose substitution.

12. The EON according to claim 11, wherein the orphan nucleotide is a deoxynucleotide containing a 6-amino-5-nitro-3-yl-2(1H)-pyridone nucleic acid base, and the nucleotide at position -1 in the EON is deoxyinosine.

13. The EON according to claim 12, wherein the one or more additional modifications in the linking portion are each independently selected from phosphorothioate (PS), phosphonoacetate, phosphorodithioate, methylsulfonate (MP), sulfonyl phosphoramidate, mesyl phosphoramidate (PNms), or (1,3-dimethylimidazolidinedine-2-ylidene)phosphoamidate (PNdmi) internucleotide linkages.

14. EON according to claim 12 or 13, wherein each of the one or more additional modifications in the ribose portion is independently selected from the group consisting of one substitution or two substitutions at the 2', 3', and / or 5' positions of the ribose: -OH, -F, - Substituted or unsubstituted, linear or branched lower (C) atoms, which may be interrupted by one or more heteroatoms. １ ~C １０ ) Alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, -O-, S-, or N-alkyl, -O-, S-, or N-alkenyl, -O-, S-, or N-alkynyl, -O-, S-, or N-allyl, -O-alkyl-O-alkyl, - Methoxycin, - Aminopropoxy, - Methoxyethoxy, -Dimethylaminooxyethoxy, and - Dimethylaminoethoxyethoxy.

15. A vector comprising a nucleic acid molecule encoding an EON containing any one of sequence numbers 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, and 184, preferably a viral vector, more preferably an adeno-associated virus (AAV) vector, wherein the orphan nucleotide is cytidine or uridine, and the nucleotide at position -1 of the EON is guanosine.

16. A pharmaceutical composition comprising an EON according to any one of claims 1 to 14, or a vector according to claim 15, and a pharmaceutically acceptable carrier.

17. EON according to any one of claims 1 to 14, or the vector according to claim 15, for use in treating disorders caused by reduced GABAergic inhibition, preferably disorders caused by reduced KCC2 activity.

18. EON for use according to claim 17, wherein the disorder is chronic pain or epilepsy.

19. Use of an EON according to any one of claims 1 to 14, or a vector according to claim 15, in the manufacture of a pharmaceutical product for treating disorders caused by reduced GABAergic inhibition, preferably disorders caused by reduced KCC2 activity.

20. The use according to claim 19, wherein the disorder is chronic pain or epilepsy.

21. A method for editing an SLC12A5 polynucleotide, comprising contacting the SLC12A5 polynucleotide with an EON capable of resulting in ADAR-mediated editing of a target adenosine in a codon encoding an amino acid related to the phosphorylation of the protein KCC2 encoded by the SLC12A5, thereby editing the SLC12A5 polynucleotide.

22. A method for treating a disorder caused by reduced GABAergic inhibition, preferably by reduced KCC2 activity, in an individual in need thereof, comprising contacting an SLC12A5 polynucleotide in a target cell with an EON capable of causing ADAR-mediated editing of adenosine to inosine at a target adenosine in a codon encoding an amino acid related to the phosphorylation of the protein KCC2 encoded by the SLC12A5, thereby treating the individual.

23. The method according to claim 21 or 22, wherein the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5, or the target adenosine is the first nucleotide of the codon encoding threonine at position 1030 of the KCC2a isoform encoded by SLC12A5.

24. A method for treating a disorder caused by reduced GABAergic inhibition, preferably a disorder caused by reduced KCC2 activity, comprising administering to an individual in need of such treatment a therapeutically effective amount of EON according to any one of claims 1 to 14, the vector according to claim 15, or the pharmaceutical composition according to claim 16.

25. The method according to any one of claims 21 to 24, wherein the disorder is chronic pain or epilepsy.

26. A method for deaminating target adenosine in an SLC12A5 mRNA precursor or mRNA molecule in cells, (i) the step of providing the cells with the EON according to any one of claims 1 to 14, (ii) The step of causing the cells to take up the EON, (iii) The step of annealing the EON with the SLC12A5 mRNA precursor or mRNA molecule, (iv) The endogenous ADAR enzyme deaminates the target adenosine in the target RNA molecule to inosine, and optionally, (v) The step of identifying the presence of the inosine in the target RNA molecule A method that includes this.

27. The method according to claim 26, wherein the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5, or the target adenosine is the first nucleotide of the codon encoding threonine at position 1030 of the KCC2a isoform encoded by SLC12A5.

28. Step (v) is a) A step of determining the sequence of the SLC12A5 mRNA precursor or mRNA molecule, b) A step of evaluating the presence of a KCC2 protein encoded by SLC12A5 having a lower phosphorylation rate, preferably a step of evaluating the presence of a KCC2 protein that is not phosphorylated at position 1007 in the KCC2b isoform or at position 1030 in the KCC2a isoform, or c) A step of evaluating, preferably, the level of GABAergic inhibition in the cells using functional readings. The method according to claim 26 or 27, including the method described in claim 26 or 27.

29. A nucleic acid molecule for editing a target adenosine in a human SLC12A5 mRNA precursor or mRNA molecule, wherein the target region is sequence number 105, and the target adenosine is the first nucleotide of the codon encoding threonine at position 1007 of the KCC2b isoform encoded by SLC12A5.

30. The nucleic acid molecule according to claim 29, wherein the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 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, and 184.

31. The nucleic acid molecule according to claim 29 or 30, further comprising at least one non-naturally occurring chemical modification and / or comprising one or more additional non-naturally occurring chemical modifications in the ribose, ligation, or base portion, wherein the orphan nucleotide, which is a nucleotide in the nucleic acid directly opposite the target adenosine in the target region, is not a cytidine comprising a 2'-OMe ribose substitution.

32. The nucleic acid molecule according to any one of claims 29 to 31, wherein the one or more additional modifications in the linking portion are each independently selected from PS, phosphonoacetate, phosphorodithioate, MP, sulfonylphosphoamide, PNms, or PNdmi nucleotide linkages.

33. The nucleic acid molecule according to claim 31 or 32, wherein each of the one or more additional modifications in the ribose moiety is independently selected from the group consisting of one substitution or two substitutions at the 2', 3', and / or 5' positions of the ribose: -OH, -F, - Substituted or unsubstituted, linear or branched lower (C1-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl molecules, which 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, - Methoxycin, - Aminopropoxy, - Methoxyethoxy, -Dimethylaminooxyethoxy, and - Dimethylaminoethoxyethoxy.

34. A vector comprising a nucleotide sequence encoding a nucleic acid molecule according to claim 29 or 30, wherein the orphan nucleotide is cytidine or uridine, and the nucleotide at position -1 in the EON is guanosine.

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