Antisense oligonucleotides targeting adenosine kinase

Abstract

Antisense oligonucleotides which are useful in the treatment of a range of neurological disorders, such as epilepsy or neuropathic pain. [Solution] The present invention provides antisense oligonucleotide compounds that target adenosine kinase pre-mRNA. Compositions and methods for treating neurological disorders using the antisense oligonucleotides of the invention are provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides novel antisense oligonucleotide compounds that target adenosine kinase, which are useful in the treatment of neurological disorders such as epilepsy or neuropathic pain. [Background technology]

[0002] background Epilepsy is a chronic neurological disorder characterized by severe recurrent spontaneous seizures that affects approximately 50 million people worldwide.

[0003] Currently available anti-epileptic drugs suppress seizures in two-thirds of patients, but likely do not affect the underlying pathophysiological functions. The remaining third of epilepsy patients are either drug-resistant or suffer from severe side effects from currently available drugs. Brain surgery, vagus nerve stimulation, intracranial stimulation, and ketogenic diets offer alternatives to avoid seizures in patients who do not respond adequately to drug treatment, but are only available to a limited number of drug-resistant epilepsy patients, so the majority remain without effective treatment options.

[0004] The development of epilepsy is thought to involve, among other things, altered expression of ion channels and neurotransmitter receptors, synaptic remodeling, inflammation, gliosis, and neuronal death. However, our understanding of the cellular and molecular mechanisms remains incomplete. With the exception of resective surgery, there are no treatments to prevent, modify, or cure epilepsy ("antiepileptogenic agents"). Similarly, there are no such treatments for acquired epilepsy following status epilepticus (SE) or brain injury, such as stroke or trauma, that likely lead to brain damage and epilepsy. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there remains a high unmet need for treatments or preventative measures that specifically target the processes that lead to epilepsy, neuropathic pain, and other neurological disorders that likely result in brain damage, and that overcome some of the problems listed above. [Means for solving the problem]

[0006] Adenosine and Adenosine Kinase Adenosine is a well-characterized endogenous anticonvulsant and seizure terminator in the brain. Adenosine influences seizure generation (ictogenesis), the development and progression of epilepsy (epileptogenesis). Maladaptive changes in adenosine metabolism, specifically increased expression of the astroglial enzyme adenosine kinase (ADK), play a major role in epileptogenesis. (Weltha et al, 2019, The role of adenosine in epilepsy, Brain Res Bull 2019 September, page 1-22.)

[0007] ADK plays a central role in regulating intracellular and interstitial concentrations of the purine nucleoside adenosine, thereby exerting potent cardioprotective and neuroprotective effects. The expression of adenosine kinase undergoes rapid coordinated changes in the brain after epileptic seizures or stroke, resulting in an acute surge of adenosine, which serves to minimize brain damage. Two ADK isoforms are expressed in mammalian cells that differ in their N-terminus. The long isoform (AdK-L) contains an extra 20-21 amino acids instead of the first four amino acids of the AdK-short (AdK-S) isoform. The N-terminal extension of AdK-L functions as a nuclear localization signal. Thus, of the two isoforms, AdK-L is targeted to the nucleus, whereas AdK-S is restricted to the cytoplasm. (Cui et al,2011,Molecular Characterization of Chinese Hamster Cells Mutants Affected in Adenosine Kinase and Showing Novel Genetic and Biochemical Characteristics,BMC Biochemistry 2011.)

[0008] Furthermore, disregulation of ADK expression and the resulting disruption of adenosine homeostasis are implicated in a wide range of neurological and neuropsychiatric pathologies. In the brain, ADK is predominantly expressed in astrocytes, and astroglial ADK is a promising target for predicting and preventing epileptic seizures. Astrogliosis and associated overexpression of ADK have also been identified in a rat model of severe traumatic brain injury (TBI) induced by lateral fluid percussion injury. Furthermore, ADK expression levels critically determine the vulnerability of the brain to the effects of stroke. Sleep and sleep intensity are also enhanced by adenosine and its receptor agonists, whereas antagonists such as caffeine and theophylline induce wakefulness. According to Boison et al., the association between ADK overexpression and cognitive impairment may be a pathological link to neurological conditions in which ADK overexpression is either confirmed (epilepsy) or suspected (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis). The adenosine hypothesis of schizophrenia posits that impaired function of adenosine signaling may contribute to the pathophysiological features of schizophrenia. In diabetes, adenosine homeostasis is critically altered in several tissues.

[0009] Furthermore, adenosine receptor signaling homeostasis is crucial for regulating inflammation and the release of pro-inflammatory cytokines. Also, adenosine receptor signaling homeostasis is critical for chronic inflammatory responses in IBD. The role of the adenosine / ADK regulatory system in cancer may depend on the type of cancer. ADK activity was found to be reduced in hepatoma cells, suggesting that increased adenosine may provide a selective advantage for liver cancer. (Boison et al., 2013, Adenosine Kinase: Exploitation for Therapeutic Gain, Pharmacol Rev 65: 906-943, July 2013.)

[0010] Adenosine Receptors Activation of inhibitory adenosine A1 receptors is beneficial in epilepsy, chronic pain, and cerebral ischemia, while inhibition of facilitatory A2A receptors has strong neuroprotective effects. (Boison et al, 2008, Adenosine as a neuromodulator in neurological diseases, Curr Opin Pharmacol, 2008 February.)

[0011] Adenosine is a neuromodulator that acts through the most abundant inhibitory adenosine A1 receptor (A1R) and the less abundant but widespread facilitatory A2AR. A1R is usually assumed to play a major role in neuroprotection, since it reduces glutamate release and hyperpolarizes neurons. (Rodrigo A. Cunha, 2005, Neuroprotection by adenosine in the brain: From A1 receptor activation to A2A receptor blockade, Purinergic Signalling (2005) 1:111-134.)

[0012] Restoring A3AR signaling in the spinal cord by inhibiting adenosine kinase or activating A3AR with intrathecal selective A3AR agonists prevents the establishment of chemotherapy-induced neuropathic pain (CINP). (Wahlman et al,2018,Chemotherapy-induced pain is promoted by enhanced spinal adenosine kinase levels via astrocyte-dependent mechanisms,Pain.2018 Jun;159(6):1025-1034..)

[0013] Epilepsy, neuroprotection, and psychiatric disorders Adenosine has anticonvulsant and neuroprotective properties. (Patodia et al,2020,Adenosine kinase and adenosine receptors A1R and A2AR in temporal lobe epilepsy are involved with hippocampal sclerosis and an association exists with risk factors for SUDEP,Epilepsia,page 787-797.)

[0014] Local adenosine augmentation therapy using adenosine kinase inhibitors has proven effective in reducing seizures in both animal models and human brain tissues excised from patients with intractable epilepsy of various etiologies. In addition to reducing seizures, adenosine augmentation therapy can also alleviate comorbidities such as torpor, cognition, or depression. Transgenic mice with reduced ADK were resistant to epileptogenesis induced by acute brain injury. (Wang et al, 2020, Role of Adenosine Kinase Inhibitor in Adenosine Augmentation Therapy for Epilepsy: A Potential Novel Drug for Epilepsy, Current Drug Targets, abstract.)

[0015] According to Boison et al. 2006, adenosine is an inhibitory modulator of brain activity with neuroprotective and anticonvulsant properties. Therefore, cell-based delivery of adenosine shows considerable promise as a novel therapy for epilepsy and stroke. (Boison et al, 2013,Adenosine kinase, epilepsy and stroke: mechanisms and therapies, Trends Pharmacol Sci, Abstract.) Adenosine kinase also plays a developmental role in mediating adult behaviors related to neuropsychiatric disorders. (Osborne et al, 2018,Developmental role of adenosine kinase for the expression of sex-dependent neuropsychiatric behaviour, Neuropharmacology, 2018 Octoberds Pharmacol Sci, Abstract) Schizophrenia, autism, ADHD

[0016] Augmentation of adenosine by pharmacological inhibition of adenosine kinase exerts antipsychotic-like activity in mice, as studied by Hai-Ying Shen et al. 2012. Furthermore, overexpression of ADK in transgenic mice was linked to attention deficits associated with schizophrenia. (Hai-Ying Shen et al. 2012,Adenosine augmentation ameliorates psychotic and cognitive endophenotypes of schizophrenia,J Clin Invest,page 2567-2577.)

[0017] pain According to Otsuguro et al. 2015, adenosine kinase inhibitors are potential candidates for suppressing pain. (Otsuguro et al,.2015,An adenosine kinase inhibitor,ABT-702,inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat,neuropharmacology volume 97,abstract.) Inhibitors of adenosine kinase enhance the extracellular concentration of the inhibitory neuromodulator adenosine at sites of tissue hyperexcitability, producing antinociceptive effects in animal models of pain and inflammation. Furthermore, adenosine kinase inhibitors produce specific antihyperalgesic effects. (Jarvis et al,2002,Comparison of the ability of adenosine kinase inhibitors and adenosine receptor agonists to attenuate thermal hyperalgesia and reduce motor performance in rats,Pharmacology Biochemistry and Behavior vol 73,abstract.)

[0018] Adenosine kinase inhibitors have demonstrated antinociceptive activity in various animal models of pain, and the novel adenosine kinase inhibitor A-134974 potently reduces tactile allodynia. (Zhu et al, 2001, A-134974: a novel adenosine kinase inhibitor, relieves tactile allodynia via spinal sites of action in peripheral nerve injured rats, Brain Research vol 905, abstract.) Adenosine kinase inhibitors have also been shown to provide effective antinociceptive, anti-inflammatory, and anticonvulsant activity in animal models, suggesting their potential therapeutic utility for pain, inflammation, epilepsy, and other central and peripheral nervous system disorders that are potentially associated with cellular trauma and inflammation. (Gomtsyan et al, 2004, Non-nucleoside inhibitors of adenosine kinase, Current Pharmaceutical Design, abstract.)

[0019] According to Bauser et al. 2004, adenosine kinase inhibition is an attractive therapeutic approach for several pathologies, such as neurodegeneration, stroke, ischemia, inflammation, and pain. (Bauser er al, 2004, Discovery and optimization of 2-aryl oxazolo-pyrimidines as adenosine kinase inhibitors using liquid phase parallel synthesis, Bioorganic & Medicinal Chemistry Letters, abstract.)

[0020] encephalitis Rasmussen's encephalitis is a rare neurological disorder characterized by unilateral inflammation of the cerebral cortex and other structures, most notably the hippocampus, progressive cognitive decline, and drug-resistant focal epilepsy. Luan et al. propose that overexpression of adenosine kinase is a common pathological hallmark of Rasmussen's encephalitis, and that upregulation of neuronal A1R in Rasmussen's encephalitis is crucial to prevent the spread of seizures. Furthermore, adenosine acts as an endogenous neuromodulator that has anticonvulsant and anti-inflammatory effects and can restore cognitive function when cognition is impaired secondary to epilepsy. Disruption of adenosine homeostasis has been linked to epilepsy, inflammation, and cognitive dysfunction. Alterations of adenosine receptors and the major adenosine-removing enzyme ADK have been shown to contribute to the disruption of adenosine homeostasis in epilepsy. (Luan et al,2017,Upregulation of Neuronal Adenosine A1 Receptor in Human Rasmussen Encephalitis,J Neuropathol Exp Neurol vol 76,page 720-731.)

[0021] Vascular Development Targeting adenosine kinase to increase intracellular adenosine promotes endothelial proliferation and migration in vitro and vascular sprouting ex vivo. In addition, endothelial-specific adenosine kinase knockout mice have increased retinal vascular development, accelerated wound healing, and were protected from hindlimb ischemic injury. (Xu et al.,2017,Intracellular adenosine regulates epigenetic programming in endothelial cells to promote angiogenesis,EMBO Molecular Medicine,page 1263-1278.)

[0022] cancer A study by Huang et al. 2015 suggested that adenosine kinase is involved in glioma progression, and that increased adenosine kinase levels in peritumoral tissues may be associated with epilepsy in gliomas. (Huang et al, 2015, Adenosine deaminase and adenosine kinase expression in human glioma and their correlation with glioma-associated epilepsy, Molecular Medicine Reports 12, page 6509-6516.)

[0023] Diabetes, inflammation, cardiovascular disorders, renal disorders, and pulmonary disorders According to Pye et al 2014, adenosine provides anti-inflammatory effects in cardiovascular diseases through activation of adenosine A2A receptors, but the physiological actions of adenosine may be limited due to its phosphorylation by adenosine kinase. Treatment with the adenosine kinase inhibitor ABY702 reduced blood glucose levels in diabetic mice, albuminuria and glomerular injury markers, nephrinuria and podocalyxin excretion levels in diabetic mice. In addition, indicators of oxidative stress were reduced. (Pye et al, 2014,Adenosine Kinase Inhibition Protects The Kidney Against Streptozotocin-Induced Diabetes Through Anti-inflammatory and Anti-oxidant Mechanisms,Pharmacol Res.)

[0024] Activation of A1 adenosine receptors protects against acute kidney injury by improving renal hemodynamic alterations and reducing tubular necrosis, and its inhibition may promote the clearance of toxins or drug metabolites in chronic kidney disease mode. (Pandey et al, 2021,"Adenosine an old player with new possibilities in kidney diseases": Preclinical evidence and clinical perspectives, Life Sciences vol 265, abstract.)

[0025] In many therapeutic areas, such as neuropathic pain, stroke, asthma, chronic obstructive pulmonary disease (COPD), and sleep promotion, modulation of adenosine function has been considered as a therapeutic option. (Knutsen et al, 2007, Therapeutic Areas I: Central Nervous System, Pain, Metabolic Syndrome, Urology, Gastrointestinal and Cardiovascular, Comprehensive Medicinal Chemistry II, 2007, https: / / www.sciencedirect.com / topics / medicine-and-dentistry / adenosine-kinase-inhibitor, accessed 21-4-2021.)

[0026] Summary of the Invention There is a high unmet medical need for improved treatment of neurological diseases, as many of these diseases cannot be adequately treated and currently available treatments cause severe side effects. The compounds of the present invention are potent inhibitors of ADK and are therefore useful for treating neurological diseases, such as epilepsy. In some embodiments, the compounds of the present invention inhibit both the short and long isoforms of ADK. [Brief description of the drawings]

[0027] Text description of the illustration image024.gif. [Figure 1] Ranking of ADK-LS antisense oligonucleotides based on ADK-LS knockdown potency from highest to lowest level of knockdown. Horizontal dotted line depicts the level of ADK-LS in mock-treated control cells (no knockdown of ADK-LS at all). Black line represents 70% knockdown and grey line represents 80% knockdown. Vertical dotted line indicates cutoff for ADK-LS antisense oligonucleotides selected for further testing. n, N=1, 1-2, mean ± SEM. [Diagram 2] Ranking of selected ADK-LS antisense oligonucleotides based on ADK-LS knockdown potency from highest to lowest level of knockdown. The horizontal dotted line depicts the level of ADK-LS in mock-treated control cells (no knockdown of ADK-LS at all), and the grey line indicates 80% knockdown. n, N = 2, 3–4, mean ± SEM. [Diagram 3] Dose-response study. The horizontal dotted line depicts the levels of ADK-LS in mock-treated control cells (no knockdown of ADK-LS at all), and the grey line indicates 80% knockdown. n, N=1–2, 2–4, mean ± SEM. [Figure 4] Dose-response curves and IC50 values, 3-parameter nonlinear curve fitting, n,N=2,4, all technical replicates are depicted. Horizontal dotted line represents 50% knockdown. [Diagram 5] Differential gene expression analysis of cells treated with SEQ ID NO:21. Volcano plots show transcript levels between SEQ ID NO:21 and mock treated cells, correlating the change in RNA expression between antisense oligonucleotide treated and mock treated groups along with the significance of differential expression. The x-axis represents the relative change in expression while the y-axis represents the significance. Each dot represents a specific transcript. Black dots represent non-significant changes while grey dots present significance values. ADK is highlighted with a black ring. n=3 [Figure 6]Differential gene expression analysis of cells treated with SEQ ID NO: 71. Volcano plots show transcript levels between SEQ ID NO: 71 and mock treated cells, correlating the change in RNA expression between antisense oligonucleotide treated and mock treated groups along with the significance of differential expression. The x-axis represents the relative change in expression while the y-axis represents the significance. Each dot represents a specific transcript. Black dots represent non-significant changes while grey dots present significance values. ADK is highlighted with a black ring. n=3 [Figure 7] Normalized mRNA expression values ​​for ADK (both isoforms), n=3 [Figure 8] In silico analysis of potential off-targets of antisense oligonucleotide SEQ ID NO:21 to predict all potential target sites within the spliced ​​transcriptome (cytoplasm, columns 1-4) and the unspliced ​​transcriptome (nucleus, columns 5-8). This was performed for 1) perfect match binding sites in the target mRNA for the antisense oligonucleotide (SEQ ID NO:21) described above, and 2) binding sites with 1, 2, 3, or 4 mismatches (INDELs). The resulting list of predicted off-targets was compared to the RNA sequencing data (table above, 3 nM, and table below, 30 nM) to determine whether any of the predicted off-target mRNAs (row 1) were expressed in the dataset (row 2), and then to assess whether any of the expressed off-target transcripts were differentially expressed (row 3) and whether such transcripts were upregulated (rows 4 and 5) or downregulated (rows 6 and 7) in the dataset. [Figure 9]In silico analysis of potential off-targets of antisense oligonucleotide SEQ ID NO: 71 to predict all potential target sites within the spliced ​​(cytoplasmic, columns 1-4) and unspliced ​​(nuclear, columns 5-8) transcriptome. This was performed for 1) perfect match binding sites in the target mRNA for the antisense oligonucleotide (SEQ ID NO: 71) described above, and 2) binding sites with 1, 2, 3, or 4 mismatches (INDELs). The resulting list of predicted off-targets was compared to the RNA sequencing data (table above, 3 nM, and table below, 30 nM) to determine whether any of the predicted off-target mRNAs (row 1) were expressed in the dataset (row 2), and then to assess whether any of the expressed off-target transcripts were differentially expressed (row 3) and whether such transcripts were upregulated (rows 4 and 5) or downregulated (rows 6 and 7) in the dataset. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description of the Invention In describing embodiments of the present invention, specific terminology is utilized for the sake of clarity, however, the present invention is not intended to be limited to the specific terminology so selected, and each specific term is understood to include all technical equivalents that function in a similar manner to accomplish a similar purpose.

[0029] The term "therapeutically effective amount" or "effective amount" or "effective dose" refers to an amount of a therapeutic agent that confers a desired therapeutic effect on an individual in need of that agent. The effective amount may vary from individual to individual depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the method of administration, assessment of the individual's medical condition, and other relevant factors.

[0030] The term "treatment" refers to the administration of any therapeutic pharmaceutical agent, including, herein, an antisense oligonucleotide, that partially or completely cures or reduces one or more symptoms or characteristics of a given disease.

[0031] The term "adenosine kinase transcript" in the context of the present invention is a pre-mRNA or mRNA or other transcript encoding at least one isoform of adenosine kinase, i.e. adenosine kinase pre-mRNA SEQ ID NO: 1.

[0032] The term "compound" as used herein refers to a compound that includes an oligonucleotide according to the present invention. In some embodiments, the compound may include other elements other than the oligonucleotide of the present invention. Such other elements may be, in a non-limiting example, a delivery vehicle conjugated or otherwise bound to the oligonucleotide.

[0033] "Antisense oligonucleotide" refers to a single-stranded oligonucleotide having a nucleobase sequence that allows hybridization to a corresponding region or segment of a target nucleic acid. In some cases, the antisense oligonucleotide of the present invention is a "mixmer" and in some cases, the antisense oligonucleotide of the present invention is a "gapmer."

[0034] A "mixmer" is an antisense oligonucleotide that contains a mix of nucleoside analogs, such as LNA, and DNA nucleosides, with the proviso that the antisense oligonucleotide does not contain an internal region with multiple nucleosides, such as a contiguous stretch of 4 or 5 DNA nucleotides or less. Mixmers are not capable of recruiting RNAses, such as RNAse H, but rather exert their effect by binding to target RNA and blocking its normal function.

[0035] A "gapmer" is an antisense oligonucleotide comprising a contiguous stretch of at least 6 or 7 DNA nucleotides flanked by stretches of nucleotides comprising affinity-enhancing nucleotide analogs such as LNA nucleosides. Gapmers are capable of recruiting RNAses such as RNAse H, and the nucleosides that make up the internal region are chemically distinct from the nucleoside or nucleosides that make up the outer wings.

[0036] "Nucleoside analogues" are described, for example, in Freier & Altmann; Nucl. Acid. Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and examples of suitable and preferred nucleoside analogues are provided in WO 2007 / 031091, which is hereby incorporated by reference.

[0037] "5-methylcytosine" refers to a cytosine modified with a methyl group attached to the 5' position. 5-methylcytosine is a modified nucleobase that often replaces cytosine in antisense oligonucleotides. In the oligonucleotides of the present invention, it is within the scope of the present invention to replace cytosine with 5-methylcytosine.

[0038] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH~)~-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring.

[0039] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside that includes a 2'-MOE modified sugar moiety.

[0040] "Locked nucleic acid" or "LNA", often referred to as inaccessible RNA, is a modified RNA nucleobase. The ribose moiety of an LNA nucleobase is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. LNA oligonucleotides exhibit substantially increased affinity for their complementary strands compared to traditional DNA or RNA oligonucleotides. In some aspects, a bicyclic nucleoside analog is an LNA nucleotide, and thus these terms may be used interchangeably, and in such embodiments, both are characterized by the presence of a linker group (e.g., a bridge) between the C2' and C4' of the ribose sugar ring. When used in the context of the present invention, the terms "LNA unit", "LNA monomer", "LNA residue", "locked nucleic acid unit", "locked nucleic acid monomer", or "locked nucleic acid residue" refer to a bicyclic nucleoside analog. LNA units are described, inter alia, in WO 99 / 14226, WO 00 / 56746, WO 00 / 56748, WO 01 / 25248, WO 02 / 28875, WO 03 / 006475, WO 2015 / 071388, and WO 03 / 095467.

[0041] "Beta-D-oxy LNA" is a preferred LNA variant.

[0042] "Bicyclic nucleic acid" or "BNA" or "BNA nucleoside" refers to a nucleic acid monomer having a bridge connecting two carbon atoms between the 4' and 2' positions of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugars include, but are not limited to, A) pt-L-methyleneoxy (4'-CH2-0-2') LNA, (B) PD-methyleneoxy (4'-CH2-0-2') LNA, (C) ethyleneoxy (4'-(CH2)2-0-2') LNA, (D) aminooxy (4'-CH2-0-N(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-0-2') LNA.

[0043] As used herein, LNA compounds include, but are not limited to, compounds that have at least one bridge between the 4' and 2' sugar positions. with the proviso that each of the bridges independently contains 1 or 2 to 4 linking groups independently selected from -[C(R~)(R2)],,-,-, -C(R~)=C(R2)-, -C(R~)=N, -C(=NREM)-, -C(=0)-, -C(=S)-, -0-, -Si(Ri)q-, -S(=0)--, and N(R&)-, where x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R& and R2 is independently H, a protecting group, hydroxyl, C≫C≫alkyl, substituted C≫, and for the (-CHz-) group connecting the 2' oxygen atom and the 4' carbon atom, the term methyleneoxy(4'-CH&-0-2')LNA is used.

[0044] Furthermore, for bicyclic sugar moieties with an ethylene bridging group at this position, ethyleneoxy(4'-CH&CH&-0-2')LNA is used. As used herein, nL-methyleneoxy(4'-CH&-0-2'), an isomer of methyleneoxy(4'-CH&-0-2')LNA is also included in the definition of LNA.

[0045] In some embodiments, the nucleoside unit is an LNA unit selected from the list of beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio-LNA, 5'-methyl-LNA, beta-D-ENA, and alpha-L-ENA.

[0046] "cEt" or "constrained ethyl" means a bicyclic sugar moiety containing a bridge connecting the 4'-carbon and the 2'-carbon, the bridge having the formula: 4'-CH(CHq)-0-2'.

[0047] "Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. cEt and some of its properties are described in Pallan et al. Chem Commun (Camb). 2012, August 25; 48(66): 8195-8197.

[0048] "Tricyclo(tc)-DNA" belongs to a class of conformationally constrained DNA analogs that exhibit enhanced binding to DNA and RNA. Structures and methods of production can be found in Renneberg et al. Nucleic Acids Res. 2002 Jul 1;30(13):2751-2757.

[0049] "2'-fluoro" as referred to herein is a nucleoside containing a fluoro group at the 2' position of the sugar ring. 2'-Fluorinated nucleotides are described in Peng et al. J Fluor Chem. 2008 September; 129(9): 743-766.

[0050] A "2'-O-methyl" as referred to herein is a nucleoside that includes a sugar that includes an -OCH3 group at the 2' position of the sugar ring.

[0051] The "conformationally restricted nucleosides" (CRNs) referred to herein and methods for their synthesis are described in WO 2013 / 036868, which is hereby incorporated by reference. CRNs are sugar-modified nucleosides in which a chemical bridge connects the C2' and C4' carbons of the ribose, similar to LNAs. However, the C2'-C4' bridge of CRNs is one carbon longer than that of LNA molecules. The chemical bridge of the ribose of CRNs locks the ribose in a fixed position, thus restricting the flexibility of the nucleobase and phosphate groups. CRN substitutions in RNA or DNA-based oligonucleotides have the advantages of increased hybridization affinity and enhanced resistance to nuclease degradation.

[0052] An "unlocked nucleic acid" or "UNA," as referred to herein, is typically an unlocked nucleic acid in which the C2-C3C-C bond of the ribose has been removed to form an unlocked "sugar" residue (see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which is hereby incorporated by reference, and Snead et al. Molecular Therapy--Nucleic Acids (2013) 2, e103;).

[0053] A "therapeutic RNA compound" in the context of the present invention is a compound that comprises a contiguous nucleotide sequence complementary to a target RNA. Therapeutic RNA compounds can be double-stranded small interfering RNA (siRNA or dsRNA) or single-stranded antisense oligonucleotides. By binding to a target RNA, therapeutic RNA compounds are capable of blocking or modulating the expression of the target RNA. Therapeutic RNA compounds can be chemically modified with nucleotide analogs for affinity enhancement or with internucleotide bonds to increase the stability of the compound. Therapeutic RNA compounds can also contain methylated cytosines to inhibit immune stimulation.

[0054] A "motif" in the context of the present invention is the unmodified sequence of an antisense oligonucleotide. SEQ ID NOs: 83-161 are the motif sequences of the modified antisense oligonucleotide compounds of SEQ ID NOs: 2-80.

[0055] "Target region" refers to a portion of a target nucleic acid targeted by one or more antisense compounds. The target region is part of the present invention. That is, SEQ ID NOs: 164-205 are target regions having sequences suitable for targeting therapeutic antisense oligonucleotides according to the present invention.

[0056] As used herein, "targeted delivery" refers to delivery in which the antisense oligonucleotide is either formulated to facilitate efficient delivery at a specific tissue or cell, or is otherwise modified, for example to include a targeting moiety, or is otherwise modified to facilitate uptake in a specific target cell.

[0057] The antisense oligonucleotide of the present invention is designed to target adenosine kinase (ADK). As used herein, the term "adenosine kinase-associated neurological disease" refers to a disease in which the disease pathology is associated with upregulation of adenosine kinase activity or in which downregulation of adenosine kinase activity is beneficial for the treatment of the disease.

[0058] compound The human ADK gene encodes 14 transcripts, 10 of which code for proteins, and are therefore potential targets for antisense oligonucleotides or siRNAs. Several ASOs have been designed to target the ADK pre-mRNA (SEQ ID NO: 1).

[0059] In its broadest sense, the invention provides an antisense oligonucleotide or siRNA comprising a sequence of 10-30 nucleotides in length complementary to adenosine kinase (ADK) pre-mRNA (SEQ ID NO: 1), with the proviso that the antisense oligonucleotide comprises at least one affinity-enhancing nucleotide analog, and the antisense oligonucleotide comprises at least one phosphorothioate or analog internucleoside linkage. In some embodiments, the antisense oligonucleotide of the invention has an alternative to phosphorothioate internucleoside linkages, e.g., the backbone can be another type of backbone, e.g., phosphodiester linkages, phosphotriester linkages, methylphosphonate linkages, phosphoramidate linkages, or combinations thereof. In preferred embodiments, the alternative nucleoside backbone is suitable for medical uses of the antisense oligonucleotide.

[0060] In some embodiments, the antisense oligonucleotides of the invention are designed to target two or more protein-coding ADK forms. In preferred embodiments, the antisense oligonucleotides of the invention are designed to target at least two protein-coding ADK RNAs. In the most preferred embodiments, the antisense oligonucleotides of the invention are designed to target ADK pre-mRNA to downregulate, e.g., knock down, at least the expression of ADK-S and ADK-L.

[0061] For example, the ASO may be any of nucleotides 26138 to 26158 (SEQ ID NO: 164), 28854 to 28871 (SEQ ID NO: 165), 31591 to 31612 (SEQ ID NO: 166), 49618 to 49648 (SEQ ID NO: 167), 73335 to 73350 (SEQ ID NO: 168), 107401 to 107420 (SEQ ID NO: 169), 120681 to 120698 (SEQ ID NO: 170), 131066 to 131085 (SEQ ID NO: 171), 131102 to 131121 (SEQ ID NO: 172), 157279 to 157300 (SEQ ID NO: 173), 163465 ~163495 (SEQ ID NO: 174), 182053-182069 (SEQ ID NO: 175), 229825-229843 (SEQ ID NO: 176), 230316-230332 (SEQ ID NO: 177), 230388-230405 (SEQ ID NO: 178), 230484-230505 (SEQ ID NO: 179), 243036-243055 (SEQ ID NO: 180), 243075-243090 (SEQ ID NO: 181), 266808-266823 (SEQ ID NO: 182), 267374-267393 (SEQ ID NO: 183), 267615-267634 (SEQ ID NO: 184), 2 88247 to 288266 (SEQ ID NO: 185), 302286 to 302305 (SEQ ID NO: 186), 370312 to 370331 (SEQ ID NO: 187), 374190 to 374206 (SEQ ID NO: 188), 404971 to 404990 (SEQ ID NO: 189), 405025 to 405044 (SEQ ID NO: 190), 411523 to 411541 (SEQ ID NO: 191), 431656 to 431673 (SEQ ID NO: 192), 434586 to 434605 (SEQ ID NO: 193), 438147 to 438189 (SEQ ID NO: 194), 438340 to 438359 (SEQ ID NO: 1 95), 441016-441035 (SEQ ID NO: 196), 449173-449194 (SEQ ID NO: 197), 451654-451686 (SEQ ID NO: 198), 494676-494696 (SEQ ID NO: 199), 512508-512527 (SEQ ID NO: 200), 512544-512563 (SEQ ID NO: 201), 519054-519071 (SEQ ID NO: 202), 531984-532003 (SEQ ID NO: 203), 532784-532822 (SEQ ID NO: 204) and 540164-557611 (SEQ ID NO: 205).Exemplary sequences of ASOs are listed in Table 1. ASOs were designed to be gapmers that recruit RNAseH for target RNA cleavage. In some embodiments, the antisense oligonucleotides of the invention are complementary to any one of SEQ ID NOs: 164-205. In some embodiments, the antisense oligonucleotides of the invention are complementary to any one of SEQ ID NOs: 164-205 and are capable of modulating, downregulating, or knocking down the expression of both ADK-L and ADK-S. In some embodiments, the antisense oligonucleotides of the invention consist of or include a motif selected from any one of SEQ ID NOs: 83-161. In some embodiments, the antisense oligonucleotides of the invention consist of or include a motif selected from any one of SEQ ID NOs: 83-161 and include at least one affinity-modifying nucleotide analog and at least one modified internucleoside linkage, such as a phosphorothioate linkage.

[0062] In some embodiments, the antisense oligonucleotides of the invention are gapmers, and the antisense oligonucleotides contain a contiguous stretch of at least 5 contiguous DNA nucleotides. The size of the antisense oligonucleotides for medical purposes is important, and therefore the antisense oligonucleotides of the invention are designed to be useful for such uses. In some embodiments, the antisense oligonucleotides of the invention are 10-30 nucleotides in length, and in some embodiments, the antisense oligonucleotides are 14-20, such as 14-19 nucleotides in length.

[0063] The efficacy of antisense oligonucleotides depends on their stability, affinity to target RNA, and other factors. The presence of affinity-enhancing nucleoside analogs, such as LNA, in antisense oligonucleotides provides such advantages. In preferred embodiments, the affinity-enhancing nucleoside analogs used in the antisense oligonucleotides of the present invention are selected from the list of LNA, tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2' methoxyethyl (2'MOE), 2' cyclic ethyl (cET), UNA, 2' fluoro, and conformationally restricted nucleosides (CRN). In some embodiments, such oligonucleotides may include a combination of LNA, DNA, and one or more of tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2' methoxyethyl (2'MOE), 2' cyclic ethyl (cET), UNA, 2' fluoro, and conformationally restricted nucleosides (CRN).

[0064] In some embodiments, the antisense oligonucleotides of the present invention contain at least one LNA. In some embodiments, the antisense oligonucleotides of the present invention contain 20-55% LNA. In some embodiments, the antisense oligonucleotides of the present invention are LNA / DNA oligos, but further contain one or more nucleosides that are any one of tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2'methoxyethyl (2'MOE), 2'cyclic ethyl (cET), UNA, 2'fluoro, and conformationally restricted nucleosides (CRN).

[0065] In some preferred embodiments, the antisense oligonucleotides of the present invention comprise LNA, wherein the LNA is beta-D-oxy LNA.

[0066] Table 1 contains non-limiting examples of ASO design for selected sequences. The same method is applicable to any other sequences disclosed herein. Gapmers were constructed to contain locked nucleic acid LNA (uppercase). For example, gapmers can have beta-deoxy LNA at the 5' and 3' ends and can have a phosphorothioate backbone. However, LNA can also be substituted with any other nucleotide analog and the backbone can be other types of backbone {e.g., phosphodiester linkage, phosphotriester linkage, methylphosphonate linkage, phosphoramidate linkage, or combinations thereof). In the compound names in Table 1, uppercase letters represent modified nucleotides, e.g., LNA nucleotides (either beta-D-oxy, alpha-L-oxy, beta-D-amino, or beta-D-thio LNA, or other modified nucleotides, e.g., cEt, cMOE, UNA, or ENA), and lowercase letters represent DNA nucleotides. Thus, the sequence represented by TCTttcctacttaaGG (SEQ ID NO: 30) represents a 3-11-2 16mer modified nucleotide-DNA-modified nucleotide gapmer having a 5'-T and a 3'-G, e.g., a 3-11-2 LNA-DNA-LNA gapmer. Some ASOs may be alternating flank gapmers as described elsewhere herein. In some embodiments, selected examples of alternating flank gapmers having a 9 nucleotide gap are SEQ ID NOs: 5, 21, and 51.

[0067] In some embodiments, the antisense oligonucleotides of the present invention are designed such that all internucleoside linkages are phosphorothioate linkages. In some embodiments, the present invention provides a series of potent antisense oligonucleotides, the antisense oligonucleotides being any one of SEQ ID NOs: 2-80. In some embodiments, the present invention provides antisense oligonucleotides selected from the list of SEQ ID NOs: 4, 12, 20, 21, 37, 50, 51, 53, 59, 60, 63, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80, as well as conjugates comprising such antisense oligonucleotides, compositions comprising such antisense oligonucleotides, and their intended use in the treatments described herein. Additionally, methods of treatment using the antisense oligonucleotides of the present invention are also encompassed by the present invention.

[0068] In Table 1, the listed ASOs are always drawn in a 5'→3' direction. Thus, the 5' end of the ASO hybridizes to the mRNA "end" number in the table, and the 3' end of the ASO hybridizes to the pre-mRNA "start" number in the table. In some embodiments, the antisense oligonucleotides of the invention comprise or consist of a motif of any one of SEQ ID NOs: 83-161. In some embodiments, the antisense oligonucleotides of the invention comprise or consist of a compound of any one of SEQ ID NOs: 2-80.

[0069] [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] In "ASO Compounds", capital letters are nucleotide analogs, e.g., LNA, such as beta-deoxy-LNA. Lower case letters represent DNA. C can be 5'-methyl-cytosine. In one embodiment, all internucleoside linkages in SEQ ID NOs: 2-80 are phosphorothioate. In one embodiment, all internucleoside linkages in SEQ ID NOs: 2-80 are phosphorothioate, capital letters are LNA, such as beta-deoxy-LNA, lower case letters represent DNA, and C is 5'-methyl-cytosine.

[0073] In some embodiments, the compound of the present invention is an siRNA. In some embodiments, the siRNA comprises modified nucleotides. In some embodiments, the modified nucleotides are deoxy-nucleotides, 3'-terminal deoxythymidine (dT) nucleotides, 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-0-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-0-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexyl toll-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or a 5' phosphate mimic, nucleotides containing vinyl phosphonate, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups, and combinations thereof.

[0074] In some embodiments, the siRNA of the invention comprises modified nucleotides selected from 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases.

[0075] Compositions and Uses The compounds of the invention are for use in compositions, such as the pharmaceutical compositions of the invention, for use as pharmaceutical agents, and for the treatment, mitigation, amelioration, preemptive treatment, prevention, disease modifying or curative treatment of diseases disclosed herein, such as neurological disorders including epilepsy. In some embodiments, the anti-adenosine kinase compounds of the invention prevent, disease modify, cure, reduce symptoms of disease, including improving seizure control and reducing anxiety and depression and cognitive impairment.

[0076] The compounds of the invention are, in some embodiments, included in compositions, such as pharmaceutical compositions, for the treatment of diseases where modulation of adenosine kinase activity is beneficial for preventative, curative, or disease-modifying treatment, prevention, mitigation, or amelioration of the disease or disease parameters. In some embodiments, the treatment, prevention, mitigation, or amelioration is curative. In some embodiments, the treatment, prevention, mitigation, or amelioration is disease modifying. In some embodiments, the treatment, prevention, mitigation, or amelioration is preventative.

[0077] Diseases that may be treated, alleviated, ameliorated, preemptively, or prophylactically treated with the compounds and compositions include, by way of non-limiting example, diseases of the central nervous system (CNS) or peripheral nervous system (PNS), including neurological, neurodegenerative, neurodevelopmental, or psychiatric disorders. In some embodiments, the antisense oligonucleotides or compositions of the invention are for use as neuroprotective agents.

[0078] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of CNS or PNS diseases, neurological disorders, neurodegenerative disorders, neurodevelopmental disorders, central and peripheral nervous system diseases associated with cellular trauma and inflammation, neuronal injury, hippocampal injury, traumatic brain injury, memory disorders, hippocampal sclerosis, Parkinson's disease, multiple sclerosis, acute spinal cord injury, amyotrophic lateral sclerosis, ataxia, Bell's palsy, Charcot-Marie-Tooth, headache, Houghton's headache, migraine, Pick's disease, progressive supranuclear palsy, multisystem degeneration, motor neuron diseases, Huntington's disease, prion diseases, Creutzfeldt-Jakob disease, corticobasal degeneration, primary progressive aphasia, or a symptom or effect thereof.

[0079] In some embodiments, the antisense oligonucleotide or composition of the invention is for use in the treatment of epilepsy.

[0080] In some embodiments, the antisense oligonucleotide or composition of the invention is for use in the treatment of stroke.

[0081] In some embodiments, the antisense oligonucleotides or compositions of the present invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of epilepsy and / or seizures, preferably treatment-resistant epilepsy, acquired, genetic, and / or idiopathic epilepsy, therapy-resistant epileptic syndromes, drug-resistant epilepsy, pharmacologically resistant focal epilepsy, spontaneous seizures, therapy-resistant seizures, focal epilepsy, generalized epilepsy, or status epilepticus.

[0082] In some embodiments, the antisense oligonucleotide or composition of the present invention is used to treat epilepsy, drug resistant epilepsy, drug resistant focal epilepsy, seizures, spontaneous seizures, therapy resistant seizures, focal epilepsy, preferably said focal epilepsy is focused on the frontal, parietal, occipital or temporal lobe, generalized epilepsy, preferably said generalized epilepsy is selected from absence seizures, myoclonic seizures, tonic clonic seizures, tonic seizures, atonic seizures, clonic seizures and convulsions, status epilepticus status, epileptogenic induced by acute brain injury, autosomal dominant nocturnal frontal lobe epilepsy, slow wave sleep The compound is for use in the treatment, palliative, pre-emptive treatment or prevention of chronic spike and wave episodes in the early stages of seizures, Dravet syndrome, post-stroke epilepsy, epileptic encephalopathy, laughter epilepsy, absence seizures, benign neonatal seizures, Jeavons syndrome, juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome, mesial temporal lobe epilepsy, myoclonic astatic epilepsy, Ohtahara syndrome, Panayiotopoulos syndrome, PCDH19 syndrome, benign childhood epilepsy with centrotemporal spikes, Sturge-Weber syndrome, symptomatic focal epilepsy, transient epileptic amnesia and West syndrome, and / or glioma-associated epilepsy.

[0083] In some embodiments, the antisense oligonucleotides or compositions of the present invention are for use in the treatment, alleviation, pre-emptive treatment, or prevention of pain, preferably wherein the pain is chronic pain, neuropathic pain, chemotherapy-induced neuropathic pain, migraine, headache, hyperalgesia, allodynia, and / or fibromyalgia.

[0084] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment of pain.

[0085] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, alleviation, pre-emptive treatment, or prevention of pain, chronic pain, neuropathic pain, chemotherapy-induced neuropathic pain, migraine including migraine with aura and migraine without aura, primary headache, tension headache, cluster headache, Horton's headache, chronic daily headache, sinus headache, post-traumatic headache, exercise headache, hemicrania continua, hypnic headache, hyperalgesia, heat hyperalgesia, allodynia, tactile allodynia, and / or fibromyalgia.

[0086] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of a psychiatric disorder, a cognitive disorder, a sleep disorder, a cardiovascular disorder, a respiratory disorder, a cancer, a renal disorder, an inflammation, or a metabolic disorder.

[0087] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of a psychiatric disorder, a neuropsychiatric disorder, anxiety, depression, bipolar disorder, attention deficit hyperactivity disorder, attention deficit disorder, autism, Asperger's syndrome, Tourette's syndrome, schizophrenia, paranoid schizophrenia, anaphylactic schizophrenia, catatonic schizophrenia, undifferentiated schizophrenia, residual schizophrenia, simple schizophrenia, or unspecified schizophrenia.

[0088] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of cognitive impairment, cognitive impairment, dementia, Alzheimer's disease, vascular dementia, frontotemporal dementia, or Lewy body dementia.

[0089] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of a sleep disorder.

[0090] In some embodiments, the antisense oligonucleotide or composition of the present invention is for use as a sleep regulator.

[0091] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in promoting sleep.

[0092] In some embodiments, the antisense oligonucleotides or compositions of the present invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of cardiovascular disorders, peripheral arterial disease, post-operative atrial fibrillation, heart failure, chronic heart failure, intracerebral hemorrhage-induced brain injury, stroke, cerebral ischemia, or ischemia.

[0093] In some embodiments, the antisense oligonucleotide or composition of the invention is for use in the treatment, mitigation, pre-emptive treatment, or prevention of a respiratory disorder, asthma, or chronic obstructive pulmonary disease.

[0094] In some embodiments, the antisense oligonucleotides or compositions of the present invention are for use in the treatment, mitigation, preemptive treatment, or prevention of cancer, nervous system cancer, glioma, glioblastoma, liver cancer, or cancer metastasis.

[0095] In some embodiments, the antisense oligonucleotide or composition of the invention is for use in the treatment, mitigation, pre-emptive treatment, or prevention of renal damage, renal injury, renal inflammation, albuminuria, or glomerular injury.

[0096] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, alleviation, pre-emptive treatment, or prevention of inflammation.

[0097] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of inflammatory disorders, oxidative stress, inflammation, apoptosis, arthritis, osteoarthritis, rheumatoid arthritis and pain associated with these conditions, encephalitis, meningitis, human Rasmussen's encephalitis, inflammation of the cerebral cortex and / or hippocampus, progressive cognitive decline, colitis, ulcerative colitis, or inflammatory bowel disease.

[0098] In some embodiments, the antisense oligonucleotide or composition of the present invention is for use in the treatment, mitigation, preemptive treatment, or prevention of a metabolic disorder, preferably diabetes, more preferably type 1 or type 2 diabetes.

[0099] In some embodiments, the antisense oligonucleotides or compositions of the present invention are for use in the treatment, mitigation, pre-emptive treatment, or prevention of Prader-Willi syndrome, Angelman syndrome, neurofibromatosis, angiogenesis related diseases, promoted angiogenesis, retinal disorders, preferably diabetic retinopathy, or hearing loss.

[0100] In some embodiments, the antisense oligonucleotides or compositions of the invention are administered systemically, intrathecally, intraventricularly into the CNS, or intravenously.

[0101] In some embodiments, the antisense oligonucleotides or compositions of the invention are for use in combination with one or more other active pharmaceutical ingredients for the treatment of any one of the diseases of the invention.

[0102] According to one embodiment, the present invention relates to the use of the antisense oligonucleotide according to the present invention, wherein the other active pharmaceutical ingredient is an ingredient prepared for the treatment of a disease according to the present invention.

[0103] According to one embodiment, the present invention relates to the use of an antisense oligonucleotide according to the present invention, wherein the other pharmaceutical ingredient is an antisense oligonucleotide.

[0104] According to one embodiment, the present invention relates to the use of an antisense oligonucleotide according to the present invention, wherein the other pharmaceutical ingredient is an antisense oligonucleotide targeting miR-27b or miR-134 or both.

[0105] In some embodiments, the present invention relates to a pharmaceutical composition comprising an effective dose of an antisense oligonucleotide according to the present invention and a pharma- ceutically acceptable carrier. In some such embodiments, the antisense oligonucleotide according to the present invention is conjugated, i.e., conjugated to a delivery vehicle, or to another therapeutic molecule, or to a molecule that in some way enhances the efficacy of the antisense oligonucleotide according to the present invention.

[0106] According to one embodiment, the present invention relates to a pharmaceutical composition comprising an effective dosage amount of an antisense oligonucleotide according to the present invention, wherein said antisense oligonucleotide is the only active pharmaceutical ingredient.

[0107] In some embodiments, the anti-adenosine kinase compounds may be advantageously used in conjunction with other therapies for certain diseases treated by the anti-adenosine kinase compositions, hi some embodiments, the anti-adenosine kinase compounds of the present invention are for use in combination with other therapies for the neurological diseases listed herein.

[0108] In some embodiments, the anti-adenosine kinase compounds of the invention are for use in the treatment, mitigation, amelioration, pre-emptive, prophylactic, disease-modifying or curative treatment of neurological diseases, particularly epilepsy, pain, or stroke, in combination with other therapies for the treatment, mitigation, amelioration, pre-emptive, prophylactic, disease-modifying or curative treatment of neurological diseases, particularly epilepsy, pain, or stroke, or comorbidities thereof.

[0109] Thereby, the anti-adenosine kinase antisense oligonucleotide of the present invention is for use in combination with one or more other therapies. In some embodiments, the other therapy is an anti-miR-27b antisense oligonucleotide. In some embodiments, the other therapy is an anti-miR-134 antisense oligonucleotide. In some embodiments, the other therapy induces the Nrf-2 / ARE pathway in a mammal, such as a human. In some embodiments, the anti-adenosine kinase antisense oligonucleotide composition should be used in combination with one or more of an anti-miR27b antisense oligonucleotide, an anti-miR-134 antisense oligonucleotide, and an Nrf-2 / ARE pathway-inducing therapy.

[0110] In some embodiments, the adenosine kinase targeted antisense oligonucleotide of the present invention is to be used in a composition in which it is the only active ingredient, and in some embodiments, it is for use in a composition containing other active pharmaceutical ingredients.

[0111] The present invention provides a pharmaceutical composition comprising an anti-adenosine kinase antisense oligonucleotide compound of the invention and further comprising a pharma- ceutically acceptable carrier.

[0112] In some embodiments, the pharmaceutical compositions of the invention comprise an anti-adenosine kinase antisense oligonucleotide as the only active pharmaceutical ingredient. In some embodiments, there is one or more active pharmaceutical ingredients in the pharmaceutical compositions of the invention.

[0113] Dosage The expression "effective dose" refers to the dose of a drug that achieves a desired effect. In the context of the present invention, the desired effect is a reduction in adenosine kinase activity. A reduction in adenosine kinase activity can be measured, for example, by measuring the level of adenosine kinase when using oligonucleotides that result in the degradation of either ADKmRNA or ADKpremRNA.

[0114] The compounds of the invention are for use in effective dosage amounts and the compositions include an effective dosage amount of a compound of the invention.

[0115] In some embodiments, the dosage of compound administered at each administration, eg, unit dose, is in the range of 0.001 mg / kg to 25 mg / kg.

[0116] In some embodiments, an effective dose is a dose sufficient to downregulate adenosine kinase or its activity to a significant level, e.g., to a level that is of therapeutic benefit to the subject, over the period between successively administered doses.

[0117] The pharmaceutical compositions of the present invention may in some embodiments be prepared for administration to provide an initial dose build-up phase, which may then be followed by a maintenance dose scheme, depending on the disease pathology, to maintain a concentration of the compound in the subject, e.g., in a target tissue of the subject, such that the disease is successfully treated. The efficacy of the dose may be measured, for example, by observation of a disease parameter indicative of the disease state, or, depending on the target tissue, by observation of various tissue parameters such as adenosine kinase activity, or alternatively, measurable disease state-dependent parameters in plasma.

[0118] Drug Delivery Various delivery systems are known and can be used to administer the therapeutic agents of the present invention. Methods of administration include, but are not limited to, subcutaneous, intravenous, parenteral, nasal, pulmonary, rectal, vaginal, intrauterine, intraurethral, ​​ocular, aural, dermal, intradermal, intramuscular, intraperitoneal, epidural, intracerebroventricular, intrathecal, or oral, or direct administration to the brain or cerebrospinal fluid. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous tissues (e.g., oral mucosa, rectal and intestinal mucosa, etc.), with or without other biologically active agents. Administration can be systemic or local. It may also be desirable to administer the compositions of the present invention to the central nervous system by any suitable route, including intracerebroventricular and intraspinal administration. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya or other reservoir approach. Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer and a formulation with an aerosolizing agent. Preferably, the therapeutic agent is delivered to the CNS or PNS.

[0119] Delivery means include inhalation delivery, intramuscular delivery directly into muscle by syringe or mini osmotic pump, intraperitoneal administration by syringe or mini osmotic pump directly into peritoneum, subcutaneous administration by syringe directly under skin, intraventricular administration by injection or directly into ventricle using small catheter attached to osmotic pump.In addition, implants (e.g., small silicone implants) can be made that are placed directly into muscle or on the spinal cord.It may be desirable to administer the composition of the present invention locally to the area requiring treatment.This can be achieved, for example, but not by way of limitation, by topical application, by injection, by using a catheter, by using a suppository, or by using an implant, which can be a porous, non-porous, or gelatinous material, including a membrane or fiber such as a silastic membrane.

[0120] Pharmaceutical CompositionsThe present invention also provides pharmaceutical compositions. Such compositions may include a therapeutically effective amount of a therapeutic agent, such as a therapeutically effective amount of an antisense oligonucleotide or siRNA of the present invention, such as any one of SEQ ID NOs: 2-80, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" may be defined as approved by a regulatory agency. The regulatory agency may be, for example, the European Medicines Agency, a federal or state government, or may be listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "therapeutically effective amount" may be defined as an amount of a therapeutic agent that results in a clinically significant inhibition, amelioration, or reversal of the occurrence or presence of a disorder or disease. The term "carrier" may refer to a diluent, adjuvant, excipient, or medium used in administering a therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water may be a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions can also contain wetting or emulsifying agents or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation will be suitable for the mode of administration.The composition for intravenous administration may be a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of the injection. The ingredients may be supplied separately or mixed together in a unit dosage form, for example as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachet, labeled with the quantity of active agent. When the composition is administered by injection, it may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration. EXAMPLES

[0121] Working Example Example 1. For example, the synthesis of oligonucleotides containing LNA nucleotides is well known in the literature. LNA monomer and oligonucleotide synthesis can be performed using the methods mentioned in Examples 1 and 2 of WO 2007 / 11275. Assessment of the stability of LNA oligonucleotides in human or rat plasma can be performed using the methods mentioned in Example 4 of WO 2007 / 112754. Treatment of cultured cells with LNA modified antisense oligonucleotides can be performed using the methods mentioned in Example 6 of WO 2007 / 11275.

[0122] Example 2. RNA isolation and expression analysis from cultured cells and tissues is performed using the methods mentioned in Example 10 of WO 2007 / 112754. RNAseq-based transcriptional profiling from cultured cells and tissues is performed using the methods mentioned in (Djebali et al. Nature 489:101-108 or Chu et al. Nucleic Acid Ther. 22:271-274 or Wang et al. Nature Reviews Genetics 10:57-63).

[0123] Example 3: Cell culture The adherent human breast adenocarcinoma cell line MCF7 (ECACC number 86012803) was purchased from ATCC (catalog number HTB-22™) and plated in Nunc™ EasYFlask™ cell culture flasks (catalog number 159910, Thermo Fischer Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (catalog number F4135, Sigma Aldrich, St. Louis, MO, USA), 1% non-essential amino acids (catalog number 11140050, Thermo Fischer Scientific, Waltham, MA, USA), 1% L-glutamine (catalog number G7513, Sigma Aldrich, St. Louis, MO, USA), and 1% penicillin / streptomycin (catalog number P4333, Sigma Aldrich, St. Louis, MO, USA). Cells were maintained in Eagle's Minimum Essential Medium (catalog no. M2279, Sigma Aldrich, St. Louis, MO, USA) supplied with 5% CO2 medium. Cells were kept in a humidified 5% CO2 incubator at 37°C and passaged twice weekly.

[0124] Example 4: Primary screening of antisense oligonucleotide compound library A library of 79 antisense oligonucleotides was designed to adenosine kinase, both long and short isoforms (ADK-LS). Antisense oligonucleotides were synthesized by IDT (Coralville, Iowa, USA) and diluted to a stock concentration of 500 μM in nuclease-free water (catalog number AM9938, Thermo Fischer Scientific, Waltham, MA, USA) under sterile conditions. Resuspended oligonucleotides were stored at -20°C.

[0125] The day before transfection, 1.25 × 10 MCF7 cells were plated in 24-well Nunc™ Cell-Culture Treated Multidishes (catalog no. 142475, Thermo Fischer Scientific, Waltham, MA, USA). 5Cells / well were seeded. On the day of transfection, cell media was removed 1 h prior to transfection and 475 μL of maintenance medium was added. All oligonucleotides were diluted in Opti-MEM (catalog no. 31985-070, Thermo Fischer Scientific, Waltham, MA, USA) to a final well concentration of 10 nM. Lipofectamine™ RNAiMAX (catalog no. 13778150, Thermo Fischer Scientific, Waltham, MA, USA) was diluted in Opti-MEM to a final well concentration of 1.5 μL. Equal volumes of RNAiMAX and antisense oligonucleotide solutions were combined and incubated for 5 min, after which 25 μL of the mixture was added to the well. As experimental controls, both scrambled control oligonucleotide and RNAiMAX mock-treated cells were used. 48 h after transfection, RNA extraction was performed using the RNeasy mini kit (catalog no. 74106, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed using Superscript IV reverse transcriptase (catalog number 18090010, Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer's instructions using random hexamer primers (catalog number SO142, Thermo Fischer Scientific, Waltham, MA, USA), including gDNA removal with ezDNase™ (catalog number 11766051, Thermo Fischer Scientific, Waltham, MA, USA). qPCR was performed on a QuantStudio 6 Flex (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's instructions using Taqman assays (Table 1) synthesized by Integrated DNA Technologies (Newark, NJ, USA) and TaqMan™ Universal Master Mix II, no UNG (catalog number 4440040, Thermo Fischer Scientific, Waltham, MA, USA).All assays were designed to be exon spanning, specificity was confirmed by primer blasting, and primer efficiency was tested using five-fold dilution. Hprt1 was used as a housekeeping gene. The ADK assay used detects all mRNA variants.

[0126] [Table 4]

[0127] All data were calculated in Microsoft Excel and visualized in Prism version 9.1.1 (GraphPad, San Diego, CA, USA). qPCR results were analyzed using the ΔΔCt method, with mock-treated cells with RNAiMAX only as reference. The first screening was performed with 2 technical replicates with n = 1 (Figure 1), depicted as mean ± SEM. The top 11 candidates were selected and the levels of ADK-LS mRNA knockdown were confirmed in follow-up experiments (n, N = 2, 3–4), depicted as mean ± SEM in Figure 2.

[0128] Example 5: Dose range for knockdown of ADK-LS with selected antisense oligonucleotides Transfections and qPCR were performed as in Example 4, except that the antisense oligonucleotide concentrations were either 5, 1, or 0.2 nM. Experiments were repeated to provide 1-2 biological replicates with 1-2 technical replicates each.

[0129] FIG. 3 shows the results of a dose-response study.

[0130] Example 6: IC of selected ADK-LS antisense oligonucleotides in cultured cell lines 50 Determining Values Transfection and qPCR were performed as in Example 6, except that cells were transfected with a range of antisense oligonucleotide concentrations from 90 nM to 0.004 nM in 3-fold dilutions. Relative levels of ADK-LS determined by qPCR were plotted against log(M) in Graphpad Prism (version 9.0.2, Graphpad GraphPad Software). Dose-response curves were fitted using a 3-parameter nonlinear fit and IC50 values ​​were calculated in nM. Experiments were repeated, giving 2 biological replicates with 2 technical replicates each. Figure 4 shows the dose-response curves and IC50 values ​​for ADK-LS antisense oligonucleotides. 50 Indicates the value.

[0131] Example 7: RNA sequencing in cultured cell lines Cells were transfected as in the previous experiments, except that the antisense oligonucleotide concentrations were 3 and 30 nM, respectively. Experiments were repeated to provide three biological replicates. RNA was isolated from cell pellets using the miRNeasy Mini Kit (Cat. No. 217004, Qiagen) and contaminating genomic DNA was removed using the RNase-Free DNase Set (Cat. No. 79254, Qiagen). Final RNA quality was assessed using an RNA Nano chip on a Bioanalyzer 2100 (Cat. No. 5067-1511, Agilent technologies, Santa Clara, CA, USA). Isolated RNA samples were rRNA-depleted and prepared for sequencing using the SMARTer Stranded Total RNA Sample Prep Kit-HI Mammalian (Cat. No. 38229000, Takara Bio Europa). rRNA depletion was performed using RiboGone, residual RNA was purified using AMPure XP beads (catalog no. A63881, Beckman Coulter, Brea, CA, USA), and library construction was performed according to the manufacturer's protocol. Final libraries were size selected (150–500 bp) on Pippin Prep (Sage Science, Inc. Beverly, MA, USA), quality controlled on a Bioanalyzer 2100 using Qubit and high sensitivity chips (Agilent), and quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA). RNA sequencing was performed on a Novaseq 6000 S4 at Novogene (Cambridge, UK).

[0132] Sequencing data were preprocessed by removing adapter sequences and trimming low quality bases with Phred scores below 20 using Trim Galore (version 0.4.1). 1 Quality control was performed using to ensure high quality data.

[0133] STAR 2 Quantification of gene expression was performed by mapping the filtered reads to the human genome (hg19) using the software FeatureCounts to quantify the number of reads and Gencode V37 3 Gene expression levels were mapped to each gene using gene annotations from 4 Differential expression analysis was performed in ref. 5 Predicted gene targets were found for each antisense oligonucleotide by in silico analysis using GGGenome from RefSeq. The sequence of each antisense oligonucleotide was matched against both mature spliced ​​mRNA sequences (splice) and unspliced ​​pre-mRNA sequences (pre-splice) from RefSeq, allowing a total of three insertions, deletions, or mismatches. The sum of the insertions, deletions, and mismatches for each antisense oligonucleotide match was expressed as a "distance" (d) that represents the quality of the predicted target site. d=0 means a perfect match of binding between the antisense oligonucleotide and the (pre)mRNA, and d=3 means three insertions, deletions, or mismatches. Predicted mRNA and pre-mRNA antisense oligonucleotide targeting was compared with gene expression and differential expression analysis from RNA-seq to estimate which genes are differentially expressed due to antisense oligonucleotide off-targeting. All plotting was done in R.

[0134] 1 Philip Ewels and others,'MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report',Bioinformatics,32.19(2016),3047-48<https: / / doi.org / 10.1093 / bioinformatics / btw354> . 2 Alexander Dobin and others,'STAR: Ultrafast Universal RNA-Seq Aligner',Bioinformatics,29.1(2013),15-21<https: / / doi.org / 10.1093 / bioinformatics / bts635> . 3 Yang Liao, Gordon K Smyth, and Wei Shi,'FeatureCounts:An Efficient General Purpose Program for Assigning Sequence Reads to Genomic Features',Bioinformatics,30.7(2014),923-30<https: / / doi.org / 10.1093 / bioinformatics / btt656> . 4 Michael I Love, Wolfgang Huber, and Simon Anders, 'Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2', Genome Biology, 15.12 (2014), 550<https: / / doi.org / 10.1186 / s13059-014-0550-8> . 5 Tokuyuki Yoshida and others,'Evaluation of Off-Target Effects of Gapmer Antisense Oligonucleotides Using Human Cells',Genes to Cells,24.12(2019),827-35<https: / / doi.org / https: / / doi.org / 10.1111 / gtc.12730> .

[0135] To assess the effect of antisense oligonucleotide treatment on ADK expression, expression levels were normalized and compared across samples (Figure 7).

[0136] To assess the effect of ADK-LS antisense oligonucleotides on the whole transcriptome, differential gene expression analysis was performed and the resulting data were visualized in a volcano plot (Figure 5 (sequence number 21) and Figure 6 (sequence number 71)).

[0137] To determine whether the changes in RNA expression could be attributed to either 1) a direct effect by targeting other sequences in the transcriptome or 2) a downstream secondary consequence of a direct effect, an initial in silico analysis was performed using antisense oligonucleotide sequences that predicted all potential target sites 1) within the spliced ​​transcriptome (cytoplasm) and 2) within the unspliced ​​transcriptome (nucleus). This was done for target sites with either 0, 1, 2, or 3 insertions, deletions, or mismatches (collectively referred to as distance (d)). A distance of 0 was observed only with antisense oligonucleotides that bound to ADK RNA. The results are depicted in Figure 8 (SEQ ID NO: 21) and Figure 9 (SEQ ID NO: 71).

[0138] Embodiment 1. A therapeutic RNA compound comprising a contiguous nucleotide sequence of 10-30 nucleotides in length that is complementary to a nucleic acid sequence within an adenosine kinase transcript.

[0139] 2 The therapeutic RNA compound of embodiment 1, which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% complementary to a nucleic acid sequence within an adenosine kinase transcript.

[0140] 3 The therapeutic RNA compound according to embodiment 1 or 2, wherein the adenosine kinase transcript is SEQ ID NO:1.

[0141] 4. The therapeutic RNA compound of embodiments 1-3, wherein the therapeutic RNA compound is complementary to any one of SEQ ID NOs: 164-205.

[0142] 5. The therapeutic RNA compound according to any one of embodiments 1 to 4, wherein the compound is capable of reducing adenosine kinase protein expression in human cells expressing the adenosine kinase protein.

[0143] 6. The therapeutic RNA compound of embodiment 5, wherein ADK protein expression is reduced by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to ADK protein expression in human cells not exposed to the compound.

[0144] 7. A therapeutic RNA compound according to any one of embodiments 1 to 6, capable of reducing ADK transcript (e.g., mRNA) expression in a human or mammalian cell expressing the ADK transcript.

[0145] 8 The therapeutic RNA compound of embodiment 7, wherein ADK transcript expression is reduced by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% compared to ADK transcript expression in a human cell not exposed to the compound.

[0146] 9. The therapeutic RNA compound according to any one of embodiments 1 to 8, wherein the compound is an antisense oligonucleotide or an siRNA.

[0147] 10. The antisense oligonucleotide or siRNA of embodiment 9, wherein the antisense oligonucleotide or siRNA comprises one or more affinity-enhancing nucleoside analogues.

[0148] 11 The antisense oligonucleotide or siRNA of embodiment 10, wherein one or more of the nucleoside analogs in the antisense oligonucleotide include 2'-0-alkyl RNA, 2'-0-methyl RNA (2'-OMe), 2'-alkoxy-RNA, 2'-0-methoxyethyl-RNA (2'-MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro ANA, bicyclic nucleoside analogs (LNA), or combinations thereof, or one or more of the nucleoside analogs in the siRNA include 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0149] 12. The antisense oligonucleotide or siRNA according to embodiment 10 or 11, wherein one or more nucleoside analogues are affinity-enhancing 2' sugar-modified nucleosides.

[0150] 13. The antisense oligonucleotide or siRNA of embodiment 12, wherein the affinity-enhancing 2' sugar-modified nucleoside is LNA.

[0151] 14. The antisense oligonucleotide or siRNA according to embodiment 13, wherein the LNA is selected from the group consisting of constrained ethyl nucleoside (cEt), 2',4'-constrained 2'-0-methoxyethyl (cMOE), uL-LNA, P-DLNA, 2'-0,4'-C-ethylene-bridged nucleic acid (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof.

[0152] 15. The antisense oligonucleotide or siRNA according to any one of the preceding embodiments, wherein the antisense oligonucleotide or siRNA comprises one or more 5'-methyl-cytosine nucleobases.

[0153] 16. An antisense oligonucleotide complementary to ADK pre-mRNA (SEQ ID NO: 1) comprising a sequence of 10 to 30 nucleotides in length, wherein the antisense oligonucleotide has at least one affinity-enhancing nucleotide analogue.

[0154] 17. The antisense oligonucleotide of embodiment 16, wherein said antisense oligonucleotide comprises at least one internucleoside linkage selected from phosphorothioate, or any of phosphodiester, phosphotriester, methylphosphonate, or phosphoramidate linkages.

[0155] 18. The antisense oligonucleotide according to embodiment 16 or 17), wherein the antisense oligonucleotide is complementary to both ADK-L and ADK-Spre-mRNA.

[0156] 19. The antisense oligonucleotide according to any one of embodiments 16 to 18, wherein the antisense oligonucleotide is capable of downregulating, eg, knocking down, expression of ADK-L and ADK-S.

[0157] 20. The antisense oligonucleotide according to any one of embodiments 16 to 19, comprising a motif complementary to any one of SEQ ID NOs: 164 to 205.

[0158] 21 The antisense oligonucleotide according to embodiment 20, wherein the motif is any one of SEQ ID NOs: 83 to 161.

[0159] 22. The antisense oligonucleotide according to any one of embodiments 16 to 21, wherein the antisense oligonucleotide is a gapmer and contains a stretch of at least 5 consecutive DNA nucleotides.

[0160] 23. The antisense oligonucleotide according to any one of embodiments 16 to 22, wherein the antisense oligonucleotide comprises a sequence of 14 to 20 nucleotides in length.

[0161] 24. The antisense oligonucleotide according to any one of embodiments 16 to 23, wherein the affinity enhancing nucleotide analogue is selected from the list of LNA, tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2'methoxyethyl (2'MOE), 2'cyclic ethyl (cET), UNA, 2'fluoro and conformationally restricted nucleosides (CRN).

[0162] 25. The antisense oligonucleotide according to any one of embodiments 16 to 24, wherein the antisense oligonucleotide comprises at least one LNA.

[0163] 26. The antisense oligonucleotide according to any one of embodiments 16 to 25, wherein the antisense oligonucleotide comprises 20 to 55% LNA.

[0164] 27. The antisense oligonucleotide of embodiment 26, wherein the antisense oligonucleotide further comprises one or more nucleosides that are any one of tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2'methoxyethyl (2'MOE), 2'cyclic ethyl (cET), UNA, 2'fluoro, and conformationally restricted nucleosides (CRN).

[0165] 28. The antisense oligonucleotide according to any one of embodiments 16 to 27, wherein the LNA is beta-D-oxy LNA.

[0166] 29. The antisense oligonucleotide according to any one of embodiments 16 to 28, wherein all internucleoside linkages are phosphorothioate linkages and all modified cytosines are 5'-methyl-cytosines.

[0167] 30. The antisense oligonucleotide according to any one of embodiments 16 to 29, wherein the antisense oligonucleotide is any one of SEQ ID NOs:2 to 80, for example, any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:80.

[0168] 31. A composition comprising a therapeutic RNA compound according to any one of embodiments 1 to 15 or an antisense oligonucleotide according to any one of embodiments 16 to 30.

[0169] 32. A therapeutic RNA compound according to any one of embodiments 1 to 15 or an antisense oligonucleotide according to any one of embodiments 16 to 30 or a composition according to embodiment 31 for use as a pharmaceutical agent.

[0170] 33 A therapeutic RNA compound according to any one of embodiments 1 to 15 or an antisense oligonucleotide according to any one of embodiments 16 to 30 or a composition according to embodiment 31, wherein said therapeutic RNA, antisense oligonucleotide or composition is for use as a pharmaceutical agent, preferably said pharmaceutical agent is for use in the preventative, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of a disease in which modulation of ADK activity is beneficial.

[0171] 34. A therapeutic RNA compound according to any one of embodiments 1 to 15 or an antisense oligonucleotide according to any one of embodiments 16 to 30 or a composition according to embodiment 31, wherein said therapeutic RNA, antisense oligonucleotide or composition is for use in reducing or knocking down expression of an ADK, such as ADK L / S, in a cell or in an individual, e.g. in a human or mammal.

[0172] 35. The therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31, wherein the use is for the preventative, curative or disease-modifying treatment, mitigation, amelioration, pre-emptive treatment or prevention of a CNS or PNS disease.

[0173] 36. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31, wherein said antisense oligonucleotide or said composition is for use in the preventative, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of a disease of the CNS or PNS, such as a psychiatric neurological disorder, a neurodegenerative disorder or a neurodevelopmental disorder.

[0174] 37. A therapeutic RNA compound, an antisense oligonucleotide or a composition according to any one of embodiments 1 to 31 for use as a neuroprotective agent.

[0175] 38 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of a disease of the CNS or PNS, a psychiatric disorder, a neurological disorder, a neurodegenerative disorder, a neurodevelopmental disorder, a central and peripheral nervous system disease associated with cellular trauma and inflammation, neuronal injury, hippocampal injury, traumatic brain injury, memory impairment, hippocampal sclerosis, Parkinson's disease, multiple sclerosis, acute spinal cord injury, amyotrophic lateral sclerosis, ataxia, Bell's palsy, Charcot-Marie-Tooth disease, headache, Houghton's headache, migraine, Pick's disease, progressive supranuclear palsy, multisystem degeneration, motor neuron diseases, Huntington's disease, prion diseases, Creutzfeldt-Jakob disease, corticobasal degeneration, primary progressive aphasia or a symptom or effect thereof.

[0176] 39. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment of epilepsy.

[0177] 40. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment of stroke.

[0178] 41. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31, wherein said antisense oligonucleotide or said composition is for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of epilepsy and / or seizures, preferably treatment-resistant epilepsy, acquired, genetic and / or idiopathic epilepsy, therapy-resistant epileptic syndromes, drug-resistant epilepsy, pharmacologically resistant focal epilepsy, spontaneous seizures, therapy-resistant seizures, focal epilepsy, generalized epilepsy or status epilepticus status.

[0179] 42 epilepsy, drug-resistant epilepsy, drug-resistant focal epilepsy, seizures, spontaneous seizures, therapy-resistant seizures, focal epilepsy, preferably said focal epilepsy is focused on the frontal, parietal, occipital or temporal lobe, generalized epilepsy, preferably said generalized epilepsy is selected from absence seizures, myoclonic seizures, tonic-clonic seizures, tonic seizures, atonic seizures, clonic seizures and convulsions, status epilepticus status, epileptogenic induced by acute brain injury, autosomal dominant nocturnal frontal lobe epilepsy, persistent spike-and-wave pattern during slow wave sleep, Dravet syndrome, post-stroke epilepsy, epileptic encephalopathy, laughter epilepsy, absence seizures, benign neonatal seizures, Jeavons syndrome 32. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of idiopathic encephalopathy syndrome, juvenile myoclonic epilepsy, Landau-Kleffner syndrome, Lennox-Gastaut syndrome, mesial temporal lobe epilepsy, myoclonic astatic epilepsy, Ohtahara syndrome, Panayiotopoulos syndrome, PCDH19 syndrome, benign childhood epilepsy with centrotemporal spikes, Sturge-Weber syndrome, symptomatic focal epilepsy, transient epileptic amnesia and West syndrome, and / or glioma-associated epilepsy.

[0180] 43. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of pain, preferably wherein said pain is chronic pain, neuropathic pain, chemotherapy-induced neuropathic pain, migraine, headache, hyperalgesia, allodynia and / or fibromyalgia.

[0181] 44. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment of pain.

[0182] 45. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1-31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of pain, chronic pain, neuropathic pain, chemotherapy-induced neuropathic pain, migraine including migraine with aura and migraine without aura, primary headache, tension headache, cluster headache, Horton's headache, chronic daily headache, sinus headache, post-traumatic headache, exercise headache, hemicrania continua, hypnic headache, hyperalgesia, heat hyperalgesia, allodynia, tactile allodynia and / or fibromyalgia.

[0183] 46. ​​A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventative, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of a psychiatric disorder, a cognitive disorder, a sleep disorder, a cardiovascular disorder, a respiratory disorder, cancer, a renal disorder, an inflammation or a metabolic disorder.

[0184] 47 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of a psychiatric disorder, a neuropsychiatric disorder, anxiety, depression, bipolar disorder, attention deficit hyperactivity disorder, attention deficit disorder, autism, Asperger's syndrome, Tourette's syndrome, schizophrenia, paranoid schizophrenia, anaphylactic schizophrenia, catatonic schizophrenia, undifferentiated schizophrenia, residual schizophrenia, simple schizophrenia or unspecified schizophrenia.

[0185] 48. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of cognitive impairment, cognitive impairment, dementia, Alzheimer's disease, vascular dementia, frontotemporal dementia or dementia with Lewy bodies.

[0186] 49. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of a sleep disorder.

[0187] 50. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use as a sleep regulator.

[0188] 51. A therapeutic RNA compound, an antisense oligonucleotide or a composition according to any one of embodiments 1 to 31 for use in promoting sleep.

[0189] 52 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prophylaxis of cardiovascular disorders, peripheral arterial disease, post-operative atrial fibrillation, heart failure, chronic heart failure, intracerebral hemorrhage induced brain injury, stroke, cerebral ischemia or ischemia.

[0190] 53. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventative, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of a respiratory disorder, asthma or chronic obstructive pulmonary disease.

[0191] 54 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventative, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of cancer, nervous system cancer, glioma, glioblastoma, liver cancer or cancer metastasis.

[0192] 55. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventative, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of renal impairment, renal injury, renal inflammation, albuminuria or glomerular injury.

[0193] 56. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, mitigation, pre-emptive treatment or prevention of inflammation.

[0194] 57 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease-modifying treatment, palliative, pre-emptive treatment or prevention of inflammatory disorders, oxidative stress, inflammation, apoptosis, arthritis, osteoarthritis, rheumatoid arthritis and pain associated with these conditions, encephalitis, meningitis, human Rasmussen's encephalitis, inflammation of the cerebral cortex and / or hippocampus, progressive cognitive decline, colitis, ulcerative colitis or inflammatory bowel disease.

[0195] 58. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the treatment, mitigation, pre-emptive, curative or disease-modifying treatment or prevention of a metabolic disorder, preferably diabetes, more preferably type 1 or type 2 diabetes.

[0196] 59 A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in the preventive, curative or disease modifying treatment, palliative, pre-emptive treatment or prevention of Prader-Willi syndrome, Angelman syndrome, neurofibromatosis, angiogenesis related diseases, angiogenesis promotion, retinal disorders, preferably diabetic retinopathy or hearing loss.

[0197] 60. The therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 59, wherein the antisense oligonucleotide or composition is administered systemically, subcutaneously, nasally, intrathecally, intraventricularly into the CNS, or intravenously.

[0198] 61. A therapeutic RNA compound, antisense oligonucleotide or composition according to any one of embodiments 1 to 60 for use in combination with one or more other active pharmaceutical ingredients for the preventive, curative or disease-modifying treatment of any of the diseases according to any one of embodiments 33 to 60.

[0199] 62. Use according to embodiment 61, in which the other active pharmaceutical ingredient is an ingredient intended for the preventive, curative or disease-modifying treatment of a disease according to any one of embodiments 33 to 60.

[0200] 63. The use according to embodiment 61 or 62, wherein the other pharmaceutical ingredient is an antisense oligonucleotide targeting miR-27b or miR-134.

[0201] 64. A pharmaceutical composition comprising an effective dosage of the antisense oligonucleotide according to any one of embodiments 1 to 63 and a pharma- ceutically acceptable carrier.

[0202] 65. A pharmaceutical composition comprising an effective dosage amount of a therapeutic RNA compound, e.g., an antisense oligonucleotide, according to any one of embodiments 1 to 64, wherein the antisense oligonucleotide is the only active pharmaceutical ingredient.

[0203] 66. The pharmaceutical composition according to embodiment 64 or 65, wherein the composition is for the use according to any one of embodiments 33 to 63.

[0204] 67. The pharmaceutical composition according to any one of embodiments 64 to 66, wherein the composition is for intrathecal or intraventricular administration.

[0205] 68. The pharmaceutical composition according to embodiment 67, wherein the composition is administered by a pump, preferably the pump is a minipump, more preferably the minipump is a miniosmotic pump.

[0206] 69. The pharmaceutical composition according to any one of embodiments 67 to 69, wherein the composition is for intraventricular administration facilitated by an intraventricular catheter, preferably said catheter is attached to a reservoir, preferably said reservoir is an Ommaya reservoir.

[0207] 70 The composition is administered for 1 day, 2 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 days.

[0208] 71. The pharmaceutical composition according to any one of embodiments 64 to 68, wherein the composition is administered at intervals of 1 to 200 days, 10 to 190 days, 20 to 180 days, 30 to 170 days, 40 to 160 days, 50 to 150 days, 60 to 140 days, 70 to 130 days, 80 to 120 days, 90 to 110 days, or preferably at intervals of about 100 days.

[0209] 72. An antisense oligonucleotide or composition according to any one of embodiments 1 to 31 for use in a method for treating a disease according to any one of embodiments 33 to 60.

[0210] 73. A method for the treatment of a disease according to any one of embodiments 33 to 60 by using a therapeutic RNA compound according to any one of embodiments 1 to 30, an antisense oligonucleotide, or a composition according to embodiment 31, or a pharmaceutical composition according to any one of embodiments 64 to 71.

[0211] 74. The use according to any one of embodiments 33 to 63 or the method according to embodiment 73, wherein the treatment is preventative, curative or disease-modifying.

[0212] 75. A method for diagnosing a disease according to any one of embodiments 33 to 59 by using an antisense oligonucleotide according to any one of embodiments 16 to 30 or a composition according to embodiment 31.

Claims

1. A therapeutic RNA compound comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within an adenosine kinase transcript.

2. The therapeutic RNA compound of claim 1 , wherein the adenosine kinase transcript is SEQ ID NO:

1.

3. 2. The therapeutic RNA compound of claim 1, wherein the compound is an antisense oligonucleotide complementary to ADK pre-mRNA (SEQ ID NO: 1), the antisense oligonucleotide comprises at least one affinity-enhancing nucleotide analog, and the antisense oligonucleotide comprises at least one internucleoside linkage selected from a phosphorothioate linkage, a phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, or a phosphoramidate linkage.

4. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide is complementary to both ADK-L and ADK-S pre-mRNAs.

5. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide is capable of downregulating, eg, knocking down, the expression of ADK-L and ADK-S.

6. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide comprises a sequence of 14 to 20 nucleotides in length.

7. 4. The antisense oligonucleotide of claim 3, wherein the affinity-enhancing nucleotide analogue is selected from the list of LNA, tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2'-methoxyethyl (2'-MOE), 2'-cyclic ethyl (cET), UNA, 2'-fluoro and conformationally restricted nucleoside (CRN).

8. The antisense oligonucleotide of claim 3 , wherein the antisense oligonucleotide comprises at least one LNA.

9. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide is complementary to one of SEQ ID NOs: 164-205.

10. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide comprises or consists of any one of SEQ ID NOs: 83 to 161.

11. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide is any one of SEQ ID NOs: 2 to 80.

12. 4. The antisense oligonucleotide of claim 3, wherein the antisense oligonucleotide is selected from any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:37, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:

80.

13. 11. The antisense oligonucleotide of claim 10, wherein all internucleoside linkages are phosphorothioate, all modified nucleotides are LNA, such as beta-d-oxy LNA, and LNA cytosines are 5-methylcytosines.

14. 10. A therapeutic RNA compound according to claim 1 or an antisense oligonucleotide according to claim 3 for use in reducing or knocking down the expression of ADK in a cell or in an individual.

15. 10. The therapeutic RNA compound of claim 1 or the antisense oligonucleotide of claim 3, wherein the compound is for use as a pharmaceutical agent.

16. 10. The therapeutic RNA compound of claim 1 or the antisense oligonucleotide of claim 3, wherein the compound is for use in the preventative, curative, or disease-modifying treatment, mitigation, amelioration, pre-emptive treatment, or prevention of a CNS or PNS disease.

17. 10. The therapeutic RNA compound of claim 1 or the antisense oligonucleotide of claim 3, wherein the compound is for use in the preventative, curative, or disease-modifying treatment, palliative, ameliorative, pre-emptive treatment, or prevention of epilepsy or neuropathic pain.

18. A therapeutic RNA compound according to claim 1 or an antisense oligonucleotide according to claim 3 for use in reducing or knocking down expression of ADK L / S in a human or mammal.

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