PIKFYVE antisense oligonucleotides

Abstract

The present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing same, and methods for treating, inhibiting, suppressing, and preventing neurological disorders therewith. One embodiment is a single stranded ASO that suppresses expression of PIKFYVE, the ASO having a nucleobase sequence that includes at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-500. The nucleobase sequence of the ASO may include 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-500.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 202,717, filed June 22, 2021, which is incorporated herein by reference.

[0002] The present disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing same, and methods for treating, inhibiting, suppressing, and preventing neurological or neurodegenerative diseases. [Background technology]

[0003] Many neurodegenerative disorders in patients are difficult to effectively treat, especially when the pathology of the neurodegenerative disorder in a particular patient is not fully understood.

[0004] WO 2016 / 210372 discloses a method of treating a neurodegenerative disease by administering a PIKFYVE inhibitor. There remains a need for effective treatments for many neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 210372 Summary of the Invention

[0006] The present invention relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing same, and their use in the treatment of neurodegenerative disorders.

[0007] One embodiment is a single stranded ASO that inhibits expression of PIKFYVE, the ASO having a nucleobase sequence that includes at least 12 or 15 consecutive nucleobases of the nucleobase sequence of any of SEQ ID NOs: 1-500. The nucleobase sequence of the ASO may include 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of the nucleobase sequence of any of SEQ ID NOs: 1-500. The ASO may be any of SEQ ID NOs: 1-500.

[0008] Another embodiment is an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-500. The oligonucleotide may include 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-500.

[0009] In certain embodiments, at least one internucleoside linkage is a modified internucleoside linkage, which may be a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. At least one of the nucleosides may be a modified nucleobase.

[0010] In other embodiments, at least one nucleoside of the ASO can be a modified sugar moiety, which can be a bicyclic sugar moiety or which can include a 2'-O-methoxyethyl group. In certain aspects, the bicyclic sugar moiety includes a 4'-CH(R)-O-2' bridge, where the R groups are independently H, C, 1-12 alkyl, or a protecting group.

[0011] In yet other embodiments, the ASO is a gapmer (e.g., an MOE gapmer), wherein the gap segment can consist of 8-12 linked deoxynucleosides, a 5' wing segment of 3-5 linked nucleosides, and a 3' wing segment of 3-5 linked nucleosides. In certain aspects, the gap segment can be disposed between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment comprise a modified sugar moiety (e.g., a sugar moiety having a 2'-O-methoxyethyl group).

[0012] In other embodiments, the oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-500.

[0013] Another embodiment is a pharmaceutical composition comprising a PIKFYVE ASO of the invention and one or more pharma- ceutically acceptable carriers, diluents, and / or excipients. In one embodiment, the pharmaceutical composition is suitable for parenteral administration, e.g., intraventricular injection or intrathecal administration.

[0014] Yet another embodiment is a method of inhibiting, suppressing, or preventing expression of PIKFYVE in a patient (e.g., a patient having a neurological or neurodegenerative disease) by administering (e.g., by intracerebroventricular injection or intrathecal administration) a PIKFYVE ASO or a pharmaceutical composition described herein (e.g., an effective amount thereof) to the patient.

[0015] Yet another embodiment is a method of treating a subject with a neurological or neurodegenerative disease by administering a therapeutically effective amount of PIKFYVE ASO or a pharmaceutical composition described herein.In one embodiment, the disease is amyotrophic lateral sclerosis (ALS) (e.g., C9orf72-associated ALS).In another embodiment, the disease is frontotemporal dementia (FTD), such as FTD with TDP-43 pathology or FTD with tau pathology.In yet another embodiment, the disease is C9orf72-associated FTD (C9-FTD).In yet another embodiment, the disease is microtubule-associated protein tau (MAPT)-associated FTD (MAPT-FTD), e.g., FTD with V337M MAPT mutation.

[0016] Yet another embodiment is a method of treating a subject having a PIKFYVE disease or disorder by administering a therapeutically effective amount of a PIKFYVE ASO or pharmaceutical composition described herein. [Brief description of the drawings]

[0017] A more complete understanding of the present invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which:

[0018] [Figure 1] PIKFYVE ASO screening in HeLa cells measuring relative mRNA expression levels of ASOs 1-33 (SEQ ID NOs: 1-33) versus a control (NCASO).

[0019] [Diagram 2] 1 is a bar graph showing the inhibitory effect of various PIKFYVE ASOs in neonatal transgenic hPIKFYVE BAC mice.

[0020] [Diagram 3]1 is a bar graph showing changes in PIKFYVE mRNA and protein in non-human primates (NHPs) with and without ASO-520 or artificial cerebrospinal fluid (aCSF) treatment.

[0021] [Figure 4A] Graph showing the survival of control motor neurons in the presence of non-coding ASO (NC ASO) or motor neurons from C9ALS patients in the presence of (i) NC ASO or (ii) AS-20 (SEQ ID NO: 20).

[0022] [Figure 4B] 1 is a bar graph showing hazard ratios for control motor neurons in the presence of NC ASO, or motor neurons from C9ALS patients in the presence of (i) NC ASO or (ii) AS-520 (SEQ ID NO: 520).

[0023] [Figure 5A] 2 is a graph showing survival probability of cortical neurons from FTD patients with MAPT V337V or V337M in the presence of NC ASO or AS-520 (SEQ ID NO: 520).

[0024] [Figure 5B] 1 is a bar graph showing hazard ratios for cortical neurons from controls, C9orf72-related FTD (C9-FTD), sporadic FTD (sFTD), and microtubule-associated protein tau (MAPT)-related FTD (MAPT-FTD) patients treated with NC ASO or AS-520. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0026] definition

[0027] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", "may", and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or phrases that do not exclude the possibility of additional acts or structures.

[0028] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents.

[0029] The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements present herein, whether or not explicitly stated.

[0030] As used herein, "2'-deoxynucleoside" means a nucleoside that contains a 2'-H(H) furanosyl sugar moiety as found in naturally occurring deoxyribonucleic acid (DNA) and nucleobases. In certain embodiments, a 2'-deoxynucleoside can contain a modified nucleobase and a furanosyl sugar moiety or can contain an RNA nucleobase (uracil) and a furanosyl sugar moiety.

[0031] As used herein, "2'-substituted nucleoside" refers to a nucleoside that includes a 2'-substituted sugar moiety. As used herein, "2'-substituted" with respect to the sugar moiety means a sugar moiety that includes at least one 2'-substituent group other than H or OH.

[0032] As used herein, an "antisense molecule" means an oligomeric nucleic acid or oligomeric duplex capable of achieving at least one antisense activity.

[0033] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of a particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the expression "about 2 to about 4" also discloses a range of "2 to 4". The term "about" may refer to plus or minus 10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1. Other meanings of "about" are apparent from the context, such as rounding, e.g., "about 1" may mean 0.5 to 1.4.

[0034] With respect to the recitation of numerical ranges herein, each intervening numerical value to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the numerical values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numerical values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0035] As used herein, "bicyclic sugar" or "bicyclic sugar moiety" refers to a modified sugar moiety that includes two rings, where a second ring is formed via a bridge connecting two of the atoms in the first ring, thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not include a furanosyl moiety. As used herein, "bicyclic nucleoside" or "BNA" refers to a nucleoside that includes a bicyclic sugar moiety.

[0036] As used herein, "chiral enriched population" refers to a plurality of molecules of the same molecular formula, in which the number or percentage of molecules in the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules that would be expected to contain the same particular stereochemical configuration at the same particular chiral center in the population, if the particular chiral center is stereorandom. A molecular chiral enriched population that has multiple chiral centers in each molecule can contain one or more stereorandom chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound that contains a modified oligonucleotide.

[0037] As used herein, "complementary" with respect to an oligonucleotide means that at least 70% of the nucleobases or one or more regions of the oligonucleotide and the nucleobases or one or more regions of another nucleic acid can hydrogen bond with each other when the nucleobase sequences of the oligonucleotide and the other nucleic acid are aligned in opposite directions. Complementary nucleobases means nucleobases that can form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have complementary nucleobases at every nucleoside. Rather, some mismatches are allowed. As used herein, "fully complementary" or "100% complementary" with respect to an oligonucleotide means that the oligonucleotide is complementary to the other oligonucleotide or nucleic acid at every nucleoside of the oligonucleotide.

[0038] As used herein, "gapmer" refers to a modified oligonucleotide that includes an internal region having multiple nucleosides that support RNase H cleavage, located between external regions having one or more nucleosides, where the nucleosides that comprise the internal region are chemically distinct from the nucleoside or nucleosides that comprise the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings." Unless otherwise specified, "gapmer" refers to a sugar motif. Unless otherwise specified, the sugar moieties of the nucleosides of the gapmer gap are unmodified 2'-deoxyfuranosyl. Thus, the term "MOE gapmer" refers to a gapmer having both wing 2'-MOE nucleoside sugar motifs and a 2'-deoxynucleoside gap. Unless otherwise specified, MOE gapmers may include one or more modified internucleoside linkages and / or modified nucleobases, and such modifications do not necessarily follow the gapmer pattern of sugar modifications. Exemplary MOE gapmers are shown in Table 2 below.

[0039] In certain embodiments, an oligonucleotide comprises one or more modified sugar and / or unmodified sugar moieties arranged along the oligonucleotide or a region thereof in a defined pattern or sugar motif, in certain examples, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0040] In certain embodiments, the modified oligonucleotide comprises or consists of a region having a gap motif defined by two external regions or "wings" and a central or internal region or "gap". The three regions of the gapmer motif include the "5' wing", the "gap", and the "3' wing", which form a contiguous sequence of nucleosides, where at least a portion of the sugar moieties of the nucleosides of each wing are different from at least a portion of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing closest to the gap (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) are different from the sugar moieties of the adjacent gap nucleosides, thus defining a boundary between the wings and the gap (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are identical to each other. In certain embodiments, the gap comprises one or more nucleosides having a sugar moiety that is different from the sugar moieties of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

[0041] In certain embodiments, a gapmer wing comprises 1 to 5 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0042] In certain embodiments, the gapmer gap comprises between 7 and 12 nucleosides (e.g., 10 nucleosides). In certain embodiments, each nucleoside of the gapmer gap is an unmodified 2'-deoxynucleoside.

[0043] In certain embodiments, the gapmer is a deoxy gapmer. In embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2'-deoxynucleosides and the nucleosides on the wing side of each wing / gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0044] In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside throughout the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif, and each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of the uniformly modified oligonucleotide comprises the same 2' modification.

[0045] As used herein, "inhibit" refers to the ability to substantially antagonize, hinder, prevent, suppress, arrest, slow, impede, alter, eliminate, halt, or reverse the progression or severity of the activity of a particular pathogen (e.g., an infectious pathogen) or disease.

[0046] As used herein, "internucleoside bond" refers to a covalent bond between adjacent nucleosides in an oligonucleotide.As used herein, "modified internucleoside bond" refers to any internucleoside bond other than a phosphodiester internucleoside bond.A "phosphorothioate bond" is a modified internucleoside bond in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside bond is replaced with a sulfur atom.

[0047] In certain embodiments, the nucleosides of modified oligonucleotides can be linked together using any internucleoside bond. Two major classes of internucleoside linkage groups are defined by the presence or absence of phosphorus atoms. Representative internucleoside linkages containing phosphorus include, but are not limited to, phosphodiester linkages (also referred to as unmodified or natural linkages), phosphotriesters, methyl phosphonates or other alkyl phosphonates, phosphoramidates, and phosphorothioates, and phosphorodithioates. Representative internucleoside linkage groups containing non-phosphate include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to natural phosphate linkages, can be used to alter (usually increase) the nuclease resistance of oligonucleotides. Methods for the preparation of phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0048] Representative internucleoside linkages with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides containing internucleoside linkages with chiral centers can be prepared as a population of modified oligonucleotides containing stereorandom internucleoside linkages or as a population of modified oligonucleotides containing phosphorothioate linkages of a specific stereochemical configuration. In certain embodiments, the population of modified oligonucleotides contains phosphorothioate internucleoside linkages, where all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be produced using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, as is well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides containing one or more specific phosphorothioate internucleoside linkages of a specific, independently selected stereochemical configuration. In certain embodiments, a particular configuration of phosphorothioate linkages is present in at least 65% of the molecules in the population. In certain embodiments, a particular configuration of phosphorothioate linkages is present in at least 70% of the molecules in the population. In certain embodiments, a particular configuration of phosphorothioate linkages is present in at least 80% of the molecules in the population. In certain embodiments, a particular configuration of phosphorothioate linkages is present in at least 90% of the molecules in the population. In certain embodiments, a particular configuration of phosphorothioate linkages is present in at least 99% of the molecules in the population.Such chirally enriched populations of modified oligonucleotides can be produced using synthetic methods known in the art, such as those described in Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc.Acid.Res.42, 13456 (2014); Chapter 10 of Locked Nucleic Acid Aptamers in Nucleic Acid and Peptide Aptamers: Methods and Protocols v 535, 2009 by Barciszewski et al., editor Gunter Mayerand; and WO2017 / 015555. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one designated phosphorothioate in the (Sp) configuration. In another embodiment, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one designated phosphorothioate in the (Rp) configuration.

[0049] As used herein, "MOE" means methoxyethyl. "2'-MOE" means a -OCH2CH2OCH3 group at the 2' position of the furanosyl ring.

[0050] A "neurological disease" is a disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, the neurological disease can be a neurodegenerative disease. The neurodegenerative disease can cause, for example, the degeneration of motor neurons. The neurological disease can be, for example, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, or frontotemporal dementia. Further examples of neurological diseases include, but are not limited to, for example, Parkinson's disease, multiple sclerosis, peripheral myopathy, Rasmussen's encephalitis, attention deficit hyperactivity disorder, autism, central pain syndrome, anxiety, and / or depression.

[0051] Neurological diseases may involve abnormal endosomal trafficking.For example, endosomal pathways and endosomes are necessary components for the recycling or destruction of membrane-bound proteins, the transport of Golgi-associated proteins, and the extracellular release of proteins in exosomes.These processes support neurotransmission, for example, promoting the balance between the recycling and degradation of synaptic vesicles or neurotransmitter receptors.

[0052] Neurological diseases may be accompanied by abnormal lysosomal degradation. Altered lysosomal degradation may be present in neurological diseases, such as neurodegenerative diseases. Cathepsin imbalance due to aging and aging-related diseases may cause harmful effects on central nervous system (CNS) neurons, and lysosomes become the site of unfolding and partial degradation of membrane proteins or their precursors, which can then be expelled from cells or released from dead cells and accumulate as pathological entities.

[0053] A health care professional may diagnose a subject as having a disease associated with motor neuron degeneration by evaluating one or more symptoms of motor neuron degeneration. To diagnose a neurological disease, a physical exam may be followed by a thorough neurological examination. A neurological examination may evaluate motor and sensory skills, nerve function, hearing and speech, vision, coordination and balance, mental status, and changes in mood or behavior. Non-limiting symptoms of diseases associated with neurological diseases include weakness in the arms, legs, feet, or ankles, slurred speech, difficulty lifting the front of the foot and toes, weakness or clumsiness in the hands, muscle paralysis, muscle rigidity, involuntary spasms or writhing movements (chorea), involuntary persistent muscle contractures (dystonia), bradykinesia, loss of motility, impaired posture and balance, lack of flexibility, tingling in parts of the body, electric shock sensations when moving the head, and weakness in the arms, shoulders, and tongue. Symptoms may include convulsions, difficulty swallowing, difficulty breathing, difficulty chewing, partial or complete loss of vision, double vision, slow or abnormal eye movements, tremors, unsteady gait, fatigue, memory loss, dizziness, difficulty thinking or concentrating, difficulty reading and writing, misunderstanding spatial relationships, disorientation, depression, anxiety, difficulty making decisions and judgments, loss of impulse control, difficulty planning and carrying out routine tasks, aggression, irritability, social withdrawal, mood swings, dementia, changes in sleep habits, wandering, and changes in appetite.

[0054] Tests may be performed to determine diseases and disorders that may have symptoms similar to neurological diseases, measure muscle involvement, and evaluate neuronal degeneration. Non-limiting examples of tests are electromyography (EMG), nerve conduction velocity tests, blood, urine, or other substance laboratory tests, magnetic resonance imaging (MRI), magnetic resonance spectroscopy, muscle or nerve biopsy, transcranial magnetic stimulation, genetic screening, x-ray, fluoroscopy, angiography, computed tomography (CT), positron emission tomography, cerebrospinal fluid analysis, subarachnoid contrast CT scan, electroencephalography, electronystagmus, evoked responses, polysomnography, thermography, and ultrasound. A medical professional may also evaluate the patient's family history of diseases related to motor neuron degeneration and make a diagnosis based in part on the family history of neurological diseases. A health care professional may diagnose a disease related to a neurological disease in a subject after one or more symptoms appear.

[0055] Neurodegenerative diseases cause the progressive destruction of neurons and affect neuronal signal transduction. For example, neurodegeneration can be amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Friedreich's ataxia, Lewy body disease, Parkinson's disease, spinal muscular atrophy, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0056] Diseases associated with the degeneration of motor neurons can be pathologies that cause the progressive destruction of motor neurons, impeding neuronal signaling to muscle, leading to muscle weakness and wasting.In healthy individuals, upper motor neurons transmit signals from the brain to lower motor neurons in the brainstem and spinal cord, which then transmit the signals to muscle to generate voluntary muscle activity.The destruction of upper and lower motor neurons can affect activities such as breathing, speaking, swallowing, and walking, and over time these functions can be lost.Examples of motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0057] Neuronal hyperexcitability can occur when receptors for the excitatory neurotransmitter glutamate (glutamate receptors), such as NMDA and AMPA receptors, are overactivated by excess glutamate or by other compounds or neurotransmitters that act on glutamate receptors. Excitotoxicity can result from neuronal hyperexcitability. Excitotoxicity is a pathological process in which nerve cells are damaged or destroyed by excessive stimulation. Excessive stimulation can result in high levels of calcium ions (Ca 2+ ) can enter the cell. 2+ The influx of ATP activates many enzymes, including phospholipases, endonucleases, and proteases, such as calpains, which can damage cellular structures such as components of the cytoskeleton, membranes, and DNA.

[0058] Neuronal hyperexcitability may be involved in spinal cord injury, stroke, traumatic brain injury, hearing loss (due to noise overexposure or ototoxicity), epilepsy, painful neuropathy, attention deficit hyperactivity disorder, autism, central pain syndromes, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, schizophrenia, Rasmussen's encephalitis, Huntington's disease, alcoholism or withdrawal, especially rapid benzodiazepine withdrawal, and Huntington's disease. Another common condition that causes excessive glutamate concentrations around neurons is hypoglycemia. Blood glucose is the primary way glutamate is removed from the intersynaptic space of NMDA and AMPA receptor sites.

[0059] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that includes modifications, such as substituents, that do not form a bridge between two atoms of the sugar to form a second ring.

[0060] As used herein, "nucleobase" refers to an unmodified or modified nucleobase. As used herein, an "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a "modified nucleobase" is an atomic group other than unmodified A, T, C, U, or G that can pair with at least one unmodified or modified nucleobase. "5-methylcytosine" or "mC" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, a "nucleobase sequence" refers to the sequential sequence of contiguous nucleobases in a nucleic acid or oligonucleotide, independent of any sugar or nucleoside linkage modifications.

[0061] In certain embodiments, modified oligonucleotide comprises one or more nucleosides that comprise unmodified nucleobases.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that comprise modified nucleobases.In certain embodiments, modified oligonucleotide comprises one or more nucleobases that do not comprise nucleobases, which are referred to as abasic nucleosides.

[0062] In certain embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobases are 2-aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynylcytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp).Modified nucleobases can also include bases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288, and those disclosed in Chapters 6 and 15 of Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166, and 442-443.

[0063] As used herein, "nucleoside" refers to a compound containing a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. As used herein, "modified nucleoside" refers to a nucleoside containing a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides that lack a nucleobase. "Linked nucleosides" are nucleosides linked in a contiguous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0064] As used herein, "oligomeric compound" refers to an oligonucleotide and, optionally, one or more additional features, such as a conjugate group or a terminal group. An oligomeric compound may or may not be paired with a second oligomeric compound that is complementary to the first oligomeric compound. A "single-stranded oligomeric compound" is an oligomeric compound that is not paired. The term "oligomeric duplex" refers to a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a "duplexed oligomeric compound."

[0065] As used herein, "oligonucleotide" refers to a single strand of linked nucleosides linked via internucleoside linkages, where each nucleoside and internucleoside linkage may be modified or unmodified. The internucleoside linkage may be any linkage described herein. Unless otherwise indicated, an oligonucleotide consists of 8 to 50 linked nucleosides. As used herein, "modified oligonucleotide" refers to an oligonucleotide in which at least one nucleoside or internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" refers to an oligonucleotide that does not contain any nucleoside or internucleoside modification.

[0066] "PIKFYVE", also known in the art as "phosphatidylinositol-3-phosphate 5-kinase type III" or "PIPKIII", is a FYVE finger-containing phosphoinositide kinase encoded by the PIKFYVE gene. PIKFYVE is a highly evolutionarily conserved lipid kinase that also has protein kinase activity that regulates endomembrane homeostasis and plays a role in biogenesis of endosomal carrier vesicles from early endosomes. PIKFYVE-mediated conversion of PI3P to PI(3,5)P2 blocks recruitment of the protein EEA1. Recruitment is blocked because PIP3 is required to form a platform with RAB5 that allows EEA1 to anchor to early endosomes. EEA1 then promotes fusion with endocytic vesicles and other endosomal vesicles.

[0067] As used herein, "PIKFYVE disease or disorder" includes lysosomal degradation diseases and disorders mediated by PIKFYVE. For example, PIKFYVE disease or disorder includes, but is not limited to, amyloid diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis, and chronic hemodialysis-associated amyloid), multiple sclerosis, and MPS disorders (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). In some embodiments, the disease is an autoimmune disease (such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, adult Still's disease, Behcet's syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman's disease, etc.), or a CNS disease (such as spongiform encephalopathies (Creutzfeldt-Jakob disease, Kuru, Mad Cow Disease)). The compositions and methods of the disclosure can be used for the treatment of an individual having a lysosomal storage disease, comprising administering to a subject in need of treatment a therapeutically effective amount of a PIKfyve ASO or pharmaceutical composition described herein. In some embodiments, the ASOs and compositions of the disclosure alter the biogenesis, function, or dynamics of the endosomal or lysosomal system in a manner that reduces or inhibits the activity of PIKfyve and reduces the amount of material abnormally stored in lysosomes in lysosomal storage diseases. In some embodiments, the ASOs and compositions target, reduce, or inhibit the activity of PIKfyve, thus altering the biogenesis, function, or dynamics of the endoplasmic reticulum or Golgi apparatus in a manner that reduces the amount of material abnormally stored in lysosomes in lysosomal storage diseases. In other embodiments, the disease is a neurological disorder.

[0068] As used herein, "sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. A superscript prime (') is used to describe the numbering of the sugar of a nucleoside or nucleotide (nucleobase positions are numbered without a prime). When describing the sugar alone, no prime is used. As used herein, "unmodified sugar moiety" refers to a 2-OH(H) furanosyl moiety found in RNA (an "unmodified RNA sugar moiety"), or a 2-H(H) moiety found in DNA (an "unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen at each of positions 1, 3, and 4, one oxygen at position 3, and two hydrogens at position 5. As used herein, "modified sugar moiety" or "modified sugar" refers to a modified furanosyl sugar moiety or sugar surrogate. As used herein, a modified furanosyl sugar moiety refers to a furanosyl sugar that contains a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic and non-bicyclic sugars.

[0069] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety that includes a furanosyl ring bearing one or more substituents, none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents may be at any position of the furanosyl, including, but not limited to, substituents at the 2-, 4-, and / or 5-positions. In certain embodiments, one or more of the non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of suitable 2-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 2-F, 2-OCH3 ("OMe" or "O-methyl"), and 2-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2'-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, OC 1-10 Alkoxy, OC 1-10 Substituted alkoxy, OC 1-10 Alkyl, OC 1-10 Substituted alkyl, S-alkyl, N(R m)-Alkyl, O-Alkenyl, S-Alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ) or OCH2C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C 1-10 and alkyl, where the 2-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), and alkyl. Examples of suitable 5-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 5-methyl (R or S), 5-vinyl, and 5-methoxy. In certain embodiments, non-bicyclic modified sugar moieties include multiple non-bridging sugar substituents, such as 2-F-5-methyl sugar moieties.

[0070] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside is selected from the group consisting of F, NH, N, OCF, OCH, O(CH)NH, CHCH=CH, OCHCH=CH, OCHCHOCH, O(CH)SCH, O(CH)ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (OCH2C(=O)-N(R m )(R n )) wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-10 It is an alkyl.

[0071] In certain embodiments, the 2'-substituted nucleoside non-bicyclic modified nucleoside comprises a sugar moiety that includes a non-linear 2'-substituent selected from F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA").

[0072] In certain embodiments, 2'-substituted non-bicyclic modified nucleosides comprise a sugar moiety that includes a non-bridging 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

[0073] Certain modified sugar moieties include a substituent that bridges two atoms of a furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety includes a bridge between 4 furanose ring atoms and 2 furanose ring atoms. Examples of such 4 to 2 bridged sugar substituents include 4-CH2-2, 4-(CH2)2-2, 4-(CH2)3-2, 4-CH2-O-2 ("LNA"), 4-CH2-S-2, 4-(CH2)2-O-2 ("ENA"), 4-CH(CH3)-O-2 (referred to as "constrained ethyl" or "cEt"), 4-CH2-O-CH2-2, 4-CH2-N( R)-2, 4-CH(CH2OCH3)-O-2 ("constrained MOE" or "cMOE") and its analogues, 4-C(CH3)(CH3)-O-2 and its analogues, 4-CH2-N(OCH3)-2 and its analogues, 4-CH2-ON(CH3)-2, 4-CH2-C(H)(CH3)-2, 4-CH2-C(=CH2)-2 and its analogues, 4-C(R a R b )-N(R)-O-2, 4-C(R a R b )-ON(R)-2, 4-CH2-ON(R)-2, and 4-CH2-N(R)-O-2, where each R, R a , and R b are independently H, a protecting group, or C 1-12It is an alkyl.

[0074] In certain embodiments, such 4 to 2 bridges are independently -[C(R a )(R b )] n -,-[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x- , and -N(R a )-, x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R a and R b are independently H, a protecting group, a hydroxyl, C 1-12 Alkyl, substituted C 1-12 Alkyl, C 1-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, Substituted C 2-12 Alkynyl, C 5-20 Aryl, Substituted C 5-20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C 5-7 alicyclic radical, substituted C5-7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); each J1 and J2 is independently H, C 1-12 Alkyl, substituted C 1-12 Alkyl, C 2-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, Substituted C 2-12 Alkynyl, C 5-20 Aryl, Substituted C 5-20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-12 Aminoalkyl, substituted C 1-12 aminoalkyl, or a protecting group.

[0075] The addition of the bicyclic sugar moiety is a well-known technical field, and the technical field is well-known. al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al. al.,J.Org.Chem.,1998,63,10035-10039;Srivastava et al.,J.Am.Chem.Soc.,20017,129,8362-8379;Wengel et a.,US Patent No. 7,053,207;Imanishi et al.,US Patent No. 6,268,490;Imanishi et al. US Patent No. 6,770,748;Imanishi et al.,USRE44,779;Wengel et al.,US Patent No. 6,794,499;Wengel et al.,US Patent No. 6,670,461;Wengel et al.,US Patent No. 7,034,133;Wengel et al., U.S. Patent No. 8,080,644; Wengel et al., U.S. Patent No. 8,034,909; Wengel et al., U.S. Patent No. 8,153,365; Wengel et al., U.S. Patent No. 7,572,582; and Ramasamy et al., U.S. Patent No. 6,525,191; Torsten et al., WO2004 / 106356; Wengel et al., WO1999 / 014226; Seth et al., WO2007 / 134181; Seth et al., U.S. Patent No. 7,547,684; Seth et al., U.S. Patent No. 7,666,854; Seth et al., U.S. Patent No. 8,088,746; Seth et al., U.S. Patent No. 7,750,131; Seth et al., U.S. Patent No. 8,030,467; Seth et al., U.S. Patent No. 8,268,980; Seth et al.See, U.S. Patent No. 8,546,556; Seth et al., U.S. Patent No. 8,530,640; Migawa et al., U.S. Patent No. 9,012,421; Seth et al., U.S. Patent No. 8,501,805; and Allerson et al., U.S. Patent Publication No. US2008 / 0039618 and Migawa et al., U.S. Patent No. US2015 / 0191727.

[0076] As used herein, "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject may be human or non-human. In particularly preferred embodiments, the subject or patient is human. The subject or patient may also undergo other forms of treatment. In one embodiment, the patient has a neurological disorder due to a mutation in the C9ORF72 gene (e.g., the patient may be haploinsufficient for the C9ORF72 gene (e.g., reducing C9ORF72 protein activity by 50% or more), or the C9ORF72 gene may contain a GGGGCC repeat expansion (e.g., (GGGGCC) n The variable "n" may be at least 30.

[0077] "Therapeutically effective amount" or "effective dose" or "effective amount", as used interchangeably herein, means a dose of a drug effective for a period of time necessary to achieve a desired therapeutic result, unless otherwise defined. An effective dose may be determined by one of skill in the art and may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the drug to elicit a desired response in the individual. As used herein, the term may also refer to an amount effective to produce a desired in vivo effect in an animal, mammal, or human, such as reducing and / or inhibiting the function of a receptor. A therapeutically effective amount may be administered in one or more administrations (e.g., the agent may be administered therapeutically as a preventative treatment or at any stage of disease progression, before or after symptoms, etc.), applications, or doses, and is not intended to be limited to a particular formulation, combination, or route of administration. It is within the scope of this disclosure that a drug may be administered at various times during the course of treatment of a subject. The administration times and doses used will depend on multiple factors, such as the goal of the treatment (e.g., therapeutic or preventative), the condition of the subject, and can be readily determined by one of skill in the art.

[0078] As used herein, the term "treat" or "treating" a subject refers to administering a composition or agent described herein to a subject such that at least one symptom of a disease or disorder is cured, alleviated, mitigated, altered, treated, reduced, ameliorated, or improved. Treatment includes administering an amount effective to alleviate, alleviate, alter, treat, reduce, ameliorate, and / or improve one or more symptoms associated with a disease or disorder. The treatment may inhibit the worsening or exacerbation of symptoms associated with a disease or disorder.

[0079] The therapeutic methods described herein may include administering to a subject in need thereof a composition comprising an effective amount of one or more antisense oligonucleotides that treats a neurological disease by inhibiting or suppressing PIKFYVE expression. The one or more antisense oligonucleotides may reduce or inhibit neurodegeneration. The one or more antisense oligonucleotides may reduce neuronal hyperexcitability. Reducing PIKFYVE mRNA and PIKFYVE protein levels inhibits neurodegeneration by promoting toxic TDP-43 aggregates, DPR aggregates (e.g., in C9ORF72-ALS patients), and nuclear retention of TDP-43. Delivering an ASO targeting PIKFYVE mRNA as described herein reduces PIKFYVE protein levels.

[0080] The composition may inhibit kinase activity by inhibiting expression of the kinase. The composition of the invention may inhibit the activity or expression of PIKFYVE kinase. One or more antisense oligonucleotides may be combined with a small molecule therapeutic agent (e.g., apilimod and / or YM201636).

[0081] The present disclosure provides oligonucleotides (modified or unmodified) that can be used to regulate PIKFYVE expression. Table 1 shows the general sequence of bases for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of the present disclosure (5' to 3'): [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

[0082] In one embodiment, the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-500 in Table 1. In some embodiments, the modified oligonucleotides are at least 80%-100% identical (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%, or any numerical range or value between any of the foregoing values) to any of the sequences comprising or consisting of SEQ ID NOs: 1-500.

[0083] The sequences shown in Table 1 can be used to design antisense molecules for inhibition of PIKFYVE expression. For example, gapmer oligonucleotides can be designed using the sequences in Table 1 and can include a 5' wing of about 3-5 nucleotides, a 3' wing of about 3-5 nucleotides, and a gap region comprising 8-12 contiguous deoxyribonucleosides of any one of the sequences in Table 1. In one embodiment, oligonucleotides of the disclosure include gapmers having a gap segment of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-136 in Table 1 adjacent to the 5' and 3' wing segments, wherein the gap segment is located between the 5' and 3' wing segments, and each wing segment comprises a modified sugar. In one embodiment, the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length. In yet another embodiment, an oligonucleotide of the disclosure comprises a 5' wing segment comprising a modified sugar and having a nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-500, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-500, followed by a 3' wing segment comprising a modified sugar and having a nucleobase sequence of the last 3-5 nucleobases of the same sequence corresponding to SEQ ID NOs: 1-500. Table 2 shows MOE gapmers of the disclosure.

[0084] The 5' and / or 3' wings may comprise the following compounds: 2'-OMe, 2'-MOE, LNA, or DNA, used alone or in combination with each other. The backbone linkages of the 5' and / or 3' wings may be phosphorothioate or a mixture of phosphodiester and phosphorothioate. The linkages in the gap region may be phosphorothioate.

[0085] In some embodiments, the oligonucleotide is single stranded, hi some embodiments, the oligonucleotide comprises or is complexed with a moiety that neutralizes the charge on the oligonucleotide to facilitate uptake and transport across a cell membrane.

[0086] In one embodiment, each ASO in Table 1 has the following 5-10-5 motif: * 2MOE-2MOE-2MOE-2MOE-N * N * N * N * N * N * N * N * N * N * 2MOE-2MOE-2MOE * 2MOE * 2MOE, (i) 2MOE is a nucleobase having a 2'-OCHCH-OCH group (i.e., 2'-MOE), (ii) N is a nucleobase, and (iii) an asterisk ( * ) indicates a phosphorothioate bond, and (iv) a dash (-) indicates a phosphodiester bond. Table 2 below shows this motif in SEQ ID NOs: 1-33 (herein SEQ ID NOs: 501-533).

[0087] Table 2: Base sequence in PIKFYVE antisense oligonucleotides (ASO) (Gapmer design: 5'-5 2'-methoxyethyl ribose nucleotides-10 DNA nucleotides-5 2'-methoxyethyl ribose nucleotides-3', capital letters indicate 2'-methoxyethyl ribose nucleosides, lowercase letters indicate DNA nucleosides, asterisks ( * ) are phosphorothioate linkages, linkages without an asterisk are phosphodiester linkages) (note that the table below shows 2'MOE wings, but alternative wings including 2'-OMe or LNA (locked nucleic acid) are also envisaged). [Table 2]

[0088] The PIKFYVE kinase antisense or inhibitory nucleic acids of the present disclosure can inhibit expression associated with PIKFYVE and thus its activity. The PIKFYVE kinase antisense or inhibitory nucleic acids can include any combination of oligonucleotides set forth in Table 2 and sequences that are 98%-99% identical thereto.

[0089] The PIKFYVE ASOs described herein, such as SEQ ID NOs:501-533, inhibit PIKFYVE mRNA expression with minimal off-target binding.

[0090] Therapeutic methods may include any number of modes of administering the disclosed compositions. Modes of administration may include aqueous, lipid, oily, or other solutions, simulated cerebrospinal fluid solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, etc. Usually, the ASO of the present disclosure is administered directly to the CNS of the subject. Therefore, the formulation or composition is sterile and more preferably suitable for injection. The following formulations and methods are merely examples and are in no way limiting.

[0091] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The preparations may be provided in unit-dose or multi-dose sealed containers such as ampoules and vials, and may be stored for injection as liquids that only require the addition of sterile liquid excipients, such as water, immediately before use, or in freeze-dried (lyophilized) state.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.The preparations may be provided in pre-filled syringes.

[0092] The additional therapeutic agent(s) may be administered simultaneously or sequentially with the one or more antisense or inhibitory nucleic acids and compositions disclosed. Sequential administration includes administration before or after the one or more antisense or inhibitory nucleic acids or compositions disclosed. In some embodiments, the additional therapeutic agent may be administered in the same composition as the one or more antisense or inhibitory nucleic acids disclosed. In other embodiments, there may be a time interval between administration of the additional therapeutic agent and administration of the one or more antisense or inhibitory nucleic acids disclosed. In some embodiments, administration of the additional therapeutic agent together with the one or more antisense or inhibitory nucleic acids disclosed may allow the other therapeutic agent to be administered at a lower dose and / or at less frequent intervals. When used in combination with one or more other active ingredients, the one or more antisense or inhibitory nucleic acids of the present disclosure and the other active ingredients may each be used at a lower dose than when used alone. Thus, the pharmaceutical compositions of the present disclosure include pharmaceutical compositions that contain one or more other active ingredients in addition to one or more antisense or inhibitory nucleic acids of the present disclosure. The above combination includes the combination of one or more antisense or inhibitory nucleic acids of the present disclosure with not only one other active compound, but also two or more other active compounds.For example, the compound of the present disclosure can be combined with various drugs for treating neurological diseases.Antisense oligonucleotides can be covalently linked to another oligonucleotide, for example, a target other than PIKFYVE.Antisense oligonucleotides can be covalently linked to antibodies.

[0093] One or more of the disclosed antisense or inhibitory nucleic acids can be combined with anticholinergics, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, analgesics, and / or stimulants, including, but not limited to, the following: Additional types of therapies and treatments include, but are not limited to, digital communication devices, feeding tubes, mechanical ventilation, nutritional support, deep brain stimulation, occupational therapy, physical therapy, and / or speech therapy.

[0094] The disclosed composition(s) may be incorporated into a pharmaceutical composition suitable for administration to a subject (e.g., a patient, which may be human or non-human). The pharmaceutical composition may include a carrier (e.g., a pharma- ceutically acceptable carrier). Any suitable carrier may be used in connection with the present disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of the compositions of the present invention.

[0095] The pharmaceutical composition may comprise a therapeutically effective amount or a prophylactically effective amount of antisense oligonucleotide. The therapeutically effective amount of the composition may be determined by those skilled in the art and may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to induce a desired response in the individual. A therapeutically effective amount is also an amount in which the toxic or harmful effects of one or more antisense or inhibitory nucleic acids of the present disclosure are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, at a dosage and for a period of time necessary to achieve a desired prophylactic result. Usually, a prophylactic dose is used in a subject before or at an early stage of a disease, so that the prophylactically effective amount will be less than the therapeutically effective amount.

[0096] A pharmaceutical composition may include one or more pharma- ceutically acceptable carriers. As used herein, the term "pharma-ceutically acceptable carrier" refers to a non-toxic, inert, solid, semi-solid, or liquid filler, diluent, encapsulant, or any kind of formulation auxiliary. Some examples of substances that can function as pharma-ceutically acceptable carriers include sugars, such as but not limited to lactose, glucose, and sucrose; starches, such as but not limited to corn starch and potato starch; cellulose and its derivatives, such as but not limited to sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients, such as but not limited to powdered tragacanth, malt, gelatin, talc, cocoa butter, and suppository wax; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. Oils, including but not limited to, glycols such as propylene glycol, esters, such as but not limited to ethyl oleate, and ethyl laurate, buffers, including but not limited to agar, magnesium hydroxide, and aluminum hydroxide, alginic acid, pyrogen free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as release agents, coating agents, preservatives, and antioxidants may also be present in the composition, at the discretion of the formulator.

[0097] The route by which one or more of the disclosed antisense or inhibitory nucleic acids are administered and the form of the composition will determine the type of carrier used.

[0098] The pharmaceutical composition of the present disclosure can be administered in many ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, for example, intrathecal, intraventricular, or intraventricular administration. In one embodiment, the antisense or inhibitory nucleic acid is administered as an intravenous, intraperitoneal, or bolus injection, or administered directly to the target organ. In another embodiment, the antisense or inhibitory nucleic acid is administered as a bolus injection intrathecally or intraventricularly.

[0099] Carriers for systemic administration generally include at least one of a solvent, diluent, lubricant, binder, disintegrant, colorant, flavorant, sweetener, antioxidant, preservative, glidant, vehicle, suspending agent, wetting agent, surfactant, combinations thereof, etc. All carriers are optional in the composition.

[0100] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate, sodium carbonate, sugar alcohols such as glycerin, mannitol, and sorbitol.

[0101] Suitable lubricants include liquid lubricants such as silica, talc, stearic acid and its magnesium and calcium salts, calcium sulfate, and polyethylene glycol, as well as vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and theobroma oil. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0102] Suitable binders include polyvinylpyrrolidone, magnesium aluminum silicate, starches such as corn starch and potato starch, gelatin, tragacanth, and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in the systemic composition is typically about 5 to about 50%.

[0103] Suitable disintegrants include agar, alginic acid and its sodium salt, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of one or more disintegrants in the systemic composition is generally from about 0.1% to about 10%.

[0104] Suitable coloring agents include colorants such as the FD&C dyes. If used, the amount of coloring agent in the systemic or topical composition is typically about 0.005 to about 0.1%.

[0105] Suitable flavorings include menthol, peppermint, and fruit flavors. When used in systemic or topical compositions, the amount of flavoring(s) is typically about 0.1 to about 1.0%.

[0106] Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically from about 0.1 to about 5%.

[0107] Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.

[0108] Suitable lubricants include silicon dioxide. The amount of lubricant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0109] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oil, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent in a systemic or topical composition is typically about 0 to about 100%.

[0110] Suitable suspending agents include AVICEL RC-591 (FMC Corporation of Philadelphia, Pa.) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0111] Suitable surfactants include lecithin, polysorbate 80, and sodium lauryl sulfate, including TWEEN® from Atlas Powder Company, Wilmington, Delaware. Suitable surfactants include those disclosed in CTFA Cosmetic Ingredient Handbook, 1992, pp. 587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.

[0112] Compositions and formulations for parenteral, intrathecal, intraventricular, or intracerebroventricular administration may also include sterile aqueous solutions that may further contain other suitable additives, including but not limited to buffers, diluents, and penetration enhancers, carrier compounds, and other pharma- ceutical acceptable carriers or excipients. For example, an intrathecal cerebrospinal fluid (CSF) catheter can be used to deliver the antisense formulations of the present disclosure. The catheter can be inserted into the L3 or L4 vertebra. The distal end of the catheter is advanced intrathecally to approximately the LI vertebra. The antisense oligonucleotide is dissolved in saline, sterilized by filtration, and administered at 0.33 ml / min in a volume of 1.0 ml, followed by a 0.5 ml rinse of sterile water. The total infusion time is 4.5 minutes.

[0113] Compositions for parenteral administration usually contain 0.1% to 10% of active substance and 90% to 99.9% of carriers, including diluents and solvents.

[0114] The amount of carrier used with the disclosed compounds is sufficient to provide a practical amount of the composition for administration per unit dose of drug.Techniques and compositions for preparing dosage forms useful in the method of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0115] In vivo testing of the candidate antisense or inhibitory nucleic acid may be performed by means known to those skilled in the art. For example, one or more candidate antisense or inhibitory nucleic acids may be administered to a mammal, such as a mouse or rabbit. A dose of the candidate antisense or inhibitory nucleic acid may be administered to the mammal by any route deemed suitable. The animal may then be monitored using conventional methods and criteria for signs of reduced or improved motor neuron activity and / or expression or activity of the PIKFYVE gene or protein, respectively. If desired, the results obtained in the presence of the candidate antisense or inhibitory nucleic acid may be compared with results in a control animal not treated with the candidate antisense or inhibitory nucleic acid. Dosage testing may be performed in or in conjunction with the methods described herein to identify one or more antisense or inhibitory nucleic acids capable of treating a neurological disease and / or to perform any subsequent testing of the candidate antisense or inhibitory nucleic acid in vivo. A person skilled in the art may determine the appropriate dose of one or more antisense or inhibitory nucleic acids. The dose may be determined by monitoring the subject for signs of inhibition or improvement of the disease. The dose may be increased or decreased to obtain the desired frequency of treatment. The toxicity and efficacy of one or more antisense or inhibitory nucleic acids can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, determining the lethal dose for 50% of the population (LD50) and the therapeutically effective dose for 50% of the population (ED50). The dose ratio of LD50 / ED50 is the therapeutic index, which indicates the ratio of toxicity to therapeutic effect. Delivery systems can be designed to help prevent adverse side effects by delivering one or more antisense or inhibitory nucleic acids to specific targets, for example, specifically to neurons of the motor nervous system or central nervous system. The optimal dose of one or more antisense or inhibitory nucleic acids can be determined based on the results of clinical electrophysiology or electromyography, for example, to analyze peripheral nerve excitability.

[0116] Dosages for use in humans may be determined by evaluating data obtained from animal tests and cell culture assays. A dose that exhibits little or no toxicity and includes the ED50 is preferred. This dose may vary depending on the dosage form and route of administration. For any antisense or inhibitory nucleic acid used in the methods described herein, the dose may be estimated first in cell culture. The dose may be formulated in an animal model that includes the concentration of the test compound that achieves half-maximal inhibition of symptoms (LD50) as determined in cell culture. Such information obtained from cell culture and animal models may be used to more accurately determine effective doses in humans.

[0117] The present invention has multiple aspects, which are illustrated by the following non-limiting examples. EXAMPLES

[0118] Small molecule inhibitors of the PIKFYVE kinase and antisense oligonucleotides (ASOs) that suppress PIKFYVE expression can prevent degeneration of neurons in humans and mice harboring mutations in the C9ORF72 gene that cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

[0119] ASOs are an attractive treatment option for neurodegenerative diseases because they are easily delivered to the CNS and have relatively low peripheral exposure. These properties maximize target engagement in the CNS and minimize undesired target engagement or off-target effects in the periphery.

[0120] The present disclosure provides a novel antisense oligonucleotide (ASO) sequence targeting the PIKFYVE gene that can suppress PIKFYVE expression in human cells. PIKFYVE ASO can also restore the survival rate of motor neurons from sporadic ALS patients. In addition, PIKFYVE ASO can reduce the level of neurotoxic dipeptide repeat protein aggregates derived from C9ORF72 repeat sequence expansion in mice in vivo.

[0121] Example 1 To identify ASO sequences that suppress PIKFYVE expression in human cells, ASOs were designed and synthesized as MOE gapmers (see Table 2) containing sugar and linkage modifications that increase nuclease resistance and melting temperature while maintaining the ability to use as a substrate for RNase H. The ability of each ASO to suppress PIKFYVE RNA levels was tested by transfecting them into human embryonic kidney 293T cells with Lipofectamine2000 at a concentration of 100 nM and measuring PIKFYVE expression 7 days after transfection. As a control, NCASO was used. Relative PIKFYVE expression shown is the average of three technical replicates, and values ​​were calculated by normalizing to the GAPDH control. Taken together, these results show that several PIKFYVE ASOs (SEQ ID NOs: 1-33, corresponding to ASOs 1-33 in the figure) suppress PIKFYVE expression in human cells, as shown in Figure 1.

[0122] Example 2 The suppression of off-target genes, including CNTN5, was computationally predicted for various ASOs described herein and the results are shown in Table 3. [Table 3]

[0123] For ASO-520 (SEQ ID NO: 520), five (5) off-target gene candidates were predicted from sequence analysis (compared to 25 for Tofersen). Two of these genes have very low expression in the brain and are undetectable in induced neurons. To evaluate the actual off-target suppression of the remaining three genes (ZNF385D, ERC2, and AKAP6), the effect of ASO-520 treatment on the expression of these three genes and PIKFYVE was tested by qPCR in patient-derived neuronal lines. ASO-520 did not significantly affect their expression at a dose that reduced PIKFYVE by 50%.

[0124] Example 3 In the study, neonatal transgenic hPIKFYVE BAC mice were administered 25 μg of negative control ASO or test compound by intracerebroventricular (ICV) injection at P1 (postnatal day 1) and tissue samples were collected 14 days after treatment. As shown in Figure 2, the ASOs tested were potent PIKFYVE inhibitors. At doses of ASO-520 ranging from 0.0004 μg to 25 μg, a dose-dependent reduction in PIKFYVE mRNA levels was observed in mice.

[0125] Example 4 The efficacy of PIKFYVE inhibition was evaluated using a TDP-43 mouse model that develops neurodegeneration, motor disorders, and paralysis. Wils et al., “TDP-43 Transgenic Mice Develop Spastic Paralysis and Neuronal Inclusions Characteristics of ALS and Frontotemporal Lobar Degeneration.” PNAS 107(8):3858-63, 2010.

[0126] Mice were further genetically modified to delete one copy of PIKFYVE, which significantly restored motor function in TDP-43 mice, increased mean survival by 28%, and reduced the risk of death (hR: hazard ratio) by 73%. The deletion did not cause motor, cognitive, or health impairments in wild-type (WT) mice.

[0127] Intracerebroventricular injection of 25 μg of mPIKFYVE-targeting ASO (5 μg / μl concentration in the central nervous system) on postnatal day 1 significantly reduced PIKFYVE expression by approximately 50% compared to negative control (NC) ASO. This PIKFYVE ASO treatment significantly restored motor function and viability in TDP-43 mice to a level similar to that of gene deletion and did not change function in WT mice. A five-fold lower dose of 5 μg of PIKFYVE ASO also significantly restored motor function and viability to a level similar to that of gene deletion, indicating that ASO has at least a five-fold therapeutic window in this model.

[0128] Histological analysis showed that the number of pathological pTDP-43 aggregates elevated in TDP-43 mice was significantly reduced by mPIKFYVE ASO treatment and fully restored to the level of WT mice. The overall localization of TDP-43, which is pathologically localized in the cytoplasm in TDP-43 mice, is significantly relocalized to the nucleus upon treatment. In PIKFYVE ASO-treated mice, the number of motor neurons in the lateral motor column of the spinal cord anterior horn region was fully restored to the level of WT.

[0129] Example 5 ASO-520 (35 mg) was administered intrathecally to non-human primates every other week for 2 weeks (twice). As a result, as shown in Figure 3, PIKFYVE mRNA was reduced, PIKFYVE was inhibited by 80%, and no adverse events (including brain and spinal cord histopathology) were observed. (One outlier was excluded from the RNA-seq data.)

[0130] Example 6 The ability of motor neurons to survive in the presence of non-coding ASO or AS-520 (SEQ ID NO: 520) was measured. Figure 4A shows the survival rate of control motor neurons in the presence of non-coding ASO (NC ASO) or motor neurons from C9ALS patients in the presence of (i) NC ASO or (ii) AS-520. In the presence of ASO-520, more motor neurons from C9ALS patients survived than in the presence of NC ASO. Figure 4B shows the hazard ratio of control motor neurons in the presence of NC ASO or motor neurons from C9ALS patients in the presence of (i) NC ASO or (ii) AS-520 (SEQ ID NO: 520). The hazard ratio of motor neurons from C9ALS patients in the presence of AS-20 was significantly lower than the hazard ratio in the presence of NC ASO.

[0131] The ability of FTD patient-derived cortical neurons to survive in the presence of non-coding ASO or AS-520 (SEQ ID NO: 520) was measured. Figure 5A is a graph showing the survival probability of FTD patient-derived cortical neurons using MAPT V337V or V337M in the presence of NC ASO or AS-520 (SEQ ID NO: 520). AS-520 increased the survival probability of MAPT V337M cortical neurons compared to NC ASO. Figure 5B is a bar graph showing the hazard ratios of control-derived cortical neurons, C9orf72-related FTD (C9-FTD), sporadic FTD (sFTD), and microtubule-associated protein tau (MAPT)-related FTD (MAPT-FTD) patients treated with NC ASO or AS-520. AS-520 significantly reduced the hazard ratios in C9-FTD, sFTD, and MAPT-FTD cortical neurons.

[0132] The present disclosure provides ASOs that suppress PIKFYVE expression in human cells, and accompanying data suggest that these ASOs may be able to prevent neurodegeneration in ALS and FTD patients.

[0133] The foregoing description and drawings should be considered only as illustrative of the principles of the invention. The invention is not intended to be limited by the preferred embodiment, but may be embodied in various ways apparent to those skilled in the art. Many applications of the invention will readily occur to those skilled in the art. It is therefore not desired to limit the invention to the specific examples disclosed, or to the exact construction and operation shown and described. Rather, all suitable modifications and equivalents may be utilized within the scope of the invention. All references cited herein are incorporated by reference in their entirety.

Claims

1. A single-stranded antisense oligonucleotide that suppresses the expression of PIKFYVE, comprising a nucleobase sequence as set forth in SEQ ID NO: 20, wherein the antisense oligonucleotide is 20 nucleotides in length, said antisense oligonucleotide.

2. The antisense oligonucleotide according to claim 1, wherein at least one internucleoside linkage is a modified internucleoside linkage.

3. The antisense oligonucleotide according to claim 2, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage.

4. The antisense oligonucleotide according to claim 2, wherein at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.

5. The antisense oligonucleotide according to claim 1, wherein at least one nucleoside comprises a modified nucleobase.

6. The antisense oligonucleotide according to claim 5, wherein the modified nucleobase is 5-methylcytosine.

7. The antisense oligonucleotide according to claim 1, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.

8. The antisense oligonucleotide according to claim 7, wherein the modified sugar moiety comprises a 2'-O-methoxyethyl group.

9. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide is a gapmer.

10. The antisense oligonucleotide is a gap segment consisting of 10 to 12 linked deoxynucleosides, a 5' wing segment consisting of 4 to 5 linked nucleosides, a 3' wing segment consisting of 4 to 5 linked nucleosides, and the gap segment is located between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment comprise a modified sugar moiety, the antisense oligonucleotide according to claim 1.

11. The antisense oligonucleotide according to claim 10, wherein each nucleoside of each wing segment comprises a modified sugar moiety.

12. The antisense oligonucleotide according to claim 10, wherein the nucleoside constituting each wing segment contains at least two different modified sugar moieties.

13. The antisense oligonucleotide according to claim 10, wherein the nucleoside constituting each wing segment contains the same modified sugar moiety.

14. The antisense oligonucleotide according to claim 13, wherein the modified sugar moiety contains a 2'-O-methoxyethyl group.

15. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide suppresses the expression of PIKFYVE by at least 80%.

16. A composition comprising the antisense oligonucleotide according to claim 1 and a pharmaceutically acceptable carrier, diluent, and / or excipient.

17. A single-stranded antisense oligonucleotide having a length of 20 nucleotides that suppresses the expression of PIKFYVE, wherein the antisense oligonucleotide has a nucleobase sequence as set forth in SEQ ID NO: 20, and the first 3 to 5 nucleotides at the 5' end ("5' wing segment") contain a modified sugar, the last 3 to 5 nucleotides at the 3' end ("3' wing segment") contain a modified sugar, and the remaining nucleotides contain an unmodified gap segment.

18. The antisense oligonucleotide according to claim 17, wherein the modified sugar contains 2'-OMe, 2'-MOE, LNA, or any combination thereof.

19. The antisense oligonucleotide according to claim 17, wherein the backbone linkages of the 5' wing segment, the 3' wing segment, and the gap segment contain a mixture of phosphorothioate linkages and phosphodiester linkages.

20. The antisense oligonucleotide according to claim 17, wherein the antisense oligonucleotide contains a moiety that neutralizes the charge on the antisense oligonucleotide.

21. A composition comprising the antisense oligonucleotide according to claim 17 and a pharmaceutically acceptable carrier, diluent, and / or excipient.

22. A single-stranded antisense oligonucleotide of 20 nucleotides in length that suppresses the expression of PIKFYVE, wherein the antisense oligonucleotide comprises a nucleotide sequence as set forth in SEQ ID NO:

520.

23. A composition comprising the antisense oligonucleotide according to Claim 22, a pharmaceutically acceptable carrier, diluent, and / or excipient.

24. A pharmaceutical composition for use as a medicament for inhibiting or suppressing the expression of PIKFYVE in a subject in need thereof, the pharmaceutical composition comprising the antisense oligonucleotide according to any one of Claims 1 to 15, 17 to 20, and 22.

25. A pharmaceutical composition for use as a medicament in the treatment of a neurological or neurodegenerative disease in a subject in need thereof, the pharmaceutical composition comprising the antisense oligonucleotide according to any one of Claims 1 to 15, 17 to 20, and 22.

26. The pharmaceutical composition according to Claim 25, wherein the neurological or neurodegenerative disease is associated with neuronal hyperexcitability.

27. The pharmaceutical composition according to Claim 25, wherein the neurological or neurodegenerative disease comprises familial and sporadic amyotrophic lateral sclerosis (ALS), familial and sporadic frontotemporal dementia (FTD), progressive supranuclear palsy, Alzheimer's disease, chronic traumatic encephalopathy, Parkinson's disease, Charcot-Marie-Tooth disease types 2A and 4B, Huntington's disease, dementia, transmissible spongiform encephalopathy, spinal bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia, or Creutzfeldt-Jakob disease.

28. The pharmaceutical composition according to Claim 25, wherein the neurological or neurodegenerative disease is associated with haploinsufficiency related to the C9ORF72 gene.

Images