PIKFYVE antisense oligonucleotide

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 Jun. 22, 2021, which is incorporated herein by reference.

[0002] This disclosure relates to PIKFYVE antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods of treating, inhibiting, suppressing, and preventing neurological or neurodegenerative diseases thereby.

Background Art

[0003] Many neurodegenerative disorders in patients are difficult to treat effectively, 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 neurodegenerative diseases by administering a PIKFYVE inhibitor. Effective treatments for many neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are still needed.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

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

[0007] One embodiment is a single-stranded ASO that suppresses PIKFYVE expression, wherein the ASO has a nucleic acid sequence containing at least 12 or 15 consecutive nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs. 1 to 500. The nucleic acid sequence of the ASO may contain 30, 25, 24, 23, 22, 21, or 20 consecutive nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs. 1 to 500. The ASO may be any of the SEQ ID NOs. 1 to 500.

[0008] Another embodiment is an oligonucleotide comprising 12 to 30 linked nucleosides and having a nucleic acid sequence containing 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 nucleic acid bases from any of the nucleic acid sequences of SEQ ID NOs. 1 to 500. The oligonucleotide may contain 25, 24, 23, 22, 21, or 20 consecutive nucleic acid bases from any of the nucleic acid sequences of SEQ ID NOs. 1 to 500.

[0009] In certain embodiments, at least one nucleoside bond is a modified nucleoside bond, which may be a phosphorothioate nucleoside bond or a phosphodiester nucleoside bond. At least one of the nucleosides may be a modified nucleic acid base.

[0010] In other embodiments, at least one nucleoside of ASO may be a modified sugar moiety, which may be a bicyclic sugar moiety or may contain a 2'-O-methoxyethyl group. In certain embodiments, the bicyclic sugar moiety contains a 4'-CH(R)-O-2' bridge, where the R group is independently H, C 1-12 It is an alkyl group or a protecting group.

[0011] In yet another embodiment, the ASO is a gapmer (e.g., a MOE gapmer), and the gap segment may consist of 8 to 12 bonded deoxynucleosides, a 5'-wing segment consisting of 3 to 5 bonded nucleosides, and a 3'-wing segment consisting of 3 to 5 bonded nucleosides. In a particular embodiment, the gap segment may be positioned between the 5'-wing segment and the 3'-wing segment, and the nucleosides of each wing segment include 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 nucleic acid base sequence containing 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 nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs. 1 to 500.

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

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

[0015] A further embodiment is a method of treating a subject having a neurological or neurodegenerative disease by administering a therapeutically effective amount of PIKFYVE ASO or any of the pharmaceutical compositions 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), such as FTD with the V337M MAPT mutation.

[0016] Another embodiment is a method for treating a subject having PIKFYVE disease or disorder by administering a therapeutically effective amount of PIKFYVE ASO or the pharmaceutical composition described herein. [Brief explanation of the drawing]

[0017] A more complete understanding of the present invention and many of its associated advantages will be readily apparent, as they are better understood by referring to the following detailed description, which is considered in conjunction with the accompanying drawings.

[0018] [Figure 1] This shows PIKFYVE ASO screening in HeLa cells, measuring the relative mRNA expression levels of ASO 1-33 (SEQ ID NOs: 1-33) compared to the control (NCASO).

[0019] [Figure 2] This bar graph shows the inhibitory effects of various PIKFYVE ASOs in neonatal transgenic hPIKFYVE BAC mice.

[0020] [Figure 3]This bar graph shows the changes in PIKFYVE mRNA and PIKFYVE protein in non-human primates (NHPs) treated with and untreated with ASO-520 or artificial cerebrospinal fluid (aCSF).

[0021] [Figure 4A] This graph shows the survival rates of control motor neurons in the presence of non-coding ASOs (NC ASOs), or motor neurons from C9ALS patients in the presence of (i) NC ASOs or (ii) AS-20 (Sequence ID 20).

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

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

[0024] [Figure 5B] This bar graph shows the hazard ratios for 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. [Modes for carrying out the invention]

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this specification, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials described herein may also be used in carrying out or testing the present invention. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The terms used herein are intended to describe specific 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 their variations, when used herein, are intended to be open transitional phrases, terms, or words that do not preclude the possibility of further actions or structures.

[0028] Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" refer to multiple objects.

[0029] This disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements present herein, whether expressly described herein or otherwise.

[0030] As used herein, “2'-deoxynucleoside” means a nucleoside containing a 2'-H(H) furanosyl sugar moiety, as found in natural deoxyribonucleic acid (DNA) and nucleoside bases. In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleoside base and a furanosyl sugar moiety, or it may contain an RNA nucleoside base (uracil) and a furanosyl sugar moiety.

[0031] As used herein, “2'-substituted nucleoside” means a nucleoside containing a 2'-substituted sugar moiety. As used herein, with respect to the sugar moiety, “2'-substituted” means a sugar moiety containing at least one 2'-substituent other than H or OH.

[0032] As used herein, “antisense molecule” means an oligomeric nucleic acid or oligomeric double strand capable of achieving at least one antisense activity.

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

[0034] In the enumeration of numerical ranges as described herein, each numerical value having a similar degree of precision between them is explicitly intended. For example, in the range 6–9, the numerical values ​​7 and 8 are intended in addition to 6 and 9, and in the range 6.0–7.0, 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 explicitly intended.

[0035] As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety containing two rings, in which the second ring is formed via a bridge connecting two 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 contain a furanosyl moiety. As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside containing a bicyclic sugar moiety.

[0036] As used herein, “chiral-enriched population” means a group of molecules of the same molecular formula in which, given that a particular chiral center is stereorandom, the number or proportion of molecules in the population containing a particular stereochemical configuration at a particular chiral center is greater than the number or proportion of molecules in the population that are expected to contain the same particular stereochemical configuration at the same particular chiral center. A molecular chiral-enriched population having multiple chiral centers in each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound containing a modified oligonucleotide.

[0037] As used herein, “complementary” with respect to oligonucleotides means that at least 70% of the nucleic acid bases or one or more regions of the oligonucleotide and at least 70% of the nucleic acid bases or one or more regions of another nucleic acid can hydrogen bond with each other when the nucleic acid base sequences of the oligonucleotide and the other nucleic acid are aligned in opposite directions. Complementary nucleic acid bases mean nucleic acid bases that can form hydrogen bonds with each other. Examples of complementary nucleic acid base 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 do not need to have complementary nucleic acid bases at each nucleoside. Rather, some mismatches are acceptable. As used herein, “perfectly complementary” or “100% complementary” with respect to oligonucleotides means that the oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0038] As used herein, “gapmer” means a modified oligonucleotide containing an internal region with multiple nucleosides that assist in RNase H cleavage, located between external regions having one or more nucleosides, where these nucleosides containing the internal region are chemically distinct from the nucleosides or multiple nucleosides containing the external region. The internal region may be called a “gap,” and the external region may be called a “wing.” Unless otherwise specified, “gapmer” refers to a sugar motif. Unless otherwise specified, the sugar portion of the nucleoside in the gap of a gapmer is unmodified 2'-deoxyfuranosyl. Therefore, the term “MOE gapmer” refers to a gapmer having a gap between the sugar motifs of 2'-MOE nucleosides and a 2'-deoxynucleoside in both wings. Unless otherwise specified, a MOE gapmer may contain one or more modified nucleoside-to-modified bonds and / or modified nucleic acid bases, and such modifications do not necessarily have to follow a gapmer pattern of sugar modification. Table 2 below shows an example of a MOE gapmer.

[0039] In certain embodiments, the oligonucleotide comprises one or more modified sugars and / or unmodified sugar moieties arranged along the oligonucleotide or its region 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, i.e., "wings," and a central or internal region, i.e., a "gap." The three regions of the gapmer motif include a "5' wing," a "gap," and a "3' wing," which form a continuous sequence of nucleosides, where at least a portion of the sugar moieties of the nucleosides in each wing differs from at least a portion of the sugar moieties of the nucleosides in the gap. Specifically, at least the sugar moieties of the nucleosides in each wing closest to the gap (the 3'-side nucleosides of the 5'-wing and the 5'-side nucleosides of the 3'-wing) differ from the sugar moieties of the adjacent gap nucleosides, thus defining the boundary between the wing 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 different from that 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, the wings of the gapmer contain 1 to 5 nucleosides. In certain embodiments, each nucleoside in each wing of the gapmer is a modified nucleoside.

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

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

[0044] In certain embodiments, the modified oligonucleotide includes or consists of a region having a fully modified sugar motif. In such embodiments, each nucleoside in the fully modified region of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, each nucleoside in the entire modified oligonucleotide contains a modified sugar moiety. In certain embodiments, the modified oligonucleotide includes or consists of a region having a fully modified sugar motif, and each nucleoside within the fully modified region contains the same modified sugar moiety, which is referred to herein as a homogeneously modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a homogeneously modified oligonucleotide. In certain embodiments, each nucleoside in a homogeneously modified oligonucleotide contains the same 2' modification.

[0045] As used herein, “inhibit” means the ability to substantially antagonize, block, prevent, suppress, deter, delay, interfere with, alter, eliminate, halt, or reverse the progression or severity of activity of a particular pathogen (e.g., an infectious pathogen) or disease.

[0046] As used herein, “nucleoside bond” means a covalent bond between adjacent nucleosides within an oligonucleotide. As used herein, “modified nucleoside bond” means any nucleoside bond other than a phosphate diester nucleoside bond. “Phosphothioate bond” is a modified nucleoside bond in which one of the non-bridged oxygen atoms of a phosphodiester nucleoside bond is replaced with a sulfur atom.

[0047] In certain embodiments, the nucleosides of modified oligonucleotides may be linked together using any internucleoside bond. Two main classes of internucleoside bonds are defined by the presence or absence of a phosphorus atom. Representative internucleoside bonds containing phosphorus include, but are not limited to, phosphodiester bonds (also referred to as unmodified or native bonds), phosphotriesters, methylphosphonates or other alkylphosphonates, phosphoramidates, and phosphorothioates, and phosphorodithioates. Representative internucleoside bonds containing non-phosphoric acid 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)-). Compared to natural phosphate bonds, modified nucleoside interbonding can be used to alter (usually increase) the nuclease resistance of oligonucleotides. Methods for preparing phosphate-containing and non-phosphate-containing nucleoside interbonding are well known to those skilled in the art.

[0048] Representative nucleoside bonds having a chiral center include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides containing nucleoside bonds having a chiral center can be prepared as a group of modified oligonucleotides containing stereorandom nucleoside bonds, or as a group of modified oligonucleotides containing phosphorothioate bonds of a specific stereochemical configuration. In certain embodiments, the group of modified oligonucleotides contains phosphorothioate nucleoside bonds, in which case all of the phosphorothioate nucleoside bonds are stereorandom. Such modified oligonucleotides can be produced using synthetic methods that result in a random selection of the stereochemical structure of each phosphorothioate bond. Nevertheless, as is well understood by those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereochemistry. In certain embodiments, the group of modified oligonucleotides is enriched with respect to modified oligonucleotides containing one or more specific phosphorothioate nucleoside bonds of a specific, independently selected stereochemical configuration. In certain embodiments, a particular configuration of a particular phosphorothioate bond is present in at least 65% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate bond is present in at least 70% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate bond is present in at least 80% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate bond is present in at least 90% of the molecules in the population. In certain embodiments, a particular configuration of a particular phosphorothioate bond is present in at least 99% of the molecules in the population.Such a population of chiral-enriched modified oligonucleotides can be produced using synthetic methods known in the art, for example, the methods 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 with modified oligonucleotides having at least one indicated phosphorothioate in (Sp) configuration. In other embodiments, the population of modified oligonucleotides is enriched with modified oligonucleotides having at least one indicated phosphorothioate in (Rp) configuration.

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

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

[0051] Neurological disorders can involve abnormal endosomal transport. For example, endosomal pathways and endosomes are essential components for the recycling or disruption of membrane-bound proteins, the transport of Golgi-associated proteins, and the extracellular release of proteins from exosomes. These processes assist neurotransmission and, for example, promote the balance between the recycling and degradation of synaptic vesicles or neurotransmitter receptors.

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

[0053] Healthcare professionals may diagnose a subject as having a disorder associated with motor neuron degeneration by evaluating one or more symptoms of motor neuron degeneration. A thorough neurological examination may be performed after a physical examination to diagnose a neurological disorder. The neurological examination may assess motor and sensory skills, neurological function, hearing and speech, vision, coordination and balance, mental state, and changes in mood or behavior. Non-limiting symptoms of disorders associated with neurological disorders include weakness of the arms, legs, feet, or ankles; slurred speech; difficulty lifting the forefoot and toes; weakness or clumsiness of the hands; muscle paralysis; muscle rigidity; involuntary spasms or distressing movements (chorea); involuntary, persistent muscle contractures (dystonia); bradykinesia; loss of automatism; postural and balance disturbances; lack of flexibility; tingling in parts of the body; electric shock sensations when moving the head; and weakness of 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 or writing, misrepresentation of spatial relationships, disorientation, depression, anxiety, difficulty making decisions and judgments, loss of impulse control, difficulty planning and performing routine tasks, aggression, irritability, withdrawal, mood swings, dementia, changes in sleep patterns, wandering, and changes in appetite.

[0054] Tests may be performed to identify diseases and disorders that may present with symptoms similar to neurological disorders, measure muscle involvement, and assess neuronal degeneration. Non-exclusive examples of tests include electromyography (EMG), nerve conduction velocity testing, clinical examination of blood, urine, or other substances, 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-enhanced CT scan, electroencephalography, electrooculography, evoked responses, polysomnography, thermography, and ultrasound. Healthcare professionals may also evaluate the patient's family history of diseases associated with motor neuron degeneration and make a diagnosis based in part on a family history of neurological disorders. Healthcare professionals may also diagnose disorders associated with the neurological disorder in question after the onset of one or more symptoms.

[0055] Neurodegenerative diseases result in the progressive destruction of neurons and affect neuronal signaling. For example, neurodegeneration can include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Huntington's disease, Friedreich's ataxia, Lewy body dementia, Parkinson's disease, spinal muscular atrophy, primary ALS, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0056] Diseases associated with motor neuron degeneration can be conditions that result in the progressive destruction of motor neurons, disrupting neuronal signaling to muscles and causing 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 are then transmitted to the muscles, resulting in voluntary muscle activity. Destruction of upper and lower motor neurons affects activities such as breathing, speaking, swallowing, and walking, and these functions can be lost over time. Examples of motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

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

[0058] Neuronal hyperexcitability may be associated with spinal cord injury, stroke, traumatic brain injury, hearing loss (due to excessive noise exposure or ototoxicity), epilepsy, painful neuropathy, attention deficit hyperactivity disorder, autism, central pain syndrome, 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 alcohol withdrawal, particularly rapid benzodiazepine withdrawal, and Huntington's disease. Another common condition that causes excessive glutamate concentration around neurons is hypoglycemia. Blood glucose is the primary way of removing glutamate 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 the two atoms of the sugar in order to form a second ring.

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

[0061] In certain embodiments, the modified oligonucleotide comprises one or more nucleosides containing unmodified nucleic acid bases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides containing modified nucleic acid bases. In certain embodiments, the modified oligonucleotide comprises one or more nucleic acid bases that do not contain nucleic acid bases, referred to as debased nucleosides.

[0062] In certain embodiments, the modified nucleic acid base 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 nucleic acid bases 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-azouracil, 6-azocytosine, 6-azocymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and 8-az. and other 8-substituted purines, 5-halos, particularly 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-methyl4-N-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases are selected. Further modified nucleic acid bases include tricyclic pyrimidines, such as 1,3-diazaphenoxadin-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxadin-2-one (G-clamp). Modified nucleic acid bases may also include bases in which a purine or pyrimidine base is substituted with another heterocyclic base, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further examples of nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, 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 Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166, and Chapters 6 and 15 of 442-443.

[0063] As used herein, “nucleoside” means a compound containing a nucleic acid base and a sugar moiety. The nucleic acid base and sugar moiety are, independently, either unmodified or modified. As used herein, “modified nucleoside” means a nucleoside containing a modified nucleic acid base and / or a modified sugar moiety. Modified nucleosides include debasic nucleosides that lack a nucleic acid base. “Linked nucleosides” are nucleosides linked in a continuous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0064] As used herein, “oligomer compound” means an oligonucleotide and, optionally, one or more additional features such as a conjugated group or terminal group. An oligomer compound may or may not pair with a second oligomer compound complementary to a first oligomer compound. A “single-stranded oligomer compound” is an oligomer compound that does not pair. The term “oligomer double-stranded” means a double-stranded structure formed by two oligomer compounds having complementary nucleic acid base sequences. Each oligomer compound in an oligomer double-stranded structure may be called a “duplicated oligomer compound.”

[0065] As used herein, “oligonucleotide” means a single-stranded linked nucleoside linked via nucleoside-to-nucleoside bonds, where each nucleoside and nucleoside-to-nucleoside bond may be modified or unmodified. The nucleoside-to-nucleoside bond may be any bond described herein. Unless otherwise indicated, an oligonucleotide consists of 8 to 50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide in which at least one nucleoside or nucleoside-to-nucleoside bond is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not contain any nucleoside modifications or nucleoside-to-nucleoside modifications.

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

[0067] As used herein, “PIKFYVE disease or disorder” includes lysosomal degradation disorders and PIKFYVE-mediated disorders. For example, PIKFYVE disease or disorder includes, but is not limited to, amyloid disorders (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis, and chronic hemodialysis-associated amyloidosis), 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, Behçet's syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease, etc.) or a CNS disease (such as spongiform encephalopathy (Creutzfeldt-Jakob disease, Kuru disease, mad cow disease)). The compositions and methods of the present disclosure can be used for the treatment of individuals with lysosomal storage disorders, comprising administering a therapeutically effective amount of the PIKfyve ASO or pharmaceutical composition described herein to a subject requiring treatment. In some embodiments, the ASOs and compositions of the present disclosure alter the biosynthesis, function, or dynamics of the endosomal or lysosomal system in a manner that reduces or inhibits the activity of PIKfyve and decreases the amount of material abnormally stored in lysosomes in lysosomal storage disorders. In some embodiments, ASOs and compositions alter the biosynthesis, function, or dynamics of the endoplasmic reticulum or Golgi apparatus by targeting, reducing, or inhibiting the activity of PIKfyve and thus reducing the amount of substance abnormally stored in lysosomes in lysosomal storage disorders. In other embodiments, the disorder is a neurological disorder.

[0068] As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. A superscript prime symbol (') is used to indicate the numbering of sugars in a nucleoside or nucleotide (nucleic acid base positions are numbered without a prime). The prime symbol is not used when describing sugars only. As used herein, “unmodified sugar moiety” means a 2-OH(H) furanosyl moiety found in RNA ("unmodified RNA sugar moiety") or a 2-H(H) moiety found in DNA ("unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen atom at positions 1, 3, and 4, one oxygen atom at position 3, and two hydrogen atoms at position 5. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or sugar substitute. As used herein, a modified furanosyl sugar moiety means a furanosyl sugar containing a non-hydrogen substituent in place of at least one hydrogen atom 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 comprising a furanosyl ring having one or more substituents, none of which bridge two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridged substituents may be at any position on the furanosyl ring, including, but not limited to, substituents at position 2, 4, and / or 5. In certain embodiments, one or more non-bridged substituents of the non-bicyclic modified sugar moiety are branched. Examples of 2-substituents suitable for the non-bicyclic modified sugar moiety 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'-substituents are halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, OC 1-10 Alkoxy, OC 1-10 Substituted alkoxy, OC 1-10 Alkyl, OC 1-10 Substitutive alkyl, S-alkyl, N(R m)-Alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkyleneyl-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 ), where each R m and R n is independently H, an amino protecting group, or substituted or unsubstituted C 1-10 alkyl, and the 2-substituents are independently one or more substituents selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl and may be further substituted. Examples of suitable 4'-substituents for the non-bicyclically modified sugar moiety include, but are not limited to, alkoxy (e.g., methoxy), and alkyl. Examples of suitable 5-substituents for the non-bicyclically modified sugar moiety include, but are not limited to, 5-methyl (R or S), 5-vinyl, and 5-methoxy. In certain embodiments, the non-bicyclically modified sugar moiety includes a plurality of non-bridging sugar substituents, such as a 2-F-5-methyl sugar moiety.

[0070] In certain embodiments, the 2'-substituted non-bicyclically modified nucleoside includes a sugar moiety having a non-linear 2'-substituent selected from F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH_{2}CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)-N(R m )(R n )), where each R m and R n is independently H, an amino protecting group, or substituted or unsubstituted C1-10 It is alkyl.

[0071] In certain embodiments, the 2'-substituted nucleoside non-bicyclic modified nucleoside comprises a sugar moiety containing a non-linear 2'-substituted 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, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety containing a non-crosslinked 2'-substituent selected from F, OCH3, and OCH2CH2OCH3.

[0073] Certain modified sugar moieties include substituents that bridge 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 a 4-furanose ring atom and a 2-furanose ring atom. Examples of such 4-2 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 "restricted 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 Examples include )-ON(R)-2, 4-CH2-ON(R)-2, and 4-CH2-N(R)-O-2, but are not limited to these, and in the formula each R, R a , and R b These are independently H, a protecting group, or C 1-12It is alkyl.

[0074] In certain embodiments, such 4 to 2 crosslinks 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 )- independently contains 1 to 4 linking groups selected from, where x is 0, 1, or 2, and n is 1, 2, 3, or 4, and each R a and R b These are independently H, protecting group, hydroxyl, and C. 1-12 Alkyl, substituted C 1-12 Alkyl, C 1-12 Alkenyl, substituted C 2-12 Alkenil, C 2-12 Alkinyl, Substitute C 2-12 Alkinyl, C 5-20 Aryl substitution C 5-20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C 5-7 Alicyclic radicals, substituted C5-7 alicyclic radicals, halogens, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where each J1 and J2 independently consists of H and C 1-12 Alkyl, substituted C 1-12 Alkyl, C 2-12 Alkenyl, substituted C 2-12 Alkenil, C 2-12 Alkinyl, Substitute C 2-12 Alkinyl, C 5-20 Aryl substitution C 5-20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-12 Aminoalkyl, substituted C 1-12 It is an aminoalkyl group 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 al., U.S. Patent No. 7,053,207; Imanishi et al., U.S. Patent No. 6,268,490; Imanishi et al., U.S. Patent No. 6,770,748; Imanishi et al., USRE44,779; Wengel et al., U.S. Patent No. 6,794,499; Wengel et al., U.S. Patent No. 6,670,461; Wengel et al., U.S. Patent No. 7,034,133; Wengel et 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 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., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice), non-human primates (e.g., monkeys such as crab-eating macaques or rhesus macaques, chimpanzees, etc.), and humans. In some embodiments, the subject may be human or non-human. In a particularly preferred embodiment, the subject or patient is human. The subject or patient may receive 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 has haploinsufficiency with respect to the C9ORF72 gene (e.g., a reduction of 50% or more in C9ORF72 protein activity) or the C9ORF72 gene has GGGGCC repeat sequence elongation (e.g., C9ORF72 contains (GGGGCC) n It may include hexanenucleotide extensions. The variable "n" can be at least 30.

[0077] As used herein, “therapeutic dose,” “effective dose,” or “effective amount” means, unless otherwise defined, the dose of a drug that is effective over the period necessary to achieve the desired therapeutic outcome. The effective dose may be determined by those skilled in the art and may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the drug’s ability to induce the desired response in the individual. As used herein, this term may also refer to the amount effective to produce a desired in vivo effect in an animal, mammal, or human, such as reducing and / or inhibiting receptor function. The therapeutic dose may be administered in one or more doses (e.g., as a prophylactic measure, or therapeutically at any stage of disease progression, before or after symptoms appear), topically, or in doses, and is not intended to be limited to any particular formulation, combination, or route of administration. It is within the scope of this disclosure that the drug may be administered at various points in time during the course of treatment of the subject. The timing and dose used may depend on several factors, such as the goal of treatment (e.g., treatment or prevention) and the condition of the subject, and can be readily determined by those skilled in the art.

[0078] As used herein, the terms “to treat” or “to treat” a subject mean administering a composition or agent described herein to a subject such that at least one symptom of a disease or disorder is cured, reduced, alleviated, altered, treated, reduced, remitted, or improved. Treatment includes administering an amount effective to reduce, alleviate, alter, treat, reduce, remit, and / or improve one or more symptoms associated with the disease or disorder. This treatment may suppress the worsening or exacerbation of symptoms associated with the disease or disorder.

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

[0080] This composition can inhibit kinase activity by inhibiting kinase expression. The composition of the present invention can inhibit the activity or expression of PIKFYVE kinase. One or more antisense oligonucleotides can be combined with small molecule therapeutics (e.g., appilimod and / or YM201636).

[0081] This disclosure provides oligonucleotides (modified or unmodified) that can be used to modulate PIKFYVE expression. Table 1 shows the typical base sequences (5' to 3' direction) for the PIKFYVE antisense oligonucleotides or inhibitory nucleic acids of this disclosure: [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 a modified oligonucleotide having a nucleic acid sequence comprising 12 to 30 linked nucleosides and containing 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 nucleotide bases of any of the nucleic acid sequences of SEQ ID NOs. 1 to 500 in Table 1. In some embodiments, the modified oligonucleotide is at least 80% to 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 aforementioned values) to any of the sequences comprising or comprising SEQ ID NOs. 1 to 500.

[0083] Antisense molecules for inhibiting PIKFYVE expression can be designed using the sequences shown in Table 1. For example, a gapmer oligonucleotide can be designed using the sequences in Table 1 and may include a 5' wing of about 3 to 5 nucleotides, a 3' wing of about 3 to 5 nucleotides, and a gap region containing 8 to 12 consecutive deoxyribonucleosides of any one of the sequences in Table 1. In one embodiment, the oligonucleotide of the present disclosure includes a gapmer 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 consecutive nucleotide bases of any of the nucleic acid base sequences of SEQ ID NOs. 1 to 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 contains a modified sugar. In one embodiment, the gap segment is a nucleoside of length 8 to 10, and each wing segment is a modified nucleoside of length 3 to 5. In yet another embodiment, the oligonucleotide of the present disclosure comprises a 5' wing segment containing a modified sugar and having the nucleic acid sequence of the first 3 to 5 nucleic acid bases of any of SEQ ID NOs. 1 to 500, followed by a gap of the next 8 to 12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs. 1 to 500, followed by a 3' wing segment containing a modified sugar and having the nucleic acid sequence of the last 3 to 5 nucleic acid bases of the same sequence corresponding to SEQ ID NOs. 1 to 500. Table 2 shows the MOE gapmers of the present disclosure.

[0084] The 5' and / or 3' wings may contain the following compounds: 2'-OMe, 2'-MOE, LNA, or DNA, either alone or in combination with each other. The skeletal bonds of the 5' and / or 3' wings may be phosphorothioates or mixtures of phosphodiesters and phosphorothioates. Bonds in the gap regions may be phosphorothioates.

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

[0086] In one embodiment, each ASO in Table 1 is the following 5-10-5 motif: 2MOE * 2MOE-2MOE-2MOE-2MOE-N * N * N * N * N * N * N * N * N * N * 2MOE-2MOE-2MOE * 2MOE * It has 2MOE, (i) 2MOE is a nucleic acid base having a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) N is a nucleic acid base, and (iii) asterisk ( * (iv) indicates a phosphorothioate bond, and (iv) a dash (-) indicates a phosphodiester bond. Table 2 below shows this motif represented by Sequence IDs 1-33 (Sequence IDs 501-533 in this specification).

[0087] Table 2: Base sequences of PIKFYVE antisense oligonucleotides (ASOs) (Gapmer design: 5'-5 2'-methoxyethylribose nucleotides-10 DNA nucleotides-5 2'-methoxyethylribose nucleotides-3', uppercase letters are 2'-methoxyethylribose nucleosides, lowercase letters are DNA nucleosides, asterisk( * () indicates a phosphorothioate bond, while bonds without an asterisk are phosphodiester bonds. (Note that although the table below shows the 2'MOE wing, alternative wings including 2'-OMe or LNA (locked nucleic acid) are also conceivable.) [Table 2]

[0088] The PIKFYVE kinase antisense or inhibitory nucleic acids of this disclosure can inhibit PIKFYVE-related expression and, consequently, its activity. The PIKFYVE kinase antisense or inhibitory nucleic acids may include any combination of oligonucleotides listed in Table 2 and sequences that are 98% to 99% identical thereto.

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

[0090] The therapeutic method may include any number of modes of administration of the disclosed composition. Modes of administration may include aqueous, lipid, oily, or other solutions, simulated cerebrospinal fluid solutions, oil-in-water emulsions, liposomes, aqueous, or oily suspensions. Typically, the ASO of this disclosure is administered directly to the target CNS. Therefore, the formulation or composition is sterile and more preferably suitable for injection. The following formulations and methods are merely examples and are not limiting.

[0091] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that are isotonic with the blood of the recipient to whom the formulation is intended, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be provided in sealed containers of one or more doses, such as ampoules and vials, and may be stored immediately before use for injection as a liquid requiring only the addition of a sterile liquid excipient, such as water, or in a freeze-dried state. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulations may be provided in pre-filled syringes.

[0092] Further therapeutic agents may be administered simultaneously with or sequentially to one or more disclosed antisense or inhibitory nucleic acids and compositions. Sequential administration includes administration before or after one or more disclosed antisense or inhibitory nucleic acids or compositions. In some embodiments, additional therapeutic agents may be administered in the same composition as one or more disclosed antisense or inhibitory nucleic acids. In other embodiments, there may be a time interval between the administration of the additional therapeutic agent and the administration of one or more disclosed antisense or inhibitory nucleic acids. In some embodiments, administering additional therapeutic agents together with one or more disclosed antisense or inhibitory nucleic acids may allow for lower doses and / or less frequent administration intervals for other therapeutic agents. When used in combination with one or more other active ingredients, one or more antisense or inhibitory nucleic acids and other active ingredients of this disclosure may be used in lower doses than when used individually. Accordingly, the pharmaceutical compositions of this disclosure include pharmaceutical compositions containing one or more other active ingredients in addition to one or more antisense or inhibitory nucleic acids of this disclosure. The above combinations include not only combinations of one or more antisense or inhibitory nucleic acids of this disclosure with one other active compound, but also combinations of two or more other active compounds. For example, the compounds of this disclosure may be combined with various drugs for treating neurological disorders. Antisense oligonucleotides may be covalently bonded to other oligonucleotides, for example, targets other than PIKFYVE. Antisense oligonucleotides may be covalently bonded to antibodies.

[0093] One or more of the disclosed antisense or inhibitory nucleic acids may be combined with, but are not limited to, anticholinergics, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, analgesics, and / or stimulants. 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 compositions(s) may be incorporated into pharmaceutical compositions suitable for administration to a subject (e.g., a patient, which may be human or non-human). The pharmaceutical compositions may include a carrier (e.g., a pharmaceutically acceptable carrier). Any suitable carrier can be used in relation to this disclosure, and such carriers are well known in the art. The choice of carrier is determined in part by the specific use of the composition (e.g., administration to animals) and the specific method used to administer the composition. Thus, a wide variety of suitable formulations of the compositions of the present invention exist.

[0095] A pharmaceutical composition may contain a therapeutically effective or prophylactically effective amount of antisense oligonucleotide. The therapeutically effective amount of a composition may be determined by those skilled in the art and may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the composition's ability to induce a desired response in the individual. The therapeutically effective amount is also the amount in which the toxic or adverse effects of one or more antisense or inhibitory nucleic acids of this disclosure outweigh the therapeutically beneficial effects. "Prophylactically effective amount" means the effective amount in dose and duration required to achieve the desired prophylactic outcome. Typically, a prophylacticly effective amount will be less than a therapeutically effective amount, as prophylactic doses are used in subjects before or at an early stage of the disease.

[0096] A pharmaceutical composition may contain one or more pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert, solid, semi-solid, or liquid filler, diluent, encapsulant, or any kind of compounding aid. Some examples of substances that can function as pharmaceutically acceptable carriers are 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; and peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. Oils, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate, but not limited to these, buffering agents such as agar, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffer, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Release agents, coating agents, preservatives and antioxidants may also be present in the composition at the discretion of the compounder.

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

[0098] The pharmaceutical compositions of this disclosure can be administered in many ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, for example, intrathecal, intraventricular, or lateral ventricle administration. In one embodiment, the antisense or inhibitory nucleic acid is administered as an intravenous, intraperitoneal, or bolus injection, or directly to the target organ. In another embodiment, the antisense or inhibitory nucleic acid is administered as a bolus injection into the spinal cavity or ventricle.

[0099] Generally, carriers for systemic administration include at least one of the following: solvents, diluents, lubricants, binders, disintegrants, colorants, fragrances, sweeteners, antioxidants, preservatives, lubricants, solvents, suspending agents, wetting agents, surfactants, and combinations thereof. All carriers are optional in the composition.

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

[0101] Suitable lubricants include liquid lubricants such as silica, talc, stearic acid, and their 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 the whole-body composition or topical composition is usually 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 whole composition is usually about 5 to about 50%.

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

[0104] Suitable colorants include FD&C dyes. When used, the amount of colorant in the whole-body or topical composition is typically about 0.005 to 0.1%.

[0105] Suitable fragrances include menthol, peppermint, and fruit flavors. When used in a systemic or topical composition, the amount of fragrance(s) is typically about 0.1% to 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 about 0.1 to 5%.

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

[0108] A suitable lubricant is silicon dioxide. The amount of lubricant(s) in the whole-body composition or topical composition is usually about 1 to 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 the whole-body or topical composition is usually about 0 to about 100%.

[0110] Suitable suspensions include AVICEL RC-591 (manufactured by FMC Corporation, Philadelphia, PA) and sodium alginate. The amount of suspension(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, as well as TWEEN® manufactured by Atlas Powder Company in 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 whole-body composition or topical composition is typically about 0.1% to about 5%.

[0112] Compositions and formulations for parenteral, intrathecal, intraventricular, or lateral intraventricular administration may also include sterile aqueous solutions further containing buffers, diluents, and other suitable additives, including but not limited to osmotic enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients. For example, antisense formulations of this disclosure can be delivered using an intrathecal cerebrospinal fluid (CSF) catheter. 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 a rate of 0.33 ml / min in a 1.0 ml volume, followed by rinsing with 0.5 ml of sterile water. The total infusion time is 4.5 minutes.

[0113] Compositions for parenteral administration typically contain 0.1% to 10% of the active substance and 90% to 99.9% of a carrier, which includes a diluent and a solvent.

[0114] The amount of carrier used with the disclosed compound is sufficient to provide a practical amount of composition for administration per unit dose of the drug. Techniques and compositions for producing useful dosage forms 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 candidate antisense or inhibitory nucleic acids may be carried out by means known to those skilled in the art. For example, one or more candidate antisense or inhibitory nucleic acids may be administered to mammals such as mice or rabbits. A certain dose of a candidate antisense or inhibitory nucleic acid may be administered to a mammal by any route deemed suitable. The animals can then be monitored using conventional methods and criteria for signs of decreased or increased motor neuron activity and / or expression or activity of the PIKFYVE gene or protein. If necessary, results obtained in the presence of the candidate antisense or inhibitory nucleic acid can be compared with results obtained in control animals not treated with the candidate antisense or inhibitory nucleic acid. Dosage studies may be carried out in or in connection with the methods described herein to identify one or more antisense or inhibitory nucleic acids that can treat neurological diseases, and / or to carry out any subsequent in vivo testing of candidate antisense or inhibitory nucleic acids. Those skilled in the art can determine an appropriate dose of one or more antisense or inhibitory nucleic acids. The dose may be determined by monitoring the subjects for signs of suppression or improvement of the disease. The dose may be increased or decreased to obtain the desired treatment frequency. The toxicity and efficacy of one or more antisense or inhibitory nucleic acids may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the lethal dose (LD50) and the therapeutically effective dose (ED50) for 50% of the population. The LD50 / ED50 dose ratio is a therapeutic index indicating the ratio of toxicity to therapeutic effect. Delivery systems may be designed to help prevent adverse side effects by delivering one or more antisense or inhibitory nucleic acids to specific targets, for example, by specifically delivering them to neurons in the motor or central nervous system. The optimal dose of one or more antisense or inhibitory nucleic acids may be determined, for example, based on the results of clinical electrophysiology or electromyography to analyze peripheral nerve excitability.

[0116] The dose for human use may be determined by evaluating data obtained from animal studies and cell culture assays. A dose containing an ED50 that exhibits little to no toxicity is preferred. This dose may be varied 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 first be estimated in a cell culture. Formulation may be carried out at a dose determined in a cell culture in an animal model containing the concentration (LD50) of the test compound that achieves up to half of the suppression of symptoms. Such information obtained from cell cultures and animal models may be used to more accurately determine the effective dose in humans.

[0117] The present invention has multiple embodiments, as shown by the following non-limiting embodiments. [Examples]

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

[0119] ASOs are an attractive treatment option for neurodegenerative diseases due to their easy delivery to the central nervous system and relatively low peripheral exposure. These characteristics maximize target engagement in the central nervous system and minimize undesirable target engagement or off-target effects in the periphery.

[0120] This 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 viability of motor neurons derived from sporadic ALS patients. Furthermore, PIKFYVE ASO can reduce the levels of neurotoxic dipeptide repeat protein aggregates derived from C9ORF72 repeat sequence elongation 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 containing sugar and binding modifications that increase nuclease resistance and melting temperature while maintaining their ability to be used as substrates for RNase H (see Table 2). The ability of each ASO to suppress PIKFYVE RNA levels was tested by transfecting human embryonic kidney 293T cells with Lipofectamine 2000 at a concentration of 100 nM and measuring PIKFYVE expression 7 days after transfection. NCASO was used as a control. The relative PIKFYVE expressions shown are the average of three technical replicates, and the values ​​were calculated by normalizing to the GAPDH control. In summary, these results indicate that several PIKFYVE ASOs (sequences 1-33, corresponding to ASO1-33 in the figure) suppress PIKFYVE expression in human cells, as shown in Figure 1.

[0122] Example 2 For the various ASOs described herein, the suppression of off-target genes such as CNTN5 was predicted by computer. The results are shown in Table 3. [Table 3]

[0123] For ASO-520 (SEQ ID NO: 520), sequence analysis (compared to 25 for Tofersen) predicted five off-target gene candidates. Two of these genes had very low expression in the brain and were undetectable in induced neurons. To evaluate the actual off-target suppression for 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-induced neuron 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 either a negative control ASO or the test compound via intraventricular (ICV) injection at P1 (postnatal day 1), and tissue samples were collected 14 days after treatment. As shown in Figure 2, the tested ASO was a potent PIKFYVE inhibitor. A dose-dependent reduction in PIKFYVE mRNA levels was observed in mice at ASO-520 doses ranging from 0.0004 μg to 25 μg.

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

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

[0127] Intracerebroventricular injection of 25 μg of mPIKFYVE-targeting ASO (at a concentration of 5 μg / μl 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 survival in TDP-43 mice to levels similar to those achieved by gene deletion, without altering function in wild-type mice. A five-fold lower dose of 5 μg of PIKFYVE ASO also significantly restored motor function and survival to levels similar to those achieved by gene deletion. This indicates that ASO has at least a five-fold therapeutic concentration range in this model.

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

[0129] Example 5 35 mg of ASO-520 was administered intrathecally to non-human primates every other week for two weeks (two doses). As shown in Figure 3, PIKFYVE mRNA was reduced, PIKFYVE was suppressed 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 rates of control motor neurons in the presence of non-coding ASO (NC ASO), or motor neurons derived from C9ALS patients in the presence of (i) NC ASO or (ii) AS-520. More motor neurons derived from C9ALS patients survived in the presence of ASO-520 than in the presence of NC ASO. Figure 4B shows the hazard ratios for control motor neurons in the presence of NC ASO, or motor neurons derived from C9ALS patients in the presence of (i) NC ASO or (ii) AS-520 (SEQ ID NO: 520). The hazard ratio for motor neurons derived 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-associated FTD (C9-FTD), sporadic FTD (sFTD), and microtubule-associated protein tau (MAPT)-associated 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] This disclosure provides ASOs that suppress PIKFYVE expression in human cells. Accompanying data suggest that these ASOs may be able to prevent neurodegeneration in patients with ALS and FTD.

[0133] The foregoing description and drawings should be considered only as illustrative examples of the principles of the present invention. The present invention is not intended to be limited by preferred embodiments and may be implemented in various ways that will be apparent to those skilled in the art. Many applications of the present invention will readily come to mind for those skilled in the art. Therefore, it is not desirable to limit the present invention to the specific embodiments disclosed or to the exact structures and operations illustrated and described. Rather, any suitable modifications and equivalents may be utilized within the scope of the present invention. All references cited herein are incorporated by reference in their entirety. In certain embodiments, for example, the following are provided: (Item 1) A single-stranded antisense oligonucleotide that suppresses the expression of PIKFYVE, wherein the antisense oligonucleotide has a nucleic acid base sequence that includes at least 12 or 15 consecutive nucleic acid bases of any of the nucleic acid base sequences of SEQ ID NOs. 1 to 500. (Item 2) The antisense oligonucleotide according to item 1, wherein the antisense oligonucleotide has one nucleic acid base sequence from sequence number 1 to 500. (Item 3) The antisense oligonucleotide according to item 1 or 2, wherein the antisense oligonucleotide has 18 to 20 linked nucleosides. (Item 4) An antisense oligonucleotide as described in any of the preceding items, wherein at least one nucleoside bond is a modified nucleoside bond. (Item 5) The antisense oligonucleotide described in item 4, wherein at least one modified nucleoside bond is a phosphorothioate nucleoside bond. (Item 6) The antisense oligonucleotide described in item 4, wherein each modified nucleoside bond is a phosphorothioate nucleoside bond. (Item 7) An antisense oligonucleotide as described in any of the preceding items, wherein at least one nucleoside bond is a phosphodiester nucleoside bond. (Item 8) The antisense oligonucleotide described in item 7, wherein at least one nucleoside bond is a phosphorothioate bond and at least one nucleoside bond is a phosphodiester bond. (Item 9) An antisense oligonucleotide as described in any of the preceding items, wherein at least one nucleoside contains a modified nucleic acid base. (Item 10) The antisense oligonucleotide described in item 9, wherein the modified nucleic acid base is 5-methylcytosine. (Item 11) The antisense oligonucleotide according to any of the preceding items, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. (Item 12) The antisense oligonucleotide according to item 11, wherein the modified sugar moiety comprises a 2'-O-methoxyethyl group. (Item 13) The antisense oligonucleotide according to any of the preceding items, wherein the antisense oligonucleotide is a gapmer. (Item 14) The antisense oligonucleotide is A gap segment consisting of 8 to 12 linked deoxynucleosides. A 5' wing segment consisting of 3 to 5 linked nucleosides. It contains a 3' wing segment consisting of 3 to 5 linked nucleosides, The antisense oligonucleotide according to item 13, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and each wing segment contains a nucleoside-modified sugar moiety. (Item 15) Each nucleoside in each wing segment contains a modified sugar moiety, as described in item 14, an antisense oligonucleotide. (Item 16) The antisense oligonucleotide according to item 14, wherein each nucleoside constituting a wing segment comprises at least two different modified sugar moieties. (Item 17) The antisense oligonucleotide described in item 14, wherein each nucleoside constituting the wing segment contains the same modified sugar moiety. (Item 18) The antisense oligonucleotide according to item 15, wherein the modified sugar moiety comprises a 2'-O-methoxyethyl group. (Item 19) The antisense oligonucleotide described above is an antisense oligonucleotide as described in any of the preceding items, comprising 15 to 50 nucleosides. (Item 20) The antisense oligonucleotide according to any of the preceding items, wherein the antisense oligonucleotide has a nucleic acid base sequence comprising at least 12 or 15 consecutive nucleic acid bases of any of the nucleic acid base sequences of Sequence ID No. 501 to 533. (Item 21) The antisense oligonucleotide described above is the antisense oligonucleotide described in any of the preceding items, having the sequence of sequence numbers 501 to 533. (Item 22) The antisense oligonucleotide described above is the antisense oligonucleotide described in any of the preceding items, having the sequence of Sequence ID No. 520. (Item 23) A pharmaceutical composition comprising an antisense oligonucleotide as described in any one of the preceding items, and a pharmaceutically acceptable carrier, diluent, and / or excipient. (Item 24) The pharmaceutical composition according to item 23, wherein the pharmaceutical composition is formulated for parenteral administration. (Item 25) The pharmaceutical composition according to item 23, wherein the pharmaceutical composition is formulated for intraventricular injection. (Item 26) A method for treating a subject having a neurological or neurodegenerative disease requiring treatment, comprising administering a therapeutically effective amount of an antisense oligonucleotide described in any one of items 1 to 22 or a pharmaceutical composition described in any one of items 23 to 25. (Item 27) The method described in item 26, wherein the neurological disorder is associated with neuronal hyperexcitability. (Item 28) The method described in item 26, wherein the neurological disorder is associated with abnormal endosomal transport. (Item 29) The method described in item 26, wherein the neurological disorder is associated with abnormal lysosome transport. (Item 30) The method according to item 26, wherein the neurological disease is selected from the group consisting of 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, infectious spongiform encephalopathy, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia, and Creutzfeldt-Jakob disease. (Item 31) The method according to item 26, wherein the neurological disease is familial amyotrophic lateral sclerosis. (Item 32) The method according to item 26, wherein the neurological disorder is sporadic amyotrophic lateral sclerosis. (Item 33) The method according to item 26, wherein the neurological disorder is familial frontotemporal dementia. (Item 34) The method according to item 26, wherein the neurological disorder is sporadic frontotemporal dementia. (Item 35) The method according to item 26, wherein the neurological disorder is frontotemporal dementia with TDP-43 pathology. (Item 36) The method according to item 26, wherein the neurological disorder is frontotemporal dementia with tau pathology. (Item 37) The method according to any one of items 26 to 36, wherein the subject is haploinsufficient with respect to the C9ORF72 gene. (Item 38) The method according to any one of items 26 to 36, wherein the subject has GGGGCC repeat sequence extension in C90RF72. (Item 39) The aforementioned subject is (GGGGCC) in C90RF72 n The method according to any one of items 26 to 36, having a hexanucleotide elongation, wherein n is at least 30. (Item 40) The method described in any one of items 26 to 36, wherein the subject has C9orf72-associated frontotemporal dementia. (Item 41) The method according to any one of items 26 to 36, wherein the subject has microtubule-associated protein tau (MAPT)-associated frontotemporal dementia. (Item 42) The method described in item 41, wherein the patient has the V337M MAPT mutation. (Item 43) A method for inhibiting or suppressing the expression of PIKFYVE in a patient with a neurological or neurodegenerative disease, comprising administering an effective amount of an antisense oligonucleotide described in any one of items 1 to 22, or a pharmaceutical composition described in any one of items 23 to 25. (Item 44) Oligonucleotides comprising 12 to 30 linked nucleosides and having a nucleic acid base sequence containing 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 nucleic acid bases from any of the nucleic acid base sequences of SEQ ID NOs. 1 to 533.

Claims

1. A single-stranded antisense oligonucleotide that suppresses the expression of PIKFYVE, comprising the nucleic acid base sequence described in Sequence ID No. 20, wherein the antisense oligonucleotide has a length of 20 nucleotides.

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

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

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

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

6. The antisense oligonucleotide according to claim 5, wherein the modified nucleic acid base 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, It contains a 3' wing segment consisting of 4 to 5 linked nucleosides, The antisense oligonucleotide according to claim 1, wherein the gap segment is located between the 5' wing segment and the 3' wing segment, and each wing segment comprises a sugar moiety modified with a nucleoside.

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

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

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

14. The antisense oligonucleotide according to claim 13, wherein the modified sugar moiety comprises 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 for suppressing the expression of PIKFYVE, wherein the antisense oligonucleotide has the nucleic acid base sequence described in Sequence ID No. 20, 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 comprises 2'-OMe, 2'-MOE, LNA, or any combination thereof.

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

20. The antisense oligonucleotide according to claim 17, wherein the antisense oligonucleotide includes a portion 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 having a length of 20 nucleotides for suppressing the expression of PIKFYVE, wherein the antisense oligonucleotide comprises the nucleotide sequence described in Sequence ID No.

520.

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

24. A pharmaceutical composition for use as an agent for inhibiting or suppressing the expression of PIKFYVE in a subject requiring inhibition or suppression of PIKFYVE expression, comprising an antisense oligonucleotide according to any one of claims 1 to 15, 17 to 20, and 22.

25. A pharmaceutical composition for use as a drug in the treatment of a neurological or neurodegenerative disease in a subject requiring treatment for a neurological or neurodegenerative disease, comprising an 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 disease or neurodegenerative disease is associated with neuronal hyperexcitability.

27. The pharmaceutical composition according to claim 25, wherein the neurological disease or neurodegenerative disease includes 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, infectious spongiform encephalopathy, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia, or Creutzfeldt-Jakob disease.

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

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