SYF2 antisense oligonucleotides

Abstract

The present invention relates to SYF2 antisense oligonucleotides (ASOs), pharmaceutical compositions containing them and methods of treating, inhibiting, suppressing, and preventing neurological diseases with them.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 482,514, filed January 31, 2023, and U.S. Provisional Application No. 63 / 513,063, filed July 11, 2023, each of which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present invention relates to SYF2 antisense oligonucleotides (ASOs), pharmaceutical compositions containing the same, and methods for treating, inhibiting, suppressing and preventing neurological diseases or neurodegenerative diseases using the same. Background of the Invention

[0004] Many neurodegenerative disorders in patients are difficult to treat effectively, particularly when the pathology of a particular patient's neurodegenerative disorder is not fully understood.

[0005] International Publication No. WO 2021 / 150840 discloses a method for treating neurodegenerative diseases by administering a SYF2 inhibitor.

[0006] There remains a need for effective treatments for many neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Summary of the Invention

[0007] The present invention relates to SYF2 antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and their use in treating neurodegenerative disorders.

[0008] One embodiment is a single-chain ASO that suppresses the expression of SYF2, wherein the ASO has a nucleobase sequence comprising at least 12 or 15 consecutive nucleobases of any nucleobase sequence of SEQ ID NO: 1-1052 (such as 1-266, 533-573 and 615-833). The nucleobase sequence of the ASO may comprise up to 30, 25, 24, 23, 22, 21, 20, 18, 17, 16 or 15 consecutive nucleobases of any nucleobase sequence of SEQ ID NO: 1-1052 (such as 1-266, 533-573 and 615-833). The ASO may also be any one of SEQ ID NO: 1-1052 (such as 1-266, 533-573 and 615-833).

[0009] Another embodiment is a single-chain ASO comprising any one of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833. Yet another embodiment is a single-chain ASO comprising any one of the nucleobase sequences of SEQ ID NOs: 1-50, 533-573, and 615-833. Yet another embodiment is a single-chain ASO comprising any one of the nucleobase sequences of SEQ ID NOs: 267-316, 574-614, and 834-1052.

[0010] Another embodiment is a single chain ASO comprising the sequence of any one of SEQ ID NOs: 1- 1052. Yet another embodiment is a single chain ASO of any one of SEQ ID NOs: 1-1052.

[0011] Yet another embodiment is an oligonucleotide comprising or consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833. The oligonucleotide may comprise up to 25, 24, 23, 22, 21, 20, 18, 17, 16, or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833.

[0012] Yet another embodiment is an oligonucleotide comprising any one of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833. In one embodiment, the oligonucleotide has 12 to 30 linked nucleosides.

[0013] In certain embodiments, at least one internucleoside linkage is a modified internucleoside linkage, and the modified internucleoside linkage can be a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. At least one nucleoside can also be a modified nucleobase.

[0014] In other embodiments, at least one nucleoside of the ASO may be a modified sugar moiety, wherein the modified sugar moiety may be a bicyclic sugar moiety, or the modified sugar moiety may comprise 2'-O-methoxyethyl, 2'-F, or 2'-O-hexadecyl. In certain aspects, the bicyclic sugar moiety comprises a 4'-CH(R)-O-2' bridge, wherein the R groups are independently H, C, or D. 1-12In one embodiment, the bicyclic sugar moiety comprises a 4-(CH2)2-O-2 bridge ("ENA" or ethylene-bridged nucleic acid).

[0015] In other embodiments, the ASO is a gapmer (e.g., a MOE gapmer), wherein the gap segment can consist of 8 to 12 linked deoxynucleosides, the 5' wing segment consists of 3 to 5 linked nucleosides, and the 3' wing segment consists of 3 to 5 linked nucleosides. In certain aspects, the gap segment can be positioned between the 5' wing segment and the 3' wing segment, wherein the nucleosides of each wing segment comprise a modified sugar moiety (e.g., a sugar moiety having a 2'-O-methoxyethyl group).

[0016] In other embodiments, the oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833.

[0017] Another embodiment is a pharmaceutical composition comprising the SYF2 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, such as intracerebroventricular injection or intrathecal administration.

[0018] Yet another embodiment is a method of treating a subject suffering from a neurological disease or a neurodegenerative disease by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein.

[0019] Yet another embodiment is a method of treating a subject suffering from a SYF2 disease or disorder by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 2B) after transfection with negative control (NC) ASO or one of ASO-1 to ASO-50 (25 nM) from Table 2B, the expression level of human SYF2 (normalized to GAPDH). ASO-20, ASO-21 and ASO-22 significantly reduced human SYF2 expression. Figure 1-6 In the table, **** refers to p < 0.0001, *** refers to p < 0.001, ** refers to p < 0.01, and * refers to p < 0.05.

[0021] Figure 2 2B) after transfection with NC ASO or one of ASO-51 to ASO-74 (25 nM) from Table 2B. ASO-53, ASO-58, and ASO-60 significantly reduced human SYF2 expression.

[0022] Figure 3 2B) after transfection with NC ASO or one of ASO-75 to ASO-100 (25 nM) from Table 2B. ASO-83, ASO-84, ASO-90, ASO-92, ASO-96, and ASO-97 significantly reduced human SYF2 expression.

[0023] Figure 4 is a bar graph showing the expression level of human SYF2 (normalized to GAPDH) in HeLa cells after transfection with NC ASO or one of ASO-101 to ASO-121 (25 nM) from Table 2B. ASO-120 significantly reduced human SYF2 expression.

[0024] Figure 5 Bar graph showing the expression levels of SYF2 (normalized to HPRT) in iPSC-derived cortical neurons (Ngn2-induced neurons, Ngn2-iNs) after treatment with NC ASO or ASO-19, ASO-24, ASO-25, ASO-32, ASO-35, ASO-42, ASO-43, ASO-63, ASO-69, ASO-70, ASO-80, ASO-86, and ASO-120 via gymnosis.

[0025] Figure 6 Bar graph showing the expression levels of human SYF2 (normalized to HPRT) in Ngn2-iN treated with 10 μM NC ASO or one of ASO-122 to ASO-219. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of any conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting. The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0027] definition

[0028] As used herein, the terms "comprises," "including," "having," "has," "can," "contains," "may," and variations thereof are intended to represent open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0029] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0030] The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0031] As used herein, "2'-deoxynucleoside" means a nucleoside comprising a 2'-H(H) furanosyl sugar moiety as found in naturally occurring deoxyribonucleic acids (DNA) and nucleobases. In certain embodiments, the 2'-deoxynucleoside may comprise a modified nucleobase and a furanosyl sugar moiety, or may comprise an RNA nucleobase (uracil) and a furanosyl sugar moiety.

[0032] As used herein, "2'-substituted nucleosides" means nucleosides comprising a 2'-substituted sugar moiety. As used herein, "2'-substituted" with respect to a sugar moiety means a sugar moiety comprising at least one 2'-substituent other than H or OH.

[0033] As used herein, "antisense molecule" means an oligomeric nucleic acid or oligomeric duplex capable of exerting at least one antisense activity.

[0034] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with the measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the expression "about 2 to about 4" also discloses the range "2 to 4." The term "about" can refer to plus or minus 10% of the specified number. For example, "about 10%" can mean a range of 9% to 11%, and "about 1" can mean 0.9-1.1. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" can also mean 0.5 to 1.4.

[0035] For the recitation of numerical ranges herein, each number therebetween is expressly contemplated with equal precision. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0036] As used herein, "bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein 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 comprise a furanosyl moiety. As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

[0037] As used herein, "chirally enriched population" means a plurality of molecules having the same molecular formula, wherein the number or percentage of molecules containing a particular stereochemical configuration at a particular chiral center within the population is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. A population of chirally enriched molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are compounds comprising modified oligonucleotides.

[0038] As used herein, "complementary" with respect to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof can form hydrogen bonds with each other when the nucleobase sequences of the oligonucleotide and the other nucleic acid are arranged in opposite directions. Complementary nucleobases mean nucleobases that can form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T); adenine (A) and uracil (U); cytosine (C) and guanine (G); and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids do not need to have nucleobase complementarity at each nucleoside. On the contrary, some mismatches are tolerated. As used herein, "completely complementary" or "100% complementary" with respect to an oligonucleotide means that an oligonucleotide is complementary to another oligonucleotide or nucleic acid at each nucleoside of an oligonucleotide.

[0039] As used herein, " gapmer " refers to a modified oligonucleotide comprising an internal region with a plurality of nucleosides supporting RNase H cleavage, wherein the internal region is between an external region with one or more nucleosides, wherein the nucleosides comprising the internal region are chemically different from the one or more nucleosides comprising the external region. The internal region may be referred to as a "gap," and the external region may be referred to as a "wing." Unless otherwise indicated, a "gapmer" refers to a sugar motif. Unless otherwise indicated, the sugar moiety of the nucleosides in the gap of a gapmer is an unmodified 2'-deoxyfuranosyl group. Therefore, the term "MOE gapmer" refers to a gapmer having a sugar motif of a 2'-MOE nucleoside in two wings and a gap of a 2'-deoxynucleoside. Unless otherwise indicated, a MOE gapmer may include one or more modified internucleoside linkages and / or modified core bases, and such modifications may not necessarily follow a sugar-modified gapmer pattern. Tables 2A and 2B below provide exemplary MOE-gapmers.

[0040] As used herein, "inhibit" refers to the ability to substantially antagonize, inhibit, prevent, curb, restrain, slow, destroy, alter, eliminate, stop, or reverse the progression or severity of the activity of a particular agent (e.g., an infectious agent) or disease.

[0041] As used herein, the term "internucleoside linkage" is a covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, "modified internucleoside linkage" means any internucleoside linkage other than a phosphodiester internucleoside linkage. A "phosphorothioate linkage" is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of the phosphodiester internucleoside linkage is replaced by a sulfur atom.

[0042] As used herein, "MOE" refers to methoxyethyl. "2'-MOE" refers to the -OCH2CH2OCH3 group at the 2' position of the furanosyl ring.

[0043] A "neurological disease" is any disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, a neurological disease can be a neurodegenerative disease.

[0044] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that includes a modification, eg, a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

[0045] As used herein, "nucleobase" means an unmodified nucleobase or a modified nucleobase. As used herein, an "unmodified nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U) or guanine (G). As used herein, a "modified nucleobase" is a group of atoms that can pair with at least one unmodified nucleobase or modified nucleobase, except unmodified A, T, C, U or G. "5-methylcytosine" or "mC" is a modified nucleobase. Universal base is a modified nucleobase that can pair with any one of five unmodified nucleobases. As used herein, a "nucleobase sequence" means the order of consecutive nucleobases in a nucleic acid or oligonucleotide that is independent of any sugar or internucleoside linkage modification.

[0046] As used herein, "nucleoside" refers to a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each independently unmodified or modified. As used herein, "modified nucleoside" means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. "Linked nucleosides" are nucleosides linked in a continuous sequence (i.e., without additional nucleosides between the linked nucleosides).

[0047] As used herein, "oligomeric compound" means an oligonucleotide and optionally one or more additional features, such as conjugate groups or terminal groups. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound, or may be unpaired. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligoduplex" means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligoduplex may be referred to as a "double-stranded oligomeric compound."

[0048] As used herein, "oligonucleotide" means a chain of linked nucleosides connected by internucleoside linkages, wherein each nucleoside and internucleoside linkage can be modified or unmodified. The internucleoside linkage can be any linkage described herein. Unless otherwise indicated, an oligonucleotide consists of 8-50 linked nucleosides. As used herein, "modified oligonucleotide" means an oligonucleotide in which at least one nucleoside or internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" means an oligonucleotide that does not contain any nucleoside modifications or internucleoside modifications.

[0049] "SYF2", also referred to in the art as "SYF2 pre-mRNA splicing factor", encodes a nuclear protein that interacts with cyclin D-type binding protein 1, which is considered to be a cell cycle regulator during the G1 / S transition. SYF2 has been shown to reduce TDP-43 aggregation and mislocalization and cryptic exon inclusion, while also rescuing C9ORF72 and ensuring survival of sporadic ALS neurons. TDP-43 has been shown to reduce the incorporation of cryptic exons that lead to non-productive RNA transcripts. Therefore, the loss or reduction of nuclear TDP-43 in motor neurons will result in reduced mRNA levels of many genes, including STMN2, which transcribes microtubule-binding proteins. The loss of STMN2 has been shown to impair neurite growth in motor neurons.

[0050] As used herein, "SYF2 disease or disorder" includes diseases and disorders of lysosomal degradation mediated by SYF2. For example, SYF2 diseases or disorders include, but are not limited to, amyloid diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis, and chronic hemodialysis-associated amyloid), multiple sclerosis, and MPS disorders (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). In some embodiments, the disease is an autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, adult Still's disease, Behcet's syndrome, familial Mediterranean fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman disease) or a CNS disorder (e.g., spongiform encephalopathy (Creutzfeld-Jakob, Kuru, Mad Cow)). The compositions and methods of the present disclosure can be used to treat an individual suffering from a lysosomal storage disease, comprising administering to a subject in need of treatment a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein. In some embodiments, the ASOs and compositions of the present disclosure reduce or inhibit the activity of SYF2 and alter the biogenesis, function, or dynamics of the endosomal or lysosomal system in a manner that reduces the abundance of abnormally stored substances in lysosomes in lysosomal storage diseases. In some embodiments, ASOs and compositions target, decrease or inhibit the activity of SYF2, thereby altering the biogenesis, function or dynamics of the endoplasmic reticulum or Golgi apparatus in a manner that reduces the abundance of abnormally stored materials in lysosomes in lysosomal storage diseases. In other embodiments, the disease is a neurological disorder.

[0051] As used herein, "sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. A superscript prime (') is used to describe the numbering of sugars in a nucleoside or nucleotide (nucleobase positions are numbered without a prime). When describing only sugars, a prime is not used. As used herein, "unmodified sugar moiety" means a 2-OH (H) furanosyl moiety as found in RNA ("unmodified RNA sugar moiety"), or a 2-H (H) moiety as found in DNA ("unmodified DNA sugar moiety"). The unmodified sugar moiety has one hydrogen at positions 1, 3, and 4, one oxygen at position 3, and two hydrogens at position 5. As used herein, "modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety or sugar surrogate. As used herein, a modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent replacing at least one hydrogen of the unmodified sugar moiety. In certain embodiments, the modified furanosyl sugar moiety is a 2-substituted sugar moiety.Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.

[0052] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including but not limited to mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees, etc.), and humans). In some embodiments, the subject can be human or non-human. In preferred embodiments, the subject or patient is human. The subject or patient may be receiving other forms of treatment.

[0053] Unless otherwise defined, "therapeutically effective amount" or "effective dose" or "effective amount" as used interchangeably herein means a dosage of a drug that is effective to achieve the desired therapeutic result within the necessary time period. An effective dose can be determined by those skilled in the art and can vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the drug to elicit the desired response in the individual. This term as used herein can also refer to an amount that is effective to produce the desired in vivo effect (such as reducing and / or inhibiting the function of a receptor) in an animal, mammal, or human. A therapeutically effective amount can be administered in one or more administrations (e.g., an agent can be administered as a preventive treatment or therapeutically at any stage of disease progression, before or after symptoms, etc.), applications, or administrations, and is not intended to be limited to a specific formulation, combination, or route of administration. Within the scope of this disclosure, the drug can be administered at different times during the course of treatment of the subject. The time of administration and the dosage used will depend on a variety of factors, such as the therapeutic goal (e.g., treatment versus prevention), the condition of the subject, etc., and can be readily determined by those skilled in the art.

[0054] As used herein, the term "treat" or "treating" a subject refers to administering a composition or agent described herein to a subject such that at least one symptom of a disease or condition is cured, alleviated, mitigated, altered, remedied, reduced, improved, or ameliorated. Treatment includes administering an amount effective to alleviate, mitigate, alter, remedy, reduce, ameliorate, and / or improve one or more symptoms associated with a disease or condition. Treatment can inhibit the worsening or worsening of symptoms associated with a disease or condition.

[0055] antisense oligonucleotides

[0056] In certain embodiments, the oligonucleotide comprises one or more types of modified sugars and / or unmodified sugar moieties arranged in a defined pattern or sugar motif along the oligonucleotide or a region thereof. In some cases, such sugar motifs include, but are not limited to, any sugar modifications discussed herein.

[0057] In certain embodiments, the modified oligonucleotide comprises a district with a gapmer motif or consists of the district, and the gapmer motif is limited by two external districts or " wings " and a central district or an internal district or " gap ". The three districts of the gapmer motif include " 5 '-wing ", " gap " and " 3 '-wing ", which form a continuous sequence of nucleosides, wherein at least some sugar moieties of the nucleosides of each wing are different from at least some sugar moieties of the nucleosides of the gap. Specifically, the sugar moieties of at least the closest gap to the nucleosides of each wing (the most 3 ' nucleosides of the 5 '-wing and the most 5 ' nucleosides of the 3 '-wing) are different from the sugar moieties of the adjacent gap nucleosides, thereby defining the boundary between the wing and the gap (that is, the wing / gap junction). In certain embodiments, the sugar moieties in the gap are identical to each other. In certain embodiments, the gap includes one or more nucleosides having a sugar moiety that is different from the sugar moiety of the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of two wings are identical to each other (symmetrical gapmers). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

[0058] In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides.In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0059] In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides (eg, 10 nucleosides). In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxynucleoside.

[0060] In certain embodiments, the gapmer is a deoxygapmer. In embodiments, the nucleoside on the gap side of each wing / gap junction is an unmodified 2'-deoxynucleoside, and the nucleoside on the wing side 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.

[0061] In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif. In such embodiments, each nucleoside in the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif, wherein each nucleoside in the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of the uniformly modified oligonucleotide comprises the same 2'-modification.

[0062] In certain embodiments, the nucleosides of the modified oligonucleotide can be linked together using any internucleoside linkage. The two major categories of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates, phosphotriesters, methylphosphonates or other alkylphosphonates, phosphoramidates, phosphorothioates, and phosphorodithioates containing phosphodiester bonds (also known as unmodified or naturally occurring linkages). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thiocarbamates (-OC(=O)(NH)-S-); siloxanes (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared to naturally occurring phosphate linkages, modified internucleoside linkages can be used to alter, and generally increase, the nuclease resistance of oligonucleotides. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0063] Representative internucleoside linkages with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages with chiral centers can be prepared as modified oligonucleotide populations comprising stereorandom internucleoside linkages, or modified oligonucleotide populations comprising phosphorothioate linkages of specific stereochemical configurations. In certain embodiments, modified oligonucleotide populations comprise phosphorothioate internucleoside linkages, wherein all phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be produced using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, as is well known to those skilled in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a definite stereoconfiguration. In certain embodiments, modified oligonucleotide populations are enriched in modified oligonucleotides comprising one or more specific phosphorothioate internucleoside linkages in specific, independently selected stereochemical configurations. In certain embodiments, the specific configuration of a specific phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the specific configuration of specific thiophosphate linkage is present in at least 70% of the molecule in the colony. In certain embodiments, the specific configuration of specific thiophosphate linkage is present in at least 80% of the molecule in the colony. In certain embodiments, the specific configuration of specific thiophosphate linkage is present in at least 90% of the molecule in the colony. In certain embodiments, the specific configuration of specific thiophosphate linkage is present in at least 99% of the molecule in the colony. Such chiral enriched modified oligonucleotide populations can be produced using synthetic methods known in the art, such as those described in Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); Locked Nucleic Acid Aptamers Chapter 10, Nucleic Acid and Peptide Aptamers: Methods and Protocols v535, 2009, Barciszewski et al., Gunter Mayer and ed.; and WO 2017 / 015555. In certain embodiments, the modified oligonucleotide population is enriched for modified oligonucleotides having at least one (Sp) configuration of a specified phosphorothioate. In another embodiment, the modified oligonucleotide population is enriched for modified oligonucleotides having at least one (Rp) configuration of a specified phosphorothioate.

[0064] In certain embodiments, the modified oligonucleotide comprises one or more nucleosides containing unmodified nucleobases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides containing modified nucleobases. In certain embodiments, the modified oligonucleotide comprises one or more nucleosides that do not comprise nucleobases, referred to as abasic nucleosides.

[0065] In certain embodiments, the modified nucleobase is selected from the group consisting of a 5-substituted pyrimidine, a 6-azapyrimidine, an alkyl or alkynyl substituted pyrimidine, an alkyl substituted purine, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from the group consisting of: 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-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, 8-aza and other 8-substituted Purines, 5-halo (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-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, hybrid bases, size-extended bases and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenthiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp). Modified nucleobases may also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone. Additional nucleobases 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 those disclosed in Chapters 6 and 15, Antisense Drug Technology , Crooke ST ed., CRC Press, 2008, 163-166 and 442-443.

[0066] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety comprising a furanosyl ring with one or more substituents, wherein no substituent bridges the two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents may be located at any position of the furanosyl group, including but not limited to substituents at positions 2, 4, and / or 5. In certain embodiments, one or more non-bridging substituents of the non-bicyclic modified sugar moiety are branched. Examples of 2-substituents suitable for non-bicyclic modified sugar moieties include but are not limited to: 2-F, 2-OCH3 ("OMe" or "O-methyl") and 2-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2-substituents are selected from: halogen, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O 1-10 Alkoxy, OC 1-10 Substituted alkoxy, O-C1 -10 Alkyl, OC 1-10 Substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylene-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 are independently H, an amino protecting group, or a substituted or unsubstituted C 1-10Alkyl, and the 2-substituent may be further substituted by one or more substituents independently selected from the following: hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4'-substituents suitable for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy) and alkyl. Examples of 5-substituents suitable for non-bicyclic modified sugar moieties include, but are not limited to, 5-methyl (R or S), 5-vinyl and 5-methoxy. In certain embodiments, the non-bicyclic modified sugar moiety comprises more than one non-bridging sugar substituent, such as 2-F-5-methyl sugar moiety and the like.

[0067] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, -O-(C H2) n CH3 (wherein n is 13-17, and in a preferred embodiment, 15) and N-substituted acetamide (OCH2C(=O)-N(R m )(R n )), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C 1-10 In one embodiment, the 2'-substituent is -O-hexadecyl (C16).

[0068] In certain embodiments, the 2'-substituted nucleoside is a non-bicyclic modified nucleoside comprising a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, OCF3, OCH3, OC H2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(C H3)2, and OCH2C(=O)-N(H)CH3 ("NMA").

[0069] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2'-substituent selected from the group consisting of F, OCH3, and OCH2CH2OCH3. In one embodiment, the 2'-substituent is F. In another embodiment, the 2'-substituent is OCH3(methoxy).

[0070] Some modified sugar moieties include two atoms of bridging furanosyl ring to form the second ring, thereby producing the substituent of bicyclic sugar moiety.In some such embodiments, bicyclic sugar moiety includes bridge between 4 furanose ring atoms and 2 furanose ring atoms.The example of such 4 to 2 bridged sugar substituents includes but is not limited to: 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(called " 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 analogs, 4-C(CH3)(CH3)-O-2 and its analogs, 4-CH2-N(OCH3)-2 and its analogs, 4-CH2-ON(CH3)-2, 4-CH2-C(H)(CH3)-2, 4-CH2-C(=CH2)-2 and its analogs, 4-C(R a R b )-N(R)-O-2、4-C(R a R b )-ON(R)-2, 4-CH2-ON(R)-2 and 4-CH2-N(R)-O-2, where R, R a and R b Each independently is H, a protecting group or C 1-12 In one embodiment, the modified sugar moiety comprises a 4-(CH2)2-O-2 bridge (wherein "4" represents 4 furanose ring atoms and "2" represents 2 furanose ring atoms) ("ENA" or ethylene-bridged nucleic acid).

[0071] In certain embodiments, such 4 to 2 bridges independently comprise 1 to 4 linking groups independently selected from: -[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 )-; wherein: x is 0, 1 or 2; n is 1, 2, 3 or 4; each Ra and R b are independently H, a protecting group, a hydroxyl group, a C 1-12 Alkyl, substituted C 1-12 Alkyl, C 1-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, substituted C 2-12 Alkynyl, C 5-20 Aryl, substituted C 5-20 Aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, C 5-7 alicyclic, substituted C5-7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfinyl (S(=O)-J1); and each J1 and J2 are independently H, C 1-12 Alkyl, substituted C 1-12 Alkyl, C 2-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, substituted C 2-12 Alkynyl, C 5-20 Aryl, substituted C 5-20 Aryl, acyl (C (= O) -H), substituted acyl, heterocyclic, substituted heterocyclic, C 1-12 Aminoalkyl, substituted C 1-12 aminoalkyl or a protecting group.

[0072] Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; Albaek et al., J. Org. Chem., 2006, 71, 7731-7740; Singh et 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., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. m. 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., U.S.RE44,779; 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 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., WO 2004 / 106356; Wengel et al., WO 1999 / 014226; Seth et al., WO 2007 / 134181; Seth et al., U.S. Patent No. 7,547,684; Seth et al., U.S. Patent No. 7,666,854; Seth et al., U.S. Patent No. 8,088,746; Seth et al., U.S. Patent No. 7,750,131; Seth et al., U.S. Patent No. 8,030,467; Seth et al., U.S. Patent No. 8,268,980; Seth et al., 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.

[0073] Another modification of an ASO involves chemically linking the ASO to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the ASO. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); an aliphatic chain, such as dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0074] The present disclosure provides oligonucleotides (modified or unmodified) that can be used to regulate SYF2 expression. Tables 1A and 1B provide the (5' to 3') universal sequences of the bases of SYF2 antisense oligonucleotides or inhibitory nucleic acids. It is predicted that the ASO sequence SEQ ID NO: 1-37 effectively skips exon 5 of SYF2 and generates a stop codon to cause premature termination of SYF2 translation. It is predicted that the ASO sequence SEQ ID NO: 38-50 effectively skips exon 2 of SYF2 and makes major structural changes to the protein, thereby rendering it non-functional. As determined by the method described in Hagedorn et al., Nucleic Acid Therapeutics, 2022, 32 (3): 151-162 (DOI: 10.1089 / n at / 2021 / 0071), the ASO sequence SEQ ID NO: 51-139 has a low acute neurotoxicity score. ASO sequences SEQ ID NOs: 140-266 had a lower number of predicted off-target binding sites as determined by the GGGenome search tool using 2 mismatches / gaps.

[0075] Table 1A

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Table 1B

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] In one embodiment, the present disclosure provides a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-266, 533-573, and 615-833 in Tables 1A and 1B. In some embodiments, the modified oligonucleotide is at least 80% to 100% (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%; or any numerical range or value between any of the foregoing values) identical to any sequence comprising or consisting of SEQ ID NOs: 1-266, 533-573, and 615-833.

[0092] The sequences provided in Tables 1A and 1B can be used to design antisense molecules for inhibiting SYF2 expression. For example, gapmer oligonucleotides can be designed using the sequences in Tables 1A and 1B and can comprise a 5' wing of about 3-5 nucleotides, a 3' wing of about 3-5 nucleotides, and a gap region comprising 8-12 consecutive deoxyribonucleosides of any of the sequences in Tables 1A and 1B. In one embodiment, the oligonucleotides of the present disclosure include gapmers having a gap segment of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any one of the core base sequences of SEQ ID NO: 1-266, 533-573 and 615-833 in Tables 1A and 1B; flanked by 5' and 3' wing segments, wherein the gap segment is between the 5' wing segment and the 3' wing segment, and wherein each of the wing segments includes a modified sugar. In one embodiment, the length of the gap segment is 8-10 nucleosides, and the length of each wing segment is 3-5 modified nucleosides. In yet another embodiment, an oligonucleotide of the present disclosure comprises a 5' wing segment comprising a modified sugar and having a nucleobase sequence of the first 3-5 nucleobases of any one of SEQ ID NOs: 1-266, 533-573, and 615-833, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-266, 533-573, and 615-833, followed by a 3' wing segment comprising a modified sugar and having a nucleobase sequence of the last 3-5 nucleobases of the same sequence corresponding to SEQ ID NOs: 1-266, 533-573, and 615-833. Tables 2A and 2B provide MOE gapmers of the present disclosure.

[0093] The 5' and / or 3' wings can comprise the following chemistries: 2'-OMe, 2'-MOE, LNA, or DNA, used alone or in combination. The backbone linkages of the 5' and / or 3' wings can be phosphorothioates or a mixture of phosphodiester and phosphorothioate. The linkages in the gap region can be phosphorothioates.

[0094] In some embodiments, the oligonucleotide is single stranded.In some embodiments, the oligonucleotide comprises or is complexed with a moiety that neutralizes the charge on the oligonucleotide to facilitate uptake and transfer across cell membranes.

[0095] In one embodiment, each nucleobase in each 18-mer listed in Table 1A is a 2MOE nucleobase and each internucleotide linkage is a phosphorothioate linkage.The ASO may be a splice-switching ASO.

[0096] In one embodiment, each of the 20-mer ASOs in Tables 1A and 1B has 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*2MOE, wherein (i) 2MOE is a nucleobase having a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) N is a nucleobase, (iii) asterisk (*) refers to a phosphorothioate linkage, and (iv) dash (-) refers to a phosphodiester linkage. The ASO can be an RNase H-dependent ASO.

[0097] In another embodiment, each of the 20-mer ASOs in Tables 1A and 1B has 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*2MOE, wherein (i) 2MOE is a nucleobase having a 2'-OCH2CH2-OCH3 group (i.e., 2'-MOE), (ii) N is a nucleobase except that each cytosine (C) is present as 5-methyldeoxycytidine (Me-dC), (iii) asterisks (*) refer to phosphorothioate linkages, and (iv) dashes (-) refer to phosphodiester linkages. The ASO can be an RNase H-dependent ASO.

[0098] Table 2A below shows SEQ ID NOs: 1-266 and 533-573 (referred to as SEQ ID NOs: 267-532 and 574-614) having certain motifs, including certain modified 2MOE nucleobases and phosphorothioate linkages. SEQ ID NOs: 1-37, 38-50, 51-139, 140-266, 533-573 correspond to SEQ ID NOs: 267-303, 304-316, 317-405, 406-532, and 574-614, respectively, having specific motifs.

[0099] Table 2A: Sequence of bases in the SYF2 antisense oligonucleotide (ASO). (Gapmer design: 5′-five 2′-methoxyethyl ribonucleotides–ten DNA nucleotides–five 2′-methoxyethyl ribonucleotides–3′; uppercase letters are 2′-methoxyethyl ribonucleosides; lowercase letters are DNA nucleosides; asterisks (*) are phosphorothioate linkages; linkages without asterisks are phosphodiester linkages) (Note that the table below provides a 2′-MOE wing; however, alternative wings comprising 2′-OMe or LNA (locked nucleic acid) are contemplated).

[0100] Table 2A

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Table 2B below shows SEQ ID NOs: 615-833 (referred to as SEQ ID NOs: 834-1052) having specific motifs including modified 2MOE nucleobases and phosphorothioate linkages. SEQ ID NOs: 615-833 correspond to SEQ ID NOs: 834-1052, respectively, having the motifs.

[0110] Table 2B shows the example of antisense oligonucleotide of the present invention, and it has incorporated modified base as described herein and other modifications.These monomeric units use known oligonucleotide synthesis nomenclature to describe, to indicate non-standard monomeric units, for example, as described by Integrated DNA Technologies (Iowa, US).For example, in the sequence provided in Table 2B, non-standard monomeric units are surrounded by forward slash " / ", and the asterisk "*" between the units represents PS linkage, and lacks asterisk to represent PO linkage.Format " / i2MOErN / " (and " / 2MOErN / " in the first and last nucleotide) refers to 2'-methoxyethyl ribonucleotide, and wherein N refers to specific core base. " 5 " in the first nucleotide refers to 5' end, and " 3 " in the last nucleotide refers to 3' end. " iMe-dC " refers to 5-methyl deoxycytidine.

[0111] Table 2B

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The SYF2 antisense or inhibitory nucleic acids disclosed herein can inhibit the expression of SYF2 and, therefore, inhibit activities associated with SYF2.SYF2 antisense or inhibitory nucleic acids can include any combination of the oligonucleotides listed in Tables 2A and 2B and sequences that are 98%-99% identical thereto.

[0128] Treatment

[0129] Yet another embodiment is a method of treating a subject suffering from a SYF2 disease or disorder by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein.

[0130] One embodiment is a method for treating a subject suffering from a neurological disease or a neurodegenerative disease by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition as described herein. The neurological disease can be a neurodegenerative disease. For example, a neurodegenerative disease can cause motor neuron degeneration. For example, the neurological disease can be amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, or frontotemporal dementia. Further examples of neurological diseases 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, and / or depression.

[0131] Neurological diseases may be associated with abnormal endosomal trafficking. For example, endosomal pathways and endosomes are essential components for the recycling or degradation of membrane-bound proteins, the transport of Golgi-associated proteins, and the extracellular release of proteins in exosomes. These processes contribute to neurotransmission and drive the balance between recycling and degradation of synaptic vesicles or neurotransmitter receptors, for example.

[0132] Neurological diseases may be associated with abnormal lysosomal degradation. Alterations in lysosomal degradation may be present in neurological diseases, such as neurodegenerative diseases. Imbalance of cathepsins during aging and age-related diseases may have deleterious effects on central nervous system (CNS) neurons, and lysosomes may be sites of unfolding and partial degradation of membrane proteins or their precursors, which are subsequently expelled from the cell or released from dying cells and accumulate as pathological entities.

[0133] Healthcare professionals can diagnose a subject with a disorder related to motor neuron degeneration by evaluating one or more of these symptoms. To diagnose a neurological disorder, a physical exam may be followed by a thorough neurological examination. This examination may assess motor and sensory skills, nerve function, hearing and speech, vision, coordination and balance, mental status, and changes in mood or behavior. Non-limiting symptoms of illnesses related to a neurological disorder may include weakness in the arms, legs, feet, or ankles; slurred speech; difficulty lifting the front of the foot and toes; weakness or clumsiness of the hands; muscle paralysis; muscle stiffness; involuntary jerking or writing movements (chorea); involuntary, persistent muscle contractions (dystonia); bradykinesia; loss of automatic movements; impaired posture and balance; lack of flexibility; tingling in parts of the body; electric shock sensations with head movements; twitching of the arms, shoulders, and tongue; 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; misunderstanding spatial relationships; disorientation; depression; anxiety; difficulty making decisions and judgments; loss of impulse control; difficulty planning and performing familiar tasks; aggression; irritability; social withdrawal; mood swings; dementia; changes in sleep habits; confusion; and changes in appetite.

[0134] Tests can be performed to diagnose diseases and conditions that may have symptoms similar to those of a neurological disease, to measure muscle involvement, and to assess neuronal degeneration. Non-limiting examples of tests are electromyography (EMG); nerve conduction velocity studies; laboratory tests of blood, urine, or other substances; magnetic resonance imaging (MRI); magnetic resonance spectroscopy; muscle or nerve biopsy; transcranial magnetic stimulation; genetic screening; X-rays; fluoroscopy; angiography; computed tomography (CT); positron emission tomography; cerebrospinal fluid analysis; intrathecal contrast-enhanced CT scan; electroencephalogram (EEG); electronystagmography; evoked responses; polysomnography; thermography; and ultrasound. Healthcare professionals can also assess a patient's family history of motor neuron degeneration-related diseases and make a diagnosis based in part on a family history of neurological diseases. Healthcare professionals can diagnose diseases associated with a neurological disease after a subject develops one or more symptoms.

[0135] Neurodegenerative diseases result in the progressive destruction of neurons, thereby affecting neuronal signaling. Examples of neurodegenerative diseases include amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Friedreich's ataxia, Lewy body disease, Parkinson's disease, spinal muscular atrophy, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0136] Diseases associated with motor neuron degeneration may be conditions that result in the progressive destruction of motor neurons, thereby interfering with neuronal signaling to muscles, leading to muscle weakness and wasting. In healthy individuals, upper motor neurons transmit signals from the brain to lower motor neurons in the brainstem and spinal cord, which then transmit the signals to the muscles, producing voluntary muscle activity. Destruction of upper and lower motor neurons can affect activities such as breathing, speaking, swallowing, and walking, and over time, these functions may be lost. Examples of motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0137] Neuronal hyperexcitation can occur when receptors for the excitatory neurotransmitter glutamate (glutamate receptors), such as NMDA receptors and AMPA receptors, are overactivated by excess glutamate or other compounds or neurotransmitters that act on glutamate receptors. Excitotoxicity can be caused by neuronal hyperexcitation. Excitotoxicity is a pathological process in which nerve cells are damaged or die due to overstimulation. Excessive stimulation can cause high levels of calcium ions (Ca 2+ ) enters the cell. 2+The influx of phospholipases into cells activates a variety of enzymes, including phospholipases, endonucleases, and proteases, such as calpains, which damage cellular structures, such as components of the cytoskeleton, cell membranes, and DNA.

[0138] Neuronal hyperexcitation 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 withdrawal from benzodiazepines, and Huntington's disease. Other common conditions that lead to excessive glutamate concentrations around neurons are hypoglycemia. Blood glucose is the primary method for removing glutamate from the intersynaptic cleft of NMDA and AMPA receptor sites.

[0139] The methods of treatment described herein may include administering to a subject in need thereof a composition comprising an effective amount of one or more antisense oligonucleotides that treat a neurological disorder by inhibiting SYF2 expression. The one or more antisense oligonucleotides may reduce or inhibit neurodegeneration. The one or more antisense oligonucleotides may reduce neuronal hyperexcitability.

[0140] The composition can inhibit SYF2 activity or expression.One or more antisense oligonucleotides can be combined with an additional therapeutic agent.

[0141] Treatment methods may include any number of ways of administering the disclosed compositions. Administration may include aqueous, lipid, oily, or other solutions, solutions in simulated cerebrospinal fluid, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, and the like. Typically, the ASOs of the present disclosure will be administered directly to the CNS of the subject. Therefore, the formulations or compositions will be sterile and more preferably suitable for injection. The following formulations and methods are exemplary only and are in no way limiting.

[0142] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Preparations may be present in unit dose or multi-dose sealed containers, such as ampoules and vials, and may be stored as liquids or under freeze-dried (lyophilized) conditions, requiring only the immediate addition of sterile liquid excipients, such as water for injection, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Preparations may be provided in prefilled syringes.

[0143] Additional therapeutic agents may be administered simultaneously or sequentially with the disclosed one or more antisense or inhibitory nucleic acids and compositions. Sequential administration includes administration before or after the disclosed one or more antisense or inhibitory nucleic acids or compositions. In some embodiments, one or more additional therapeutic agents may be administered in the same composition as the disclosed one or more antisense or inhibitory nucleic acids. In other embodiments, there may be a time interval between the administration of the additional therapeutic agent and the disclosed one or more antisense or inhibitory nucleic acids. In some embodiments, administering the additional therapeutic agent together with the disclosed one or more antisense or inhibitory nucleic acids allows for lower doses of the other therapeutic agents and / or administration at less frequent time intervals. When used in combination with one or more other active ingredients, the one or more antisense or inhibitory nucleic acids of the present disclosure and the other active ingredients can be used at lower doses than when each is used alone. Therefore, pharmaceutical compositions of the present disclosure include those containing one or more other active ingredients in addition to the one or more antisense or inhibitory nucleic acids of the present disclosure. The above-mentioned combinations include combinations of the one or more antisense or inhibitory nucleic acids of the present disclosure not only with one other active compound, but also with two or more other active compounds. For example, the compounds of the present disclosure can be combined with various drugs to treat neurological diseases.Antisense oligonucleotides can be covalently linked to another oligonucleotide, such as an oligonucleotide with a target other than SYF2.Antisense oligonucleotides can be covalently linked to antibodies.

[0144] One or more of the disclosed antisense or inhibitory nucleic acids can be combined with, but are not limited to, anticholinergics, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, analgesics, and / or stimulants. Other types of therapy and treatment 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.

[0145] The disclosed compositions can be incorporated into pharmaceutical compositions suitable for administration to a subject (e.g., a patient, which can be human or non-human). The pharmaceutical composition can include a carrier (e.g., a pharmaceutically acceptable carrier). Any suitable carrier can be used in the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will be determined in part by the specific use of the composition (e.g., administration to an animal) and the specific method used to administer the composition. Thus, the compositions of the present invention have a variety of suitable formulations.

[0146] The pharmaceutical composition may comprise a therapeutically effective amount or a prophylactic effective amount of the antisense oligonucleotide. The therapeutically effective amount of the composition can be determined by those skilled in the art and can vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of the composition to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of one or more antisense or inhibitory nucleic acids of the present disclosure are offset by the therapeutically beneficial effects. A "prophylactic effective amount" refers to an amount that effectively achieves the desired prophylactic result within the necessary dosage and time period. Typically, since a prophylactic dose is used for a subject before the disease or in the early stages of the disease, the prophylactic effective amount will be lower than the therapeutically effective amount.

[0147] The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary. Some examples of materials that can serve as pharmaceutically acceptable carriers, at the discretion of the formulator, 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; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffers such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions, and other nontoxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, and release agents, coating agents, preservatives, and antioxidants may also be present in the composition.

[0148] The route of administration of the disclosed antisense or inhibitory nucleic acid(s) and the form of the composition will dictate the type of vector used.

[0149] The pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is needed and the area to be treated. Administration can be parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal, intracerebroventricular, or intraventricular administration. In one embodiment, the antisense or inhibitory nucleic acid is administered intravenously, intraperitoneally, or as a bolus or directly into the target organ. In another embodiment, the antisense or inhibitory nucleic acid is administered as a bolus intrathecally or intraventricularly.

[0150] Carriers for systemic administration typically include at least one of the following: solvents, diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, etc. All carriers in the composition are optional.

[0151] Suitable diluents include sugars such as glucose, lactose, dextrose and sucrose; glycols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols such as glycerol; mannitol; and sorbitol.

[0152] Suitable lubricants include silicon dioxide, talc, stearic acid and its magnesium and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter. The amount of lubricant in the systemic or local composition is generally about 5% to about 10%.

[0153] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches, such as corn starch, potato starch; gelatin; gum tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder in the systemic composition is generally from about 5% to about 50%.

[0154] Suitable disintegrants include agar, alginic acid and its sodium salt, effervescent mixtures, cross-linked carboxymethylcellulose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays and ion exchange resins.The amount of disintegrant in systemic compositions is generally from about 0.1% to about 10%.

[0155] Suitable coloring agents include colorants such as FD&C dyes.When used, coloring agents are generally present in systemic or topical compositions at levels of from about 0.005% to about 0.1%.

[0156] Suitable flavoring agents include menthol, mint, and fruit flavors.When used, flavoring agents are generally present in systemic or topical compositions at levels of from about 0.1% to about 1.0%.

[0157] Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of antioxidant in the systemic or topical compositions is generally from about 0.1% to about 5%.

[0158] Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate.The amount of preservative in systemic or topical compositions is generally from about 0.01% to about 5%.

[0159] Suitable glidants include silicon dioxide.The amount of glidant in the systemic or topical composition is generally from about 1% to about 5%.

[0160] Suitable solvents include water, isotonic saline, ethyl oleate, glycerol, hydroxylated castor oil, alcohols such as ethanol, and phosphate buffered saline.The amount of solvent in a systemic or topical composition is generally from about 0 to about 100%.

[0161] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate.The amount of suspending agent in systemic or topical compositions is generally from about 1% to about 8%.

[0162] Suitable surfactants include lecithin, polysorbate 80, and sodium lauryl sulfate, as well as TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the CTFA Cosmetic Ingredient Handbook, 1992, pages 587-592; Remington's Pharmaceutical Sciences, 15th edition, 1975, pages 335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American edition, pages 236-239. The amount of surfactant in the systemic or topical composition is generally about 0.1% to about 5%.

[0163] Compositions and preparations for parenteral, intrathecal, intraventricular or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. For example, intrathecal cerebrospinal fluid (CSF) catheters may be used to deliver antisense preparations of the present disclosure. The catheter may be inserted into the L3 or L4 vertebra. The distal end of the catheter extends to approximately the L1 vertebra in the intrathecal space. The antisense oligonucleotides are dissolved in saline, sterilized by filtration, and administered at 0.33 ml / min with a 1.0 ml volume, followed by rinsing with 0.5 ml sterile water. The total infusion time is 4.5 minutes.

[0164] Compositions for parenteral administration generally contain from 0.1% to 10% of active substance and from 90% to 99.9% of carrier (including diluent and solvent).

[0165] The amount of carrier used in conjunction with the disclosed compounds is sufficient to provide the actual amount of the composition for administration per unit dose of the drug. Techniques and compositions for preparing dosage forms useful in the methods of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, ed. Banker and Rhodes (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd ed., (1976).

[0166] In vivo testing of candidate antisense or inhibitory nucleic acid can be carried out by means known to those of ordinary skill in the art. For example, candidate's one or more antisense or inhibitory nucleic acid can be applied to mammals, such as mice or rabbits. Candidate's antisense or inhibitory nucleic acid of a certain dose can be applied to mammals by any approach that is considered suitable. Conventional methods and standards can then be used to monitor the expression or activity of animal motor neuron activity and / or SYF2 gene or protein respectively or to determine whether the activity reduces or improves. If desired, the result obtained in the presence of candidate antisense or inhibitory nucleic acid can be compared with the result in a control animal not treated with candidate antisense or inhibitory nucleic acid. Dosage studies can be performed in any subsequent test of candidate antisense or inhibitory nucleic acid in vivo for identifying one or more antisense or inhibitory nucleic acid capable of treating neurological diseases as described herein or in combination with the method. Those skilled in the art of medicine can determine the appropriate dosage of one or more antisense or inhibitory nucleic acid. Dosage can be determined by monitoring the disease suppression or improvement sign of the experimenter. Dosage can be increased or decreased to obtain the required treatment frequency. The toxicity and efficacy of one or more antisense or inhibitory nucleic acids can be determined by the standard pharmaceutical procedures in cell culture or experimental animals (e.g., animals), such as, determining 50% population lethal dose (LD50) and 50% population therapeutic effective dose (ED50). The dosage ratio of LD50 / ED50 is a therapeutic index, and represents the ratio between toxic effect and therapeutic effect. Delivery system can be designed to help prevent toxic side effects by delivering one or more antisense or inhibitory nucleic acids to specific targets (e.g., specifically delivered to motor or central nervous system neurons). For example, the optimal dose of one or more antisense or inhibitory nucleic acids can be determined based on the result of clinical electrophysiology or electromyography, to analyze the excitability of peripheral nerves.

[0167] The dosage for humans can be determined by evaluating the data obtained from animal studies and cell culture assays. Preferably, the dosage has minimal toxicity or no toxicity and includes an ED50. The dosage can vary according to the dosage form and route of administration. For any antisense or inhibitory nucleic acid used in the methods described herein, the dosage can be initially estimated in cell culture. The dosage can be formulated in an animal model, comprising the concentration of the test compound that achieves a half-maximal inhibition (LD50) of the symptoms as measured in cell culture. Such information obtained from cell culture and animal models can be used to more accurately determine a useful dosage for humans.

[0168] In another embodiment, the antisense oligonucleotides of the present invention are expressed by a transgene, for example as antisense RNA transcripts. The transgene can be administered to a subject in the form of a DNA expression construct engineered to express the antisense RNA transcript in the subject. The DNA expression construct can be administered directly or using a viral vector (e.g., a recombinant AAV (rAAV) vector) or other suitable vector. Viral vectors used in gene therapy protocols include, but are not limited to, retroviruses, other RNA viruses (e.g., poliovirus or Sindbis virus), adenoviruses, adeno-associated viruses (AAV), herpes viruses, SV40, vaccinia, lentiviruses, and other DNA viruses.

[0169] The present invention has various aspects, which are illustrated by the following non-limiting examples.

[0170] Example

[0171] ASOs are an attractive therapeutic option for neurodegenerative diseases because they are easily delivered to the central nervous system and their exposure to the periphery is relatively low. These properties maximize target engagement in the central nervous system and minimize undesired target engagement or off-target effects in the periphery.

[0172] The present disclosure provides novel antisense oligonucleotide (ASO) sequences targeting the SYF2 gene that can inhibit SYF2 expression in human cells. SYF2 ASOs can also rescue the survival of motor neurons from sporadic ALS patients, while also rescuing TDP-43 mislocalization, neurodegeneration, and NMJ loss, and resulting in improved motor function.

[0173] To identify ASO sequences that suppress SYF2 expression in human cells, ASOs were designed (see Table 2) and synthesized as MOE gapmers containing sugar and linkage modifications that increase nuclease resistance and melting temperature while retaining the ability to serve as a substrate for RNase H. As a control, NCASO was used, and values ​​were normalized to GapdH.

[0174] The present disclosure provides ASOs that suppress SYF2 expression in human cells. These ASOs can prevent neurodegeneration in patients with ALS and FTD.

[0175] Example 1

[0176] Human SYF2 ASO Screening in HeLa Cells (ASO 1-121)

[0177] Antisense oligonucleotides against human SYF2 were evaluated in HeLa cells targeting genomic regions including sequences within the untranslated regions (UTRs) as well as intronic and exonic regions. TM 25 nM of a 2′-MOE gapmer chemistry ( Figure 1-Figure 3 ) or 2'-Ome gapmer chemistry ( Figure 4 ) were transfected with antisense oligonucleotides. Cells were harvested 72 hours after transfection. Negative control (NC) ASOs were tested as controls.

[0178] qRT-PCR analysis of human SYF2 levels in HeLa cells treated with ASO-1 to ASO-121 was performed. mRNA levels were determined using the ddCT method, with human GAPDH as the reference gene and a negative control ASO with a scrambled sequence (NCASO) as the reference group. SYF2 mRNA levels in all tested conditions were compared to cells treated with the NC ASO using a one-way ANOVA. The results are shown in Figure 1 (ASO-1 to ASO-50), Figure 2 (ASO-51 to ASO-74), Figure 3 (ASO-75-100) and Figure 4 (ASO-101 to ASO-121). P values ​​are represented as follows: ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

[0179] Example 2

[0180] Selection of Syf2 ASO in Ngn2-iN

[0181] Selected sequences (ASO-19, ASO-24, ASO-25, ASO-32, ASO-35, ASO-42, ASO-43, ASO-63, ASO-69, ASO-70, ASO-80, ASO-86, and ASO-120 corresponding to SEQ ID NOs: 633, 638, 639, 646, 649, 656, 657, 677, 683, 684, 694, 700, and 734) were validated in human iPSC-derived cortical neurons (Ngn2-induced neurons, Ngn2-iNs) based on their potency in reducing SYF2 mRNA levels and their conservation in non-human primates (NHPs). Gymnotic delivery was performed using 10 uM of antisense oligonucleotides. Cells were harvested 7 days after gymnosis.

[0182] qRT-PCR analysis of human SYF2 levels was performed in Ngn2-iN treated with ASOs. mRNA levels were determined using the ddCT method, with human HPRT as the reference gene and a negative control ASO with scrambled sequence (NC ASO) as the reference group. SYF2 mRNA levels in all tested conditions were compared with cells treated with NC ASOs using a one-way ANOVA. Results are shown in Figure 5 P values ​​are indicated as follows: ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

[0183] Example 3

[0184] Human SYF2 ASO Screening in Ngn2-iN (ASO 122-219)

[0185] A script was developed that systematically examined the entire target gene sequence and identified 20 nucleotide sequences that met certain design criteria, including off-target prediction and evaluation of sequence conservation in non-human primates.

[0186] 98 sequences were synthesized and evaluated in Ngn2-iN, and the sequences were selected as having the lowest number of predicted off-targets and being conserved in non-human primates. 10 uM of antisense oligonucleotides were used for naked delivery. Cells were harvested 7 days after naked delivery.

[0187] qRT-PCR analysis of human SYF2 levels was performed in Ngn2-iN treated with ASOs. mRNA levels were determined using the ddCT method, with human HPRT as the reference gene and a negative control ASO with a scrambled sequence (NC ASO) as the reference group. SYF2 mRNA levels in all tested conditions were compared with cells treated with NC ASOs using a one-way ANOVA. Results are shown in Figure 6 P values ​​are indicated as follows: ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05. ASO-122, ASO-123, ASO-125, ASO-135, ASO-136, ASO-150, ASO-159, ASO-160, ASO-165, ASO-171 , ASO-185, ASO-189, ASO-191, ASO-196, ASO-197, ASO-198, ASO-199, ASO-201 and ASO-211 (corresponding to SEQ ID NO: 736, 737, 739, 749, 750, 764, 773, 774, 779, 785, 799, 803, 805, 810, 811, 812, 813, 815 and 825) significantly reduced the SYF2 expression level.

[0188] The foregoing description and accompanying drawings should be considered as merely illustrative of the principles of the present invention. The present invention is not intended to be limited to the preferred embodiments and can be implemented in a variety of ways that are clear to those skilled in the art. Those skilled in the art will readily appreciate the various applications of the present invention. Therefore, it is not intended to limit the present invention to the specific embodiments disclosed or the precise construction and operation shown and described. On the contrary, all suitable modifications and equivalents that fall within the scope of the present invention may be adopted. All references cited herein are incorporated by reference.

Claims

1. A single-stranded antisense oligonucleotide that suppresses the expression of SYF2, wherein the antisense oligonucleotide has a nucleobase sequence comprising at least 12 or 15 consecutive nucleobases of any nucleobase sequence of SEQ ID NOs: 1-1052.

2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 1-266, 533-573, and 615-833.

3. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide has 18 to 20 linked nucleosides.

4. The antisense oligonucleotide of any preceding claim, wherein at least one internucleoside linkage is a modified internucleoside linkage.

5. The antisense oligonucleotide of claim 4, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.

6. The antisense oligonucleotide of claim 4, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.

7. The antisense oligonucleotide of any one of the preceding claims, wherein at least one internucleoside linkage is a phosphodiester internucleoside linkage.

8. The antisense oligonucleotide of claim 7, wherein at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.

9. The antisense oligonucleotide of any one of the preceding claims, wherein at least one nucleoside comprises a modified nucleobase.

10. The antisense oligonucleotide of claim 9, wherein the modified nucleobase is 5-methylcytosine.

11. The antisense oligonucleotide of any one of the preceding claims, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.

12. The antisense oligonucleotide of claim 11, wherein the modified sugar moiety comprises a 2'-O-methoxyethyl group.

13. The antisense oligonucleotide of claim 11, wherein the modified sugar moiety comprises 2'-F.

14. The antisense oligonucleotide of claim 11, wherein the modified sugar moiety comprises a 2'-O-hexadecyl group.

15. The antisense oligonucleotide of any one of the preceding claims, wherein the antisense oligonucleotide is a gapmer.

16. The antisense oligonucleotide of claim 15, wherein the antisense oligonucleotide comprises: a gap segment consisting of 8 to 12 linked deoxynucleosides; a 5' wing segment consisting of 3 to 5 linked nucleosides; and a 3' wing segment consisting of 3 to 5 linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and wherein the nucleosides of each wing segment comprise a modified sugar moiety.

17. The antisense oligonucleotide of claim 16, wherein each nucleoside of each wing segment comprises a modified sugar moiety.

18. The antisense oligonucleotide of claim 16, wherein the nucleosides comprising each wing segment comprise at least two different modified sugar moieties.

19. The antisense oligonucleotide of claim 16, wherein the nucleosides comprising each wing segment comprise the same modified sugar moiety.

20. The antisense oligonucleotide of claim 17, wherein the modified sugar moiety comprises a 2'-O-methoxyethyl group.

21. The antisense oligonucleotide of any one of the preceding claims, wherein the antisense oligonucleotide comprises 15 to 50 nucleosides.

22. The antisense oligonucleotide of any one of the preceding claims, wherein the antisense oligonucleotide is one of SEQ ID NOs: 267-316, 574-614, and 834-1052.

23. A pharmaceutical composition comprising the antisense oligonucleotide according to any one of the preceding claims and a pharmaceutically acceptable carrier, diluent and / or excipient.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is formulated for parenteral delivery.

25. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is formulated for intracerebroventricular injection.

26. A method of treating a subject in need thereof suffering from a neurological disease or a neurodegenerative disease, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 23.

27. The method of claim 26, wherein the neurological disease is associated with neuronal hyperexcitability.

28. The method of claim 26, wherein the neurological disease is associated with abnormal endosomal trafficking.

29. The method of claim 26, wherein the neurological disease is associated with abnormal lysosomal trafficking.

30. The method of claim 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, Chuck-Marie-Dews disease 2A and 4B, Huntington's disease, dementia, transmissible spongiform encephalopathy, spinobulbar muscular atrophy, dentatorubral-pallidular Lewy body atrophy, spinocerebellar ataxia, and Creutzfeldt-Jakob disease.

31. An oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-1052.

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