SYF2 antisense oligonucleotides

Abstract

The present invention relates to SYF2 antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods for using them to treat, inhibit, suppress, and prevent neurological disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 363,729, filed April 28, 2022, which is incorporated by reference in its entirety.

[0002] The present invention relates to SYF2 antisense oligonucleotides (ASOs), pharmaceutical compositions containing them, and methods for using them to treat, inhibit, suppress, and prevent neurological or neurodegenerative diseases. [Background technology]

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

[0004] International Publication No. WO2021 / 150840 discloses a method for treating neurodegenerative diseases by administering a SYF2 inhibitor. SYF2 binds to the pre-mRNA splicing complex, and there is evidence suggesting that SYF2 is not essential for splicing, but regulates the nuclear transport of mRNA.

[0005] 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

[0006] The present invention relates to SYF2 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 inhibits expression of SYF2, 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: 1-121. The nucleobase sequence of the ASO can include up to 30, 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-121. The ASO can also be any of SEQ ID NOs: 1-121.

[0008] Another embodiment is an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-121. The oligonucleotide may include up to 25, 24, 23, 22, 21, or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-121.

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

[0010] In other embodiments, at least one nucleoside of the ASO may be a modified sugar moiety, which may be a bicyclic sugar moiety or which may include a 2'-O-methoxyethyl group, a 2'-F, or a 2'-O-hexadecyl group. In certain aspects, the bicyclic sugar moiety includes a 4'-CH(R)-O-2' bridge, where the R groups are independently H, C, 1-12 In one embodiment, the bicyclic sugar moiety comprises a 4-(CH2)2-O-2 bridge ("ENA" or ethylene bridged nucleic acid).

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

[0012] In other embodiments, the oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-121.

[0013] Another embodiment is a pharmaceutical composition comprising a SYF2 ASO of the invention and one or more pharma- ceutically acceptable carriers, diluents, and / or excipients. In one embodiment, the pharmaceutical composition is suitable for parenteral administration, such as intraventricular injection or intrathecal administration.

[0014] Yet another embodiment is a method of treating a subject having a neurological or neurodegenerative disease by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein.

[0015] Yet another embodiment is a method of treating a subject having a SYF2 disease or disorder by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein.

[0016] The invention will be more fully appreciated and many of the attendant advantages will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, in which: [Brief description of the drawings]

[0017] [Figure 1A] SYF2 ASO screen in HeLa cells measuring fold change normalized to GadpH for ASO51-74 (SEQ ID NO:51-74) relative to control (NCASO), ASO 20 (SEQ ID NO:20), and ASO 21 (SEQ ID NO:21). [Figure 1B] SYF2 ASO screen in HeLa cells measuring fold change normalized to GadpH for ASO75-100 (SEQ ID NO:75-100) relative to control (NCASO), ASO 20 (SEQ ID NO:20), and ASO 21 (SEQ ID NO:21). [Figure 1C] SYF2 ASO screen in HeLa cells measuring fold change normalized to GadpH for ASO101-121 (SEQ ID NO:101-121) relative to control (NCASO) and ASO 21 (SEQ ID NO:21). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In the event of any conflict, the present specification, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0019] definition The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," "may," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or phrases that do not exclude the possibility of additional acts or structures.

[0020] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements present herein, whether or not explicitly stated.

[0022] As used herein, "2'-deoxynucleoside" refers to a nucleoside containing a 2'-H(H) furanosyl sugar moiety as found in naturally occurring deoxyribonucleic acid (DNA) and nucleobase. In certain embodiments, a 2'-deoxynucleoside can contain a modified nucleobase and a furanosyl sugar moiety or a RNA nucleobase (uracil) and a furanosyl sugar moiety.

[0023] As used herein, "2'-substituted nucleoside" refers to a nucleoside that contains a 2'-substituted sugar moiety. As used herein, "2'-substituted" with reference to the sugar moiety means that the sugar moiety includes at least one 2'-substituent group other than H or OH.

[0024] As used herein, an "antisense molecule" means an oligomeric nucleic acid or oligomeric duplex capable of achieving at least one antisense activity.

[0025] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of 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 phrase "about 2 to about 4" also discloses a range of "2 to 4". The term "about" may refer to ±10% of the indicated number. For example, "about 10%" may indicate a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, thus, for example, "about 1" may also mean 0.5 to 1.4.

[0026] When numerical ranges are recited herein, each intervening numerical value to the same degree of precision is expressly contemplated. For example, in the range 6 to 9, the numerical values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numerical values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0027] As used herein, "bicyclic sugar" or "bicyclic sugar moiety" refers to a modified sugar moiety that contains two rings, where the second ring is formed via a bridge that connects two of the atoms in the first ring to form a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not include a furanosyl moiety. As used herein, "bicyclic nucleoside" or "BNA" refers to a nucleoside that includes a bicyclic sugar moiety.

[0028] As used herein, "chirally enriched population" refers to a plurality of molecules of the same molecular formula, wherein the number or percentage of molecules in the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules in the population that would be expected to contain the same particular stereochemical configuration at the same particular chiral center if the particular chiral center were sterically random. A chirally enriched population of molecules with multiple chiral centers within each molecule may contain one or more stereo-random chiral centers. In certain embodiments, the molecule is a modified oligonucleotide. In certain embodiments, the molecule is a compound that includes a modified oligonucleotide.

[0029] 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 the other nucleic acid or one or more regions thereof can hydrogen bond with each other when the nucleobase sequence of the oligonucleotide and the nucleobase sequence of the other nucleic acid are aligned in opposite directions. Complementary nucleobases means nucleobases that can form hydrogen bonds with each other. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have complementary nucleobases at each nucleoside. Rather, some mismatches are allowed. As used herein, "fully complementary" or "100% complementary" with respect to an oligonucleotide means that the oligonucleotide is complementary to the other oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0030] As used herein, "gapmer" refers to a modified oligonucleotide that includes an internal region having multiple nucleosides that are positioned between external regions having one or more nucleosides and that support RNase H cleavage, and the nucleosides that comprise the internal region are chemically distinct from the nucleoside(s) that comprise the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings." Unless otherwise indicated, "gapmer" refers to a sugar motif. Unless otherwise indicated, the sugar moieties of the nucleosides of the gapmer gap are unmodified 2'-deoxyfuranosyl. Thus, the term "MOE gapmer" refers to a gapmer having a 2'-MOE nucleoside sugar motif in both wings and also having a 2'-deoxynucleoside gap. Unless otherwise indicated, MOE gapmers may include one or more modified internucleoside linkages and / or modified nucleobases, although such modifications do not necessarily follow a gapmer pattern of sugar modifications. Table 2 below provides exemplary MOE gapmers.

[0031] As used herein, "inhibition" refers to the ability to substantially antagonize, prohibit, prevent, suppress, limit, delay, hinder, alter, remove, halt, or reverse the progression or severity of the activity of a particular agent (such as an infectious agent) or disease.

[0032] As used herein, the term "internucleoside bond" refers to the covalent bond between adjacent nucleosides in an oligonucleotide.As used herein, "modified internucleoside bond" refers to any internucleoside bond other than phosphodiester internucleoside bond."Phosphorothioate bond" refers to a modified internucleoside bond in which one of the non-bridging oxygen atoms of phosphodiester internucleoside bond is replaced with a sulfur atom.

[0033] As used herein, "MOE" means methoxyethyl. "2'-MOE" means a -OCH2CH2OCH3 group at the 2' position of a furanosyl ring.

[0034] A "neurological disorder" is any disorder that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, the neurological disorder can be a neurodegenerative disorder.

[0035] As used herein, "non-bicyclic modified sugar moiety" means a modified sugar moiety that includes modifications such as substituents that do not form a bridge between two atoms of the sugar to form a second ring.

[0036] As used herein, "nucleobase" refers to an unmodified or modified nucleobase. As used herein, "unmodified nucleobase" refers to adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, "modified nucleobase" refers to a group of atoms other than unmodified A, T, C, U, or G that can pair with at least one unmodified or modified nucleobase. "5-methylcytosine" or "mC" is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, "nucleobase sequence" refers to the order of consecutive nucleobases in a nucleic acid or oligonucleotide, independent of any sugar or nucleoside linkage modification.

[0037] As used herein, "nucleoside" refers to a compound containing a nucleobase and a sugar moiety. Each of the nucleobase and sugar moieties is independently unmodified or modified. As used herein, "modified nucleoside" refers to a nucleoside containing a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides in which the nucleobase is missing. "Linked nucleosides" are nucleosides that are linked in a contiguous sequence (i.e., there are no additional nucleosides between the linked ones).

[0038] As used herein, "oligomeric compound" refers to an oligonucleotide and, optionally, one or more additional features, such as a conjugate group or a terminal group. An oligomeric compound may or may not be paired with a second oligomeric compound that is complementary to the first oligomeric compound. A "single-stranded oligomeric compound" is an unpaired oligomeric compound. The term "oligomeric duplex" refers to a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a "double-stranded oligomeric compound".

[0039] As used herein, "oligonucleotide" refers to a single strand of linked nucleosides linked via internucleoside linkages, each nucleoside and internucleoside linkage may be modified or unmodified. The internucleoside linkages may be any of those described herein. Unless otherwise indicated, an oligonucleotide consists of 8 to 50 linked nucleosides. As used herein, "modified oligonucleotide" refers to an oligonucleotide in which at least one nucleoside or internucleoside linkage is modified. As used herein, "unmodified oligonucleotide" refers to an oligonucleotide that does not contain any nucleoside or internucleoside modification.

[0040] "SYF2", also known in the art as "SYF2 pre-MRNA splicing factor", encodes a nuclear protein that interacts with cyclin D-type binding protein 1, a thought cell cycle regulator at the G1 / S phase transition. SYF2 has been shown to reduce TDP-43 aggregation and mislocalization and cryptic exon incorporation, as well as rescue C9ORF72 and ensure survival of sporadic ALS neurons. TDP-43 has been shown to reduce cryptic exon incorporation that leads to non-productive RNA transcripts. As a result, loss or reduction of nuclear TDP-43 in motor neurons reduces the mRNA levels of many genes, including STMN2, which transcribes microtubule-associated proteins. Loss of STMN2 has been shown to impair neurite outgrowth in motor neurons.

[0041] As used herein, "SYF2 disease or disorder" includes lysosomal degradation diseases and disorders mediated by SYF2. For example, SYF2 disease or disorder includes, but is not limited to, amyloid diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, type 2 diabetes, diabetic amyloidosis, and chronic hemodialysis-associated amyloid), multiple sclerosis, and MPS disorders (such as MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS IVA, MPS IVB, MPS VI, MPS VII, or MPS IX). In some embodiments, the disease is an autoimmune disorder (such as multiple sclerosis, rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, adult quiescent disease, Behcet's syndrome, familial idiopathic mesenchymal fever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronic bronchiectasis, Castleman's disease, etc.), or a CNS disorder (such as spongiform encephalopathies (Creutzfeldt-Jakob, kuru, mad cow)). The compositions and methods of the disclosure can be used to treat individuals with lysosomal storage diseases, including administering to a subject in need of treatment a SYF2 ASO or pharmaceutical composition described herein. In some embodiments, the ASOs and compositions of the disclosure reduce or inhibit the activity of SYF2 and alter the biogenesis, function, or dynamics of the endosomal or lysosomal system to reduce the abundance of material abnormally stored in lysosomes in lysosomal storage diseases. In some embodiments, the ASOs and compositions target, reduce or inhibit the activity of SYF2, thereby altering the biogenesis, function or dynamics of the endoplasmic reticulum or Golgi apparatus to reduce the abundance of material abnormally stored in lysosomes in lysosomal storage diseases. In other embodiments, the disease is a neurological disorder.

[0042] As used herein, "sugar moiety" refers to an unmodified sugar moiety or a modified sugar moiety. A superscript prime (') is used to represent the numbering of the sugar within a nucleoside or nucleotide (nucleobase positions are numbered without a prime). When representing only the sugar, no prime is used. As used herein, "unmodified sugar moiety" refers to a 2-OH(H) furanosyl moiety found in RNA (an "unmodified RNA sugar moiety"), or a 2-H(H) moiety found in DNA (an "unmodified DNA sugar moiety"). An unmodified sugar moiety has one hydrogen at each of the 1', 3', and 4' positions, one oxygen at the 3' position, and two hydrogens at the 5' position. As used herein, "modified sugar moiety" or "modified sugar" refers to a modified furanosyl sugar moiety or sugar surrogate. As used herein, a modified furanosyl sugar moiety refers to a furanosyl sugar that contains a non-hydrogen substituent in place of at least one hydrogen of 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 and non-bicyclic modified sugars.

[0043] "Subject" and "patient", as used interchangeably herein, 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), and humans). In some embodiments, the subject may be human or non-human. In preferred embodiments, the subject or patient is human. The subject or patient may be undergoing other forms of therapy.

[0044] A "therapeutically effective amount" or "effective dose" or "effective amount," as used interchangeably herein, unless otherwise defined, refers to a dose of a drug effective for a period of time necessary to obtain a desired therapeutic result. An effective dose may be determined by one of skill in the art and may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the individual. As used herein, the term may also refer to an amount effective to produce a desired in vivo effect, such as reducing and / or inhibiting the function of a receptor in an animal, mammal, or human. A therapeutically effective amount may be administered in one or more administrations (e.g., the agent may be administered as a prophylactic treatment or therapeutically, at any stage of the progression of a disease, before or after the onset of symptoms, etc.), application, or dose, and is not intended to be limited to a particular formulation, combination, or route of administration. It is within the scope of this disclosure that a drug may be administered at various times during the course of treatment of a subject. The time of administration and the dose used will depend on several factors, such as the purpose of the treatment (e.g., treatment, prevention), the condition of the subject, and can be readily determined by one of skill in the art.

[0045] As used herein, the term "treating" or "treating" a subject refers to administering a composition or agent described herein to a subject to cure, alleviate, relieve, alter, treat, reduce, ameliorate, or improve at least one symptom of a disease or disorder. Treating includes administering an amount effective to alleviate, alleviate, alter, treat, reduce, ameliorate, and / or improve one or more symptoms associated with a disease or disorder. Treatment may inhibit the exacerbation or worsening of symptoms associated with a disease or disorder.

[0046] Antisense oligonucleotides In certain embodiments, oligonucleotides contain one or more types of modified sugar and / or unmodified sugar moieties arranged along the oligonucleotide or regions thereof in a defined pattern or sugar motif, in certain examples, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.

[0047] In certain embodiments, the modified oligonucleotide comprises or consists of a region having a gapmer motif defined by two external regions or "wings" and a central or internal region or "gap". The three regions of the gapmer motif include the "5' wing", the "gap", and the "3' wing", which form a contiguous sequence of nucleosides, where at least a portion of the sugar moieties of each nucleoside of the wing are different from at least a portion of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides closest to the gap in each wing (the 3'-most nucleoside of the 5'-wing and the 5'-most nucleoside of the 3'-wing) are different from the sugar moieties of the adjacent gap nucleosides, thus defining 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 that is different from the sugar moieties of one or more other nucleosides in the gap. In certain embodiments, the sugar motifs of the two wings are identical to each other (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing is different from the sugar motif of the 3'-wing (asymmetric gapmer).

[0048] In certain embodiments, the wings of a gapmer comprise from 1 to 5 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0049] In certain embodiments, the gapmer gap comprises between 7 and 12 nucleosides (e.g., 10 nucleosides). In certain embodiments, each nucleoside of the gapmer gap is an unmodified 2'-deoxynucleoside.

[0050] In certain embodiments, the gapmer is a deoxy gapmer. In certain embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2'-deoxynucleosides and the nucleosides on the wing side of each wing / gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2'-deoxynucleoside. In certain embodiments, each nucleoside of each wing of the gapmer is a modified nucleoside.

[0051] In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside throughout the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the modified oligonucleotide comprises or consists of a region with a fully modified sugar motif, where each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of the uniformly modified oligonucleotide comprises the same 2' modification.

[0052] In certain embodiments, the nucleosides of modified oligonucleotides can be linked together using any internucleoside bond. Two major classes of internucleoside linking groups are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester linkages (also referred to as unmodified or natural linkages), phosphotriesters, methylphosphonates or other alkylphosphonates, phosphoramidates, and phosphorothioates, and phosphates including phosphorodithioates. Representative non-phosphate-containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters, thionocarbamates (-OC(=O)(NH)-S-), siloxanes (-O-SiH2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to natural phosphate linkages, can be used to alter (usually increase) the nuclease resistance of oligonucleotides. Methods for the preparation of phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0053] Representative internucleoside linkages with chiral centers include, but are not limited to, alkylphosphonates and phosphorothioates. Modified oligonucleotides containing internucleoside linkages with chiral centers can be prepared as a population of modified oligonucleotides containing sterically random internucleoside linkages or as a population of modified oligonucleotides containing phosphorothioate linkages in a specific stereochemical configuration. In certain embodiments, the population of modified oligonucleotides contains phosphorothioate internucleoside linkages, where all of the phosphorothioate internucleoside linkages are sterically random. Such modified oligonucleotides can be generated using a synthetic method that results in random selection of the stereochemical configuration of each phosphorothioate linkage. Nevertheless, as will be appreciated by those skilled in the art, each individual oligonucleotide molecule of each individual phosphorothioate has a defined stereochemical configuration. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides containing one or more specific phosphorothioate internucleoside linkages in a specific, independently selected stereochemical configuration. In certain embodiments, a particular arrangement of phosphorothioate linkages is present in at least 65% of the molecules in the population. In certain embodiments, a particular arrangement of phosphorothioate linkages is present in at least 70% of the molecules in the population. In certain embodiments, a particular arrangement of phosphorothioate linkages is present in at least 80% of the molecules in the population. In certain embodiments, a particular arrangement of phosphorothioate linkages is present in at least 90% of the molecules in the population. In certain embodiments, a particular arrangement of phosphorothioate linkages is present in at least 99% of the molecules in the population.Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, such as those described in Oka et al., JACS 125, 8307 (2003); Wan et al., Nuc. Acid. Res. 42, 13456 (2014); Chapter 10 of Locked Nucleic Acid Aptamers in Nucleic Acid and Peptide Aptamers: Methods and Protocols v535, 2009 by Barciszewski et al., editor Gunter Mayerand; and WO2017 / 015555. In certain embodiments, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one of the indicated phosphorothioates in the (Sp) configuration. In another embodiment, the population of modified oligonucleotides is enriched for modified oligonucleotides having at least one of the indicated phosphorothioates in the (Rp) configuration.

[0054] In certain embodiments, modified oligonucleotide comprises one or more nucleosides that contain unmodified nucleobase.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that contain modified nucleobase.In certain embodiments, modified oligonucleotide comprises one or more nucleosides that do not contain nucleobase, which are called abasic nucleosides.

[0055] In certain embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and O-6 substituted purines. In certain embodiments, the modified nucleobase is selected from 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-hydroxyl, 8-aza. and other 8-substituted purines, 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G-clamp).Modified nucleobases can also include bases in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further examples of the nucleobase include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288, and those disclosed in Chapters 6 and 15 of Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, 163-166 and 442-443.

[0056] In certain embodiments, the modified sugar moiety is a non-bicyclic modified sugar moiety that includes a furanosyl ring bearing one or more substituents, none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non-bridging substituents may be at any position, including, but not limited to, substituents at the 2-, 4-, and / or 5-positions of the furanosyl. In certain embodiments, one or more of the non-bridging substituents of the non-bicyclic modified sugar moiety is branched. Examples of suitable 2-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 2-F, 2-OCH3 ("OMe" or "O-methyl"), and 2-O(CH2)2OCH3 ("MOE"). In certain embodiments, the 2-substituent is halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, OC 1-10 Alkoxy, OC 1-10 Substituted alkoxy, OC 1-10 Alkyl, OC 1-10 Substituted alkyl, S-alkyl, N(R m )-Alkyl, O-Alkenyl, S-Alkenyl, N(R m)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl (alkylenyl)-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(R m )(R n ), or OCH2C(=O)-N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or an unsubstituted C 1-10 and alkyl, where the 2-substituent may be further substituted with one or more substituents independently selected from among hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro(NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl, and alkynyl. Examples of suitable 4'-substituents for non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), and alkyl. Examples of suitable 5-substituents for non-bicyclic modified sugar moieties include, but are not limited to, 5-methyl (R or S), 5-vinyl, and 5-methoxy. In certain embodiments, non-bicyclic modified sugar moieties include multiple non-bridging sugar substituents, such as, for example, 2-F-5-methyl sugar moieties.

[0057] In certain embodiments, the 2'-substituted non-bicyclic modified nucleoside is selected from the group consisting of F, NH, N, OCF, OCH, O(CH)NH, CHCH=CH, OCHCH=CH, OCHCHOCH, O(CH)SCH, O(CH)ON(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, -O-(CH2) n CH3 (wherein n is 13 to 17, and in a preferred embodiment, n is 15), and N-substituted acetamide (OCH2C(=O)-N(R m )(R n )) wherein each R m and R nare independently H, an amino protecting group, or a substituted or unsubstituted C 1-10 In one embodiment, the 2'-substituent is -O-hexadecyl (C16).

[0058] In certain embodiments, the 2'-substituted nucleoside non-bicyclic modified nucleoside comprises a sugar moiety containing a non-bridging 2'-substituent selected from F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, and OCH2C(=O)-N(H)CH3 ("NMA").

[0059] In certain embodiments, 2'-substituted non-bicyclic modified nucleosides comprise a sugar moiety that includes a non-bridging 2'-substituent selected from F, OCH3, and OCH2CH2OCH3. In one embodiment, the 2'-substituent is F. In another embodiment, the 2'-substituent is OCH3 (methoxy).

[0060] 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 4 and 2 furanose ring atoms. Examples of such 4 to 2 bridged sugar substituents include 4-CH2-2, 4-(CH2)2-2, 4-(CH2)3-2, 4-CH2-O-2 ("LNA"), 4-CH2-S-2, 4-(CH2)2-O-2 ("ENA"), 4-CH(CH3)-O-2 (referred to as "constrained ethyl" or "cEt"), 4-CH2-O-CH2-2, 4-CH2-N (R)-2, 4-CH(CH2OCH3)-O-2 ("constrained MOE" or "cMOE") and its analogues, 4-C(CH3)(CH3)-O-2 and its analogues, 4-CH2-N(OCH3)-2 and its analogues, 4-CH2-ON(CH3)-2, 4-CH2-C(H)(CH3)-2, 4-CH2-C(=CH2)-2 and its analogues, 4-C(R a R b )-N(R)-O-2, 4-C(R a R b)-ON(R)-2, 4-CH2-ON(R)-2, and 4-CH2-N(R)-O-2, where each R, R a , and R b are independently H, a protecting group, or C 1-12 In one embodiment, the modified sugar moiety comprises a 4-(CH2)2-O-2 bridge, where "4" represents 4 furanose ring atoms and "2" represents 2 furanose ring atoms ("ENA" or ethylene bridged nucleic acid).

[0061] In certain embodiments, such 4 to 2 bridges are independently -[C(R a )(R b )] n -,-[C(R a )(R b )] n -O-, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a )-, wherein x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R a and R b are independently H, a protecting group, a hydroxyl, C 1-12 Alkyl, substituted C 1-12 Alkyl, C 1-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, Substituted C 2-12 Alkynyl, C 5-20 Aryl, Substituted C 5-20 Aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C 5-7an alicyclic radical, a substituted C5-7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where each J1 and J2 is independently H, C 1-12 Alkyl, substituted C 1-12 Alkyl, C 2-12 Alkenyl, substituted C 2-12 Alkenyl, C 2-12 Alkynyl, Substituted C 2-12 Alkynyl, C 5-20 Aryl, Substituted C 5-20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C 1-12 Aminoalkyl, substituted C 1-12 aminoalkyl, or a protecting group.

[0062] Additional bicyclic sugar moiety, well-known technical field, 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. 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.,US Patent No. 7,053,207、Imanishi et al.,US Patent No. 6,268,490、Imanishi et al. US Patent No. 6,770,748、Imanishi et al.,US.RE44,779、Wengel et al.,US Patent No. 6,794,499、Wengel et al.,US Patent No. 6,670,461、Wengel et al.,US Patent No. 7,034,133、Wengel et al., U.S. Patent No. 8,080,644, Wengel et al., U.S. Patent No. 8,034,909, Wengel et al., U.S. Patent No. 8,153,365, Wengel et al., U.S. Patent No. 7,572,582, and Ramasamy et al., U.S. Patent No. 6,525,191, Torsten et al., WO2004 / 106356, Wengel et al., WO1999 / 014226, Seth et al., WO2007 / 134181, Seth et al., U.S. Patent No. 7,547,684, Seth et al., U.S. Patent No. 7,666,854, Seth et al., U.S. Patent No. 8,088,746, Seth et al., U.S. Patent No. 7,750,131, Seth et al., U.S. Patent No. 8,030,467, Seth et al., U.S. Patent No. 8,268,980, Seth et al.See, U.S. Patent No. 8,546,556, Seth et al., U.S. Patent No. 8,530,640, Migawa et al., U.S. Patent No. 9,012,421, Seth et al., U.S. Patent No. 8,501,805, and U.S. Patent Publications Allerson et al., US2008 / 0039618 and Migawa et al., US2015 / 0191727.

[0063] Another modification of the ASO involves chemically linking to the ASO one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the ASO. Such moieties include lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553-6556), choline acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids, 1997, 1:106-1070), and choline triglycerides (Citrus et al., 1997, 1:106-1070). Res., 1992, 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kavanov et al., FEBS Lett., 1990, 259:327-330; Svinrcuk et al., Biochmie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shaa et al., Nucl. Acids, 1999, 20:533-538), Res., 1990,18:3777-3783), polyamine 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), palmityl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237), or octadecylamine or hexylaminocarbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0064] The present disclosure provides oligonucleotides (modified or unmodified) that can be used to regulate SYF2 expression. Table 1 provides the general sequence of bases (5' to 3') for the SYF2 antisense oligonucleotides or inhibitory nucleic acids of the present disclosure: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0065] In one embodiment, the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having 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: 1-121 in Table 1. In some embodiments, the modified oligonucleotides are at least 80%-100% identical (i.e., 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100%, or any numerical range or numerical value between any of the foregoing values) to any of the sequences comprising or consisting of SEQ ID NOs: 1-121.

[0066] The sequences provided in Table 1 can be used to design antisense molecules for inhibiting SYF2 expression. For example, gapmer oligonucleotides can be designed using the sequences of Table 1 and can include a 5'-wing of about 3-5 nucleotides, a 3'-wing of about 3-5 nucleotides, and a gap region comprising 8-12 consecutive deoxyribonucleosides of any one of the sequences in Table 1. In one embodiment, oligonucleotides of the disclosure include gapmers having a gap segment of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs: 1-121 in Table 1, flanked by a 5' wing segment and a 3' wing segment, where the gap segment is located between the 5' wing segment and the 3' wing segment, and where each of the wing segments comprises a modified sugar. In one embodiment, the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length. In yet another embodiment, an oligonucleotide of the disclosure comprises a 5' wing segment comprising a modified sugar and having a nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs: 1-121, followed by a gap of 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs: 1-121, 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: 1-121. Table 2 shows MOE gapmers of the disclosure.

[0067] The 5' and / or 3' wings may comprise the following chemistries: 2'-OMe, 2'-MOE, LNA, or DNA, either by themselves or in combination with each other. The backbone linkages of the 5' and / or 3' wings may be phosphorothioate or a mixture of phosphodiester and phosphorothioate. The linkages in the gap region may be phosphorothioate.

[0068] In some embodiments, the oligonucleotide is single stranded, hi some embodiments, the oligonucleotide comprises or is complexed with a moiety that neutralizes the charge on the oligonucleotide to facilitate uptake and transport across a cell membrane.

[0069] In one embodiment, each of the ASOs in Table 1 has the following 5-10-5 motif: * 2MOE-2MOE-2MOE-2MOE-N * N * N * N * N * N * N * N * N * N * 2MOE-2MOE-2MOE * 2MOE * 2MOE, where (i) 2MOE is a nucleobase having a 2'-OCHCH-OCH group (i.e., 2'-MOE), (ii) N is a nucleobase, and (iii) an asterisk ( * ) indicates a phosphorothioate bond, and (iv) a dash (-) indicates a phosphodiester bond. Table 2 below shows this motif on SEQ ID NOs: 1-20 and 102-121 (SEQ ID NOs: 122-141 and 142-161).

[0070] Table 2: Sequence of bases in SYF2 antisense oligonucleotides (ASOs). (Gapmer design: 5'-5 2'-methoxyethyl ribose nucleotides-10 DNA nucleotides-5 2'-methoxyethyl ribose nucleosides-3', capital letters are 2'-methoxyethyl ribose nucleosides, lowercase letters are DNA nucleosides, asterisks ( * ) are phosphorothioate linkages, linkages without an asterisk are phosphodiester linkages) (note that in the table below 2'MOE wings are shown, but alternative wings are contemplated, including 2'-OMe or LNA (locked nucleic acid)). [Table 2-1] [Table 2-2]

[0071] The SYF2 antisense or inhibitory nucleic acids of the present disclosure can inhibit the expression of SYF2 and thus SYF2-related activity. The SYF2 antisense or inhibitory nucleic acids can include any combination of the oligonucleotides set forth in Table 2 and sequences that are 98%-99% identical thereto.

[0072] Treatment method Yet another embodiment is a method of treating a subject having a SYF2 disease or disorder by administering a therapeutically effective amount of a SYF2 ASO or pharmaceutical composition described herein.

[0073] One embodiment is a method of treating a subject with a neurological or neurodegenerative disease by administering a therapeutically effective amount of SYF2 ASO or pharmaceutical composition described herein. The neurological disease may be a neurodegenerative disease. The neurodegenerative disease may result in, for example, the degeneration of motor neurons. The neurological disease may be, for example, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, or frontotemporal dementia. Further examples of neurological diseases include, but are not limited to, for example, Parkinson's disease, multiple sclerosis, peripheral myopathy, Rasmussen's encephalitis, attention deficit hyperactivity disorder, autism, central pain syndrome, anxiety, and / or depression.

[0074] Neurological diseases may be associated with abnormal endosomal trafficking. For example, endosomal pathways and endosomes are components necessary 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 support neurotransmission, for example, driving the balance between the recycling and degradation of synaptic vesicles or neurotransmitter receptors.

[0075] Neurological diseases may be associated with abnormal lysosomal degradation. Alterations in lysosomal degradation may be present in neurological diseases, such as neurodegenerative diseases. Cathepsin imbalance during aging and age-related diseases may induce deleterious effects on neurons of the central nervous system (CNS), and lysosomes may be the site of unfolding and partial degradation of membrane proteins or their precursors, which are then expelled from the cell or released from dying cells and accumulate as pathological entities.

[0076] A medical professional may diagnose a subject as having a disease associated with motor neuron degeneration by evaluating one or more symptoms of motor neuron degeneration. To diagnose a neurological disease, a physical examination may be followed by a thorough neurological examination. A neurological examination may evaluate motor and sensory abilities, nerve function, hearing and speech, vision, coordination and balance, mental status, and changes in mood or behavior. Non-limiting symptoms of diseases associated with neurological diseases include weakness in the arms, legs, feet, or ankles, slurred speech, difficulty lifting the front of the foot and toes, weakness or clumsiness in the hands, muscle paralysis, muscle stiffness, involuntary spasms or writing movements (chorea), involuntary persistent muscle contractures (dystonia), bradykinesia, loss of automatic movements, impaired posture and balance, lack of flexibility, tingling in parts of the body, electric shock irritation with head movement, and tingling in the arms, shoulders, and tongue. Symptoms may include convulsions, difficulty swallowing, difficulty breathing, difficulty chewing, partial or complete loss of vision, double vision, slow or abnormal eye movements, tremors, unsteady gait, fatigue, memory loss, dizziness, difficulty thinking or concentrating, difficulty reading and writing, misunderstanding spatial relationships, disorientation, depression, anxiety, difficulty making decisions and judgments, loss of impulse control, difficulty planning and carrying out familiar tasks, aggression, irritability, social withdrawal, mood swings, dementia, changes in sleep habits, wandering, and changes in appetite.

[0077] Tests may be performed to regularize diseases and disorders that may have similar symptoms to neurological diseases, measure muscle involvement, and evaluate neuronal degeneration. Non-limiting examples of tests are electromyogram (EMG), nerve conduction velocity test, blood, urine, or other substance laboratory tests, magnetic resonance imaging (MRI), magnetic resonance spectroscopy, muscle or nerve biopsy, transcranial magnetic stimulation, genetic screening, x-ray, fluoroscopy, angiography, computed tomography (CT), positron emission tomography, cerebrospinal fluid analysis, intrathecal contrast-enhanced CT scan, electroencephalography, electronystagmus, evoked responses, polysomnography, thermography, and ultrasound. The medical professional may also evaluate the patient's family history of diseases related to motor neuron degeneration and make a diagnosis based in part on the family history of neurological diseases. The medical professional may diagnose a disease related to neurological diseases in a subject after the presentation of one or more symptoms.

[0078] Neurodegenerative diseases result in the progressive destruction of neurons, which affects neuronal signal transduction.For example, neurodegeneration can be amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Friedreich's ataxia, Lewy body disease, Parkinson's disease, spinal muscular atrophy, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0079] Diseases related to the degeneration of motor neurons can be pathologies that cause the progressive destruction of motor neurons, which interfere 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 signals to muscles to cause voluntary muscle activity.The destruction of upper and lower motor neurons can affect activities such as breathing, speaking, swallowing, and walking, and over time these functions can be lost.Examples of motor neuron diseases include, but are not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, and pseudobulbar palsy.

[0080] Neuronal hyperexcitability can occur when receptors for the excitatory neurotransmitter glutamate (glutamate receptors), such as NMDA and AMPA receptors, are overactivated by excess glutamate or by other compounds or neurotransmitters that act on glutamate receptors. Excitotoxicity can result from neuronal hyperexcitability. Excitotoxicity is a pathological process in which nerve cells are damaged or killed by excessive stimulation. Excessive stimulation can result in high levels of calcium ions (Ca 2+ ) can enter the cell. 2+ The influx of IL-1 activates a large number of enzymes, including phospholipases, endonucleases, and proteases such as calpains, which can damage cellular structures such as components of the cytoskeleton, membranes, and DNA.

[0081] Neuronal hyperexcitability may be involved in spinal cord injury, stroke, traumatic brain injury, hearing loss (due to overexposure to noise or ototoxicity), epilepsy, painful neuropathy, attention deficit hyperactivity disorder, autism, central pain syndromes, neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, frontotemporal dementia, schizophrenia, Rasmussen's encephalitis, Huntington's disease, alcoholism or withdrawal, especially excessive withdrawal from benzodiazepines, and Huntington's disease. Another common condition that causes excessive glutamate concentrations around neurons is hypoglycemia. Blood glucose is the primary way that glutamate is removed from the intersynaptic space of NMDA and AMPA receptor sites.

[0082] The therapeutic method described herein may comprise administering to a subject in need of treatment a composition comprising an effective amount of one or more antisense oligonucleotides that inhibit SYF2 expression and thereby treat neurological disease.The one or more antisense oligonucleotides may reduce or inhibit neurodegeneration.The one or more antisense oligonucleotides may reduce neuronal hyperexcitability.

[0083] The composition may inhibit kinase activity by inhibiting expression of the kinase. The composition may inhibit activity or expression of SYF2. One or more antisense oligonucleotides may be used in combination with a small molecule therapeutic agent, such as apilimod and / or YM201636.

[0084] The treatment method may include any number of ways of administering the disclosed composition. The administration methods may include aqueous solutions, lipid solutions, oily solutions, or other solutions, solutions in simulated cerebrospinal fluid, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, and the like. Usually, the ASO of the present disclosure is administered directly to the CNS of the subject. Thus, the formulation or composition will be sterile and more preferably suitable for injection. The following formulations and methods are merely exemplary and are in no way limiting.

[0085] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.The preparations may be provided in unit-dose or multi-dose sealed containers such as ampoules and vials, and may be stored for injection as liquid or in a freeze-dried (lyophilized) state that only requires the addition of a sterile liquid excipient, such as water, immediately before use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.The preparations may be provided in pre-filled syringes.

[0086] The additional therapeutic agent(s) may be administered simultaneously or sequentially with the one or more antisense or inhibitory nucleic acids and compositions disclosed. Sequential administration includes administration before or after the one or more antisense or inhibitory nucleic acids or compositions disclosed. In some embodiments, the additional therapeutic agent(s) may be administered in the same composition as the one or more antisense or inhibitory nucleic acids disclosed. In other embodiments, there may be a time interval between administration of the additional therapeutic agent and the one or more antisense or inhibitory nucleic acids disclosed. In some embodiments, administering the additional therapeutic agent with the one or more antisense or inhibitory nucleic acids disclosed may allow the other therapeutic agent to be administered at a lower dose and / or at a shorter interval. When combined with one or more other active ingredients, the one or more antisense or inhibitory nucleic acids and other active ingredients of the present disclosure may be used at a lower dose than when each is used alone. Thus, the pharmaceutical compositions of the present disclosure include pharmaceutical compositions that contain one or more other active ingredients in addition to one or more antisense or inhibitory nucleic acids of the present disclosure. The above combinations include combining one or more antisense or inhibitory nucleic acids of the present disclosure with not only one other active compound, but two or more other active compounds. For example, the compounds of the present disclosure may be used in combination with various drugs to treat neurological diseases.Antisense oligonucleotides may be covalently linked to other oligonucleotides, such as those with targets other than SYF2.Antisense oligonucleotides may be covalently linked to antibodies.

[0087] One or more of the disclosed antisense or inhibitory nucleic acids can be used in combination with, but not limited to, anticholinergics, anticonvulsants, antidepressants, benzodiazepines, congestives, muscle relaxants, analgesics, and / or stimulants. Additional types of therapies and treatments include, but are not limited to, digital communication devices, feeding tubes, ventilators, nutritional supplements, deep brain stimulation, occupational therapy, physical therapy, and / or speech therapy.

[0088] The disclosed composition(s) can be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which can be human or non-human). The pharmaceutical composition can include a carrier (e.g., a pharma- ceutically acceptable carrier). Any suitable carrier can be used within the context of the present disclosure, and such carriers are well known in the art. The choice of carrier is determined, in part, by the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Thus, there are a wide variety of suitable formulations of the compositions of the present invention.

[0089] The pharmaceutical composition may comprise a therapeutically effective amount or a prophylactically effective amount of the antisense oligonucleotide. The therapeutically effective amount of the composition may be determined by those skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to induce a desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or harmful effects of one or more antisense or inhibitory nucleic acids of the present disclosure outweigh the beneficial effects of treatment. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired prophylactic result. Usually, the prophylactically effective amount is less than the therapeutically effective amount, since the prophylactic dose is used before or at an early stage of the disease in the subject.

[0090] Pharmaceutical compositions may include one or more pharma- ceutically acceptable carriers. The term "pharma-ceutically acceptable carrier" as used herein means any type of non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. Some examples of substances that can serve as pharma-ceutically acceptable carriers include sugars, such as but not limited to lactose, glucose, and sucrose; starches, such as but not limited to corn starch and potato starch; cellulose and its derivatives, such as but not limited to sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as but not limited to cocoa butter and suppository wax; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. ethyl alcohol, and phosphate buffers, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as release agents, coating agents, preservatives, and antioxidants may also be present in the composition, at the discretion of the formulator.

[0091] The route by which one or more of the disclosed antisense or inhibitory nucleic acids are to be administered and the form of the composition will determine the type of carrier used.

[0092] Depending on whether local or systemic treatment is desired and the area to be treated, the pharmaceutical composition of the present disclosure can be administered in many ways.Administration can be intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or parenteral administration, including intracranial, for example, intrathecal, intraventricular, or intraventricular administration.In one embodiment, antisense or inhibitory nucleic acid is administered intravenously, intraperitoneally, or as a bolus injection, or directly into the target organ.In another embodiment, antisense or inhibitory nucleic acid is administered intrathecally or intraventricularly as a bolus injection.

[0093] Carriers for systemic administration typically include at least one of a solvent, diluent, lubricant, binder, disintegrant, colorant, flavorant, sweetener, antioxidant, preservative, glidant, vehicle, suspending agent, wetting agent, surfactant, combinations thereof, etc. All carriers are optional in the composition.

[0094] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols such as glycerin; mannitol; and sorbitol.

[0095] Suitable lubricants include liquid lubricants such as silica, talc, stearic acid and its magnesium and calcium salts, calcium sulfate, as well as polyethylene glycols and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0096] Suitable binders include polyvinylpyrrolidone, magnesium aluminum silicate, starches such as corn starch and potato starch, gelatin, tragacanth, and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in the systemic composition is usually about 5 to about 50%.

[0097] Suitable disintegrants include agar, alginic acid and its sodium salt, effervescent mixture, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in the systemic composition is usually about 0.1 to about 10%.

[0098] Suitable coloring agents include colorants such as the FD&C dyes. If used, the amount of coloring agent in the systemic or topical composition is typically about 0.005 to about 0.1%.

[0099] Suitable flavors include menthol flavors, peppermint flavors, and fruit flavors. The amount of flavor(s) when used in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0100] Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically from about 0.1 to about 5%.

[0101] Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.

[0102] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0103] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oil, alcohols such as ethanol, and phosphate buffers. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.

[0104] Suitable suspending agents include AVICEL RC-591 (FMC Corporation of Philadelphia, Pa.) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0105] Suitable surfactants include lecithin, polysorbate 80, and sodium lauryl sulfate, as well as TWEENS from Atlas Powder Company, Wilmington, Delaware.Suitable surfactants include those disclosed in CTFA Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp.335-337; and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp.236-239.The amount of surfactant(s) in the systemic or topical composition is usually about 0.1% to about 5%.

[0106] Compositions and formulations for parenteral, intraspinal, intraventricular, or intracerebroventricular administration may include sterile aqueous solutions that further contain other suitable additives, including but not limited to buffers, diluents, and penetration enhancers, carrier compounds, and other pharma- ceutical acceptable carriers or excipients. For example, an intrathecal cerebrospinal fluid (CSF) catheter can be used to deliver the antisense formulations of the present disclosure. The catheter can be inserted at the L3 or L4 vertebra. The distal end of the catheter extends within the intrathecal space to approximately the L1 vertebra. The antisense oligonucleotide is dissolved in saline, filter sterilized, and administered at 0.33 ml / min in a volume of 1.0 ml, followed by a 0.5 ml flush of sterile water. The total infusion time is 4.5 minutes.

[0107] Compositions for parenteral administration usually contain 0.1% to 10% of active substance and 90% to 99.9% of carrier, including diluents and solvents.

[0108] The amount of carrier used in combination with the disclosed compounds is sufficient to provide a practical amount of the composition for administration per unit dose of drug.Techniques and compositions for preparing dosage forms useful in the method of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0109] In vivo testing of the candidate antisense or inhibitory nucleic acid may be performed by means known to those skilled in the art. For example, one or more of the candidate antisense or inhibitory nucleic acids may be administered to a mammal, such as a mouse or rabbit. The mammal may be administered a dose of the candidate antisense or inhibitory nucleic acid by any route deemed appropriate. The animal may then be monitored for signs of reduction or improvement of motor neuron activity and / or expression or activity of the SYF2 gene or protein, respectively, using conventional methods and criteria. If desired, the results obtained in the presence of the candidate antisense or inhibitory nucleic acid may be compared with the results in a control animal not treated with the candidate antisense or inhibitory nucleic acid. Dosage testing may be performed with or in conjunction with the methods described herein and / or any additional testing of the candidate antisense or inhibitory nucleic acid to identify one or more antisense or inhibitory nucleic acids capable of treating a neurological disease. Those skilled in the art of medicine may determine the appropriate dose of one or more antisense or inhibitory nucleic acids. The dose may be determined by monitoring the subject for signs of inhibition or improvement of the disease. The dose may be increased or decreased to obtain the desired frequency of treatment. The toxicity and efficacy of one or more antisense or inhibitory nucleic acids may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, determining the dose lethal to 50% of the population (LD50) and the dose therapeutically effective to 50% of the population (ED50). The dose ratio LD50 / ED50 is the therapeutic index, which indicates the ratio of toxic to therapeutic effects. Delivery systems may be designed to help prevent toxic side effects by specifically delivering one or more antisense or inhibitory nucleic acids to specific targets, for example, motor neurons or central nervous system neurons. For example, the optimal dose of one or more antisense or inhibitory nucleic acids for analyzing excitability in peripheral nerves may be determined based on the results of clinical electrophysiology or electromyography.

[0110] Dosages for use in humans may be determined by evaluating data obtained from animal tests and cell culture assays. A preferred dose is one that produces little or no toxicity, including the ED50. Doses may vary depending on the formulation and route of administration. For any antisense or inhibitory nucleic acid used in the methods described herein, a dose may be estimated initially in cell culture. A dose may be formulated in an animal model that contains a concentration of the test compound that achieves half-maximal inhibition of symptoms (LD50) as determined in cell culture. Such information obtained from cell cultures and animal models may be used to more accurately determine useful doses in humans.

[0111] In another embodiment, the antisense oligonucleotide of the present invention is expressed from a transgene, for example, as an antisense RNA transcript. The transgene may be administered to a subject in a DNA expression construct modified to express antisense RNA transcript in the subject. The DNA expression construct may be administered directly or using a viral vector (e.g., recombinant AAV (rAAV) vector) or other suitable vector. Viral vectors that have been used in gene therapy protocols include retroviruses, polioviruses or other RNA viruses such as Sindbis virus, adenoviruses, adeno-associated viruses (AAV), herpes viruses, SV40, vaccinia, lentiviruses, and other DNA viruses.

[0112] The present invention has multiple aspects, which are illustrated by the following non-limiting examples. EXAMPLES

[0113] ASOs are an attractive therapeutic option for neurodegenerative diseases because they are easily delivered to the CNS and have relatively little peripheral exposure. These properties maximize target engagement in the CNS while minimizing unwanted target engagement or off-target effects in the periphery.

[0114] The present disclosure provides novel antisense oligonucleotide (ASO) sequences targeting the SYF2 gene that can suppress the expression of SYF2 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 improving motor function.

[0115] 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 maintaining the ability to be used as a substrate for RNaseH. Taken together, these results show that several SYF2 ASOs suppress the expression of SYF2 in human cells (HeLa), as shown in Figures 1A-1C. NCASO was used as a control and values ​​were normalized to GapdH. Figure 1A shows the SYF2 ASO screen in HeLa cells measuring fold change normalized to GadpH for ASOs 50-74 (SEQ ID NOs: 50-74). Figure 1B shows the SYF2 ASO screen in HeLa cells measuring fold change normalized to GadpH for ASOs 75-100 (SEQ ID NOs: 75-100). FIG. 1C shows SYF2 ASO screening in HeLa cells measuring fold change normalized to GadpH for ASO101-121 (SEQ ID NOs: 101-121).

[0116] Thus, the present disclosure provides ASOs that suppress SYF2 expression in human cells, and accompanying data suggest that these ASOs may be able to prevent neurodegeneration in ALS and FTD patients.

[0117] The above description and drawings should be considered as illustrative only of the principles of the invention. The invention is not intended to be limited by the preferred embodiment, but may be implemented in various ways apparent to those skilled in the art. Numerous applications of the invention will readily occur to those skilled in the art. It is therefore not desired to limit the invention to the specific examples disclosed, or to the exact construction and operation shown and described. Rather, all suitable modifications and equivalents falling within the scope of the invention may be utilized. All references cited herein are incorporated by reference.

Claims

1. A single-stranded antisense oligonucleotide that inhibits expression of SYF2, said antisense oligonucleotide having a nucleobase sequence comprising at least 12 or 15 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 1-121.

2. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide has a nucleobase sequence of any one of SEQ ID NOs: 1 to 161.

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

4. 10. The antisense oligonucleotide of any of the preceding claims, 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 internucleotide linkage is a phosphorothioate internucleoside linkage.

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

8. 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. 2. The antisense oligonucleotide of any 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. 2. The antisense oligonucleotide of any preceding claim, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.

12. 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 a 2'-F.

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

15. 2. The antisense oligonucleotide of any preceding claim, wherein the antisense oligonucleotide is a gapmer.

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

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

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

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

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

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

22. 10. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of the preceding claims and a pharma- ceutically acceptable carrier, diluent and / or excipient.

23. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for parenteral delivery.

24. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for intraventricular injection.

25. 23. A method of treating a subject having a neurological or neurodegenerative disease in need of treatment comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 22.

26. 26. The method of claim 25, wherein the neurological disorder is associated with neuronal hyperexcitability.

27. 26. The method of claim 25, wherein the neurological disorder is associated with abnormal endosomal trafficking.

28. 26. The method of claim 25, wherein the neurological disorder is associated with abnormal lysosomal trafficking.

29. 26. The method of claim 25, 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 2A and 4B, Huntington's disease, dementia, transmissible spongiform encephalopathies, spinal-bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, spinocerebellar ataxia, and Creutzfeldt-Jakob disease.

30. 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: 1-121.

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