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Definitions

• This application relates generally to methods of treating neurological diseases with antisense oligonucleotides, in particular, antisense oligonucleotides with one or more spacers that target a transcript.
• Motor neuron diseases are a class of neurological diseases that result in the degeneration and death of motor neurons - those neurons which coordinate voluntary movement of muscles by the brain. Motor neuron diseases may be sporadic or inherited, and may affect upper motor neurons and/or lower motor neurons. Motor neuron diseases include amyotrophic lateral sclerosis, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, and post-polio syndrome.
• ALS Amyotrophic lateral sclerosis
• ALS is a group of motor neuron diseases affecting about 15,000 individuals in the United States of America. ALS is characterized by degeneration and death of upper and lower motor neurons, resulting in loss of voluntary muscle control. Motor neuron death is accompanied by muscle fasciculation and atrophy. Early symptoms of ALS include muscle cramps, muscle spasticity, muscle weakness (for example, affecting an arm, a leg, neck, or diaphragm), slurred and nasal speech, and difficulty chewing or swallowing. Loss of strength and control over movements, including those necessary for speech, eating, and breathing, eventually occur.
• ALS occurs in individuals of all ages, but is most common in individuals between 55 to 75 years of age, with a slightly higher incidence in males. ALS can be characterized as sporadic or familial. Sporadic ALS appears to occur at random and accounts for more than 90% of all incidences of ALS. Familial ALS accounts for 5-10% of all incidences of ALS.
• FTD refers to a spectrum of progressive neurodegenerative diseases caused by loss of neurons in frontal and temporal lobes of the brain.
• FTD is the third most common form of dementia (following Alzheimer’s disease and dementia with Lewy bodies), and the second most common form of dementia in individuals below 65 years of age.
• FTD is estimated to affect 20,000 to 30,000 individuals in the United States of America.
• FTD is characterized by changes in behavior and personality, and language dysfunction.
• Forms of FTD include behavioral variant FTD (bvFTD), semantic variant primary progressive aphasia (svPPA), and nonfluent variant primary progressive aphasia (nfvPPA).
• ALS with FTD is characterized by symptoms associated with FTD, along with symptoms of ALS such as muscle weakness, atrophy, fasciculation, spasticity, speech impairment (dysarthria), and inability to swallow (dysphagia). Individuals usually succumb to FTD within 5 to 10 years, while ALS with FTD often results in death within 2 to 3 years of the first disease symptoms appearing.
• ALS amyotrophic lateral sclerosis
• FTD frontotemporal dementia
• ALS with FTD Alzheimer’s disease
• AD Alzheimer’s disease
• PD Parkinson’s disease
• Huntington’s disease progressive supranuclear palsy
• brain trauma spinal cord injury
• corticobasal degeneration CBD
• nerve injuries e.g., brachial plexus injuries
• neuropathies e.g., chemotherapy induced neuropathy
• TDP43 proteinopathies e.g., chronic traumatic encephalopathy, Perry Syndrome, Dementia with Lewy body in association with Alzheimer’s disease, Parkinson’s disease with or without dementia
• LATE Limbic-predominant age- related TDP-43 encephalopathy
• RNA-binding protein transactive response DNA-binding protein 43 (TDP-43) is involved in fundamental RNA processing activities including RNA transcription, splicing, and transport.
• TDP-43 binds to thousands of pre-messenger RNA/mRNA targets, with high affinity for GU-rich sequences, including autoregulation of its own mRNA via binding to 3’ untranslated region. Reduction in TDP-43 from an otherwise normal adult nervous system alters the splicing or expression levels of more than 1,500 RNAs, including long intron-containing transcripts. See Melamed et al., Nat Neurosci. (2019), 22(2): 180-190.
• TDP-43 has been shown to regulate expression of the neuronal growth-associated factor Stathmin-2 (STMN2).
• STMN2 encodes a protein necessary for normal motor neuron outgrowth and repair.
• STMN2 encodes a protein necessary for normal motor neuron outgrowth and repair.
• TDP-43 disruption is shown to drive premature polyadenylation and aberrant splicing in intron 1 of stathmin-2 pre-mRNA, producing a non-functional mRNA. See Melamed (2019).
• oligonucleotides comprising one or more spacers and comprising a sequence that is between 85 and 98% complementary to an equal length portion of a STMN2 transcript.
• the present disclosure provides STMN2 oligonucleotides that target a STMN2 transcript (for example, a STMN2 transcript comprising a cryptic exon).
• the oligonucleotides target a transcript for the treatment of neurological diseases, including motor neuron diseases, and/or neuropathies.
• STMN2 oligonucleotides can be used to treat PD, ALS, FTD, and ALS with FTD.
• the present disclosure provides a compound comprising a modified oligonucleotide comprising a sequence that is between 85 and 98% complementary to an equal length portion of any one of SEQ ID NO: 1339 or SEQ ID NO: 1341, a sequence having 90% identity thereof, or to a 15 to 50 contiguous nucleobase portion thereof, wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is a non-natural linkage, and further wherein the oligonucleotide comprises a spacer.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides.
• the oligonucleotide comprises a segment with at most 10, 9, or 8 linked nucleosides. In various embodiments, the oligonucleotide comprises a segment with at most 7 linked nucleosides. In certain embodiments, the oligonucleotide comprises a segment with at most 6, 5, 4, 3, or 2 linked nucleosides. In certain embodiments, every segment of the oligonucleotide comprises at most 7 linked nucleosides.
• the oligonucleotide comprises a sequence that shares at least 85% identity with an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-
• the oligonucleotide comprises a sequence that shares at least 90% identity with an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664. In various embodiments, the oligonucleotide comprises a sequence that shares 95% identity with an equal length portion of any one of SEQ ID NOs: 1451-1664. In various embodiments, the oligonucleotide comprises a sequence that shares 100% identity with an equal length portion of any one of SEQ ID NOs: 1451-1664.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion within any one of positions 144-168, 173-197, 185-209, or 237-261 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 144-164, 144-166, 145-167, 146-166, 146-168, 147-165, or 148-168 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 173-191, 173-193, 173-195, 173-197, 175-195, 175-197, 177-197, or 179-197 of SEQ ID NO:
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 185-205, 187-209, 189-209, or 191-209 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 237-255, 237-259, 239-259, 239-261, 241-261, or 243-261 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 6, 5, 4, 3, or 2 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 144-168, 173-197, 185-209, or 237-261 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 6, 5, 4, 3, or 2 linked nucleosides, and wherein the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of nucleobases within any one of positions 144-164, 144-166, 145-167, 146-166, 146-168, 147-165, 148-168, 173-191, 173-193, 173-195, 173-197, 175-195, 175-197, 177-197, 179-197, 185-205, 187-209, 189-209, 191-209, 237-255, 237-259, 239-259, 239-261, 241-261, or 243-261 of SEQ ID NO: 1339.
• the oligonucleotide comprises a segment with at most 11 linked nucleosides or at most 7 linked nucleosides, and wherein the oligonucleotide comprises a sequence that shares at least 85% identity with an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664.
• the oligonucleotide comprises a segment with at most 6, 5, 4, 3, or 2 linked nucleosides, and wherein the oligonucleotide comprises a sequence that shares at least 85% identity with an equal length portion of any one of SEQ ID NOs: 36, 55, 144, 173, 177, 181, 185, 197, 203, 209, 215, 237, 244, 252, 380, 385, 390, 395, 400, 928, 947, 1036, 1065, 1069, 1073, 1077, 1089, 1095, 1101, 1107, 1129, 1136, 1144, 1272, 1277, 1282, 1287, or 1292.
• the oligonucleotide comprises a segment with at most 6, 5, 4, 3, or 2 linked nucleosides, and wherein the oligonucleotide comprises a sequence that shares at least 90% identity with an equal length portion of any one of SEQ ID NOs: 36, 55, 144, 173, 177, 181, 185, 197, 203, 209, 215, 237, 244, 252, 380, 385, 390, 395, 400, 928, 947, 1036, 1065, 1069, 1073, 1077, 1089, 1095, 1101, 1107, 1129, 1136, 1144, 1272, 1277, 1282, 1287, or 1292.
• the oligonucleotide is at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 oligonucleotide units in length. In various embodiments, the oligonucleotide is at least 19 oligonucleotide units in length.
• the spacer is a nucleoside-replacement group comprising a non-sugar substitute that is incapable of linking to a nucleotide base.
• the spacer is located between positions 10 and 15 of the oligonucleotide. In various embodiments, the spacer is located between positions 7 and 11 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a second spacer, wherein the second spacer is located between positions 14 and 22 of the oligonucleotide. In various embodiments, the spacer and the second spacer are separated by at least 5 nucleobases, at least 6 nucleobases, or at least 7 nucleobases in the oligonucleotide.
• the spacer is located between positions 7 and 9 of the oligonucleotide, and wherein the second spacer is located between positions 15 and 18 of the oligonucleotide. In various embodiments, the spacer is located at position 8 of the oligonucleotide, and wherein the second spacer is located at position 16 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a third spacer, wherein the third spacer is located between positions 21 and 24 of the oligonucleotide. [0020] In various embodiments, the spacer is located between positions 2 and 5 of the oligonucleotide.
• the oligonucleotide further comprises a second spacer, wherein the second spacer is located between positions 8 and 12 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a third spacer, wherein the third spacer is located between positions 18 and 22 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a second spacer and a third spacer, wherein the three spacers are located at positions in the oligonucleotide such that each segment of the oligonucleotide has at most 7 linked nucleosides. In various embodiments, at least two of the three spacers are adjacent to a guanine nucleobase. In various embodiments, each of the at least two of the three spacers immediately precede a guanine nucleobase.
• each of the first, second or third spacers is a nucleoside- replacement group comprising a non-sugar substitute wherein the non-sugar substitute does not contain a ketone, aldehyde, ketal, hemiketal, acetal, hemiacetal, aminal or hemiaminal moiety and is incapable of forming a covalent bond with a nucleotide base.
• each of the first, second or third spacers is independently represented by Formula (X), wherein:
• Ring A is an optionally substituted 4-8 member monocyclic cycloalkyl group or a 4-8 member monocyclic heterocyclyl group, wherein the heterocyclyl group contains 1 or 2 heteroatoms selected from O, S and N, provided that A is not capable of forming a covalent bond to a nucleobase; and the symbol represents the point of connection to an internucleoside linkage.
• each of the first, second or third spacers is independently represented by Formula (Xa), wherein: Formula (Xa).
• ring A is an optionally substituted 4-8 member monocyclic cycloalkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl; or a 4-8 member monocyclic heterocyclyl group, selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, 1,4-dioxanyl, pyrolidinyl, piperidinyl, piperazinyl, morpholinyl and azepanyl.
• ring A is tetrahydrofuranyl.
• ring A is tetrahydropyranyl.
• each of the first, second or third spacers is independently represented by Formula I, wherein: Formula (I)
• X is selected from -CFh— and -0-; and n is 0, 1, 2 or 3.
• each of the first, second or third spacers is independently represented by Formula F, wherein: Formula (F)
• each of the first, second or third spacers is independently represented by Formula (la), wherein: Formula (la); and n is 0, 1, 2 or 3.
• each of the first, second or third spacers is independently represented by Formula (la’), wherein: Formula (la’); and n is 0, 1, 2 or 3.
• each of the first, second or third spacers is independently represented by Formula II, wherein: Formula (II); and
• X is selected from -CFh- and -0-.
• each of the first, second or third spacers is independently represented by Formula IF, wherein: Formula (IF); and X is selected from -CFh- and -0-.
• each of the first, second or third spacers is independently represented by Formula (Ha), wherein: Formula (Ila).
• each of the first, second or third spacers is independently represented by Formula (Ha’), wherein: Formula (Ha’).
• each of the first, second or third spacers is independently represented by Formula III, wherein: Formula (III); and X is selected from -CFh- and -O-.
• each of the first, second or third spacers is independently represented by Formula IIF, wherein: Formula (IIF); and X is selected from -CFh- and -0-.
• each of the first, second or third spacers is independently represented by Formula (Ilia), wherein: Formula (Ilia).
• each of the first, second or third spacers is independently represented by Formula (Ilia’), wherein: Formula (Ilia’).
• the oligonucleotide comprising the spacer has a GC content of at least 10%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 20%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 25%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 30%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 40%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 50%.
• the oligonucleotide is between 12 and 40 oligonucleotide units in length.
• At least one (i.e., one or more) nucleoside linkage of the oligonucleotide is independently selected from the group consisting of a phosphodiester linkage, a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3-methoxypropyl phosphonate linkage, a methylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoramidothioate linkage, a thiophosphorodiamidate linkage, a phosphorodiamidate (e.g ., comprising a phosphorodiamidate morpholino (PMO), 3' amino ribose, or 5' amino ribos
• PMO phosphorodiamid
• one or more nucleoside linkages that link a base at position 3 or position 4 of the oligonucleotide are phosphodiester linkages. In various embodiments, only one nucleoside linkage that links a base at position 3 or position 4 of the oligonucleotide is a phosphodiester linkage. In various embodiments, nucleoside linkages that link bases at both position 3 and position 4 of the oligonucleotide are phosphodiester linkages. In various embodiments, one or more bases immediately preceding a spacer in the oligonucleotide are linked through phosphodiester bonds.
• only the base immediately preceding the spacer in the oligonucleotide is linked to the spacer through a phosphodiester bond.
• the base immediately preceding the spacer in the oligonucleotide is further linked to a further preceding base through a phosphodiester bond.
• the oligonucleotide comprises a second spacer, wherein a base immediately preceding the second spacer is linked to a further preceding base through a phosphodiester bond.
• one or more bases immediately succeeding a spacer in the oligonucleotide are linked through phosphodiester bonds. In various embodiments, only the base immediately succeeding the spacer in the oligonucleotide is linked to the spacer through a phosphodiester bond. In various embodiments, two bases immediately preceding the spacer in the oligonucleotide are linked through phosphodiester bonds. In various embodiments, one or more bases immediately preceding a spacer in the oligonucleotide are linked through phosphodiester bonds and wherein one or more bases immediately succeeding the spacer in the oligonucleotide are linked through phosphodiester bonds.
• one base immediately preceding the spacer and one base immediately succeeding the spacer are linked through phosphodiester bonds.
• the oligonucleotide includes a second spacer, and wherein one or more bases immediately preceding the second spacer in the oligonucleotide are linked through phosphodiester bonds and wherein one or more bases immediately succeeding the second spacer in the oligonucleotide are linked through phosphodiester bonds.
• one base immediately preceding the second spacer and one base immediately succeeding the second spacer are linked through phosphodiester bonds.
• the oligonucleotide comprises a range of bases that are linked through phosphodiester bonds, the range of bases comprising at least two bases.
• the oligonucleotide comprises a range of bases that are linked through phosphodiester bonds, the range of bases comprising at least five bases. In various embodiments, the oligonucleotide comprises two or more spacers, and wherein the range of bases are positioned between the at least two spacers. [0044] Additionally disclosed herein is a compound comprising an oligonucleotide comprising a nucleobase sequence that shares at least 90% identity to an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392- 1664.
• an oligonucleotide comprising a nucleobase sequence that shares at least 90% identity to an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664.
• the nucleobase sequence shares at least 95% identity to an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664.
• the nucleobase sequence shares at least 100% identity to an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664.
• the oligonucleotide is any one of a 19mer, 21mer, 23mer, or 25mer.
• an internucleoside linkage of the oligonucleotide is a modified intemucleoside linkage.
• the modified internucleoside linkage of the oligonucleotide is a phosphorothioate linkage.
• all intemucleoside linkages of the oligonucleotide are phosphorothioate linkages.
• the phosphorothioate linkage is in one of a Rp configuration or a rip configuration.
• the oligonucleotide comprises at least one modified sugar moiety.
• the modified sugar moiety is one of a 2'-OMe modified sugar moiety, bicyclic sugar moiety, 2’-0-(2-methoxyethyl) (MOE), 2'-deoxy-2'-fluoro nucleoside, 2’-fluoro-P-D- arabinonucleoside, locked nucleic acid (LNA), a tricyclic nucleic acid, constrained ethyl 2’-4’- bridged nucleic acid (cEt), ri'-cEt, tcDNA, hexitol nucleic acids (HNA), and tricyclic analog (e.g, tcDNA).
• MOE 2-methoxyethyl
• LNA locked nucleic acid
• cEt constrained ethyl 2’-4’- bridged nucleic acid
• HNA hexitol nucleic acids
• tricyclic analog e.g, tcDNA
• the oligonucleotide exhibits at least a 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase of full length STMN2 protein. In various embodiments, the oligonucleotide exhibits at least a 100% increase of full length STMN2 protein. In various embodiments, the oligonucleotide exhibits at least a 200% increase of full length STMN2 protein. In various embodiments, the oligonucleotide exhibits at least a 300% increase of full length STMN2 protein. In various embodiments, the oligonucleotide exhibits at least a 400% increase of full length STMN2 protein.
• increase of the full length STMN2 protein is measured in comparison to a reduced level of full length STMN2 protein achieved using a TDP43 antisense oligonucleotide.
• the oligonucleotide exhibits at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% rescue of full length STMN2 protein.
• the oligonucleotide exhibits at least a 50%, 60%, 70%, 80%, or 90% reduction of a STMN2 transcript with a cryptic exon.
• a method of treating a neurological disease and/or a neuropathy in a patient in need thereof comprising administering to the patient an oligonucleotide of any of the oligonucleotides disclosed above.
• the neurological disease selected from the group consisting of: amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), ALS with FTD, Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, progressive supranuclear palsy (PSP), brain trauma, spinal cord injury, corticobasal degeneration (CBD), nerve injuries (e.g., brachial plexus injuries), neuropathies (e.g., chemotherapy induced neuropathy), and TDP43 proteinopathies (e.g., chronic traumatic encephalopathy, Perry Syndrome, Dementia with Lewy body in association with Alzheimer’s disease, Parkinson’s disease with or without dementia, and Limbic-predominant age-related TDP- 43 encephalopathy (LATE)).
• ALS amyotrophic lateral sclerosis
• FTD frontotemporal dementia
• ALS with FTD Alzheimer’s disease
• AD Alzheimer’s disease
• PD Parkinson’s disease
• Huntington’s disease progressive supran
• the neurological disease is ALS. In various embodiments, the neurological disease is FTD. In various embodiments, the neurological disease is ALS with FTD. In various embodiments, the neuropathy is chemotherapy induced neuropathy. [0048] Additionally disclosed is a method of restoring axonal outgrowth and/or regeneration of a neuron, the method comprising exposing the motor neuron to an oligonucleotide of any of the oligonucleotides disclosed above. Additionally disclosed is a method of increasing, promoting, stabilizing, or maintaining STMN2 expression and/or function in a neuron, the method comprising exposing the cell to an oligonucleotide of any of the oligonucleotides disclosed above.
• the neuron is a neuron of a patient in need of treatment of a neurological disease and/or a neuropathy.
• the neuropathy is chemotherapy induced neuropathy.
• the exposing is performed in vivo or ex vivo.
• the exposing comprises administering the oligonucleotide to a patient in need thereof.
• the oligonucleotide is administered topically, parenterally, intrathecally, intrathalamically, intracistemally, orally, rectally, buccally, sublingually, vaginally, pulmonarily, intratracheally, intranasally, transdermally, or intraduodenally.
• the oligonucleotide is administered orally. In various embodiments, a therapeutically effective amount of the oligonucleotide is administered intrathecally, intrathalamically or intracistemally. In various embodiments, the patient is a human.
• a pharmaceutical composition comprising the oligonucleotide of any one of the oligonucleotides disclosed above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
• the pharmaceutical composition is suitable for topical, intrathecal, intrathalamic, intracisternal, intracerebroventricular, parenteral, oral, pulmonary, intratracheal, intranasal, transdermal, rectal, buccal, sublingual, vaginal, or intraduodenal administration.
• the neurological disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), ALS with FTD,
• Alzheimer’s disease AD
• Parkinson’s disease PD
• Huntington’s disease progressive supranuclear palsy (PSP)
• brain trauma spinal cord injury
• corticobasal degeneration CBD
• nerve injuries e.g., brachial plexus injuries
• neuropathies e.g., chemotherapy induced neuropathy
• TDP43 proteinopathies e.g., chronic traumatic encephalopathy, Perry Syndrome, Dementia with Lewy body in association with Alzheimer’s disease, Parkinson’s disease with or without dementia, and Limbic-predominant age-related TDP-43 encephalopathy (LATE)
• the neurological disease is ALS.
• the neurological disease is FTD.
• the neurological disease is ALS with FTD.
• the neuropathy is chemotherapy induced neuropathy.
• the pharmaceutical composition is administered topically, parenterally, orally, pulmonarily, rectally, buccally, sublingually, vaginally, intratracheally, intranasally, intracisternally, intrathecally, intrathalamically, intravenously, intramuscularly, transdermally, or intraduodenally.
• intrathecally intrathalamically intracerebroventricularly, or intracisternally.
• a therapeutically effective amount of the oligonucleotide is administered intrathecally, intrathalamically or intracisternally.
• the patient is human.
• a method for treating a neurological disease in a subject in need thereof comprising administering to the subject an oligonucleotide comprising a segment with at most 7 linked nucleosides, and wherein oligonucleotide shares at least 85% identity with any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664, or a pharmaceutically acceptable salt thereof; wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is independently selected from the group consisting of: a phosphodiester linkage, a phosphorothioate linkage, an alkyl phosphate linkage,
• oligonucleotide comprising a segment with at most 7 linked nucleosides, and wherein oligonucleotide shares at least 85% identity with any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664, or a pharmaceutically acceptable salt thereof; wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is independently selected from the group consisting of: a phosphodiester linkage, a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3- methoxypropyl phosphonate linkage, a methyl
• oligonucleotide comprising a segment with at most 7 linked nucleosides, and wherein oligonucleotide shares at least 85% identity with any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664, or a pharmaceutically acceptable salt thereof; wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is independently selected from the group consisting of: a phosphodiester linkage, a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3- methoxypropyl phosphonate linkage, a methyl
• a method for treating ALS with FTD in a subject in need thereof comprising administering to the subject an oligonucleotide comprising a segment with at most 7 linked nucleosides, and wherein oligonucleotide shares at least 85% identity with any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664, or a pharmaceutically acceptable salt thereof; wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is independently selected from the group consisting of: a phosphodiester linkage, a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotri ester linkage, an alkylphosphonate linkage, a 3-methoxypropyl phosphonate linkage,
• one or more nucleoside linkages that link a base at position 3 or position 4 of the oligonucleotide are phosphodiester linkages. In various embodiments, only one nucleoside linkage that links a base at position 3 or position 4 of the oligonucleotide is a phosphodiester linkage. In various embodiments, nucleoside linkages that link bases at both position 3 and position 4 of the oligonucleotide are phosphodiester linkages. In various embodiments, one or more bases immediately preceding a spacer in the oligonucleotide are linked through phosphodiester bonds.
• only the base immediately preceding the spacer in the oligonucleotide is linked to the spacer through a phosphodiester bond.
• the base immediately preceding the spacer in the oligonucleotide is further linked to a further preceding base through a phosphodiester bond.
• the oligonucleotide comprises a second spacer, wherein a base immediately preceding the second spacer is linked to a further preceding base through a phosphodiester bond.
• one or more bases immediately succeeding a spacer in the oligonucleotide are linked through phosphodiester bonds. In various embodiments, only the base immediately succeeding the spacer in the oligonucleotide is linked to the spacer through a phosphodiester bond. In various embodiments, two bases immediately preceding the spacer in the oligonucleotide are linked through phosphodiester bonds. In various embodiments, one or more bases immediately preceding a spacer in the oligonucleotide are linked through phosphodiester bonds and wherein one or more bases immediately succeeding the spacer in the oligonucleotide are linked through phosphodiester bonds.
• one base immediately preceding the spacer and one base immediately succeeding the spacer are linked through phosphodiester bonds.
• the oligonucleotide includes a second spacer, and wherein one or more bases immediately preceding the second spacer in the oligonucleotide are linked through phosphodiester bonds and wherein one or more bases immediately succeeding the second spacer in the oligonucleotide are linked through phosphodiester bonds.
• one base immediately preceding the second spacer and one base immediately succeeding the second spacer are linked through phosphodiester bonds.
• the oligonucleotide comprises a range of bases that are linked through phosphodiester bonds, the range of bases comprising at least two bases.
• the oligonucleotide comprises a range of bases that are linked through phosphodiester bonds, the range of bases comprising at least five bases. In various embodiments, the oligonucleotide comprises two or more spacers, and wherein the range of bases are positioned between the at least two spacers. In various embodiments, the oligonucleotide is any one of a 19mer, 21mer, 23mer, or 25mer.
• At least one (i.e., one or more) internucleoside linkage of the oligonucleotide is a phosphorothioate linkage. In various embodiments, all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.
• an oligonucleotide and a pharmaceutically acceptable excipient comprising a sequence that is between 85 and 98% complementary to an equal length portion of any one of SEQ ID NO: 1339 or SEQ ID NO: 1341, a sequence having 90% identity thereof, or to a 15 to 50 contiguous nucleobase portion thereof, wherein the oligonucleotide comprises a spacer and wherein the oligonucleotide is capable of increasing, restoring, or stabilizing expression of the STMN2 mRNA capable of translation of a functional STMN2 and/or activity and/or function of STMN2 protein in a cell or a human patient of an immune-mediated demyelinating disease, and wherein the level of increase, restoration, or stabilization of expression and/or activity and/or function is sufficient for use of the oligonucleotide as a medicament for the treatment of the immune-mediated demyelinating disease.
• the oligonucleotide comprises one or more chiral centers and/or double bonds.
• the oligonucleotide exist as stereoisomers selected from geometric isomers, enantiomers, and diastereomers.
• a method of treating a neurological disease and/or a neuropathy in a patient in need thereof comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed above, in combination with a second therapeutic agent.
• the second therapeutic agent is selected from Riluzole (Rilutek), Edaravone (Radicava), rivastigmine, donepezil, galantamine, selective serotonin reuptake inhibitor, antipsychotic agents, cholinesterase inhibitors, memantine, benzodiazepine antianxiety drugs, AMX0035 (ELYBRIO), ZILUCOPLAN (RA101495), pridopidine, dual AON intrathecal administration (e.g, BIIBG67, BIIBG78, and BUB 105), B IIBIOO, levodopa/carbidopa, dopaminergic agents (e.g, ropinirole, pramipexole, rotigotine), medroxyprosterone, KCNQ2/KCNQ3 openers (e.g., retigabine, XEN1101, QRL-101), anticonvulsants and psychostimulant agents, and/or a therapy (e
• a method of treating a neurological disease and/or a neuropathy in a patient in need thereof comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition disclosed above, wherein at least one (i.e., one or more) nucleoside linkage of the oligonucleotide is a non-natural linkage, wherein the oligonucleotide comprises a spacer, and wherein the oligonucleotide further comprises a targeting or conjugate moiety selected from cholesterol, lipoic acid, panthothenic acid, polyethylene glycol, and an antibody for crossing the blood brain barrier.
• the spacer is a nucleoside-replacement group comprising a non sugar substitute that is incapable of linking to a nucleotide base. In various embodiments, the spacer is located between positions 10 and 15 of the oligonucleotide. In various embodiments, the spacer is located between positions 7 and 11 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a second spacer, wherein the second spacer is located between positions 14 and 22 of the oligonucleotide.
• the spacer and the second spacer are separated by at least 5 nucleobases, at least 6 nucleobases, or at least 7 nucleobases in the oligonucleotide. In various embodiments, the spacer is located between positions 7 and 9 of the oligonucleotide, and wherein the second spacer is located between positions 15 and 18 of the oligonucleotide. In various embodiments, the spacer is located at position 8 of the oligonucleotide, and wherein the second spacer is located at position 16 of the oligonucleotide.
• the oligonucleotide further comprises a third spacer, wherein the third spacer is located between positions 21 and 24 of the oligonucleotide. In various embodiments, the spacer is located between positions 2 and 5 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a second spacer, wherein the second spacer is located between positions 8 and 12 of the oligonucleotide. In various embodiments, the oligonucleotide further comprises a third spacer, wherein the third spacer is located between positions 18 and 22 of the oligonucleotide.
• the oligonucleotide further comprises a second spacer and a third spacer, wherein the three spacers are located at positions in the oligonucleotide such that each segment of the oligonucleotide has at most 7 linked nucleosides.
• At least two of the three spacers are adjacent to a guanine nucleobase. In various embodiments, each of the at least two of the three spacers immediately precede a guanine nucleobase.
• each of the first, second or third spacers is a nucleoside-replacement group comprising a non-sugar substitute wherein the non-sugar substitute does not contain a ketone, aldehyde, ketal, hemiketal, acetal, hemiacetal, aminal or hemiaminal moiety and is incapable of forming a covalent bond with a nucleotide base.
• each of the first, second or third spacers is independently represented by Formula (X), wherein:
• Ring A is an optionally substituted 4-8 member monocyclic cycloalkyl group or a 4-8 member monocyclic heterocyclyl group, wherein the heterocyclyl group contains 1 or 2 heteroatoms selected from O, S and N, provided that A is not capable of forming a covalent bond to a nucleobase; and the ⁇ symbol represents the point of connection to an internucleoside linkage.
• each of the first, second or third spacers is independently represented by Formula (Xa), wherein: Formula (Xa).
• ring A is an optionally substituted 4-8 member monocyclic cycloalkyl group selected from cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl; or a 4-8 member monocyclic heterocyclyl group, selected from oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, 1,4-dioxanyl, pyrolidinyl, piperidinyl, piperazinyl, morpholinyl and azepanyl.
• ring A is tetrahydrofuranyl.
• ring A is tetrahydropyranyl.
• each of the first, second or third spacers is independently represented by Formula (I), wherein:
• X is selected from -CFh- and -O-; and n is 0, 1, 2 or 3.
• X is selected from -CFh- and -O-; and n is 0, 1, 2 or 3.
• Formula (F) wherein:
• X is selected from -CFh- and -O-; and n is 0, 1, 2 or 3.
• each of the first, second or third spacers is independently represented by Formula (la), wherein:
• each of the first, second or third spacers is independently represented by Formula (la’), wherein:
• each of the first, second or third spacers is independently represented by Formula II, wherein:
• Formula (II); and X is selected from -CH2- and -0-.
• each of the first, second or third spacers is independently represented by Formula IF, wherein:
• X is selected from -CH2- and -0-.
• each of the first, second or third spacers is independently represented by Formula (Ha), wherein: Formula (Ila).
• each of the first, second or third spacers is independently represented by Formula (Ha’), wherein: Formula (Ha’).
• each of the first, second or third spacers is independently represented by Formula III, wherein: Formula (III); and X is selected from -CH2- and -O-.
• each of the first, second or third spacers is independently represented by Formula IIF, wherein: Formula (IIF); and X is selected from -CH2- and -0-.
• each of the first, second or third spacers is independently represented by Formula (Ilia), wherein:
• each of the first, second or third spacers is independently represented by Formula (Ilia’), wherein: [0084]
• the oligonucleotide comprising the spacer has a GC content of at least 10%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 20%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 25%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 30%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 40%. In various embodiments, the oligonucleotide comprising the spacer has a GC content of at least 50%.
• FIG. l is a schematic depiction of portions of the STMN2 transcript and STMN2 antisense oligonucleotides that are designed to target certain portions of the STMN2 transcript.
• FIG. 2 is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 6 different STMN2 parent oligonucleotides (SEQ ID NO: 36, SEQ ID NO: 55, SEQ ID NO: 177, SEQ ID NO: 203, SEQ ID NO: 244, and SEQ ID NO: 395).
• FIG. 3 is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 6 different STMN2 parent oligonucleotides (SEQ ID NO: 173, SEQ ID NO: 181, SEQ ID NO: 197, SEQ ID NO: 215, SEQ ID NO: 385, and SEQ ID NO: 400).
• FIG. 4 is a bar graph showing the results of RT-qPCR analysis of STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 6 different STMN2 parent oligonucleotides (SEQ ID NO: 173, SEQ ID NO: 181, SEQ ID NO: 197, SEQ ID NO: 215, SEQ ID NO: 385, and SEQ ID NO: 400).
• FIG. 5 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 6 different STMN2 parent oligonucleotides (SEQ ID NO: 185, SEQ ID NO: 209, SEQ ID NO: 237, SEQ ID NO: 252, SEQ ID NO: 380, and SEQ ID NO: 390).
• FIG. 5B is a bar graph showing the results of RT-qPCR analysis of STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 6 different STMN2 parent oligonucleotides (SEQ ID NO: 185, SEQ ID NO: 209, SEQ ID NO: 237, SEQ ID NO: 252, SEQ ID NO: 380, and SEQ ID NO: 390).
• FIG. 6A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 2 different STMN2 parent oligonucleotides (SEQ ID NO: 144 and SEQ ID NO: 237) over two duplicate experiments.
• FIG. 6B is a bar graph showing the results of RT-qPCR analysis of STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 2 different STMN2 parent oligonucleotides (SEQ ID NO: 144 and SEQ ID NO: 237) over two duplicate experiments.
• FIG. 7A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 5 different STMN2 parent oligonucleotides (SEQ ID NO: 36, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, and SEQ ID NO: 185).
• SEQ ID NO: 36 SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, and SEQ ID NO: 185.
• 7B is a bar graph showing the results of RT-qPCR analysis of STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 5 different STMN2 parent oligonucleotides (SEQ ID NO: 36, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181, and SEQ ID NO: 185).
• FIG. 8 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 5 different STMN2 parent oligonucleotides (SEQ ID NO: 197, SEQ ID NO: 203, SEQ ID NO: 237, SEQ ID NO: 380, and SEQ ID NO: 395).
• FIG. 8B is a bar graph showing the results of RT-qPCR analysis of STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 5 different STMN2 parent oligonucleotides (SEQ ID NO: 197, SEQ ID NO: 203, SEQ ID NO: 237, SEQ ID NO: 380, and SEQ ID NO: 395).
• FIG. 9A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels in the presence of 3 different STMN2 parent oligonucleotides (SEQ ID NO: 144, SEQ ID NO: 173, and SEQ ID NO: 237).
• SEQ ID NO: 144 SEQ ID NO: 173, and SEQ ID NO: 237
• 9B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript in the presence of 3 different STMN2 parent oligonucleotides (SEQ ID NO: 144, SEQ ID NO: 173, and SEQ ID NO: 237).
• FIG. 10A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 181 STMN2 parent oligonucleotide.
• FIG. 10B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 181 STMN2 parent oligonucleotide.
• FIG. 11 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• FIG. 11 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• IB is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• 12A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 197 STMN2 parent oligonucleotide.
• FIG. 12B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 197 STMN2 parent oligonucleotide.
• FIG. 13A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a
• SEQ ID NO: 144 STMN2 parent oligonucleotide SEQ ID NO: 144 STMN2 parent oligonucleotide.
• FIG. 13B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 144 STMN2 parent oligonucleotide.
• FIG. 14A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 14A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• 14B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 15A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 15A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 15B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 16 is a protein blot and quantified bar graph showing the normalized quantity of
• STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript for 2 different STMN2 parent oligonucleotides SEQ ID NO: 173 and SEQ ID NO: 237).
• FIG. 17A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 17B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 18A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• FIG. 18B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• FIG. 18B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 185 STMN2 parent oligonucleotide.
• 19A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 19B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 19B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• 20A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 20B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 237 STMN2 parent oligonucleotide.
• FIG. 21 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 21B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 173 STMN2 parent oligonucleotide.
• FIG. 22A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels using different variants of a SEQ ID NO: 144 STMN2 parent oligonucleotide.
• FIG. 22B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript using different variants of a SEQ ID NO: 144 STMN2 parent oligonucleotide.
• FIG. 23 is a bar graph showing reversal of cryptic exon induction using SEQ ID NO: 237 STMN2 parent oligonucleotide even in view of increasing proteasome inhibition.
• FIG. 24 shows the dose response curve illustrating increasing restoration of full length STMN2 transcript with increasing concentrations of STMN2 AON.
• FIG. 25A shows a protein blot assay demonstrating the qualitative increase of full length STMN2 protein in response to higher concentrations of STMN2 AON.
• FIG. 25B shows the quantitated levels of full length STMN2 protein normalized to GAPDH in response to different concentrations of STMN2 AON.
• FIG. 26A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including a SEQ ID NO: 144 AON, a SEQ ID NO: 144 AON with two spacers (SEQ ID NO: 1589), a SEQ ID NO: 173 AON, a SEQ ID NO: 173 with two spacers (SEQ ID NO: 1590), a SEQ ID NO: 237 AON, and a SEQ ID NO: 237 AON with two spacers (SEQ ID NO: 1591).
• FIG. 26B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 siRNA and TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including a SEQ ID NO: 144 AON, a SEQ ID NO: 144 AON with two spacers (SEQ ID NO: 1589), a SEQ ID NO: 173 AON, a SEQ ID NO: 173 with two spacers (SEQ ID NO: 1590), a SEQ ID NO: 237 AON, and a SEQ ID NO: 237 AON with two spacers (SEQ ID NO: 1591).
• FIG. 27A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 173, SEQ ID NO: 1608, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1611, SEQ ID NO: 1612, SEQ ID NO: 1613, SEQ ID NO: 1614, SEQ ID NO: 1615, SEQ ID NO: 1596, SEQ ID NO: 1597, and SEQ ID NO: 1418.
• FIG. 27B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 173, SEQ ID NO: 1608, SEQ ID NO: 1609, SEQ ID NO: 1610, SEQ ID NO: 1611, SEQ ID NO: 1612, SEQ ID NO: 1613, SEQ ID NO: 1614, SEQ ID NO: 1615, SEQ ID NO: 1596, SEQ ID NO: 1597, and SEQ ID NO: 1418.
• FIG. 28A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 173, SEQ ID NO: 1632, SEQ ID NO: 1346, SEQ ID NO: 1631, SEQ ID NO: 1353, and SEQ ID NO: 1598.
• FIG. 28B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 173, SEQ ID NO: 1632, SEQ ID NO: 1346, SEQ ID NO: 1631, SEQ ID NO: 1353, and SEQ ID NO: 1598. [00134] FIG.
• 29A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 173 and SEQ ID NO: 1610.
• FIG. 29B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 173 and SEQ ID NO: 1610.
• FIG. 30A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 185 and SEQ ID NO: 1635.
• FIG. 30B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 185 and SEQ ID NO: 1635.
• FIG. 31 A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 1347, SEQ ID NO: 1633, and SEQ ID NO: 1634.
• FIG. 3 IB is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 1347, SEQ ID NO: 1633, and SEQ ID NO: 1634.
• FIG. 32A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 197, SEQ ID NO: 1617, SEQ ID NO: 1618, and SEQ ID NO: 1619.
• FIG. 32B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 197, SEQ ID NO: 1617, SEQ ID NO: 1618, and SEQ ID NO: 1619.
• FIG. 33A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 252, SEQ ID NO: 1650, SEQ ID NO: 1434, SEQ ID NO: 1651, and SEQ ID NO: 1620.
• FIG. 33B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 252, SEQ ID NO: 1650, SEQ ID NO: 1434, SEQ ID NO: 1651, and SEQ ID NO: 1620.
• FIG. 34A is a bar graph showing the results of RT-qPCR analysis of STMN2 transcript with cryptic exon mRNA levels in the presence of TDP43 antisense, and reduction of the STMN2 transcript with cryptic exon mRNA levels across different dosages of STMN2 AONs including SEQ ID NO: 1434 and SEQ ID NO: 1620.
• FIG. 34B is a bar graph showing the results of RT-qPCR analysis of TDP43 and STMN2 full-length mRNA levels in the presence of TDP43 antisense, and restoration of the full-length STMN2 transcript across different dosages of STMN2 AONs including SEQ ID NO: 1434 and SEQ ID NO: 1620.
• FIG. 35 is a bar graph showing normalized STMN2 protein levels following treatment with TDP43 antisense and restoration using STMN2 AONs including SEQ ID NO: 144, SEQ ID NO: 1589, SEQ ID NO: 173, SEQ ID NO: 1616, SEQ ID NO: 237, and SEQ ID NO: 1591.
• oligonucleotides capable of targeting a region of a transcript transcribed from a gene.
• such oligonucleotides target a STMN2 transcript.
• oligonucleotides including antisense oligonucleotide sequences, and methods for treating neurological diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia, and/or neuropathies such as chemotherapy induced neuropathy, using same.
• the oligonucleotides target a cryptic exon sequence of STMN2 transcripts, thereby reducing levels of STMN2 transcripts with the cryptic exon sequence.
• compositions comprising STMN2 oligonucleotides that target a region of STMN2 transcripts that comprise a cryptic exon, for treating neurological diseases and/or neuropathies; and manufacture of medicaments containing a disclosed STMN2 oligonucleotide that targets a region of STMN2 transcripts that comprise a cryptic exon to be used in treating a neurological disease and/or neuropathy.
• the terms “treat,” “treatment,” “treating,” and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect.
• the effect may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease.
• treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) inhibiting the disease, i.e., preventing the disease from increasing in severity or scope; (b) relieving the disease, i.e., causing partial or complete amelioration of the disease; or (c) preventing relapse of the disease, i.e., preventing the disease from returning to an active state following previous successful treatment of symptoms of the disease or treatment of the disease.
• Preventing includes delaying the onset of clinical symptoms, complications, or biochemical indicia of the state, disorder, disease, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, disease, or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder, disease, or condition. “Preventing” includes prophylactically treating a state, disorder, disease, or condition in or developing in a subject, including prophylactically treating clinical symptoms, complications, or biochemical indicia of the state, disorder, disease, or condition in or developing in a subject.
• compositions refers to a composition comprising at least one biologically active compound, for example, a STMN2 antisense oligonucleotide (AON), as disclosed herein formulated together with one or more pharmaceutically acceptable excipients.
• AON STMN2 antisense oligonucleotide
• “Individual,” “patient,” or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or non-human primates, and most preferably humans.
• the compounds of the invention can be administered to a mammal, such as a human, but can also be other mammals such as an animal in need of veterinary treatment, e.g ., domestic animals (e.g, dogs, cats, and the like), farm animals (e.g, cows, sheep, pigs, horses, and the like) and laboratory animals (e.g, rats, mice, guinea pigs, non-human primates, and the like).
• the mammal treated in the methods of the invention is desirably a mammal in whom modulation of STMN2 expression and/or activity is desired.
• STMN2 also known as Superior Cervical Ganglion- 10 Protein, Stathmin-Like 2, SCGN10, SCG10, Neuronal Growth-Associated Protein, Neuron-Specific Growth-Associated Protein, or Protein SCG10 (Superior Cervical Ganglia NEAR Neural Specific 10) refers to the gene or gene products (e.g, protein or mRNA transcript (including pre-mRNA) encoded by the gene) identified by Entrez Gene ID No. 11075 and allelic variants thereof, as well as orthologs found in non-human species (e.g, non-human primates or mice).
• gene or gene products e.g, protein or mRNA transcript (including pre-mRNA) encoded by the gene
• STMN2 transcript refers to a STMN2 transcript comprising a cryptic exon.
• STMN2 transcript comprising a cryptic exon can be a STMN2 pre-mRNA sequence or a STMN2 mature RNA sequence.
• STMN2 transcript comprising a cryptic exon refers to a STMN2 transcript that includes one or more cryptic exon sequences.
• STMN2 oligonucleotide refers to an oligonucleotide that is capable of increasing, restoring, or stabilizing full- length STMN2 activity e.g., full length STMN2 expression, for example, full length STMN2 mRNA and/or full length STMN2 protein expression.
• a STMN2 oligonucleotide reduces the level of mature STMN2 transcripts with a cryptic exon by targeting a STMN2 transcript comprising a cryptic exon.
• the STMN2 oligonucleotide reduces the level of mature STMN2 transcripts with a cryptic exon by repressing premature polyadenylation of STMN2 pre-mRNA and/or increasing, restoring, or stabilizing activity or function of STMN2.
• a STMN2 oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of a transcript comprising a sequence at least 90% identity to SEQ ID NO: 1339 or SEQ ID NO: 1341, or a contiguous 15 to 50 nucleobase portion of SEQ ID NO: 1339 or SEQ ID NO: 1341.
• STMN2 oligonucleotides are characterized by having one or more spacers, where each spacer divides up the STMN2 oligonucleotide into segments of linked nucleosides.
• STMN2 oligonucleotides have two spacers.
• STMN2 oligonucleotides have two segments of linked nucleosides separated by one spacer.
• STMN2 oligonucleotides have three segments of linked nucleosides separated by two spacers. In such embodiments, STMN2 oligonucleotides have one segment with at most 7 linked nucleosides.
• a STMN2 oligonucleotide may have, from the 5’ to the 3’ end, 5 linked nucleosides, followed by a spacer, 10 linked nucleosides, followed by a second spacer, and 8 linked nucleosides.
• the first segment of 5 linked nucleosides satisfies the one segment with at most 7 linked nucleosides.
• STMN2 oligonucleotides have three spacers that divide the STMN2 oligonucleotide into four segments.
• each of the four segments of the STMN2 oligonucleotide have at most 7 linked nucleosides.
• STMN2 oligonucleotide encompasses a “STMN2 parent oligonucleotide,” a “STMN2 oligonucleotide with one or more spacers” (e.g., STMN2 oligonucleotide with two spacers or a STMN2 oligonucleotide with three spacers), a “STMN2 oligonucleotide variant with one or more spacers.”
• STMN2 oligonucleotides include oligonucleotides comprising a sequence of any one of SEQ ID NOs: 1-466, SEQ ID NO: 893- 1338, SEQ ID NOs: 1342-1366, and SEQ ID NOs: 1392-1664.
• STMN2 parent oligonucleotide refers to an oligonucleotide that targets a STMN2 transcript with a cryptic exon and is capable of increasing, restoring, or stabilizing full- length STMN2 activity e.g., full length STMN2 expression, for example, full length STMN2 mRNA and/or full length STMN2 protein expression.
• STMN2 parent oligonucleotides do not include a spacer.
• Examples of STMN2 parent oligonucleotides include oligonucleotides comprising a sequence of any one of SEQ ID NOs: 1-446 and SEQ ID NOs: 893-1338. As described hereafter, STMN2 oligonucleotide with spacers and STMN2 oligonucleotide variants are described in relation to a corresponding STMN2 parent oligonucleotide.
• STMN2 oligonucleotide variant refers to a STMN2 oligonucleotide that represents a modified version of a corresponding STMN2 parent oligonucleotide.
• a STMN2 oligonucleotide variant represents a shortened version of a STMN2 parent oligonucleotide.
• a STMN2 oligonucleotide variant is any one of a 15mer, 16mer, 17mer, 18mer 19mer, 20mer, 21mer, 22mer or 23mer.
• STMN2 oligonucleotide variants include oligonucleotides comprising a sequence of any one of SEQ ID NOs: 1342-1366 or SEQ ID NOs: 1392-1521.
• STMN2 oligonucleotide variants comprise one or more spacers.
• Such STMN2 oligonucleotide variants comprise a sequence of any one of SEQ ID NOs: 1342-1366 and SEQ ID NOs: 1392-1416.
• oligonucleotide with one or more spacers refers to an oligonucleotide with at least one spacer.
• An oligonucleotide with one or more spacers can, in various embodiments, include one spacer, two spacers, three spacers, four spacer, five spacers, six spacers, seven spacers, eight spacers, nine spacers, or ten spacers.
• an oligonucleotide comprising one or more spacers includes at least one segment with at most 7 linked nucleosides.
• an oligonucleotide comprising a spacer can include a segment with 7 linked nucleosides, followed by a spacer, a second segment with 9 linked nucleosides, followed by a second spacer, and a third segment with 7 linked nucleosides.
• the first segment of 7 linked nucleosides and the third segment of 7 linked nucleosides each represents segments with at most 7 linked nucleosides.
• an oligonucleotide comprising a spacer can include a segment with 10 linked nucleosides, followed by a spacer, a second segment with 10 linked nucleosides, followed by a second spacer, and a third segment with 3 linked nucleosides.
• the third segment of 3 linked nucleosides represents the segment with at most 7 linked nucleosides.
• an oligonucleotide with one or more spacers includes multiple segments with at most 7 linked nucleosides.
• every segment of an oligonucleotide with one or more spacers has at most 7 linked nucleosides.
• the oligonucleotide may be a 23mer and include two spacers that divide the 23mer into three separate segments of 7 linked nucleosides each. Therefore, each segment of the oligonucleotide has at most 7 linked nucleosides.
• STMN2 oligonucleotides comprising one or more spacers are described in reference to a corresponding STMN2 parent oligonucleotide or a corresponding STMN2 oligonucleotide variant.
• Example STMN2 oligonucleotides comprising one or spacers include any of SEQ ID NOs: 1417-1420 and SEQ ID NOs: 1451-1664.
• the term “therapeutically effective amount” means the amount of an oligonucleotide that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician.
• the oligonucleotide comprises a sequence that is between 85 and 98% complementary to an equal length portion of a transcript comprising a sequence at least 90% identity to SEQ ID NO: 1339 or SEQ ID NO: 1341, or a contiguous 15 to 50 nucleobase portion of SEQ ID NO: 1339 or SEQ ID NO: 1341.
• the oligonucleotide is administered in therapeutically effective amounts to treat and/or prevent a disease, condition, disorder, or state, for example, a neurological disease and/or a neuropathy.
• a therapeutically effective amount of an oligonucleotide is the quantity required to achieve a desired therapeutic and/or prophylactic effect, such as an amount which results in the prevention of or a decrease in the symptoms associated with a disease associated with reduced STMN2 activity in the motor neurons.
• a STMN2 oligonucleotide that targets a STMN2 transcript refers to a STMN2 oligonucleotide that binds to a STMN2 transcript.
• Example regions of a STMN2 transcript are shown in Table 1, which depicts sequences corresponding to regions of branch points ( e.g ., branch point 1, 2, and 3) a 3’ splice acceptor region, an ESE binding region, TDP43 binding sites, a cryptic exon, and a Poly A region.
• the oligonucleotide binds to a region of a STMN2 transcript with a cryptic exon, the region being located less than 75 nucleobases upstream or downstream to any of the branch points (e.g., branch point 1, 2, and 3) a 3’ splice acceptor region, an ESE binding region, TDP43 binding sites, a cryptic exon, and a Poly A region.
• branch points e.g., branch point 1, 2, and 3
• pharmaceutically acceptable salt(s) refers to salts of acidic or basic groups that may be present in a STMN2 oligonucleotide used in the present compositions.
• a STMN2 oligonucleotide included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
• the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesul
• a STMN2 oligonucleotide included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
• Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, and lithium salts.
• Pharmaceutically acceptable salts of the disclosure include, for example, pharmaceutically acceptable salts of STMN2 oligonucleotides that include a sequence of any of SEQ ID NOs: 1- 466, SEQ ID NO: 893-1338, SEQ ID NOs: 1342-1366, and SEQ ID NOs: 1392-1664.
• a STMN2 oligonucleotide of the disclosure may contain one or more chiral centers, groups, linkages, and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers.
• stereoisomers when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S” (or “R/E or “S p”) depending on the configuration of substituents around the stereogenic atom, for example, a stereogenic carbon, phosphorous, or sulfur atom.
• one or more linkages of the compound may have a Rp or Sp configuration (e.g ., one or more phosphorothioate linkages have either a Rp or Sp configuration).
• the configuration of each phosphorothioate linkage may be independent of another phosphorothioate linkage (e.g., one phosphorothioate linkage has a Rp configuration and a second phosphorothioate linkage has a Sp configuration).
• the STMN2 oligonucleotide can have a mixed configuration of phosphorothioate linkages.
• the STMN2 oligonucleotide may have five phosphorothioate linkages in a Rp configuration, followed by fifteen phosphorothioate linkages in a Sp configuration, followed by five phosphorothioate linkages in a Rp configuration.
• Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “( ⁇ )” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.
• Individual stereoisomers of a STMN2 oligonucleotide of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns.
• Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase super critical fluid chromatography, chiral-phase simulated moving bed chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent.
• Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
• STMN2 oligonucleotide disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.
• the disclosure also embraces isotopically labeled compounds of the invention (i.e., isotopically labeled STMN2 oligonucleotide) which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number abundantly found in nature.
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as 2 H, 3 H, U C, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 33 P, 35 S, 18 F, and 36 C1, respectively.
• Certain isotopically labeled disclosed compounds are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3 H), carbon-14 (i.e ., 14 C), or 35 S methionine isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
• 2’-0-(2-methoxyethyl) refers to an O-methoxyethyl modification of the T position of a furanose ring.
• a 2’-0-(2- methoxyethyl) is used interchangeably as “2’-0-methoxyethyl” in the present disclosure.
• a sugar moiety in a nucleoside modified with 2’-MOE is a modified sugar.
• 2’-MOE nucleoside (also 2’-0-(2-methoxyethyl) nucleoside) means a nucleoside comprising a T -MOE modified sugar moiety.
• T substituted nucleoside means a nucleoside comprising a substituent at the T -position of the furanose ring other than H or OH.
• T substituted nucleosides include nucleosides with bicyclic sugar modifications.
• 5-methyl cytosine means a cytosine modified with a methyl group attached to the 5 position.
• a 5-methyl cytosine (5-MeC) is a modified nucleobase.
• bicyclic sugar means a furanose ring modified by the bridging of two atoms.
• a bicyclic sugar is a modified sugar.
• bicyclic nucleoside also BNA means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4’-carbon and the 2’-carbon of the sugar ring.
• cap structure or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
• cEt or “constrained ethyl” means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4’-carbon and the 2’-carbon, wherein the bridge has the formula: 4’ -CHICLE) — 0-2’.
• constrained ethyl nucleoside means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH3) — O-T bridge.
• cEt can be modified.
• the cEt can be ri-cEt (in an S- constrained ethyl 2’-4’-bridged nucleic acid).
• the cEt can be R- cEt.
• integerucleoside linkage refers to the covalent linkage between adjacent nucleosides in an oligonucleotide.
• non-natural linkage refers to a “modified intemucleoside linkage.”
• oligonucleotide in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or intemucleoside linkages that are immediately adjacent to each other.
• contiguous nucleobases means nucleobases that are immediately adjacent to each other in a sequence. As an example to the contrary, two nucleosides separated by a spacer are not contiguous.
• locked nucleic acid or “LNA” or “LNA nucleosides” means nucleic acid monomers having a bridge (e.g ., methylene, ethylene, aminooxy, or oxyimino bridge) connecting two carbon atoms between the 4’ and T position of the nucleoside sugar unit, thereby forming a bicyclic sugar.
• bicyclic sugar include, but are not limited to (A) a-L- Methyleneoxy (4’-CH 2— 0-2’) LNA, (B) b-D-Methyleneoxy (4’-CH 2— 0-2’) LNA, (C)
• Examples of 4’ -T bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: — [C(Ri)( R2)]n — , — [C(Ri)(R2)]n — O — , — C(RIR2) — N(Ri) — O — or — C(RIR2) — O — N(Ri) — .
• bridging groups encompassed with the definition of LNA are 4’-CH 2 -2’, 4’-(CH 2 )2-2’, 4’-(CH 2 )3-2’, 4’-CH 2— 0-2’, 4’-(CH 2 )2— O- 2’, 4’- CH2 — O — N(Ri)-2’ and 4’- CH2 — N(Ri) — 0-2’- bridges, wherein each Ri and R2 is, independently, H, a protecting group or C1-C12 alkyl.
• LNAs in which the 2’-hydroxyl group of the ribosyl sugar ring is connected to the 4’ carbon atom of the sugar ring, thereby forming a bridge to form the bicyclic sugar moiety.
• the bridge can be a methylene ( — CFh — ) group connecting the T oxygen atom and the 4’ carbon atom, for which the term methyleneoxy (4’-CH2 — 0-2’) LNA is used.
• ethyleneoxy (d’-CFLCFh — 0-2’) LNA is used.
• A-L-methyleneoxy (4’-CH2-0-2’) an isomer of methyleneoxy (4’-CH2 — 0-2’) LNA is also encompassed within the definition of LNA, as used herein.
• a “spacer” refers to a nucleoside-replacement group (e.g ., a non nucleoside group that replaces a nucleoside present in a STMN2 parent oligonucleotide).
• the spacer is characterized by the lack of a nucleotide base and by the replacement of the nucleoside sugar moiety with a non-sugar substitute.
• the non-sugar substitute group of a spacer lacks an aldehyde, ketone, acetal, ketal, hemiacetal or hemiketal group.
• the non-sugar substitute group of a spacer is thus capable of connecting to the 3’ and 5’ positions of the nucleosides adjacent to the spacer through an internucleoside linker as described herein, but not capable of forming a covalent bond with a nucleotide base (i.e., not capable of linking a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide).
• a STMN2 oligonucleotide with a spacer is described in relation to a STMN2 parent oligonucleotide, wherein the spacer replaces a nucleoside of the STMN2 parent oligonucleotide.
• a spacer cannot hybridize to a nucleoside comprising a nucleobase at the corresponding position of a STMN2 transcript, within the numerical order of the length of the AON oligonucleotide (i.e., if the spacer is positioned after nucleoside 4 of an AON (i.e., at position 5 from the 5’ -end), the spacer is not complementary to the nucleoside (A,
• mismatch or a “non-complementary group” refers to the case when a group (e.g., nucleobase) of a first nucleic acid is not capable of pairing with the corresponding group (e.g., nucleobase) of a second or target nucleic acid.
• modified intemucleoside linkage refers to a substitution or any change from a naturally occurring intemucleoside linkage (e.g., a phosphodiester intemucleoside bond).
• modified nucleobase means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of a modified nucleobase include 5-methyl cytosine, pseudouridine, or 5-methoxyuridine.
• An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
• a “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
• a universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.
• Modified nucleosides include abasic nucleosides, which lack a nucleobase. However, modified nucleosides do not include spacers or other groups that are incapable of linking a nucleobase.
• linked nucleosides are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are presented between those that are linked).
• an oligonucleotide may have different segments of linked nucleosides connected through a spacer.
• the spacer i.e., nucleoside replacement
• the spacer is not considered a nucleoside and therefore, divides up the oligonucleotide into two segments of linked nucleosides.
• the oligonucleotide may have a first segment of Y linked nucleosides (e.g, Y nucleosides that are connected in a contiguous sequence), followed by a spacer, and then a second segment of Z linked nucleosides.
• Y and Z linked nucleosides is described in either the 5’ to 3’ direction or the 3’ to 5’ direction.
• modified oligonucleotide means an oligonucleotide comprising at least one (i.e., one or more) modified intemucleoside linkage, modified sugar, and/or modified nucleobase.
• modified sugar or “modified sugar moiety” means a modified furanosyl sugar moiety or a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide.
• monomer means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.
• motif means the pattern of unmodified and modified nucleosides in an antisense compound.
• natural sugar moiety means a sugar moiety found in DNA (2’-H) or RNA (2’-OH).
• naturally occurring intemucleoside linkage means a 3’ to 5’ phosphodiester linkage.
• non-complementary nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
• nucleic acid refers to molecules composed of monomeric nucleotides.
• a nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, non-coding RNA, small interfering ribonucleic acids (siRNA), short-hairpin RNA (shRNA), and microRNAs (miRNA).
• RNA ribonucleic acids
• DNA deoxyribonucleic acids
• siRNA small interfering ribonucleic acids
• shRNA short-hairpin RNA
• miRNA microRNAs
• nucleobase complementarity refers to a nucleobase that is capable of base pairing with another nucleobase.
• adenine (A) is complementary to thymine (T).
• adenine (A) is complementary to uracil (U).
• complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
• nucleobase sequence means the order of nucleobases independent of any sugar, linkage, and/or nucleobase modification.
• nucleoside refers to a nucleobase linked to a sugar.
• nucleoside also includes a “modified nucleoside” which has independently, a modified sugar moiety and/or modified nucleobase.
• nucleoside mimetic includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, ortricyclo sugar mimetics, e.g ., non-furanose sugar units.
• Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by a phosphorodiamidate or other non-phosphodiester linkage).
• Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only.
• the tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.
• “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or intemucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-intemucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
• nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
• oligomeric compound or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
• oligonucleotide means a polymer of one or more segments of linked nucleosides each of which can be modified or unmodified, independent one from another.
• hotspot region is a range of nucleobases on a target nucleic acid amenable to oligomeric compound-mediated modulation of the splicing of the target nucleic acid.
• hybridization means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases.
• increasing the amount of activity refers to more transcriptional expression, more accurate splicing resulting in full length mature mRNA and/or protein expression, and/or more activity relative to the transcriptional expression or activity in an untreated or control sample.
• Antisense therapeutics are a class of nucleic acid-based compounds that can be used to modulate a transcript, such as mRNA.
• antisense therapeutics comprise one or more spacers and can be used to modulate a transcript that is transcribed from a gene, such as a STMN2 pre-mRNA comprising a cryptic exon.
• Antisense therapeutics may be single- or double-stranded deoxyribonucleic acid (DNA)- based, ribonucleic acid (RNA)-based, or DNA/RNA chemical analogue compounds.
• antisense therapeutics are designed to include a sequence that is complementary or nearly complementary to an mRNA or pre-mRNA sequence transcribed from a given gene in order to promote binding between the antisense therapeutic and the pre-mRNA or mRNA.
• antisense therapeutics act by binding to an mRNA or pre-mRNA, thereby inhibiting protein translation, altering pre-mRNA splicing into mature mRNA ( e.g by preventing appropriate proteins such as splicing activator proteins from binding), and/or causing destruction of mRNA.
• the antisense therapeutic sequence is complementary to a portion of a targeted gene’s or mRNA’s sense sequence.
• antisense therapeutics described herein are oligonucleotide-based compounds that include an oligonucleotide sequence complementary to a pre-mRNA sense, or a portion thereof, and one or more spacers.
• antisense therapeutics described herein can also be nucleotide chemical analog-based compounds.
• an oligonucleotide such as disclosed herein, may be an oligonucleotide sequence of 5 to 100 oligonucleotide units in length, for example, 10 to 60 oligonucleotide units in length, for example, 12 to 50 oligonucleotide units in length, 14 to 40 oligonucleotide units in length, 10 to 30 oligonucleotide units in length, for example, 14 to 30 oligonucleotide units in length, for example, 14 to 25 or 15 to 22 oligonucleotide units in length, or 18, 19, 20, 21, 22, 23, 24, or 25 oligonucleotide units in length.
• an “oligonucleotide unit” refers to either a nucleoside (e.g., a nucleoside which includes a sugar and/or a nucleobase) or a nucleoside-replacement group (e.g., a spacer) of the oligonucleotide.
• the oligonucleotides are 25 oligonucleotide units in length.
• the oligonucleotides are 23 oligonucleotide units in length.
• the oligonucleotides are 21 oligonucleotide units in length.
• the oligonucleotides are 19 oligonucleotide units in length.
• the oligonucleotide is at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 oligonucleotide units in length.
• the oligonucleotide is at least 18 oligonucleotide units in length.
• the oligonucleotide is at least 19 oligonucleotide units in length.
• the oligonucleotide is at least 20 oligonucleotide units in length.
• the oligonucleotide is at least 21 oligonucleotide units in length. In various embodiments, the oligonucleotide is at least 22 oligonucleotide units in length. In various embodiments, the oligonucleotide is at least 23 oligonucleotide units in length. In various embodiments, the oligonucleotide is at least 24 oligonucleotide units in length. In various embodiments, the oligonucleotide is at least 25 oligonucleotide units in length.
• AONs may include chemically modified nucleosides (for example, 2’-0-methylated nucleosides or 2’-0-(2-methoxyethyl) nucleosides) as well as modified intemucleoside linkages (for example, phosphorothioate linkages).
• chemically modified nucleosides for example, 2’-0-methylated nucleosides or 2’-0-(2-methoxyethyl) nucleosides
• modified intemucleoside linkages for example, phosphorothioate linkages
• AONs described herein include oligonucleotide sequences that are complementary to RNA sequences, such as STMN2 mRNA sequences.
• AONs described herein can include chemically modified nucleosides and modified intemucleoside linkages (for example, phosphorothioate linkages).
• AONs described herein include one or more spacers.
• the oligonucleotides comprise one or more spacers.
• the oligonucleotides comprise one spacer.
• the oligonucleotides comprise two spacers.
• the oligonucleotide includes 23 oligonucleotide units with 21 nucleobases and two nucleoside replacement groups (e.g., two spacers). Further embodiments of oligonucleotides with one spacer and oligonucleotides with two spacers are described herein.
• an antisense oligonucleotide can be, but is not limited to, inhibitors of a gene transcript (for example, shRNAs, siRNAs, PNAs, LNAs, T -O-methyl (2Ome) antisense oligonucleotide (AON), T -0-(2-m ethoxy ethyl) (MOE) AON, or morpholino oligomers (e.g., phosphorodiamidate morpholino (PMO))), or compositions that include such compounds.
• a gene transcript for example, shRNAs, siRNAs, PNAs, LNAs, T -O-methyl (2Ome) antisense oligonucleotide (AON), T -0-(2-m ethoxy ethyl) (MOE) AON, or morpholino oligomers (e.g., phosphorodiamidate morpholino (PMO))
• PMO phosphorodiamidate morpholino
• an oligonucleotide is an antisense oligonucleotide (AON) comprising 2’Ome (e.g, a AON comprising one or more 2’Ome modified sugar), MOE (e.g, a AON comprising one or more MOE modified sugar), peptide nucleic acids (e.g, a AON comprising one or more /V-(2-ami noethyl )-glycine units linked by amide bonds or carbonyl methylene linkage as repeating units in place of a sugar-phosphate backbone), locked nucleic acids ( e.g ., a AON comprising one or more locked ribose, and can be a mixture of 2’-deoxy nucleotides or 2’Ome nucleotides), c-ET (e.g., a AON comprising one or more cET sugar), constrained methoxy ethyl (cMOE) (e.g, a AON comprising
• a AON comprises one or more intemucleoside linkage independently selected from a phosphorothioate linkage, phosphodiester linkage, phosphotriester linkage, methylphosphonate linkage, phosphoramidate linkage, a phosphoramidothioate linkage, a thiophosphorodiamidate linkage, phosphorodiamidate morpholino (PMO) (morpholino) linkage, PNA linkage, or any combination of phosphorothioate linkage, phosphodiester linkage, a phosphotriester linkage, methylphosphonate linkage, phosphoramidate linkage, a phosphoramidothioate linkage, thiophosphorodiamidate linkage, phosphorodiamidate morpholino (PMO) (morpholino) linkage, and PNA linkage.
• a STMN2 AON comprises one or more phosphorothioate linkage, phosphodiester linkage, or
• PNAs Peptide nucleic acids
• PNAs are short, artificially synthesized polymers with a structure that mimics DNA or RNA.
• PNAs include a backbone composed of repeating N-(2-aminoethyl)- glycine units linked by peptide bonds.
• PNAs described herein can be used as antisense therapeutics that bind to RNA sequences with high specificity and increase, restore, and/or stabilize levels (e.g, full length STMN2 mRNA or protein levels) and/or activity (e.g, biological activity, for example, STMN2 activity).
• Locked nucleic acids are oligonucleotide sequences that include one or more modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon. LNAs are believed to have higher Tm’s than analogous oligonucleotide sequences. In certain embodiments, LNAs described herein can be used as antisense therapeutics that bind to RNA sequences with high specificity.
• LNAs can bind to STMN2 pre-RNA and repress premature polyadenylation of STMN2 pre-mRNA, and increase, restore, and/or stabilize STMN2 levels (e.g., STMN2 mRNA or protein levels) and/or activity (e.g, biological activity, for example, STMN2 activity).
• STMN2 levels e.g., STMN2 mRNA or protein levels
• activity e.g, biological activity, for example, STMN2 activity
• Morpholino oligomers are oligonucleotide compounds that include DNA bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups.
• morpholino oligomers of the present invention can be designed to bind to specific pre-RNA sequence of interest.
• morpholino oligomers bind to STMN2 pre- RNA thereby repressing premature polyadenylation of the pre-mRNA, and increase, restore, and/or stabilize STMN2 levels (e.g, STMN2 mRNA or protein levels) and/or activity (e.g, biological activity, for example, STMN2 activity).
• STMN2 morpholino oligomers described herein can be used as antisense therapeutics that bind to STMN2 pre-mRNA sequences with high specificity and repress premature polyadenylation of STMN2 pre-mRNA, and increase, restore, and/or stabilize STMN2 levels (e.g, STMN2 mRNA or protein levels) and/or activity (e.g, biological activity, for example, STMN2 activity).
• STMN2 mRNA or protein levels e.g, STMN2 mRNA or protein levels
• activity e.g, biological activity, for example, STMN2 activity
• STMN2 morpholino oligomers described herein can also be used to bind STMN2 pre-mRNA sequences, altering STMN2 pre-mRNA splicing and STMN2 gene expression, and increase, restore, and/or stabilize STMN2 levels (e.g, STMN2 mRNA or protein levels) and/or activity (e.g, biological activity, for example, STMN2 activity).
• STMN2 levels e.g, STMN2 mRNA or protein levels
• activity e.g, biological activity, for example, STMN2 activity
• a STMN2 AON includes a sequence that is between 85 and 98% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON includes a sequence that is between 90-95% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON includes a sequence that is between 85% and 90% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON includes a sequence that is between 84% to 88% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON includes a sequence that is between 89% to 92% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON includes a sequence that is between 94% to 96% complementary to a sequence that shares at least 90% (e.g, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a region of a STMN2 transcript that includes a cryptic exon (e.g., SEQ ID NO: 1339 or SEQ ID NO: 1341).
• a STMN2 AON comprises a sequence that shares at least 85% identity with an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392-1664. In various embodiments, a STMN2 AON comprises a sequence that shares at least 90% identity with an equal length portion of any one of SEQ ID NOs: 1-466, SEQ ID NOs: 893-1338, SEQ ID NOs: 1342-1366, or SEQ ID NOs: 1392- 1664.
• the region of the STMN2 transcript targeted by the STMN2 AON is the cryptic exon sequence.
• the region of the STMN2 transcript targeted by the STMN2 AON is a sequence located upstream or downstream (e.g., 100 or 200 bases upstream or downstream) of the cryptic exon sequence.
• the STMN2 AON comprises a spacer and has a segment having at most 7 linked nucleosides.
• the STMN2 AON comprises a spacer and has a segment having at most 6, 5, 4, 3, or 2 linked nucleosides.
• STMN2 AON binding specificity can be assessed via measurement of parameters such as dissociation constant, melting temperature ®, or other criteria such as changes in protein or RNA expression levels or other assays that measure STMN2 activity or expression.
• a STMN2 AON can include a non-duplexed oligonucleotide.
• a STMN2 AON can include a duplex of two oligonucleotides where the first oligonucleotide includes a nucleobase sequence that is completely or almost completely complementary to a STMN2 pre-mRNA sequence and the second oligonucleotide includes a nucleobase sequence that is complementary to the nucleobase sequence of the first oligonucleotide.
• a STMN2 AON can target STMN2 pre-mRNAs that include a cryptic exon produced from STMN2 genes of one or more species.
• a STMN2 AON can target a STMN2 pre-mRNA, which includes a cryptic exon, of a mammalian STMN2 gene, for example, a human (i.e., Homo sapiens) STMN2 gene.
• the STMN2 AON targets a human STMN2 pre-mRNA, which includes a cryptic exon.
• the STMN2 AON includes a nucleobase sequence that is complementary to a nucleobase sequence of a STMN2 gene or a STMN2 pre-mRNA, which includes a cryptic exon, or a portion thereof.
• STMN2 AONs described herein include antisense oligonucleotides comprising the oligonucleotide sequences listed in Table 1 below:
• At least one (i.e., one or more) nucleoside linkage of the oligonucleotide sequence is independently selected from a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3- methoxypropyl phosphonate linkage, a methylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoramidothioate linkage, a thiophosphorodiamidate linkage, a phosphorodiamidate ( e.g ., comprising a phosphorodiamidate morpholino (PMO), 3’ amino ribose, or 5’ amino ribose) linkage, an aminoalkylphosphoramidate linkage, a thi
• At least one (i.e., one or more) nucleoside linkage of the oligonucleotide sequence is independently selected from a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3- methoxypropyl phosphonate linkage, a methylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoramidothioate linkage, a thiophosphorodiamidate linkage, a phosphorodiamidate ( e.g ., comprising a phosphorodiamidate morpholino (PMO), 3’ amino ribose, or 5’ amino ribose) linkage, an aminoalkylphosphoramidate linkage, a thi
• Table 3 below identifies exemplary STMN2 AON sequences: Table 3.
• Exemplary STMN2 AON Sequences, in each one or more spacers described in the present disclosure are incorporated for generation of an oligonucleotide of the present invention
• At least one (i.e., one or more) nucleoside linkage of the oligonucleotide sequence is independently selected from a phosphorothioate linkage, an alkyl phosphate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, a 3- methoxypropyl phosphonate linkage, a methylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoramidothioate linkage, a thiophosphorodiamidate linkage, a phosphorodiamidate ( e.g ., comprising a phosphorodiamidate morpholino (PMO), 3’ amino ribose, or 5’ amino ribose) linkage, an aminoalkylphosphoramidate linkage, a thi
• all intemucleoside linkages of the STMN2 AON oligonucleotides listed in Table 3 are phosphorothioate linkages (except when a spacer is present, the linkage may or may not be a phosphorothioate linkage), and each of the linked nucleosides of the oligonucleotide are 2’-0-(2-methoxyethyl) (2’-MOE) nucleosides, and each “C” is replaced with a 5-MeC.
• all intemucleoside linkages of the STMN2 AON oligonucleotides listed in Table 3 are phosphorothioate linkages, and each of the linked nucleosides of the oligonucleotide are T -0-(2-m ethoxy ethyl) (2’-MOE) nucleosides, and not all or none of the ‘C” is replaced with 5-MeC.
• a STMN2 AON targets a region of a STMN2 transcript comprising a cryptic exon sequence, the STMN2 transcript comprising the sequence provided as SEQ ID NO: 1339.
• a cryptic exon sequence within the STMN2 transcript is provided as SEQ ID NO: 1340.
• the STMN2 transcript with a cryptic exon shares between 90- 100% identity with SEQ ID NO: 1339. In various embodiments, the STMN2 transcript with a cryptic exon shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1341.
• a STMN2 transcript with a cryptic exon can comprise a pre-mRNA STMN2 transcript.
• a STMN2 transcript with a cryptic exon can comprise the sequence provided as SEQ ID NO: 1341.
• AGAATTT C AGGAT AAAACTGAAAGAAATGGC AGT AGTTT AT C AATT AATCTC AT GT A
• GAGT GACTT GCTCCTGAT C AC AA ATGCTGGCC AAGGAAGAGTCGAGTTT C AAATCT A ATGATCTTTCCACTGCACTCTAGATTCCTCATTTTGAACTATTTTTTTATTTTTTGCACT
• TGT ACC ACGTTAGGAGGAAACCCTTCTTCACAGGAGAGTGTGCCTTTGCTGC AGGGA

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