Antisense oligonucleotides targeting ACTL6B

Abstract

The present invention relates to antisense oligonucleotide splice modulators of actin-like 6B (ACTL6B). These antisense oligonucleotide splice modulators are complementary, e.g., fully complementary, to ACTL6B precursor-mRNA and can increase or restore expression of ACTL6B in TDP-43-depleted cells, such as for use in conditions and medical indications in which TDP-43 is functionally depleted.

Description

[Technical field]

[0001] The present invention relates to antisense oligonucleotide splice modulators of actin-like 6B (ACTL6B). These antisense oligonucleotide splice modulators are complementary, e.g., fully complementary, to ACTL6B precursor-mRNA and can increase or restore expression of ACTL6B in TDP-43-depleted cells, such as for use in conditions and medical indications in which TDP-43 is functionally depleted. [Background technology]

[0002] background TAR DNA-binding protein 43 (TDP-43) is a versatile RNA / DNA-binding protein involved in RNA-related metabolism. Dysregulation of TDP-43 deposits acts as inclusion bodies in the brain and spinal cord of patients with motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) (Prasad et al., Front. Mol. Neurosci., 2019).

[0003] TDP-43 depletion is shown in various diseases, termed TDP-43 pathology, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathies and chronic traumatic encephalopathy.

[0004] ACTL6B is involved in chromatin remodeling during neuronal differentiation. ACTL6B is a splice variant of ACTL6A and replaces ACTL6A once stem cells have completed their differentiation into mature neurons.

[0005] We show that splicing of ACTL6B is regulated, at least in part, by TDP-43 through a TDP-43 binding site present within the ACTL6B pre-mRNA sequence. Summary of the Invention

[0006] Summary of the Invention The inventors surprisingly determined that when TDP-43 is depleted in cells, ACTL6B mRNA splicing is altered.

[0007] Therefore, we hypothesized that by modifying the ACTL6B splicing pattern, it might be possible to ameliorate the deleterious effects of TDP-43 depletion on neuronal cells.

[0008] Here, we used an antisense oligonucleotide ACTL6B splice modulator to increase the expression of ACTL6B.

[0009] In one aspect, the present invention provides an antisense oligonucleotide actin-like 6B (ACTL6B) splice modulator, the antisense oligonucleotide splice modulator being 8 to 40 nucleotides in length and comprising a contiguous nucleotide sequence of at least 8 nucleotides in length that is complementary to ACTL6B precursor-mRNA.

[0010] In some embodiments, the antisense oligonucleotide splice modulator may be capable of increasing expression of Actin-Like 6B (ACTL6B) in TDP-43-depleted cells.

[0011] In some embodiments, the antisense oligonucleotide splice modulator may be capable of reducing expression of ACTL6B mutant polypeptides, such as splice variants of ACTL6B, in TDP-43-depleted cells.

[0012] The present inventors have surprisingly determined that in TDP-43 depleted cells, increased expression of ACTL6B splice variants containing additional exons is observed. This leads to a decrease in the production of functionally active wild-type (WT) ACTL6B polypeptide. Thus, in some embodiments, the splice variant may comprise a polypeptide sequence encoded by an additional exon compared to the wild-type ACTL6B polypeptide sequence.

[0013] In some embodiments, the mutant ACTL6B splice variant may contain an insertion, such as an insertion of about 23 amino acids, compared to the wild-type ACTL6B polypeptide sequence.

[0014] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be complementary to a splice enhancer site in the ACTL6B precursor-mRNA.

[0015] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be complementary to a sequence selected from SEQ ID NOs: 199-205.

[0016] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be complementary to a sequence selected from SEQ ID NOs: 6-97 and 190-193.

[0017] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be complementary to a sequence selected from SEQ ID NOs: 26, 28, 29, 30, 31, 32, 33, 38, 39, 46, 47, 48, 52, 53, 55 and 72.

[0018] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be complementary to a sequence selected from SEQ ID NOs:28, 29, 30, 31, 32, 33 and 47.

[0019] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be a sequence selected from SEQ ID NOs: 98-189 and 194-197, or at least 10 contiguous nucleotides thereof.

[0020] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be a sequence selected from the group consisting of SEQ ID NOs: 118, 120, 121, 122, 123, 124, 125, 130, 131, 138, 139, 140, 144, 145, 147 and 164, or at least 10 contiguous nucleotides thereof.

[0021] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be a sequence selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 125 and 139, or at least 10 contiguous nucleotides thereof.

[0022] In some embodiments, antisense oligonucleotide splice modulators may be at least 12 nucleotides in length, e.g., at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length.

[0023] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be the same length as the antisense oligonucleotide splice modulator.

[0024] In some embodiments, the antisense oligonucleotide splice modulator may comprise one or more modified nucleosides, such as 2' sugar modified nucleosides, which may be independently selected from the group consisting of 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabinonucleic acid (ANA); 2'-fluoro-ANA; locked nucleic acid (LNA), or any combination thereof.

[0025] In some embodiments, the contiguous nucleotide sequence of an antisense oligonucleotide splice modulator may comprise 2'-O-methoxyethyl-RNA (2'-MOE) nucleosides optionally linked by phosphorothioate internucleoside linkages.

[0026] In some embodiments, one or more of the modified nucleosides can be a locked nucleic acid nucleoside (LNA), for example, an LNA nucleoside selected from the group consisting of constrained ethyl nucleoside (cEt) and β-D-oxy-LNA.

[0027] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be at least 75%, such as at least 80%, at least 85%, at least 90% or at least 95% complementary to the ACTL6B precursor-mRNA sequence.

[0028] In other embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator may be perfectly complementary to the ACTL6B precursor-mRNA.

[0029] In some embodiments, the antisense oligonucleotide splice modulator may not contain a region of more than three or more than four consecutive DNA nucleosides and may not be able to mediate RNAseH cleavage.

[0030] In some embodiments, one or more or all of the internucleoside linkages in the antisense oligonucleotide splice modulator may be modified. For example, the modified internucleoside linkage may comprise a phosphorothioate linkage.

[0031] In some embodiments, the antisense oligonucleotide splice modulator may be covalently linked to at least one conjugate moiety.

[0032] In some embodiments, the antisense oligonucleotide splice modulator may be in the form of a pharma- ceutically acceptable salt, such as a sodium or potassium salt.

[0033] In another aspect, there is provided a pharmaceutical composition comprising an antisense oligonucleotide splice modulator of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0034] In another aspect, there is provided a method, e.g., an in vivo or in vitro method, for increasing ACTL6B expression in a cell, the method comprising administering to said cell an effective amount of an antisense oligonucleotide splice modulator or pharmaceutical composition of the present invention, the cell may express abnormal levels of TDP-43 or exhibit depleted levels of TDP-43.

[0035] In another aspect, the present invention provides a method for treating or preventing a disease in a subject comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide splice modulator or pharmaceutical composition of the invention to a subject suffering from or susceptible to said disease.

[0036] In another aspect, there is provided an antisense oligonucleotide splice modulator or a pharmaceutical composition of the invention for use as a medicament.

[0037] In another aspect, there is provided an antisense oligonucleotide splice modulator or a pharmaceutical composition of the invention for use in the treatment or prevention of a disease in a subject.

[0038] In another aspect, there is provided an antisense oligonucleotide splice modulator or a pharmaceutical composition of the invention for preparing a medicament for treating or preventing a disease in a subject.

[0039] In all aspects of the invention, the disease may be a neurological disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0040] In certain embodiments, the disease may be a neurological disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). [Brief description of the drawings]

[0041]

Figure 1

[0042] Detailed Description We identified that splicing of ACTL6B is affected by TDP-43, which may result in the production of non-functional or hypofunctional ACTL6B in TDP-43 cells.

[0043] Without wishing to be bound by theory, it is believed that in TDP-43 depleted cells, ACTL6B may be spliced ​​to include an additional exon. This additional exon may be 23 amino acids long. The inclusion of this additional exon may result in the formation of a polypeptide that is functionally less active than wild-type ACTL6B. Such alternatively spliced ​​polypeptides are referred to herein as "mutant ACTL6B polypeptides", "ACTL6B splice variants" or "ACTL6B splice variants".

[0044] The present inventors have also determined that antisense oligonucleotide splice modulators can be used to reduce the production of ACTL6B splicing variants. In this specification, the antisense oligonucleotide splice modulators of the present invention can also be referred to as the oligonucleotides of the present invention or the antisense oligonucleotides of the present invention.

[0045] The oligonucleotide splice modulators of the present invention can target splice enhancer sites in the ACTL6B precursor-mRNA, which can reduce alternative splicing, thereby increasing normal splicing and the production of wild-type ACTL6B protein.

[0046] Enhanced wild-type ACTL6B expression is desirable for treating a wide range of disorders characterized by or caused by reduced expression of ACTL6B, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0047] Splice Adjustment The antisense oligonucleotides of the present invention are ACTL6B splice modulators, i.e., the antisense oligonucleotides of the present invention affect the splicing of ACTL6B pre-mRNA. As used herein, the oligonucleotides of the present invention may be referred to as "antisense oligonucleotide splice modulators."

[0048] In some embodiments, the antisense oligonucleotide splice modulators of the invention may be complementary to ACTL6B precursor-mRNA.

[0049] In some embodiments, the ACTL6B precursor-mRNA may have the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is provided herein as a reference sequence, and it will be understood that the target precursor-mRNA may be an allelic variant of SEQ ID NO: 1, for example, an allelic variant that includes one or more polymorphisms.

[0050] In some embodiments, the antisense oligonucleotide splice modulator may be capable of increasing the expression of ACTL6B in TDP-43-depleted cells. It is predicted herein that exposure to the antisense oligonucleotide splice modulator of the present invention increases the expression of wild-type, i.e., normally spliced, ACTL6B.

[0051] Without wishing to be bound by theory, it is believed that the antisense oligonucleotide splice modulators of the present invention may increase normal splicing of ACTL6B precursor-mRNA, which would result in an increase in the amount of normally spliced ​​mature ACTL6B mRNA, which would in turn result in an increase in the amount of wild-type ACTL6B protein.

[0052] As used herein, the terms "wild-type" and "normally spliced" are used interchangeably.

[0053] In some embodiments, the wild-type (i.e., normally spliced) mature ACTL6B mRNA sequence can have the sequence of SEQ ID NO: 2, or a fragment or variant thereof. SEQ ID NO: 2 is provided herein as a reference sequence, and it will be understood that normally spliced ​​ACTL6B mRNA can be an allelic variant of SEQ ID NO: 2, for example, an allelic variant comprising one or more polymorphisms.

[0054] In some embodiments, the wild-type ACTL6B protein may have the sequence of SEQ ID NO: 3, or a fragment or variant thereof. SEQ ID NO: 3 is provided herein as a reference sequence, and it will be understood that the wild-type ACTL6B protein may be an allelic variant of SEQ ID NO: 3, for example, an allelic variant comprising one or more polymorphisms.

[0055] As used herein, the term "increasing expression of wild-type ACTL6B" is understood to mean increasing normally spliced ​​ACTL6B mRNA levels, increasing wild-type ACTL6B protein levels, or increasing normally spliced ​​ACTL6B mRNA levels and wild-type ACTL6B protein levels.

[0056] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can increase normal splicing of ACTL6B precursor-mRNA by at least about 10% compared to a control. More preferably, the antisense oligonucleotide splice modulator of the present invention can increase normal splicing of ACTL6B precursor-mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to a control.

[0057] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can increase the amount of wild-type ACTL6B protein by at least about 10% compared to a control. More preferably, the antisense oligonucleotide splice modulator of the present invention can increase the amount of wild-type ACTL6B protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to a control.

[0058] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can increase the normal splicing of ACTL6B precursor-mRNA and increase the amount of wild-type ACTL6B protein by at least about 10% compared to the control.More preferably, the antisense oligonucleotide splice modulator of the present invention can increase the normal splicing of ACTL6B precursor-mRNA and increase the amount of wild-type ACTL6B protein by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to the control.

[0059] Preferably, the antisense oligonucleotide splice modulators of the invention increase the amount of wild-type ACTL6B by decreasing the expression of ACTL6B mutant polypeptides in TDP-43-depleted cells.

[0060] The ACTL6B mutant polypeptide can be a splicing variant of ACTL6B. As used herein, the term "splice variant" or "splice variant" includes, but is not limited to, variant mature mRNA that contains additional exons compared to wild-type ACTL6B mature mRNA sequence. Wild-type ACTL6B mature mRNA sequence can be SEQ ID NO:2.

[0061] In some embodiments, the inclusion of an additional exon in the ACTL6B mature mRNA sequence compared to the wild-type ACTL6B polypeptide sequence may result in an insertion in the translated polypeptide sequence. The wild-type ACTL6B polypeptide may have the sequence of SEQ ID NO:3.

[0062] In some embodiments, the insertion may be about 23 amino acids compared to the wild-type ACTL6B polypeptide sequence. In other embodiments, the insertion may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids compared to the wild-type ACTL6B polypeptide sequence. The wild-type ACTL6B polypeptide may have the sequence of SEQ ID NO:3.

[0063] In some embodiments, the mutant polypeptide may be encoded by the nucleotide sequence of SEQ ID NO: 4, or a fragment or variant thereof. SEQ ID NO: 4 is provided herein as a reference sequence, and it will be understood that a nucleic acid sequence encoding a mutant ACTL6B polypeptide may be an allelic variant of SEQ ID NO: 4, such as an allelic variant comprising one or more polymorphisms.

[0064] In other embodiments, the ACTL6B mutant polypeptide may have the sequence of SEQ ID NO: 5, or a fragment or variant thereof. SEQ ID NO: 5 is provided herein as a reference sequence, and it will be understood that the mutant ACTL6B polypeptide may be an allelic variant of SEQ ID NO: 5, such as an allelic variant comprising one or more polymorphisms.

[0065] As used herein, the term "reducing the expression of an ACTL6B mutant" is understood to mean reducing alternatively spliced ​​ACTL6B mature mRNA levels, reducing mutant ACTL6B polypeptide levels, or reducing alternatively spliced ​​ACTL6B mature mRNA levels and reducing mutant ACTL6B polypeptide levels.

[0066] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can reduce the level of alternatively spliced ​​ACTL6B mature mRNA by at least 10% compared to a control. More preferably, the antisense oligonucleotide splice modulator of the present invention can reduce the level of alternatively spliced ​​ACTL6B mature mRNA by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to a control.

[0067] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can reduce mutant ACTL6B polypeptide levels by at least 10% compared to a control. More preferably, the antisense oligonucleotide splice modulator of the present invention can reduce mutant ACTL6B polypeptide levels by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to a control.

[0068] In some embodiments, the antisense oligonucleotide splice modulator of the present invention can reduce alternatively spliced ​​ACTL6B mature mRNA levels and reduce wild-type ACTL6B polypeptide levels by at least 10% compared to a control. More preferably, the antisense oligonucleotide splice modulator of the present invention can reduce alternatively spliced ​​mature ACTL6B mRNA levels and reduce mutant ACTL6B polypeptide levels by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% or more compared to a control.

[0069] Control When the term "control" is used in reference to measuring the effect of an antisense oligonucleotide splice modulator, it is generally understood that the control is a cell that has not been exposed to the antisense oligonucleotide splice modulator of the invention.

[0070] Alternatively, increased expression of wild-type ACTL6B or decreased expression of ACTL6B mutant may be determined by reference to the amount of wild-type and / or mutant ACTL6B mRNA and / or polypeptide expressed prior to exposure to an antisense oligonucleotide splice modulator of the invention.

[0071] In other embodiments, the control can be cells treated with a non-targeting oligonucleotide.

[0072] In some embodiments, the control can be a mock transfection, for example, where the cells are treated with PBS.

[0073] Oligonucleotides As used herein, the term "oligonucleotide" is defined as a molecule that contains two or more covalently linked nucleosides, as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0074] Oligonucleotides are usually produced in the laboratory by solid phase chemical synthesis followed by purification and isolation.When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of covalently linked nucleotides or nucleosides, or their modification.Antisense oligonucleotide splice modulators of the present invention are artificial, chemically synthesized, and typically purified or isolated.Antisense oligonucleotide splice modulators of the present invention can include one or more modified nucleosides, such as 2' sugar modified nucleosides.Antisense oligonucleotide splice modulators of the present invention can include one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages.

[0075] In some embodiments, the antisense oligonucleotide splice modulators of the invention are single-stranded oligonucleotides.

[0076] In some embodiments, the antisense oligonucleotide splice modulators of the invention are 8-40 nucleotides in length.

[0077] In some embodiments, the antisense oligonucleotide splice modulators of the invention are 8-40 nucleotides in length and comprise a contiguous nucleotide sequence of 8-40 nucleotides.

[0078] In some embodiments, antisense oligonucleotide splice modulators of the invention are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0079] In some embodiments, the antisense oligonucleotide splice modulators of the invention are at least 12 nucleotides in length.

[0080] In some embodiments, the antisense oligonucleotide splice modulators of the invention are at least 14 nucleotides in length.

[0081] In some embodiments, the antisense oligonucleotide splice modulators of the invention are at least 16 nucleotides in length.

[0082] In some embodiments, the antisense oligonucleotide splice modulators of the invention are at least 18 nucleotides in length.

[0083] Preferably, the antisense oligonucleotide splice modulators of the invention are 16-20 nucleotides in length.

[0084] More preferably, the antisense oligonucleotide splice modulators of the invention are 18 to 20 nucleotides in length.

[0085] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" as used herein refers to a region of the antisense oligonucleotide splice modulator of the present invention that is complementary to a target nucleic acid, which may be or may include an oligonucleotide motif sequence. This term is used interchangeably with the term "contiguous nucleobase sequence" herein.

[0086] Antisense oligonucleotide splice modulators comprise a contiguous nucleotide sequence and may optionally comprise a nucleotide linker region that can be used to attach additional nucleotides, such as functional groups (e.g., conjugate groups), to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0087] It is understood that the contiguous nucleotide sequence of an antisense oligonucleotide splice modulator cannot be longer than the antisense oligonucleotide splice modulator itself, and that the antisense oligonucleotide splice modulator cannot be shorter than the contiguous nucleotide sequence.

[0088] In some embodiments, the entire nucleotide sequence of an antisense oligonucleotide splice modulator of the invention is a contiguous nucleotide sequence.

[0089] A contiguous nucleotide sequence is a sequence of nucleotides in an antisense oligonucleotide splice modulator of the invention that is complementary to a target nucleic acid, target sequence or target site sequence, and in some cases is completely complementary to a target nucleic acid, target sequence or target site sequence.

[0090] In some embodiments, the contiguous nucleotide sequence is between 8 and 40 nucleotides in length.

[0091] In some embodiments, the contiguous nucleotide sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

[0092] In some embodiments, the contiguous nucleotide sequence is at least 12 nucleotides in length.

[0093] In some embodiments, the contiguous nucleotide sequence is at least 14 nucleotides in length.

[0094] In some embodiments, the contiguous nucleotide sequence is at least 16 nucleotides in length.

[0095] In some embodiments, the contiguous nucleotide sequence is at least 18 nucleotides in length.

[0096] In a preferred embodiment, the contiguous nucleotide sequence is 16 to 20 nucleotides in length.

[0097] More preferably, the contiguous nucleotide sequence is 18 to 20 nucleotides in length.

[0098] In some embodiments, the antisense oligonucleotide splice modulators of the invention consist of a contiguous nucleotide sequence.

[0099] In some embodiments, the antisense oligonucleotide splice modulators of the invention are contiguous nucleotide sequences.

[0100] Antisense oligonucleotide splice modulators targeting ACTL6B precursor-mRNA The antisense oligonucleotide splice modulators of the invention comprise a contiguous nucleotide sequence that is complementary to ACTL6B precursor-mRNA.

[0101] ACTL6B precursor-mRNA can be described as the target of the contiguous nucleotide sequence or the target of the antisense oligonucleotide splice modulator. In other words, the antisense oligonucleotide splice modulator targets ACTL6B precursor-mRNA.

[0102] In some embodiments, the target sequence may have the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is provided herein as a reference sequence, and it will be understood that the ACTL6B precursor-mRNA sequence may be an allelic variant of SEQ ID NO: 1, for example an allelic variant that includes one or more polymorphisms. This applies equally to all sequences identified herein as target sequences.

[0103] In one aspect, the present invention relates to an antisense oligonucleotide splice modulator, the antisense oligonucleotide splice modulator being 8 to 40 nucleotides in length and comprising a contiguous nucleotide sequence of at least 8 nucleotides in length that is complementary to SEQ ID NO:1.

[0104] In some embodiments, the antisense oligonucleotide splice modulators of the invention comprise a contiguous sequence that is at least about 75% complementary, at least about 80% complementary, at least about 85% complementary, at least about 90% complementary, at least about 95% complementary, or fully complementary (i.e., 100% complementary) to SEQ ID NO: 1, where complementarity is determined over the length of the contiguous nucleotide sequence.

[0105] In some embodiments, antisense oligonucleotide splice modulators of the invention comprise a contiguous sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary or fully complementary (i.e., 100% complementary) to SEQ ID NO:1.

[0106] In some embodiments, the antisense oligonucleotide splice modulators of the invention comprise a contiguous sequence which may contain one, two or three mismatches between the contiguous nucleotide sequence and the target nucleic acid.

[0107] In a preferred embodiment, the oligonucleotide of the present invention or its contiguous nucleotide sequence is fully complementary (100% complementary) to SEQ ID NO: 1 over the length of the contiguous nucleotide sequence.

[0108] In one embodiment, the contiguous nucleotide sequence is complementary to a splice enhancer site in the ACTL6B precursor-mRNA.

[0109] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:199.

[0110] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:199, such as at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or 100% complementary.

[0111] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is fully complementary (ie, 100% complementary) to SEQ ID NO:199.

[0112] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:200.

[0113] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:200, such as at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or 100% complementary.

[0114] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is perfectly complementary (ie, 100% complementary) to SEQ ID NO:200.

[0115] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:201.

[0116] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:201, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98% or 100% complementary.

[0117] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is perfectly complementary (ie, 100% complementary) to SEQ ID NO:201.

[0118] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:202.

[0119] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:202, such as at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or 100% complementary.

[0120] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is perfectly complementary (ie, 100% complementary) to SEQ ID NO:202.

[0121] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:203.

[0122] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:203, such as at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98% or 100% complementary.

[0123] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is perfectly complementary (ie, 100% complementary) to SEQ ID NO:203.

[0124] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:204.

[0125] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:204, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98% or 100% complementary.

[0126] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is perfectly complementary (ie, 100% complementary) to SEQ ID NO:204.

[0127] One aspect of the invention relates to an antisense oligonucleotide splice modulator comprising a contiguous nucleotide sequence of 8 to 40 nucleotides in length that is complementary to SEQ ID NO:205.

[0128] In some embodiments, the antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence which is at least 90% complementary to SEQ ID NO:205, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98% or 100% complementary.

[0129] In a preferred embodiment, an antisense oligonucleotide splice modulator of the invention comprises a contiguous sequence that is fully complementary (ie, 100% complementary) to SEQ ID NO:205.

[0130] In one embodiment, the target sequence is SEQ ID NO: 199. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO:199.

[0131] In one embodiment, the target sequence is SEQ ID NO: 200. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 200.

[0132] In one embodiment, the target sequence is SEQ ID NO: 201. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 201.

[0133] In one embodiment, the target sequence is SEQ ID NO: 202. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 202.

[0134] In one embodiment, the target sequence is SEQ ID NO: 203. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 203.

[0135] In one embodiment, the target sequence is SEQ ID NO: 204. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 204.

[0136] In one embodiment, the target sequence is SEQ ID NO: 205. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 205.

[0137] In some embodiments, the antisense oligonucleotide splice modulators of the invention are selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 , SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192 and SEQ ID NO:193.

[0138] In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55 and SEQ ID NO:72. In other words, in some embodiments, the contiguous nucleic acid is complementary to a sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:55 and SEQ ID NO:72.

[0139] In some embodiments, the target sequence is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:47. In other words, in some embodiments, the contiguous nucleic acid is complementary to a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:47.

[0140] In one embodiment, the target sequence is SEQ ID NO: 26 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 26 or a fragment thereof.

[0141] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:26.

[0142] In one embodiment, the target sequence is SEQ ID NO: 28 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 28 or a fragment thereof.

[0143] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:28.

[0144] In one embodiment, the target sequence is SEQ ID NO: 29 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 29 or a fragment thereof.

[0145] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:29.

[0146] In one embodiment, the target sequence is SEQ ID NO: 30 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 30 or a fragment thereof.

[0147] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:30.

[0148] In one embodiment, the target sequence is SEQ ID NO: 31 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 31 or a fragment thereof.

[0149] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:31.

[0150] In one embodiment, the target sequence is SEQ ID NO: 32 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 32 or a fragment thereof.

[0151] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:32.

[0152] In one embodiment, the target sequence is SEQ ID NO: 33 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 33 or a fragment thereof.

[0153] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:33.

[0154] In one embodiment, the target sequence is SEQ ID NO: 38 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 38 or a fragment thereof.

[0155] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:38.

[0156] In one embodiment, the target sequence is SEQ ID NO: 39 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 39 or a fragment thereof.

[0157] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:39.

[0158] In one embodiment, the target sequence is SEQ ID NO: 46 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 46 or a fragment thereof.

[0159] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:46.

[0160] In one embodiment, the target sequence is SEQ ID NO: 47 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 47 or a fragment thereof.

[0161] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:47.

[0162] In one embodiment, the target sequence is SEQ ID NO: 48 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 48 or a fragment thereof.

[0163] In another embodiment, the contiguous nucleotide sequence can be completely complementary to SEQ ID NO:48.

[0164] In one embodiment, the target sequence is SEQ ID NO: 52 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 52 or a fragment thereof.

[0165] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:52.

[0166] In one embodiment, the target sequence is SEQ ID NO: 53 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 53 or a fragment thereof.

[0167] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:53.

[0168] In one embodiment, the target sequence is SEQ ID NO: 55 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 55 or a fragment thereof.

[0169] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:55.

[0170] In one embodiment, the target sequence is SEQ ID NO: 72 or a fragment thereof. In other words, in some embodiments, the contiguous nucleic acid is complementary to SEQ ID NO: 72 or a fragment thereof.

[0171] In another embodiment, the contiguous nucleotide sequence can be fully complementary to SEQ ID NO:72.

[0172] In some embodiments, any fragment of the target sequence may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 consecutive nucleotides thereof, preferably at least 10 consecutive nucleotides.

[0173] Complementarity The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U).

[0174] Oligonucleotides may contain nucleosides having modified nucleobases, for example, 5-methylcytosine is often substituted for cytosine, and thus it will be understood that the term "complementary" encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al., 2012, Accounts of Chemical Research, 45, 2055 and Bergstrom, 2009, Curr. Protoc. Nucleic Acid Chem., 37, 1.4.1).

[0175] The term "% complementary" as used herein refers to the proportion (in percent) of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) over a contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned nucleobases (from Watson-Crick base pairs) that are complementary between two sequences (when aligned between the target sequence 5'-3' and the oligonucleotide sequence from 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in the calculation of the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of a nucleobase will be disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairing is retained (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating percent identity).

[0176] In the present invention, the term "complementary" requires that the antisense oligonucleotide splice modulator or its contiguous nucleotide sequence is at least about 75% complementary, at least about 80% complementary, at least about 85% complementary, at least about 90% complementary or at least about 95% complementary to a target sequence, e.g., ACTL6B precursor-mRNA. In some embodiments, the antisense oligonucleotide splice modulator or a contiguous sequence thereof may be at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% complementary or 100% complementary to a target sequence, e.g., ACTL6B precursor-mRNA.

[0177] In some embodiments, the antisense oligonucleotide splice modulators of the present invention or their contiguous nucleotide sequences may contain one, two, three or more mismatches, where a mismatch is a nucleotide within the antisense oligonucleotide splice modulator or its contiguous nucleotide sequence that does not base pair with its target.

[0178] The term "fully complementary" refers to 100% complementarity.

[0179] In some embodiments, the antisense oligonucleotide splice modulator is perfectly complementary to the target sequence.

[0180] In some embodiments, the contiguous nucleotide sequence is perfectly complementary to the target sequence.

[0181] identity As used herein, the term "identity" refers to the proportion of nucleotides (expressed as a percentage) of a contiguous nucleotide sequence within a nucleic acid molecule (e.g., an antisense oligonucleotide splice modulator) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence.

[0182] Thus, the percentage of identity is calculated by counting the number of aligned nucleobases that are identical (matching) between two sequences (in the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator of the present invention and in the reference sequence), dividing that number by the total number of nucleotides in the contiguous nucleotide sequence, and multiplying by 100. Thus, percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

[0183] It is therefore understood that a relationship exists between identity and complementarity, such that a contiguous nucleotide sequence within an antisense oligonucleotide splice modulator of the invention which is complementary to a target sequence also shares a percentage of identity with said target sequence.

[0184] Hybridization The term "hybridize" or "hybridize" as used herein should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between two nucleic acid strands is the strength of hybridization. This is often described in terms of melting temperature (Tm), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. In physiological conditions, Tm is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents the binding affinity and is related to the dissociation constant (Kd) of the reaction by ΔG°=-RTln(Kd), where R is the gas constant and T is the absolute temperature. Thus, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction with an aqueous concentration of 1M, pH of 7, and temperature of 37°C. The hybridization of oligonucleotides to target nucleic acids is a spontaneous reaction, and in spontaneous reactions, ΔG° is less than zero. ΔG° can be experimentally measured by using isothermal titration calorimetry (ITC) method, for example, as described in Hansen et al., 1965, Chem.Comm.36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available equipment is available for measuring ΔG°. ΔG° can also be estimated numerically using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95-1460-1465, using appropriately derived thermodynamic parameters as described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.

[0185] In some embodiments, the antisense oligonucleotide splice modulators of the invention hybridize to a target nucleic acid with a predicted ΔG° value of less than −10 kcal for an oligonucleotide that is 10-30 nucleotides in length.

[0186] In some embodiments, the degree or strength of hybridization is measured by the Gibbs free energy ΔG° at standard conditions. Antisense oligonucleotide splice modulators may hybridize to target nucleic acids with estimated ΔG° values ​​in the range of less than -10 kcal for oligonucleotides of 8 to 30 nucleotides in length, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal. In some embodiments, antisense oligonucleotide splice modulators hybridize to target nucleic acids with estimated ΔG° values ​​of -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal, or -16 to -27 kcal, such as -18 to -25 kcal.

[0187] Antisense Oligonucleotide Splice Modulators The antisense oligonucleotides of the invention are antisense oligonucleotide splice modulators that contain a contiguous nucleotide sequence complementary to ACTL6B precursor-mRNA.

[0188] In some embodiments, the contiguous nucleotide sequence is SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145 , SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, A sequence selected from the group consisting of sequence number 171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196 and SEQ ID NO:197, or a fragment thereof.

[0189] In some embodiments, a fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of a contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

[0190] In some embodiments, the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:164, or a fragment thereof.

[0191] In some embodiments, a fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of a contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

[0192] In some embodiments, the contiguous nucleotide sequence is a sequence selected from the group consisting of SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:139, or a fragment thereof.

[0193] In some embodiments, a fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of a contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

[0194] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 118 or a fragment thereof.

[0195] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 118 or a fragment thereof.

[0196] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 120 or a fragment thereof.

[0197] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 120 or a fragment thereof.

[0198] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 121 or a fragment thereof.

[0199] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 121 or a fragment thereof.

[0200] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 122 or a fragment thereof.

[0201] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 122 or a fragment thereof.

[0202] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 123 or a fragment thereof.

[0203] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 123 or a fragment thereof.

[0204] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 124 or a fragment thereof.

[0205] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 124 or a fragment thereof.

[0206] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 125 or a fragment thereof.

[0207] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 125 or a fragment thereof.

[0208] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 130 or a fragment thereof.

[0209] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 130 or a fragment thereof.

[0210] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 131 or a fragment thereof.

[0211] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 131 or a fragment thereof.

[0212] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 138 or a fragment thereof.

[0213] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 138 or a fragment thereof.

[0214] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 139 or a fragment thereof.

[0215] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 139 or a fragment thereof.

[0216] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 140 or a fragment thereof.

[0217] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 140 or a fragment thereof.

[0218] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 144 or a fragment thereof.

[0219] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 144 or a fragment thereof.

[0220] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 145 or a fragment thereof.

[0221] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 145 or a fragment thereof.

[0222] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 147 or a fragment thereof.

[0223] In one embodiment the contiguous nucleotide sequence consists of SEQ ID NO: 147 or a fragment thereof.

[0224] In one embodiment, the contiguous nucleotide sequence comprises SEQ ID NO: 164 or a fragment thereof.

[0225] In one embodiment, the contiguous nucleotide sequence consists of SEQ ID NO: 164 or a fragment thereof.

[0226] In some embodiments, a fragment may be at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 or at least 17 contiguous nucleotides of a contiguous nucleotide sequence, preferably at least 10 contiguous nucleotides thereof.

[0227] Nucleotides and Nucleosides Nucleotide and nucleoside are the building blocks of oligonucleotide and polynucleotide, and in the present invention, include both naturally occurring nucleotide and nucleoside and non-naturally occurring nucleotide and nucleoside.Naturally, nucleotide, such as DNA and RNA nucleotide, comprises a ribose sugar moiety, a nucleic acid base moiety and one or more phosphate groups (not present in nucleoside).Nucleoside and nucleotide can also be referred to interchangeably as "unit" or "monomer".

[0228] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that is modified compared to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or (nucleo) base moieties.

[0229] Advantageously, antisense oligonucleotide splice modulators of the invention may contain one or more modified nucleosides.

[0230] In some embodiments, the antisense oligonucleotide splice modulator or its contiguous nucleotide sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid. Advantageously, high affinity modified nucleosides are used.

[0231] Advantageously, one or more of the modified nucleosides of the antisense oligonucleotide splice modulators of the present invention may include a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the terms "nucleoside analog" or modified "unit" or modified "monomer". Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing. Exemplary modified nucleosides that may be used in the antisense oligonucleotide splice modulators of the present invention include LNA, 2'-O-MOE, 2'oMe and morpholino nucleoside analogs.

[0232] Modified internucleoside linkages Advantageously, antisense oligonucleotide splice modulators of the invention contain one or more modified internucleoside linkages.

[0233] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides to each other as commonly understood by those skilled in the art. The antisense oligonucleotide splice modulators of the present invention may therefore contain one or more modified internucleoside linkages, for example, one or more phosphorothioate internucleoside linkages.

[0234] In some embodiments, at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 90% or more of the internucleoside linkages in the antisense oligonucleotide splice modulator of the present invention or in its contiguous nucleotide sequence are phosphorothioate. In some embodiments, all of the internucleoside linkages in the antisense oligonucleotide splice modulator of the present invention or in its contiguous nucleotide sequence are phosphorothioate.

[0235] In a further embodiment, the antisense oligonucleotide splice modulator of the invention or its contiguous nucleotide sequence comprises at least one modified internucleoside linkage. Advantageously, at least 75%, for example all, of the internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages.

[0236] Advantageously, all internucleoside linkages of the contiguous nucleotide sequence of an antisense oligonucleotide splice modulator of the invention may be phosphorothioate linkages, or all internucleoside linkages of an antisense oligonucleotide splice modulator of the invention may be phosphorothioate linkages.

[0237] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1.

[0238] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

[0239] The nucleobase moieties may be designated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine.

[0240] Modified Oligonucleotides Antisense oligonucleotide splice modulators of the invention may be modified oligonucleotides.

[0241] The term "modified oligonucleotide" describes the oligonucleotide that comprises one or more sugar-modified nucleosides and / or modified internucleoside linkages.The term "chimeric oligonucleotide" is a term used in the literature to describe the oligonucleotide that comprises sugar-modified nucleosides and DNA nucleosides.In some embodiments, it may be advantageous for the antisense oligonucleotide splice modulator of the present invention to be or comprise chimeric oligonucleotides.

[0242] In some embodiments, the antisense oligonucleotide splice modulator of the present invention or its contiguous nucleotide sequence may contain modified nucleobases that function as the indicated nucleobases in base pairing, for example, 5-methylcytosine may be used instead of methylcytosine. Inosine may be used as a universal base.

[0243] It is understood that the consecutive nucleobase sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0244] The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into an oligonucleotide sequence is commonly referred to as the oligonucleotide design.

[0245] In one embodiment, the antisense oligonucleotide splice modulators of the invention comprise at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 modified nucleosides.

[0246] Suitable modifications are described herein under the headings "modified nucleosides," "high affinity modified nucleosides," "sugar modifications," "2' sugar modifications," and "Locked Nucleic Acids (LNA)."

[0247] High-affinity modified nucleosides A high affinity modified nucleoside is a modified nucleoside that, when incorporated into an oligonucleotide, increases the affinity of the oligonucleotide for its complementary target, for example as measured by melting temperature (Tm). The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature of +0.5 to +12°C, more preferably +1.5 to +10°C, and most preferably +3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213).

[0248] sugar modification Antisense oligonucleotide splice modulators of the invention may contain one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0249] A number of nucleosides with modifications in the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0250] Such modifications include, for example, those in which the ribose ring structure is modified by replacing it with a hexose ring (HNA), or a bicyclic ring, typically having a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring, typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0251] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

[0252] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or a nucleoside that contains a 2' linked biradicle that can form a bridge between the 2' carbon and a second carbon of the ribose ring, such as an LNA (2'-4' biradicle bridged) nucleoside.

[0253] In fact, much attention has been paid to the development of 2' sugar-substituted nucleosides, and many 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides.For example, 2' modified sugars can confer improved binding affinity and / or increased nuclease resistance to oligonucleotides.Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA nucleosides. For further examples, see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 203-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrative examples of some 2'-substituted modified nucleosides. [ka]

[0254] In the context of the present invention, 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides such as LNA.

[0255] In one embodiment, the antisense oligonucleotide splice modulator of the present invention can comprise one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides.Preferably, the antisense oligonucleotide splice modulator of the present invention comprises one or more 2' sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA (2'oMe), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (2'MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid (ANA), 2'-fluoro-ANA and LNA nucleoside.It is advantageous if one or more of the modified nucleosides are locked nucleic acid (LNA).

[0256] Locked Nucleoside (LNA) Nucleoside "LNA nucleosides" are 2' modified nucleosides that contain a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of said nucleoside, restricting or fixing the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixing the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into an oligonucleotide to a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0257] Non-limiting, exemplary LNA nucleosides include those described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al. al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0258] Further non-limiting exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1: [ka]

[0259] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0260] A particularly advantageous LNA is beta-D-oxy-LNA.

[0261] Morpholino Oligonucleotides In some embodiments, the antisense oligonucleotide splice modulator of the present invention comprises or consists of morpholino nucleosides (i.e., morpholino oligomers and phosphorodiamidate morpholino oligomers (PMOs)). Splice-modulating morpholino oligonucleotides have been approved for clinical use, see, for example, eteplirsen, a 30-nucleotide morpholino oligonucleotide that targets frameshift mutations in DMD and is used to treat Duchenne muscular dystrophy. Morpholino oligonucleotides have nucleases attached to a six-membered morpholine ring rather than a ribose, such as, for example, a methylene morpholine ring linked via a phosphorodiamidate group, as shown in the following diagram of four consecutive morpholino nucleotides: [ka]

[0262] In some embodiments, an antisense oligonucleotide splice modulator of the invention can be a morpholino, for example, from 8 to 40 nucleotides in length, such as from 16 to 20 nucleotides in length, for example, from 18 to 20 nucleotides in length.

[0263] RNase H activity and recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, the oligonucleotide has an initial rate, measured in pmol / l / min, of at least 5%, e.g., at least 10%, at least 20%, or more than 20% of the initial rate determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers and having phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). Recombinant RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland, for use in determining RNase H activity.

[0264] DNA oligonucleotides are known to effectively recruit RNaseH, such as gapmer oligonucleotides that contain regions of 2' sugar-modified nucleosides, typically high affinity 2' sugar-modified nucleosides, such as 2-O-MOE and / or LNA-containing regions of DNA nucleosides (typically at least 5 or 6 consecutive DNA nucleosides) flanked at 5' and 3'. For effective function as splice modulators, degradation of precursor-mRNA is undesirable, and thus it is preferable to avoid RNaseH degradation of the target. Thus, the antisense oligonucleotide splice modulator of the present invention is preferably not a gapmer oligonucleotide that recruits RNaseH.

[0265] RNaseH recruitment can be avoided by limiting the number of consecutive DNA nucleotides in antisense oligonucleotide splice modulators, so mixmer and totalmer designs can be used.Advantageously, in some embodiments, the antisense oligonucleotide splice modulator of the present invention or its consecutive nucleotide sequence does not contain more than 3 consecutive DNA nucleosides.More advantageously, in some embodiments, the antisense oligonucleotide splice modulator of the present invention or its consecutive nucleotide sequence does not contain more than 4 consecutive DNA nucleosides.More advantageously, in some embodiments, the antisense oligonucleotide splice modulator of the present invention or its consecutive nucleotide sequence does not contain more than 2 consecutive DNA nucleosides.

[0266] Mixmar and Totalmar For use as splice modulators, it is often advantageous to use antisense oligonucleotides that do not recruit RNAaseH and do not cause destruction of target precursor RNA. Because RNaseH activity requires a continuous sequence of DNA nucleotides, recruitment of RNaseH can be prevented by designing oligonucleotides that do not contain a region of more than three or more than four consecutive DNA nucleosides. This can be achieved by using antisense oligonucleotides or consecutive nucleoside regions thereof in mixmer designs that contain sugar-modified nucleosides, such as 2' sugar-modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides. Mixmers are exemplified herein by every other design having 5' and 3' terminal LNA nucleosides, where the nucleosides alternate between one LNA nucleoside and one DNA nucleoside, e.g., LDLDLDLDLDLDLDLL, and every third design such as LDDLDDLDDLDDLDDL, where every third nucleoside is an LNA nucleoside.

[0267] In one embodiment, a mixmer may comprise or consist of alternating nucleosides of 1, 2 or 3 consecutive DNA nucleosides followed by 1 or 2 consecutive LNA nucleosides.

[0268] A totalmer is an oligonucleotide or contiguous nucleotide sequence thereof that does not contain DNA or RNA nucleosides and may, for example, contain only 2'-O-MOE nucleosides, such as a full MOE phosphorothioate, e.g., MMMMMMMMMMMMMMMMMMMM, where M=2'-O-MOE, or may, for example, contain only 2'oMe nucleosides that have been reported to be effective for therapeutic use.

[0269] Alternatively, the mixmer may comprise a mixture of modified nucleosides such as MLMLMLMLMLMLMLMLML, where L=LNA and M=2'-O-MOE nucleoside.

[0270] Advantageously, the internucleosides in the mixmers and totalmers may be phosphorothioates or the majority of the nucleoside linkages in the mixmers may be phosphorothioates.

[0271] Mixmers and totalmers may contain other internucleoside linkages, such as, by way of example, phosphodiester or phosphorodithioate.

[0272] In some embodiments, the antisense oligonucleotide splice modulator is or comprises an oligonucleotide mixmer or totalmer. In some embodiments, the contiguous nucleotide sequence is a mixmer or totalmer.

[0273] Region D' or D" in the oligonucleotide Antisense oligonucleotide splice modulators of the invention may, in some embodiments, comprise a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, e.g., a mixmer or totalmer region, and additional 5' and / or 3' nucleosides. The additional 5' and / or 3' nucleosides may be complementary, e.g., fully complementary, to the target nucleic acid, or may not be complementary, e.g., fully complementary. Such additional 5' and / or 3' nucleosides may be referred to herein as regions D' and D".

[0274] The addition of region D' or D" can be used for the purpose of linking a contiguous nucleotide sequence, such as a mixmer or totalmer, to a conjugate moiety or another functional group. When used for linking, the contiguous nucleotide sequence with the conjugate moiety can serve as a biocleavable linker. Alternatively, the contiguous nucleotide sequence with the conjugate moiety can be used to provide exonuclease protection or to facilitate synthesis or production.

[0275] Region D' or D" can independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides, such as DNA or RNA or base-modified versions thereof. The D' or D' region can serve as a nuclease-sensitive biocleavable linker (see definition of linker). In some embodiments, the additional 5' and / or 3' terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biocleavable linkers suitable for use as region D' or D" are disclosed in WO 2014 / 076195, including by way of example phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in International Publication No. WO2015 / 113922, where they are used to link multiple antisense constructs within a single oligonucleotide.

[0276] In one embodiment, the antisense oligonucleotide splice modulators of the invention may contain regions D' and / or D" in addition to the contiguous nucleotide sequence which may constitute a mixmer or totalmer.

[0277] In some embodiments, the internucleoside linkage located between region D' or D" and the mixmer or totalmer region can be a phosphodiester bond.

[0278] Conjugates The present invention encompasses an antisense oligonucleotide splice modulator covalently attached to at least one conjugate moiety, which in some embodiments may be referred to as a conjugate of the invention.

[0279] In some embodiments, the present invention provides an antisense oligonucleotide splice modulator covalently linked to at least one conjugate moiety.

[0280] The term "conjugate" as used herein refers to an antisense oligonucleotide splice modulator that is covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or a third region). The conjugate moiety may optionally be covalently attached to the antisense oligonucleotide splice modulator via a linker group such as region D' or D".

[0281] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.

[0282] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate (e.g., GalNAc), a cell surface receptor ligand, a drug substance, a hormone, a lipophile, a polymer, a protein, a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0283] Linker Bond or linker is the connection between two atoms, which connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds.Conjugate moiety can be attached to antisense oligonucleotide splice modulator directly or through a linking moiety (e.g., linker or tether).Linker serves to covalently link the third region, e.g., conjugate moiety (region C), to the first region, e.g., the antisense oligonucleotide splice modulator or the continuous nucleotide sequence (region A) that is complementary to target nucleic acid.

[0284] In some embodiments of the invention, the conjugates or antisense oligonucleotide splice modulators of the invention may optionally comprise a linker region (second region or region B and / or region Y) located between the antisense oligonucleotide splice modulator or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

[0285] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally or similar to those encountered in a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidizing or reducing conditions or oxidizing or reducing agents, as well as salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In some embodiments, the nuclease-sensitive linker comprises 1-5 nucleosides, such as DNA nucleosides that contain at least two consecutive phosphodiester bonds. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195.

[0286] Region Y refers to a linker that is not necessarily biocleavable but primarily serves to covalently attach the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may comprise chain structures or oligomers of repeating units such as ethylene glycol, amino acid units or aminoalkyl groups. Antisense oligonucleotide splice modulator conjugates of the invention may be constructed from the following region elements AC, ABC, ABYC, AYBC or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, such as, for example, a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In some embodiments, the linker (region Y) is a C6 aminoalkyl group.

[0287] salt The term "salt" as used herein conforms to its commonly understood meaning, namely, an ionic assembly of anions and cations.

[0288] In some embodiments, the antisense oligonucleotide splice modulators of the present invention may be in the form of a pharma- ceutically acceptable salt. In other words, the present invention provides pharma- ceutically acceptable salts of the antisense oligonucleotide splice modulators of the present invention.

[0289] In some embodiments, the pharma- ceutically acceptable salt may be a sodium or potassium salt.

[0290] The present invention provides pharma- ceutically acceptable sodium salts of the antisense oligonucleotide splice modulators of the invention.

[0291] The present invention provides pharma- ceutically acceptable sodium salts of the antisense oligonucleotide splice modulators of the invention.

[0292] Delivery of antisense oligonucleotide splice modulators The present invention provides an antisense oligonucleotide splice modulator of the present invention, wherein said antisense oligonucleotide splice modulator is encapsulated in a lipid-based delivery vehicle, covalently linked to a dendrimer, or encapsulated in a dendrimer, or conjugated to an aptamer.

[0293] This may be for the purpose of delivering the antisense oligonucleotide splice modulators of the invention to targeted cells and / or improving the pharmacokinetics of the antisense oligonucleotide splice modulators.

[0294] Examples of lipid-based delivery vehicles include oil-in-water emulsions, micelles, liposomes, and lipid nanoparticles.

[0295] Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising an antisense oligonucleotide splice modulator of the invention and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0296] The present invention provides pharmaceutical compositions comprising an antisense oligonucleotide splice modulator of the invention and a pharma- ceutically acceptable salt.

[0297] For example, the salt may include a metal cation, such as a sodium or potassium salt.

[0298] The invention provides a pharmaceutical composition of the invention, said pharmaceutical composition comprising an antisense oligonucleotide splice modulator of the invention and an aqueous diluent or solvent.

[0299] The invention provides solutions, such as phosphate buffered saline solutions, of the antisense oligonucleotide splice modulators of the invention. In some embodiments, the solutions, such as phosphate buffered saline solutions, of the invention are sterile solutions.

[0300] Methods for increasing ACTL6B expression The present invention provides a method for enhancing, upregulating or restoring wild-type ACTL6B expression in a cell, such as a cell expressing ACTL6B, comprising administering to the cell an effective amount of an antisense oligonucleotide splice modulator of the present invention or a pharmaceutical composition of the present invention.

[0301] In some embodiments, the method is an in vitro method.

[0302] In some embodiments, the method is an in vivo method.

[0303] In some embodiments, the cell is an animal cell, preferably a mammalian cell, such as a mouse cell, a rat cell, a hamster cell or a monkey cell, or preferably a human cell.

[0304] In some embodiments, the cell is a mammalian cell.

[0305] In some embodiments, the cells are human cells.

[0306] In some embodiments, the cell is part of or derived from a subject that is affected with or susceptible to disease associated with the reduced expression of wild-type ACTL6B.Such diseases include but are not limited to amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine disease such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0307] treatment The term "treatment" as used herein refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Thus, it will be recognized that the treatment referred to herein may, in some embodiments, be prophylactic.

[0308] The invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide splice modulator of the invention or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

[0309] The disease may be associated with reduced expression of wild-type ACTL6B.

[0310] In some embodiments, the present invention provides a method for treating or preventing a disease associated with reduced expression of wild-type ACTL6B, comprising administering a therapeutically or prophylactically effective amount of an antisense oligonucleotide splice modulator of the present invention or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to a disease associated with reduced expression of wild-type ACTL6B.

[0311] In one embodiment, the disease is a neurological disorder.

[0312] In one embodiment, the disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0313] In some embodiments, the subject is an animal, preferably a mammal such as a mouse, rat, hamster, or monkey or human.

[0314] In some embodiments, the subject is a human.

[0315] The present invention provides an antisense oligonucleotide splice modulator of the invention for use as a pharmaceutical.

[0316] The invention provides an antisense oligonucleotide splice modulator of the invention for the preparation of a medicament.

[0317] The present invention provides antisense oligonucleotide splice modulators of the invention for use in therapy.

[0318] The present invention provides a pharmaceutical composition of the invention for use as a medicament.

[0319] The invention provides a pharmaceutical composition of the invention for the preparation of a medicament.

[0320] The invention provides pharmaceutical compositions of the invention for use in therapy.

[0321] The present invention provides an antisense oligonucleotide splice modulator of the invention for use as a pharmaceutical in the treatment of a neurological disorder.

[0322] The present invention provides an antisense oligonucleotide splice modulator of the invention for use as a medicament in the treatment of a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0323] The invention provides the use of an antisense oligonucleotide splice modulator of the invention for the preparation of a medicament for the treatment or prevention of a neurological disorder.

[0324] The present invention provides the use of an antisense oligonucleotide splice modulator of the present invention for the preparation of a medicament for the treatment or prevention of a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0325] The invention provides a pharmaceutical composition of the invention for use as a medicament in the treatment of a neurological disorder.

[0326] The present invention provides a pharmaceutical composition of the invention for use as a medicament in the treatment of a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0327] The present invention provides the use of a pharmaceutical composition of the present invention for the preparation of a medicament for the treatment or prevention of a neurological disorder.

[0328] The present invention provides a use of the pharmaceutical composition of the present invention for the preparation of a medicament for the treatment or prevention of a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy.

[0329] Administration The antisense oligonucleotide splice modulators of the invention or pharmaceutical compositions of the invention may be administered topically (such as to the skin, inhalation, eye or ear) or enterally (such as orally or through the digestive tract) or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0330] In a preferred embodiment, the antisense oligonucleotide splice modulator of the invention or the pharmaceutical composition of the invention is administered by a parenteral route, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular administration. In one embodiment, the antisense oligonucleotide splice modulator of the invention is administered intracerebrally or intracerebroventricularly. In another embodiment, the antisense oligonucleotide splice modulator of the invention is administered intrathecally.

[0331] The present invention also provides the use of a described antisense oligonucleotide splice modulator of the invention or a pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intrathecal administration.

[0332] The present invention also provides the use of a described antisense oligonucleotide splice modulator of the invention or a pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intracerebral or intraventricular administration.

[0333] The present invention also provides the use of a described antisense oligonucleotide splice modulator of the invention or a pharmaceutical composition of the invention for the preparation of a medicament in a dosage form for intracerebroventricular administration.

[0334] Combination therapy In some embodiments, the antisense oligonucleotide splice modulators of the invention or the pharmaceutical compositions of the invention are for use in combination treatment with one or more other therapeutic agents. EXAMPLES

[0335] Example 1: Identification of ACTL6B as a novel target of TDP43 mRNA splice regulation. One hallmark of ALS disease is the presence of cytoplasmic aggregated TDP43 protein in a small proportion of patients' neurons. The consequence of the cytoplasmic aggregation of TDP43 is that TDP43 is depleted in the cell nucleus, where it cannot perform its normal function.

[0336] TDP43 has been shown to affect mRNA splicing. To identify new genes whose mRNAs are regulated by the presence of TDP43, we knocked down TDP43 in a neuronal model. We performed RNA sequencing on the cells and de novo transcript analysis to identify affected genes with novel splice patterns.

[0337] Human glutamatergic neurons (Fujifilms) were seeded at 60,000 viable cells together with 10,000 live astrocytes (Fujifilms) in 200 μl of culture medium in 96-well plates coated with laminin and poly(ethyleneimine) solution (Sigma Aldrich) (day −1).

[0338] To knock down TDP-43, Compound A (SEQ ID NO: 198) was added to the culture medium at 5 μM on day 0, and in other wells, PBS was added instead as a control. Half of the cell culture medium was replaced three times a week during the entire experiment (days 2, 5, 7, 10, 12, 14 and 17). Cells were harvested on day 20 using Magnapure lysis buffer (Roche) and RNA was isolated with MagNA pure 96 system (Roche) according to the manufacturer's instructions, including a DNase treatment step. NGS libraries were prepared from 100 ng of total RNA using KAPA mRNA HyperPrep Kit Illumina® Platforms (Roche). Libraries were subjected to paired-end sequencing on a NovaSeq6000 sequencer (Illumina) with a read length of 150 bp. Data analysis was performed using CLC Genomics Workbench 21. The data were first analyzed by performing giant gap mapping analysis using the hg38 genome assembly for subsequent transcript discovery. Predicted novel splice events were investigated by manual visual inspection to identify actual splice events.

[0339] Upon loss of TDP43, a new 69-base pair exon was found to be included in ACTL6B. The first and last bases of the new exon are 100,650,643 and 100,650,575, according to the hg38 human gene annotation, in which ACTL6B is arranged in a negative orientation.

[0340] Figure 1 shows a screenshot from CLC Genomics Workbench software, where NGS read mapping from ACTL6B gene can be seen. The arrow indicates the inclusion of a new exon in the sample treated with compound A (SEQ ID NO: 198). About 99% of all mRNA in this sample contains this exon. For untreated samples, the inclusion of this exon is seen in less than 0.5% of reads.

[0341] Example 2: Rescue of incorrect ACTL6B mRNA splicing caused by lack of TDP43 using ASO. In this example, we demonstrate the ability of ASO to induce proper splicing for ACTL6B, a target of TDP43. Human glutamatergic neurons (Fujifilms) were seeded with 60,000 live cells in 96-well plates coated with laminin and poly(ethyleneimine) solution (Sigma Aldrich) in 200 μl of culture medium together with 10,000 live astrocytes (Fujifilms) (day -1). To knock down TDP-43, compound A (SEQ ID NO: 198) was added to the culture medium at 5 μM on day 0 (except for four control wells per plate). Half of the cell culture medium was replaced three times a week during the entire experiment (days 2, 5, 7, 9, 12, 14, 16 and 19). ASO targeting the hidden ACTL6B exon was added to the culture medium at 10 μM on day 5. A total of 96 different ASOs were added (SEQ ID NOs: 98-189 and 194-197). At least 12 wells per plate received only Compound A (SEQ ID NO: 198) to serve as a baseline standard. Experiments were performed in duplicate. A total of four 96-well plates were used.

[0342] Cells were harvested on day 20 using Magnapure lysis buffer (Roche) and RNA was isolated with the MagNA pure 96 system (Roche) according to the manufacturer's instructions, including a DNase treatment step. Purified RNA was denatured at 90 for 30 s prior to cDNA synthesis. cDNA was generated using the iScript Advanced cDNA Synthesis kit for RT-qPCR (Biorad) according to the manufacturer's instructions.

[0343] Measurement of expression levels of target genes was performed by droplet digital PCR using the QX1 system (Bio-Rad) with QX1 software standard version. The PCR probe assay used to measure expression of normally spliced ​​target mRNA was designed to span two exons between which a new "mutated" exon would arise.

[0344] Expression values ​​from two replicate experiments are shown in Table 1. The following PCR probe assays synthesized by Integrated DNA technologies (IDT) were used: TARDBP Primer 1: CAGCTCATCCTCAGTCATGTC, Primer 2: GATGGTGTGACTGCAAACTTC, Probe: / 5Cy5 / CAGCGCCCCACAAACACTTTTCT / 3IAbRQSp / ) ACTL6B wt(ex4~ex5): Primer 1: TCTGAGCCAAACCTGCAC, Primer 2: ATCAGCTCTGTCAGCTTCTCC, Probe: / 5HEX / CGAGGCTCC / ZEN / GTGGAACACACG / 3IABkFQ / )

[0345] The following CY5.5-labeled HPRT1 probe was purchased from BioRad: dHsaCPE13136107. Table 1: Production of TDP43 and ACTL6B WT after exposure to oligonucleotides The data shown in Table 2 was normalized to the expression of the housekeeping gene HPRT1 and finally to the average expression value of control wells (PBS) that did not receive TDP43 knockdown or CA repeat ASO. The average expression for all of the given conditions is shown in the last column. KD ("Knockdown" describes wells that received only treatment with a gapmer ASO that degrades TDP43 mRNA). [Table 1-1]

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

[0346] array SEQ ID NO:1 ACTL6B precursor-mRNA GRCh38.p13, chromosome 7: 100,643,097-100,656,448 reverse strand SEQ ID NO:2 ACTL6B wild-type mature mRNA sequence SEQ ID NO:3 ACTL6B wild-type protein sequence MSGGVYGGDEVGALVFDIGSFSVRAGYAGEDCPKADFPTTVGLLAAEEGGGLELEGDKEKKGKIFHIDTNALHVPRDGAEVMSPLKNGMIEDWECFRAILDHTYSK HVKSEPNLHPVLMSEAPWNTRAKREKLTELMFEQYNIPAFFLCKTAVLTAFANGRSTGLVLDSGATHTTAIPVHDGYVLQQGIVKSPLAGDFISMQCRELFQEMAID IIPPYMIAAKEPVREGAPPNWKKKEKLPQVSKSWHNYMCNEVIQDFQASVLQVSDSPYDEQVAAQMPTVHYEMPNGYNTDYGAERLRIPEGLFDPSNVKGLSGNTML GVGHVVTTSIGMCDIDIRPGLYGSVIVTGGNTLLQGFTDRLNRELSQKTPPSMRLKLIASNSTMERKFSPWIGGSILASLGTFQQMWISKQEYEEGGKQCVERKCP* SEQ ID NO:4 ACTL6B mutant mature mRNA GAGTCCCGCCCCGCCAGGGATCCCGGGAGCTGTCCGGCCGCCTCGGTGCTGATCCCGCCACCGCCCACGGGCCGCTAGCAGCGCAGCGGGCACTATGAGCGGGGGCGTCTACGGCGGAGATGAGGTGGGGGCGCTGGTCTTTGACATTGGCTCCTTCTCAGTCCGCGCTGGGTACGCTGGGGAGGACTGTCCCAAGGCTGACTTCCCCACCACAGTGGGGCTGCTGGCCGCGGAGGAGGGGGGCGGGCTGGAGCTGGAGGGGGACAAAGAGAAGAAAGGGAAGATCTTCCACATCGACACCAATGCCCTGCACGTGCCTCGGGATGGAGCGGAGGTCATGTCGCCCCTCAAGAATGGCATGATCGAGGACTGGGAGTGCTTCCGAGCCATCCTGGATCACACCTACAGCAAACACGTCAAGTCTGAGCCAAACCTGCACCCAGTGCTCATGTCCGAGGCTCCG GCTAGTTTCGAACTCCTGGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGC SEQ ID NO:5 ACTL6B mutant protein sequence MSGGVYGGDEVGALVFDIGSFSVRAGYAGEDCPKADFPTTVGLLAAEEGGGLELEGDKEKKGKIFHIDTNALHVPRDGAEVMSPLKNGMIEDWECFRAILDHTYSKHVKSEP NLHPVLMSEAPASFELLGSSNPPASASQSAGIIGWNTRAKREKLTELMFEQYNIPAFFLCKTAVLTAFANGRSTGLVLDSGATHTTAIPVHDGYVLQQGIVKSPLAGDFISMQ CRELFQEMAIDIIPPYMIAAKEPVREGAPPNWKKKEKLPQVSKSWHNYMCNEVIQDFQASVLQVSDSPYDEQVAAQMPTVHYEMPNGYNTDYGAERLRIPEGLFDPSNVKGL SGNTMLGVGHVVTTSIGMCDIDIRPGLYGSVIVTGGNTLLQGFTDRLNRELSQKTPPSMRLKLIASNSTMERKFSPWIGGSILASLGTFQQMWISKQEYEEGGKQCVERKCP* SEQ ID NO:6-97 ASO target sequence SEQ ID NO: 98-189 ASO sequences SEQ ID NO: 190-193 ASO target sequence SEQ ID NO: 194-197 ASO sequences SEQ ID NO:198 TCCACACTGAACAAACC SEQ ID NO:199 Target area 1 GAGACAGCATCTCACTATGTTGTCCAGGCTAGTTTCGAACTCCTGGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTAAGCCACCCTGTCTGGCCTGTGCAAATGTTTTAAATGCATGTGTGTGACTGTGAGTGTGAATATATGTGTATGTGTGTGTGAGTTTGTGTGTGAATGTGAGTATAT Sequence number 200 target area 2 ACTCCTGGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTAAGCCACCCTGTCTGGCCTGTGCAAATGTTTTAAATGCATGTGTGTGACTGTGAGTGTGAATATATGGTTAGTGTGTGTGTGAGT SEQ ID NO:201 target area 3 TCCTGGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTAAGCCACCCTGTCTGGCCTGTGCAAATGTTTTAAATGCATGTGTGTGTGACTGTGAGT SEQ ID NO:202 target area 4 TCCTGGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTAAGCCACCCTGTCTGGCC SEQ ID NO:203 target area 5 GGGTTCAAGCAATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGCGTAA SEQ ID NO:204 target area 6 AAGCAATCCTCCTGCCTCGGCC SEQ ID NO:205 target area 7 CTGGGATTATAGGCGTAA

Claims

1. 1. An antisense oligonucleotide actin-like 6B (ACTL6B) splice modulator, comprising: An antisense oligonucleotide splice modulator of actin-like 6B (ACTL6B), wherein the antisense oligonucleotide splice modulator is 8 to 40 nucleotides in length and comprises a contiguous nucleotide sequence of at least 8 nucleotides that is complementary to ACTL6B precursor-mRNA, and wherein the antisense oligonucleotide splice modulator is capable of increasing expression of ACTL6B in TDP-43-depleted cells.

2. a) ACTL6B is encoded by the nucleotide sequence of SEQ ID NO: 2 or a fragment or variant thereof; and / or b) The antisense oligonucleotide splice modulator of claim 1, wherein the ACTL6B protein has the sequence of SEQ ID NO: 3 or a fragment or variant thereof.

3. the antisense oligonucleotide splice modulator is capable of reducing the expression of an ACTL6B mutant polypeptide in TDP-43-depleted cells, and optionally the ACTL6B mutant polypeptide is a splicing variant of ACTL6B, and optionally The ACTL6B mutant polypeptide comprises a polypeptide sequence encoded by an additional exon when compared to a wild-type ACTL6B polypeptide sequence, and optionally the ACTL6B mutant polypeptide comprises an insertion when compared to the wild-type ACTL6B polypeptide sequence, and optionally 3. The antisense oligonucleotide splice modulator of claim 1 or 2, wherein the insertion is an insertion of about 23 amino acids.

4. a) the ACTL6B mutant polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 4 or a fragment or variant thereof; and / or b) The antisense oligonucleotide splice modulator of claim 3, wherein the ACTL6B mutant polypeptide has the sequence of SEQ ID NO: 5 or a fragment or variant thereof.

5. a) the contiguous nucleotide sequence is complementary to a splice enhancer site in the ACTL6B precursor-mRNA, and / or b) the contiguous nucleotide sequence is complementary to SEQ ID NO: 204, and / or c) the contiguous nucleotide sequence is complementary to a sequence selected from SEQ ID NOs: 6-35, 37, 38, 40, 43-45, 48-97 and 190-193, and optionally the contiguous nucleotide sequence is complementary to a sequence selected from SEQ ID NOs: 26, 28, 29, 30, 31, 32, 33, 38, 46, 48, 52, 53, 55 and 72; and optionally the contiguous nucleotide sequence is complementary to a sequence selected from SEQ ID NOs: 28, 29, 30, 31, 32 and 33; and / or d) the contiguous nucleotide sequence is a sequence selected from SEQ ID NOs: 98-127, 129, 130, 132, 135-137, 140-189 and 194-197 or at least 10 contiguous nucleotides thereof, and optionally the contiguous nucleotide sequence is a sequence selected from SEQ ID NOs: 118, 120, 121, 122, 123, 124, 125, 130, 140, 144, 145, 147 and 164 or at least 10 contiguous nucleotides thereof, and optionally the contiguous nucleotide sequence is a sequence selected from SEQ ID NOs: 120, 121, 122, 123, 124 and 125 or at least 10 contiguous nucleotides thereof; and / or e) the contiguous nucleotide sequence is at least 12 nucleotides in length, and optionally the contiguous nucleotide sequence is at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length; and / or f) said contiguous nucleotide sequence is the same length as said antisense oligonucleotide splice modulator; and / or g) The antisense oligonucleotide splice modulator of claim 3, wherein the antisense oligonucleotide splice modulator is isolated, purified or manufactured.

6. a) the contiguous nucleotide sequence comprises one or more modified nucleosides, and / or b) the antisense oligonucleotide splice modulator is a morpholino antisense oligonucleotide, and / or c) the one or more modified nucleosides are 2' sugar modified nucleosides, e.g., 2'-O-alkyl-RNA; 2'-O-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-O-methoxyethyl-RNA (2'-MOE); 2'-amino-DNA; 2'-fluoro-RNA; 2'-fluoro-DNA; arabinonucleic acid (ANA); 2'-fluoro-ANA; locked nucleic acid (LNA), or any combination thereof; and optionally 2. The antisense oligonucleotide splice modulator of claim 1, wherein the 2' sugar modified nucleoside is an affinity-enhancing 2' sugar modified nucleoside.

7. a) the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator comprises 2'-O-methoxyethyl-RNA (2'-MOE) nucleosides, and optionally all nucleosides of said contiguous nucleotide sequence are 2'-O-methoxyethyl-RNA (2'-MOE) nucleosides optionally linked by phosphorothioate internucleoside linkages; and / or b) one or more of said modified nucleosides are locked nucleic acid nucleosides (LNA), e.g., LNA nucleosides selected from the group consisting of constrained ethyl nucleosides (cEt) and β-D-oxy-LNA, and optionally 2. The antisense oligonucleotide splice modulator of claim 1, wherein the contiguous nucleotide sequence of the antisense oligonucleotide splice modulator comprises or consists of LNA nucleosides and DNA nucleosides.

8. a) the contiguous nucleotide sequence is at least 75% complementary to the ACTL6B precursor-mRNA sequence, and optionally the contiguous nucleotide sequence is at least 80%, at least 85%, at least 90%, or at least 95% complementary to the ACTL6B precursor-mRNA sequence; or b) the contiguous nucleotide sequence is completely complementary to the ACTL6B precursor-mRNA, and / or c) the contiguous nucleotide sequence contains one, two, three or more mismatches to the ACTL6B precursor-mRNA sequence, and / or d) the Gibbs free energy of said antisense oligonucleotide splice modulator with respect to a complementary target RNA is less than about -10 ΔG, such as less than about -15 ΔG, for example less than about -17 ΔG; and / or e) the antisense oligonucleotide splice modulator does not contain a region of more than three or more than four consecutive DNA nucleosides, and / or f) the antisense oligonucleotide splice modulator is incapable of mediating RNAse H cleavage, and / or g) The antisense oligonucleotide splice modulator of claim 1, wherein the antisense oligonucleotide splice modulator or its contiguous nucleotide sequence is a mixmer or a totalmer.

9. A method for treating a leukemia, comprising: (a) administering to a subject a leukemia, comprising administering to a subject a leukemia, a leukemia, or an ... b) the cytosine bases present in said antisense oligonucleotide splice modulator or its contiguous nucleotide sequence are 5-methylcytosine, and / or c) one or more of the internucleoside linkages located between nucleosides on said contiguous nucleotide sequence are modified, and / or d) at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the internucleoside linkages located between nucleosides on said contiguous nucleotide sequence are modified; and / or e) one or more or all of said modified internucleoside linkages comprise a phosphorothioate linkage; and / or f) all internucleoside linkages present in said antisense oligonucleotide splice modulator are phosphorothioate internucleoside linkages, and / or g) The antisense oligonucleotide splice modulator of claim 1, wherein the antisense oligonucleotide splice modulator is covalently linked to at least one conjugate moiety.

10. the antisense oligonucleotide splice modulator is in the form of a pharmaceutically acceptable salt, and optionally 2. The antisense oligonucleotide splice modulator of claim 1, wherein the pharmaceutically acceptable salt is a sodium salt or a potassium salt.

11. A pharmaceutical composition comprising the antisense oligonucleotide splice modulator of claim 1 and a pharmaceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

12. 12. An in vitro method for increasing ACTL6B expression in a cell, the method comprising administering to the cell an effective amount of the antisense oligonucleotide splice modulator of claim 1 or the pharmaceutical composition of claim 11, wherein the cell expresses abnormal levels of TDP-43 or exhibits depleted levels of TDP-43.

13. 12. An antisense oligonucleotide splice modulator according to claim 1 or a pharmaceutical composition according to claim 11 for use as a medicament.

14. 12. The antisense oligonucleotide splice modulator of claim 1 or the pharmaceutical composition of claim 11 for use in treating or preventing a disease in a subject.

15. the disease is a neurological disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), progressive supranuclear palsy (PSP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer's disease, Parkinson's disease, autism, hippocampal sclerosis dementia, Down's syndrome, Huntington's disease, polyglutamine diseases such as spinocerebellar ataxia 3, myopathy and chronic traumatic encephalopathy, and optionally 15. The antisense oligonucleotide splice modulator for use according to claim 14, wherein the disease is a neurological disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

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