JP7051683B2 - Antisense oligomer for the treatment of tuberous sclerosis - Google Patents

Description

This application claims the benefit of US Provisional Patent Application No. 62 / 267,212 filed December 14, 2015, which provisional application is incorporated herein by reference in its entirety.

<Sequence list>
This application includes a sequence listing, which is submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. A copy of ASCII made on 12 December 2016 is 47991-706_601_SL. It is a file name of txt and has a size of 1,663,708 bytes. The aforementioned files were created on December 12, 2016 and are incorporated herein by reference in their entirety.

Tuberous sclerosis (TSC) is a disorder characterized by the growth of benign tumors in multiple organ systems (Au, K., et al., J. Child Neurol., 2004, 19: 699-709). Tumors of the central nervous system (CNS) are the leading contributors to morbidity and mortality, followed by renal disease. Patients may suffer from brain abnormalities that can include seizures, intellectual disability, and developmental delays, as well as skin, lung, kidney, and heart abnormalities. Disability affects 25,000 to 40,000 individuals in the United States and approximately 1 to 2 million worldwide, with an assessed prevalence of 1 in 6,000 newborns. Presumed.

TSC is a genetic disease with an autosomal dominant inheritance pattern caused by a hereditary defect or mutation in de novo that occurs on the two genes TSC1 and TSC2. TSC is present if only one of the genes is affected. The TSC1 gene on chromosome 9 produces a protein called hamartin. Discovered in 1993, the TSC2 gene is located on chromosome 16 and produces the protein tuberin. Scientists believe that these proteins act in the complex as growth inhibitors by inhibiting the activation of a dominant evolutionarily conserved kinase called mTOR. Loss of mTOR regulation occurs in cells lacking either hamartin or tuberin, which causes the abnormal differentiation and development as seen in TSC brain lesions, and the production of enlarged cells.

The present invention provides compositions and methods for treating tuberous sclerosis, wherein the composition is constitutive splicing at the intron splice site of a retained intron-containing pre-mRNA (RIC pre-mRNA) of TSC2. Contains antisense oligomers (ASO) that promote. The invention further comprises compositions and methods for increasing the production of mature TSC2 mRNA and, in turn, TSC2 protein in the cells of the subject in need, eg, the cells of the subject who can benefit from increased production of TSC2 protein. I will provide a. The above method results in tuberous sclerosis caused by deletion of all genes in addition to mutations within the TSC2 gene, including missense, splicing, frameshift and nonsense mutations that result in a deficiency of tuberin protein production. It may be used to treat a subject having.

The present specification discloses, in a particular embodiment, a method of treating a subject's nodular sclerosis in need by increasing the expression of a target protein or functional RNA by the subject's cells. , The cell has a retained intron-containing mRNA precursor (RIC mRNA precursor), the RIC mRNA precursor is a retained intron, an exon adjacent to a 5'splice site, a 3'splice site. Containing an exson flanking the RIC mRNA precursor, wherein the RIC mRNA precursor encodes a target protein or functional RNA, wherein the method encodes the subject's cells with the RIC mRNA precursor encoding the target protein or functional RNA. Contains contact with an antisense oligomer (ASO) that is complementary to the target portion of the cell, whereby the retained intron is constitutively spliced from the RIC mRNA precursor encoding the target protein or functional RNA. Increases the level of mRNA encoding the target protein or functional RNA and increases the expression of the target protein or functional RNA in the subject's cells. In some embodiments, the specification also discloses a method of increasing the expression of a target protein, which is a tuberin, by cells having a retained intron-containing mRNA precursor (RIC mRNA precursor). , The RIC mRNA precursor comprises a retained intron, an exson flanking the 5'splice site of the retained intron, and an exson flanking the 3'splice site of the retained intron. The RIC mRNA precursor encodes a tuberin protein, the method comprising contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the tuberin protein. Retained introns are constitutively spliced from the RIC mRNA precursor encoding the tuberin protein, thereby increasing the level of mRNA encoding the tuberin protein in the cell and expressing the tuberin protein. To increase. In some embodiments, the target protein is tuberin. In some embodiments, the target protein or functional RNA functionally increases or replaces the target protein or functional RNA that is deficient in quantity or activity in the subject, the compensatory protein or compensation. It is a functional RNA. In some embodiments, the cells are in or derived from a subject having a disease caused by a lack of amount or activity of the tuberin protein. In some embodiments, the deficiency in the amount of target protein is caused by haplodeficiency of the target protein, where the subject does not produce the first allogeneic gene encoding the functional target protein, the target protein. It has two allelic genes, or a second allelic gene encoding a non-functional target protein, and the antisense oligomer binds to the target portion of the RIC pre-mRNA transcribed from the first allelic gene. In some embodiments, the subject has a disease caused by a disorder due to a lack of quantity or function of the target protein, wherein the subject has (a) (i) the target protein in the wild. It is produced at a reduced level compared to the production from the type allelic gene, and (ii) the target protein is produced in a less functional form compared to the equivalent wild type protein, or (iii) the target protein is produced. The first mutant allelic gene that is not produced and (b) (i) the target protein is produced at a reduced level compared to the production from the wild-type allelic gene, and (ii) the target protein is an equivalent wild-type protein. It has a second mutated allelic gene, which is produced in a reduced function as compared to, or (iii) no target protein is produced, wherein the subject is the first mutated allelic gene a. iii. The second mutation allele is b. i. Or b. ii. Here, the subject is the second mutation allele b. iii. When, the first mutation allele is a. i. Or a. ii. Here, the RIC mRNA precursor is a. i. Or a. ii. The first mutation allele and / or b. i. Or b. ii. Is transcribed from the second mutation allele. In some embodiments, the target protein is produced in a reduced function as compared to an equivalent wild-type protein. In some embodiments, the target protein is produced in a fully functional form as compared to an equivalent wild-type protein. In some embodiments, the target portion of the RIC mRNA precursor is within the region from +6 to the 5'splice site of the retained intron to -16 relative to the 3'splice site of the retained intron. Located in the retained intron. In some embodiments, the target portion of the RIC mRNA precursor is within the region from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron. Located in the retained intron. In some embodiments, the target portion of the RIC pre-mRNA is in the following internal retained introns: (a) +6 to +500, +6 to +495 for the 5'splice site of the retained intron. , Or a region of +6 to +100; or (b) a region of -16 to -500, -16 to -400, or -16 to -100 with respect to the retained intron's 3'splice site. In some embodiments, the target portion of the RIC pre-mRNA is within: (a) the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron; or (b). ) The region of + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to SEQ ID NO: 1. It is encoded by a gene sequence that has sequence identity. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 2-8. , 99%, or 100% sequences with sequence identity. In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from the gene encoding the functional RNA or target protein. .. In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by regulating aberrant splicing due to mutations in the gene encoding the target protein or functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing of the full-length pre-mRNA or partial splicing of the wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. In some embodiments, the target protein produced is a full-length protein or a wild-type protein. In some embodiments, the total amount of mRNA encoding the target protein or functional RNA produced in cells contacted with the antisense oligomer is that of the mRNA encoding the target protein or functional RNA produced in control cells. Compared to the total amount, about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to About 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to About 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, It increases at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times. In some embodiments, the total amount of target protein produced by cells in contact with the antisense oligomer is about 1.1 to about 10 times, about 10 times, as compared to the total amount of target protein produced by control cells. 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1. 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Double, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times , About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times , At least about 5 times, or at least about 10 times. In some embodiments, the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, Alternatively, it consists of 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100 of the target portion of the RIC mRNA precursor encoding the protein. % Complementary. In some embodiments, the target portion of the RIC mRNA precursor is within a sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, relative to any one of SEQ ID NO: 9-5096. Includes a nucleotide sequence that is 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9-5096. In some embodiments, the cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA is retained by more than one. Introns are included, and where the antisense oligomer binds to the most abundant retained introns in the population of RIC pre-mRNA. In some embodiments, binding of the antisense oligomer to the most abundant retained intron was retained by two or more from a population of RIC pre-mRNA that produce mRNA encoding the target protein or functional RNA. Induces intron splicing out. In some embodiments, the cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA is retained by more than one. It comprises an intron, where the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNA. In some embodiments, binding of the antisense oligomer to the second abundant retained intron is the retention of two or more from the population of RIC pre-mRNA that produce mRNA encoding the target protein or functional RNA. Induces splicing out of the intron. In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disorder is tuberous sclerosis. In some embodiments, the target protein and RIC mRNA precursor are encoded by the TSC2 gene. In some embodiments, the method further comprises the step of assessing TSC2 protein expression. In some embodiments, the antisense oligomer binds to the target portion of the Tuberin RIC mRNA precursor, where the target portion is SEQ ID NOS: 49, 50, 51, 52, 53, 54, 55, 56, and. It is in the sequence selected from 57. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a fetus, embryo, or child. In some embodiments, the cells are Exvivo. In some embodiments, the antisense oligomer is administered topically to the skin of a subject, delivered to the lungs, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous. It is administered by intramuscular injection. In some embodiments, the exons -3e to -1e flanking the 5'splice site and the nine nucleotides at +1 to +6 of the retained intron are identical to the corresponding wild type sequence. In some embodiments, the 16 nucleotides at -15 to -1 of the retained intron and + 1e of the exon flanking the 3'splice site are identical to the corresponding wild type sequence.

The present specification discloses antisense oligomers used in the above methods in certain embodiments.

In certain embodiments, it comprises a sequence having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity for any one of SEQ ID NO: 9-5096. Antisense oligomers are disclosed herein.

The present specification discloses, in a specific embodiment, a pharmaceutical composition comprising the above antisense oligomers and excipients. In some embodiments, the present specification also includes topical administration to the skin, lung delivery to the lungs, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject in need by administering the pharmaceutical composition by injection is described.

As used herein, in certain embodiments, expression of a target protein or functional RNA by cells to treat nodular sclerosis of a subject associated with a deficient protein or deficient functional RNA. A composition comprising an antisense oligomer for use in a method of increasing protein is disclosed, wherein the deficient protein or functional RNA is deficient in quantity or activity in the subject, where anti. The sense oligomer enhances the constitutive splicing of the retained intron-containing mRNA precursor (RIC mRNA precursor), which encodes the target protein or functional RNA, where the target protein is: (a) deficient. A protein; or (b) a compensatory protein that functionally increases or replaces a deficient protein in a subject, where the functional RNA is: (a) the deficient RNA; Or (b) a functional RNA that functionally enhances or replaces or compensates for the deficient functional RNA in the subject, where the RIC mRNA precursor is flanking the retained intron and 5'splice site. The retained intron, which comprises an exson that is adjacent to the 3'splice site, is spliced from the RIC mRNA precursor encoding the target protein or functional RNA, thereby the target protein in the subject. Or increase the production or activity of functional RNA. In some embodiments, the specification also discloses a composition comprising an antisense oligomer for use in a method of treating a disease associated with a tuberin protein in a subject, wherein the method is subject. In the process of increasing the expression of the tuberin protein by the cells of the cell, to the intron in which the cell was retained, the exson adjacent to the 5'splice site of the retained intron, and the 3'splice site of the retained intron. Having a retained intron-containing mRNA precursor (RIC mRNA precursor), including adjacent exons, the RIC mRNA precursor encodes a tuberin protein, the method of contacting the cell with an antisense oligomer. Containing, thereby the retained intron is constitutively spliced from the transcript of the RIC mRNA precursor encoding the tuberin protein, thereby in the cell of the subject, of the mRNA encoding the tuberin protein. Includes steps to increase levels and increase expression of tuberin protein. In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disorder is tuberous sclerosis. In some embodiments, the target protein and RIC mRNA precursor are encoded by the TSC2 gene. In some embodiments, the antisense oligomer is a retained intron in the region from +6 to the 5'splice site of the retained intron to -16 relative to the 3'splice site of the retained intron. Targets some of the RIC pre-mRNA in. In some embodiments, the antisense oligomer targets a portion of the RIC mRNA precursor, which is located in the inner retained intron below: (a) against the 5'splice site of the retained intron. +6 to +100 regions; (b) -16 to -100 regions relative to the retained intron's 3'splice site. In some embodiments, the antisense oligomer is from about 500, 400, 300, 200, or 100 nucleotides downstream of the 5'splice site of at least one retained intron, the 3'of at least one retained intron. Target a portion of the RIC mRNA precursor within a region up to about 100, 200, 300, 400, or 500 nucleotides upstream of the splice site. In some embodiments, the target portion of the RIC pre-mRNA is within: (a) the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron; or (b). ) The region of + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to SEQ ID NO: 1. It is encoded by a gene sequence that has sequence identity. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 2-8. , 99%, or 100% sequences with sequence identity. In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from the gene encoding the target protein or functional RNA. In some embodiments, the antisense oligomer does not increase the amount of functional RNA or functional protein by regulating aberrant splicing due to mutations in the target protein or gene encoding the functional RNA. In some embodiments, the RIC pre-mRNA was produced by full-length pre-mRNA, or partial splicing from wild-type pre-mRNA. In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. In some embodiments, the target protein produced is a full-length protein or a wild-type protein. In some embodiments, the retained intron is a rate-determining intron. In some embodiments, the retained intron is the most abundant retained intron in the RIC pre-mRNA. In some embodiments, the retained intron is the second most abundant retained intron in the RIC pre-mRNA. In some embodiments, the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, Alternatively, it consists of 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100 of the target portion of the RIC mRNA precursor encoding the protein. % Complementary. In some embodiments, the antisense oligomer binds to the target portion of the Tuberin RIC mRNA precursor, where the target portion is within a sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, relative to any one of SEQ ID NO: 9-5096. Includes a nucleotide sequence that is 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9-5096.

The present specification discloses, in a specific embodiment, a pharmaceutical composition comprising the above antisense oligomers and excipients. In some embodiments, the present specification also includes topical administration to the skin, lung delivery to the lungs, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject in need by administering the pharmaceutical composition by injection is described.

In certain embodiments, the pharmaceutical composition is disclosed herein, the composition being an antisense oligomer that hybridizes to the target sequence of the deficient TSC2 mRNA transcript, and a pharmaceutically acceptable embodiment. The agent is included, where the deficient TSC2 mRNA transcript comprises retained introns, and the antisense oligomer induces splicing out of the retained introns from the deficient TSC2 mRNA transcript. In some embodiments, the deficient TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor. In some embodiments, the target portion of the transcript of the TSC2 RIC pre-mRNA is from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron. Is in a retained intron within the area of. In some embodiments, the transcript of the TSC2 RIC pre-mRNA is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 relative to SEQ ID NO: 1. It is encoded by a gene sequence having% or 100% sequence identity. In some embodiments, the TSC2 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to any one of SEQ ID NO: 2-8. Includes sequences with%, 99%, or 100% sequence identity. In some embodiments, the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, Alternatively, it contains 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% of the target portion of the transcript of the TSC2 RIC mRNA precursor. , Or 100% complementary. In some embodiments, the target portion of the transcript of the TSC2 RIC mRNA precursor is within the sequence selected from SEQ ID NO: 5097-5105. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, relative to any one of SEQ ID NO: 9-5096. Includes a nucleotide sequence that is 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9-5096. In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

In certain embodiments, treatment of the TSC2 mRNA transcript that is deficient to facilitate the removal of retained introns to produce a well-processed TSC2 mRNA transcript that encodes the functional form of the tuberin protein. Disclosed herein are methods of (a) contacting a subject's target cells with an antisense oligomer and (b) a transcript of TSC2 mRNA deficient in the antisense oligomer. In the step of hybridizing to, the deficient TSC2 mRNA transcript can encode the functional form of the tuberin protein and comprises at least one retained intron. The step of removing at least one retained intron from the TSC2 mRNA transcript that is deficient to produce a well-processed TSC2 mRNA transcript that encodes the functional form of the verin protein, and (d). It comprises translating the functional form of the tuberin protein from a well-treated transcript of TSC2 mRNA. In some embodiments, the retained intron is the entire retained intron. In some embodiments, the deficient TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor.

The present specification discloses, in a particular embodiment, a method of treating a subject having a disease caused by a lack of amount or activity of a tuberin protein, the method of which is SEQ ID NO: 9-5096. Sequence identity of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% for any one. Including a step of administering an antisense oligomer containing a nucleotide sequence having a substance to a subject.

<Embedded by citation>
All publications, patents, and patent applications referred to herein are by reference to the extent that they are specifically and individually directed to include the individual publications, patents, or patent applications by reference. Incorporated herein.
The present application includes the following inventions.
[Item 1] A method for treating tuberous sclerosis in a subject by increasing the expression of a target protein or functional RNA in the cells of the subject.
Here, the cell has a retained intron-containing pre-mRNA (RIC pre-mRNA), and the RIC pre-mRNA is a retained intron, an exon adjacent to a 5'splice site, and a 3'splice. Containing exons adjacent to the site, the RIC pre-mRNA encodes the target protein or functional RNA.
The method is
The subject's cells are contacted with an antisense oligomer (ASO) complementary to the target portion of the RIC pre-mRNA encoding the target protein or functional RNA, whereby the retained intron is the target protein. Alternatively, it is constitutively spliced from a RIC pre-mRNA encoding a functional RNA, thereby increasing the level of the target protein or mRNA encoding the functional RNA in the subject's cells, thereby increasing the level of the target protein or functional RNA. A method of increasing the expression of.
[Item 2] A method for increasing the expression of a target protein, which is tuberin, by a cell having a retained intron-containing pre-mRNA (RIC pre-mRNA).
The RIC pre-mRNA includes a retained intron, an exon adjacent to the 5'splice site of the retained intron, an exon adjacent to the 3'splice site of the retained intron, and the RIC pre-mRNA Encodes the tuberin protein and
The method is
It involves contacting the cell with an antisense oligomer (ASO) that is complementary to the target portion of the RIC mRNA precursor encoding the tuberin protein, whereby the retained intron is the RIC mRNA encoding the tuberin protein. A method of constitutively splicing from a precursor, thereby increasing the level of mRNA encoding the Tubelin protein and increasing the expression of the Tubelin protein in the cell.
[Item 3] The method according to item 1, wherein the target protein is tuberin.
[Item 4] The target protein or functional RNA functionally increases or replaces the target protein or functional RNA that is deficient in quantity or activity in the subject, and is a compensatory protein or compensatory functional RNA. The method according to item 1.
[Item 5] The method of item 2, wherein the cells are in or derived from a subject having a disease caused by a lack of amount or activity of tuberin protein.
[Item 6] The deficiency in the amount of the target protein is caused by the haplodeficiency of the target protein, where the subject is given a first allelic gene encoding a functional target protein, a second in which the target protein is not produced. Item 1 to having a second allelic gene encoding an allelic or non-functional target protein, the antisense oligomer binding to the target portion of the RIC pre-mRNA transcribed from the first allelic gene. The method according to any one of 5.
[Item 7] The subject has a disease caused by a disorder caused by a lack of the amount or function of the target protein.
Here, the subject is
a.
i. Target proteins are produced at reduced levels compared to those produced from wild-type alleles,
ii. The target protein is produced in a less functional form compared to the equivalent wild-type protein, or
iii. No target protein is produced,
The first mutation allele and
b.
i. Target proteins are produced at reduced levels compared to those produced from wild-type alleles,
ii. The target protein is produced in a less functional form compared to the equivalent wild-type protein, or
iii. No target protein is produced,
Has a second mutant allele and
Here, the subject is the first mutation allele a. iii. The second mutation allele is b. i. Or b. ii. Here, the subject is the second mutation allele b. iii. When, the first mutation allele is a. i. Or a. ii. Here, the RIC mRNA precursor is a. i. Or a. ii. The first mutation allele and / or b. i. Or b. ii. The method according to any one of items 1 to 5, which is transcribed from the second mutation allele.
[Item 8] The method according to item 7, wherein the target protein is produced in a form with reduced function as compared with an equivalent wild-type protein.
[Item 9] The method according to item 7, wherein the target protein is produced in a sufficiently functional form as compared to an equivalent wild-type protein.
[Item 10] The target portion of the RIC mRNA precursor was retained within the region from +6 to the 5'splice site of the retained intron to -16 relative to the 3'splice site of the retained intron. The method according to any one of items 1 to 9 in the intron.
[Item 11] The target portion of the RIC mRNA precursor was retained within the region from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron. The method according to any one of items 1 to 9 in the intron.
[Item 12] The target portion of the RIC mRNA precursor is
(A) Regions +6 to +500, +6 to +495, or +6 to +100, or +6 to +100 relative to the 5'splice site of the retained intron.
(B) Regions -16 to -500, -16 to -400, or -16 to -100, relative to the 3'splice site of the retained intron.
The method according to any one of items 1 to 9, which is in the retained intron inside the.
[Item 13] The target portion of the RIC mRNA precursor is
(A) Regions + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, or
(B) Regions + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron,
The method according to any one of items 1 to 9 inside.
[Item 14] The RIC mRNA precursor has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to SEQ ID NO: 1. The method according to any one of items 1 to 13, which is encoded by a gene sequence having.
[Item 15] The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% with respect to any one of SEQ ID NO: 2-8. , Or the method of any one of items 1-14, comprising a sequence having 100% sequence identity.
[Item 16] The antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from the gene encoding the functional RNA or target protein, item 1. The method according to any one of 15 to 15.
[Item 17] Antisense oligomers do not increase the amount of target protein or functional RNA by regulating aberrant splicing due to mutations in the gene encoding the target protein or functional RNA, items 1-15. The method according to any one of.
[Item 18] The item 1 to 17, wherein the RIC pre-mRNA is produced by partial splicing of a full-length pre-mRNA or partial splicing of a wild-type pre-mRNA. Method.
[Item 19] The method according to any one of items 1 to 18, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.
[Item 20] The method according to any one of claims 1 to 19, wherein the produced target protein is a full-length protein or a wild-type protein.
[Item 21] The total amount of mRNA encoding the target protein or functional RNA produced in the cells contacted with the antisense oligomer is compared with the total amount of mRNA encoding the target protein or functional RNA produced in the control cells. Then, about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times. , About 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times , About 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, About 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 The method according to any one of items 1 to 20, wherein the method is increased by a factor of, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.
[Item 22] The total amount of target protein produced by the cells contacted with the antisense oligomer is about 1.1 to about 10 times, about 1.5 times, the total amount of the target protein produced by the control cells. ~ About 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 ~ About 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about about The method according to any one of items 1 to 21, which increases by 5 times, or at least about 10 times.
[Item 23] The method according to any one of items 1 to 22, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 24] The antisense oligomer comprises either phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety, any of items 1-23. The method described in one.
[Item 25] The method according to any one of items 1 to 24, wherein the antisense oligomer contains at least one modified sugar moiety.
[Item 26] The method according to item 25, wherein each sugar moiety is a modified sugar moiety.
[Item 27] The antisense oligomer includes 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. , 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 -15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11- 15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12- The method according to any one of items 1 to 26, which comprises 15 nucleobases.
[Item 28] The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the RIC mRNA precursor encoding the protein. The method according to any one of items 1 to 27.
[Item 29] The method according to any one of items 1 to 28, wherein the target portion of the RIC mRNA precursor is in a sequence selected from SEQ ID NO: 5097 to 5105.
[Item 30] The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, with respect to any one of SEQ ID NO: 9 to 5096. The method of any one of claims 1-29, comprising a nucleotide sequence having 96%, 97%, 98%, or 99% sequence identity.
31. The method of any one of Items 1-29, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9-5096.
[Item 32] The cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA comprises two or more retained introns. And, here, the method according to any one of items 1-31, wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre-mRNA.
[Item 33] Binding of antisense oligomers to the most abundant retained introns is the splicing of two or more retained introns from a population of RIC pre-mRNAs that produce mRNA encoding the target protein or functional RNA. 32. The method of item 32, which induces out.
[Item 34] The cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA comprises two or more retained introns. And, here, the method according to any one of items 1-31, wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNA.
[Item 35] Binding of the antisense oligomer to the second most abundant retained intron is the binding of two or more retained introns from a population of RIC pre-mRNAs that produce mRNA encoding the target protein or functional RNA. 34. The method of claim 34, which induces splicing out of.
[Item 36] The method according to any one of items 5 to 35, wherein the disease is a disease or a disorder.
37. The method of item 36, wherein the disease or disorder is tuberous sclerosis.
38. The method of claim 37, wherein the target protein and RIC mRNA precursor are encoded by the TSC2 gene.
[Item 39] The method according to any one of items 1 to 38, wherein the method further comprises a step of evaluating TSC2 protein expression.
[Item 40] The antisense oligomer binds to the target portion of the Tuberin RIC mRNA precursor, where the target portion is selected from SEQ ID NOs: 49, 50, 51, 52, 53, 54, 55, 56, and 57. The method according to any one of items 1 to 39 in the sequence.
[Item 41] The method according to any one of items 1 to 40, wherein the subject is a human.
[Item 42] The method according to any one of items 1 to 40, wherein the subject is an animal other than a human.
[Item 43] The method according to any one of items 1 to 41, wherein the subject is a fetus, an embryo, or a child.
[Item 44] The method according to any one of items 1-42, wherein the cell is Exvivo.
[Item 45] The antisense oligomer is administered by topical administration to the skin of the subject, lung delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. The method according to any one of items 1-42, which is administered.
[Item 46] Exons -3e to -1e adjacent to the 5'splice site and nine nucleotides at +1 to +6 of the retained intron are identical to the corresponding wild type sequences, items 1-45. The method described in any one.
[Item 47] The 16 nucleotides at -15 to -1 of the retained intron and + 1e of the exon adjacent to the 3'splice site are identical to the corresponding wild type sequence, any one of items 1-46. The method described in one.
[Item 48] An antisense oligomer used in any one of the methods of items 1 to 39.
[Item 49] Antis comprising sequences with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity for any one of SEQ ID NO: 9-5096. Sense oligomer.
[Item 50] A pharmaceutical composition comprising the antisense oligomer and excipient of item 48 or 49.
[Item 51] The pharmaceutical composition according to item 50 by topical administration to the skin, lung delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject by administering an object.
[Item 52] To be used in a method of increasing the expression of a target protein or functional RNA by cells to treat tuberous sclerosis in a subject associated with a deficient protein or deficient functional RNA. A composition comprising the antisense oligomer of
Here, the deficient protein or functional RNA is deficient in quantity or activity in the subject, where the antisense oligomer is a retained intron-containing mRNA encoding the target protein or functional RNA. Enhances constitutive splicing of precursors (RIC mRNA precursors),
Here, the target protein is
(A) Missing protein or
(B) Compensating protein that functionally increases or replaces the deficient protein in the subject.
Here, the functional RNA is
(A) Missing RNA or
(B) Functional RNA that functionally enhances or replaces or compensates for the functional RNA that is deficient in the subject.
Here, the RIC pre-mRNA comprises a retained intron, an exon flanking the 5'splice site, and an exon flanking the 3'splice site, where the retained intron is the target protein or functional. A composition spliced from an RNA-encoding RIC pre-mRNA, thereby increasing the production or activity of a target protein or functional RNA in a subject.
[Item 53] A composition comprising an antisense oligomer for use in a method of treating a disease associated with a tuberin protein in a subject.
The method is a step of increasing the expression of the tuberin protein by the subject's cells, the cell-retained intron, the exson adjacent to the 5'splice site of the retained intron, and the retained intron. It has a retained intron-containing mRNA precursor (RIC mRNA precursor), including exson adjacent to the 3'splice site of the RIC mRNA precursor, which encodes a tuberin protein, a step and anti-cell. The step of contacting with the sense oligomer, whereby the retained intron is constitutively spliced from the transcript of the RIC mRNA precursor encoding the tuberin protein, thereby in the subject's cells. A composition comprising a step of increasing the level of mRNA encoding the verin protein and increasing the expression of the tuberin protein.
[Item 54] The composition according to item 53, wherein the disease is a disease or disorder.
55. The composition of item 54, wherein the disease or disorder is tuberous sclerosis.
56. The composition of claim 55, wherein the target protein and RIC mRNA precursor are encoded by the TSC2 gene.
[Item 57] The antisense oligomer is an RIC in the retained intron within the region from +6 for the 5'splice site of the retained intron to -16 for the 3'splice site of the retained intron. The composition according to any one of items 52 to 56, which targets a part of the pre-mRNA.
[Item 58] The antisense oligomer targets a portion of the RIC mRNA precursor, which is:
(A) Region +6 to +100 with respect to the 5'splice site of the retained intron,
(B) The composition according to any one of items 52-57, located in the retained intron within the region -16 to -100 relative to the 3'splice site of the retained intron.
[Item 59] The antisense oligomer is from about 500, 400, 300, 200, or 100 nucleotides downstream of the 5'splice site of at least one retained intron to the 3'splice site of at least one retained intron. The composition according to any one of items 52-56, which targets a portion of the RIC mRNA precursor within a region up to about 100, 200, 300, 400, or 500 nucleotides upstream.
[Item 60] The target portion of the RIC mRNA precursor is
(A) Regions + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, or
(B) Regions + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron,
The composition according to any one of items 52 to 56, which is contained in the above.
[Item 61] The RIC mRNA precursor has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to SEQ ID NO: 1. The composition according to any one of items 52 to 60, which is encoded by a gene sequence having.
[Item 62] The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% with respect to any one of SEQ ID NO: 2-8. , Or the composition according to any one of items 52-61, comprising a sequence having 100% sequence identity.
[Item 63] The antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from the gene encoding the target protein or functional RNA, items 52- 62. The composition according to any one of 62.
[Item 64] The antisense oligomer does not increase the amount of functional RNA or functional protein by regulating aberrant splicing due to mutations in the target protein or gene encoding the functional RNA, items 52-. 62. The composition according to any one of 62.
[Item 65] The composition according to any one of items 52 to 64, wherein the RIC pre-mRNA is produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.
[Item 66] The composition according to any one of items 52 to 65, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.
[Item 67] The composition according to any one of items 52 to 66, wherein the produced target protein is a full-length protein or a wild-type protein.
[Item 68] The composition according to any one of items 52 to 67, wherein the retained intron is a rate-determining intron.
[Item 69] The composition according to any one of items 52 to 68, wherein the retained intron is the most abundant retained intron in the RIC pre-mRNA.
[Item 70] The composition according to any one of items 52 to 68, wherein the retained intron is the second most abundant retained intron in the RIC pre-mRNA.
[Item 71] The composition according to any one of items 52 to 70, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 72] The composition according to any one of items 52 to 71, wherein the antisense oligomer is an antisense oligonucleotide.
[Item 73] The antisense oligomer comprises either phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety, any of items 52-72. The composition according to one.
[Item 74] The composition according to any one of items 52 to 73, wherein the antisense oligomer comprises at least one modified sugar moiety.
[Item 75] The composition according to item 74, wherein each sugar moiety is a modified sugar moiety.
[Item 76] The antisense oligomer includes 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. , 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 -15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11- 15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12- The composition according to any one of items 52 to 75, which comprises 15 nucleobases.
[Item 77] The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the RIC mRNA precursor encoding the protein. The composition according to any one of items 52 to 76.
[Item 78] The antisense oligomer binds to the target portion of the Tuberin RIC mRNA precursor, where the target portion is any of items 52-77 within the sequence selected from SEQ ID NO: 5097-5105. The composition according to one.
[Item 79] The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, relative to any one of SEQ ID NO: 9-5096. The composition according to any one of claims 52 to 78, comprising a nucleotide sequence having 96%, 97%, 98%, or 99% sequence identity.
[Item 80] The composition according to any one of items 52 to 78, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9 to 5096.
[Item 81] A pharmaceutical composition comprising any of the antisense oligomers and excipients of the compositions of items 52-80.
Item 82. The pharmaceutical composition according to item 81, by topical administration to the skin, lung delivery to the lung, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A method of treating a subject by administering an object.
[Item 83] An antisense oligomer that hybridizes to the target sequence of the deficient TSC2 mRNA transcript, including an intron that retains the deficient TSC2 mRNA transcript, and is deficient in antisense oligomers. With antisense oligomers, which induce splicing out of retained introns from the transcript of TSC2 mRNA.
Including pharmaceutically acceptable excipients,
Pharmaceutical composition.
[Item 84] The pharmaceutical composition according to item 83, wherein the deficient TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor.
[Item 85] The target portion of the transcript of the TSC2 RIC pre-mRNA is within the region from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron. The pharmaceutical composition according to item 83 or 84, which is in the retained intron of.
[Item 86] TSC2 RIC mRNA precursor transcripts are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 relative to SEQ ID NO: 1. The pharmaceutical composition according to any one of items 83 to 85, which is encoded by a gene sequence having% sequence identity.
[Item 87] The TSC2 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 with respect to any one of SEQ ID NO: 2-8. %, The pharmaceutical composition according to any one of items 83-86, comprising a sequence having 100% sequence identity.
[Item 88] The pharmaceutical composition according to any one of items 83 to 87, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 89] The pharmaceutical composition according to any one of items 83 to 88, wherein the antisense oligomer is an antisense oligonucleotide.
[Item 90] The antisense oligomer comprises either phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety, any of items 83-89. The pharmaceutical composition according to one.
[Item 91] The pharmaceutical composition according to any one of items 83 to 90, wherein the antisense oligomer contains at least one modified sugar moiety.
[Item 92] The antisense oligomer includes 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, and 8 to 20 nucleobases. , 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 -15 nucleobases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11- 15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12- The pharmaceutical composition according to any one of items 83 to 91, which comprises 15 nucleobases.
[Item 93] The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100 with respect to the target portion of the transcript of the TSC2 RIC mRNA precursor. % The pharmaceutical composition according to any one of items 83-92, which is complementary.
[Item 94] The pharmaceutical composition according to any one of items 83 to 93, wherein the target portion of the transcript of the TSC2 RIC mRNA precursor is in a sequence selected from SEQ ID NO: 5097 to 5105.
[Item 95] The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, with respect to any one of SEQ ID NO: 9 to 5096. The pharmaceutical composition according to any one of claims 83 to 94, comprising a nucleotide sequence having 96%, 97%, 98%, or 99% sequence identity.
[Item 96] The pharmaceutical composition according to any one of items 83 to 94, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 9 to 5096.
[Item 97] The pharmaceutical composition is formulated for any one of items 83 to 95, which is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. The pharmaceutical composition according to description.
[Item 98] Treatment of deficient TSC2 mRNA transcripts to facilitate removal of retained introns to produce fully treated TSC2 mRNA transcripts that encode the functional morphology of the tuberin protein. It ’s a way to trigger
The method is
(A) A step of contacting the antisense oligomer with the target cells of the subject,
(B) In the step of hybridizing to the transcript of TSC2 mRNA deficient in antisense oligomer, the transcript of deficient TSC2 mRNA can encode the functional form of the tuberin protein, at least. A process that includes one retained intron, and
(C) The step of removing at least one retained intron from the TSC2 mRNA transcript that is deficient to produce a well-processed TSC2 mRNA transcript that encodes the functional form of the tuberin protein.
(D) A method comprising translating the functional form of a tuberin protein from a well-treated transcript of TSC2 mRNA.
Item 99. The method of item 98, wherein the retained intron is the entire retained intron.
[Item 100] The method of item 98 or 99, wherein the deficient TSC2 mRNA transcript is a transcript of the TSC2 RIC mRNA precursor.
[Item 101] A method of treating a subject having a disease caused by a lack of amount or activity of tuberin protein.
The method is
SEQ ID NO: At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% for any one of 9 to 5096. , Or a method comprising administering to a subject an antisense oligomer comprising a nucleotide sequence having 99% sequence identity.

The novel features of the invention are specified in particular within the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description illustrating exemplary embodiments in which the principles of the invention are utilized and the accompanying drawings. ..
FIG. 6 shows a schematic representation of a typical retained intron-containing (RIC) pre-mRNA transcript. The 5'splice site consensus sequence is an underlined letter (letter) from -3e to -1e and from +1 to +6 (numbers labeled "e" are exons and unlabeled numbers are introns). Is a nucleotide; uppercase: exon part, and lower case: intron part). The 3'splice site consensus sequence is an underlined letter (letter is nucleotide) from -15 to -1 and + 1e (numbers labeled "e" are exons and unlabeled numbers are introns). (Uppercase: exon part, and lowercase: intron part). Intron target regions for ASO screening range from nucleotide +6 to the 5'splice site (left arrow) of the retained intron to nucleotide-16 to the 3'splice site (right arrow) of the retained intron. including. In embodiments, the intron target region for ASO screening comprises nucleotides +6 to +100 for the 5'splice site of the retained intron and nucleotides -16 to -100 for the 3'splice site of the retained intron. .. Exon target sites are + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, and + 2e to -4e nucleotides in the exon adjacent to the 3'splice site of the retained intron. include. "N" or "N" represents a nucleotide and "y" represents a pyrimidine. The illustrated sequence represents a consensus sequence for the mammalian splice site, and the individual introns and exons need not match the consensus sequence at all positions. A schematic diagram of a targeted augmentation (TANGO) method of nuclear gene output is illustrated. FIG. 2A shows cells divided into nuclei and cytoplasmic compartments. In the nucleus, transcripts of pre-mRNA of target genes consisting of exons (rectangular) and introns (connecting lines) are spliced to produce mRNA, which is transported to the cytoplasm and translated into the target protein. To. For this target gene, splicing of intron 1 is inefficient, and retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus and degrades when transported to the cytoplasm of the target protein. It does not lead to production. A schematic diagram of a targeted augmentation (TANGO) method of nuclear gene output is illustrated. FIG. 2B shows an example of similar cells divided into nuclei and cytoplasmic compartments. Treatment with antisense oligomers (ASOs) promotes splicing of intron 1 and results in an increase in mRNA, which in turn is translated into high levels of target protein. The intron retention in the TSC2 gene with intron 4 is shown in detail. Identification of intron retention events in the TSC2 gene using RNA sequencing (RNAseq) is visualized and shown in the UCSC Genome Browser. The upper panel shows the reading density corresponding to the TSC2 transcript expressed in HCN (human cortical neurons) and localized in either the cytoplasm (top) or the nuclear fraction (bottom). At the bottom of this panel, a graphical representation of the TSC2 gene is shown on a scale. The reading density is shown as a peak. The highest reading density corresponds to exons (black boxes), while no readings are observed for most of the introns (lines with arrowheads) in any cell fractionation. Higher read densities compared to the cytoplasmic fraction were detected in introns 4, 25/26, and 31/32 (indicated by arrows) in the nuclear fraction, which are introns 4, 25/26, and 31 /. It is shown that the splicing efficiency of 32 is low, resulting in intron retention. Transcripts of retained intron-containing mRNA precursors are retained in the nucleus and not transported to the cytoplasm. The reading density of intron 4 in HCN is shown in detail in the panel below. The TSC2 gene IVS 4 ASO walk (walk) is shown. An ASO walk performed on TSC2 IVS 4 targeting sequences immediately downstream of the 5'splice site or upstream of the 3'splice site using a 2'-OMe ASO, PS backbone is illustrated. ASO was designed to cover these regions by repositioning 5 nucleotides at a time. The TSC2 exon-intron structure is shown to scale. Intron retention in the TSC2 gene with introns 25 and 26 is shown in detail. Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC Genome Browser, as described in the examples herein. The reading densities of introns 25 and 26 in HCN are shown in detail in the panel below. Introns 25 and 26 are adjacent to exons 26, which are alternative spliced exons. This is evidenced in the graphical representation of TSC2 transcripts and RNAseq data, thus the rectangle representing exon 26 is present in some transcripts while not in other transcripts and in the cytoplasmic fraction. The reading density corresponding to exons 26 is significantly lower than the constitutively spliced exons in TSC2. The reading density for the intron 26 is shown in detail in the lower panel showing 51% intron retention as calculated by bioinformatics analysis. ASO walks of TSC2 genes IVS 25 and 26 are shown. Performed on TSC2 IVS 25 and 26 using a 2'-OMe ASO, PS backbone, targeting sequences immediately downstream of the 5'splice site of intron 25 or upstream of the 3'splice site of intron 26. A graphical representation of the ASO walk is shown. The intron region of the splice site adjacent to the alternative exon 26 is not targeted to avoid affecting the inclusion level of the exon 26. ASO was designed to cover these regions by repositioning 5 nucleotides at a time. The TSC2 exon-intron structure is shown to scale. Intron retention in the TSC2 gene with introns 31 and 32 is shown in detail. Intron retention in the TSC2 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC Genome Browser, as described in the examples herein. The reading densities of the introns 31 and 32 are shown in detail in the lower panel. Introns 31 and 32 are adjacent to exons 32, which are alternative spliced exons. This is evidenced in the schematic representation of TSC2 transcripts and RNAseq data, thus the rectangle representing exons 32 is present in some transcripts while not in other transcripts and in the cytoplasmic fraction. The reading density corresponding to exons 32 is significantly lower than the constitutively spliced exons in TSC2. The reading density for the intron 31 is shown in detail in the lower panel showing 43% intron retention as calculated by bioinformatics analysis. ASO walks of TSC2 genes IVS 31 and 32 are shown. Performed on TSC2 IVS 31 and 32 using a 2'-OMe ASO, PS backbone, targeting sequences immediately downstream of the 5'splice site of intron 31 or upstream of the 3'splice site of intron 32. A graphical representation of the ASO walk is shown. The intron region of the splice site adjacent to the alternative exon 32 is not targeted to avoid affecting the inclusion level of the exon 32. ASO was designed to cover these regions by repositioning five nucleotides at a time, with the exception of TSC2-IVS32-33 and TSC2-IVS32-51. The TSC2 exon-intron structure is shown to scale. The schematic diagram of the ReSeq gene of TSC2 intron 4 corresponding to NM_000548 is shown. The intron retention rate (PIR) of the circled intron is shown. Represents a typical graph showing an average (n = 3) fold change in the expression level of TSC2 mRNA in the absence of intron 4 in ARPE-19 cells treated with 80 nM of indicated ASO on sham-treated cells for 24 hours. .. The data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 25 corresponding to NM_000548 is shown. The intron retention rate (PIR) of the circled intron is shown. Represents a typical graph showing an average (n = 3) fold change in the expression level of TSC2 mRNA in the absence of intron 25 in ARPE-19 cells treated with 80 nM of indicated ASO on sham-treated cells for 24 hours. .. The data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 26 corresponding to NM_000548 is shown. The intron retention rate (PIR) of the circled intron is shown. The schematic diagram of the ReSeq gene of TSC2 intron 31 corresponding to NM_000548 is shown. The intron retention rate (PIR) of the circled intron is shown. Represents a typical graph showing an average (n = 3) fold change in the expression level of TSC2 mRNA in the absence of intron 31 in ARPE-19 cells treated with 80 nM of indicated ASO on sham-treated cells for 24 hours. .. The data are normalized to RPL32 expression. The schematic diagram of the ReSeq gene of TSC2 intron 32 corresponding to NM_000548 is shown. The intron retention rate (PIR) of the circled intron is shown.

Individual introns in the primary transcript of a protein-encoding gene with two or more introns are spliced from the primary transcript with different efficiencies. In most cases, only fully spliced mRNA is transported through the nuclear pores for subsequent cytoplasmic translation. Unspliced or partially spliced transcripts are detectable in the nucleus. Nuclear accumulation of transcripts that are not fully spliced is generally thought to be a mechanism that prevents the accumulation of potentially harmful mRNAs in the cytoplasm that can be translated into proteins. For some genes, the least efficient intron splicing is a rate-determining post-transcriptional step in gene expression prior to translation in the cytoplasm.

The present invention is one that is rate-determining to the nuclear stage of gene expression in order to increase the production of a stable state of fully spliced mature mRNA and, therefore, to increase the level of translated protein. Provided are compositions and methods for upregulating the above retained splicing of TSC2 introns. These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at the intron splice sites of retained intron-containing TSC2 pre-mRNA that accumulate in the nucleus. Therefore, in embodiments, the methods of the invention are used to increase the TSC2 protein to treat diseases resulting from TSC2 deficiency.

In other embodiments, the methods of the invention are used to increase TSC2 production in order to treat a subject's disease in need. In an embodiment, the subject has a disease in which TSC2 is not necessarily deficient for the wild type, but the increase in TSC2 nevertheless alleviates the disease. In embodiments, the disease is caused by TSC2 haploinsufficiency. In embodiments, the disease is an autosomal dominant disorder. In embodiments, the disease is an autosomal recessive disorder.

<Tuberous sclerosis>
Tuberous sclerosis is a disease characterized by tumor growth in a multi-organ system (Au et al., J. Child Neurol. 2004, 19, 699-709). Tumors are usually benign, but sometimes malignant. Approximately 90% of cases of tuberous sclerosis represent cortical nodules; facial fibrous hemangiomas and angiomyolipoma of the kidney occur in more than 80% of cases. In addition, approximately 80% of cases represent subependymal nodules, approximately 50% of cases represent rhabdomyoma of the heart, and 51% to approximately 88% of cases represent nail / subungual fibroma. Tumors of the central nervous system (CNS) are a major contributor to subsequent morbidity and mortality with renal disease (Au et al., J. Child Neurol. 2004, 19, 699-709).

Tuberous sclerosis is a genetic disease with an autosomal dominant pattern and a high mutation rate (Au et al., J. Child Neurol. 2004, 19, 699-709). The binding of tuberous sclerosis to the chromosomal regions 9q34.3 and 16p13.3 led to the identification of the TSC1 and TSC2 genes respectively. More than 69% of cases of tuberous sclerosis result from haploinsufficiency of TSC2, and approximately two-thirds of patients suffer from mutations or deletions in de novo (Au et al., J. et al., J. Mol. Child Neurol. 2004, 19, 699-709). Serious disease is thought to require a reduction in the "second hit" of other alleles. The prevalence of tuberous sclerosis is about 1 in 5,800 live births per year, or approximately 1 in 300 live births in the United States, with 200 cases per year from the results of TSC2 mutations. Occurs. Diseases are described, for example, by OMIM # 61354 (Online Mendelian Inheritance in Man, Johns Hopkins University, 1966-2015), which is incorporated herein by reference.

The TSC2 gene encodes a protein called tuberin. The TSC2 gene contains 41 exons, two of which are alternative spliced and spans approximately 40 kb on chromosome 16 (Au et al., J. Child Neurol. 2004, 19, 699-709). Tuberin is a 200 kDa protein that forms a complex with the TSC1 gene product, hamartin. The tuberin-hamartin complex plays a role in regulating cell proliferation, transport, cell size, cell adhesion, and migration through various signal cascades. For example, the tuberin-hamartin complex functions in the phosphatidylinositol-3-kinase and protein B pathway (PI3K / PKB) that regulate translation. Specifically, the tuberin-hamartin complex value suppresses cell proliferation and translation by suppressing mTOR kinase activity, resulting in suppression of p70 ribosomal protein S6 kinase (S6K) 1 and eukaryotic initiation factor 4E binding protein. It results in activation of 1 (4E-BP1). In addition, the GAP domain of tuberin hydrolyzes guanosine triphosphate (GTP) bound to the small G protein Rheb, a rich Ras homolog in the brain, thus preventing mTOR activation.

In addition, tuberin and hamartin regulate cell proliferation and cell adhesion via the MAP kinase (MAPK) and E-cadherin-beta-catenin pathways, respectively. Numerous mutations were described for TSC2 (Au et al., J. Child Neurol. 2004, 19, 699-709) when first discovered by identifying a loss of heterozygosity in the portion of chromosome 16p13. Mutations include frameshifts and protein shortenings, nonsense mutations, mutations that affect splicing, inframe mutations, deletions, insertions, duplications, and large deletions and transpositions. Mutations in TSC2 that result in shortened protein products are unable to form the Tuberin-Hamartin complex, thus resulting in loss of regulation of cell proliferation. The role of the tuberous-hamartin complex as a major regulator of multiple signaling pathways involves multi-organ systems, including neurological and neuroreactive abnormalities such as seizures, intellectual disability, and developmental delay. It is consistent with the broad spectrum of clinical findings among patients with tuberous sclerosis (Au et al., J. Child Neurol. 2004, 19, 699-709).

<Retained intron-containing mRNA precursor (RIC mRNA precursor)>
In embodiments, the methods herein utilize the presence of a retained intron-containing pre-mRNA (RIC pre-mRNA) that is transcribed from the TSC2 gene and encodes a tuberin protein in the nucleus of the cell. Splicing of specific TSC2 RIC mRNA precursor species to produce mature, fully spliced TSC2 mRNA is induced using ASOs that stimulate the splicing out of retained introns. The resulting mature TSC2 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of tuberous sclerosis in the patient's cells and alleviating the symptoms of tuberous sclerosis. This method is known as Targeted Enhancement (TANGO) of nuclear gene output, further described below.

<Transcript product of TSC2 nucleus>
As described in the examples herein, the TSC2 gene was analyzed for intron retention events and retention of introns 4, 25, 26, 31, and 32 was observed. RNA sequencing (RNAseq) visualized by the UCSC Genome Browser showed TSC2 transcripts expressed on HCN (human cortical neurons) cells and was localized to either the cytoplasm or the nuclear fraction. No readings were observed for the majority of introns in either fraction. However, higher reading densities were detected in introns 4, 25, 26, 31, and 32 of the nuclear fraction compared to the cytoplasmic fraction, which is the splicing efficiency of introns 4, 25, 26, 31, and 32. Is low, indicating that it results in intron retention. Transcripts of retained intron-containing pre-mRNA accumulate primarily in the nucleus and are not translated into tuberin proteins. The reading density for the AST intron 26 is shown in detail in the lower panel showing 51% intron retention as calculated by bioinformatics analysis. The intron retention (PIR) values for the intron 26 were obtained by averaging four values (87, 83, 20 and 12). Each value used one of four different algorithms and the reading density for AST intron 31 as determined in renal epithelial cells is detailed in the lower panel showing 43% intron retention. .. The value of the intron retention rate (PIR) of the intron 31 was obtained by averaging four values (78, 71, 16 and 8). Each value was determined in renal epithelial cells using one of four different algorithms. Introns 4 and 32 were not mapped. Analysis of ENCODE data to identify intron retention events (eg, Tirgner, et al., 2012, "Deep sequencing of subcellular RNA fractions shows spinning to be predominant genome 22 (9): described in 1616-25) did not identify introns 25, 26, or 31 as retained and did not map introns 4 and 32.

Table 1 shows the target sequences of the transcript of the TSC2 RIC mRNA precursor by sequence ID, and the sequence ID, which are useful for increasing the production of tuberin protein by targeting the region of the TSC2 RIC mRNA precursor. Provides an unrestricted list of per ASO. In embodiments, other ASOs useful for these purposes are identified, for example, using the methods described herein.

In some embodiments, the ASO disclosed herein targets the RIC mRNA precursor transcribed from the TSC2 genomic sequence. In some embodiments, ASO targets transcripts of RIC pre-mRNA from the TSC2 genomic sequence containing retained introns. In some embodiments, ASO targets the transcript of the RIC pre-mRNA with SEQ ID NO: 1. In some embodiments, the ASO targets a transcript of the RIC pre-mRNA of SEQ ID NO: 1, which comprises a retained intron. In some embodiments, the ASO disclosed herein targets the sequence of the TSC2 RIC mRNA precursor. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 3, 24, 29, or a combination thereof, where the intron numbering is mRNA in NM_001318829. Corresponds to an array. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 4, 25, 26, 31, 32, or combinations thereof, where the intron numbering is. , NM_000548 corresponds to the mRNA sequence. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 4, 25, 30, or a combination thereof, where the intron numbering is mRNA in NM_001077183. Corresponds to an array. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 4, 25, 26, 31, or combinations thereof, where the intron numbering is NM_001114382. Corresponds to the mRNA sequence in. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 22, 27, 28, or combinations thereof, where the intron numbering is mRNA in NM_001318831. Corresponds to an array. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 4, 25, 30, or a combination thereof, where the intron numbering is mRNA in NM_001318832. Corresponds to an array. In some embodiments, ASO targets transcripts of TSC2 RIC mRNA precursors, including introns retained at 3, 24, 29, or a combination thereof, where the intron numbering is mRNA in NM_001318827. Corresponds to an array.

In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 2. In some embodiments, the ASO sequences a TSC2 RIC pre-mRNA according to SEQ ID NO: 2, comprising retained intron 24, retained intron 3, retained intron 29, or a combination thereof. Target. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 3. In some embodiments, the ASO comprises a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31, or a combination thereof, the TSC2 RIC according to SEQ ID NO: 3. Target sequences of pre-mRNA. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 4. In some embodiments, the ASO sequences a TSC2 RIC pre-mRNA according to SEQ ID NO: 4, comprising retained intron 25, retained intron 4, retained intron 30, or a combination thereof. Target. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 5. In some embodiments, the ASO comprises a retained intron 25, a retained intron 26, a retained intron 4, a retained intron 31, or a combination thereof, the TSC2 RIC according to SEQ ID NO: 5. Target sequences of pre-mRNA. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 6. In some embodiments, the ASO sequences a TSC2 RIC pre-mRNA according to SEQ ID NO: 6, comprising retained intron 27, retained intron 28, retained intron 22, or a combination thereof. Target. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 7. In some embodiments, the ASO sequences a TSC2 RIC pre-mRNA according to SEQ ID NO: 7, comprising retained intron 25, retained intron 4, retained intron 30, or a combination thereof. Target. In some embodiments, the ASO targets the sequence of the TSC2 RIC mRNA precursor according to SEQ ID NO: 8. In some embodiments, the ASO sequences a TSC2 RIC pre-mRNA according to SEQ ID NO: 8, comprising retained intron 24, retained intron 3, retained intron 29, or a combination thereof. Target. In some embodiments, the ASO disclosed herein targets SEQ ID NO: 5097-5105. In some embodiments, the ASO has a sequence according to any one of SEQ ID NO: 9-5096.

In some embodiments, the ASO targets exon 24 or exon 25 of the TSC2 RIC mRNA precursor, including the retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets an exon 24 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. In some embodiments, the ASO is an exon 24 about 2 to about 75 or about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing the retained intron 24. Target the sequence. In some embodiments, the ASO targets an exon 25 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 24. In some embodiments, the ASO is an exon about 2 to about 150 or about 2 to about 147 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 24. Target 25 sequences.

In some embodiments, the ASO targets the intron 24 of the TSC2 RIC pre-mRNA, including the retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets the intron 24 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA, including the retained intron 24. In some embodiments, the ASO targets about 6 to about 497 nucleotide downstream (or 3') intron 24 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. .. In some embodiments, the ASO targets the intron 24 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including the retained intron 24. In some embodiments, the ASO targets about 16 to about 496 nucleotide upstream (or 5') intron 24 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. ..

In some embodiments, the ASO targets exon 3 or exon 4 of the TSC2 RIC mRNA precursor, including retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets an exon 3 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets an exon 3 sequence approximately 4 to approximately 94 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. .. In some embodiments, the ASO targets an exon 4 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 2 to about 127 nucleotide downstream (or 3') exon 4 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. ..

In some embodiments, the ASO targets the intron 3 of the TSC2 RIC pre-mRNA, including the retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets the intron 3 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 6 to about 435 nucleotide downstream (or 3') intron 3 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. .. In some embodiments, the ASO targets the intron 3 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 16 to about 432 nucleotide upstream (or 5') intron 3 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. ..

In some embodiments, the ASO targets exon 29 or exon 30 of the TSC2 RIC pre-mRNA, including retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets an exon 29 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. In some embodiments, the ASO targets an exon 29 sequence approximately 4 to approximately 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. .. In some embodiments, the ASO targets an exon 30 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. In some embodiments, the ASO targets about 2 to about 102 nucleotide downstream (or 3') exon 30 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. ..

In some embodiments, the ASO targets the intron 29 of the TSC2 RIC mRNA precursor, including the retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318829. In some embodiments, the ASO targets the intron 29 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA, including retained intron 29. In some embodiments, the ASO targets about 6 to about 492 nucleotide downstream (or 3') intron 29 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. .. In some embodiments, the ASO targets the intron 29 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including retained intron 29. In some embodiments, the ASO is an intron about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 29. Target 29 sequences.

In some embodiments, the ASO targets exon 32 or exon 33 of the TSC2 RIC mRNA precursor, including the retained intron 32, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 32 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO targets an exon 32 sequence about 4 to about 49 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO targets an exon 33 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO targets an exon 33 sequence about 2 to about 102 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 32.

In some embodiments, the ASO targets the intron 32 in the TSC2 RIC mRNA precursor, including the retained intron 32, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the intron 32 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO targets an intron 32 sequence approximately 6 to about 495 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO targets the intron 32 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 32. In some embodiments, the ASO is an intron 32 about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 32. Target the sequence.

In some embodiments, the ASO targets exon 25 or exon 26 of the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 25 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets the intron 25 in the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the intron 25 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 6 to about 497 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets the intron 25 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 16 to about 498 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets exon 26 or exon 27 of the TSC2 RIC mRNA precursor, including the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 26 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 27 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26.

In some embodiments, the ASO targets the intron 26 in the TSC2 RIC mRNA precursor, including the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the intron 26 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an intron 26 sequence approximately 6 to about 500 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets the intron 26 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets the intron 26 sequence approximately 16 to about 496 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26.

In some embodiments, the ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor, including the retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets the intron 4 in the TSC2 RIC mRNA precursor, including the retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets an intron 4 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 6 to about 435 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets the intron 4 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 16 to about 432 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets exon 31 or exon 32 of the TSC2 RIC mRNA precursor, including the retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the exon 31 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31. In some embodiments, the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets an exon 32 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 52 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31.

In some embodiments, the ASO targets the intron 31 in the TSC2 RIC mRNA precursor, including the retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_000548. In some embodiments, the ASO targets the intron 31 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets the intron 31 sequence approximately 6 to about 331 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets the intron 31 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets the intron 31 sequence approximately 16 to about 333 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 31.

In some embodiments, the ASO targets exon 25 or exon 26 of the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an exon 25 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 142 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets the intron 25 in the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, the ASO targets the intron 25 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 6 to about 497 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets the intron 25 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 16 to about 499 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor, including retained intron 4, where the intron numbering is consistent with the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets the intron 4 in the TSC2 RIC mRNA precursor, including the retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, the ASO targets an intron 4 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 6 to about 435 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets the intron 4 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 16 to about 432 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets exon 30 or exon 31 of the TSC2 RIC mRNA precursor, including the retained intron 30, where the intron numbering is consistent with the mRNA sequence in NM_001077183. In some embodiments, the ASO targets the exon 30 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. In some embodiments, the ASO targets an exon 30 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. In some embodiments, the ASO targets the exon 31 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence about 2 to about 102 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30.

In some embodiments, the ASO targets the intron 30 in the TSC2 RIC mRNA precursor, including the retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001077183. In some embodiments, the ASO targets the intron 30 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 30. In some embodiments, the ASO targets the intron 30 sequence approximately 6 to about 492 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 30. In some embodiments, the ASO targets the intron 30 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including the retained intron 30. In some embodiments, the ASO is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') of the intron 30 from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. Target the sequence.

In some embodiments, the ASO targets exon 25 or exon 26 of the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 25 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 112 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets the intron 25 in the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets the intron 25 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 6 to about 497 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets the intron 25 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 16 to about 498 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets exon 26 or exon 27 of the TSC2 RIC mRNA precursor, including the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 26 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 26 sequence about 4 to about 111 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 27 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an exon 27 sequence about 2 to about 147 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26.

In some embodiments, the ASO targets the intron 26 in the TSC2 RIC mRNA precursor, including the retained intron 26, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets the intron 26 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets an intron 26 sequence approximately 6 to about 500 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets the intron 26 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26. In some embodiments, the ASO targets the intron 26 sequence approximately 16 to about 496 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 26.

In some embodiments, the ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor, including retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets intron 4 in the TSC2 RIC mRNA precursor, including retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an intron 4 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 6 to about 435 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets the intron 4 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 16 to about 432 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets exon 31 or exon 32 of the TSC2 RIC mRNA precursor, including the retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets an exon 31 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets an exon 31 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets an exon 32 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31. In some embodiments, the ASO targets an exon 32 sequence about 2 to about 102 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31.

In some embodiments, the ASO targets the intron 31 in the TSC2 RIC mRNA precursor, including the retained intron 31, where the intron numbering corresponds to the mRNA sequence in NM_001114382. In some embodiments, the ASO targets the intron 31 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets an intron 31 sequence approximately 6 to about 492 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO targets the intron 31 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 31. In some embodiments, the ASO is an intron 31 about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 31. Target the sequence.

In some embodiments, the ASO targets exon 27 or exon 28 of the TSC2 RIC mRNA precursor, including the retained intron 27, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an exon 27 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets an exon 27 sequence about 4 to about 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets an exon 28 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets an exon 28 sequence about 2 to about 52 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 27.

In some embodiments, the ASO targets the intron 27 in the TSC2 RIC mRNA precursor, including the retained intron 27, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets the intron 27 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets an intron 27 sequence approximately 6 to about 331 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets the intron 27 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 27. In some embodiments, the ASO targets the intron 27 sequence approximately 16 to about 333 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 27.

In some embodiments, the ASO targets exon 28 or exon 29 of the TSC2 RIC pre-mRNA, including retained intron 28, where intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an exon 28 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO targets an exon 28 sequence about 4 to about 49 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO targets an exon 29 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO targets an exon 29 sequence about 2 to about 102 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 28.

In some embodiments, the ASO targets the intron 28 in the TSC2 RIC mRNA precursor, including the retained intron 28, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets the intron 28 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO targets an intron 28 sequence approximately 6 to about 495 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO targets the intron 28 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. In some embodiments, the ASO is about 16 to about 500 or about 36 to about 500 nucleotides upstream (or 5') of the intron 28 from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 28. Target the sequence.

In some embodiments, the ASO targets exon 22 or exon 23 of the TSC2 RIC mRNA precursor, including retained intron 22, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets an exon 22 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets an exon 22 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets an exon 23 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets an exon 23 sequence about 2 to about 142 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 22.

In some embodiments, the ASO targets the intron 22 in the TSC2 RIC mRNA precursor, including the retained intron 22, where the intron numbering corresponds to the mRNA sequence in NM_001318831. In some embodiments, the ASO targets the intron 22 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets an intron 22 sequence approximately 6 to about 497 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets the intron 22 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 22. In some embodiments, the ASO targets an intron 22 sequence approximately 16 to about 499 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 22.

In some embodiments, the ASO targets exon 25 or exon 26 of the TSC2 RIC mRNA precursor, which comprises the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an exon 25 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 25 sequence about 4 to about 74 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an exon 26 sequence about 2 to about 142 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets the intron 25 in the TSC2 RIC mRNA precursor, including the retained intron 25, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets the intron 25 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 6 to about 497 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets the intron 25 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25. In some embodiments, the ASO targets an intron 25 sequence approximately 16 to about 499 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 25.

In some embodiments, the ASO targets exon 4 or exon 5 of the TSC2 RIC mRNA precursor, including retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an exon 4 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 4 sequence about 4 to about 94 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an exon 5 sequence about 2 to about 127 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets the intron 4 in the TSC2 RIC mRNA precursor, including the retained intron 4, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets an intron 4 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 6 to about 435 nucleotides downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets the intron 4 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4. In some embodiments, the ASO targets an intron 4 sequence approximately 16 to about 432 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 4.

In some embodiments, the ASO targets exon 30 or exon 31 of the TSC2 RIC mRNA precursor, including the retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets the exon 30 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. In some embodiments, the ASO targets an exon 30 sequence approximately 4 to approximately 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 30. .. In some embodiments, the ASO targets the exon 31 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. In some embodiments, the ASO targets an exon 31 sequence about 2 to about 102 nucleotides downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. ..

In some embodiments, the ASO targets the intron 30 in the TSC2 RIC mRNA precursor, including the retained intron 30, where the intron numbering corresponds to the mRNA sequence in NM_001318832. In some embodiments, the ASO targets the intron 30 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 30. In some embodiments, the ASO targets about 6 to about 492 nucleotide downstream (or 3') intron 30 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 30. .. In some embodiments, the ASO targets the intron 30 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including the retained intron 30. In some embodiments, the ASO is about 16 to about 500, or about 36 to about 500 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 30. , Intron 30 sequence is targeted.

In some embodiments, the ASO targets exon 24 or exon 25 of the TSC2 RIC mRNA precursor, including the retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an exon 24 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. In some embodiments, the ASO targets about 4 to about 74 nucleotide upstream (or 5') exon 24 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. .. In some embodiments, the ASO targets an exon 25 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 24. In some embodiments, the ASO targets about 2 to about 147 nucleotide downstream (or 3') exon 25 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 24. ..

In some embodiments, the ASO targets the intron 24 in the TSC2 RIC mRNA precursor, including the retained intron 24, where the intron numbering corresponds to the mRNA sequence in NM_0013188827. In some embodiments, the ASO targets the intron 24 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA, including the retained intron 24. In some embodiments, the ASO targets about 6 to about 497 nucleotide downstream (or 3') intron 24 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. .. In some embodiments, the ASO targets the intron 24 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including the retained intron 24. In some embodiments, the ASO targets about 16 to about 496 nucleotide upstream (or 5') intron 24 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 24. ..

In some embodiments, the ASO targets exon 3 or exon 4 of the TSC2 RIC mRNA precursor, including retained intron 3, where intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an exon 3 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 4 to about 69 nucleotide upstream (or 5') exon 3 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. .. In some embodiments, the ASO targets an exon 4 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 2 to about 127 nucleotide downstream (or 3') exon 4 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. ..

In some embodiments, the ASO targets the intron 3 in the TSC2 RIC mRNA precursor, including the retained intron 3, where the intron numbering corresponds to the mRNA sequence in NM_0013188827. In some embodiments, the ASO targets the intron 3 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 6 to about 499 nucleotide downstream (or 3') intron 3 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. .. In some embodiments, the ASO targets the intron 3 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. In some embodiments, the ASO targets about 16 to about 497 nucleotide upstream (or 5') intron 3 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 3. ..

In some embodiments, the ASO targets exon 29 or exon 30 of the TSC2 RIC mRNA precursor, including the retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_001318827. In some embodiments, the ASO targets an exon 29 sequence upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. In some embodiments, the ASO targets an exon 29 sequence approximately 4 to approximately 184 nucleotides upstream (or 5') from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. .. In some embodiments, the ASO targets an exon 30 sequence downstream (or 3') from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. In some embodiments, the ASO targets about 2 to about 102 nucleotide downstream (or 3') exon 30 sequences from the 3'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. ..

In some embodiments, the ASO targets the intron 29 in the TSC2 RIC mRNA precursor, including the retained intron 29, where the intron numbering corresponds to the mRNA sequence in NM_0013188827. In some embodiments, the ASO targets the intron 29 sequence downstream (or 3') from the 5'splice site of the TSC2 RIC pre-mRNA, including retained intron 29. In some embodiments, the ASO targets about 6 to about 492 nucleotide downstream (or 3') intron 29 sequences from the 5'splice site of the TSC2 RIC pre-mRNA containing retained intron 29. .. In some embodiments, the ASO targets the intron 29 sequence upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA, including retained intron 29. In some embodiments, the ASO is about 16 to about 500, or about 36 to about 500 nucleotides upstream (or 5') from the 3'splice site of the TSC2 RIC pre-mRNA containing the retained intron 29. , Intron 29 sequence is targeted.

The degree of intron retention can be expressed as percent intron retention (PIR), i.e., the percentage of transcripts in which a given intron is retained. Briefly, the PIR can be calculated as the percentage of the average number of reads that map to the exon-intron connection to the sum of the average readings of the exon-intron connection and the readings of the exon-exon connection.

<Tubelin protein expression>
More than 69% of cases of tuberous sclerosis result from haploinsufficiency of TSC2, and approximately two-thirds of patients suffer from mutations or deletions in de novo (Au, K. et al. , J. Child Neurol., 2004, 19: 699-709).

In embodiments, the methods described herein are used to increase the production of functional tuberin protein. As used herein, the term "functional" refers to the amount of activity or function of the tuberin protein required to eliminate one or more symptoms of tuberous sclerosis. In embodiments, the method is used to increase the production of partially functional tuberin protein. As used herein, the term "partially functional" refers to the activity or function of a tuberin protein that is less than the amount of activity or function required to eliminate or prevent one or more symptoms of the disease or disease. Refers to the quantity. In some embodiments, the partially functional protein or RNA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 compared to the fully functional protein or RNA. %, At least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less activity.

In an embodiment, the method is a method of increasing the expression of a tuberin protein by the cells of a subject having an RIC mRNA precursor encoding the tuberin protein, wherein the subject has an amount of tuberin protein activity. It has tuberous sclerosis due to a deficiency, and the deficiency in the amount of tuberin protein is due to haploinsufficiency of the tuberin protein. In such an embodiment, the subject has a first allele encoding a functional tuberin protein and a second allele from which the tuberin protein is not produced. In another such embodiment, the subject has a first allele encoding a functional tuberin protein and a second allele encoding a non-functional tuberin protein. In another such embodiment, the subject has a first allele encoding a functional tuberin protein and a second allele encoding a partially functional tuberin protein. In any of these embodiments, the antisense oligomer binds to the target portion of the RIC pre-mRNA transcribed from the first allogeneic gene (encoding the functional tuberin protein), thereby RIC mRNA. It induces constitutive splicing of retained introns from the precursor, resulting in increased levels of mature mRNA encoding functional Tubelin protein and increased Tubelin protein expression in the subject's cells.

In an embodiment, the subject has a first allelic gene encoding a functional tuberin protein and a second allelic gene encoding a partially functional tuberin protein, wherein the antisense oligomer is a first. The target portion of the RIC pre-mRNA transcribed from the allelic gene (encoding the functional tuberin protein) or the RIC pre-mRNA transcribed from the second allelic gene (encoding the partially functional tuberin protein) It binds to the target portion of the RIC mRNA precursor, thereby inducing constitutive splicing of retained introns from the RIC pre-mRNA, increasing the level of mature mRNA encoding the tuberin protein, and functionally or functionally in the subject's cells. It results in increased expression of partially functional tuberin protein.

In a related embodiment, the method is a method using ASO to increase expression of protein or functional RNA. In embodiments, ASO is used to increase the expression of tuberin protein in the cells of a subject having an RIC mRNA precursor encoding the tuberin protein, wherein the subject is of the amount or function of the tuberin protein. Have a deficiency, eg tuberous sclerosis.

In embodiments, the transcript of the protein-encoding RIC pre-mRNA that causes the disease or disease is targeted by the ASOs described herein. In some embodiments, the transcript of the RIC pre-mRNA that encodes the protein, which is not the cause of the disease, is targeted by ASO. For example, diseases caused by mutations or deletions of the first protein in a particular pathway are ameliorated by targeting the RIC mRNA precursor encoding the second protein, thereby increasing the production of the second protein. Can be done. In some embodiments, the activity of the second protein can compensate for a mutation or deficiency of the first protein, which is the cause of the disease or disease.

In embodiments, the subject is
a. The first mutant allele,
i) Tuberin protein is produced at reduced levels compared to production from wild-type alleles,
ii) A first mutant allele in which the tuberin protein is produced in a less functional form compared to an equivalent wild-type protein, or ii) the tuberin protein or functional RNA is not produced;
And b. The second mutation allele,
i) Tuberin protein is produced at reduced levels compared to production from wild-type alleles,
ii) The tuberin protein is produced in a less functional form compared to the equivalent wild-type protein, or ii) the tuberin protein is not produced, with a second mutated allele, where.
The RIC mRNA precursor is transcribed from the first and / or second allele. In these embodiments, the ASO is constitutive of the retained intron from the RIC mRNA precursor by binding to the target portion of the RIC mRNA precursor transcribed from the first or second allelic gene. It induces splicing, causes an increase in the level of mRNA encoding the tuberin protein, and results in an increase in the expression of the target protein or functional RNA in the subject's cells. In these embodiments, the target protein or functional RNA with increased expression levels resulting from constitutive splicing of retained introns from the RIC pre-mRNA is functional compared to comparable wild-type proteins. It can be either in a low form (partially functional) or with full functionality compared to an equivalent wild-type protein (fully functional).

In embodiments, levels of mRNA encoding the tuberin protein are produced in control cells, eg, treated with antisense oligomers that are not treated with antisense oligomers or that do not bind to the target portion of the TSC2 RIC mRNA precursor. Compared to the amount of TSC2-encoding mRNA, it increases 1.1-fold to 10-fold.

In embodiments, the disease resulting from a lack of amount or activity of the tuberin protein is not a disease resulting from alternative splicing or aberrant splicing of retained introns targeted by ASO. In embodiments, the disease resulting from a lack of amount or activity of the tuberin protein is not a disease resulting from alternative splicing or abnormal splicing of the retained introns in the RIC mRNA precursor encoding the tuberin protein. In embodiments, alternative splicing or aberrant splicing can occur in the pre-mRNA transcribed from the gene. However, the compositions and methods of the invention do not prevent or modify this alternative or anomalous splicing.

In embodiments, subjects treated using the methods of the invention express a partially functional tuberin protein from one allele, the partially functional tuberin protein being a frameshift mutation, nonsense. It is due to a mutation, a missense mutation, or a partial gene deletion. In an embodiment, a subject treated using the method of the invention expresses a non-functional tuberin protein from one allele, the non-functional tuberin protein is a frame in one allele. It is due to a shift mutation, nonsense mutation, missense mutation, or partial gene deletion. In embodiments, subjects treated using the methods of the invention have a TSC2 total gene deletion in one allele.

In embodiments, the subject has a TSC2 missense mutation selected from R611Q / W and R905W. In embodiments, the subject has a TSC2 deletion mutation in exon 38 for at least 6 amino acids. In embodiments, the subject has a TSC2 substitution mutation in arginine 611 for tryptophan or glutamine. In embodiments, those known in the art and described, for example, in literature such as previously referenced (Au, K. et al., J. Child Neurol., 2004, 19: 699-709). Subjects with any TSC2 mutation are treated using the methods and compositions of the invention.

<Use of TANGO to increase expression of tuberin protein>
As mentioned above, in embodiments, targeted enhancement of nuclear gene output (TANGO) is used in the methods of the invention to increase expression of the tuberin protein. In these embodiments, the retained intron-containing pre-mRNA (RIC pre-mRNA) that encodes the tuberin protein is in the nucleus of the cell. Cells with a TSC2 RIC pre-mRNA containing a retained intron encoding a tuberin protein, an exon flanking the 5'splice site, and an exon flanking the 3'splice site are targeted at the RIC mRNA precursor. In contrast, it is contacted with a complementary antisense oligomer (ASO). Hybridization of ASO to the target portion of the RIC mRNA precursor enhances splicing of the retained intron splice site (5'splice site or 3'splice site) and increases subsequent target protein production.

The terms "pre-mRNA" and "pre-mRNA transcript" are used interchangeably and may also refer to a pre-mRNA species containing at least one intron. In embodiments, the pre-mRNA or pre-mRNA transcript comprises a 5'-7-methylguanosine cap and / or a poly A terminal. In embodiments, the pre-mRNA or pre-mRNA transcript comprises both a 5'-7-methylguanosine cap and a poly A terminal. In some embodiments, the transcript of the pre-mRNA does not contain a 5'-7-methylguanosine cap and / or a poly A terminal. If not translated into protein (or transported from the nucleus to the cytoplasm), the transcript of the pre-mRNA is a non-productive messenger RNA (mRNA) molecule.

As used herein, a "retained intron-containing pre-mRNA" ("RIC mRNA pre-mRNA") is a transcript of a pre-mRNA containing at least one retained intron. The RIC pre-mRNA comprises a retained intron, an exon flanking the 5'splice site of the retained intron, and an exon flanking the 3'splice site of the retained intron, encoding the target protein. The "RIC mRNA precursor encoding the target protein" is understood to encode the target protein when fully spliced. A "retained intron" is a pre-mRNA transcript when one or more other introns, such as adjacent introns, encoded by the same gene are spliced out of the same pre-mRNA transcript. It is an intron that exists in. In some embodiments, the retained intron is the most abundant intron in the RIC pre-mRNA encoding the target protein. In embodiments, retained introns are the most abundant introns in a population of RIC pre-mRNA transcribed from a gene encoding a target protein in a cell, and the population of RIC pre-mRNA is a population of two or more. Includes pre-mRNA. In embodiments, the antisense oligomers targeted to the most abundant introns in the population of RIC pre-mRNA encoding the target protein are within the population containing retained introns to which the antisense oligomers are targeted or bound. Induces splicing out of two or more retained introns. In embodiments, the mature mRNA encoding the target protein is produced thereby. The terms "mature mRNA" and "fully spliced mRNA" are fully processed mRNAs that encode the target protein (eg, mRNA that is transported from the nucleus to the cytoplasm and translated into the target protein), or complete. Used interchangeably herein to describe functional RNA processed in. The term "productive mRNA" can also be used to describe fully processed mRNA encoding a target protein. In embodiments, the target region is in the most abundant retained introns in the RIC pre-mRNA precursor population that encodes the tuberin protein. In embodiments, the most abundant retained introns in the RIC pre-mRNA encoding the tuberin protein are introns 4, 25, 26, 31, 32, 25 and 26, or 31 and 32. In embodiments, the most abundant retained intron in the RIC pre-mRNA encoding the tuberin protein is intron 26. In embodiments, the most abundant retained intron in the RIC pre-mRNA encoding the tuberin protein is intron 31. In some embodiments, the most abundant retained intron in the RIC mRNA precursor encoding the tuberin protein is intron 26, and the second most abundant retained intron in the RIC mRNA precursor encoding the tuberin protein. The intron that was made is the intron 31.

In embodiments, retained introns are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, An intron identified as a retained intron based on retention retention of at least about 45%, or at least about 50%. In embodiments, the retained introns are, ie, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about 5% to about 80%. , About 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% ~ About 25%, about 5% ~ about 20%, about 5% ~ about 15%, about 10% ~ about 100%, about 10% ~ about 95%, about 10% ~ about 90%, about 10% ~ about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65% , About 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% ~ About 85%, about 15% ~ about 80%, about 15% ~ about 75%, about 15% ~ about 70%, about 15% ~ about 65%, about 15% ~ about 60%, about 15% ~ about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35% , About 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 65%, about 20% ~ About 60%, about 20% ~ about 55%, about 20% ~ about 50%, about 20% ~ about 45%, about 20% ~ about 40%, about 20% ~ about 35%, about 20% ~ about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75% , About 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about An intron identified as a retained intron based on retention retention rates of 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%. ..

As used herein, variants such as the terms "comprise", or "comprises" or "comprising" are listed features (eg, in the case of antisense oligomers). , Defined nucleic acid base sequence), but should be understood to indicate that it does not exclude any other features. Thus, as used herein, the term "comprising" is inclusive and includes additional unlisted features (eg, in the case of antisense oligomers, the presence of additional unlisted nucleobases). ) Is not excluded.

In any embodiment of the compositions and methods provided herein, "comprising" can be replaced with "consisting essentally" or "consisting of". be. The phrase "consisting essentially" requires the specified features (eg, nucleic acid base sequences) as well as features that do not substantially affect the claimed properties or functions of the invention. Used in the specification. As used herein, the term "consisting" is used to indicate the presence of a single listed feature (eg, a nucleobase sequence) (as a result, an antisense consisting of the identified nucleobase sequence). In the case of oligomers, the presence of additional unlisted nucleobases is excluded).

In embodiments, the target region is in a retained intron, which is the second most abundant intron in the RIC pre-mRNA encoding the tuberin protein. For example, the second most abundant retained intron can be targeted rather than the most abundant retained intron. The factors are the uniqueness of the nucleotide sequence of the second most abundant retained intron, the ease of ASO design targeting a specific nucleotide sequence, and / or the amount of protein produced by targeting the intron by ASO. Is an increase in. In embodiments, the retained intron is the second most abundant intron in the RIC mRNA precursor population transcribed from the gene encoding the target protein in the cell, and the RIC mRNA precursor population is more than one. Includes retained introns. In embodiments, the second abundant intron-targeting antisense oligomer in the RIC pre-mRNA precursor population encoding the target protein is in a population comprising retained introns to which the antisense oligomer is targeted or bound. Induces splicing out of two or more retained introns. Thereby, in the embodiment, a fully spliced (mature) RNA encoding the target protein is produced. In embodiments, the second most retained intron in the RIC pre-mRNA encoding the tuberin protein is intron 4, 25, 26, 31, 32, 25 and 26, or 31 and 32. In some embodiments, the second most abundant intron in the RIC pre-mRNA encoding the tuberin protein is intron 31.

In embodiments, the ASO is complementary to a target region within an intron in which the RIC pre-mRNA is not retained. In embodiments, the target portion of the RIC mRNA precursor is: within the region +6 to +100 relative to the 5'splice site of the unretained intron; or within the region -16 to -100 relative to the 3'splice site of the unretained intron. It is in. In embodiments, the target portion of the RIC pre-mRNA is within the region from +100 to the 5'splice site of the unretained intron to -100 to the 3'splice site of the unretained intron. As used to locate a region or sequence, "within" is understood to include residues at the listed positions. For example, the region +6 to +100 contains residues at positions +6 and +100. Thereby, in the embodiment, a fully spliced (mature) RNA encoding the target protein is produced.

In an embodiment, the intron carrying the RIC pre-mRNA is an intron spliced inefficiently. As used herein, "inefficiently spliced" refers to the splicing site adjacent to the retained intron as compared to the frequency of splicing at another splicing site in the RIC pre-mRNA. It can indicate that the frequency of splicing at the 5'splice site or the 3'splice site) is relatively low. The term "inefficiently spliced" can also refer to the relative rate or kinetics of splicing at the splicing site. This "inefficiently spliced" intron can be spliced or removed at a slower rate compared to other introns in the RIC pre-mRNA.

In embodiments, the -3e to -1e of exons adjacent to the 5'splice site and the +1 to +6 9-nucleotide sequences of the retained introns are identical to the corresponding wild-type sequences. In embodiments, the + 15e 16 nucleotide sequence of the exon flanking the -15 to -1 and 3'splice sites of the retained intron is identical to the corresponding wild-type sequence. As used herein, "wild-type sequence" refers to a nucleotide sequence for the TSC2 gene in the published reference genome deposited in the NCBI repository of biological and scientific information. As used herein, "wild-type sequence" refers to the standard sequence for the TSC2 gene found in NCBI Gene ID 7249. Further, as used herein, the nucleotide position indicated by "e" is in the sequence of the exon in which the nucleotide is adjacent to an exon (eg, an exon flanking the 5'splice site, or an exon flanking the 3'splice site). Indicates that it exists in.

In this method, cells are contacted with ASO, which is complementary to some of the pre-mRNA encoding the tuberin protein, resulting in increased expression of TSC2. As used herein, "contacting" or administering a cell refers to a method of bringing the ASO into immediate proximity to the cell so that the ASO interacts with the cell. Cells that are brought into contact with ASO take up or transport ASO into the cells. The method comprises contacting the disease or disease-related or disease-related cells with any of the ASOs described herein. In some embodiments, the ASO targets the ASO to the cell type, enhances contact between the ASO and the disease or disease-related or disease-related cell, or incorporates the ASO. Can be further modified or attached to another molecule (eg, covalently attached) to enhance.

As used herein, the terms "increasing protein production" or "increasing expression of a target protein" mean increasing the amount of protein translated from mRNA in a cell. The "target protein" can be a protein for which increased expression / production is desired.

In embodiments, ASO, which is complementary to the target site of the transcript of the TSC2 RIC pre-mRNA, is contacted with cells expressing the TSC2 RIC pre-mRNA to bring the tuberin protein encoded by the pre-mRNA (eg, for example). It results in a measurable increase in the amount of target protein). Methods of measuring or detecting protein production will be apparent to those of skill in the art and include all known methods such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

In embodiments, by contacting the cells with ASO, which is complementary to the target site of the transcript of TSC2 RIC mRNA, the amount of protein produced by the cells in the absence of ASO / treatment is compared. It results in an increase in the amount of tuberin protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In embodiments, the total amount of tuberin protein produced by cells contacted with the antisense oligomer is about 1.1 to about 10 times, as compared to the amount of target protein produced by the control compound. About 1.5 times to about 10 times, about 2 times to about 10 times, about 3 times to about 10 times, about 4 times to about 10 times, about 1.1 times to about 5 times, from about 1.1 times About 6 times, about 1.1 times to about 7 times, about 1.1 times to about 8 times, about 1.1 times to about 9 times, about 2 times to about 5 times, about 2 times to about 6 times, About 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 Double to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least It is increased by about 2.5 times, at least about 3 times, at least about 3.5 times, at least 4 times, at least about 5 times, or at least about 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the target site of the RIC mRNA precursor.

In some embodiments, contacting cells with ASO, which is complementary to the target site of the TSC2 RIC mRNA precursor, results in an increase in the amount of TSC2 encoding mRNA, including mature mRNA encoding the target protein. .. In some embodiments, the amount of mRNA encoding the tuberin protein, or mature mRNA encoding the tuberin protein, is compared to the amount of protein produced by the cells in the absence of ASO / treatment. Increase by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In embodiments, the total amount of RNA encoding the tuberin protein, or mature mRNA encoding the tuberin protein, produced in the cells contacted with the antisense oligomer is the untreated cell, eg, untreated cell. Or about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, compared to the amount of mature RNA produced in cells treated with the control compound. , About 3 to about 10 times, about 4 to about 10 times, about 1.1 times to about 5 times, about 1.1 times to about 6 times, about 1.1 times to about 7 times, about 1. 1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to 9 times, 3 to 6 times, 3 to 7 times, 3 to 8 times, 3 to 9 times, 4 to 7 times, 4 times About 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times , At least 4 times, at least about 5 times, or at least about 10 times. The control compound may be, for example, an oligonucleotide that is not complementary to the target site of the TSC2 RIC mRNA precursor.

<Constitutive splicing of retained introns from RIC pre-mRNA>
The methods and antisense oligonucleotide compositions provided herein are, for example, mRNA encoding the tuberin protein, or tu, in a subject having tuberous sclerosis resulting from a lack of amount or activity of the tuberin protein. It is useful for increasing intracellular expression of tuberin protein by increasing the level of mature mRNA encoding the verin protein. In particular, methods and compositions as described herein induce constitutive splicing of retained introns from transcripts of the TSC2 RIC mRNA precursor encoding the Tubelin protein, thereby Tubelin. Increased levels of protein-encoding mRNA or mature mRNA encoding tuberin protein increase expression of tuberin protein.

Constitutive splicing of retained introns from the RIC pre-mRNA correctly removes the retained introns from the RIC pre-mRNA. This retained intron has a wild-type splice sequence. Constitutive splicing, as used herein, is RIC mRNA transcribed from a gene or allelic gene with a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from a gene or allelic gene. Does not include alternative splicing of retained introns from precursors. For example, constitutive splicing of retained introns induced using the methods provided herein and antisense oligonucleotides does not correct aberrant splicing in pre-mRNA or selective splicing of pre-mRNA. It does not affect splicing, resulting in increased expression of the target protein or functional RNA.

In embodiments, constitutive splicing correctly removes retained introns from the TSC2 RIC pre-mRNA. This TSC2 RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, and encodes a fully functional target protein or functional RNA. The gene or allele is free of mutations that cause alternative or alternative splicing of retained introns.

In some embodiments, constitutive splicing of retained introns from the TSC2 RIC mRNA precursor encoding the TSC2 protein correctly removes the retained introns from the TSC2 RIC mRNA precursor encoding the tuberin protein. .. The TSC2 RIC pre-mRNA is transcribed from a gene or allele that produces a target gene or functional RNA at reduced levels compared to production from a wild-type allele. And the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron. In these embodiments, correct removal of constitutively spliced retained introns results in the production of a target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA. ..

In other embodiments, constitutive splicing correctly removes retained introns from the TSC2 RIC pre-mRNA. This TSC2 RIC pre-mRNA is transcribed from a gene or allele encoding a target protein or functional RNA produced in a reduced form as compared to an equivalent wild-type protein or functional RNA. And the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron. In these embodiments, the correct removal of constitutively spliced retained introns is partially functional when compared to a partially functional target protein, or equivalent wild-type protein or functional RNA. It results in the production of functional RNA that is targeted.

"Correct removal" of retained introns by constructive splicing refers to the removal of all introns without removing any part of the exon.

In embodiments, the antisense oligomers as described herein or used in the methods described herein regulate alternative splicing or aberrant splicing of pre-mRNA transcribed from the TSC2 gene. By doing so, the amount of mRNA encoding the tuberin protein, or the amount of tuberin protein is not increased. The regulation of alternative splicing or aberrant splicing uses any known method of analyzing the sequence and length of RNA species, eg, by RT-PCR, and the methods described separately herein and in the literature. Can be measured using. In embodiments, the regulation of alternative or abnormal splicing is determined based on an increase or decrease in the amount of the species of interest spliced at least 10% or 1.1 times. In embodiments, the regulation is at least 10% to 100% or 1.1-fold to 10 as described herein in connection with determining the increase in mRNA encoding the tuberin protein in the method of the invention. Judgment is based on an increase or decrease at a double level.

In embodiments, the method is one in which the TSC2 RIC pre-mRNA is produced by partial splicing of the wild-type TSC2 pre-mRNA. In embodiments, the method is one in which the TSC2 RIC pre-mRNA is produced by partial splicing of the full-length wild-type TSC2 pre-mRNA. In embodiments, the TSC2 RIC pre-mRNA was produced by partial splicing of the full-length TSC2 pre-mRNA. In these embodiments, the full-length TSC2 pre-mRNA is at the splicing site of the retained intron without compromising the correct splicing of the retained intron as compared to the splicing of the retained intron having a wild-type splice site sequence. Can have polymorphism.

In embodiments, the mRNA encoding the tuberin protein is a full-length mature mRNA or a wild-type mature mRNA. In these embodiments, the full-length mature mRNA is a polymorphism that does not affect the activity of the target protein or functional RNA encoded by the mature mRNA as compared to the activity of the tuberin protein encoded by the wild-type mature mRNA. May have.

<Antisense oligomer>
One aspect of the present disclosure is a composition comprising an antisense oligomer that enhances splicing by binding to a targeted portion of the TSC2 RIC pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and are targeted nucleic acids (eg, TSC2 RIC) by Watson-Crick base pair or wobble base pair (GU). MRNA precursor) Refers to an oligomer such as a polynucleotide containing a nucleobase that hybridizes to a sequence. The ASO may have an exact sequence that is complementary or nearly complementary to the target sequence (eg, sufficient complementarity to bind to the target sequence and enhance splicing at the splice site). ASO is designed to bind (hybridize) to and remain hybridized under physiological conditions to the target nucleic acid (eg, the targeted portion of the transcript of the pre-mRNA). Typically, when hybridizing to a site other than the intended (targeted) nucleic acid sequence, the ASO hybridizes to a limited number of non-target nucleic acid sequences (to a small number of sites other than the target nucleic acid). The design of the ASO takes into account the generation of nucleic acid sequences in the targeted portion of the pre-mRNA transcript, or in sufficiently similar nucleic acid sequences elsewhere in the genomic or cellular pre-mRNA or transcriptome. It can be entered, and as a result, the possibility of ASO binding to other sites and causing an "off-target" effect is limited. Antisense oligomers known to those of skill in the art, such as those in PCT International Application No. PCT / US2014 / 054151 published as WO2015 / 035991 under the title of the invention "Reducing Nonsense-Mediated mRNA Decay", are described herein. Can be used to carry out the methods that are used.

In some embodiments, the ASO is "specifically hybridizes" or "specifically" to the targeted portion of the target nucleic acid or RIC mRNA precursor. Typically, such hybridization occurs at melting temperatures (Tm) substantially above 37 ° C, preferably at least 50 ° C, and typically between 60 ° C and approximately 90 ° C. Such hybridization preferably corresponds to stringent hybridization conditions. At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to the complementary oligonucleotide.

Oligoomers, such as oligonucleotides, are "complementary" to each other when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide if hybridization occurs between one of the strands of the first polynucleotide and one of the strands of the second polynucleotide. Complementarity (to some extent that one polynucleotide is complementary to another) is the proportion (eg, percentage) of bases in opposite chains that are expected to form hydrogen bonds with each other according to the generally accepted base pairing rule. It can be quantified from the point of. The sequence of the antisense oligomer (ASO) need not be 100% complementary to the sequence of the target nucleic acid to hybridize. In certain embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, relative to the target site in the targeted target nucleic acid sequence. It can contain at least 97%, at least 98%, or at least 99% sequence complementarity. For example, 18 of the 20 nucleobases of an oligomer compound are complementary to the target site, and therefore ASOs that specifically hybridize represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases can be clustered together or interspersed with the complementary nucleobases and need not be adjacent to each other or to the complementary nucleobases. Percent complementarity of ASO with the region of the target nucleic acid is the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) can be determined by convention.

ASO does not have to hybridize to all nucleobases in the target sequence, and the nucleobases to which it hybridizes may or may not be adjacent. ASO can hybridize over one or more segments of the transcript of the mRNA precursor so that the intervening or adjacent segments are not involved in the hybridization event (eg, loop or hairpin structures can be formed). ). In certain embodiments, ASO hybridizes to non-adjacent nucleobases in the transcript of the target pre-mRNA. For example, ASO can hybridize to a nucleobase in a transcript of a pre-mRNA separated by one or more nucleobases to which ASO does not hybridize.

The ASOs described herein include nucleobases that are complementary to the nucleobases present at the target portion of the RIC mRNA precursor. The term "ASO" refers to oligonucleotides and other oligomeric molecules that include nucleic acid bases capable of hybridizing to complementary nucleic acid bases on target mRNAs, but do not contain sugar moieties such as peptide nucleic acids (PNAs). Be embodied. ASO can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three thereof. The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes a modified or substituted saccharide and / or a nucleotide having a modified backbone. In some embodiments, all of the ASO nucleotides are modified nucleotides. Chemical modifications of ASO and components of ASO that are compatible with the methods and compositions described herein are apparent to those of skill in the art, eg, US Pat. No. 8,258,109 B2, US Pat. No. 5, , 656,612, US Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer The. It can be found in 2002, 347-355, all of which are incorporated herein by reference.

ASO nucleobases are unmodified to allow hydrogen binding to naturally occurring unmodified nucleobases such as adenin, guanine, cytosine, timine and uracil, or nucleobases present on target mRNA precursors. It can be a synthesized or modified nucleobase that closely resembles the nucleobase. Examples of modified nucleobases include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

The ASOs described herein also include a skeletal structure that binds to the components of the oligomer. The terms "skeleton structure" and "oligomer bond" are used interchangeably and may refer to bonds between monomers of ASO. In naturally occurring oligonucleotides, the scaffold contains a 3'-5'phosphodiester bond that binds the sugar moiety of the oligomer. The skeletal structures or oligomeric bonds of ASOs described herein are (but not limited to) phosphorothioates, phosphorodithioates, phosphoroserenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniladates. ), Phosphoramidate and the like. For example, LaPlanche et al. , Nucleic Acids Res. 14: 9081 (1986); Stick et al. , J. Am. Chem. Soc. 106: 6077 (1984), Stein et al. , Nucleic Acids Res. 16: 3209 (1988), Zon et al. , AntiCancerDrug Design 6: 539 1991; Zon et al. , Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stick et al. , U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). In some embodiments, the skeletal structure of ASO does not contain phosphite, but comprises, for example, peptide nucleic acid (PNA), or carbamate, amide, and linear and cyclic hydrocarbon groups. The linking group contains a peptide bond. In some embodiments, the skeletal modification is a phosphorothioate bond. In some embodiments, the skeletal modification is a phosphoramidate binding.

In embodiments, the stereochemistry at each of the bonds between the linkers of the ASO backbone is random. In embodiments, the stereochemistry at each of the bonds between the linkers of the ASO backbone is controlled and not random. For example, US Patent Application Publication No. 2014/01/94610, "Methos for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, has independent chirality handedness at each phosphorus atom in nucleic acid oligomers. It describes how to select. In embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, include ASOs with non-random linkages between phosphonucleotides. In embodiments, the composition used in the method of the invention comprises pure diastereomeric ASO. In embodiments, the compositions used in the methods of the invention are 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%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100 %, About 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%. Contains ASO with diastereomeric purity of.

In embodiments, the ASO has a non-random mixture of Rp and Sp constituents in the binding between its phosphonucleotides. For example, it has been suggested that a mixture of Rp and Sp is required for antisense oligonucleotides to achieve a good balance between activity and nuclease stability (Wan, et al., 2014 "Synthesis, biophysical policies". and biological activity of nucleotide generation antisense oligonucleotides contacting chiral phosphorothioate linkages ”Nucleic Acids Research 42). In embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, are about 5-100% Rp, at least about 5% Rp. , At least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, At least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 75% Rp. It contains about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp and the remaining sp, or about 100% Rp. In embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, range from about 10% to about 100% Rp, from about 15%. About 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about 30% to about 100% Rp, about 35% to about 100% Rp, from about 40% About 100% Rp, about 45% to about 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, from about 65% About 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% about 100% Rp, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, from about 40% It contains about 60% Rp, or about 45% to about 55% Rp, and the remaining Sp.

In embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, are about 5-100% Sp, at least about 5% Sp. , At least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, At least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 75% Sp. It contains about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, and the remaining Rp, or about 100% Sp. In embodiments, ASOs used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, range from about 10% to about 100% Sp, from about 15%. About 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, from about 40% About 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, from about 65% About 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, from about 90% About 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% Includes about 60% Sp, or about 45% to about 55% Sp, and the remaining Rp.

Any of the ASOs described herein contains a sugar moiety containing ribose or deoxyribose, or a modified sugar moiety or sugar analog, including a morpholine ring, as present in naturally occurring nucleotides. obtain. An unlimited example of the modified sugar moiety is 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F. N3'-> P5'phosphoramide, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, carbamate-modified sugar, And 2'substituted groups such as bicyclic modified sugars. In some embodiments, the sugar modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar modification is an additional cross-linking as in locked nucleic acid (LNA). In some embodiments, the sugar analog contains a morpholine ring such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises modification of ribofuranosyl or 2'deoxyribofuranosyl. In some embodiments, the sugar moiety comprises a 2'4'-constrained 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2'4'-inhibition 2'-O ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricycloDNA (tDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and are described in the literature, eg, Jarver, et al. , 2014, "A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications" Nucleic Acid Therapeutics 24 (1): 37-47.

In some examples, each monomer of ASO is modified in the same way, for example, each bond in the ASO backbone comprises a phosphorothioate bond, or each ribose sugar moiety comprises a 2'O-methyl modification. Such modifications present on each of the monomeric components of ASO are referred to as "uniform modifications". In some examples, different combinations of modifications may be desired, for example, ASO may include a combination of a phosphorodiamidate bond and a sugar moiety (morpholino) containing a morpholine ring. The combination of different modifications to ASO is called "mixed modification" or "mixed chemistry".

In some embodiments, the ASO comprises one or more skeletal modifications. In some embodiments, the ASO comprises one or more sugar moiety modifications. In some embodiments, the ASO comprises one or more skeletal modifications and one or more sugar moiety modifications. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA). Any of the ASOs described herein or components of the ASO (eg, nucleobases, sugar moieties, skeletons) to achieve the desired properties or activities of the ASO or reduce the undesired properties or activities of the ASO. May be modified for. For example, ASO or one or more components of ASO enhances the binding affinity of pre-mRNA to target sequences on transcripts; reduces binding to non-target sequences; cellular nucleases (ie, RNase H). To reduce degradation by); to improve the uptake of ASO into cells and / or the nucleus of cells; to alter the pharmacokinetics or pharmacodynamics of ASO; and to regulate the half-life of ASO. Can be modified.

In some embodiments, the ASO is composed of 2'-O- (2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs composed of such nucleotides are particularly well suited to the methods disclosed herein, and oligomers with such modifications have significantly enhanced resistance and increased resistance to nuclease degradation. It has been shown to have bioavailability, which makes it suitable for oral delivery, for example, in some embodiments described herein. For example, Gary et al. , J Pharmacol Exp Ther. 2001; 296 (3): 890-7; Geary et al. , J Pharmacol Exp Ther. 2001; 296 (3): 898-904.

Methods of synthesizing ASO are known to those of skill in the art. Alternatively or in addition, ASO may be obtained from commercial sources.

Unless otherwise stated, the left end of a single-stranded nucleic acid (eg, pre-mRNA transcript, oligonucleotide, ASO, etc.) sequence is the 5'end, to the left of the single-stranded or double-stranded nucleic acid sequence. Is called the 5'direction. Similarly, the right or right end of a nucleic acid sequence (single or double strand) is the 3'end or 3'direction. Generally, a region or sequence at 5'with respect to a reference point in nucleic acid is referred to as "upstream" and a region or sequence at 3'with respect to a reference point in nucleic acid is referred to as "downstream". In general, the 5'direction or 5'end of mRNA is where the initiation or start codon is located, while the 3'end or 3'direction is where the stop codon is located. In some embodiments, nucleotides upstream of the reference point in nucleic acid may be designated by a negative number and nucleotides downstream of the reference point may be designated by a positive number. For example, a reference point (eg, an exon-exon link in mRNA) can be designated as a "zero" site, and nucleotides directly adjacent to and upstream of the reference point are "minus 1", eg, "-1". Designated, on the other hand, nucleotides directly adjacent to and downstream of the reference point are designated as "plus 1", eg "+1".

In another embodiment, the ASO is relative to the target portion of the TSC2 RIC mRNA precursor (eg, the 5'splice site) downstream of the 5'splice site of the intron retained in the TSC2 RIC mRNA precursor (3'direction). And is complementary (and binds to the target moiety) with respect to the direction indicated by the positive number (FIG. 1). In some embodiments, the ASO is a TSC2 RIC mRNA precursor within the region of +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100 with respect to the 5'splice site of the retained intron. Complementary to the target portion of. In some embodiments, the ASO is not complementary to the 5'splice site from +1 to +5 nucleotides (the first 5 nucleotides located downstream of the 5'splice site). In some embodiments, the ASO may be complementary to the target portion of the TSC2 RIC pre-mRNA within the region of +6 to +50 nucleotides relative to the 5'splice site of the retained intron. In some embodiments, the ASO is +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to the 5'splice site of the retained intron. Complementary to the target portion within the +20 region.

In some embodiments, the ASO is located upstream of the 3'splice site of the intron retained in the TSC2 RIC mRNA precursor (in the 5'direction) in the target region of the TSC2 RIC mRNA precursor (eg, in a negative number). Complementary to the designated direction) (Fig. 1). In some embodiments, the ASO is -16 to -500, -16 to -400, -16 to -300, -16 to -200, -16 to -100 for the 3'splice site of the retained intron. It is complementary to the target portion of the TSC2 RIC mRNA precursor within the region. In some embodiments, the ASO is not complementary to the 3'splice site from -1 to -15 nucleotides (the first 15 nucleotides located upstream of the 3'splice site). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC pre-mRNA within the -16 to -50 region of the retained intron's 3'splice site. In some embodiments, the ASO is -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50,-relative to the 3'splice site of the retained intron. Complementary to the target moiety, which is in the region 16 to -40, or -16 to -30.

In embodiments, the target portion of the TSC2 RIC pre-mRNA is in the region from +100 to the 5'splice site of the retained intron to -100 to the 3'splice site of the retained intron.

In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC pre-mRNA located within (upstream) the exon adjacent to the 5'splice site of the retained intron (FIG. 1). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC pre-mRNA within the + 2e to -4e regions of the exon flanking the 5'splice site of the retained intron. In some embodiments, the ASO is not complementary to the -1e to -3e nucleotides for the 5'splice site of the retained intron. In some embodiments, the ASO is -4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, with respect to the 5'splice site of the retained intron. Complementary to the target portion of the TSC2 RIC mRNA precursor, which lies within the region of -4e to -50e, -4e to -40e, -4e to -30e, or -4e to -20e.

In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC pre-mRNA located within (downstream) the exon adjacent to the 3'splice site of the retained intron (FIG. 1). In some embodiments, the ASO is complementary to the target portion of the TSC2 RIC pre-mRNA within the + 2e to -4e regions of the exon flanking the 3'splice site of the retained intron. In some embodiments, ASO is not complementary to nucleotide + 1e for the 3'splice site of the retained intron. In some embodiments, the ASO is + 2e to + 100e, + 2e to + 90e, + 2e to + 80e, + 2e to + 70e, + 2e to + 60e, + 2e to + 50e, + 2e to + 40e, + 2e to the 3'splice site of the retained intron. Complementary to the target portion of the TSC2 RIC pre-mRNA, which lies within the region of + 30e, or + 2e to + 20e. The ASO may be of any length suitable for the specific binding and effective enhancing action of splicing. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, ASO has lengths of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It may be 29, 30, 31, 32, 33, 34, 35, 45 or 50 nucleobases. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO is 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases. Bases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases , 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 To 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 Nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases Bases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases , 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, The length is 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases. In some embodiments, the ASO is a nucleotide 30 in length. In some embodiments, the ASO is a nucleotide of length 29. In some embodiments, the ASO is a nucleotide with a length of 28. In some embodiments, the ASO is a nucleotide of length 27. In some embodiments, the ASO is a nucleotide 26 in length. In some embodiments, the ASO is a nucleotide 25 in length. In some embodiments, the ASO is a nucleotide 24 in length. In some embodiments, the ASO is a nucleotide 23 in length. In some embodiments, the ASO is a nucleotide 22 in length. In some embodiments, the ASO is a nucleotide of length 21. In some embodiments, the ASO is a nucleotide 20 in length. In some embodiments, the ASO is a nucleotide with a length of 19. In some embodiments, the ASO is a nucleotide 18 in length. In some embodiments, the ASO is a nucleotide 17 in length. In some embodiments, the ASO is a nucleotide 16 in length. In some embodiments, the ASO is a nucleotide 15 in length. In some embodiments, the ASO is a nucleotide 14 in length. In some embodiments, the ASO is a nucleotide 13 in length. In some embodiments, the ASO is a nucleotide of length 12. In some embodiments, the ASO is a nucleotide 11 in length. In some embodiments, the ASO is a nucleotide 10 in length.

In some embodiments, two or more ASOs with different chemistries but complementary to the same target portion of the RIC mRNA precursor are used. In some embodiments, two or more ASOs that are complementary to different target portions of the RIC mRNA precursor are used.

In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, eg, target moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. .. Such moieties include, but are not limited to, for example, cholesterol moieties, lipid moieties such as cholesteryl moieties, fatty chains such as dodecanediol or undecyl residues, polyamine chains or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In embodiments, antisense oligonucleotides are, but are not limited to, debase nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GulNAc), or mannose. It is conjugated at a moiety containing (eg, mannose-6-phosphate), a lipid, or a polyhydrogen compound. The conjugate comprises an antisense oligonucleotide at any of several positions on a sugar, base or phosphate group, as understood in the art and described in the literature, for example using a linker. It can be linked to one or more of the nucleotides. The linker can include a divalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3'end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in US Pat. No. 8,450,467, "Carbohydrates as delivery agents for oligonucleotides," which are incorporated herein by reference.

In some embodiments, the nucleic acid targeted by ASO is a TSC2 RIC mRNA precursor expressed in cells such as eukaryotic cells. In some embodiments, the term "cell" can refer to a population of cells. In some embodiments, the cells are present in the subject. In some embodiments, the cells are isolated from the subject. In some embodiments, the cells are in Exvivo. In some embodiments, the cell is a disease or disease-related cell or cell line. In some embodiments, the cells are in vitro (eg, in cell culture).

<Pharmaceutical composition>
Pharmaceutical compositions or formulations comprising the antisense oligonucleotides of the described compositions and for use in any of the described methods are in accordance with conventional techniques well known in the pharmaceutical industry and described in published literature. Can be prepared. In embodiments, the pharmaceutical composition or formulation for treating a subject is an effective amount of an antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. , And pharmaceutically acceptable diluents. Antisense oligomers of pharmaceutical formulations may further include pharmaceutically acceptable excipients, diluents or carriers.

Pharmaceutically acceptable salts are suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit-risk ratio ( See, for example, SM Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference for this purpose). The salt can be prepared in situ during the final separation and purification of the compound, or separately by reacting the functional group of the free base with a suitable organic acid. Examples of pharmaceutically acceptable and non-toxic acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, An organic acid such as succinic acid or malonic acid, or a salt of amino groups formed by using other documented methods such as ion exchange. Other pharmaceutically acceptable salts are adipates, alginates, ascorbics, asparaginates, benzenesulfonates, benzoates, bicarbonates, borates, butyrate, sulphates, Cerebral sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate , Heptaneate, hexanenate, hydride, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate , Methan sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, Includes phosphate, picphosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate acid, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. .. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Further pharmaceutically acceptable salts include, where appropriate, counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates. Contains non-toxic ammonium, quaternary ammonium, and amine cations formed using.

In embodiments, the composition is formulated into one of many possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, softgels, suppositories, and enemas. In embodiments, the composition is formulated as a suspension in the form of an aqueous medium, a non-aqueous medium, or a mixed medium. Aqueous suspensions may further comprise certain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers. In embodiments, the pharmaceutical or composition of the invention includes, but is not limited to, solutions, emulsions, microemulsions, foams or liposome-containing formulations (eg, cationic or non-cationic liposomes).

The pharmaceutical compositions or formulations of the invention are one or more permeation enhancers, carriers, excipients, or other active or non-active agents as needed and described in literature well known or published to those of skill in the art. May contain active ingredients. In embodiments, liposomes also include sterically stable liposomes, such as liposomes containing one or more specific lipids. These particular lipids result in increased circulating life of the liposomes. In embodiments, sterically stable liposomes contain one or more glycolipids or are derivatized with one or more lipophilic polymers such as polyethylene glycol (PEG) moieties. In embodiments, the surfactant is included in a pharmaceutical formulation or composition. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present invention utilizes permeation enhancers to achieve efficient delivery of antisense oligonucleotides, eg, to aid diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In embodiments, the permeation enhancer is a surfactant, fatty acid, bile acid salt, chelating agent, or non-chelating non-surfactant.

In embodiments, the pharmaceutical formulation comprises a plurality of antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. In embodiments, the antisense oligonucleotide is administered by a method known in the art with one or more agents capable of promoting the penetration of the subject antisense oligonucleotide through the blood-brain barrier. For example, delivery of a drug by administration of an adenoviral vector to motor neurons in muscle tissue is described in US Pat. No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons", which is by reference. Incorporated herein. Delivery of the vector directly to the brain, such as the striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in US Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the center. It is described in "system patentary in brain", which is incorporated herein by reference.

In embodiments, the antisense oligonucleotide is linked or conjugated to an agent that provides the desired pharmaceutical or pharmacodynamic properties. In embodiments, antisense oligonucleotides are attached to antibodies to substances known in the art, such as transferrin receptors, to facilitate penetration or transport across the blood-brain barrier. In embodiments, the antisense oligonucleotide is linked to a viral vector, for example, to make the antisense composition more effective or to increase transport across the blood-brain barrier. In embodiments, disruption of the permeable blood-brain barrier involves sugars such as meso-erythritol, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, zulcitol, myoinositol, L (-). ) Fucose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) ramnorth, D (+) ) Meribios, D (-) ribose, adonitol, D (+) arabinose, L (-) arabinose, D (+) fucose, L (-) fucose, D (-) lyxose, L (+) lyxose, and L ( -) Helped by infusion of lyxose or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, treonine, tyrosine, valine, and taurine. Be done. Methods and substances for enhancing the penetration of the blood-brain barrier are described, for example, in US Pat. No. 4,866,042, US Pat. No. 4,866,042, US Pat. It is described in "Material for passage through the blood-brain barrier" and US Pat. No. 6,936,589 "Parental delivery systems", each of which is incorporated herein by reference.

In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, eg, targeted moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. .. Such moieties include, but are not limited to, for example, cholesterol moieties, lipid moieties such as cholesteryl moieties, fatty chains such as dodecanediol or undecyl residues, polyamine chains or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In other embodiments, antisense oligonucleotides are, but are not limited to, debase nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GulNAc), and the like. Alternatively, it is conjugated with a moiety containing mannose (eg, mannose-6-phosphate), a lipid, or a polyhydrogen compound. The conjugate comprises an antisense oligonucleotide at any of several positions on a sugar, base or phosphate group, as understood in the art and described in the literature, for example using a linker. It can be linked to one or more of the nucleotides. The linker can include a divalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3'end of the antisense oligonucleotide. Methods for preparing oligonucleotides are described, for example, in US Pat. No. 8,450,467, "Carbohydrate concentrates as delivery agents for oligonucleotides," which are incorporated herein by reference.

<Subject treatment>
Any of the compositions provided herein can be administered to an individual. "Individual" may be used interchangeably with "subject" or "patient". The individual may be a mammal, for example an animal such as a human or non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. good. In embodiments, the individual is a human. In embodiments, the individual is a fetus, embryo, or child. In other embodiments, the individual may be another eukaryote, such as a plant. In some embodiments, the compounds provided herein are administered to cells exvivo.

In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disorder, such as any of the disorders described herein. In some embodiments, the individual is at risk of having a disease, such as any of the diseases described herein. In some embodiments, the individual has an increased risk of having a disease or disorder caused by a deficiency in the amount of protein or a deficiency in protein activity. If an individual has an increased risk of having a disease or disorder caused by a lack of protein quantity or protein activity, the method comprises prophylactic or prophylactic treatment. For example, an individual may have an increased risk of having such a disease or disorder due to a family history of the disease. Typically, individuals at an increased risk of having such a disease or disorder will benefit from prophylactic treatment (eg, by preventing or delaying the onset or progression of the disease or disorder).

Suitable routes for administration of ASO of the present invention may vary depending on the cell type in which delivery of ASO is desired. Multiple tissues and organs are affected by tuberous sclerosis, and the brain, kidneys, skin, lungs and heart are the most significantly affected tissues. The ASOs of the invention can be administered topically to the patient's skin or to the lungs by pulmonary delivery. The ASO of the present invention may be administered parenterally to a patient by, for example, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery to the brain, kidneys, skin, lungs or heart is performed. In embodiments, the fetus is treated in utero, for example, by administering the ASO composition directly or indirectly (eg, via the mother) to the fetus.

<How to identify additional ASOs that enhance splicing>
Within the scope of the invention are also methods of identifying (determining) additional ASOs that enhance splicing of TSC2 RIC pre-mRNA, especially in target introns. ASOs that specifically hybridize various nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and / or degree of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site of the splicing inhibitor / silencer. Known methods in the art to identify (determine) the ASO that results in the desired effect (eg, enhancement of splicing, protein or functional RNA production) when hybridized to the target region of the intron. May be used. These methods can also be used to identify ASOs that enhance splicing of retained introns by binding to target regions in exons adjacent to retained introns or in unretained introns. can. An example of a method that can be used is provided below.

A SO A round of screening, called a "walk", can be performed using an ASO designed to hybridize to the target site of the pre-mRNA. For example, the ASO used for the ASO walk is approximately 100 nucleotides upstream of the 5'splice site of the retained intron (eg, part of an exxon sequence located upstream of the targeted / retained intron). Targeted / retained from approximately 100 nucleotides downstream of the 5'splice site of the targeted / retained intron and / or approximately 100 nucleotides upstream of the 3'splice site of the retained intron. Approximately 100 nucleotides downstream of the 3'splice site of the intron (eg, part of the exon sequence located downstream of the targeted / retained intron) are tiled every 5 nucleotides. obtain. For example, a first ASO with a length of 15 nucleotides can be designed to specifically hybridize nucleotides +6 to +20 to the 5'splice site of the targeted / retained intron. The second ASO is designed to specifically hybridize nucleotides +11 to +25 to the 5'splice site of the targeted / retained intron. ASO is designed to extend to the target region of pre-mRNA. In embodiments, the ASO can be spread more tightly, eg, every 1, 2, 3, or 4 nucleotides. In addition, ASO can be spread from 100 nucleotides downstream of the 5'splice site to 100 nucleotides upstream of the 3'splice site.

One or more ASOs, or one control ASO (an ASO with a scrambled sequence that is not expected to hybridize to a target site), is a target pre-mRNA (eg, herein), eg, by transfection. It is delivered to a disease-related cell line that expresses the RIC mRNA precursor (described separately). The effect of inducing each splicing of ASO is assessed by methods known in the art, such as reverse transcriptase (RT) -PCR, using primers that span splice junctions, as described herein. Obtain (see "Identifying Intron Retention Events"). Decreased or absent RT-PCR products produced using primers that span splice junctions in ASO-treated cells compared to control ASO-treated cells enhanced splicing of target introns. It is shown that. In some embodiments, the splicing efficiency, the ratio of spliced pre-mRNA to non-spliced pre-mRNA, the rate of splicing, or the degree of splicing is improved using the ASOs described herein. Can be done. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO has achieved the desired effect (eg, enhanced protein production). Methods known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can also be used.

ASO A second round of screening, called a "micro-walk," can be performed using ASOs designed to hybridize to target sites of pre-mRNA. The ASO used for the ASO microwalk is spread one nucleotide at a time to further refine the nucleotide acid sequence of the pre-mRNA that results in enhanced splicing when hybridized with the ASO.

Regions defined by ASOs that facilitate splicing of target introns include longer ASOs, typically 18-25 nt, in addition to ASOs spaced in 1-nt steps. , Will be investigated in more detail by the ASO "Microwalk".

As described above for ASO walks, ASO microwalks include one or more ASOs, or control ASOs (ASOs with scrambled sequences, sequences that are not expected to hybridize to the target site), eg. It is performed by hybridization by delivering the target mRNA precursor to a disease-related cell line that expresses it. The effect of inducing each splicing of ASO is assessed by methods known in the art, such as reverse transcriptase (RT) -PCR, using primers that span splice junctions, as described herein. Obtain (see "Identifying Intron Retention Events"). Decreased or absent RT-PCR products produced using primers that span splice junctions in ASO-treated cells compared to control ASO-treated cells enhanced splicing of target introns. It is shown that. In some embodiments, the splicing efficiency, the ratio of spliced pre-mRNA to non-spliced pre-mRNA, the rate of splicing, or the degree of splicing is improved using the ASOs described herein. Can be done. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO has achieved the desired effect (eg, enhanced protein production). Methods known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can also be used.

When hybridized to the region of pre-mRNA, ASO results in enhanced splicing and increased protein production in animal models, such as transgenic mouse models in which the full-length human gene is knocked in or humanization of the disease. It can be tested in vivo using a mouse model. Suitable routes for administration of ASO may vary depending on the disease and / or cell type for which delivery of ASO is desired. ASO can be administered, for example, by topical application to the skin, lung delivery to the lungs, intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. After administration, the cells, tissues, and / or organs of the model animal are evaluated, for example, for splicing (efficiency, rate, degree) and protein production by methods known in the art and methods described herein. Thereby, it can be evaluated to determine the effect of ASO treatment. Animal models can also be phenotypic or behavioral signs of disease or disease severity.

Preferred embodiments of the invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided as just one example. Many changes, changes and substitutions are conceived by those skilled in the art without departing from the invention. It should be appreciated that various alternatives of the embodiments of the invention described herein may be utilized in the practice of the invention. The following claims define the scope of the invention, and it is intended that the methods and structures within the scope of these claims and their equivalents are incorporated therein.

The present invention is more specifically exemplified by the following examples. However, it should be understood that the invention is not limited by these examples in any way.

Example 1: Identification of Intron Retention Events for TSC2 Transcripts by RNAseq Using Next Generation Seqing To reveal snapshots of transcripts produced by the TSC2 gene to identify intron retention events. The decision was used to perform a shotgun sequence decision for the entire transcriptome. To this end, polyA + RNA from the nuclear and cytoplasmic fractions of HCN (human cortical neurons), renal epithelial cells, bronchial epithelial cells, and THLE-3 (human hepatitis) cells was isolated and Illumina's TruSeq Stranded mRNA. A cDNA library was constructed using the library Prep Kit. The library was pair-end sequenced and, as a result, 100 nucleotide readings were mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The result of sequencing for TSC2 is shown in FIG. Briefly, FIG. 3 shows (UCSC Genome Information Group (Center for Biomolecular Science & Enginereing, University of California, California, Santa Cruz, Santa Cruz, Santa Cruz, E. 2015, "The UCSC Genome Browser database: 2015 updated" Nuclear Acids Research 43, Database Issue, doi: 10.1093 / nar / gku1177-mapped and visualized in the UCSC Genome Browser (described in UCSC Genome Browser). It indicates a value and the scope and number of readings can be inferred by the peak signal. The height of the peak indicates the level of expression given by the density of readings in a particular region. A schematic of TSC2 (drawn to scale) is provided by the UCSC Genome Browser (under the read signal) so that the peaks are adapted to the TSC2 exon and intron regions. Based on this indication, HCN (as shown for intron 4 in the lower figure of FIG. 3, for introns 25 and 26 in the lower figure of FIG. 5, and for introns 31 and 32 in the lower figure of FIG. 7). Five introns (4, 25 indicated by arrows) that have high reading densities in the nuclear fraction of these cells but have very low or no readings in the cytoplasmic fraction of these cells. , 26, 31, and 32) have been identified. This indicates that these introns are retained and that transcripts containing intron-4, intron-25, intron-26, intron-31, and intron-32 remain in the cell nucleus. This suggests that these retained TSC2 RIC pre-mRNA are unproductive because they are not transported to the cytoplasm.

Example 2: Designing an ASO-Walk Targeting Intron 4 of TSC2 The ASO Walk was designed to target Intron 4 using the methods described herein (FIG. 4; Table 2). .. 2'-O-Me RNA, PS in the region immediately downstream of the 5'splice site of intron 4 extending from nucleotides +6 to +58 and immediately upstream of the 3'splice site of intron 4 extending from nucleotides -16 to -68 Targeted using the skeleton, 18-mer ASO was shifted at 5-nucleotide intervals.

Example 3: Designing an ASO-Walk Targeting Introns 25 and 26 of TSC2 The ASO Walk was designed to target Introns 25 and 26 using the methods described herein (FIG. 6). Table 2). 2'-O-Me RNA, PS Targeted using the skeleton, 18-mer ASO was shifted at 5-nucleotide intervals. The intron region of the splice site adjacent to the alternative exon 26 is not targeted to avoid affecting the inclusion level of the exon 26.

Example 4: Designing an ASO-Walk Targeting Introns 31 and 32 of TSC2 The ASO Walk was designed to target Introns 31 and 32 using the methods described herein (FIG. 8). Table 2). 2'-O-Me RNA, PS Targeted using the skeleton, 18-mer ASOs were shifted at 5-nucleotide intervals (with the exception of two ASOs, TSC2-IVS32-33 and TSC2-IVS32-51). The intron region of the splice site adjacent to the alternative exon 32 is not targeted to avoid affecting the inclusion level of the exon 32.

Example 5: Improvement of splicing efficiency via ASO target of TSC2 intron 4, 25, 26, 31 or 32 increases TSC2 expression using ASO that increases transcript levels, TSC2 intron 4, 25, RT-PCR products were evaluated using radioactive RT-PCR and RT-qPCR to determine if this could be achieved by improving the splicing efficiency of 26, 31 or 32. The human retinal epithelial cell line, ARPE-19 cells (American Type Culture Collection (ATCC), USA), or the human hepatocellular carcinoma cell line, Huh-7 (NIBIOHN, Japan), or the human neuroblastoma cell line. The SK-N-AS (ATCC) was pseudotransfected or transfected with the target ASOs set forth in FIGS. 10, 12, 15, and 1-2. Cells were transfected with Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to the supplier's specifications. Briefly, ASO was sown on 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were stripped with trypsin, resuspended in complete medium and approximately 25,000 cells were added to the ASO transfection mixture. Transfection experiments were performed on three plate replicas. The final ASO concentration was 80 nM. According to the supplier's specifications, the medium was replaced 6 hours after transfection and cells were harvested in 24 hours using Cells-to-Ct RT reagent supplemented with DNAse (Thermo Fisher). The cDNA was generated with the Cells-to-Ct RT reagent (Thermo Fisher) according to the supplier's specifications. Quantitative PCR was performed using the Taqman assay with the corresponding exon-exon ligation (Thermo Fisher) probes listed in Table 1-2 to quantify the amount of splicing in the intron of interest. .. The Taqman assay was performed on the QuantStudio 7Flex Real-Time PCR system (Thermo Fisher) according to the supplier's specifications. Target gene assay values are normalized to RPL32 (deltaCt) and plate-matched pseudotransfection samples (delta-delta Ct), resulting in multiple plate changes over pseudoquantitative (2 ^-(delta-delta Ct)). Average multiple changes across mock replicas were plotted (FIG. 10, FIGS. 12 and 15). In FIGS. 10, 12, and 15, some ASOs were identified as increasing target gene expression. This suggests an increase in splicing at each target intron.

Claims (14)

An antisense oligomer (ASO) complementary to the target portion of the retained intron-containing pre-mRNA (RIC pre-mRNA) that encodes tuberin, with SEQ ID NOs: 21-23 , 25-27, 250-252. , 254 to 256, 395 to 401, 460 to 462, 464 to 467.
The RIC pre-mRNA contains retained introns and contains
By binding ASO to the target portion of the RIC pre-mRNA in cells expressing the RIC pre-mRNA, retained introns are constitutively spliced from the RIC pre-mRNA, thereby encoding tuberin intracellularly. The ASO that increases the level of mature mRNA to be produced and the target portion of the RIC pre-mRNA is within the retained intron. 1. ASO. The RIC mRNA precursor comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to the sequence selected from SEQ ID NO: 2-8. , ASO according to claim 1 or 2. The ASO according to any one of claims 1 to 3, wherein the target portion of the RIC mRNA precursor is in a sequence selected from SEQ ID NO: 5097 to 5105. The ASO according to any one of claims 1 to 4 , wherein the ASO comprises 18 to 50 nucleobases. Any of claims 1-4, wherein the ASO consists of a sequence selected from SEQ ID NOs: 21-23, 25-27, 250-252, 254-256, 395-401, 460-462, 464-467. The ASO described in item 1. Tuberin compared to comparable wild-type protein:
i) Decreased form; or ii) Fully functional form,
The ASO according to any one of claims 1 to 6, which is generated in 1. ASO is a skeletal modification comprising phosphorothioate binding or phosphorodiamidate binding; or contains a modified sugar moiety or sugar analog, phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl moiety, 2'. The ASO according to any one of claims 1 to 7, which may contain a fluoro moiety or a 2'-O-methoxyethyl moiety. A viral vector in which a polynucleotide containing the ASO according to any one of claims 1 to 8 is ligated. The ASO or claim 9 according to any one of claims 1 to 8 , which is a pharmaceutical composition for treating a disease or a disease characterized by a lack of amount or function of a tuberin protein in a subject. The pharmaceutical composition comprising the described viral vector. The pharmaceutical composition according to claim 10 , wherein the disease or disease is tuberous sclerosis. A deficiency in the amount of tuberin is caused by haploinsufficiency of tuberin, where the subject is given a first allele encoding functional tuberin and a second allele or non-functional that no tuberin is produced. The pharmaceutical composition according to claim 10 or 11 , having a second allele encoding tuberin, wherein the ASO binds to a target portion of the RIC pre-mRNA transcribed from the first allele. The subject has a first mutagen allele and a second mutagen allele, and tuberin is
a. From the first mutation allele:
i. Produced at reduced levels compared to production from wild-type alleles;
ii. Produced in a less functional form compared to comparable wild-type tuberin; or iii. Not generated, and b. From the second mutation allele:
i. Produced at reduced levels compared to production from wild-type alleles;
ii. Produced in a less functional form compared to comparable wild-type tuberin; or iii. Not generated,
Here, the subject is the first mutation allele a. iii. If the second mutation allele is b. i. Or b. ii. And the subject is the second mutation allele b. iii. If the first mutation allele is a. i. Or a. ii. The RIC mRNA precursor is a. i. Or a. ii. First mutation allele or b. i. Or b. ii. The pharmaceutical composition according to any one of claims 10 to 12 , which is transcribed from the second mutation allele. The pharmaceutical composition according to any one of claims 10 to 13 , which is for administration by subarachnoid space injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

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