JP2022106803A - Antisense oligomer for treatment of alagille syndrome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide methods for treating patients with Alagille syndrome (ALGS).

SOLUTION: The method comprises a step of bringing cells of a subject into contact with an antisense oligomer (ASO) complementary to a targeted portion of a RIC mRNA precursor encoding target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC mRNA precursor encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA and increasing the expression of the target protein or functional RNA, in the cells of the subject.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

<Cross reference>
This application is filed on December 14, 2015, in US Provisional Patent Application Nos. 62 / 267,21.
Claiming the benefit of No. 0, the provisional application is incorporated herein by reference in its entirety.

<Sequence list>
The 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 the ASCII made on December 9, 2016 is available at 47991-705_601_SL. It is a txt file name and has a size of 201,192 bytes. The aforementioned files were created on December 9, 2016 and are incorporated herein by reference in their entirety.

Alagille syndrome (ALGS), also known as arterial hepatic dysplasia, is a rare, debilitating, autosomal dominant multilineage disorder (Turnpenny and Ellard,).
Euro. J. Hum. Gen. 2012, 20, 251-257). Patients suffer from liver damage caused by abnormalities in the bile ducts. Other effects include heart disease, angiogenesis abnormalities, skeletal abnormalities, ophthalmic features, facial features, renal abnormalities, developmental retardation, and pancreatic insufficiency. The 1: 70,000 reported ALGS epidemic is believed to be underestimated due to disease variability and infestation.

Mutations in genes involved in Notch signaling have been reported to cause ALGS. Mutations in JAG1 cause ALGS1 type, while mutations in NOTCH2 cause ALGS2 type, which is not as prevalent as ALGS1 type. JAG1 encodes the JAG1 protein, which is a cell surface ligand for Notch membrane receptors. Binding of the JAG1 protein to the Notch receptor triggers a signaling cascade, resulting in transcription of genes involved in cell fate determination and differentiation.

The present invention provides compositions and methods for treating Alagille syndrome.
An antisense oligomer (A) that promotes constitutive splicing of JAG1 retained intron-containing pre-mRNA (RIC pre-mRNA) at the intron splice site.
SO) is included. The present invention further presents in the cells of a subject in need, eg, a subject who can benefit from increased production of the JAG1 protein, mature JAG1 mR.
Compositions and methods for increasing the production of NA and, in turn, the JAG1 protein are provided. The above method is a test with Alagille syndrome caused by a total gene deletion in addition to mutations in the JAG1 gene, including missense, splicing, frameshift and nonsense mutations that result in a deficiency in JAG1 protein production. It may be used to treat the body. In other embodiments, the compositions and methods of the invention are used to treat a subject with muscular dystrophy, who can benefit from increased production of the JAG1 protein.

The present specification discloses, in certain embodiments, a method of treating a subject's Alagille syndrome in need by increasing the expression of a target protein or functional RNA by the subject's cells, wherein the cells are described. , Retained intron-containing pre-mRNA (R)
IC mRNA precursor), and the RIC mRNA precursor is a retained intron, 5
It comprises an exon adjacent to a'splice site, and an exon adjacent to a 3'splice site, where the RIC pre-mRNA encodes a target protein or functional RNA, the method of which comprises the subject's cells. RI encoding a target protein or functional RNA
It involves contacting the target portion of the C-mRNA with a complementary antisense oligomer (ASO), whereby the retained intron is the target protein or functional RN.
Constitutively spliced from the RIC pre-mRNA encoding A, thereby
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, JAG1, by cells carrying a retained intron-containing pre-mRNA (RIC pre-mRNA). , The RIC pre-mRNA is a retained intron, an exon flanking the 5'splice site of the retained intron, and a 3'of the retained intron.
Containing exons adjacent to the splice site, where the RIC mRNA precursor is JAG
One protein is encoded, the method encodes a cell, a RIC encoding a JAG1 protein.
It involves contacting the target portion of the pre-mRNA with a complementary antisense oligomer (ASO), whereby the retained intron is the R encoding the JAG1 protein.
Constitutively spliced from IC pre-mRNA, thereby making JAG intracellularly
Increases the level of mRNA encoding one protein and increases the expression of JAG1 protein. In some embodiments, the target protein is JAG1. In some embodiments, the target protein or functional RNA functionally enhances or replaces or compensates for the target protein or functional RNA that is deficient in quantity or activity in the subject (compe).
nsating) A protein or a compensating 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 JAG1 protein. In some embodiments, a 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 resulting from a lack of quantity or function of the target protein, wherein the subject has a) (i) the target protein is a wild-type allele. Produce at reduced levels compared to production from, (i
i) The first mutation alliance gene, in which the target protein is produced in a reduced function compared to the equivalent wild-type protein, or (iii) the target protein is not produced, and b) (i) the target. The protein is produced at a reduced level compared to the production from the wild-type allelic gene, (ii) the target protein is produced in a reduced function compared to the equivalent wild-type protein, or (Iii) When the target protein is not produced and has a second mutated allelic gene, wherein the subject has the first mutated allelic gene of a) (iii), the second mutated allelic gene b) (i) or b) (ii), and when the subject has a second mutant allegogen of b) (iii), the first mutant allelic gene is a) ( i) or a) (ii) mutation allogene, RIC mRNA
The precursors are the first mutant alleles a) (i) or a) (ii) and / or b) (
It is transcribed from a second mutation allele that is i) or b) (ii). 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 pre-mRNA is from the +6 region of the retained intron to the 5'splice site to -1 to the retained intron's 3'splice site in the retained intron.
It is in the area of 6. In some embodiments, the target portion of the RIC pre-mRNA is (a) from +6 to +500, +6 to +400, +6 to 300, +6 to the 5'splice site of the retained intron in the retained intron. 200, or +6
Within the region from +100; or (b) -16 to -500, -16 to -400, -16 to -300, -16 to -20 for the retained intron's 3'splice site.
It is in the range of 0, or -16 to -100. In some embodiments, the RIC mRN
The target portion of the A precursor is (a) within the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron; or (b) 3 of the retained intron.
'It is within the region of + 2e to -4e in the exon adjacent to the splice site. 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 having% sequence identity. In some embodiments, the nucleotide construct is at least about 80%, 85%, 90% of SEQ ID NO: 2.
, 95%, 96%, 97%, 98%, 99% or 100% sequences having 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 the abnormal splicing that results from 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 target protein or function R
The mRNA encoding NA 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 intracellularly in contact with the antisense oligomer is the amount of mRNA encoding the target protein or functional RNA produced in the control cell. 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 times to about 5 times, about 1.1 times to about 6 times, about 1.
1 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 5 times, 2 to 6 times, 2 to 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 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,
It is increased by 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 the cells contacted with the antisense oligomer is about 1 compared to the total amount of target protein produced in the control cells.
.. 1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times
Double, 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 times to about 6
Double, 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 It is increased about 10 times. In some embodiments, the antisense oligomer comprises a skeletal modification involving a phosphorothioate or phosphorodiamidate bond. In some embodiments, the antisense oligomers are phosphorodiamidate morpholinos, locked nucleic acids, peptide nucleic acids, 2'-O-methyl,
Contains a 2'-fluoro, or 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. Nucleic acid 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 Nucleic acid bases, 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
~ 20 nucleobases, 10 ~ 15 nucleobases, 11 ~ 50 nucleobases, 11 ~ 40 nucleobases, 11 ~ 35 nucleobases, 11 ~ 30 nucleobases, 11 ~ 25 nucleobases, 11 ~ 20
Nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 1
It consists of 2 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 8 on the target portion of the protein-encoding RIC mRNA precursor.
It is 0%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary. In some embodiments, the RIC mRN
The target portion of the A precursor is in the sequence selected from SEQ ID NO: 437-439. In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, relative to any one of SEQ ID NO: 3-436.
Includes nucleotide sequences that are 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer is SEQ ID NO: 3.
Contains a nucleotide sequence selected from -436. In some embodiments, the cell comprises a population of RIC mRNA precursors transcribed from a gene encoding a target protein or functional RNA, where the population of RIC mRNA precursors is a population of two or more retained introns. 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 induces splicing out of two or more retained introns from the population of RIC mRNA precursors, targeting protein or function. Produces mRNA that encodes RNA. In some embodiments, the cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, where the RIC mRN
The population of A precursors comprises two or more retained introns, and the antisense oligomer binds to the second most abundant retained introns in the population of RIC mRNA precursors. In some embodiments, binding of the antisense oligomer to the second most abundant retained intron induces splicing out of two or more retained introns from the population of RIC mRNA precursors, targeting proteins. Or mRN encoding functional RNA
Produces A. In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disease is Alagille syndrome or muscular dystrophy. In some embodiments, the target protein and RIC mRNA precursor are encoded by the JAG1 gene. In some embodiments, the method further comprises the step of assessing JAG1 protein expression. In some embodiments, the antisense oligomer is JAG1 RIC m.
It binds to the target portion of the pre-RNA, where the target portion is SEQ ID NO: 49, 50,
It is in a sequence selected from 51, 52, 53, 54, 55, 56, and 57. In some embodiments, the subject is 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 in Exvivo. In some embodiments, the antisense oligomer is administered by intraperitoneal, intramuscular, subcutaneous, or intravenous injection of the subject.
In some embodiments, exons -3e to -1e adjacent to 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 + 1e of the exon flanking the -15 to -1 and 3'splice sites of the retained intron are identical to the corresponding wild-type sequence.

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

In the specific embodiment, the present specification describes any one of SEQ ID NO: 3-436.
Disclosed are antisense oligomers comprising sequences having at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to one.

In a particular embodiment, the specification discloses a pharmaceutical composition comprising the antisense oligomers and excipients described above.

The present specification discloses a method of treating a subject in need by administering the above-mentioned pharmaceutical composition by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection in a specific embodiment. To.

As used herein, in certain embodiments, it is used in a method of increasing the expression of a target protein or functional RNA by a cell to treat a subject's Arazil syndrome in need associated with a protein deficiency or functional RNA deficiency. A composition comprising an antisense oligomer is disclosed, wherein a protein deficiency or functional RNA deficiency is a deficiency of amount or activity in the subject and the antisense oligomer is retained encoding a target protein or functional RNA. Intron-containing mRNA precursor (RIC mRNA precursor) enhances constitutive splicing, where the target protein is (a) deficient protein; or (b) functionally enhances or replaces the deficient protein in the subject. A protein that supplements, compensates, and functional RNA is (a) a deficient RNA; or (b) a functional RNA that functionally enhances or replaces the deficient functional RNA in a subject, and here RIC. mRNA precursors include retained introns, exons adjacent to the 5'splice site, and exons adjacent to the 3'splice site, where the retained introns are RICs encoding target proteins or functional RNAs. Spliced from the mRNA precursor, thereby increasing the production or activity of the target protein or functional RNA in the subject. In some embodiments, the specification also also includes.
A composition comprising an antisense oligomer for use in a method of treating a disease associated with the JAG1 protein in a subject in need has been disclosed, wherein the method is a step of increasing the expression of the JAG1 protein by the subject's cells. Containing, where the cell is retained, including a retained intron, an exson adjacent to the 5'splice site of the retained intron, and an exson adjacent to the 3'splice site of the retained intron. It has an intron-containing mRNA precursor (RIC mRNA precursor), the RIC mRNA precursor encodes the JAG1 protein, and the method also comprises the step of contacting the cell with an antisense oligomer, thereby being retained. The intron is constitutively spliced from the transcript of the RIC mRNA precursor encoding the JAG1 protein, thereby increasing the level of the mRNA encoding the JAG1 protein in the subject's cells, targeting protein or functional RNA. Increases the expression of. In some embodiments, the disease is a disease or disorder. In some embodiments, the disease or disease is Alagille syndrome or muscular dystrophy. In some embodiments, the target protein and RIC
The pre-mRNA is encoded by the JAG1 gene. In some embodiments, the antisense oligomer is within the region of +6 relative to the 5'splice site of the retained intron to the region -16 relative to the 3'splice site of the retained intron in the retained intron. , Targets some of the RIC mRNA precursors. In some embodiments, the antisense oligomer is in the retained intron in the region of (a) +6 to +100 relative to the 5'splice site of the retained intron; or (b) the 3'splice of the retained intron. It is in the region of -16 to -100 with respect to the site.
In some embodiments, the antisense oligomer is about 100 nucleotides downstream of the 5'splice site of at least one retained intron and about 100 nucleotides upstream of the 3'splice site of at least one retained intron. Targets some of the RIC pre-mRNA within the region up to. In some embodiments, RIC mRNA
The target portion of the precursor is (a) within the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron; or (b) the 3'of the retained intron.
It is within the region of + 2e to -4e in the exon adjacent to the splice site. 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 having the sequence identity of. In some embodiments, the RIC
Pre-mRNA is at least about 80%, 85%, 90 relative to SEQ ID NO: 2.
Includes sequences with%, 95%, 96%, 97%, 98%, 99% or 100% 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 target protein or functional RNA by regulating the abnormal splicing that results from mutations in the gene encoding the target protein or functional RNA. In some embodiments, the RIC pre-mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA. In some embodiments, it encodes a target protein or functional RNA.
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-limited 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 involving a phosphorothioate or phosphorodiamidate bond. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid,
Contains a 2'-O-methyl, 2'-fluoro, or 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. Nucleic acid 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 Nucleic acid bases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 2
5 nucleobases, 10-20 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 ~
It consists of 40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 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 at the target portion of the RIC mRNA precursor encoding the protein. It is 100% complementary. In some embodiments, the antisense oligomer binds to the target portion of the JAG1 RIC mRNA precursor and
Here, the target moiety is in the sequence selected from SEQ ID NO: 437-439. 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: 3-436.
Includes nucleotide sequences that are%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer is SEQ ID NO: 3-4.
Contains a nucleotide sequence selected from 36.

The present specification discloses, in a specific embodiment, a pharmaceutical composition comprising any of the above compositions antisense oligomers and excipients. In some embodiments, the specification also describes a method of treating a subject in need by administering the pharmaceutical composition by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Will be done.

The present specification discloses a pharmaceutical composition comprising, in certain embodiments, an antisense oligomer that hybridizes to the target sequence of the deficient JAG1 mRNA transcript, and a pharmaceutically acceptable excipient. Here, the deficient JAG1 mRNA transcript comprises retained introns, and the antisense oligomer induces splicing out of the retained introns from the deficient JAG1 mRNA transcript. In some embodiments, the deficient JAG1 mRNA transcript is a transcript of the JAG1 RIC mRNA precursor. In some embodiments, the target portion of the transcript of the JAG1 RIC pre-mRNA is from the region +6 to the 5'splice site of the retained intron in the retained intron to the 3'splice site of the retained intron. It is in the area of -16. In some embodiments, the transcript of the JAG1 RIC mRNA precursor is
At least about 80%, 85%, 90%, 95%, 96% with respect to SEQ ID NO: 1.
, 97%, 98%, 99% or 100% encoded by a gene sequence having sequence identity. In some embodiments, the transcript of the JAG1 RIC mRNA precursor is SE.
At least about 80%, 85%, 90%, 95%, 96%, 97 for Q ID NO: 2.
Includes sequences with%, 98%, 99% or 100% sequence identity. In some embodiments, the antisense oligomer comprises a skeletal modification involving a phosphorothioate or phosphorodiamidate bond. In some embodiments, the antisense oligomer is an antisense oligonucleotide. In some embodiments, the antisense oligomer is
Phosphologic amidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2
Includes'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the antisense oligomer comprises at least one modified sugar moiety. Antisense oligomers are 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobases, 10-50 nucleobases, 10-40
Nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 1
0 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 ~ 2
0 nucleobases, 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases,
It contains 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the antisense oligomer is at least 80% on the target portion of the transcript of the JAG1 RIC mRNA precursor.
, At least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary. In some embodiments, the JAG1 RIC m
The target portion of the transcript of the pre-RNA is within the sequence selected from SEQ ID NO: 437-439. In some embodiments, the antisense oligomer is a SEQ ID NO.
: At least about 80%, 85%, 90%, 91%, 9 for any one of 3-436
Includes nucleotide sequences that are 2%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the antisense oligomer is a SEQ
ID NO: Contains a nucleotide sequence selected from 3-436. In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.

To the present specification, in certain embodiments, there was a deficiency to facilitate the removal of retained introns in order to produce a fully processed transcript of JAG1 mRNA that encodes the functional form of the tuberin protein. A method of inducing processing of a transcript of JAG1 mRNA has been disclosed, wherein the method is (a) contacting the antisense oligomer with the target cell of the subject; (b) the transcript of JAG1 mRNA deficient in the antisense oligomer. In the step of hybridizing to, the deficient JAG1 mRNA transcript can encode the functional form of the tuberin protein and comprises at least one retained intron; (c) of the tuberin protein. Well-processed JAG1 encoding functional form
The step of removing at least one retained intron from the deficient JAG1 mRNA transcript to produce the mRNA transcript; and (d) well-treated JA.
It comprises translating the functional form of the tuberin protein from the transcript of G1 mRNA. In some embodiments, the retained intron is the entire retained intron. In some embodiments, the deficient JAG1 mRNA transcript is JAG1 RIC mRN.
It is a transcript of A 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 the JAG1 protein, the method of which is SEQ.
ID NO: At least about 80%, 85%, 90% with respect to any one of 3-436
, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% comprising the step of administering to a subject an antisense oligomer comprising a nucleotide sequence having sequence identity. ..

<Built-in by reference>
All publications, patents, and patent applications referred to herein are by reference to the extent that each publication, patent, or patent application is specifically and individually directed to be incorporated by reference. Incorporated herein.
The present application includes the following inventions.
[Item 1] A method for treating a subject's Alagille syndrome, which is required by increasing the expression of a target protein or functional RNA by the subject's cells.
It has a retained intron-containing pre-mRNA (RIC mRNA pre-mRNA), and the RIC
Pre-mRNA includes retained introns, exons flanking the 5'splice site, and exons flanking the 3'splice site, where the RIC pre-mRNA encodes the target protein or functional RNA. However, the method involved contacting the subject's cells with an antisense oligomer (ASO) complementary to the target portion of the RIC pre-mRNA encoding the target protein or functional RNA, thereby retaining. The intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby in the subject's cells the target protein or functional RNA.
A method of increasing the level of mRNA encoding a target protein or increasing the expression of a functional RNA.
[Item 2] A method for increasing the expression of a target protein, which is JAG1, by cells having a retained intron-containing pre-mRNA (RIC pre-mRNA).
The RIC pre-mRNA 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, where the RIC The pre-mRNA encodes the JAG1 protein, the method comprising contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC pre-mRNA encoding the JAG1 protein, thereby retaining. A method of constructively splicing the resulting intron from a RIC mRNA precursor encoding the JAG1 protein, thereby increasing the level of the mRNA encoding the JAG1 protein and increasing the expression of the JAG1 protein in the cell.
[Item 3] The method according to item 1, wherein the target protein is JAG1.
[Item 4] Item 1, wherein the target protein or functional RNA is a compensating protein or compensating functional RNA that functionally enhances or exchanges the target protein or functional RNA that is deficient in amount or activity in the subject. The method described.
[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 the JAG1 protein.
[Item 6] A deficiency in the amount of the target protein is caused by haplodeficiency of the target protein, in which the subject is given a first allelic gene encoding a functional target protein and a second 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 resulting from a lack of target protein amount or function, wherein the subject.
a. The first mutant allele
i. Target proteins are produced at reduced levels compared to production from wild-type alleles,
ii. The target protein is produced in a less functional form as compared to an equivalent wild-type protein, or iii. The first mutant allele, which does not produce the target protein, and b. The second mutation allele
i. Target proteins are produced at reduced levels compared to production from wild-type alleles,
ii. The target protein is produced in a less functional form as compared to an equivalent wild-type protein, or iii. It has a second mutated allele in which the target protein is not produced, and where the subject is a. iii. When having the first mutated allele of, the second mutated allele is b. i. Or b. ii. Mutant allele of the subject, b. iii.
When having a second mutated allele of, the first mutated allele is a. i. Or a. i
i. Mutant alleles of, and RIC mRNA precursors are a. i. Or a. ii.
The first mutant allele and / or b. i. Or b. ii. The method according to any one of items 1 to 5, which is transcribed from any of the second mutation alleles.
[Item 8] The method according to item 7, wherein the target protein is produced in a form in which the function is reduced as compared with the equivalent wild-type protein.
Item 9. The method of item 7, wherein the target protein is produced in a fully functional form as compared to an equivalent wild-type protein.
[Item 10] In an intron in which the target portion of the RIC mRNA precursor is retained,
The method of any one of items 1-9, which lies within the region +6 with respect to the 5'splice site of the retained intron to the region -16 with respect to the 3'splice site of the retained intron.
[Item 11] In an intron in which the target portion of the RIC mRNA precursor is retained,
(A) +6 to +500, +6 for the 5'splice site of the retained intron
Within the region from +400, +6 to 300, +6 to 200, or +6 to +100; or (b) -16 to -500,-to the 3'splice site of the retained intron.
16 to -400, -16 to -300, -16 to -200, or -16 to -100
The method according to any one of items 1 to 9, which is within the area of.
[Item 12] The target portion of the RIC mRNA precursor is
(A) + in the exon adjacent to the 5'splice site of the retained intron
Within the region 2e to -4e; or (b) + in the exon adjacent to the 3'splice site of the retained intron.
The method according to any one of items 1 to 9, which is within the region of 2e to -4e.
[Item 13] The sequence of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with respect to SEQ ID NO: 1. The method according to any one of items 1 to 12, which is encoded by a gene sequence having sex.
[Item 14] Sequence identity of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the RIC mRNA precursor with respect to SEQ ID NO: 2. The method according to any one of items 1 to 13, which comprises a sequence having.
[Item 15] Items 1-14, wherein 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. The method according to any one item.
[Item 16] Any of items 1 to 15, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by regulating the abnormal splicing resulting from mutation of the gene encoding the target protein or functional RNA. The method according to item 1.
[Item 17] The method according to any one of items 1 to 16, 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.
[Item 18] The mRNA encoding the target protein or functional RNA is a full-length mature mRN.
The method according to any one of items 1 to 17, which is A or wild-type mature mRNA.
[Item 19] The method according to any one of items 1 to 18, wherein the target protein produced is a full-length protein or a wild-type protein.
[Item 20] 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 cell. Then, about 1.1 times to about 10
Double, 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, about 1.1 Double 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
Double, 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
Double 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 The method according to any one of items 1 to 19, which is increased by about 5 times, or at least about 10 times.
[Item 21] The total amount of target protein produced by cells contacted with the antisense oligomer is approximately 1 compared to the total amount of target protein produced by control cells.
.. 1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times
Double, 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 times to about 6
Double, 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 Items 1 to 20 increased by about 10 times
The method according to any one of the above.
[Item 22] The method according to any one of items 1 to 21, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 23] Items 1-22, wherein 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. The method according to any one item.
[Item 24] The method according to any one of items 1 to 23, wherein the antisense oligomer contains at least one modified sugar moiety.
[Item 25] The method according to item 24, wherein each sugar moiety is a modified sugar moiety.
[Item 26] The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, and the like.
8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 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 to 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
Item 1 consisting of ~ 25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases
The method according to any one of 25 to 25.
[Item 27] The antisense oligomer complements the target portion of the protein-encoding RIC mRNA precursor by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. Item 1 ~
The method according to any one of 26.
[Item 28] The target portion of the RIC mRNA precursor is SEQ ID NO: 437 to 43.
The method according to any one of items 1 to 27, which is in the sequence selected from 9.
[Item 29] The antisense oligomer is any one of SEQ ID NO: 3 to 436.
On the other hand, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
The method of any one of items 1-28, comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, or 99% sequence identity.
[Item 30] The method according to any one of items 1 to 28, wherein the antisense oligomer contains a nucleotide sequence selected from SEQ ID NO: 3 to 436.
[Item 31] 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. The antisense oligomer binds to the most abundant retained intron in the population of RIC pre-mRNA, any one of items 1-30.
The method described in the section.
[Item 32] The binding of antisense oligomers to the most abundant retained introns
RIC mRN to produce mRNA encoding a target protein or functional RNA
31. The method of item 31, which induces splicing out of two or more retained introns from a population of A precursors.
[Item 33] 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. The method of any one of items 1-30, wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNA.
[Item 34] The binding of the antisense oligomer to the second most abundant retained intron produces mRNA encoding the target protein or functional RNA, RIC m.
33. The method of item 33, which induces splicing out of two or more retained introns from a population of pre-mRNA.
[Item 35] The method according to any one of items 5 to 34, wherein the disease is a disease or a disorder.
[Item 36] The disease or disorder is Alagille syndrome or muscular dystrophy, item 3.
The method according to 5.
37. The method of item 36, wherein the target protein and RIC mRNA precursor are encoded by the JAG1 gene.
[Item 38] The method according to any one of items 1 to 37, further comprising a step of evaluating JAG1 protein expression.
[Item 39] The antisense oligomer binds to the target portion of the JAG1 RIC mRNA precursor, where the target portion is SEQ ID NO: 49, 50, 51, 52, 53, 54.
, 55, 56, and 57, wherein the method according to any one of items 1-38.
[Item 40] The method according to any one of items 1 to 39, wherein the subject is a human.
[Item 41] The method according to any one of items 1 to 39, wherein the subject is an animal other than a human.
42. The method of any one of items 1-40, wherein the subject is a fetus, embryo, or child.
[Item 43] The method according to any one of items 1-41, wherein the cells are in Exvivo.
[Item 44] The method according to any one of items 1-41, wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
[Item 45] Nine nucleotides at -3e to -1e of exons adjacent to the 5'splice site and +1 to +6 of retained introns are identical to the corresponding wild-type sequences, items 1-44. The method according to any one of the above.
[Item 46] The 16 nucleotides at + 1e of the exon flanking the -15 to -1 and 3'splice sites of the retained intron are identical to the corresponding wild-type sequence.
The method according to any one of items 1 to 45.
[Item 47] An antisense oligomer used by the method according to any one of items 1 to 39.
[Item 48] SEQ ID NO: At least about 8 for any one of 3 to 436.
An antisense oligomer comprising a sequence having 0%, 85%, 90%, 95%, 97%, or 100% sequence identity.
[Item 49] A pharmaceutical composition comprising the antisense oligomer and excipient of item 47 or 48.
[Item 50] A method for treating a subject in need by administering the pharmaceutical composition by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[Item 51] A composition comprising an antisense oligomer for use in a method of increasing the expression of a target protein or functional RNA by a cell to treat a subject's Arazil syndrome in need associated with a protein deficiency or functional RNA deficiency. A protein deficiency or functional RNA deficiency here is a deficiency in quantity or activity in the subject, where the antisense oligomer is a retained intron-containing mRNA precursor (RIC mRNA) encoding a target protein or functional RNA. The constitutive splicing of the precursor) is enhanced, where the target protein is
(A) deficient protein; or (b) a protein that functionally enhances or replaces or compensates for the deficient protein in the subject.
And functional RNA
(A) deficient RNA; or (b) functional RNA that functionally enhances or replaces or compensates for deficient functional RNA in a subject.
Here, the RIC pre-mRNA includes a retained intron, an exon adjacent to the 5'splice site, and an exon adjacent to the 3'splice site, and the retained intron contains a target protein or functional RNA. A composition that is spliced from a coding RIC pre-mRNA, thereby increasing the production or activity of a target protein or functional RNA in a subject.
[Item 52] A composition comprising an antisense oligomer for use in a method of treating a disease associated with the JAG1 protein in a subject in need, wherein the method causes the expression of the JAG1 protein by the subject's cells. Including the step of increasing, where the cells are
Includes a retained intron, an exon adjacent to the 5'splice site of the retained intron, and an exon adjacent to the 3'splice site of the retained intron.
It has a retained intron-containing pre-mRNA (RIC mRNA pre-mRNA) and has RIC.
The pre-mRNA encodes the JAG1 protein, which method also involves contacting the cell with an antisense oligomer, whereby the retained intron is JAG1.
Constitutively spliced from transcripts of protein-encoding RIC pre-mRNA, thereby increasing the level of JAG1 protein-encoding mRNA and increasing the expression of target protein or functional RNA in the subject's cells. ,Composition.
53. The composition of item 52, wherein the disease is a disease or disorder.
[Item 54] The disease or disorder is Alagille syndrome or muscular dystrophy, item 5.
The composition according to 3.
55. The composition of item 54, wherein the target protein and RIC mRNA precursor are encoded by the JAG1 gene.
[Item 56] In the retained intron, the antisense oligomer is added from the region +6 to the 5'splice site of the retained intron to the retained intron 3'.
The composition according to any one of items 51 to 55, which targets a portion of the RIC mRNA precursor within the region -16 relative to the splice site.
[Item 57] In the intron in which the antisense oligomer is retained,
(A) Within the region +6 to +100 relative to the 5'splice site of the retained intron; or (b) within the region -16 to -100 relative to the 3'splice site of the retained intron of the RIC mRNA precursor. The composition according to any one of items 51 to 56, which targets a part thereof.
[Item 58] 5 of introns in which the antisense oligomer is at least one retained
An RIC located within the region from about 100 nucleotides downstream of the'splice site to about 100 nucleotides upstream of the 3'splice site of at least one retained intron.
The composition according to any one of items 51 to 57, which targets a part of pre-mRNA.
[Item 59] The target portion of the RIC mRNA precursor is
(A) + in the exon adjacent to the 5'splice site of the retained intron
Within the region 2e to -4e; or (b) + in the exon adjacent to the 3'splice site of the retained intron.
The composition according to any one of items 51 to 57, which is in the region of 2e to -4e.
[Item 60] The sequence of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with respect to SEQ ID NO: 1. The composition according to any one of items 51 to 59, which is encoded by a gene sequence having sex.
[Item 61] The sequence of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with respect to SEQ ID NO: 2. The composition according to any one of items 51 to 60, which comprises a sequence having sex.
[Item 62] Items 51-61, wherein 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. The composition according to any one item.
[Item 63] The antisense oligomer functions by regulating the abnormal splicing that results from mutations in the gene encoding the target protein or functional RNA.
The composition according to any one of items 51 to 62, which does not increase the amount of NA or functional protein.
[Item 64] The composition according to any one of items 51 to 63, wherein the RIC pre-mRNA is produced from a full-length pre-mRNA or a wild-type pre-mRNA by partial splicing.
[Item 65] The mRNA encoding the target protein or functional RNA is a full-length mature mRN.
The composition according to any one of items 51 to 64, which is A or wild-type mature mRNA.
[Item 66] The composition according to any one of items 51 to 65, wherein the target protein produced is a full-length protein or a wild-type protein.
[Item 67] The composition according to any one of items 51 to 66, wherein the retained intron is a rate-determining intron.
[Item 68] The composition according to any one of items 51 to 67, wherein the retained intron is the most abundant retained intron in the RIC pre-mRNA.
[Item 69] The composition according to any one of items 51 to 67, wherein the retained intron is the second most abundant retained intron in the RIC pre-mRNA.
[Item 70] The composition according to any one of items 51 to 69, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 71] The composition according to any one of items 51 to 70, wherein the antisense oligomer is an antisense oligonucleotide.
[Item 72] Item 51-71, wherein 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. The composition according to any one item.
[Item 73] The composition according to any one of items 51 to 72, wherein the antisense oligomer contains at least one modified sugar moiety.
[Item 74] The composition according to item 73, wherein each sugar moiety is a modified sugar moiety.
[Item 75] The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, and the like.
8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 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 to 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
Item 5 consisting of ~ 25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases
The composition according to any one of 1 to 74.
[Item 76] The antisense oligomer complements the target portion of the protein-encoding RIC mRNA precursor by at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. Item 51
9. The composition according to any one of 75.
[Item 77] Any of claims 51-76, wherein the antisense oligomer binds to a target moiety of the JAG1 RIC mRNA precursor, where the target moiety is in a sequence selected from SEQ ID NO: 437-439. The composition according to item 1.
[Item 78] The antisense oligomer is any one of SEQ ID NO: 3 to 436.
On the other hand, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
The composition according to any one of items 51 to 77, which comprises a nucleotide sequence having 95%, 96%, 97%, 98%, or 99% sequence identity.
[Item 79] The composition according to any one of items 51 to 77, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 3 to 436.
[Item 80] A pharmaceutical composition comprising any of the antisense oligomers and excipients of the compositions of items 51-79.
[Item 81] A method for treating a subject in need by administering the pharmaceutical composition of item 80 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[Item 82] A pharmaceutical composition comprising an antisense oligomer that hybridizes to the target sequence of the deficient JAG1 mRNA transcript and a pharmaceutically acceptable excipient.
A pharmaceutical composition in which the deficient JAG1 mRNA transcript comprises retained introns and the antisense oligomer induces splicing out of the retained introns from the deficient JAG1 mRNA transcript.
[Item 83] The pharmaceutical composition according to item 82, wherein the deficient JAG1 mRNA transcript is a transcript of the JAG1 RIC mRNA precursor.
[Item 84] The target portion of the transcript of the JAG1 RIC pre-mRNA is from the +6 region of the retained intron to the 5'splice site to -16 to the retained intron's 3'splice site in the retained intron. Item 82, which is in the area of
Or the pharmaceutical composition according to 83.
[Item 85] The transcript of the JAG1 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 relative to SEQ ID NO: 1.
Items 82-84 encoded by a gene sequence having% or 100% sequence identity
The pharmaceutical composition according to any one of the above.
[Item 86] The transcript of the JAG1 RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 with respect to SEQ ID NO: 2.
The pharmaceutical composition according to any one of items 82 to 85, which comprises a sequence having% or 100% sequence identity.
[Item 87] The pharmaceutical composition according to any one of items 82 to 86, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[Item 88] The pharmaceutical compound according to any one of items 82 to 87, wherein the antisense oligomer is an antisense oligonucleotide.
[Item 89] Item 82-88, wherein 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. The pharmaceutical composition according to any one of the following items.
[Item 90] The pharmaceutical composition according to any one of items 82 to 89, wherein the antisense oligomer contains at least one modified sugar moiety.
[Item 91] The antisense oligomer contains 8 to 50 nucleobases, 8 to 40 nucleobases, and the like.
8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 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 to 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
Item 82, comprising from 25 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
9. The pharmaceutical composition according to any one of 90.
[Item 92] Antisense oligomers are present in the target portion of the transcript of the JAG1 RIC mRNA precursor at least 80%, at least 85%, at least 90%, at least 9.
Items 82-9 that are 5%, at least 98%, at least 99%, or 100% complementary
The pharmaceutical composition according to any one of 1.
[Item 93] The target portion of the transcript of the JAG1 RIC mRNA precursor is the SEQ ID.
NO: The pharmaceutical composition according to any one of items 82 to 92, which is in the sequence selected from 437 to 439.
[Item 94] The antisense oligomer is any one of SEQ ID NO: 3 to 436.
On the other hand, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%,
The pharmaceutical composition according to any one of items 82 to 93, which comprises a nucleotide sequence having 95%, 96%, 97%, 98%, or 99% sequence identity.
[Item 95] The pharmaceutical composition according to any one of items 82 to 93, wherein the antisense oligomer contains a nucleotide sequence selected from SEQ ID NO: 3 to 436.
[Item 96] The pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection, according to any one of items 82 to 95. The pharmaceutical composition described.
[Item 97] Fully processed JAG1 encoding the functional morphology of the tuberin protein
A method of inducing treatment of a deficient JAG1 mRNA transcript to facilitate the removal of retained introns in order to produce the mRNA transcript.
(A) Step of contacting the antisense oligomer with the target cells of the subject;
(B) In the step of hybridizing the antisense oligomer to the transcript of the deficient JAG1 mRNA, the deficient JAG1 mRNA transcript can encode the functional form of the tuberin protein and is retained at least one. Processes, including introns;
(C) Fully processed JAG1 mRN encoding functional morphology of tuberin protein
The step of removing at least one retained intron from the deficient JAG1 mRNA transcript to produce A transcript; and (d) the function of the tuberin protein from the well-treated JAG1 mRNA transcript. A method that includes the step of translating the morphology.
[Item 98] The method of item 97, wherein the retained intron is the entire retained intron.
[Item 99] The method according to item 97 or 98, wherein the deficient JAG1 mRNA transcript is a transcript of the JAG1 RIC mRNA precursor.
[Item 100] A method of treating a subject having a disease caused by a lack of amount or activity of the JAG1 protein.
SEQ ID NO: At least about 80% and 85% with respect to any one of 3 to 436.
, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, administer an antisense oligomer containing a nucleotide sequence having sequence identity to the subject. A method, including steps.

The novel features of the present 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, which describes exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings.
FIG. 1 shows a schematic representation of an exemplary retained intron-containing (RIC) pre-mRNA transcript. The 5'splice site consensus sequence is the underlined letter (letter is nucleotide, letter is nucleotide, number from -3e to -1e and +1 to +6 (numbers with "e" are exons, numbers without "e" are introns). Uppercase letters are indicated by exon parts, lowercase letters are indicated by intron parts). The 3'splice site consensus sequence is the underlined letters (letter is nucleotide, uppercase is exon) of -15 to -1 and + 1e (numbers with "e" are exons, numbers without "e" are introns). The part and lowercase letters are indicated by the intron part). Target sites for ASO screening introns range from nucleotide +6 corresponding to the 5'splice site (left arrow) of the retained intron to nucleotide-16 corresponding to the 3'splice site (right arrow) of the retained intron. Including up to. In embodiments, the target sites for ASO screening introns are nucleotides +6 to +100 corresponding to the 5'splice site of the retained intron and nucleotides -16 to -100 corresponding to the 3'splice site of the retained intron. include. Exon target sites include + 2e to -4e nucleotides of exons flanking the 5'splice site of the retained intron and + 2e to -4e nucleotides flanking the 3'splice site of the retained intron. "N" or "N" represents a nucleotide and "y" represents a pyrimidine. The sequence shown represents the consensus sequence of the mammalian splice site, and individual introns and exons do not necessarily need to match the consensus sequence at all positions. FIG. 2A shows a schematic diagram of Targeted Augmentation of Nuclear Gene Output (TANGO). FIG. 2A shows cells divided into nuclei and cytoplasmic compartments. In the nucleus, transcripts of pre-mRNA of the target gene, 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. .. For this target gene, splicing of intron 1 is inefficient, and retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus and, when transported to the cytoplasm, does not lead to target protein production. to degrade. 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 translates into higher levels of target protein. FIG. 2B shows a schematic diagram of Targeted Augmentation of Nuclear Gene Output (TANGO). FIG. 2A shows cells divided into nuclei and cytoplasmic compartments. In the nucleus, transcripts of pre-mRNA of the target gene, 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. .. For this target gene, splicing of intron 1 is inefficient, and retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus and, when transported to the cytoplasm, does not lead to target protein production. to degrade. 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 translates into higher levels of target protein. FIG. 3 details intron retention in the JAG1 gene with intron 13. Identification of intron retention events in the JAG1 gene using RNA sequencing (RNAseq) is visualized and demonstrated in the UCSC Genome Browser. The top panel shows the read density corresponding to the JAG1 transcript expressed in THLE-3 (human liver epithelial) cells and localized in either the cytoplasm (top) or the nuclear fraction (bottom). At the bottom of this panel, a graphical representation of the JAG1 gene is shown on a scale. The reading density is shown as a peak. The highest reading density corresponds to an exon (black box), while no reading is 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 13, 18, and 23 of the nuclear fraction (indicated by arrows), which resulted in lower splicing efficiency of introns 13, 18, and 23. It has been shown to bring about intron retention. The retained intron-containing pre-mRNA transcript accumulates primarily in the nucleus and is not translated into the JAG1 protein. The reading density for intron 13 of THLE-3 cells is shown in detail in the lower panel showing 12% intron retention as calculated by bioinformatics analysis. FIG. 4 shows the JAG1 gene IVS 13 ASO walk. A graphical representation of the ASO walk performed on JAG1 IVS 13 targeting sequences immediately downstream of the 5'splice site or upstream of the 3'splice site using the 2'-OMe ASO, PS backbone. Shown. ASO was designed to cover these regions by repositioning 5 nucleotides at a time. The JAG1 exon-intron structure is shown on the scale. FIG. 5 shows a JAG1 intron 13 ASO walk evaluated by radioactive RT-PCR. At the top, a schematic (not scaled) of exon 13-intron 13-exon 14 shows the primers Formula1 (F1) and Revase1 (R1) used in the RT-PCR assay. In the central panel, a representative gel targets JAG1 sham-treated (neg, RNAiMAX only), ASO-treated SMN-control, or intron 13 at a concentration of 60 nM in ARPE-19 cells 2'-. The radioactive RT-PCR products of SMN-control, treated with O-Me ASO are shown. In the lower panel, the quantification of the band corresponding to the beta-actin-normalized JAG1 radioactive RT-PCR product from two independent experiments is plotted on a bar graph as a multiple change relative to the sham-treated product. A black line labeled 1 on the Y-axis indicates a ratio of 1 (no change). FIG. 6 shows the JAG1 intron 13 ASO walk evaluated by RT-qPCR. In the upper left, a schematic of the RT-qPCR assay shows the primer recognition site. RT-qPCR amplification results obtained using the same ASO transfection experiment evaluated by radioactive RT-PCR as shown in FIG. 3 were normalized to β-actin (upper bar graph) or RPL32 (lower bar graph). Plot against the sham-treated product to confirm the results of radioactive RT-PCR. A black line labeled 1 on the Y-axis indicates a ratio of 1 (no change). FIG. 7 details intron retention in the JAG1 gene with intron 18. Intron retention in the JAG1 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC Genome Browser, as described herein in the Examples. The reading density for intron 18 of THLE-3 cells is shown in detail in the lower panel, showing 14% intron retention as calculated by bioinformatics analysis. FIG. 8 shows the JAG1 gene IVS 18 ASO walk. An ASO walk performed on JAG1 IVS 18 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 JAG1 exon-intron structure is shown on the scale. FIG. 9 shows the JAG1 intron 18 ASO walk evaluated by radioactive RT-PCR. At the top, schematics (not to scale) of exons 18 to 20 show the primers Formula 1 (F1) and Revase 1 (R1) used in the RT-PCR assay. In the central panel, a representative gel at a concentration of 60 nM in ARPE-19 cells, JAG1 sham treatment (neg, RNAiMAX only), as described herein in the examples and in the description of FIG. The radioactive RT-PCR products of ASO-treated SMN-control or SMN-control treated with 2'-O-Me ASO targeting intron 18 are shown. The quantification of the band corresponding to the beta-actin-normalized JAG1 radioactive RT-PCR product from two independent experiments is plotted in the bar graph below as a multiple change relative to the sham-treated product. A black line labeled 1 on the Y-axis indicates a ratio of 1 (no change). FIG. 10 shows a JAG1 intron 18 ASO walk evaluated by RT-qPCR. In the upper left, a schematic of the RT-qPCR assay shows the primer recognition site. RT-qPCR amplification results obtained using the same ASO transfection experiment evaluated by radioactive RT-PCR as shown in FIG. 7 were normalized to β-actin (upper bar graph) or RPL32 (lower bar graph). Plot against the sham-treated product to confirm the results of radioactive RT-PCR. A black line labeled 1 on the Y-axis indicates a ratio of 1 (no change). FIG. 11 details intron retention in the JAG1 gene with intron 23. Intron retention in the JAG1 gene was identified by RNA sequencing (RNAseq) and visualized in the UCSC Genome Browser, as described herein in the Examples and in the description of FIG. The reading density for intron 23 of THLE-3 cells is shown in detail in the lower panel, showing 17% intron retention as calculated by bioinformatics analysis. FIG. 12 shows the JAG1 gene IVS 23 ASO walk. An ASO walk performed on JAG1 IVS 23 targeting sequences immediately downstream of the 5'splice site or upstream of the 3'splice site using the 2'-OMe ASO, PS backbone is illustrated. ASO was designed to cover these regions by repositioning 5 nucleotides at a time. The JAG1 exon-intron structure is shown on the scale.

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 process in gene expression that precedes translation in the cytoplasm.

A significant level of partially spliced transcript of the JAG1 gene, which encodes the deficient JAG1-protein in the debilitating hereditary disease Alagille syndrome, is in the nucleus of human cells. It has been discovered. These JAG1mR
NA precursor seeds include at least one retained intron. The present invention increases steady-state production of fully spliced mature mRNA and is therefore translated JAG.
1-Be rate-determining at the nuclear stage of gene expression to increase protein levels,
To provide compositions and methods for upregulating the splicing of one or more retained JAG1 introns. These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at the intron splice sites of retained intron-containing JAG1 mRNA precursors that accumulate in the nucleus. Therefore, in the embodiment, JA is used in order to treat a disease caused by a deficiency of JAG1 by using the method of the present invention.
Increases G1-protein.

In other embodiments, the methods of the invention are used to increase JAG1-production to treat the symptoms of a subject in need of it. In a plurality of embodiments, the subject is JA
Although G1 is not necessarily deficient for the wild type, an increase in JAG1 has the symptom of alleviating the disease. In some embodiments, the subject suffers from muscular dystrophy. In a plurality of embodiments, the subject is dystrophin deficient (dystrophin d).
I am suffering from efficiency). For example, Vieira et al. , 2015,
“Jugged 1 Rescues the Duchenne Muscular
Dystrophy Phenotype ”, Cell 163: 1204-1213
Describes findings suggesting that increased JAG1 expression in muscle can rescue the dystrophin-deficient phenotype in two different animal models of the disease.

<Alagille Syndrome>
Alagille syndrome (ALGS), also called arterial hepatic dysplasia, is an autosomal dominant multilineage disorder (Turnpenny and Ellard, 2012). The main clinical and pathological findings are liver disease, heart disease, vascular abnormalities, skeletal abnormalities, eye features, facial features, kidney abnormalities, growth retardation, and pancreatic insufficiency due to intrahepatic bile duct deficiency. be. The clinical findings of ALGS are based on the presence of three of the five clinical features, including liver, heart, spine, eye, or facial abnormalities, with bile duct deficiency. May fluctuate with physical diagnosis (Lu et al., 2003, 72, 1065-1070; Pe
ton et al. , Seminars Cell & Dev. Biol. 2012,
23, 450-457). The reported prevalence of 1: 70,000 ALGS is believed to be underestimated due to variability in its condition and reduced penetration.

Mutations in genes related to Notch signaling have been reported to cause ALGS. JAG1 encodes the JAG1 protein, a cell surface ligand for the Notch transmembrane receptor. The sequence of the human genome of the JAG1 gene is, for example, Jurkievic.
z, D. , Et al. , 2014, "Spectrum of JAG1 g
ene mutations in Polish patients with Al
agile syndrome, "J. Appl. Genetics 55: 3
29-336, and the amino acid sequence at Uni
ProtKB: P7854-1, Oda, T. et al. , Et al. , 1997,
"Identification and cloning of the human"
homolog (JAG1) of the rat Jugged1 gene
from the Alagille syndrome critical regi
on at 20p12, "Genomics 43 (3): disclosed in NCBI Gene ID 182 described by 376-379, all of which are incorporated herein by reference. The canonical mRNA sequence of JAG1 is the NCBI reference sequence: Duryag
ina R, et al. , 2013, “Overexpression of
Jugged-1 and it's intracellular domain in
human mesenchymal stromal cells diffuse
initially affect the interaction with hema
topoietic stone and progenitor cells, "St
em Cells Dev. 22 (20), NM described in 2736-2750.
_000214.2, both of which are incorporated herein by reference.
Binding of the JAG1 ligand to Notch induces a signal cascade that results in transcription of genes involved in cell fate determination and differentiation. Mutations in JAG1 cause ALGS1, the most prevalent disease type, while mutations in NOTCH2 cause ALGS2.

The JAG1 gene consists of 26 exons and is located on chromosome 20p11.2-20p12. The JAG1 mutation in ALGS is spread across all proteins and is approximately 60
It has a high de novo mutation rate of%. Eighty percent of mutations include frameshift mutations, nonsense mutations, and splice site mutations; total gene deletions account for 7% of mutations, and missense mutations correspond to 12% (Lu et al). , 2003). In most cases, haploinsufficiency is a mechanism that is likely to be the cause of the disease, as global gene deletion results in phenotypes similar to those found in truncating mutations and missense mutations. There is (Lu et al., 2003)
Turnpenny and Ellard, 2012).

Mutations associated with the classical ALGS phenotype, including liver disease, represented functional haploinsufficiency, resulting in a cell surface concentration of JAG1 of 50% of wild-type levels (Lu et a).
l. , 2003). In contrast, JAG1 missense mutations resulting in expression of the JAG1-G274D protein have been reported to produce two protein populations, one population exhibiting glycosylation deficiency and thus impeding transport to the cell surface. .. JAG1-G
The 274D mutation was associated with a heart-specific phenotype in the absence of liver disease, with mutation carriers greater than 50% but less than 100% of the normal concentration of JAG1 on the cell surface. Therefore, the developing liver may require less JAG1 than the developing heart (Lute).
al. , 2003).

<Muscle repair>
As mentioned above, the possibility that JAG1 rescues the dystrophin deficiency phenotype in two animal models has been described (Vieira, et al., 2015). A deficiency of muscular dystrophin in muscular dystrophy, including Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), causes progressive muscle degeneration and atrophy. In DMD, respiratory failure or heart failure eventually leads to death. In these diseases J
Although AG1 is not deficient, increased levels of JAG1 can stimulate muscle cell proliferation and repair. In multiple embodiments, the methods and compositions of the invention are used to increase JAG1 production to stimulate muscle repair in the subject in need thereof. In some embodiments, the subject suffers from muscular dystrophy. In a plurality of embodiments, the muscular dystrophy is DMD, BMD, or limb band, congenital, facioscapulohumeral, muscle tone, oropharyngeal muscle, distal,
Or Emery-Dreifs type muscular dystrophy. In some embodiments, the subject suffers from dystrophin deficiency. In embodiments, the dystrophin deficiency is DMD or BMD. In a plurality of embodiments, the subject is dysferin (encoded by the DYSF gene), emerin (encoded by the EMD gene), DUX4, (DMP).
Myotonic dystrophy protein kinase (MDPK) (encoded by the K gene)
) Or myotonin protein kinase (MT-PK), also known as dystrophia myotonica protein kinase (DMK), cellular nucleic acid-binding protein (encoded by the CNBP gene), or polyadenylation-binding nuclear protein (encoded by the PABPN1 gene). Polyadenylation binding protein 2 (PABPN1)
It has muscular dystrophy caused by a deficiency in PANP-2).

<Retained intron-containing mRNA precursor (RIC mRNA precursor)>
In multiple embodiments, the method presented herein is a retained intron-containing mRNA that is transcribed from the JAG1 gene and encodes the JAG1-protein in the cell nucleus.
Take advantage of the presence of precursors (RIC mRNA precursors). Splicing of specific JAG1 RIC mRNA precursor seeds to produce mature, fully spliced JAG1 mRNA is induced using ASOs that stimulate the splicing out of retained introns. The resulting mature JAG1 mRNA is transported to the cytoplasm and translated, thereby increasing the amount of JAG1-protein in the subject's cells and alleviating the symptoms of Alagille syndrome. This method, described in more detail below, is known as increased targeting of nuclear gene output (TANGO).

Transcript of JAG1 nucleus As described in the examples herein, the JAG1 gene was analyzed for intron retention events and retention of introns 13, 18 and 23 was observed. RNA sequencing (RNAseq) visualized by the UCSC Genome Browser showed JAG1 transcripts expressed on THLE-3 (human hepatic epithelial) cells and was localized to either the cytoplasm or the nuclear fraction.
No readings were observed in the majority of introns in either fraction. However, the higher read densities compared to the cytoplasmic fraction are the introns 13 and 18 of the nuclear fraction.
Detected at and 23, this indicates that the splicing efficiencies of introns 13, 18 and 23 are low, resulting in intron retention. The retained intron-containing pre-mRNA transcript accumulates primarily in the nucleus and is not translated into the JAG1 protein. The reading densities for introns 13, 18 and 23 were 12%, respectively.
It showed 14% and 17% intron retention.

Table 1 shows the target sequences of the JAG1 RIC mRNA precursor transcript by sequence ID and the ASO by sequence ID, which are useful for increasing the production of the JAG1 protein by targeting the region of the JAG1 RIC mRNA precursor. , Provides a non-limiting list. In multiple embodiments, other ASOs useful for this purpose are identified, for example, using the methods described herein.

In some embodiments, the ASOs disclosed herein target RIC mRNA precursors transcribed from the JAG1 genomic sequence. In some embodiments, the ASO targets a transcript of the RIC pre-mRNA from the JAG1 genomic sequence containing the retained intron. In some embodiments, the ASO is a RIC with SEQ ID NO: 1.
Target transcripts of pre-mRNA. In some embodiments, the ASO targets a transcript of a SEQ ID NO: 1 RIC pre-mRNA containing a retained intron. In some embodiments, the ASO disclosed herein is the RIC of JAG1.
Target sequences of pre-mRNA. In some embodiments, the ASO is 13, 18,
Targeting transcripts of JAG1's RIC mRNA precursor, including introns retained at 23 or a combination thereof, where introns are numbered NM_000214.
Consistent with the mRNA sequence in.

In some embodiments, the ASO is the R of JAG1 represented by SEQ ID NO: 2.
Target the sequence of IC pre-mRNA. In some embodiments, the ASO sequences the RIC mRNA precursor of JAG1 according to SEQ ID NO: 2, which comprises retained intron 13, retained intron 18, retained intron 23, or a combination thereof. Target. In some embodiments, the ASO disclosed herein is
Targets SEQ ID NOs: 437-439. In some embodiments, ASO
Has a sequence according to any one of SEQ ID NO: 3-436.

In some embodiments, the ASO is the RI of JAG1 containing the retained intron 18.
Targeting the C mRNA precursor exon 18 or exon 19, the intron numbering is consistent with the mRNA sequence in NM_000214. In some embodiments, the ASO targets an exon 18 sequence upstream (or 5') of the 5'splice site of the JAG1 RIC pre-mRNA containing retained intron 18.
In some embodiments, the ASO is a JAG1 RIC containing a retained intron 18.
Target the exon 18 sequence about 4 to about 99 nucleotides upstream (or 5') of the 5'splice site of the pre-mRNA. In some embodiments, the ASO targets the exon 19 sequence downstream (or 3') of the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 18. In some embodiments, A
SO targets the exon 19 sequence about 2 to about 7 nucleotides downstream (or 3') from the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 18.

In some embodiments, the ASO is the RI of the JAG1 containing the retained intron 18.
Targeting intron 18 in the C mRNA precursor, where intron numbering corresponds to the mRNA sequence in NM_000214. In some embodiments, ASO
Targets the intron 18 sequence downstream (or 3') of the 5'splice site of the JAG1 RIC pre-mRNA containing retained intron 18. In some embodiments, the ASO targets the intron 18 sequence approximately 16 to about 239 nucleotides downstream (or 3') from the 5'splice site of the JAG1 RIC pre-mRNA containing retained intron 18. do. In some embodiments, the ASO targets the intron 18 sequence upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA containing retained intron 18. In some embodiments, the ASO targets the intron 18 sequence approximately 16 to about 253 nucleotides upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA containing retained intron 18. do.

In some embodiments, the ASO is the RI of the JAG1 containing the retained intron 13.
Targeting the C mRNA precursor exon 13 or exon 14, the intron numbering is consistent with the mRNA sequence in NM_000214. In some embodiments, the ASO targets the exon 13 sequence upstream (or 5') of the 5'splice site of the JAG1 RIC pre-mRNA containing the retained intron 13.
In some embodiments, the ASO is a JAG1 RIC containing a retained intron 13.
Target the exon 13 sequence about 14 to about 131 nucleotides upstream (or 5') of the 5'splice site of the pre-mRNA. In some embodiments, the ASO targets the exon 14 sequence downstream (or 3') of the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 13. In some embodiments,
The ASO is an exon 14 about 2 to about 147 nucleotides downstream (or 3') from the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 13.
Target the sequence.

In some embodiments, the ASO is the RI of the JAG1 containing the retained intron 13.
Targeting intron 13 in the C mRNA precursor, where intron numbering corresponds to the mRNA sequence in NM_000214. In some embodiments, ASO
Targets the intron 13 sequence downstream (or 3') of the 5'splice site of the JAG1 RIC pre-mRNA containing the retained intron 13. In some embodiments, the ASO targets the intron 13 sequence approximately 16 to about 440 nucleotides downstream (or 3') from the 5'splice site of the JAG1 RIC pre-mRNA containing retained intron 13. do. In some embodiments, the ASO targets the intron 13 sequence upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 13. In some embodiments, the ASO targets the intron 13 sequence approximately 16 to approximately 441 nucleotides upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA containing retained intron 13. do.

In some embodiments, the ASO is the RI of the JAG1 containing the retained intron 23.
Targeting the C mRNA precursor exon 23 or exon 24, the intron numbering is consistent with the mRNA sequence in NM_000214. In some embodiments, the ASO targets the exon 23 sequence upstream (or 5') of the 5'splice site of the JAG1 RIC pre-mRNA containing the retained intron 23.
In some embodiments, the ASO is a JAG1 RIC containing a retained intron 23.
Target the exon 23 sequence about 4 to about 216 nucleotides upstream (or 5') of the 5'splice site of the pre-mRNA. In some embodiments, the ASO targets the exon 24 sequence downstream (or 3') of the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 23. In some embodiments,
The ASO is an exon 24, about 2 to about 114 nucleotides downstream (or 3') from the 3'splice site of the JAG1 RIC pre-mRNA containing the retained intron 23.
Target the sequence.

In some embodiments, the ASO is the RI of the JAG1 containing the retained intron 23.
Targeting the intron 23 in the C mRNA precursor, where the intron numbering corresponds to the mRNA sequence in NM_000214. In some embodiments, ASO
Targets the intron 23 sequence downstream (or 3') of the 5'splice site of the JAG1 RIC pre-mRNA containing the retained intron 23. In some embodiments, the ASO targets the intron 23 sequence approximately 6 to about 121 nucleotides downstream (or 3') of the 5'splice site of the JAG1 RIC pre-mRNA containing retained intron 23. do. In some embodiments, the ASO targets the intron 23 sequence upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA, including the retained intron 23. In some embodiments, the ASO targets the intron 23 sequence approximately 16 to approximately 121 nucleotides upstream (or 5') of the 3'splice site of the JAG1 RIC pre-mRNA containing retained intron 23. do.

It is understood that intron numbering can vary with respect to different JAG1 isoform sequences. Those skilled in the art will appreciate the intron sequences provided herein, or NM_0.
By using the intron number obtained for the mRNA sequence in 00214.2, the number of the corresponding intron in any JAG1 isoform can be determined. Those skilled in the art will further appreciate the intron sequences provided herein, or NM_0.
By using the intron number obtained for the mRNA sequence at 00214.2, the method of the invention can be used to determine the sequence of adjacent exons in any JAG1 isoform for targeting. In multiple embodiments, the compositions and methods of the invention are used to increase the expression of any known JAG1 isoform.

JAG1 protein expression As mentioned above, the JAG1 mutation in ALGS extends to all proteins and has a high de novo mutation rate of approximately 60%. Of the characteristic mutations, 80% include frameshift mutations, nonsense mutations, and splice site mutations; total gene deletions account for 7% of mutations, and missense mutations correspond to 12%. (Lu et)
al. , 2003).

In multiple embodiments, the methods described herein are used to increase the production of the functional JAG1 protein. As used herein, "functional"
The term refers to the amount of activity or function of the JAG1 protein required to eliminate one or more symptoms of the disease being treated, such as Alagille syndrome or muscular dystrophy. In embodiments, the method is used to increase the production of partially functional JAG1 protein. As used herein, the term "partially functional" refers to any amount of activity or function of the JAG1 protein required to eliminate or prevent one or more symptoms of a disease or disease. .. In some embodiments, the partially functional protein or RNA is at least 10%, at least 20%, at least 30%, at least 4
0%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less active compared to a fully functional protein or RNA Will have.

In multiple embodiments, the method is a RIC mRNA encoding a JAG1-protein.
A method of increasing the expression of JAG1-protein by the cells of a subject having a precursor, wherein the subject has Alagille syndrome due to a lack of activity of the JAG1 protein and the amount of the JAG1 protein. Deficiency is caused by haploinsufficiency of JAG1-protein. In such an embodiment, the subject has a first allele encoding a functional JAG1 protein and a second allele that does not produce the JAG1 protein. In another such embodiment, the subject has a first allele encoding a functional JAG1 protein and a second allele encoding a non-functional JAG1 protein. In another such embodiment, the subject has a first allele encoding a functional JAG1 protein and a second allele encoding a partially functional JAG1 protein. In any of these embodiments, the antisense oligomer binds to the target site of the RIC mRNA precursor transcribed from the first allelic gene (encoding the functional JAG1 protein), thereby RIC mRNA precursor. It induces constitutive splicing of retained introns from the body and results in an increase in the amount of mature mRNA encoding the functional JAG1 protein and an increase in JAG1 protein expression in the cells of the subject.

In an embodiment, the subject has a first allelic gene encoding a functional JAG1 protein and a second allelic gene encoding a partially functional JAG1 protein, the first allelic gene (functional JAG1 protein). Antisense oligomers are located on the target portion of the RIC mRNA precursor transcribed from) or on the target portion of the RIC mRNA precursor transcribed from the second allelic gene (encoding the partially functional JAG1 protein). It binds, thereby inducing constitutive splicing of retained introns from the RIC mRNA precursor, increasing the level of mature mRNA encoding the JAG1 protein and functional or partially functional JAG1 in the subject's cells. It results in increased protein expression.

In a related embodiment, the method is a method that uses ASO to increase expression of protein or functional RNA. In embodiments, ASO is used to increase JAG1 protein expression in the cells of a subject having a RIC pre-mRNA encoding the JAG1 protein, wherein the subject lacks the amount or function of the JAG1 protein, eg. Have Alagille syndrome.

In embodiments, a protein-encoding RIC mRN that causes the disease or disease.
Transcripts of A precursor are targeted by the ASOs described herein. In some embodiments, the transcript of the protein-encoding RIC pre-mRNA, which is not the cause of the disease, is targeted by ASO. For example, a disease resulting from a mutation or deficiency of the first protein in a particular pathway is an RI encoding a second protein.
It can be improved by targeting the C-mRNA precursor, which increases the production of the second protein. 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 first protein is the protein deficient in muscular dystrophy and the second protein is JAG1. In an embodiment, the first protein is dystrophin and the second protein is JAG1.

In the embodiment, the subject is

a.
i) The JAG1 protein is produced at reduced levels compared to production from wild-type alleles.
ii) A first mutant allele in which the JAG1 protein is produced in a reduced function compared to an equivalent wild-type protein, or ii) the JAG1 protein or functional RNA is not produced; and b.
i) The JAG1 protein is produced at reduced levels compared to production from wild-type alleles.
ii) JAG1 protein is produced in a less functional form compared to an equivalent wild-type protein, or ii) JAG1 protein is not produced, with a second mutation allele.
Here, the RIC pre-mRNA is transcribed from the first and / or second allele. In these embodiments, the ASO binds to the target portion of the RIC pre-mRNA transcribed from the first or second allele to RIC.
Induces constitutive splicing of retained introns from pre-mRNA and JAG1
It causes an increase in the level of mRNA encoding the protein and 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 equivalent wild-type proteins. It can be either a low form (partially functional) or a form that has full functionality compared to an equivalent wild-type protein (fully functional).

In embodiments, levels of mRNA encoding the JAG1 protein were produced in control cells, eg, treated with antisense oligomers that were not treated with antisense oligomers or that did not bind to the target portion of the JAG1 RIC mRNA precursor. JAG1
Compared to the amount of mRNA encoding, it increases 1.1 to 10-fold.

In embodiments, the disease resulting from a lack of JAG1 protein amount or activity is ASO.
Is not a disease resulting from alternative or abnormal splicing of retained introns targeted. In embodiments, the disease resulting from a lack of amount or activity of the JAG1 protein is not a disease resulting from alternative splicing or abnormal splicing of retained introns in the RIC mRNA precursor encoding the JAG1 protein. In embodiments, alternative or aberrant splicing can occur in pre-mRNA transcribed from the gene. However, the compositions and methods of the present invention do not prevent or modify this alternative or abnormal splicing.

In embodiments, subjects treated using the methods of the invention express a partially functional JAG1 protein from one allele, which partial functional JAG1 protein is a frameshift mutation, a nonsense mutation, It is due to a missense mutation or a partial gene deletion. In embodiments, subjects treated using the methods of the invention express a non-functional JAG1 protein from one allele, which non-functional JAG1 protein is a frameshift mutation, nonsense in one allele. Due to mutations, missense mutations, or partial gene deletions. In an embodiment, a subject treated using the method of the invention is 1
One allele has a JAG1 total gene deletion.

In the embodiment, the subject is G274D, L37S, R184H, P163L, P871.
It has a JAG1 missense mutation selected from R, C234Y, C664S, P810L, or R937Q. In an embodiment, the subject is a JAG1 deletion mutation 1485-148.
It has 6 del CT. In an embodiment, the subject is a JAG1 duplicate mutation 414-415d.
Has upGT. In embodiments, Lu, et al., Known in the art and, eg, referenced above. , 2003, Penton, et al. , 2012. Subjects with any JAG1 mutation described in the literature are treated with the methods and compositions of the present invention.

Use of TANGO to Increase JAG1 Protein Expression As mentioned above, in embodiments, targeted enhancement of nuclear gene output (TANGO) is used in the methods of the invention to increase JAG1 protein expression. used. In these embodiments, a retained intron-containing mRN encoding the JAG1 protein
The A precursor (RIC mRNA precursor) is in the nucleus of the cell. Cells having a JAG1 RIC pre-mRNA containing a retained intron encoding a PC-2 protein, an exon flanking the 5'splice site, and an exon flanking the 3'splice site are targeted portions of the RIC mRNA precursor. Complementary to antisense oligomers (AS)
Contact with O). 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 that contains 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, pre-mRNA or mRNA
Precursor transcripts contain 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, "retained intron-containing pre-mRNA" (
"RIC mRNA precursor") is an mRN containing at least one retained intron.
It is a transcript of A precursor. The RIC pre-mRNA is the retained intron, the exon adjacent to the 5'splice site of the retained intron, and the 3'of the retained intron.
It contains an exon adjacent to the splice site and encodes a 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 the population of RIC pre-mRNA transcribed from genes encoding target proteins in cells.
The pre-mRNA population contains two or more retained introns. In embodiments, the most abundant intron-targeted antisense oligomers in the population of RIC mRNA precursors encoding the target protein are within the population containing retained introns to which the antisense oligomer is targeted or bound. Induces splicing out of two or more retained introns. In embodiments, a mature mR encoding a target protein
NA 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. As used interchangeably herein, as described in the processed functional RNA. 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 retained intron, which is the most abundant intron in the population of RIC pre-mRNA encoding the JAG1 protein. In embodiments, the RIC encoding the JAG1 protein
The most retained intron in the pre-mRNA population is intron 23.

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%, At least about 45%, or at least about 5
An intron identified as a retained intron based on a retention retention rate of 0%. In embodiments, retained introns are from about 5% to about 100% and from 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% to about 25
%, About 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, About 10% to about 80%, about 1
0% 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 1
00%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15
% To about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%
, About 15% to about 60%, about 15% to 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 1
5% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 10
0%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20%
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% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20 % To about 40%, about 20
% To about 35%, about 20% to 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 25% to about 5
An intron identified as a retained intron based on retention retention rates of 0%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%. ENCODE data (eg, Tilgn) to help identify retained introns
er, et al. , 2012, "Deep Sequencing of sub
llular RNA fractions shows splicing to b
e predominantly co-transcriptional in th
e human genome but ineffect for lcRN
As ”Genome Research 22 (9): described in 1616-25) can be used.

As used herein, the term "comprise", or "
Variants such as "comprises" or "comprising"
It should be understood to indicate that it contains the listed features (eg, in the case of antisense oligomers, the defined nucleobase sequence) but 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" essentially consists of "consisting essen."
It can be replaced with "tally" or "consisting of". The phrase "consisting essentially" requires a feature that does not substantially affect the properties or functions of the invention as claimed, as well as the identified features (eg, nucleic acid sequences). 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, from the nucleobase sequence identified). In the case of antisense oligomers consisting of, 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 population of RIC pre-mRNA encoding the JAG1 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 particular nucleotide sequence, and /
Or an increase in protein production resulting from targeting introns by ASO. In embodiments, the retained intron is the second most abundant intron in the RIC mRNA precursor population transcribed from the gene encoding the intracellular target protein, and the RIC mRNA precursor population is two or more. Includes retained introns. In an embodiment, the antisense oligomer that targets the second most abundant intron in the RIC mRNA precursor population encoding the target protein is in a population that includes retained introns that the antisense oligomer targets or binds to. 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 an embodiment, the second most abundant intron in the RIC pre-mRNA precursor population encoding the JAG1 protein is intron 18. In an embodiment, the second most abundant intron in the RIC pre-mRNA precursor population encoding the JAG1 protein is intron 13.

In embodiments, the ASO is complementary to a target region within an intron in which the RIC mRNA precursor 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 site for the RIC mRNA precursor is within a region of +100 relative to the 5'splice site of the unretained intron to a region of -100 relative 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 embodiments, the retained intron of the RIC pre-mRNA is an inefficiently spliced intron. 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 (splicing site adjacent to the retained intron). It can be pointed out 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 velocity or kinetics of splicing at the splice 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 + 1e 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 sequences" are NCs of biological and scientific information.
BI repository (National Center for Biotechnology)
y Information, National Library of Medical
ine, 8600 Rockville Pike, Bethesda, MD USA
Refers to the nucleotide sequence for the JAG1 gene in the published reference genome deposited (operated by 20894). As used herein, "wild-type sequence" refers to the standard sequence obtained with NCBI Gene ID 182. Further, as used herein, the nucleotide position indicated by "e" is that the nucleotide is exon (eg, for example.
It is shown to be present in the sequence of exons adjacent to the 5'splice site or exons adjacent to the 3'splice site).

The method comprises contacting the cells with ASO, which is complementary to a portion of the pre-mRNA encoding the JAG1 protein, resulting in increased expression of JAG1. As used herein, "contacting" or administering to 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 a disease or disease-related or disease-related cell 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 the embodiment, the cells expressing the JAG1 RIC mRNA precursor are the JAG1 RIC.
The step of contacting ASO, which is complementary to the target portion of the transcript of the pre-mRNA, results in a measurable increase in the amount of JAG1 protein (eg, target protein) encoded by the pre-mRNA. How to measure or detect protein production
All known methods will be apparent to those of skill in the art and include, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

In embodiments, the step of contacting cells with ASO, which is complementary to the target portion of the transcript of the JAG1 RIC mRNA precursor, results in the amount of protein produced by the cells in the absence of ASO / treatment. By comparison, at least 10, 20, 30, 40
, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 45
It results in an increase in the amount of JAG1 protein produced, by 0, 500, or 1000%. In the embodiment, JA produced by cells contacted with antisense oligomers.
The total amount of G1 protein is 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 target protein produced by 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 to 7 times, 1.1 to 8 times, 1.1 to 9 times, 2 to 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 About 8 times to about 8 times, about 3 times to about 9 times, about 4 times to about 7 times, 4 times to about 8 times, about 4 times 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,
It increases at least about 4 times, at least about 5 times, or at least about 10 times. The control compound can be, for example, an oligonucleotide that is not complementary to the target portion of the RIC mRNA precursor.

In some embodiments, the step of contacting the cell with ASO, which is complementary to the target portion of the transcript of the JAG1 RIC mRNA precursor, results in a mRNA encoding JAG1 comprising a mature mRNA encoding the target protein. Brings an increase in quantity. In some embodiments, the amount of mRNA encoding the JAG1 protein, or mature mRNA encoding the JAG1 protein, is at least 10 compared to the amount of protein produced by the cells in the absence of ASO / treatment. , 20, 30, 40, 50, 60, 80,
100, 150, 200, 250, 300, 350, 400, 450, 500, or 1
Increase by 000%. In embodiments, the total amount of RNA encoding the JAG1 protein produced in cells contacted with the antisense oligomer, or mature mRNA encoding the JAG1 protein, is determined by untreated cells, such as untreated cells or Approximately 1. Compared to the amount of mature RNA produced intracellularly treated with the control compound.
1 to 10 times, 1.5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 1.1 to 5 times , About 1.1 times to 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 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 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times Double. The control compound is
For example, it can be an oligonucleotide that is not complementary to the target portion of the JAG1 RIC mRNA precursor.

Constitutive splicing of retained introns from RIC pre-mRNA The methods and antisense oligonucleotide compositions provided herein are JAG1.
Protein-encoding mRNA, or JAG1 protein-encoding mature mRNA
By increasing the level of JAG1 protein, for example, it is useful to increase the expression of JAG1 protein in cells in a subject having Alagille syndrome caused by a lack of amount or activity of JAG1 protein. In particular, methods and compositions as described herein induce constitutive splicing of retained introns from transcripts of the JAG1 RIC mRNA precursor encoding the JAG1 protein, thereby producing the JAG1 protein. The level of the mRNA encoding or the mature mRNA encoding the JAG1 protein is increased, and the expression of the JAG1 protein is increased.

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

In embodiments, constitutive splicing makes the retained intron JAG1 RIC.
Correctly removed from the pre-mRNA, where the JAG1 RIC pre-mRNA is transcribed from a wild-type gene or allelic gene, or a polymorphic gene or allelic gene to produce a fully functional target protein or functional RNA. The coding gene or allele is free of mutations that cause selective splicing or aberrant splicing of retained introns.

In some embodiments, the JAG1 RIC mRNA encoding the JAG1 protein
Constitutive splicing of retained introns from the precursor correctly removes retained introns from the JAG1 RIC mRNA precursor encoding the JAG1 protein, where the JAG1 RIC mRNA precursor is from the wild-type allelic gene. Transcribed from a gene or allelic gene that produces a target gene or functional RNA at a reduced level compared to production, and the gene or allelic gene has mutations that cause selective or aberrant splicing of retained introns. Do not. In these embodiments, the correct removal of constitutively spliced retained introns results in the correct removal.
It 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 makes the retained intron JAG1 RI.
Correctly removed from the C pre-mRNA, where the JAG1 RIC pre-mRNA encodes a target protein or functional RNA produced in a reduced form as compared to an equivalent wild-type protein or functional RNA. Transcribed from a gene or allele
The gene or allele is free of mutations that cause alternative or alternative splicing of retained introns. In these embodiments, the correct removal of constitutively spliced retained introns results in partial functional target proteins, or equivalent wild-type proteins or functional RNAs when compared. It results in the production of functional RNA that is functional.

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

In embodiments, antisense oligomers as described herein or used in the methods described herein regulate alternative splicing or abnormal splicing of pre-mRNA transcribed from the JAG1 gene. By doing so, the amount of mRNA encoding the JAG1 protein, or the amount of the JAG1 protein is not increased. The regulation of alternative splicing or abnormal splicing uses known methods of analyzing the sequence and length of RNA species, eg, by RT-PCR, and using the methods described separately herein and in the literature. Can be measured. In embodiments, the regulation of alternative splicing or abnormal splicing is determined based on an increase or decrease of at least 10% or 1.1 fold of the amount of spliced species of interest. In embodiments, the regulation is at least 10% to 100% or 1.1 to 10 fold levels, as described herein with respect to determining the increase in mRNA encoding the JAG1 protein in the methods of the invention. Judgment is based on the increase or decrease of.

In an embodiment, the method is such that the JAG1 RIC mRNA precursor is wild-type JAG1 m.
It is a method produced by partial splicing of pre-RNA. In an embodiment, the method is one in which the JAG1 RIC pre-mRNA is produced by partial splicing of the full-length wild-type JAG1 pre-mRNA. In the embodiment, JAG1 RI
The C pre-mRNA was produced by partial splicing of the full-length JAG1 pre-mRNA. In these embodiments, the full-length JAG1 pre-mRNA at the splicing site of the retained intron does not compromise 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 JAG1 protein is a full-length mature mRNA or a wild-type mature mRNA. In these embodiments, the full-length mature mRNA has 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 JAG1 protein encoded by the wild-type mature mRNA. Can be done.

<Antisense oligomer>
One aspect of the disclosure is a composition comprising an antisense oligomer that enhances splicing by binding to a target portion of the JAG1 RIC pre-mRNA. As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and are targeted nucleic acids (eg, JAG1 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 can 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 a target nucleic acid (eg, the target portion of a transcript of a pre-mRNA) and remain hybridized under physiological conditions. Typically, when ASO hybridizes to a site other than the intended (targeted) nucleic acid sequence, it 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 may take into account the generation of nucleic acid sequences at the target portion of the transcript of the pre-mRNA, or of sufficiently similar nucleic acid sequences elsewhere in the pre-mRNA or transcriptome of the genome or cells. It can, and as a result, the possibility of ASO binding to other sites and causing an "off-target" effect is limited. For example, the title of the invention "Reducing Nonsense-Me"
Antisense oligomers known to those of skill in the art, such as those in PCT International Application No. PCT / US2014 / 054151 published as WO 2015/035091 of "diated mRNA Decay", are used to carry out the methods described herein. Can be done.

In some embodiments, the ASO is "specifically hybridizes" or "specifically" to the target 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 severe 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 (one polynucleotide is complementary to another to some extent)
Can be quantified in terms of the percentage (eg, percentage) of bases in opposite chains that are expected to form hydrogen bonds with each other, according to generally accepted base pairing rules. The sequence of antisense oligomer (ASO) need not be 100% complementary to the sequence of its 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. At least 97%
, At least 98%, or at least 99% sequence complementarity can be included. For example, 18 of the 20 nucleobases of an oligomeric 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 need not be co-clustered or interspersed with complementary nucleobases and adjacent to each other or complementary nucleobases. Percent complementarity of ASO with regions of target nucleic acids is known in the BLAST program (basic local alignment search tool) and in the art.
rBLAST program (Altschul et al., J. Mol. Bio
l. , 1990, 215, 403-410; Zhang and Madden
, Genome Res. , 1997, 7, 649-656) can be determined by convention.

The ASO does not have to hybridize to all nucleobases in the target sequence, and the nucleobases to which it hybridizes may be adjacent or non-adjacent. A
SO can hybridize over one or more segments of the transcript of pre-mRNA 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, the ASO hybridizes to a non-adjacent nucleobase in the transcript of the target pre-mRNA. For example, ASO can hybridize to a nucleobase in a transcript of pre-mRNA, which is 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" includes oligonucleotides and nucleic acid bases capable of hybridizing to complementary nucleobases on target mRNAs, but other oligomeric molecules that do not contain sugar moieties such as peptide nucleic acids (PNAs). Materialize. ASO
Can include naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three thereof. The term "naturally occurring nucleotides"
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 compatible with the methods and compositions described herein will be apparent to those of skill in the art, eg, US Pat. No. 8,258,109.
No. B2, US Patent No. 5,656,612, US Patent Publication No. 2012/019072
8. And Dias and Stein, Mol. Cancer The. 200
It can be found in 2,347-355, 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,
Included are 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 backbone contains a 3'-5'phosphodiester bond that binds the sugar moiety of the oligomer. The skeletal structures or oligomeric bonds of ASO described herein are (but not limited to) phosphorothioate, phosphorodithioate, phosphoroserenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilate. ), Phosp
horamidate) and the like. For example, LaPlanche et al. , N
uclic Acids Res. 14: 9081 (1986); Stick e
t al. , J. Am. Chem. Soc. 106: 6077 (1984)
, Stein et al. , Nucleic Acids Res. 16:32
09 (1988), Zon et al. , Anti Cancer Drug
Design 6: 539 (1991); Zon et al. ， Oligonu
cleotides and Analogues: A Practical App
roach, pp. 87-108 (F. Eckstein, Ed., Oxf
ord University Press, Oxford England (19)
91)); Stec et al. , US Pat. No. 5,151,510; Ulm
anand Peyman, Chemical Reviews 90: 543
(1990). In some embodiments, the skeletal structure of ASO does not contain phosphite, but includes, for example, peptide nucleic acids (PNAs), or carbamates, amides, 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 bond.

In embodiments, the stereochemistry at each of the bonds between the phosphonucleotides of the ASO backbone is random. In embodiments, the stereochemistry at each of the bonds between the phosphonucleotides of the ASO backbone is controlled and non-random. For example, US Patent Publication No. 2014/0194610, incorporated herein by reference, "Methods for the Synthe".
"sis of Functionalized Nucleic Acids" describes a method of independently selecting the handedness of chirality at each phosphorus atom in a nucleic acid oligomer. 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 phosphorus nucleotides. In embodiments, the composition used in the methods 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 9
0% 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
Includes ASOs with diastereomeric purity of about 100%, about 98% to about 100%, or about 99% to about 100%.

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 needs to achieve a balance between good activity and nuclease stability in antisense oligonucleotides (Wan, et al., 2014, "Synthe".
sis, biophysical products and biological
al activity of generation generation antisen
Seoligonucleotides contouring chiral ph
osphorothioate linkages, "Nucleic Acids R
escape 42 (22): 13456-13468, incorporated herein by reference). 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 2
0% 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 80% R
p, 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, A used in the methods of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein.
SO is about 10% to about 100 Rp, about 15% to 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 1
00% Rp, about 40% to 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, about 65
% To 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 % To about 100% Rp
, About 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp
, About 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% R
Includes p 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 55% Sp About 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least It contains about 95% Sp and the remaining Rp, or about 100% Sp. In embodiments, the ASO used in the method of the invention, including, but not limited to, any of the ASOs specified in Table 1 herein, is about 1.
0% to about 100% Sp, about 15% to 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% S
p, about 40% to 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, about 65% to about 10
0% 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, about 90% to 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% to About 60% Sp, or about 45% to about 55% Sp
And the remaining Rp.

Any of the ASOs described herein contain 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 a modified sugar moiety is 2'-O-
Methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;N3'->P5'phosphoramide,2'dimethylaminooxy Ethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinin
idium), 2'-O-guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars and the like. In some embodiments, the sugar modification is
It is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar modification is an additional cross-linking, such as in locked nucleic acids (LNAs). 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 was suppressed by 2'4'(c).
Onstrained) Includes 2'O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt 2', 4 suppressed 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 literature, eg, Jarve
r, et al. , 2014, "A Chemical View of Olig
oncleotides for Exon Skipping and Relat
ed Drug Applications, "Nucleic Acid Thera
peutics 24 (1): 37-47, which is incorporated herein by reference for this purpose.

In some examples, each monomer of ASO is modified in the same way, for example, each bond in the backbone of ASO 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 is 2'MO
Includes E-modified and phosphorothioate backbones. 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 a component of the ASO (eg, nucleobase, sugar moiety, backbone) 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). ) Reduces degradation by; improves the uptake of ASO into cells and / or the nucleus of cells; pharmacokinetics or potency of ASO (
Pharmacodynamics) can be modified; and modified to regulate the half-life of ASO.

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 to nuclease degradation and increased resistance. It has been shown to have bioavailability, which makes it suitable for oral delivery, for example, in some embodiments described herein. For example, Geary
et al. , J Pharmacol Exp Ther. 2001; 296 (
3): 890-7; Gary et al. , J Pharmacol Exp
The. 2001; 296 (3): 898-904.

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

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- or double-stranded nucleic acid sequence. Is called the 5'direction. Similarly, the right end or right direction 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 a nucleic acid is referred to as "upstream" and a region or sequence at 3'with respect to a reference point in a 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, the nucleotides upstream of the reference point in the nucleic acid are designated by a negative number.
Nucleotides downstream of the reference point can be specified by a positive number. For example, a reference point (eg, an exon exon linkage in mRNA) can be designated as a "zero" site, and nucleotides directly adjacent to and upstream of the reference point can be designated as "minus 1", eg, "-1". On the other hand, nucleotides directly adjacent to and downstream of the reference point are "plus 1",
For example, it is specified as "+1".

In another embodiment, the ASO is JAG1 RIC mRN downstream (3'direction) of the 5'splice site of the intron retained in the JAG1 RIC mRNA precursor.
It is complementary (and binds to) the target portion of the A precursor (eg, with respect to the 5'splice site, in the direction indicated by the positive number) (FIG. 1). In some embodiments, AS
O is the region +6 to +500, +6 for the 5'splice site of the retained intron.
It is complementary to the target portion of the JAG1 RIC mRNA precursor, which is within +400, +6 to +300, +6 to +200, or +6 to +100. In some embodiments,
ASO is not complementary to 5'splice sites 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 JAG1 RIC pre-mRNA located within the region between nucleotides +6 and +50 with respect to the 5'splice site of the retained intron. In some embodiments, the ASO is the region +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 for the 5'splice site of the retained intron. Complementary to the target portion within +20 from.

In some embodiments, the ASO is the target region of the JAG1 RIC mRNA precursor (eg, negative) located upstream of the 3'splice site of the intron retained in the JAG1 RIC mRNA precursor (5'direction (reactive)). It is complementary to the direction specified by the number (Fig. 1). In some embodiments, the ASO is 3 of the retained introns.
'Regions for splice sites -16 to -500, -16 to -400, -16 to-
JAG1 RIC m within 300, -16 to -200, or -16 to -100
It is complementary to the target portion of the pre-RNA. 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,
ASO is complementary to the target portion of the JAG1 RIC mRNA precursor within the region -16 to -50 relative to the 3'splice site of the retained intron. In some embodiments, the ASO is the region -16 to -90 for the 3'splice site of the retained intron.
, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16
Complementary to the target portion, which is within -40 or -16 to -30.

In embodiments, the target portion of the JAG1 RIC pre-mRNA resides 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.

In some embodiments, the ASO is complementary to the target portion of the JAG1 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 within the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron JAG1 R.
It is complementary to the target portion of the IC pre-mRNA. In some embodiments, the ASO is not complementary to nucleotides -1e to -3e for the 5'splice site of the retained intron. In some embodiments, the ASO is the region of the retained intron with respect to the 5'splice site-4e to -100e, -4e to -90e, -4e to -80e,-.
4e to -70e, -4e to -60e, -4e to -50e, -4e to -40e,-
It is complementary to the target portion of the JAG1 RIC mRNA precursor, which is within 4e to -30e or -4e to -20e.

In some embodiments, the ASO is complementary to the target portion of the JAG1 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 in the region + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron JAG1.
It is complementary to the target portion of the RIC mRNA precursor. In some embodiments, the ASO is
It 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 + 7 for the retained intron's 3'splice site.
0e, + 2e to + 60e, + 2e to + 50e, + 2e to + 40e, + 2e to + 3
It is complementary to the target portion of the JAG1 RIC mRNA precursor, which lies within the region of 0e, 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, 1
6, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 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 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30.
Nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50
Nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25
Nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to
40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 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, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 1
3 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 ~ 3
0 nucleobases, 14-25 nucleobases, 14-20 nucleobases, 15-50 nucleobases,
15-40 nucleobases, 15-35 nucleobases, 15-30 nucleobases, 15-25 nucleobases, 15-20 nucleobases, 20-50 nucleobases, 20-40 nucleobases, 20 ~
35 nucleobases, 20-30 nucleobases, 20-25 nucleobases, 25-50 nucleobases, 25-40 nucleobases, 25-35 nucleobases, or 25-30 nucleobase lengths Is. 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 of length 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 of length 25. In some embodiments,
ASO is a nucleotide with a length of 24. In some embodiments, the ASO is 23 in length.
Nucleotides. 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 19 in length. 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, A
SO is a nucleotide with a length of 15. 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 12 nucleotide in length. In some embodiments, the ASO is a nucleotide of length 11. In some embodiments, the ASO is a nucleotide of length 10.

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, the RIC
Two or more ASOs that are complementary to different target portions of the pre-mRNA 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, a cholesterol moiety, a cholesteryl moiety, such as an aliphatic chain such as a dodecanediol or undecyl residue, a polyamine or polyethylene glycol chain, or a lipid moiety, such as adamantan acetic acid. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In embodiments, antisense oligonucleotides are, but are not limited to, debased nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose ( For example, it conjugates with a moiety containing mannose 6-phosphate), a lipid, or a polyhydrogen compound. As understood in the art and described in the literature, for example, using a linker, the conjugate comprises an antisense oligonucleotide at any of several positions on a sugar, base or phosphate group. It can be linked to one or more of any nucleotides. The linker may 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 include, for example, US Pat. No. 8,450,467.
No. "Carbohydrate conjugates as delivery ag"
Ents for oligonucleotides, which are incorporated herein by reference.

In some embodiments, the nucleic acid targeted by ASO is a JAG1 RIC mRNA precursor expressed intracellularly, such as in eukaryotic cells. In some embodiments, the term "cell" may also 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 exvivo. In some embodiments, the cell is a disease-related cell or disease-related cell, or cell line. In some embodiments, the cells are in vitro (eg, in cell culture).

<Pharmaceutical composition>
A pharmaceutical composition or pharmaceutical formulation comprising the antisense oligonucleotide of the described composition for use in any of the above methods can be prepared according to prior art well known in the pharmaceutical industry and is also in the published literature. Have been described. In embodiments, the pharmaceutical composition or pharmaceutical formulation for treating a subject is of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. Contains effective amounts 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 that do not have excessive toxic, irritating, allergic, etc., and are suitable for an appropriate benefit-risk ratio. (For example, SM Berge, et al., J. Pharmac
Eutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose). The salts can be prepared in situ during the final separation and purification of the compound, or separately by reacting the free radical function with the appropriate organic acid. Examples of pharmaceutically acceptable and non-toxic acid addition salts are inorganic acids such as hydrogen chloride, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid. , Organic acids such as succinic acid or malonic acid, or salts of amino groups formed by using other documented methods such as ion exchange. Other pharmaceutically acceptable salts are adipate, alginate, ascorbate, asparagate, benzenesulfonate, benzoate, bicarbonate, borate, butyrate, cypress acid, Brain sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate , Heptaneate, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate , Methansulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, Includes phosphates, picphosphates, pivalates, propionates, stearate, succinates, sulfates, tartaric 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 are, 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 contain, for example, substances that increase the viscosity of the suspension, including sodium carboxymethyl cellulose (sorbitol and / or dextran). The suspension may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the invention include, but are not limited to, solutions, emulsions, microemulsions, foam or liposome-containing formulations (eg, cationic or non-cationic liposomes).

The pharmaceutical composition or pharmaceutical formulation of the present invention is one or more permeation enhancers, carriers, excipients, or as described as needed by those skilled in the art or as described in the published literature. It may also contain other active or inactive ingredients. In embodiments, liposomes also include sterically stable liposomes, such as liposomes containing one or more specific lipids. These particular lipids result in liposomes with enhanced circulating lifespan. 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. Drug products,
The use of surfactants in 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 an embodiment, the pharmaceutical formulation comprises a plurality of antisense oligonucleotides. In embodiments, antisense oligonucleotides are administered in combination with other drugs or therapeutic agents. In embodiments, the antisense oligonucleotide is administered by any method known in the art with one or more agents capable of facilitating the penetration of the subject antisense oligonucleotide through the blood-brain barrier. To. For example, delivery of a drug by administering an adenovirus vector to motor neurons in muscle tissue is incorporated herein by reference in US Pat. No. 6,632,427, "Adenovira."
l-vector-mediated gene transfer into medul
It is described in "lary motor neurons". Brain, eg striatum, thalamus,
Delivery of the vector directly to the hippocampus, or substantia nigra, is described, for example, in US Pat. No. 6,756,523, "Adenovirus vector's for," which is incorporated herein by reference.
the transfer of foreign genes into cells of
the central nervous system system palticularly in
It is described in "brain".

In embodiments, the antisense oligonucleotide is bound or conjugated to an agent that provides the desired pharmacological or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is bound to an antibody of a substance known in the art, such as a transferrin receptor, that facilitates penetration or transport across the blood-brain barrier. In embodiments, the antisense oligonucleotide is bound to a viral vector, for example, to make the antisense compound more effective or to increase transport across the blood-brain barrier. In embodiments, disruption of the permeable blood-brain barrier involves sugars such as mesoerythritol, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, zulcitol, myo-inositol, L (-). Fructose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) ramnorth, D (+) Meribios, D (-) ribose, adonitol, D (+) arabitol, L (-) arabitol, D (+) fucose, L (-) fucose, D (-) xylose, L (+) xylose, and L (-) ) Promoted by injecting lyxose, or amino acids such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Will be done. Methods and substances for enhancing the permeability of the blood-brain barrier are described, for example, in US Pat. No. 4,866,042, “Method fo”.
r the delivery of genetic axial axes t
he blood brain barrier, US Pat. No. 6,294,520 "M"
attrial for passage through the blood-braille
n barrier, and US Pat. No. 6,936,589 “Parenteral”
It is described in "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, target moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, a cholesterol moiety, a cholesteryl moiety, such as an aliphatic chain such as a dodecanediol or undecyl residue, a polyamine or polyethylene glycol chain, or a lipid moiety, such as adamantan acetic acid. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In embodiments, antisense oligonucleotides are, but are not limited to, debased nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GluNAc), N-Ac glucosamine (GalNAc), or mannose (eg, mannose). Mannose-6-phosphate), lipids, or conjugates containing polyhydrogen compounds. Conjugates are optional, including antisense oligonucleotides at any of several positions on a sugar, base or phosphate group, for example using a linker, as understood in the art and described in the literature. Can be linked to one or more of the nucleotides of. The linker may 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 include, for example, US Pat. No. 8,450,467 "C.
arbohydrate conjugates as delivery agents
It is described in "for oligonucleotides" and 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 is a mammal,
For example, it may be a human or an animal such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. 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 diseases 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 is at increased risk of having a disease or disorder caused by an inadequate amount of protein or an inadequately active protein. If an individual has an increased risk of having a disease or disorder caused by an inadequate amount of protein or an inadequately active protein, 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 the 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 exacerbation 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. Many tissues and organs are affected by Alagille syndrome, with the liver being the most significantly affected tissue. The ASO of the present invention may be administered parenterally to a patient. In embodiments, the ASOs of the invention are administered to a patient by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery is to the heart or liver. In embodiments, the fetus is treated in utero, eg, by administering the ASO composition directly or indirectly (eg, via the mother) to the fetus.

In the treatment of subjects with muscular dystrophy, the compositions of the invention are administered directly (eg, locally by injection or locally at the treatment site) or systemically (eg, parenterally or orally), intranasally. Any suitable means, including oral administration, or inhalation, intestinal, topical, intrauterine, vagina, sublingual, rectal, intramuscular, intrathoracic, intraventricular, intraperitoneal, ocular, intravenous Alternatively, it may be provided to muscle cells by subcutaneous means.

Methods to Identify Additional ASOs that Enhance Splicing Within the scope of the present invention, there are also methods to identify (determine) additional ASOs that enhance splicing of pre-mRNA of JAG1 RIC, 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 also block or interfere with the binding site of the splicing inhibitor / silencer. Any method known in the art is used to identify (determine) an ASO that, when hybridized to a target region of an intron, produces the desired effect (eg, increased splicing, protein or functional RNA production). May be used. These methods are also used to identify ASOs that enhance splicing of retained introns by binding to target regions in exons adjacent to retained introns or in non-retained introns. Can be done. An example of a method that can be used is provided below.

A with an ASO designed to hybridize to the target region of the pre-mRNA
A one-round screening called SO "walk" may be performed. 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 the 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. It can be spread every 5 nucleotides up to 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). For example, a first A with a length of 15 nucleotides.
SO can be designed to specifically hybridize nucleotides +6 to +20 to the 5'splice site of the targeted / retained intron. The second ASO is nucleotide +1 for the 5'splice site of the targeted / retained intron.
It is designed to specifically hybridize from 1 to +25. ASO is mRN
It is designed to cover the target area of the A precursor. In the embodiment, the ASO is more densely
For example, it can be spread every 1, 2, 3, or 4 nucleotides. In addition, ASO is from 100 nucleotides downstream of the 5'splice site to 10 upstream of the 3'splice site.
It can be spread up to 0 nucleotides.

One or more ASOs, or one control ASO (an ASO having a scrambled sequence that is not expected to hybridize to a target region), can be a target mRNA precursor (eg, herein), eg, by transfection. RIC mRNA described separately in
It is delivered to disease-related cell lines that express (precursor). 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. (See "Identifying Intron Preservation Events"). Decreased or absent RT-PCR products produced using primers that extend to splice junctions in ASO-treated cells as compared to control ASO-treated cells enhances splicing of target introns. It shows that it was done. In some embodiments, splicing efficiency, the ratio of spliced pre-mRNA to non-spliced pre-mRNA, rate of splicing, or degree of splicing is improved using the ASOs described herein. sell. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO 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.

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

The region defined by the ASO that facilitates splicing of the target intron is 1-n.
Investigate in more detail by ASO "microwalks", including ASOs spaced in t-steps, as well as longer ASOs, typically 18-25 nt.

One or more ASOs, or control ASs, as described above for ASO walks
ASO microwalk by delivering O (ASO with a scrambled sequence, a sequence not expected to hybridize to the target region) to a disease-related cell line expressing the target pre-mRNA, eg, by transfection. Is executed. AS
The effect of inducing each splicing of O is assessed by methods known in the art, such as reverse transcriptase (RT) -PCR, using primers that span splice junctions, as described herein. (See "Identifying Intron Preservation Events").
Decreased or absent RT-PCR products produced using primers that extend to splice junctions in ASO-treated cells as compared to control ASO-treated cells enhances splicing of target introns. It shows that it was done. In some embodiments, splicing efficiency, the ratio of spliced pre-mRNA to non-spliced pre-mRNA, rate of splicing, or degree of splicing is improved using the ASOs described herein. sell. Target mRN
The amount of protein or functional RNA encoded by the A precursor can also be evaluated to determine if each ASO 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, which results in enhanced splicing and increased protein production when hybridized to the region of pre-mRNA, is an animal model, eg, a transgenic mouse model in which a 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 in which delivery of ASO is desired. ASO is, for example, intraperitoneal injection, intramuscular injection,
It can be administered by subcutaneous or intravenous injection. After administration, model animal cells, tissues,
And / or organs determine the effectiveness of ASO treatment by methods known in the art and methods described herein, eg, by assessing splicing (efficiency, rate, degree) and protein production. Can be evaluated for. Animal models can also be phenotypic or behavioral signs of disease or disease severity.

While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many changes, changes, and substitutions will be made to those skilled in the art without departing from the present invention. It should be understood that various alternatives of embodiments of the invention described herein may be utilized in the practice of the invention. The following claims define the scope of the invention.
It is intended that the methods and structures and their equivalents within the scope of this claim are incorporated therein.

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

<Example 1: Identification of intron retention event of JAG1 transcript by RNAseq using next-generation sequencing>
Next-generation sequencing was used to identify intron retention events, and whole transcriptome shotgun sequencing was performed to reveal a snapshot of the transcript produced by the JAG1 gene. For this purpose, polyA + RNA from the nucleus and cytoplasmic fractions of THLE-3 (human liver epithelium) cells was isolated and Illumina's T.
ruSeq Stranded mRNA library Prep Kit using c
A DNA library was constructed. Pair-end sequencing for the library (pair-e)
nd sequence), and as a result, the human genome (February 2009, GRCh
It resulted in a reading of 100 nucleotides mapped to 37 / hg19 assembly). The result of sequencing for JAG1 is shown in FIG. Briefly, Figure 3 shows UCS.
C Genome Informatics Group (Center for Biom)
olecular Science & Engineering, University
of California, Santa Cruz, 1156 High Street
t, Santa Cruz, CA 95064) and operated by, for example, Ros
enbloom, et al. , 2015, "The UCSC Genome Browser"
ser database: 2015 update, "Nucleic Acids R"
escape 43, Database Issue (doi: 10.1093 / n
Ar / gku1177), showing the visualized and mapped reads using the UCSC Genome Browser, the range and number of reads can be inferred by the peak signal. The height of the peak indicates the level of expression given by the reading density in a particular region. Schematic diagram of JAG1 by UCSC Genome Browser (under the read signal) so that the peaks are adapted to the JAG1 exon and intron regions.
(Drawn to scale) is provided. Based on this indication, it has a high read density in the nuclear fraction of THLE-3 cells (intron 1 in the diagram below FIG. 3).
With respect to 3, as shown with respect to intron 18 in the diagram below FIG. 7 and with respect to intron 23 in the diagram below FIG. 11), the readings are very low or none in the cytoplasmic fraction of these cells. Two introns (13, 18 and 23 indicated by arrows) were identified. This is because these introns are retained and the intron-13, intron-18 and intron-23 containing transcripts remain in the cell nucleus and are not carried out to the cytoplasm. J
It is shown that the AG1 RIC mRNA precursor is suggested to be unproductive.

<Example 2: Design of ASO walk targeting intron 13 of JAG1>
The ASO walk was designed to target the intron 13 using the methods described herein (FIG. 4; Table 2). The region immediately downstream of the 5'splice site of intron 13 extending from nucleotides +6 to +69 and the region immediately upstream of the 3'splice site of intron 13 extending from nucleotides -16 to -68 are (1ASO, JAG1-IVS1).
With the exception of 3 + 52, targets were used with) 2'-O-Me RNA, PS backbone, and 18-mer ASO shifted at 5-nucleotide intervals (FIG. 4; Table 2).

<Example 3: Improvement of splicing efficiency by ASO targeting of JAG1 intron 13 increases transcription level>
The method described herein was used to determine if increased JAG1 expression could be achieved by improving the splicing efficiency of the JAG1 intron 13 using ASO (FIG. 5). .. For this purpose, each of the target ASOs listed in FIG. 4 and Table 2, either sham-transfected ARPE-19 cells or independently using RNAiMAX (RiM) (Invitrogen) delivery reagents, Alternatively, it was transfected with a non-targeted SMN-ASO control. 48 hours (as shown in FIG. 5) 60
Experiments were performed using nM ASO. The results of radioactive RT-PCR show some target ASOs (+6, SEQ ID NO: 135; +11, SEQ ID NO: 136; +21.
, SEQ ID NO: 138; +26, SEQ ID NO: 139; +31, SEQ I
D NO: 140; +36, SEQ ID NO: 141; +52; -16, SEQ ID
NO: 301; -36, SEQ ID NO: 297; -41, SEQ ID NO: 296
-46, SEQ ID NO: 295; and -51, SEQ ID NO: 294) increase the level of JAG1 transcript compared to pseudotransfected or untargeted ASO (FIG. 5). I showed that. The intensity of the band corresponding to the JAG1 PCR product from the cells transfected with the target ASO was normalized to β-actin and plotted against the normalized JAG1 PCR product from the sham-treated cells. The results of this analysis show that some of the target ASOs increase PKD1 transcript levels by a factor of 2 to 6 (FIG. 5). These results were confirmed by RT-qPCR using primers for the rest of the JAG1 transcript and show the same trend of JAG1 upregulation as demonstrated by the magnification change plot in FIG. 6 (top normal for beta actin). The one that has been normalized, the one whose lower part has been normalized to RPL32). In short, these results
It is confirmed that improving the rate-determining intron splicing efficiency in the JAG1 gene using ASO leads to an increase in gene expression.

<Example 4: Design of ASO walk targeting intron 18 of JAG1>
The ASO walk was designed to target the intron 18 using the methods described herein (FIG. 8; Table 2). The region immediately downstream of the 5'splice site extending from nucleotides +15 to +67 of intron 18 and the region immediately upstream of the 3'splice site extending from nucleotides -16 to -68 of intron 18 are 2'-O-Me RNA, PS
Skeletons were targeted using 18-mer ASOs shifted at 5-nucleotide intervals (FIG. 8; Table 2).

<Example 5: Improvement of splicing efficiency by ASO targeting of JAG1 intron 18 increases transcription level>
The method described herein was used to determine if increased expression of JAG1 could be achieved by improving the splicing efficiency of the JAG1 intron 18 using ASO (FIG. 9). For this purpose, RNAiMAX (RiM) (In) independently
The ARPE-19 cells were sham-transfected using the delivery reagent of vitrogen), transfected with each of the target ASOs shown in FIG. 8 and Table 2, or transfected with a non-targeted SMN-ASO control. It was perfect. Experiments were performed using 60 nM ASO for 48 hours (as shown in FIG. 5). Radio RT-PCR results show that some targeted ASOs (+15; +20; and -26, SEQ ID NO: 107) were pseudotransfected or increased JAG1 transcript levels compared to non-targeted ASOs. It is shown that it was made (Fig. 9). JAG1 PC from cells transfected with target ASO
The intensity of the band corresponding to the R product was normalized to β-actin and plotted against the normalized JAG1 PCR product from sham-treated cells. The results of this analysis show that the three target ASOs increase JAG1 transcript levels by at least 1.5-fold (
FIG. 9). These results show RT-q using primers for other parts of JAG1 transcript.
It shows the same trend of JAG1 upregulation confirmed by PCR and demonstrated by the magnification change plot in FIG. 10 (upper part normalized to beta actin, lower part normalized to RPL32). In short, these results indicate that ASO can be used to improve the rate-determining intron splicing efficiency in the JAG1 gene.
It is confirmed that it leads to an increase in gene expression.

A second method can be used to determine if ASO can be used to achieve increased splicing efficiency of the JAG1 target gene intron. Briefly, ARPE-19 cells (American Type Cultu), which are epithelial cells of the human retina.
reCollection (ATCC), USA), or Huh-7 (NIBIOHN (Japan)), a human hepatocytoma cell line, or S, a human neuroblastoma cell line.
KN-AS (ATCC) is either pseudotransfected or transfected with the target ASOs listed in Tables 1 and 2. Lip the cells according to the supplier's specifications
offectamine RNAiMax Transfection Reagent (Thermo Fis)
Transfect using her). Sow ASO on a 96-well tissue culture plate and
Combine with RNAiMax diluted with Opti-MEM. Cells are exfoliated with trypsin, resuspended in complete medium and approximately 25,000 cells are added to the ASO transfection mixture. Transfection experiments are performed on three plate replicas. The final ASO concentration is 80 nM. According to the supplier's specifications, the medium is replaced 6 hours after transfection and cells are harvested in 24 hours using a Cells-to-Ct RT reagent supplemented with DNAse (Thermo Fisher). Cell-to-Ct RT Reagent (Thermo Fisher), according to the supplier's specifications.
). Taqman with a probe spanning the corresponding exon-exon Fisher to quantify the amount of splicing in the intron of interest.
Quantitative PCR is performed using the assay. Taqman assay, QuantSt
udio 7Flex Real-Time PCR System (Thermo Fisher)
) Perform according to the supplier's specifications above. The target gene assay value is RPL32 (ΔCt).
) And plate-matched pseudotransfection samples (ΔΔCt)
Generate a multiple change with respect to the quantification of the simulated mock (2 ^-(ΔΔCt)).

<Example 6: Design of ASO walk targeting intron 23 of JAG1>
The ASO walk was designed to target the intron 23 using the methods described herein (FIG. 12; Table 2). The region immediately downstream of the 5'splice site extending from nucleotides +6 to +58 of intron 23 and the region immediately upstream of the 3'splice site extending from nucleotides -16 to -68 of intron 23 are 2'-O-Me RNA, PS
Skeletons were targeted using 18-mer ASOs shifted at 5-nucleotide intervals (FIG. 12; Table 2).

Claims (100)

A method of treating a subject's Arazil syndrome in need by increasing the expression of a target protein or functional RNA by the subject's cells, wherein the cells are retained intron-containing mRNA precursors (RIC mRNA precursors). ), And the RIC mRNA
Precursors include retained introns, exons flanking the 5'splice site, and exons flanking the 3'splice site, where the RIC mRNA precursor is:
Encoding the target protein or functional RNA, the method comprises contacting the subject's cells with an antisense oligomer (ASO) complementary to the target portion of the RIC pre-mRNA encoding the target protein or functional RNA. , Thereby, the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby causing the target protein or functional RN within the subject's cells.
A method of increasing the level of mRNA encoding A and increasing the expression of a target protein or functional RNA. A method of increasing the expression of a target protein, which is JAG1, by cells having a retained intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC
Pre-mRNA includes retained introns, exons adjacent to the 5'splice site of the retained intron, and exons adjacent to the 3'splice site of the retained intron, where the RIC pre-mRNA. The body encodes the JAG1 protein,
The method comprises contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the JAG1 protein, thereby.
Retained introns are constitutively spliced from RIC mRNA precursors encoding the JAG1 protein, thereby increasing intracellular levels of mRNA encoding the JAG1 protein and increasing expression of the JAG1 protein. .. The method of claim 1, wherein the target protein is JAG1. The method of claim 1, wherein the target protein or functional RNA is a compensating protein or compensating functional RNA that functionally enhances or replaces the target protein or functional RNA that is deficient in quantity or activity in the subject. .. The method of claim 2, wherein the cells are in or derived from a subject having a disease caused by a lack of amount or activity of the JAG1 protein. A deficiency in the amount of target protein is caused by haplodeficiency of the target protein, where the subject is given a first allelic gene encoding a functional target protein and a second allelic gene in which the target protein is not produced, or Any of claims 1-5, which has 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. The method according to item 1. The subject has a disease caused by a disorder resulting from a lack of target protein amount or function, where the subject.
a. The first mutant allele
i. Target proteins are produced at reduced levels compared to production from wild-type alleles,
ii. The target protein is produced in a less functional form as compared to an equivalent wild-type protein, or iii. The first mutant allele, which does not produce the target protein, and b. The second mutation allele
i. Target proteins are produced at reduced levels compared to production from wild-type alleles,
ii. The target protein is produced in a less functional form as compared to an equivalent wild-type protein, or iii. It has a second mutated allele in which the target protein is not produced, and where the subject is a. iii. When having the first mutated allele of, the second mutated allele is b. i. Or b. ii. Mutant allele of the subject, b. iii
.. When having a second mutated allele of, the first mutated allele is a. i. Or a
.. ii. Mutant alleles of, and RIC mRNA precursors are a. i. Or a
.. ii. The first mutant allele and / or b. i. Or b. ii. The method according to any one of claims 1 to 5, which is transcribed from any of the second mutation alleles. The method of claim 7, wherein the target protein is produced in a reduced function as compared to an equivalent wild-type protein. The method of claim 7, wherein the target protein is produced in a form that is fully functional as compared to an equivalent wild-type protein. Claim that the target portion of the RIC pre-mRNA is within the region of +6 with respect to the 5'splice site of the retained intron to the region -16 relative to the 3'splice site of the retained intron in the retained intron. The method according to any one of 1 to 9. In the intron where the target portion of the RIC mRNA precursor was retained,
(A) +6 to +500, +6 for the 5'splice site of the retained intron
Within the region from +400, +6 to 300, +6 to 200, or +6 to +100; or (b) -16 to -500,-to the 3'splice site of the retained intron.
16 to -400, -16 to -300, -16 to -200, or -16 to -10
The method according to any one of claims 1 to 9, which is within the region of 0. The target portion of the RIC mRNA precursor is
(A) + in the exon adjacent to the 5'splice site of the retained intron
Within the region 2e to -4e; or (b) + in the exon adjacent to the 3'splice site of the retained intron.
The method according to any one of claims 1 to 9, which is within the region of 2e to -4e. RIC mRNA precursor is at least about 80%, 8 relative to SEQ ID NO: 1.
The method of any one of claims 1-12, encoded by a gene sequence having 5%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. .. RIC mRNA precursor is at least about 80%, 85 relative to SEQ ID NO: 2.
The method of any one of claims 1-13, comprising a sequence having a%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Any one of claims 1-14, wherein 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. The method described in the section. 13. The method described. The method according to any one of claims 1 to 16, 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. The method according to any one of claims 1 to 17, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. The method according to any one of claims 1 to 18, wherein the target protein produced is a full-length protein or a wild-type protein. The total amount of mRNA encoding the target protein or functional RNA produced intracellularly in contact with the antisense oligomer is approximately relative to the total amount of mRNA encoding the target protein or functional RNA produced in the control cell. 1.1 times to about 10 times, about 1
.. 5 to 10 times, 2 to 10 times, 3 to 10 times, 4 to 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 times to about 8 times, about 1.1 times to about 9 times, About 2 to about 5 times, about 2 to about 6 times, about 2 to about 7
Double, 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
About 8 times to about 8 times, about 3 times to about 9 times, about 4 times to about 7 times, about 4 times to about 8 times, about 4 times to about 9 times, at least about 1.1 times, at least about 1.5 times Claims 1-19, which are multiplied, 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. The method according to any one of the above. The total amount of target protein produced by cells contacted with the antisense oligomer is from about 1.1 to about 10 times, from about 1.5 times, compared to the total amount of target protein produced by control cells. 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, about 1.1 times 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 Double, 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 7 times, about 4 to 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,
The method according to any one of claims 1 to 20, which is increased by at least about 5 times, or at least about 10 times. The method according to any one of claims 1 to 21, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. Any 1 of claims 1 to 22, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. The method described in the section. Claims 1-2, wherein the antisense oligomer comprises at least one modified sugar moiety.
The method according to any one of 3. 24. The method of claim 24, wherein each sugar moiety is a modified sugar moiety. The antisense oligomers are 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10
~ 25 nucleobases, 10 ~ 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, 1
Claims 1 to 25 consisting of 2 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
The method according to any one of the above. 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 protein-encoding RIC mRNA precursor. The method according to any one of claims 1 to 26. The method according to any one of claims 1 to 27, wherein the target portion of the RIC mRNA precursor is in a sequence selected from SEQ ID NO: 437-439. The antisense oligomer is used for any one of SEQ ID NO: 3-436.
At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96
%, 97%, 98%, or 99% sequence identity comprising a nucleotide sequence, claim 1.
The method according to any one of 28 to 28. The method according to any one of claims 1 to 28, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 3-436. RIC transcribed from a gene encoding a target protein or functional RNA
Containing a population of pre-mRNA, where the population of RIC mRNA precursors comprises two or more retained introns, the antisense oligomer binds to the most abundant retained introns in the population of RIC mRNA precursors. The method according to any one of claims 1 to 30. Binding of antisense oligomers to the most abundant retained introns splicing out two or more retained introns from a population of RIC pre-mRNA to produce mRNA encoding the target protein or functional RNA. 31. The method of claim 31. RIC transcribed from a gene encoding a target protein or functional RNA
Containing a population of pre-mRNA, where the population of RIC mRNA precursors comprises two or more retained introns, the antisense oligomer is the second most abundant retained intron in the population of RIC mRNA precursors. Any one of claims 1 to 30, which is coupled to
The method described in the section. Binding of antisense oligomers to the second most abundant retained introns of two or more retained introns from a population of RIC pre-mRNA to produce mRNA encoding the target protein or functional RNA. 33. The method of claim 33, which induces splicing out. The method according to any one of claims 5 to 34, wherein the disease is a disease or a disorder. 35. The method of claim 35, wherein the disease or disorder is Alagille syndrome or muscular dystrophy. 36. The method of claim 36, wherein the target protein and RIC mRNA precursor are encoded by the JAG1 gene. Any one of claims 1 to 37, further comprising the step of evaluating JAG1 protein expression.
The method described in the section. The antisense oligomer binds to the target portion of the JAG1 RIC mRNA precursor, where the target portion is SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56.
, And the method according to any one of claims 1 to 38, which is in the sequence selected from 57. The method according to any one of claims 1 to 39, wherein the subject is a human. The method according to any one of claims 1 to 39, wherein the subject is an animal other than a human. The method according to any one of claims 1 to 40, wherein the subject is a fetus, an embryo, or a child. The method according to any one of claims 1 to 41, wherein the cells are in Exvivo. The method according to any one of claims 1 to 41, wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. Any of claims 1-44, wherein the nine nucleotides at -3e to -1e of the exon adjacent to the 5'splice site and +1 to +6 of the retained intron are identical to the corresponding wild-type sequence. The method according to item 1. 16. The method described in. The antisense oligomer used in the method according to any one of claims 1 to 39. At least about 80% and 85% with respect to any one of SEQ ID NO: 3-436.
, 90%, 95%, 97%, or 100% sequence identity. A pharmaceutical composition comprising the antisense oligomer and excipient according to claim 47 or 48. A method of treating a subject in need by administering the pharmaceutical composition by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A composition comprising an antisense oligomer for use in a method of increasing the expression of a target protein or functional RNA by cells to treat a subject's Arazil syndrome in need associated with protein deficiency or functional RNA deficiency. Where protein deficiency or functional RNA deficiency is a deficiency in amount or activity in the subject, the antisense oligomer is a retained intron-containing mRNA precursor (RIC mRNA precursor) that encodes the target protein or functional RNA. Enhances constitutive splicing, where the target protein is
(A) deficient protein; or (b) a protein that functionally enhances or replaces or compensates for the deficient protein in a subject.
And functional RNA
(A) deficient RNA; or (b) functional RNA that functionally enhances or replaces or compensates for deficient functional RNA in a subject.
Here, the RIC pre-mRNA includes a retained intron, an exon adjacent to the 5'splice site, and an exon adjacent to the 3'splice site, and the retained intron contains the target protein or functional RNA. Spliced from the encoding RIC pre-mRNA, thereby targeting protein or functional RNA in the subject
A composition that increases the production or activity of. A composition comprising an antisense oligomer for use in a method of treating a disease associated with the JAG1 protein in a subject in need, wherein the method involves increasing the expression of the JAG1 protein by the subject's cells. Containing, where the cells were retained, including a retained intron, an exson adjacent to the 5'splice site of the retained intron, and an exson adjacent to the 3'splice site of the retained intron. Having an intron-containing mRNA precursor (RIC mRNA precursor), the RIC mRNA precursor encodes the JAG1 protein, which method also comprises contacting the cell with an antisense oligomer, thereby retaining it. Introns are constitutively spliced from transcripts of the RIC mRNA precursor encoding the JAG1 protein, thereby increasing the level of mRNA encoding the JAG1 protein in the cells of the subject and of the target protein or functional RNA. A composition that increases expression. 52. The composition of claim 52, wherein the disease is a disease or disorder. 53. The composition of claim 53, wherein the disease or disorder is Alagille syndrome or muscular dystrophy. The composition according to claim 54, wherein the target protein and the RIC mRNA precursor are encoded by the JAG1 gene. One of the RIC mRNA precursors in which the antisense oligomer is in the retained intron, from the +6 region to the 5'splice site of the retained intron to the -16 region relative to the 3'splice site of the retained intron. The composition according to any one of claims 51 to 55, which targets a portion. In the intron where the antisense oligomer was retained,
(A) Within the region +6 to +100 relative to the 5'splice site of the retained intron; or (b) within the region -16 to -100 relative to the 3'splice site of the retained intron of the RIC mRNA precursor. The composition according to any one of claims 51 to 56, which targets a part thereof. Antisense oligomers are present at least one retained intron 3'from about 100 nucleotides downstream of the 5'splice site of at least one retained intron.
The composition according to any one of claims 51 to 57, which targets a part of RIC mRNA precursor within a region up to about 100 nucleotides upstream of the splice site. The target portion of the RIC mRNA precursor is
(A) + in the exon adjacent to the 5'splice site of the retained intron
Within the region 2e to -4e; or (b) + in the exon adjacent to the 3'splice site of the retained intron.
The composition according to any one of claims 51 to 57, which is in the region of 2e to -4e. RIC mRNA precursor is at least about 80%, 8 relative to SEQ ID NO: 1.
The composition according to any one of claims 51 to 59, which is encoded by a gene sequence having 5%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. thing. RIC mRNA precursor is at least about 80%, 8 relative to SEQ ID NO: 2.
The composition according to any one of claims 51 to 60, comprising a sequence having 5%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Any one of claims 51-61, wherein the antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from a gene encoding the target protein or functional RNA. The composition according to the section. 13. The composition described. The composition according to any one of claims 51 to 63, wherein the RIC pre-mRNA is produced from a full-length pre-mRNA or a wild-type pre-mRNA by partial splicing. The composition according to any one of claims 51 to 64, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA. The composition according to any one of claims 51 to 65, wherein the target protein produced is a full-length protein or a wild-type protein. Any one of claims 51 to 66, wherein the held intron is a rate-determining intron.
The composition according to the section. The composition according to any one of claims 51 to 67, wherein the retained intron is the most abundant retained intron in the RIC pre-mRNA. The composition according to any one of claims 51 to 67, wherein the retained intron is the second most abundant retained intron in the RIC pre-mRNA. The composition according to any one of claims 51 to 69, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. The composition according to any one of claims 51 to 70, wherein the antisense oligomer is an antisense oligonucleotide. Any 1 of claims 51-71, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. The composition according to the section. The composition according to any one of claims 51 to 72, wherein the antisense oligomer contains at least one modified sugar moiety. The composition according to claim 73, wherein each sugar moiety is a modified sugar moiety. The antisense oligomers are 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10
~ 25 nucleobases, 10 ~ 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, 1
Claims 51 to 7 consisting of 2 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
The composition according to any one of 4. 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 protein-encoding RIC mRNA precursor. Claims 51 to 75
The composition according to any one of the above. The antisense oligomer binds to the target moiety of the JAG1 RIC mRNA precursor, where the target moiety is in any one of claims 51-76 within the sequence selected from SEQ ID NO: 437-439. The composition described. The antisense oligomer is used for any one of SEQ ID NO: 3-436.
At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96
5. A nucleotide sequence that comprises a nucleotide sequence that is%, 97%, 98%, or 99% sequence identity.
The composition according to any one of 1 to 77. The composition according to any one of claims 51 to 77, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 3-436. A pharmaceutical composition comprising any of the antisense oligomers and excipients of the compositions of claims 51-79. A method of treating a subject in need by administering the pharmaceutical composition of claim 80 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. A pharmaceutical composition comprising an antisense oligomer that hybridizes to the target sequence of the deficient JAG1 mRNA transcript, and a pharmaceutically acceptable excipient, wherein the deficient JAG1 mRNA transcript is retained. A pharmaceutical composition comprising an intron and an antisense oligomer that induces splicing out of retained introns from a deficient JAG1 mRNA transcript. The pharmaceutical composition according to claim 82, wherein the deficient JAG1 mRNA transcript is a transcript of the JAG1 RIC mRNA precursor. The target portion of the transcript of the JAG1 RIC pre-mRNA is in the retained intron, from the +6 region to the 5'splice site of the retained intron to the -16 region relative to the 3'splice site of the retained intron. There, claim 82 or 83
The pharmaceutical composition according to. Transcripts of JAG1 RIC mRNA precursors are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 10 relative to SEQ ID NO: 1.
The pharmaceutical composition according to any one of claims 82 to 84, which is encoded by a gene sequence having 0% sequence identity. Transcripts of JAG1 RIC mRNA precursors are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 10 relative to SEQ ID NO: 2.
The pharmaceutical composition according to any one of claims 82 to 85, which comprises a sequence having 0% sequence identity. The pharmaceutical composition according to any one of claims 82 to 86, wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond. The pharmaceutical compound according to any one of claims 82 to 87, wherein the antisense oligomer is an antisense oligonucleotide. One of claims 82 to 88, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety. The pharmaceutical composition according to the section. The pharmaceutical composition according to any one of claims 82 to 89, wherein the antisense oligomer contains at least one modified sugar moiety. The antisense oligomers are 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 Nucleobases, 9-50 nucleobases, 9-40 nucleobases, 9-35 nucleobases, 9-30 nucleobases, 9-25 nucleobases, 9-20 nucleobases, 9-15 Nucleobases, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10
~ 25 nucleobases, 10 ~ 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, 1
Claims 82-90, comprising 2-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases.
The pharmaceutical composition according to any one of the above. 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 transcript of the JAG1 RIC mRNA precursor. The pharmaceutical composition according to any one of claims 82 to 91. The target portion of the transcript of the JAG1 RIC mRNA precursor is SEQ ID NO: 437.
The pharmaceutical composition according to any one of claims 82 to 92, which is in the sequence selected from -439. The antisense oligomer is used for any one of SEQ ID NO: 3-436.
At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96
8%, 97%, 98%, or 99% containing nucleotide sequences that are sequence-identical.
The pharmaceutical composition according to any one of 2 to 93. The pharmaceutical composition according to any one of claims 82 to 93, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 3-436. The medicament according to any one of claims 82 to 95, wherein the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. Composition. JAG deficient to facilitate the removal of retained introns to produce a fully processed transcript of JAG1 mRNA that encodes the functional form of the tuberin protein.
1 A method of inducing processing of mRNA transcripts.
(A) Step of contacting the antisense oligomer with the target cells of the subject;
(B) In the step of hybridizing the antisense oligomer to the transcript of the deficient JAG1 mRNA, the deficient JAG1 mRNA transcript can encode the functional form of the tuberin protein and is retained at least one. Processes, including introns;
(C) Fully processed JAG1 mRN encoding functional morphology of tuberin protein
The step of removing at least one retained intron from the deficient JAG1 mRNA transcript to produce A transcript; and (d) the function of the tuberin protein from the well-treated JAG1 mRNA transcript. A method that includes the step of translating the morphology. The method of claim 97, wherein the retained intron is the entire retained intron. The method of claim 97 or 98, wherein the deficient JAG1 mRNA transcript is a transcript of the JAG1 RIC mRNA precursor. A method of treating a subject having a disease caused by a lack of amount or activity of the JAG1 protein.
At least about 80% and 85% with respect to any one of SEQ ID NO: 3-436.
, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% administration of an antisense oligomer containing a nucleotide sequence having sequence identity to the subject. A method, including steps.

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