JP7049247B2 - Compositions and Methods for the Treatment of Kidney Disease - Google Patents

Description

This application claims the benefit of US Provisional Patent Application No. 62 / 267,242 filed December 14, 2015, which provisional application is incorporated herein by reference in its entirety.

References to Sequence Lists This application includes a sequence listing, which is submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. A copy of ASCII made on 12 December 2016 is 47991-715_601_SL. It is a file name of txt and has a size of 4,351,059 bytes. The aforementioned files were created on December 12, 2016 and are incorporated herein by reference in their entirety.

Kidney disease is a debilitating and potentially fatal group of diseases associated with kidney damage. The kidneys are important for many functions in the body, including, but not limited to, removing waste and excess fluid, maintaining an equilibrium between salt and minerals in the blood, and purifying the blood by regulating blood pressure. .. The incidence of kidney disease often results in fluid and waste accumulation, vomiting, weakness, light sleep, and shortness of breath, and can even be fatal in the end. While kidney transplants often prevent mortality, the chances of receiving a donor's kidney are generally low.

Although there are many diseases and diseases associated with the kidney, some kidney diseases have been found to be advanced by deletions in the gene product as well as deletions in gene expression. For example, CTNS, PAX2, CYP24A1, and PPARD.

In one embodiment, herein discloses a method of treating a subject's kidney disease by increasing the expression of a target protein or functional RNA by the subject's cells, wherein the cells are retained. It has an RNA-containing mRNA precursor (RIC mRNA precursor), and the RIC mRNA precursor is a retained intron, an exson adjacent to a 5'splice site, and an exson adjacent to a 3'splice site. Where the RIC mRNA precursor encodes a target protein or functional RNA, the method complements the subject's cells with the target portion of the RIC mRNA precursor encoding the target protein or functional RNA. It comprises contacting with an antisense oligomer (ASO), whereby the retained intron is constitutively spliced from the RIC mRNA precursor encoding the target protein or functional RNA, thereby the subject's. Increases the level of mRNA encoding the target protein or functional RNA in the cell and increases the expression of the target protein or functional RNA.

In some embodiments, the kidney disease is nephropathy, delayed cystine accumulation in infants, focal segmental glomerulosclerosis 7, papillorenal syndrome, or hypercalcemia in infants. I have a disease.

In one embodiment, a method of increasing the expression of a target protein by a cell having a retained intron-containing mRNA precursor (RIC mRNA precursor) is provided herein, wherein the RIC mRNA precursor is: It contains 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, where the RIC mRNA precursor is the target protein. The method comprises contacting the cell with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the target protein, whereby the retained intron is targeted. It is constitutively spliced from the RIC mRNA precursor that encodes the protein, thereby increasing the level of the mRNA encoding the target protein and increasing the expression of the target protein in the cell, where the target protein is cystinosin ( Cystinosin), protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxysome growth factor activating receptor δ.

In some embodiments, the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome proliferator-activated receptor δ.

In some embodiments, the target protein or functional RNA functionally increases or replaces the target protein or functional RNA that is deficient in quantity or activity in the subject, the compensatory protein or compensation. It is a functional RNA.

In some embodiments, the cells are in or derived from a subject having a disease caused by a lack of amount or activity of the target protein.

In some embodiments, the deficiency in the amount of target protein is caused by haplodeficiency of the target protein, where the subject does not produce the first allogeneic gene encoding the functional target protein, the target protein. It has two allelic genes, or a second allelic gene encoding a non-functional target protein, and the antisense oligomer binds to the target portion of the RIC pre-mRNA transcribed from the first allelic gene.

In some embodiments, the subject has a disease caused by a disorder due to a lack of quantity or function of the target protein, wherein the subject produces the target protein from a wild-type allelic gene. With the first mutant allogene, which is produced at reduced levels and the target protein is produced in a less functional form compared to the equivalent wild-type protein, or the target protein is not produced. , The target protein is produced at a reduced level compared to the production from the wild-type allelic gene, and the target protein is produced in a less functional form compared to the equivalent wild-type protein, or the target protein is produced. It has a second mutated allelic gene that is not produced, where the second mutated allelic gene is (b) (i) or when the subject has the first mutated allelic gene (a) (iii). (B) (ii), where the first mutated alligator gene is (a) (i) or (a) (ii) when the subject has a second mutated allelic gene (b) (iii). ), Where the RIC mRNA precursor is the first mutated allelic gene, (a) (i) or (a) (ii), and / or (b) (i) or (b) ( It is transcribed from the second mutation alliance gene, which is 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 retained within the region from +6 to the retained intron's 5'splice site to -16 with respect to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target portion of the RIC pre-mRNA is retained within the region from +69 to the retained intron's 3'splice site to -79 relative to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target protein is cystinocin.

In some embodiments, the target portion of the RIC pre-mRNA is retained within the region from +500 to the retained intron's 5'splice site to -500 with respect to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% with respect to any one of SEQ ID NO: 8624-10068. , 98%, 99%, or 100% complementary sequences.

In some embodiments, the target portion of the RIC pre-mRNA is at least 80%, 85%, 90%, 95% in a region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10748 or SEQ ID NO: 10734. , 96%, 97%, 98%, 99%, or 100% sequences with sequence identity.

In some embodiments, the ASO is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% for any one of SEQ ID NO: 8624-10068. , Or sequences with 100% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97 for any one of SEQ ID NO: 16 or SEQ ID NO: 17. Includes sequences with%, 98%, 99%, or 100% sequence identity.

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: 3. It is encoded by a gene sequence that has sequence identity.

In some embodiments, the target protein is the paired box gene 2 protein.

In some embodiments, the target portion of the RIC pre-mRNA is retained within the region from +500 to the retained intron's 5'splice site to -500 with respect to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% with respect to any one of SEQ ID NO: 6935-8623. , 98%, 99%, or 100% complementary sequences.

In some embodiments, the target portion of the RIC pre-mRNA is at least 80%, 85%, 90%, 95% in a region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10738 or SEQ ID NO: 10740. , 96%, 97%, 98%, 99%, or 100% sequences with sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% relative to any one of SEQ ID NO: 6935-8623. , 99%, or 100% sequences with sequence identity. In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 10-15. , 99%, or 100% sequences with sequence identity.

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: 2. It is encoded by a gene sequence having the sequence identity of.

In some embodiments, the target protein is the protein cytochrome P450 family 24, subfamily A, polypeptide 1.

In some embodiments, the target portion of the RIC pre-mRNA is retained within the region from +500 to the retained intron's 5'splice site to -500 with respect to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% for any one of SEQ ID NO: 9520-10733. , 98%, 99%, or 100% complementary sequences.

In some embodiments, the target portion of the RIC mRNA precursor is at least eight of SEQ ID NO: 10748, SEQ ID NO: 10734, SEQ ID NO: 10746, SEQ ID NO: 10745, or SEQ ID NO: 10741. Regions containing adjacent nucleic acids include sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, the ASO is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% for any one of SEQ ID NO: 9520-10733. , Or sequences with 100% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 16-19. , 99%, or 100% sequences with sequence identity.

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: 4. It is encoded by a gene sequence having the sequence identity of.

In some embodiments, the target protein is the peroxisome proliferator-activated receptor δ.

In some embodiments, the target portion of the RIC pre-mRNA is retained within the region from +500 to the retained intron's 5'splice site to -500 with respect to the retained intron's 3'splice site. It is in the intron that was made.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% for any one of SEQ ID NO: 20-6952. , 98%, 99%, or 100% complementary sequences.

In some embodiments, the target portion of the RIC mRNA precursor is SEQ ID NO: 10736, SEQ ID NO: 10747, SEQ ID NO: 10739, SEQ ID NO: 10743, SEQ ID NO: 10742, SEQ ID NO :: At least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 with respect to the region containing at least 8 adjacent nucleic acids of 10737, SEQ ID NO: 10735, or SEQ ID NO: 10744. % Or 100% sequence identity.

In some embodiments, ASO is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% for any one of SEQ ID NO: 20-6952. , Or sequences with 100% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 5-9. , 99%, or 100% sequences with sequence identity.

In some embodiments, 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. It is encoded by a gene sequence having the sequence identity of.

In some embodiments, the target portion of the RIC pre-mRNA is in the inner retained intron below: the region +6 to +100 relative to the 5'splice site of the retained intron; or the retained intron. The region of -16 to -100 with respect to the 3'splice site of.

In some embodiments, the antisense oligomer is from about 100 nucleotides downstream of the 5'splice site of at least one retained intron to about 100 nucleotides upstream of the 3'splice site of at least one retained intron. Target some of the RIC pre-mRNA within the region.

In some embodiments, the target portion of the RIC pre-mRNA is within: the region + 2e to -4e in the exon adjacent to the 5'splice site of the retained intron; or the retained intron. The region of + 2e to -4e in the exon adjacent to the 3'splice site of.

In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by regulating the alternative splicing of pre-mRNA transcribed from the gene encoding the functional RNA or target protein. ..

In some embodiments, the antisense oligomer does not increase the amount of target protein or functional RNA by regulating aberrant splicing due to mutations in the gene encoding the target protein or functional RNA.

In some embodiments, the RIC pre-mRNA was produced by partial splicing of the full-length pre-mRNA or partial splicing of the wild-type pre-mRNA.

In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.

In some embodiments, the target protein produced is a full-length protein or a wild-type protein.

In some embodiments, in some embodiments, the total amount of mRNA encoding the target protein or functional RNA produced in cells contacted with the antisense oligomer is the target protein or function produced in control cells. 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 compared to the total amount of mRNA encoding the target RNA. Double, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to About 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, It increases at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

In some embodiments, the total amount of target protein produced by cells in contact with the antisense oligomer is about 1.1 to about 10 times, about 10 times, as compared to the total amount of target protein produced by control cells. 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1. 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 Double, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times , About 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times , At least about 5 times, or at least about 10 times.

In some embodiments, the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.

In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety.

In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.

In some embodiments, each sugar moiety is a modified sugar moiety.

In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, Alternatively, it consists of 12 to 15 nucleobases.

In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100 of the target portion of the RIC mRNA precursor encoding the protein. % Complementary.

In some embodiments, the cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA is retained by more than one. Introns are included, and where the antisense oligomer binds to the most abundant retained introns in the population of RIC pre-mRNA.

In some embodiments, binding of the antisense oligomer to the most abundant retained intron is the retention of two or more from the population of RIC pre-mRNA to produce mRNA encoding the target protein or functional RNA. Induces splicing out of the intron.

In some embodiments, the cell comprises a population of RIC pre-mRNA transcribed from a gene encoding a target protein or functional RNA, wherein the population of RIC pre-mRNA is retained by more than one. It comprises an intron, where the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNA.

In some embodiments, binding of the antisense oligomer to the second abundant retained intron is more than one from a population of RIC pre-mRNA to produce mRNA encoding a target protein or functional RNA. Induces the splicing out of retained introns.

In some embodiments, the disease is a disease or disorder.

In some embodiments, the disease or disorder is kidney disease.

In some embodiments, the kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants. ..

In some embodiments, the target protein and RIC mRNA precursor are encoded by a gene, where the gene is CTNS, PAX2, CYP24A1, or PPARD.

In some embodiments, the method further comprises the step of assessing protein expression.

In some embodiments, the subject is a human.

In some embodiments, the subject is a non-human animal.

In some embodiments, the subject is a fetus, embryo, or child.

In some embodiments, the cells are Exvivo.

In some embodiments, the antisense oligomer is administered by intraperitoneal, intramuscular, subcutaneous, or intravenous injection of the subject.

In some embodiments, the exons -3e to -1e flanking the 5'splice site and the nine nucleotides at +1 to +6 of the retained intron are identical to the corresponding wild type sequence.

In some embodiments, the 16 nucleotides at -15 to -1 of the retained intron and + 1e of the exon flanking the 3'splice site are identical to the corresponding wild type sequence.

In one aspect, antisense oligomers such as those used in the methods described herein are provided herein.

In one embodiment, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for any one of SEQ ID NO: 20-10733. Provided herein are antisense oligomers containing sequences with sequence identity.

In one aspect, a pharmaceutical composition comprising the antisense oligomers and excipients described herein is provided herein.

In one embodiment, there is provided herein a method of treating a subject by administering the pharmaceutical composition described herein by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. To.

In one embodiment, used in a method of increasing the expression of a target protein or functional RNA by a cell to treat a subject's kidney disease associated with a deficient protein or deficient functional RNA. Compositions comprising antisense oligomers are provided herein where the deficient protein or functional RNA is deficient in quantity or activity in the subject, where the antisense oligomer is. Enhances the constitutive splicing of retained intron-containing mRNA precursors (RIC mRNA precursors), which encode the target protein or functional RNA, where the target protein is: the deficient protein; or the test. A compensatory protein that functionally increases or replaces a deficient protein in the body, where the functional RNA is: deficient RNA; or subject's deficient function. A compensatory functional RNA that functionally increases or replaces a protein, where the RIC mRNA precursor is a retained intron, an exson adjacent to a 5'splice site, and 3'. The retained intron containing the exson adjacent to the splice site is spliced from the RIC mRNA precursor encoding the target protein or functional RNA, thereby producing or activating the target protein or functional RNA in the subject. To increase.

In some embodiments, the kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants. ..

In one embodiment, a composition comprising an antisense oligomer for use in a method of treating a disease associated with a target protein in a subject is provided herein, wherein the method of the target protein by the subject's cells. Including the step of increasing expression, the cell is adjacent to the retained intron, the exson adjacent to the 5'splice site of the retained intron, and the 3'splice site of the retained intron. Having a retained intron-containing mRNA precursor (RIC mRNA precursor), including exson, the RIC mRNA precursor encodes a target protein, the method comprising contacting the cell with an antisense oligomer. Thereby, the retained intron is constitutively spliced from the transcript of the RIC mRNA precursor encoding the target protein, thereby the level of the mRNA encoding the target protein or functional RNA within the subject's cells. And increase the expression of the target protein.

In some embodiments, the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome proliferator-activated receptor δ.

In some embodiments, the disease is a disease or disorder.

In some embodiments, the disease or disorder is kidney disease.

In some embodiments, the kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants. ..

In some embodiments, the target protein and RIC mRNA precursor are encoded by a gene, where the gene is CTNS, PAX2, CYP24A1 or PPARD.

In some embodiments, the antisense oligomer is in the region of +6 to the 5'splice site of the retained intron to -16 to the 3'splice site of the retained intron in the retained intron, RIC. Target some of the pre-mRNA.

In some embodiments, the target portion of the RIC pre-mRNA is within the region of +69 to the 5'splice site of the retained intron to -79 relative to the 3'splice site of the retained intron in the retained intron. It is in.

In some embodiments, the target protein is cystinocin.

In some embodiments, the target portion of the RIC pre-mRNA is within the region of +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. It is in.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 97%, 98% with respect to any one of SEQ ID NO: 8624-10068. , 99%, or 100% complementary sequences.

In some embodiments, the target portion of the RIC mRNA precursor is at least 80%, 85%, 90%, 95%, relative to the region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10748 or 10734. Includes sequences with 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% with respect to any one of SEQ ID NO: 8624-10068. , 99% or 100% sequence identity.

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: 16 or 17. Includes sequences with% sequence identity.

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: 3. It is encoded by a gene sequence that has sequence identity.

In some embodiments, the target protein is the paired box gene 2 protein.

In some embodiments, the target portion of the RIC pre-mRNA is within the region of +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. It is in.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% with respect to any one of SEQ ID NO: 6935-8623. , 98%, 99% or 100% complementary sequences.

In some embodiments, the target portion of the RIC mRNA precursor is at least 80%, 85%, 90%, 95%, relative to a region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10738 or 10740. Includes sequences with 96%, 97%, 98%, 99% or 100% sequence identity.

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: 6935-8623. Includes sequences with% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 10-15. , 99% or 100% sequence identity.

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: 2. It is encoded by a gene sequence that has sequence identity.

In some embodiments, the target protein is family 24, subfamily A, polypeptide 1 of the protein cytochrome P450.

In some embodiments, the target portion of the RIC pre-mRNA is within the region of +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. It is in.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97% for any one of SEQ ID NO: 9520-10733. , 98%, 99% or 100% complementary sequences.

In some embodiments, the target portion of the RIC pre-mRNA is at least 80%, 85%, relative to a region containing at least eight adjacent nucleic acids of SEQ ID NO: 10748, 10734, 10746, 10745 or 10741. Includes sequences with 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

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: 9520-10733. Includes sequences with% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 16-19. , 99% or 100% sequence identity.

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: 4. It is encoded by a gene sequence that has sequence identity.

In some embodiments, the target protein is a peroxisome proliferator-activated receptor δ.

In some embodiments, the target portion of the RIC pre-mRNA is within the region of +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. It is in.

In some embodiments, the target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 97%, 98% for any one of SEQ ID NO: 20-6952. , 99% or 100% complementary sequences.

In some embodiments, the target portion of the RIC mRNA precursor is relative to a region containing at least eight adjacent nucleic acids of SEQ ID NO: 10736, 10747, 10739, 10743, 10742, 10737, 10735, or 10744. Includes sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

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: 20-6952. Includes sequences with% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% for any one of SEQ ID NO: 5-9. , 99% or 100% sequence identity.

In some embodiments, the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% relative to SEQ ID NO: 1. It is encoded by a gene sequence that has sequence identity.

In some embodiments, the antisense oligomer is used in the retained intron.
Target a portion of the RIC pre-mRNA that is in the region +6 to +100 relative to the 5'splice site of the retained intron; or -16 to -100 relative to the 3'splice site of the retained intron.

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, the target portion of the RIC pre-mRNA is in the region + 2e to -4e in an exon adjacent to the 5'splice site of the retained intron; or to the 3'splice site of the retained intron. It is within the region of + 2e to -4e in the adjacent exon.

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, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.

In some embodiments, the target protein produced is a full-length protein or a wild-type protein.

In some embodiments, the retained intron is a rate-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 comprising a phosphorothioate bond or a phosphorodiamidate bond.

In some embodiments, the antisense oligomer is an antisense oligonucleotide.

In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety.

In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.

In some embodiments, each sugar moiety is a modified sugar moiety.

In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12 Consists of 15 nucleobases.

In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% of the target portion of the RIC mRNA precursor encoding the protein. , Or 100% complementary.

In one aspect, the specification provides a pharmaceutical composition comprising the antisense oligomers and excipients described herein.

In one aspect, the present specification treats a subject in need by administering the pharmaceutical composition described herein by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. The method is provided.

In one embodiment, the pharmaceutical composition is provided herein, wherein the pharmaceutical composition is an antisense oligomer that hybridizes to the target sequence of the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript. The deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript contained the retained intron and the antisense oligomer spliced out the retained intron from the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript. Includes antisense oligomers; and pharmaceutically acceptable excipients that induce.

In some embodiments, the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is a transcript of the CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor.

In some embodiments, the target portion of the pre-mRNA of CTNS, PAX2, CYP24A1 or PPARD is from +500 to the 5'splice site of the retained intron in the retained intron to the 3'splice of the retained intron. Within -500 regions relative to the site.

In some embodiments, the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD is at least about 80%, 85%, 90% with respect to any one of SEQ ID NO: 1-4. , 95%, 96%, 97%, 98%, 99% or 100% encoded by a gene sequence with sequence identity.

In some embodiments, the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD is at least about 80%, 85%, 90% with respect to any one of SEQ ID NO; 5-19. , 95%, 96%, 97%, 98%, 99% or 100% sequences having sequence identity.

In some embodiments, the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.

In some embodiments, the antisense oligomer is an antisense oligonucleotide.

In some embodiments, the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, 2'-fluoro, or a 2'-O-methoxyethyl moiety.

In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.

In some embodiments, the antisense oligomers are 8-50 nucleobases, 8-40 nucleobases, 8-35 nucleobases, 8-30 nucleobases, 8-25 nucleobases, 8-20. Nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 Nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases Bases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases , 11-15 nucleobases, 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12 Contains up to 15 nucleobases.

In some embodiments, the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, relative to the target portion of the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD. It is at least 98%, at least 99%, or 100% complementary.

In some embodiments, the target portion of the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD is within the sequence selected from SEQ ID NO: 10043-10748.

In some embodiments, the antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94% with respect to any one of SEQ ID NO: 20-10733. , 95%, 96%, 97%, 98%, or 99% contains nucleotide sequences that are sequence identity.

In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 20-10733.

In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

In one aspect, the specification herein is a fully treated mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD that encodes a functional form of a protein of CTNS, PAX2, CYP24A1 or PPARD (a method comprising:). Provided is a method of inducing treatment of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript to facilitate removal of retained introns, wherein the antisense oligomer is used in the subject. Contact with target cells; a step of hybridizing the antisense oligomer to the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript, wherein the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is CTNS. , PAX2, CYP24A1 or PPARD protein functional morphology, including at least one retained intron; step; CTNS, PAX2, CYP24A1 or PPARD protein functional morphology encoded, fully processed. The step of removing at least one retained intron from the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript to produce a CTNS, PAX2, CYP24A1 or PPARD mRNA transcript; and well treated. It comprises translating the functional form of the protein of CTNS, PAX2, CYP24A1 or PPARD from the mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD.

In some embodiments, the retained intron is the entire retained intron.

In some embodiments, the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is a transcript of the CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor.

In one aspect, the present specification provides a method of treating a subject having a disease caused by a lack of protein amount or activity of CTNS, PAX2, CYP24A1 or PPARD, wherein the method is SEQ ID NO: :. At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for any one of 20-10733. Including a step of administering an antisense oligomer containing a nucleotide sequence having sequence identity to a subject.

Incorporation by Citation All publications, patents, and patent applications referred to herein are herein to the extent that individual publications, patents, and patent applications are specifically and individually indicated to be incorporated by citation. Incorporated into the book.

The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description illustrating exemplary embodiments in which the principles of the invention are utilized and the accompanying drawings.

FIG. 1 shows a schematic representation of a typical retained intron-containing (RIC) pre-mRNA transcript. The 5'splice site consensus sequence is an underlined letter (letter) from -3e to -1e and from +1 to +6 (numbers labeled "e" are exons and unlabeled numbers are introns). Is a nucleotide; uppercase: exon part, and lower case: intron part). The 3'splice site consensus sequence is an underlined letter (letter is nucleotide) from -15 to -1 and + 1e (numbers labeled "e" are exons and unlabeled numbers are introns). (Uppercase: exon part, and lowercase: intron part). Intron target regions for ASO screening range from nucleotide +6 to the 5'splice site (left arrow) of the retained intron to nucleotide-16 to the 3'splice site (right arrow) of the retained intron. including. In embodiments, the intron target region for ASO screening comprises nucleotides +6 to +100 for the 5'splice site of the retained intron and nucleotides -16 to -100 for the 3'splice site of the retained intron. .. Exon target sites are + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, and + 2e to -4e nucleotides in the exon adjacent to the 3'splice site of the retained intron. include. "N" or "N" represents a nucleotide and "y" represents a pyrimidine. The illustrated sequence represents a consensus sequence for the mammalian splice site, and the individual introns and exons need not match the consensus sequence at all positions. FIG. 2A shows a typical schematic of targeted enhancement (TANGO) of nuclear gene output. FIG. 2A shows cells divided into nuclei and cytoplasmic compartments. In the nucleus, transcripts of pre-mRNA of target genes consisting of exons (rectangular) and introns (connecting lines) are spliced to produce mRNA, which is transported to the cytoplasm and translated into the target protein. Will be done. For this target gene, splicing of intron 1 is inefficient, and retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus and degrades when transported to the cytoplasm to produce the target protein. Does not lead to. FIG. 2B shows a typical schematic of targeted enhancement (TANGO) of nuclear gene output. FIG. 2B shows an example of the same cells as FIG. 2A, which was divided into nuclei and cytoplasmic compartments. Treatment with antisense oligomers (ASOs) promotes splicing of intron 1 resulting in an increase in mRNA, which in turn is translated into high levels of target protein. FIG. 3A outlines the ReSeq gene for CTNS corresponding to NM_004937 and NM_001031681. The intron retention rate (PIR) of the circled intron is shown. FIG. 3B outlines the ReSeq gene for CTNS corresponding to NM_004937 and NM_001031681. The PIR of the circled intron is shown. FIG. 3C shows the mean (n = 3) fold change in the expression level of CTNS mRNA without intron 10 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours compared to sham-treated cells. A typical graph is shown. The data are normalized to RPL32 expression. FIG. 4A outlines the ReSeq gene for CYP24A1 corresponding to NM_000782 and NM_001128915. The PIR of the circled intron is shown. FIG. 4B outlines the ReSeq gene for CYP24A1 corresponding to NM_000782 and NM_001128915. The PIR of the circled intron is shown. FIG. 4C shows the mean (n = 3) fold change in the expression level of CYP24A1 mRNA without intron 10 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours compared to sham-treated cells. A typical graph is shown. The data are normalized to RPL32 expression. FIG. 5 outlines the ReSeq gene for PAX2 corresponding to NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987 and NM_003989. FIG. 6A outlines the RefSeq gene for PPARD corresponding to PPARD: NM_006238, NM_177435, NM_001171818, NM_001171819 and NM_001171820. The intron retention rate (PIR) of the circled intron is shown (PPARD intron 3, NM_006238). FIG. 6B shows the mean (n = 3) fold change in the expression level of PPARD mRNA without intron 3 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours compared to sham-treated cells. A typical graph is shown. The data are normalized to RPL32 expression. FIG. 6C outlines the RefSeq gene for PPARD corresponding to PPARD: NM_006238, NM_177435, NM_001171818, NM_001171819 and NM_001171820. The intron retention rate (PIR) of the circled intron is shown (PPARD intron 4, NM_006238). FIG. 6D outlines the RefSeq gene for PPARD corresponding to PPARD: NM_006238, NM_177435, NM_001171818, NM_001171819 and NM_001171820. The intron retention rate (PIR) of the circled intron is shown (PPARD intron 5, NM_006238). FIG. 6E outlines the RefSeq gene for PPARD corresponding to PPARD: NM_006238, NM_177435, NM_001171818, NM_001171819 and NM_001171820. The intron retention rate (PIR) of the circled intron is shown (PPARD intron 6, NM_006238). FIG. 6F shows the mean (n = 3) fold change in the expression level of PPARD mRNA without intron 6 in ARPE-19 cells treated with 80 nM of indicated ASO for 24 hours compared to sham-treated cells. A typical graph is shown. The data are normalized to RPL32 expression. FIG. 6G outlines the RefSeq gene for PPARD corresponding to PPARD: NM_006238, NM_177435, NM_001171818, NM_001171819 and NM_001171820. The intron retention rate (PIR) of the circled intron is shown (PPARD intron 7, NM_006238).

Renal failure is a debilitating condition that is estimated to affect more than 600,000 people in the United States alone. In many cases, dialysis can extend lifespan, with an estimated 400,000 patients undergoing renal dialysis, according to a 2009 study. Moreover, while kidney transplantation is the only hope for many patients suffering from renal failure, donor pools are sufficient to treat only a small number of patients in need of it. Therefore, the probability of receiving a transplant is low, and there are few measures that can improve the condition of those who cannot receive a transplant. Therefore, there is a need for compositions and methods for treating kidney disease.

Individual introns in the primary transcript of a protein-encoding gene with more than one intron 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 unspliced transcripts is generally believed 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 post-transcriptional rate-determining step in gene expression prior to translation in the cytoplasm.

Partially spliced transcripts of significant levels of genes encoding deficient proteins in a subset of kidney disease have been found in the nucleus of human cells.
These pre-mRNA species contain at least one retained intron. The present invention is rate-determining to the nuclear stage of gene expression in order to increase the production of a stable state of fully spliced mature mRNA and, therefore, to increase the translated-protein level1 To provide compositions and methods for upregulating the splicing of one or more retained introns. These compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at the intron splice sites of retained intron-containing pre-mRNA that accumulate in the nucleus. Therefore, in embodiments, protein is increased using the methods of the invention to treat diseases caused by protein deficiency.

The kidney disease described herein that can be treated by the present invention is a disease in which the subject is deficient in the gene product, where the deficiency of the gene product causes kidney disease.

These CTNS, PAX2, CYP24A1 and PPARD pre-mRNA species contain at least one retained intron. The present invention is at the nuclear stage of gene expression to increase the production of a stable state of fully spliced mature mRNA and therefore to increase the protein levels of the translated CTNS, PAX2, CYP24A1 or PPARD. Provided are compositions and methods for upregulating the splicing of one or more retained CTNS, PAX2, CYP24A1 or PPARD introns that are rate-determining. These compositions and methods are antisense oligomers that promote constitutive splicing at the intron splicing site of retained intron-containing CTNS, PAX2, CYP24A1 or PPARD pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus. ASO) can be utilized. Therefore, in embodiments, the method of the invention is used to treat a disease caused by a deficiency of CTNS, PAX2, CYP24A1 or PPARD to increase the protein of CTNS, PAX2, CYP24A1 or PPARD.

In other embodiments, the methods of the invention can be used to increase the production of CTNS, PAX2, CYP24A1 or PPARD to treat the subject's symptoms in need. In embodiments, the subject has a disease in which CTNS, PAX2, CYP24A1 or PPARD is not necessarily deficient as compared to wild type, in which case an increase in CTNS, PAX2, CYP24A1 or PPARD nevertheless. Reduce the disease. In embodiments, the disease can be caused by haploinsufficiency of CTNS, PAX2, CYP24A1 or PPARD.

Renal Disorder Cystine Accumulation / Delayed Cystine Accumulation In some embodiments, the invention described herein can be used to treat autosomal recessive cystine accumulation in infants with cystine accumulation. .. Infant nephropathy cystine storage is characterized by the accumulation of cystine in lysosomes. Clinical manifestations of infant nephropathy cystine accumulation include hypoglycemia and electrolytes, excessive excretion of urinary proteins, slow physical development, weak bones, hypothyroidism, blindness, muscle weakness, pulmonary dysfunction and renal failure. Infant nephropathy cystine accumulation is typically diagnosed in early childhood.

In some embodiments, the invention described herein can be used to treat delayed cystine accumulation of autosomal recessive nephropathy. Late-onset cystine accumulation has similar clinical manifestations as the infant type, but the late-onset type typically manifests in either late adolescence or early adulthood.

A deficiency of the protein cystinocin (CTNS) results in the clinical manifestations found in both infant nephropathy cystine storage and late-onset cystine storage. The CTNS gene, located at 17q13.2 and spanning 12 exons, encodes the CTNS protein. Mutations in the CTNS gene that result in inadequate amounts of CTNS protein have been shown to be responsible for the progression of both infant nephropathy cystine accumulation and late-onset cystine accumulation. In one study, a G169D missense mutation was found in a patient with infantile nephropathy cystine accumulation. This missense mutation resulted in reduced levels of CTNS, providing a clear relationship between CTNS deficiency and progression of infantile nephropathy cystine accumulation.

Focal segmental glomerulosclerosis 7 / papillary renal syndrome In some embodiments, the invention described herein treats autosomal dominant renal impairment focal segmental glomerulosclerosis 7 (FSGS7). Can be used to. FSGS7 is one of the leading causes of renal failure in adults. FSGS7 characterizes edema, hypoalbuminemia, hyperlipidemia and hypertension, ultimately resulting in renal failure.

In some embodiments, the invention described herein can be used to treat papillary kidney syndrome (PAPRS), an autosomal dominant nephropathy. PAPRS is characterized by hypoplastic kidney, hypodysplasia, polycystic dysplastic kidney, giant glomerulopathy, renal failure, and vesicoureteral reflux.

Deletion of the protein paired box gene 2 (PAX2) results in the clinical manifestations found in both FSGS7 and PAPRS. The PAX2 gene, located at 10q24.31 and spanning 12 exons, encodes the PAX2 protein. Mutations in the PAX2 gene that result in inadequate amounts of PAX2 protein have been shown to be responsible for the progression of both FSGS7 and PAPRS. In one study, a missense mutation in G76S was found in five generations of families suffering from PAPRS. This missense mutation resulted in reduced levels of PAX2 protein, providing a clear relationship between PAX2 protein deficiency and progression of PAPRS.

Infant Hypercalcemia In some embodiments, the invention described herein can be used to treat infant hypercalcemia in autosomal recessive renal disease. Infant hypercalcemia can result in renal stones, bone pain, abdominal pain, nausea, vomiting, polyuria, and mental states such as depression, anxiety, cognitive dysfunction, insomnia, and coma. It is characterized by elevated levels of calcium in the blood.

A deficiency of the proteins cytochrome P450 family 24, subfamily A, polypeptide 1 (CYP24A1) results in the clinical manifestations seen in infant hypercalcemia. The CYP24A1 gene, located at 20q13.2 and spanning 2 exons, encodes the CYP24A1 protein. Mutations in the CYP24A1 gene that result in inadequate amounts of CYP24A1 protein have been shown to be responsible for the progression of infantile hypercalcemia. One study investigated a large number of children diagnosed with infantile hypercalcemia. Children with a missense mutation in R396W or E322K were shown to undergo complete ablation of CYP24A1 activity, and children with a mutation in L409S retained low but measurable levels. This finding provides a clear relationship between the reduced CYP24A1 protein and the clinical manifestations of infantile hypercalcemia.

Lipid Metabolism Dysfunction and Chronic Kidney Disease In some embodiments, the invention described herein is a lipid dysfunction, chronic, caused by a deficiency of the protein peroxysome proliferator-responsive receptor δ (PPARD). It is used to treat kidney disease (CKD), end-stage renal disease (ESRD), or cardiovascular disease (CVD). Deletions in the amount of PPARD protein located on chromosome 6 and encoded by the PPARD gene spanning 8 exons have been shown to be associated with increased dysfunction of lipid metabolism.

Retained intron-containing mRNA precursor (RIC mRNA precursor)
In embodiments, the methods of the invention are retained intron-containing pre-mRNA (RIC pre-mRNA) that are transcribed from a gene and encode a protein that is found to be deficient in the diseases described herein. Utilize the presence of introns in the cell nucleus. Splicing of specific RIC pre-mRNA species to produce mature, fully spliced mRNA is induced using ASOs that stimulate the splicing out of retained introns. The resulting mature mRNA is transported to the cytoplasm and translated, thereby increasing the amount of protein in the subject's cells and alleviating the disease or symptoms of the disease described herein. In embodiments, the methods of the invention are transcribed from the CTNS, PAX2, CYP24A1, or PPARD gene and are retained intron-containing pre-mRNA (RIC mRNA precursors) encoding the CTNS, PAX2, CYP24A1, or PPARD protein. The presence of can be utilized in the cell nucleus. Splicing of CTNS, PAX2, CYP24A1, or PPARD RIC pre-mRNA species to produce mature, fully spliced CTNS, PAX2, CYP24A1, or PPARD mRNA splicing out of retained introns. Can be induced using ASO that stimulates. The resulting mature CTNS, PAX2, CYP24A1 or PPARD mRNA is transported to the cytoplasm for translation, thereby increasing the amount of CTNS, PAX2, CYP24A1 or PPARD protein in the patient's cells. And the disease or symptoms of CNS caused by a deficiency of CTNS, PAX2, CYP24A1 or PPARD can be alleviated. This method, described in more detail below, is known as increased targeting of nuclear gene output (TANGO).

In embodiments, retained introns are at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, An intron identified as a retained intron based on retention retention of at least about 45% or at least about 50%. In embodiments, the retained introns are, ie, about 5% to about 100%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, and 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% From 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 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65% , About 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% From 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%, from 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 15% to about 35% , About 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 65%, about 20% From 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%, from 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 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35 An intron identified as a retained intron based on the percentage retention retention rate. In multiple embodiments, other ASOs useful for this purpose are identified, for example, using the methods described herein.

In embodiments, the CTNS intron numbering corresponds to the mRNA sequence in NM_001031681 or NM_001031681. In embodiments, the target site for the CTNS RIC mRNA precursor is within introns 9 and / or 10. In embodiments, the target site for the CTNS RIC pre-mRNA is within the intron 10. In embodiments, the percentage of retained introns can be 18%. In embodiments, the target site for the CTNS RIC pre-mRNA is within intron 9. In embodiments, the percentage of retained introns can be 10%. In embodiments, hybridization of ASO to the target site of the RIC mRNA precursor results in enhanced splicing at the retained intron 9 and / or 10 splice sites (5'splice sites or 3'splice sites). , Subsequent increased CTNS protein production. By reference to different CTNS isoform sequences, it is understood that the intron numbering can change. One of ordinary skill in the art can determine the corresponding number in any isoform by using the number obtained by reference to the mRNA sequence in NM_001031681 or NM_001031681 based on the intron sequence provided herein. can. One of ordinary skill in the art will further use the intron number obtained by reference to the mRNA sequence in NM_001031681 or NM_001031681 based on the intron sequence provided herein for targeting using the method of the invention. The sequence of adjacent exons in any CTNS isoform can be determined.

In embodiments, the PAX2 intron numbering corresponds to the mRNA sequence in NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987, or NM_003989. In embodiments, the target site for the PAX2 RIC mRNA precursor is within intron 1 or 2. In embodiments, hybridization of ASO to the target site of the RIC mRNA precursor results in enhanced splicing at the retained intron 1 or 2 splice site (5'splice site or 3'splice site) and thereafter. Caused increased production of PAX2 protein. By reference to different PAX2 isoform sequences, it is understood that the intron numbering can change. One of skill in the art will respond in any isoform based on the intron sequence provided herein or using the number provided for the mRNA sequence in NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987, or NM_003989. The intron number to be used can be determined. One of ordinary skill in the art will use the method of the invention based on the intron sequence provided herein or using the intron number provided for the mRNA sequence in NM_001304569, NM_000278, NM_003990, NM_003988, NM_003987, or NM_003989. , The sequence of adjacent exons in any PAX2 isoform to be targeted can also be determined.

In embodiments, the CYP24A1 intron numbering corresponds to the mRNA sequence in NM_000782 or NM_001128915. In embodiments, the target site for the CYP24A1 RIC pre-mRNA is intron 10 and / or 9 or intron 11. In embodiments, the target site for the CYP24A1 RIC pre-mRNA is within intron 9. In embodiments, the target site for the CYP24A1 RIC mRNA precursor is within intron 11. In embodiments, the percentage of retained introns can be 23%. In embodiments, the target site for the CYP24A1 RIC mRNA precursor is within the intron 10. In embodiments, the percentage of retained introns can be 50%. In embodiments, hybridization of ASO to the target site of the RIC mRNA precursor results in retained introns 10 and / or 9 or at the intron 11 splice site (5'splice site or 3'splice site). It resulted in enhanced splicing and subsequent increased CYP24A1 protein production. By reference to different CYP24A1 isoform sequences, it is understood that the intron numbering can be varied. One of ordinary skill in the art can determine the corresponding number in any isoform by using the number obtained by reference to the mRNA sequence in NM_000782 or NM_001128915, based on the intron sequence provided herein. can. One of ordinary skill in the art will further use the intron number obtained by referring to the mRNA sequence in NM_000782 or NM_001128915 based on the intron sequence provided herein for targeting using the method of the invention. The sequence of adjacent exons in any CYP24A1 isoform can be determined.

In embodiments, the PPARD intron numbering corresponds to the mRNA sequence in NM_006238, NM_177435, NM_001171818, NM_001171819, or NM_001171820. In embodiments, the target portion of the PPARD RIC pre-mRNA is intron 3, or 4, or 5, or 6, or 7, and / or intron 2, or 3, or 4, or 5, or 6, or 7, Or it is in 8. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 2. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 3. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 4. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 5. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 6. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 7. In embodiments, the target portion of the PPARD RIC mRNA precursor is within intron 8. In embodiments, hybridization of ASO to the target portion of the RIC mRNA precursor results in enhanced splicing at the retained intron splice site (5'splice site or 3'splice site), followed by PPARD protein production. To increase. It is understood that the numbering of introns may change by reference to different PPARD isoform sequences. One of skill in the art is based on the intron sequence provided herein or using the number provided for the mRNA sequence in NM_006238, NM_177435, NM_001171818, NM_001171819, or NM_001171820, and the corresponding intron in any isoform. The number can be determined. One of ordinary skill in the art is based on the intron sequence provided herein, or using the method of the invention using the intron number provided for the mRNA sequence in NM_006238, NM_177435, NM_001171818, NM_001171819, or NM_001171820. It is also possible to determine the sequence of adjacent exons in any PPARD isoform to be used.

In some embodiments, the ASO disclosed herein targets a RIC mRNA precursor transcribed from the genomic sequence of CTNS, PAX2, CYP24A1, or PPARD. In some embodiments, the ASO disclosed herein targets the sequence of the RIC mRNA precursor of CTNS, PAX2, CYP24A1, or PPARD.

In some embodiments, the ASO targets the sequence of transcripts of the RIC mRNA precursor encoded by the CTNS genomic sequence. In some embodiments, ASO targets transcripts of RIC pre-mRNA encoded by the genomic sequence of CTNS, including retained introns 9 and / or 10. In some embodiments, the ASO targets the RIC mRNA precursor encoded by SEQ ID NO: 3. In some embodiments, ASO targets the transcript of the RIC pre-mRNA with SEQ ID NO: 16 or 17. In some embodiments, the ASO targets a transcript of the RIC pre-mRNA of SEQ ID NO: 16 or 17 comprising retained introns 9 and / or 10. In some embodiments, the ASO disclosed herein targets SEQ ID NO: 10748 and / or 10734. In some embodiments, the ASO comprises the sequence of any one of SEQ ID NO: 20-6952.

In some embodiments, ASO targets the sequence of transcripts of the RIC mRNA precursor encoded by the genomic sequence of PAX2. In some embodiments, the ASO targets a transcript of the RIC pre-mRNA encoded by the genomic sequence of PAX2 containing retained introns 1 and / or 2. In some embodiments, ASO targets the transcript of the RIC pre-mRNA encoded by SEQ ID NO: 2. In some embodiments, the ASO targets the transcript of one or more of the SEQ ID NOs: 10-15 RIC pre-mRNA. In some embodiments, the ASO targets transcripts of one or more RIC pre-mRNA of SEQ ID NO: 10-15, including retained introns 1 or 2. In some embodiments, the ASO disclosed herein targets SEQ ID NO: 10740 and / or 10738. In some embodiments, the ASO comprises the sequence of any one of SEQ ID NO: 6935-8623.

In some embodiments, the ASO targets the sequence of transcripts of the RIC mRNA precursor encoded by the genomic sequence of CYP24A1. In some embodiments, the ASO targets a transcript of the RIC mRNA precursor encoded by the genomic sequence of CYP24A1 containing retained introns 10 and / or 9, or introns 11. In some embodiments, ASO targets the transcript of the RIC mRNA precursor encoded by SEQ ID NO: 4. In some embodiments, ASO targets the transcript of the RIC pre-mRNA with SEQ ID NO: 18 or 19. In some embodiments, the ASO targets the transcript of the retained intron 10 and / or 9, or the RIC pre-mRNA of SEQ ID NO: 18 or 19 comprising intron 11. In some embodiments, the ASO disclosed herein targets SEQ ID NO: 10748, 10734, 10746, 10745, and / or 10741. In some embodiments, the ASO comprises the sequence of any one of SEQ ID NO: 9520-10733.

In some embodiments, the ASO targets the sequence of transcripts of the RIC pre-mRNA encoded by the genomic sequence of PPARD. In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Target the transcript of the RIC pre-mRNA encoded by the genomic sequence of PPARD containing 7, or 8. In some embodiments, ASO targets the transcript of the RIC mRNA precursor encoded by SEQ ID NO: 1. In some embodiments, the ASO targets the transcript of one or more of the SEQ ID NO: 5-9 RIC pre-mRNA. In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Target the transcript of one or more of the SEQ ID NOs: 5-9, including 7, or 8. In some embodiments, the ASO targets SEQ ID NO: 10736, 10747, 10739, 10743, 10742, 10737, 10735, and / or 10744. In some embodiments, the ASO comprises the sequence of any one of SEQ ID NO: 20-6952.

In some embodiments, the ASO targets exons 9 and / or 10 of the CTNS RIC pre-mRNA, including retained introns 9 and / or 10. In some embodiments, the ASO targets exon 9 and / or 10 sequences upstream (or 5') of the 5'splice site of the CTNS RIC pre-mRNA, including retained introns 9 and / or 10. And.

In some embodiments, the ASO targets introns 9 and / or 10 in the RIC pre-mRNA of CTNS, including retained introns 9 and / or 10. In some embodiments, the ASO targets intron 9 and / or 10 sequences downstream (or 3') of the 5'splice site of the CTNS RIC pre-mRNA, including retained introns 9 and / or 10. And.

In some embodiments, the ASO targets intron 9 and / or 10 sequences upstream (or 5') of the 3'splice site of the CTNS RIC pre-mRNA, including retained introns 9 and / or 10. And.

In some embodiments, the ASO targets exons 10 and / or 11 in the RIC pre-mRNA of CTNS, including retained introns 9 and / or 10. In some embodiments, the ASO targets exon 10 and / or 11 sequences downstream (or 3') of the 3'splice site of the CTNS RIC pre-mRNA, including retained introns 9 and / or 10. And.

In some embodiments, the ASO targets exons 1 or 2 of the RIC pre-mRNA of PAX2, including retained introns 1 or 2. In some embodiments, the ASO targets an exon 1 or 2 sequence upstream (or 5') of the 5'splice site of the RIC pre-mRNA of PAX2, including retained intron 1 or 2.

In some embodiments, the ASO targets introns 1 or 2 in the RIC pre-mRNA of PAX2, including retained introns 1 or 2. In some embodiments, the ASO targets intron 1 or 2 sequences downstream (or 3') of the 5'splice site of the RIC pre-mRNA of PAX2, including retained intron 1 or 2.

In some embodiments, the ASO targets an intron 1 or 2 sequence upstream (or 5') of the 3'splice site of the RIC pre-mRNA of PAX2, including retained intron 1 or 2.

In some embodiments, the ASO targets exons 2 or 3 in the RIC pre-mRNA of PAX2, including retained introns 1 or 2. In some embodiments, the ASO targets exon 2 or 3 sequences downstream (or 3') of the 3'splice site of the RIC pre-mRNA of PAX2, including retained introns 1 or 2.

In some embodiments, the ASO targets exons 10 and / or 9 or exons 11 of the CYP24A1 RIC pre-mRNA, including retained introns 10 and / or 9, or introns 11. In some embodiments, the ASO contains exons 10 and / or 9 upstream (or 5') of the 5'splice site of the CYP24A1 RIC pre-mRNA, including retained introns 10 and / or 9, or introns 11. Alternatively, target the 9 or exon 11 sequence.

In some embodiments, the ASO targets introns 10 and / or 9, or introns 11 in the RIC mRNA precursor of CYP24A1, which comprises retained introns 10 and / or 9, or introns 11. In some embodiments, the ASO contains introns 10 and / or 9 retained, or introns 10 and / / downstream of the 5'splice site of the CYP24A1 RIC pre-mRNA. Alternatively, target the 9 or intron 11 sequence.

In some embodiments, the ASO contains introns 10 and / or 9 retained, or introns 10 and / / upstream of the 3'splice site of the CYP24A1 RIC pre-mRNA. Alternatively, target the 9 or intron 11 sequence.

In some embodiments, the ASO targets exons 11 and / or 10 or exons 12 in the RIC mRNA precursor of CYP24A1, which comprises retained introns 10 and / or 9, or introns 11. In some embodiments, the ASO contains exons 11 and / or 9 downstream (or 3') of the 3'splice site of the CYP24A1 RIC pre-mRNA, including retained introns 10 and / or 9, or introns 11. Alternatively, target 10 or exon 12 sequences.

In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Exons 3, or 4, or 5, or 6, or 7, and / or exons 2, or 3, or 4, or 5, or 6, or 7, the RIC mRNA precursors of PPARD, including 7, or 8. Or target 8. In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Exons 3, or 4, or 5, or 6, or 7, and / or exons 2, or 3, or 4, upstream (or 5') of the 5'splice site of PPARD, including 7, or 8 Target sequences 5, 6, or 7, or 8.

In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Intron 3, or 4, or 5, or 6, or 7, and / or intron 2, or 3, or 4, or 5, or 6, or 7, in the RIC mRNA precursor of PPARD containing 7, or 8. Or target 8. In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Intron 3 or 4, or 3, or 6, or 7, and / or intron 2, or 3, or 4, downstream of (or 3') the 5'splice site of PPARD, including 7, or 8. Target 5, or 6, or 7, or 8 sequences.

In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Intron 3 or 4, or 5, or 6, or 7, and / or intron 2, or 3, or 4, upstream of (or 5') the 3'splice site of PPARD, including 7, or 8. Target sequences 5, 6, or 7, or 8.

In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Exons 4, or 5, or 6, or 7, or 8, and / or exons 3, or 4, or 5, or 6, or 7, or 8, in the RIC mRNA precursor of PPARD, including 7, or 8. Or target 9. In some embodiments, the ASO is a retained intron 3, or 4, or 5, or 6, or 7, and / or a retained intron 2, or 3, or 4, or 5, or 6, or Exons 4, or 5, or 6, or 7, or 8, and / or exons 3 or 4 downstream (or 3') of the 3'splice site of PPARD's RIC pre-mRNA, including 7, or 8 , Or 5, or 6, or 7, or 8, or 9 sequences are targeted.

Protein Expression In embodiments, the methods described herein are used to increase the production of functional proteins. As used herein, the term "functional" refers to the amount of protein activity or function required to eliminate one or more symptoms of the disease being treated. In embodiments, the method is used to increase the production of partially functional proteins. As used herein, the term "partially functional" is the activity of a protein that is less than the amount of activity or function required to eliminate or prevent one or more symptoms of a disease or disease. Or refers to any amount of function. In some embodiments, the partially functional protein or RNA is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least. It will have 80%, 85%, at least 90%, or at least 95% less activity compared to a fully functional protein or RNA.

In embodiments, the method is a method of increasing protein expression by a subject's cells having a protein-encoding RIC mRNA precursor, wherein the subject is of the amount of protein activity described herein. Has the disease described herein due to a deletion. In some embodiments, the protein amount deletion is due to protein haploinsufficiency. In such an embodiment, the subject has a first allele encoding a functional protein and a second allele that does not produce the protein. In another such embodiment, the subject has a first allele encoding a functional protein and a second allele encoding a non-functional protein. In another such embodiment, the subject has a first allele encoding a functional protein and a second allele encoding a partially functional protein. In any of these embodiments, the antisense oligomer binds to the target site of the RIC pre-mRNA transcribed from the first allogeneic gene (encoding the functional protein), thereby the RIC pre-mRNA. Induces constitutive splicing of retained introns from and results in an increase in the amount of mature mRNA encoding a functional protein and an increase in protein expression in the cells of the subject.

In embodiments, the subject has a first allele encoding a functional protein and a second allele encoding a partially functional protein, the pre-mRNA is from the first allele. It binds to the target site of the transcribed RIC mRNA precursor, or to the target site of the RIC mRNA precursor transcribed from a second allele (encoding a partially functional protein). Thereby, it induces constitutive splicing of retained introns from the RIC pre-mRNA, increasing the amount of mature mRNA encoding the protein and increasing the expression of functional or partially functional protein in the cells of the subject. Bring.

In a related embodiment, the method is a method using ASO to increase expression of protein or functional RNA. In embodiments, ASO is used to increase the expression of the proteins described herein in the cells of a subject having a RIC mRNA precursor that encodes the protein. The subject has a lack of protein quantity or function.

In embodiments, transcripts of RIC mRNA precursors that encode the disease or disease-causing protein are targeted by the ASOs described herein. In some embodiments, the transcript of the RIC pre-mRNA that encodes the protein is not the cause of the disease targeted by ASO. For example, diseases caused by mutations or deficiencies of the first protein in a particular pathway are ameliorated by targeting the RIC mRNA precursor encoding the second protein, thereby increasing the production of the second protein. sell. In some embodiments, the function of the second protein can compensate for a mutation or deficiency of the first protein, which is the cause of the disease or disease.

In embodiments, the subject is
a. The first mutant allele, from which
i) Protein is produced at reduced levels compared to production from wild-type alleles,
ii) The protein is produced in a form with reduced function compared to the equivalent wild-type protein, or ii) the protein or functional RNA is not produced.
First mutant allele; and b. The second mutant allele, from which
i) Protein is produced at reduced levels compared to production from wild-type alleles,
ii) The protein is produced in a form with reduced function compared to the equivalent wild-type protein, or ii) the protein is not produced.
Has a second mutated allele and
Here, the RIC mRNA precursor is transcribed from the first allele and / or the second allele. In these embodiments, the ASO is constitutive of the retained intron from the RIC mRNA precursor by binding to the target portion of the RIC mRNA precursor transcribed from the first or second allelic gene. It induces splicing, causes an increase in the amount of protein-encoding mRNA in the subject's cells, and results in increased expression of the target protein or functional RNA. In these embodiments, constitutive splicing of retained introns from the RIC pre-mRNA causes the targeted protein or functional RNA with increased expression levels to function as compared to the equivalent wild-type protein (partial function). Is either a form that is low or has sufficient function compared to an equivalent wild-type protein (full function).

In embodiments, the levels of mRNA encoding the proteins described herein are treated with, for example, antisense oligomers that are not treated with antisense oligomers or that do not bind to the target portion of the RIC mRNA precursor, control cells. It increases 1.1 to 10-fold when compared to the amount of mRNA encoding the protein produced in it.

In embodiments, the disease resulting from a lack of protein quantity or activity is not a disease resulting from selective or aberrant splicing of retained introns targeted by the ASO. In embodiments, the disease resulting from a lack of protein quantity or activity is not a disease resulting from selective or aberrant splicing of retained introns in the protein-encoding RIC pre-mRNA. In embodiments, selective or aberrant splicing can occur in the pre-mRNA transcribed from the gene, but the compositions and methods of the invention do not interfere with, or interfere with, selective or aberrant splicing in the pre-mRNA. Do not fix.

In embodiments, subjects treated using the methods of the invention express partially functional proteins from one allele, and the partially functional proteins are frameshift mutations, nonsense mutations, missense mutations. Due to a mutation or partial gene deletion. In embodiments, subjects treated using the methods of the invention express non-functional proteins from one allele, where the non-functional proteins are frameshift mutations, nonsense mutations in one allele. , Missense mutation, or partial gene deletion. In embodiments, subjects treated using the methods of the invention have a total gene deletion in one allele.

Use of TANGO to Increase Protein Expression As mentioned above, in embodiments, targeted enhancement of nuclear gene output (TANGO) is used in the methods of the invention to increase protein expression. To. In these embodiments, the retained intron-containing pre-mRNA (RIC pre-mRNA) that encodes the protein is present in the cell nucleus. Cells with RIC pre-mRNA containing retained introns, exons flanking the 5'splice site, and exons flanking the 3'splice site are complementary to the target site of the RIC mRNA precursor, antisense. Contact with oligomers (ASO). ASO that hybridizes to the target site 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 to refer to any pre-mRNA species, including at least one intron. In embodiments, the pre-mRNA or pre-mRNA transcript comprises a 5'-7-methylguanosine cap and / or a poly A terminal. In embodiments, the pre-mRNA or pre-mRNA transcript comprises both a 5'-7-methylguanosine cap and a poly A terminal. In some embodiments, the transcript of the pre-mRNA does not contain a 5'-7-methylguanosine cap and / or a poly A terminal. If not translated into protein (or transported from the nucleus to the cytoplasm), the transcript of the pre-mRNA is a non-proliferative messenger RNA (mRNA) molecule.

As used herein, a "retained intron-containing pre-mRNA" ("RIC mRNA pre-mRNA") is a transcript of a pre-mRNA containing at least one retained intron. The RIC pre-mRNA comprises a retained intron, an exon flanking the 5'splice site of the retained intron, and an exon flanking the 3'splice site of the retained intron, encoding the target protein. The "RIC mRNA precursor encoding the target protein" is understood to encode the target protein when fully spliced. A "retained intron" is a pre-mRNA transcription when one or more other introns encoded by the same gene, eg, adjacent introns, are spliced out of the same pre-mRNA transcript. Any intron present in the product. In some embodiments, the retained intron is the most abundant intron among the RIC pre-mRNA encoding the target protein. In embodiments, the retained intron is the most abundant intron in the population of RIC pre-mRNA transcribed from the gene encoding the target protein in the cell, and the population of RIC pre-mRNA is more than one. Includes retained introns. In embodiments, the antisense oligomers targeted against the most abundant introns in the population of RIC pre-mRNA encoding the target protein are populations comprising retained introns targeted or bound by the antisense oligomers. In it, it induces splicing out of two or more retained introns. In embodiments, it produces mature mRNA encoding the target protein. The terms "mature mRNA" and "fully spliced mRNA" are fully processed mRNAs that encode the target protein (eg, mRNA that is transported from the nucleus to the cytoplasm and translated into the target protein), or complete. Used interchangeably herein to illustrate functional RNA processed into. The term "proliferative mRNA" can also be used to describe a fully processed mRNA that encodes a target protein. In embodiments, the target region is in a retained intron, which is the most abundant intron in the RIC pre-mRNA encoding the protein.

As used herein, variants such as the terms "comprise", or "comprises" or "comprising" are the features mentioned in detail (eg, anti). In the case of sense oligomers, it is understood that it contains the defined nucleic acid sequence) but does not preclude any other features. Accordingly, as used herein, the term "contains" is inclusive and includes additional features not mentioned in detail (eg, in the case of antisense oligomers, additional features not mentioned in detail). The presence of nucleic acid bases) is not excluded.

In any embodiment of the compositions and methods provided herein, "contains" is referred to as "consisting essentially of" or "consisting of". Can be replaced. The phrase "consisting substantially from" is used herein to require a particular feature (eg, a nucleic acid base sequence), as well as a feature that does not substantially affect the trait or function of the claimed invention. To. As used herein, the term "consisting" is used to indicate the presence of only the features mentioned in detail (eg, a nucleobase sequence) (hence, an anti consisting of a particular nucleobase sequence). In the case of sense oligomers, the presence of additional nucleobases not mentioned in detail is excluded).

In embodiments, the target region is in a retained intron, which is the second most abundant intron in the RIC pre-mRNA encoding the proteins described herein. For example, 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 the increased protein production resulting from the targeting of introns with ASO. Depending on the amount of, the second most abundant retained intron may be targeted instead of the most abundant retained intron. In embodiments, the retained intron is the second most abundant intron in the population of RIC pre-mRNA transcribed from the gene encoding the target protein in the cell, with more than one population of RIC pre-mRNA. Includes retained introns. In embodiments, the antisense oligomer targeted by the second most abundant antisense oligomer in the population of RIC mRNA precursors encoding the target protein is a retained intron to which the antisense oligomer is targeted or bound. Induces splicing out of two or more retained introns in the inclusion population. In embodiments, it produces fully spliced (mature) RNA encoding the target protein.

In embodiments, the ASO is complementary to the targeted region within the unretained intron in the RIC mRNA precursor. In embodiments, the target portion of the RIC mRNA precursor is within the region +6 to +100 relative to the 5'splice site of the unretained intron; or within the region -16 to -100 relative to the 3'splice site of the unretained intron. It is in. In embodiments, the target portion of the RIC mRNA precursor is in the region from +100 to the 5'splice site of the unretained intron to -100 to the 3'splice site of the unretained intron. .. As used to locate a region or sequence, "within" is understood to include residues at the listed positions. For example, the +6 to +100 region contains residues at +6 and +100 positions. In embodiments, it produces fully spliced (mature) RNA encoding the target protein.

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. It can refer to relatively infrequent splicing at (5'splice sites or 3'splice sites). The term "inefficiently spliced" also refers to the relative velocity or kinetics of splicing at the splicing site, where the "inefficiently spliced" intron is another intron in the RIC pre-mRNA. Can be spliced or removed at a slower rate compared to.

In embodiments, the 9 nucleotide sequences of the 5'splice site and the exons -3e to -1e flanking the retained introns +1 to +6 are identical to the corresponding wild-type sequences. In embodiments, the 16 nucleotide sequences of the retained introns -15 to -1 and the exons + 1e flanking the 3'splice site are identical to the corresponding wild-type sequences. As used herein, "wild-type sequence" refers to the nucleotide sequence of a gene in a published reference genome deposited in the NCBI repository of biological and scientific information (National Center for Biotechnology Information, National). Operated by the National Library of Medicine, 8600 Rockville Picke, Bethesda, MD USA 20894). As used herein, a nucleotide position represented by an "e" is such that the nucleotide is present in a sequence of exons (eg, an exon flanking a 5'splice site or an exon flanking a 3'splice site). Show that.

The method comprises contacting the cells with ASO complementary to a portion of the pre-mRNA encoding the protein described herein, resulting in increased expression of the protein. As used herein, "contacting" or administering a cell refers to a method of providing the ASO in the closest state to the cell so that the cell interacts with the ASO. Cells that come into contact with ASO take up cells or carry ASO to cells. The method comprises contacting the disease or disease-related or disease-related cells with ASO. 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 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" may be any protein for which increased expression / production is desired.

As a result of contacting cells expressing the RIC pre-mRNA with ASO complementary to the target portion of the transcript of the RIC pre-mRNA, the amount of protein encoded by the pre-mRNA (eg, target protein) can be measured. An increase will be brought about. Methods of measuring or detecting protein production are apparent to those of skill in the art and include known methods such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.

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

In some embodiments, cells are contacted with ASO complementary to the target portion of the transcript of the RIC mRNA precursor, resulting in the amount of protein-encoding mRNA, including mature mRNA encoding the target protein. An increase will be brought about. In some embodiments, the amount of protein-encoding mRNA, or the amount of protein-encoding mature mRNA, is at least 10, compared to the amount of protein produced by the cells in the absence of ASO / lack of treatment. Increased by 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%. In embodiments, the total amount of mRNA encoding the protein, or the total amount of mature mRNA encoding the protein produced in cells contacted with the antisense oligomer, is treated with untreated cells, eg, untreated cell control compounds. 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, from about 4 compared to the amount of mature RNA produced in the cells. About 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 About 8 times, about 3 to about 9 times, about 4 to about 7 times, 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. The control compound may be, for example, an oligonucleotide that is not complementary to the target portion of the RIC mRNA precursor.

Constitutive Splicing of Retained Introns from RIC Pre-mRNA The methods and antisense oligonucleotide compositions provided herein increase the levels of protein-encoding mRNA or protein-encoding mature mRNA. Thus, it is useful for increasing protein expression in cells, eg, subjects suffering from a disease caused by a lack of amount or activity of the protein described herein. In particular, methods and compositions as described herein induce constitutive splicing of retained introns from transcripts of protein-encoding RIC mRNA precursors, thereby encoding proteins. Increases the level of mRNA, or the level of mature mRNA that encodes the protein, and increases the expression of the protein.

Constitutive splicing of retained introns from the RIC pre-mRNA exactly removes the retained introns from the RIC pre-mRNA, where the retained introns have a wild-type splice sequence. As used herein, constitutive splicing is transcribed from a gene or allelic gene with mutations that cause alternative splicing or aberrant splicing of pre-mRNA transcribed from the gene or allelic gene. It does not include the splicing of retained introns from RIC pre-mRNA. For example, constitutive splicing of retained introns does not adjust for aberrant splicing of pre-mRNA, or as induced using the methods provided herein and antisense oligonucleotides. It does not affect alternative splicing, resulting in increased expression of target protein or functional RNA.

In embodiments, constitutive splicing precisely removes retained introns from the RIC pre-mRNA, where the RIC pre-mRNA encodes a fully functional target protein or functional RNA, the wild form. There are no mutations that are transcribed from a gene or allelic gene, or polymorphic or allelic gene, and that cause selective splicing or aberrant splicing of retained introns.

In some embodiments, constitutive splicing of retained introns from the protein-encoding RIC pre-mRNA exactly removes the retained introns from the protein-encoding RIC pre-mRNA, where the RIC Pre-mRNA was transcribed from a gene or allelic gene where the target gene or functional RNA was produced at a reduced level compared to the production from the wild-type allelic gene, and was retained in the gene or allelic gene. There are no mutations that cause selective splicing or abnormal splicing of introns. In these embodiments, accurate removal of constitutively spliced retained introns results in the production of a target protein or functional RNA that is functional when compared to an equivalent wild-type protein or functional RNA. ..

In other embodiments, constitutive splicing precisely removes the retained introns from the RIC pre-mRNA, where the RIC pre-mRNA is functional as compared to an equivalent wild-type protein or functional RNA. Mutations that are transcribed from genes or alleles encoding target proteins or functional RNAs that are produced in reduced form and that cause selective splicing or aberrant splicing of retained introns to the genes or alleles. There is no. In these embodiments, the exact removal of constitutively spliced retained introns results in partial functioning when compared to a partially functional target protein or equivalent wild-type protein or functional RNA. It results in the production of functional RNA that is targeted.

"Precise removal" of a retained intron by constructive splicing refers to the removal of the entire intron without the removal of any part of the exon.

In embodiments, antisense oligomers as described herein or used in any manner described herein are selective splicing or aberrant splicing of pre-mRNA transcribed from a gene. Does not increase the amount of mRNA encoding the protein, or the amount of protein. The regulation of alternative splicing or aberrant splicing is measured, for example, by a known method of analyzing the sequence and length of an RNA species by RT-PCR, and by using the methods described separately herein and in the literature. Can be done. In embodiments, the regulation of selective or abnormal splicing is determined based on an increase or decrease in the amount of spliced species of subject at least 10% or 1.1 times. In embodiments, the regulation is at least 10% to 100% or 1.1 to 10-fold as described herein with respect to determining the increase in mRNA encoding the protein in the method of the invention. Determined on the basis of increase or decrease.

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

In embodiments, the mRNA encoding the 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 protein encoded by the wild-type mature mRNA. obtain.

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

In some embodiments, the ASO is "specifically hybridizes" or "specifically" to the 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 stringent hybridization conditions. At a given ionic strength and pH, Tm is the temperature at which 50% of the target sequence hybridizes to the complementary oligonucleotide.

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

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

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

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

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

In embodiments, the stereochemistry at each of the bonds between the linkers of the ASO backbone is random. In embodiments, the stereochemistry at each of the bonds between the linkers of the ASO backbone is controlled and not random. For example, US Patent Application Publication No. 2014/0194610, "Methos for the Synthesis of Functionalized Nucleic Acids," which is incorporated herein by reference, has an independent chirality handedness at each phosphorus atom in the nucleic acid oligomer. Describes how to select. In embodiments, the ASO used in the method of the invention comprises an ASO having non-random linkages between phosphonucleotides. In embodiments, the composition used in the method of the invention comprises pure diastereomeric ASO. In embodiments, the compositions used in the methods of the invention are at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. , At least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100 %, About 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%. Contains ASO with diastereomeric purity of.

In embodiments, the ASO has a non-random mixture of Rp and Sp constituents in the chain between its phosphonucleotides. For example, it has been suggested that a mixture of Rp and Sp is required for antisense oligonucleotides to achieve a good balance between activity and nuclease stability (Wan, et al., 2014, "Synthesis, biophysical". Properties and biological activity of nucleotide generation antisense oligolocalids contouring chiral phosphorotheioate linkages, as described in Nucleic Acids 42. In embodiments, the ASO used in the methods of the invention is about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp. , At least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 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% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp. , Remaining Sp, or about 100% Rp. In embodiments, the ASOs used in the methods of the invention are from about 10% to about 100% Rp, from about 15% to about 100% Rp, from about 20% to about 100% Rp, from about 25% to about 25%. 100% Rp, about 30% to about 100%, about 35% to about 100% 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, or about 95% to about 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, and the rest of the Sp. including.

In embodiments, the ASO used in the methods of the invention is about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp. , At least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, At least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or It contains at least about 95% Sp and the remaining Rp, or about 100% Sp. In embodiments, the ASO used in the methods of the invention is from about 10% to about 100% Sp, from about 15% to about 100% Sp, from about 20% to about 100% Sp, from about 25% to about 25%. 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, 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 100% Sp, about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about 90% to about 100% Sp, or about 95% to about 100% Sp, from about 20% 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 rest Includes Rp.

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

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

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

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

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

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

In other embodiments, the ASO is CTNS, PAX2, CYP24A1, or PPARD downstream (in the 3'direction) of the 5'splice site of the intron retained in the CTNS, PAX2, CYP24A1, or PPARD RIC mRNA precursor. It is complementary (and binds to) the target portion of the pre-mRNA (eg, the direction indicated by a positive number with respect to the 5'splice site) (FIG. 1). In some embodiments, the ASO is 1 to +4000, 1 to +3500, 1 to +3000, 1 to +2500, 1 to +2000, 1 to +1500, and 1 to +1000 for the 5'splice site of the retained intron. 1 to +500, +2 to +4000, +2 to +3500, +2 to +3000, +2 to +2500, +2 to +2000, +2 to +1500, +2 to +1000, +2 to +500, +2 to +400, +2 to +300, +2 to +200, +6 From +4000, +6 to +3500, +6 to +3000, +6 to +2500, +6 to +2000, +6 to +1500, +6 to +1000, +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +100. Is complementary to the target portion of CTNS, PAX2, CYP24A1, or PPARD RIC RNA precursor. In some embodiments, the ASO is not complementary to the 5'splice site from +1 to +5 nucleotides (the first 5 nucleotides located downstream of the 5'splice site).
In some embodiments, ASO is applied to the 5'splice site of the retained intron and to the target portion of the CTNS, PAX2, CYP24A1, or PPARD RIC mRNA precursor within the region of +6 to +50 nucleotides. May be complementary.
In some embodiments, the ASO is +6 to +90, +6 to +80, +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +30 for the 5'splice site of the retained intron. Complementary to the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors within the +6 to +20 region.

In some embodiments, the ASO is the target region of the CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor (eg, in the direction of 5') upstream of the 3'splice site of the intron retained in the RIC mRNA precursor. , The direction specified by a negative number) is complementary (Fig. 1). In some embodiments, the ASO is -16 to -4000, -16 to -3000, -16 to -2000, -16 to -1000, -16 to-for the 3'splice site of the retained intron. Complementary to the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors within the region of 500, -16 to -400, -16 to -300, -6 to -200, or -16 to -100. be. 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 applied to the 3'splice site of the retained intron, against the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor within the region -16 to -50. It is complementary. In some embodiments, the ASO is -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to-to the 3'splice site of the retained intron. Complementary to the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors within the region of 50, -16 to -40, or -16 to -30.

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

In some embodiments, the ASO is complementary to the target portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA in an exon adjacent to the 5'splice site (upstream) of the retained intron (FIG. 1). In some embodiments, the ASO is complementary to the target portion of the RIC pre-mRNA within the + 2e to -4e regions of the exon flanking the 5'splice site of the retained intron. In some embodiments, ASO is not complementary to nucleotides -1e to -3e for the 5'splice site of the retained intron. In some embodiments, the ASO is -4e to -100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to the 5'splice site of the retained intron. Complementary to the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor within the region of -60e, -4e to -50e, -4e to -40e, -4e to -30e, or -4e to -20e. Is.

In some embodiments, the ASO is complementary to the target portion of the CTNS, PAX2, CYP24A1 or PPARD RIC pre-mRNA within an exon adjacent to the 3'splice site (downstream) of the retained intron (Figure). 1). In some embodiments, the ASO is a target portion of a CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor within the region + 2e to -4e within an exon adjacent to the 3'splice site of the retained intron. Is complementary to. In some embodiments, ASO is not complementary to nucleotide + 1e for the 3'splice site of the retained intron. In some embodiments, the ASO is + 2e to + 100e, + 2e to + 90e, + 2e to + 80e, + 2e to + 70e, + 2e to + 60e, + 2e to + 50e, + 2e to + 40e for the retained intron 3'splice site. , + 2e to + 30e, or + 2e to + 20e, complementary to the target portion of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors. The ASO may be of any length suitable for the specific binding and effective enhancing action of splicing. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, ASO has lengths of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, It may be 29, 30, 31, 32, 33, 34, 35, 45 or 50 nucleobases. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the ASO is 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases. Bases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases , 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 From 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 Nucleobases, 14-40 nucleobases, 14-35 nucleobases, 14-30 nucleobases, 14-25 nucleobases, 14-20 nucleobases, 15-50 nucleobases, 15-40 Nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases The length is a base, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases. In some embodiments, the ASO is a nucleotide 30 in length. In some embodiments, the ASO is a nucleotide of length 29. In some embodiments, the ASO is a nucleotide with a length of 28. In some embodiments, the ASO is a nucleotide of length 27. In some embodiments, the ASO is a nucleotide with a length of 26. In some embodiments, the ASO is a nucleotide 25 in length. In some embodiments, the ASO is a nucleotide 24 in length. In some embodiments, the ASO is a nucleotide of length 23. In some embodiments, the ASO is a nucleotide 22 in length. In some embodiments, the ASO is a nucleotide of length 21. In some embodiments, the ASO is a nucleotide 20 in length. In some embodiments, the ASO is a nucleotide with a length of 19. In some embodiments, the ASO is a nucleotide of length 18. In some embodiments, the ASO is a nucleotide 17 in length. In some embodiments, the ASO is a nucleotide 16 in length. In some embodiments, the ASO is a nucleotide 15 in length. In some embodiments, the ASO is a nucleotide 14 in length. In some embodiments, the ASO is a nucleotide 13 in length. In some embodiments, the ASO is a nucleotide of length 12. In some embodiments, the ASO is a nucleotide of length 11. In some embodiments, the ASO is a nucleotide 10 in length.

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

In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, eg, target moieties or other conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, cholesterol moieties, cholesteryl moieties, such as aliphatic chains such as dodecanediol or undecyl residues, polyamines or polyethylene glycol chains, or lipid moieties such as adamantane 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, debase nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GluNAc), N-Ac glucosamine (GalNAc), or mannose (eg, mannose). Mannose-6-phosphate), lipids, or moieties containing polyhydrogen compounds. The conjugate may include an antisense oligonucleotide at any of several positions on a sugar, base or phosphate group, eg, 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 are described, for example, in US Pat. No. 8,450,467, "Carbohydrates as delivery agents for oligonucleotides," which are incorporated herein by reference.

In some embodiments, the nucleic acid targeted by ASO is an intracellularly expressed RIC pre-mRNA such as 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 or disease-related cell or cell line. In some embodiments, the cells are in vitro (eg, in cell culture).

Pharmaceutical Compositions Pharmaceutical compositions or pharmaceutical formulations comprising the antisense oligonucleotides of the described compositions for use in any of the above methods can be prepared and published according to prior art well known in the pharmaceutical industry. It is also described in the literature. In embodiments, the pharmaceutical composition or pharmaceutical product for treating a subject is 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 appropriate benefit / risk ratios. (See, for example, SM Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose). 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 adipates, alginates, ascorbics, asparaginates, benzenesulfonates, benzoates, bicarbonates, borates, butyrate, sulphates, Cerebral sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate , Heptaneate, hexanenate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate , Methan sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, Includes phosphate, picphosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate acid, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. .. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Further pharmaceutically acceptable salts 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. The aqueous suspension may further contain, for example, a substance containing sodium carboxymethyl cellulose (sorbitol and / or dextran) to increase the viscosity of the suspension. The suspension may also contain stabilizers. In embodiments, pharmaceutical formulations or compositions of the invention include, but are not limited to, solutions, emulsions, microemulsions, foams 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 other active ingredients suitable and well known to those of skill in the art or described in the published literature. Alternatively, it may contain an inactive ingredient. 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. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, the present invention utilizes permeation enhancers to achieve efficient delivery of antisense oligonucleotides, eg, to aid diffusion across cell membranes and / or to enhance the permeability of lipophilic drugs. In embodiments, the permeation enhancer is a surfactant, fatty acid, bile acid salt, chelating agent, or non-chelating non-surfactant.

In 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 promoting the penetration of the subject's antisense oligonucleotide through the blood-brain barrier. For example, delivery of a drug by administering an adenovirus vector to a motor neuron in muscle tissue is incorporated herein by reference in US Pat. It is described in "neurons". Delivery of the vector directly to the brain, such as the striatum, thalamus, hippocampus, or substantia nigra, is, for example, US Pat. No. 6,756,523, "Adenovirus vectors for the transfer of," which is incorporated herein by reference. It is described in "foreign genes into cells of the central nervous system system partially in brain".

In embodiments, the antisense oligonucleotide is bound or conjugated to a drug 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 (-). Fucose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) ramnorth, D (+) Meribios, D (-) ribose, adonitol, D (+) arabinose, L (-) arabinose, D (+) fucose, L (-) fucose, D (-) lyxose, L (+) lyxose, and L (-) ) 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 materials for enhancing blood-brain barrier penetration are described, for example, in US Pat. No. 4,866,042, "Meth for for the delivery of genetic material acrylic cross the blood brain barrier," each of which is incorporated herein by reference. It is described in US Pat. No. 6,294,520 "Material for passage the blood-brain barrier" and US Pat. No. 6,936,589 "Parental delivery systems".

In embodiments, the antisense oligonucleotides of the invention are chemically bound 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, lipid moieties such as cholesterol moieties, cholesteryl moieties, eg dodecanediol or undecyl residues, fat chains such as polyamine chains or polyethylene glycol chains, or adamantane acetate. Oligonucleotides containing lipophilic moieties and methods of preparation are described in published literature. In multiple embodiments, the antisense oligonucleotide is, but is not limited to, a debased nucleotide, a polyether, a polyamine, a polyamide, a peptide, a carbohydrate, such as N-acetylgalactosamine (GluNAc), N-Ac glucosamine (GalNAc), or It is conjugated with a moiety containing mannose (eg, 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. Can be attached to one or more of the nucleotides of. The linker can include a divalent or trivalent branched linker. In embodiments, the conjugate is attached to the 3'end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in US Pat. No. 8,450,467, "Carbohydrates as delivery agents for oligonucleotides," which are incorporated herein by reference.

Subject Treatment Any of the compositions provided herein can be administered to an individual. The "individual" may be used interchangeably with a "subject" or "patient". The individual may be a mammal, for example 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 health history of the disease. Typically, individuals at 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. Numerous tissues and organs can be affected by the symptoms described herein, with the kidney being the most significantly affected tissue. The ASO of the present invention may be administered parenterally to a patient, for example, by intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection. In multiple embodiments, delivery is to the kidney. In embodiments, the fetus is treated in utero, for example by directly or indirectly (eg, via the mother) administering the ASO composition to the fetus.

Methods of Identifying Additional ASOs that Enhance Splicing Within the scope of the invention, there are also methods of identifying (determining) additional ASOs that specifically enhance RIC pre-mRNA splicing at the target intron. ASOs that specifically hybridize to various nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and / or degree of splicing of the target intron. In some embodiments, the ASO may block or interfere with the binding site of the splicing inhibitor / silencer. 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 done. 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 unretained introns. Can be done. An example of a method that can be used is provided below.

ASOs designed to hybridize to the target region of pre-mRNA can be used to perform a round of screening called the ASO "walk". 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 exon 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 ASO with a length of 15 nucleotides can be designed to specifically hybridize to nucleotides +6 to +20 to the 5'splice site of the targeted / retained intron. The second ASO is designed to specifically hybridize to nucleotides +11 to +25 to the 5'splice site of the targeted / retained intron. ASO is designed to extend to the target region of pre-mRNA. In multiple embodiments, the ASO can be spread more tightly, eg, by 1, 2, 3, or 4 nucleotides. In addition, ASO can be spread from 100 nucleotides downstream of the 5'splice site to 100 nucleotides upstream of the 3'splice site.

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

ASO A second round of screening, called a "micro-walk", can be performed using an ASO designed to hybridize to the target region of the pre-mRNA. The ASO used for the ASO microwalk is spread one nucleotide at a time to further refine the nucleotide acid sequence of the pre-mRNA that results in enhanced splicing when hybridized with the ASO. Regions defined by ASOs that facilitate splicing of target introns include ASOs located in 1-nt steps, as well as longer ASOs, typically 18-25 nt. It will be investigated in more detail by the "Microwalk". As described above for ASO walks, one or more ASOs, or control ASOs (ASOs with scrambled sequences, sequences that are not expected to hybridize to the target region), are targeted mRNAs, eg, by transfection. ASO microwalks are performed by delivering the precursor to a disease-related cell line that expresses it. The effect of inducing each splicing of ASO is assessed by methods known in the art, such as reverse transcriptase (RT) -PCR, using primers that span splice junctions, as described herein. Obtain (see "Identifying Intron Retention Events"). Decreased or absent RT-PCR products produced using primers that span splice junctions in ASO-treated cells compared to control ASO-treated cells resulted in enhanced splicing of the target intron. Is shown. In some embodiments, the splicing efficiency, ratio, ratio of spliced pre-mRNA to non-spliced pre-mRNA, rate of splicing, or degree of splicing is the ASO described herein. Can be improved. The amount of protein or functional RNA encoded by the target pre-mRNA can also be evaluated to determine if each ASO has achieved the desired effect (eg, enhanced protein production). Methods known in the art for assessing and / or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can also be used.

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

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 come to the minds of those skilled in the art without departing from the invention. It should be understood that various alternatives of the embodiments of the invention described herein may be utilized in the practice of the invention. The following claims define the scope of the invention and are intended to include methods and structures within the scope of this claim and its equivalents.
The present specification includes the following aspects of the invention.
[1]
A method of treating a subject's kidney disease 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). The RIC mRNA precursor comprises a retained intron, an exson flanking the 5'splice site, and an exson flanking the 3'splice site, wherein the RIC mRNA precursor. Encodes the target protein or functional RNA, the method of contacting the subject's cells with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the target protein or functional RNA. Containing a step of causing the retained intron to be constitutively spliced from the RIC mRNA precursor encoding the target protein or functional RNA, thereby determining the level of mRNA encoding the target protein or functional RNA. A method of increasing and increasing the expression of a target protein or functional RNA in a subject's cells.
[2]
The kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants, according to [1]. Method.
[3]
A method of increasing the expression of a target protein by cells having a retained intron-containing mRNA precursor (RIC mRNA precursor), wherein the RIC mRNA precursor is a retained intron, a retained intron. It comprises an exson flanking the 5'splice site and an exson flanking the 3'splice site of the retained intron, where the RIC mRNA precursor encodes the target protein, wherein the method comprises cells. Contains contact with an antisense oligomer (ASO) complementary to the target portion of the RIC mRNA precursor encoding the target protein, whereby the retained intron is composed of the RIC mRNA precursor encoding the target protein. Spliced to increase the level of mRNA encoding the target protein, thereby increasing the expression of the target protein in the cell, where the target protein is cystinosin, protein paired box gene 2 protein, protein cytochrome. P450 Family 24, Subfamily A, Polypeptide 1, or Peroxysome Proliferation Factor Activating Receptor δ.
[4]
The target protein is cystinocin, a protein paired box gene 2 protein, a protein cytochrome P450 family 24, subfamily A, polypeptide 1, or a peroxisome proliferator-activated receptor δ, according to [1] or [2]. Method.
[5]
The target protein or functional RNA is a compensatory protein or compensatory functional RNA that functionally increases or replaces the target protein or functional RNA that is deficient in quantity or activity in the subject, [ 1] or the method according to [2].
[6]
The method according to [3], wherein the cells are in or derived from a subject having a disease caused by a lack of amount or activity of the target protein.
[7]
A deficiency in the amount of target protein is caused by haplodeficiency of the target protein, where the subject has a first allele encoding a functional target protein, a second allele in which the target protein is not produced, or non-target protein. It has a second allele encoding a functional target protein and the antisense oligomer is a RIC transcribed from the first allele.
The method according to any one of [1]-[6], which binds to a target portion of an mRNA precursor.
[8]
The subject has a disease caused by a disorder due to a lack of quantity or function of the target protein, where the subject is.
(A) (i) Target proteins are produced at reduced levels compared to production from wild-type alleles.
(Ii) The target protein is produced or produced in a less functional form compared to an equivalent wild-type protein.
(Iii) The first mutation allele, which does not produce the target protein,
(B) (i) Target proteins are produced at reduced levels compared to production from wild-type alleles.
(Ii) The target protein is produced or produced in a less functional form compared to an equivalent wild-type protein.
(Iii) With a second mutation allele, which does not produce a target protein,
Here, when the subject has the first mutation allele (a) (iii), the second mutation allele is (b) (i) or (b) (ii), and the subject is the second. Mutant allele
(B) When having (iii), the first mutation allele is (a) (i) or (a) (ii) and the RIC pre-mRNA is (a) (i) or (a) ( Either [1]-[6] transcribed from the first mutant allele that is ii) and / or the second mutant allele that is (b) (i) or (b) (ii). The method described in one.
[9]
The method according to [8], wherein the target protein is produced in a form with reduced function as compared with an equivalent wild-type protein.
[10]
The method according to [8], wherein the target protein is produced in a fully functional form as compared to an equivalent wild-type protein.
[11]
The target portion of the RIC pre-mRNA is in the retained intron 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. 1]-The method according to any one of [10].
[12]
The target portion of the RIC pre-mRNA is in the retained intron within the region from +69 to the 3'splice site of the retained intron to -79 relative to the 5'splice site of the retained intron. 1]-The method according to any one of [10].
[13]
The method according to any one of [1]-[12], wherein the target protein is cystinocin.
[14]
The target portion of the RIC pre-mRNA is in the retained intron within the region from +500 to the 5'splice site of the retained intron to -500 relative to the 3'splice site of the retained intron. 13].
[15]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, relative to any one of SEQ ID NO: 8624-10068. Alternatively, the method according to [13] or [14], which comprises a 100% complementary sequence.
[16]
The target portion of the RIC mRNA precursor is SEQ ID NO: 10748 or SEQ
ID NO: 10734 with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in the region containing at least 8 adjacent nucleic acids. The method according to any one of [13]-[15], which comprises a sequence.
[17]
ASO is sequence identical at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NO: 8624-10068. The method according to any one of [13]-[16], which comprises a sequence having sex.
[18]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for SEQ ID NO: 16 or SEQ ID NO: 17. The method according to any one of [13]-[17], which comprises a sequence having sequence identity.
[19]
The RIC mRNA precursor is a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. The method according to any one of [13]-[18] encoded by.
[20]
The method according to any one of [1]-[12], wherein the target protein is a paired box gene 2 protein.
[21]
The target portion of the RIC pre-mRNA is in the retained intron within the region from +500 to the 5'splice site of the retained intron to -500 relative to the 3'splice site of the retained intron. 20].
[22]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, relative to any one of SEQ ID NO: 6935-8623. Alternatively, the method according to [20] or [21], which comprises a 100% complementary sequence.
[23]
The target portion of the RIC mRNA precursor is SEQ ID NO: 10738 or SEQ
ID NO: 10740 with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in the region containing at least 8 adjacent nucleic acids. The method according to any one of [20]-[22], which comprises a sequence.
[24]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to any one of SEQ ID NO: 6935-8623. The method according to any one of [20]-[23], which comprises a sequence having sequence identity of.
[25]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to any one of SEQ ID NO: 10-15. The method according to any one of [20]-[24], which comprises a sequence having sequence identity of.
[26]
The RIC mRNA precursor is a gene with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. The method according to any one of [20]-[25], which is encoded by an array.
[27]
The method according to any one of [1]-[12], wherein the target protein is the protein cytochrome P450 family 24, subfamily A, and polypeptide 1.
[28]
The target portion of the RIC pre-mRNA is in the retained intron within the region from +500 to the 5'splice site of the retained intron to -500 relative to the 3'splice site of the retained intron. 27].
[29]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, relative to any one of SEQ ID NO: 9520-10733, Alternatively, the method according to [27] or [28], which comprises a 100% complementary sequence.
[30]
The target portion of the RIC mRNA precursor is in a region containing at least eight adjacent nucleic acids of SEQ ID NO: 10748, SEQ ID NO: 10734, SEQ ID NO: 10746, SEQ ID NO: 10745, or SEQ ID NO: 10741. Any one of [27]-[29], comprising sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The method described in one.
[31]
ASO is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to any one of SEQ ID NO: 9520-10733. The method according to any one of [27]-[30], which comprises a sequence having sex.
[32]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for any one of SEQ ID NO: 16-19. The method according to any one of [27]-[31], which comprises a sequence having sequence identity of.
[33]
The RIC mRNA precursor is a gene with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. The method according to any one of [27]-[32], which is encoded by an array.
[34]
The method according to any one of [1]-[12], wherein the target protein is a peroxisome proliferator-activated receptor δ.
[35]
The target portion of the RIC pre-mRNA is in the retained intron within the region from +500 to the 5'splice site of the retained intron to -500 relative to the 3'splice site of the retained intron. 34].
[36]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, relative to any one of SEQ ID NO: 20-6952. Alternatively, the method according to [34] or [35], which comprises a 100% complementary sequence.
[37]
The target portions of the RIC mRNA precursor are SEQ ID NO: 10736, SEQ ID NO: 10747, SEQ ID NO: 10739, SEQ ID NO: 10743, SEQ ID NO: 10742, SEQ ID NO: 10737, SEQ ID NO: 10735. , Or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence with respect to the region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10744. The method according to any one of [34]-[36], which comprises a sequence having identity.
[38]
ASO is sequenced at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for any one of SEQ ID NO: 20-6952. The method according to any one of [34]-[37], which comprises a sequence having sex.
[39]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for any one of SEQ ID NO: 5-9. The method according to any one of [34]-[38], which comprises a sequence having sequence identity of.
[40]
The RIC mRNA precursor is a gene with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. The method according to any one of [34]-[39], which is encoded by an array.
[41]
The target portion of the RIC mRNA precursor is
(A) A region of +6 to +100, or a region relative to the 5'splice site of the retained intron.
(B) A region of -16 to -100 with respect to the 3'splice site of the retained intron,
The method according to any one of [1]-[10] and [13]-[40] in the retained intron inside.
[42]
The antisense oligomer is an RIC pre-mRNA in the region from about 100 nucleotides downstream of the 5'splice site of at least one retained intron to about 100 nucleotides upstream of the 3'splice site of at least one retained intron. The method according to any one of [1]-[10] and [13]-[40], which targets a part of the body.
[43]
The target portion of the RIC mRNA precursor is
(A) Regions + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, or
(B) Regions + 2e to -4e in the exon adjacent to the 3'splice site of the retained intron,
The method according to any one of [1]-[10] and [13]-[40] inside.
[44]
Antisense oligomers do 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, [1]-[43]. ] The method according to any one of.
[45]
The antisense oligomer does not increase the amount of target protein or functional RNA by regulating abnormal splicing due to mutations in the gene encoding the target protein or functional RNA, either [1]-[44]. The method described in one.
[46]
The method according to any one of [1]-[45], 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.
[47]
The method according to any one of [1]-[46], wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.
[48]
The method according to any one of [1]-[47], wherein the produced target protein is a full-length protein or a wild-type protein.
[49]
The total amount of mRNA encoding the target protein or functional RNA produced in the cells contacted with the antisense oligomer is approximately compared to the total amount of mRNA encoding the target protein or functional RNA produced in the control cells. 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1. 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to About 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about about The method according to any one of [1]-[48], which increases 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.
[50]
The total amount of target protein produced by cells in contact with the antisense oligomer is about 1.1 to about 10 times, about 1.5 to about 10 times, compared to the total amount of target protein produced by control cells. , About 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 Double, 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 The method according to any one of [1]-[49], which increases by at least about 10 times.
[51]
The method according to any one of [1]-[50], wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[52]
The antisense oligomer comprises either phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety, either [1]-[51]. The method described in one.
[53]
The method according to any one of [1]-[52], wherein the antisense oligomer contains at least one modified sugar moiety.
[54]
The method according to [53], wherein each sugar moiety is a modified sugar moiety.
[55]
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 Nucleobase, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases Bases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases , 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. The method according to any one of [1]-[54].
[56]
The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the RIC mRNA precursor encoding the protein. 1]-The method according to any one of [55].
[57]
The cell is a 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, where the antisense oligomer is the most abundant retention of the population of RIC mRNA precursors. The method according to any one of [1]-[56], which binds to the intron.
[58]
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 a target protein or functional RNA. The method of inducing, according to [57].
[59]
The cell is a RIC transcribed from a gene encoding a target protein or functional RNA.
Containing a population of pre-mRNA, where the population of RIC pre-mRNA comprises two or more retained introns, where the antisense oligomer is the second most abundant in the population of RIC pre-mRNA. The method according to any one of [1]-[56], which binds to a retained intron.
[60]
Binding of antisense oligomers to the second abundant retained intron splicing two or more retained introns from a population of RIC pre-mRNA to produce mRNA encoding a target protein or functional RNA. The method according to [59], which induces out.
[61]
The method according to any one of [6]-[60], wherein the disease is a disease or a disorder.
[62]
The method according to [61], wherein the disease or disorder is kidney disease.
[63]
The kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants, [62]. Method.
[64]
The method according to [63], wherein the target protein and the RIC mRNA precursor are encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1, or PPARD.
[65]
The method according to any one of [1]-[64], wherein the method further comprises a step of evaluating protein expression.
[66]
The method according to any one of [1]-[65], wherein the subject is a human.
[67]
The method according to any one of [1]-[65], wherein the subject is an animal other than a human.
[68]
The method according to any one of [1]-[66], wherein the subject is a fetus, an embryo, or a child.
[69]
The method according to any one of [1]-[67], wherein the cell is Exvivo.
[70]
The method according to any one of [1]-[67], wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
[71]
Exons -3e to -1e adjacent to the 5'splice site and the retained intron
The method according to any one of [1]-[70], wherein the nine nucleotides in +1 to +6 are the same as the corresponding wild type sequence.
[72]
The 16 nucleotides at -15 to -1 of the retained intron and + 1e of the exon adjacent to the 3'splice site are identical to the corresponding wild-type sequence, any one of [1]-[71]. The method described in.
[73]
An antisense oligomer used in any one of [1]-[72].
[74]
With at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 20-10733. Antisense oligomer containing the same sequence.
[75]
A pharmaceutical composition comprising the antisense oligomer of [73] or [74] and an excipient.
[76]
A method of treating a subject by administering the pharmaceutical composition according to [75] by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[77]
Antisense used in a method that increases the expression of a target protein or functional RNA by cells to treat a subject's kidney disease associated with a deficient protein or deficient functional RNA. A composition comprising an oligomer, wherein the deficient protein or functional RNA is deficient in quantity or activity in the subject, where the antisense oligomer contains the target protein or functional RNA. Enhancing the constitutive splicing of the encoded, retained intron-containing pre-mRNA (RIC pre-mRNA), where the target protein is:
(A) Missing protein or
(B) Compensating protein that functionally increases or replaces the deficient protein in the subject.
Here, the functional RNA is
(A) Missing RNA or
(B) Compensatory functional RNA that functionally increases or replaces the subject's deficient functional RNA.
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, where the retained intron is a target protein or functional RNA. A composition that is spliced from a RIC pre-mRNA that encodes, thereby increasing the production or activity of a target protein or functional RNA in a subject.
[78]
The kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants, [77]. Composition.
[79]
A composition comprising an antisense oligomer for use in a method of treating a disease associated with a target protein in a subject, wherein the method is a step of increasing the expression of the target protein by the subject's cells. A retained intron-containing mRNA precursor, including an intron in which cells are retained, 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 (RIC mRNA precursor), the RIC mRNA precursor is a step of encoding the target protein and a step of contacting the cell with the antisense oligomer, whereby the retained intron can control the target protein. Constitutively spliced from the transcript of the encoding RIC mRNA precursor, thereby increasing the level of the target protein or mRNA encoding the functional RNA and increasing the expression of the target protein in the subject's cells. A composition comprising steps.
[80]
The target protein is either cystinosin, protein paired box gene 2 protein, protein cytochrome P450 family 24, subfamily A, polypeptide 1, or peroxisome proliferator-activated receptor δ, either [77]-[79]. The composition according to one.
[81]
The composition according to [79], wherein the disease is a disease or disorder.
[82]
The composition according to [80], wherein the disease or disorder is kidney disease.
[83]
The composition according to [82], wherein the kidney disease is nephropathy cystine accumulation in infants, delayed cystine accumulation, focal segmental glomerulosclerosis 7, papillary kidney syndrome, or hypercalcemia in infants [82]. thing.
[84]
The composition according to [82] or [83], wherein the target protein and the RIC mRNA precursor are encoded by a gene, wherein the gene is CTNS, PAX2, CYP24A1 or PPARD.
[85]
The antisense oligomer comprises a portion of the RIC pre-mRNA that is in the region of +6 for the 5'splice site of the retained intron to -16 for the 3'splice site of the retained intron in the retained intron. The composition according to any one of [77]-[84] to be targeted.
[86]
The target portion of the RIC pre-mRNA is within the region of +69 for the 5'splice site of the retained intron to -79 for the 3'splice site of the retained intron in the retained intron, [77]-. The composition according to any one of [84].
[87]
The composition according to any one of [77]-[86], wherein the target protein is cystinocin.
[88]
The target portion of the RIC pre-mRNA is within the region [87] from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. The composition described.
[89]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, relative to any one of SEQ ID NO: 8624-10068. Or the composition according to [87] or [88], which comprises a 100% complementary sequence.
[90]
The target portion of the RIC mRNA precursor is at least 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to the region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10748 or 10734. The composition according to any one of [87]-[89], comprising a sequence having%, 99% or 100% sequence identity.
[91]
ASO has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 8624-10068. The composition according to any one of [87]-[90], which comprises a sequence comprising.
[92]
The RIC mRNA precursor comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity for SEQ ID NO: 16 or 17. The composition according to any one of [87]-[91], which comprises the above-mentioned sequence.
[93]
The RIC mRNA precursor is a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. The composition according to any one of [87]-[92] encoded by.
[94]
The composition according to any one of [77]-[86], wherein the target protein is a paired box gene 2 protein.
[95]
The target portion of the RIC pre-mRNA is in the retained intron, from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron, [94]. The composition described.
[96]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or for any one of SEQ ID NO: 6953-8623. The composition according to [94] or [95], which comprises a sequence that is 100% complementary.
[97]
The target portion of the RIC mRNA precursor is at least 80%, 85%, 90%, 95%, 96%, 97%, 98 relative to the region containing at least 8 adjacent nucleic acids of SEQ ID NO: 10738 or 10740. The composition according to any one of [94]-[96], comprising a sequence having%, 99% or 100% sequence identity.
[98]
ASO has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 6935-8623. The composition according to any one of [94]-[97], which comprises a sequence comprising.
[99]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% for any one of SEQ ID NO: 10-15. The composition according to any one of [94]-[98], which comprises a sequence having sequence identity.
[100]
The RIC mRNA precursor is a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. The composition according to any one of [94]-[99] encoded by.
[101]
The composition according to any one of [77]-[86], wherein the target protein is the protein cytochrome P450 family 24, subfamily A, and polypeptide 1.
[102]
The target portion of the RIC pre-mRNA is within the region [101] from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. The composition described.
[103]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or for any one of SEQ ID NO: 9520-10733. The composition according to [101] or [102], which comprises a sequence that is 100% complementary.
[104]
The target portion of the RIC pre-mRNA is at least 80%, 85%, 90%, 95%, 96 relative to the region containing at least eight adjacent nucleic acids of SEQ ID NO: 10748, 10734, 10746, 10745 or 10741. The composition according to any one of [101]-[103], comprising a sequence having%, 97%, 98%, 99% or 100% sequence identity.
[105]
ASO has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 9520-10733. The composition according to any one of [101]-[104], which comprises a sequence comprising.
[106]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% for any one of SEQ ID NO: 16-19. The composition according to any one of [101]-[105], which comprises a sequence having sequence identity.
[107]
The RIC mRNA precursor is a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. The composition according to any one of [101]-[106], encoded by.
[108]
The composition according to any one of [77]-[86], wherein the target protein is a peroxisome proliferator-activated receptor δ.
[109]
The target portion of the RIC pre-mRNA is in the retained intron, from +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron, [108]. The composition described.
[110]
The target portion of the RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or for any one of SEQ ID NO: 20-6952. The composition according to [108] or [109], which comprises a sequence that is 100% complementary.
[111]
The target portion of the RIC mRNA precursor is at least 80%, 85%, relative to the region containing at least eight adjacent nucleic acids of SEQ ID NO: 10736, 10747, 10739, 10743, 10742, 10737, 10735, or 10744. The composition according to any one of [108]-[110], comprising a sequence having 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
[112]
ASO has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 20-6952. The composition according to any one of [108]-[111], which comprises a sequence comprising.
[113]
The RIC mRNA precursor is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% for any one of SEQ ID NO: 5-9. The composition according to any one of [108]-[112], which comprises a sequence having sequence identity.
[114]
The RIC mRNA precursor is a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to SEQ ID NO: 1. The composition according to any one of [108]-[113] encoded by.
[115]
The antisense oligomer is used in the retained intron.
(A) Region +6 to +100 relative to the 5'splice site of the retained intron, or
(B) A region of -16 to -100 with respect to the 3'splice site of the retained intron,
The composition according to any one of [77]-[84] and [87]-[114], which targets a part of the RIC mRNA precursor inside the RIC mRNA.
[116]
The antisense oligomer is in the region from about 100 nucleotides downstream of the 5'splice site of at least one retained intron to about 100 nucleotides upstream of the 3'splice site of at least one retained intron. The composition according to any one of [77]-[84] and [87]-[114], which targets a part of the precursor.
[117]
The target portion of the RIC mRNA precursor is
(A) Regions + 2e to -4e in exons adjacent to the 5'splice site of the retained intron, or
(B) Regions + 2e to -4e in exons adjacent to the 3'splice site of the retained intron,
The composition according to any one of [77]-[84] and [87]-[114], which is contained in the above.
[118]
Antisense oligomers do 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, [77]-[117]. The composition according to any one.
[119]
Antisense oligomers do not increase the amount of functional RNA or functional protein by regulating the abnormal splicing resulting from mutations in the gene encoding the target protein or functional RNA, [77]-[118]. The composition according to any one.
[120]
The composition according to any one of [77]-[119], wherein the RIC pre-mRNA is produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.
[121]
The composition according to any one of [77]-[120], wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a wild-type mature mRNA.
[122]
The composition according to any one of [77]-[121], wherein the target protein produced is a full-length protein or a wild-type protein.
[123]
The composition according to any one of [77]-[122], wherein the retained intron is a rate-determining intron.
[124]
The composition according to any one of [77]-[123], wherein the retained intron is the most abundant retained intron in the RIC pre-mRNA.
[125]
The composition according to any one of [77]-[123], wherein the retained intron is the second most abundant retained intron in the RIC pre-mRNA.
[126]
The composition according to any one of [77]-[125], wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[127]
The antisense oligomer is an antisense oligonucleotide, [77]
-The composition according to any one of [126].
[128]
The antisense oligomer comprises either phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety, either [77]-[127]. The composition according to one.
[129]
The composition according to any one of [77]-[128], wherein the antisense oligomer contains at least one modified sugar moiety.
[130]
The composition according to [129], wherein each sugar moiety is a modified sugar moiety.
[131]
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 Nucleobase, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases Bases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases , 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. , [77]-[130].
[132]
The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the target portion of the RIC mRNA precursor encoding the protein. The composition according to any one of [77]-[131].
[133]
A pharmaceutical composition comprising any of the antisense oligomers and excipients of the composition of [77]-[132].
[134]
A method of treating a subject by administering the pharmaceutical composition according to [133] by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[135]
An antisense oligomer that hybridizes to the target sequence of a deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript, including an intron carrying the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript. With antisense oligomers, the oligomers induce splicing out of retained introns from deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcripts.
A pharmaceutical composition comprising a pharmaceutically acceptable excipient.
[136]
The pharmaceutical composition according to [135], wherein the deficient CTNS, PAX2, CYP24A1 or PPARD mRNA transcript is a transcript of the CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursor.
[137]
The target portion of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD is a region of +500 to the 5'splice site of the retained intron to -500 to the 3'splice site of the retained intron in the retained intron. The pharmaceutical composition according to [135] or [136], which is contained therein.
[138]
Transcripts of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors are at least about 80%, 85%, 90%, 95%, 96%, relative to any one of SEQ ID NO: 1-4. The pharmaceutical composition according to [135] or [136], which is encoded by a gene sequence having 97%, 98%, 99% or 100% sequence identity.
[139]
Transcripts of CTNS, PAX2, CYP24A1 or PPARD RIC mRNA precursors are at least about 80%, 85%, 90%, 95%, 96%, relative to any one of SEQ ID NO: 5-19. The pharmaceutical composition according to [135] or [136], comprising a sequence having 97%, 98%, 99% or 100% sequence identity.
[140]
The pharmaceutical composition according to [135], wherein the antisense oligomer comprises a skeletal modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[141]
The pharmaceutical composition according to [135], wherein the antisense oligomer is an antisense oligonucleotide.
[142]
The pharmaceutical composition according to [135], 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. ..
[143]
The pharmaceutical composition according to [135], wherein the antisense oligomer comprises at least one modified sugar moiety.
[144]
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 Nucleobase, 10-50 nucleobases, 10-40 nucleobases, 10-35 nucleobases, 10-30 nucleobases, 10-25 nucleobases, 10-20 nucleobases, 10-15 nucleobases Bases, 11-50 nucleobases, 11-40 nucleobases, 11-35 nucleobases, 11-30 nucleobases, 11-25 nucleobases, 11-20 nucleobases, 11-15 nucleobases , 12-50 nucleobases, 12-40 nucleobases, 12-35 nucleobases, 12-30 nucleobases, 12-25 nucleobases, 12-20 nucleobases, or 12-15 nucleobases. , [135].
[145]
The antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% of the target portion of the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD. , Or the pharmaceutical composition according to [135] or [136], which is 100% complementary.
[146]
The pharmaceutical composition according to [135] or [136], wherein the target portion of the transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD is in the sequence selected from SEQ ID NO: 10043-10748.
[147]
The antisense oligomer is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, relative to any one of SEQ ID NO: 20-10733. The pharmaceutical composition according to [135], comprising a nucleotide sequence having 97%, 98%, or 99% sequence identity.
[148]
The pharmaceutical composition according to [135], wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NO: 20-10733.
[149]
The pharmaceutical composition is formulated for intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection, according to any one of [135]-[148]. Pharmaceutical composition.
[150]
CTS, which facilitates the removal of retained introns to produce a fully treated mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD, which encodes the functional form of the protein of CTNS, PAX2, CYP24A1 or PPARD. A method of inducing treatment of mRNA transcripts of PAX2, CYP24A1 or PPARD, said method.
(A) 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 mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD, the mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD is the protein of CTNS, PAX2, CYP24A1 or PPARD. A process that can encode a functional form of, including at least one retained intron, and
(C) CTNS at least one retained intron to produce a fully processed mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD that encodes the functional form of the protein in CTNS, PAX2, CYP24A1 or PPARD. , PAX2, CYP24A1 or PPARD mRNA transcript and removal.
(D) A method comprising translating the functional form of a protein of CTNS, PAX2, CYP24A1 or PPARD from a fully treated mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD.
[151]
The method according to [150], wherein the retained intron is the entire retained intron.
[152]
The method according to [150] or [151], wherein the mRNA transcript of CTNS, PAX2, CYP24A1 or PPARD is a transcript of the RIC mRNA precursor of CTNS, PAX2, CYP24A1 or PPARD.
[153]
A method of treating a subject having a disease caused by a lack of protein amount or activity of CTNS, PAX2, CYP24A1 or PPARD, the SEQ ID.
NO: At least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 for any one of 20-10733. A method comprising administering to a subject an antisense oligomer comprising a nucleotide sequence having a% sequence identity.

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

Example 1: Identification of Transcript Intron Retention Events by RNAseq Using Next Generation Seqing To reveal snapshots of transcripts produced by the genes described herein to identify intron retention events. To this end, next-generation sequencing was used to perform shotgun sequencing of all transcriptomes. To this end, polyA + RNA from the nuclear and cytoplasmic fractions of renal epithelial cells was isolated and a cDNA library was constructed using Illumina's TruSeq Stranded mRNA library Prep kit. The library was pair-end sequenced, resulting in a reading of 100 nucleotides mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The mapped reads are (UCSC Genome Infomatics Group (Center for Biomolecule Science & Enginereing, Universal of California, Santa Cruz, 1156 High), eg, Santa Cruz, 1156 Hi “The UCSC Genome Browser data: 2015 update,” Nucleic Acids Research 43, Database Issue doi: 10.1093 / nar / gku1177 is described in the UCSC Genome Browser. Inferred by the signal. The height of the peak indicates the level of expression given by the reading density in a particular region. A schematic diagram of the P gene (drawn to scale) is provided by the UCSC Genome Browser (under the read signal) so that the peaks can be matched to the exon and intron regions. Based on this display, we identified introns with high reading densities in the nuclear fraction of renal epithelial cells, but very low or no reading in the cytoplasmic fraction of these cells (described herein). See Table 1 for intron retention (PIR) data for introns identified in the gene). This indicates that these introns were retained and that the intron-containing transcript remained in the nucleus, and that the retained RIC pre-mRNA is not excreted in the cytoplasm, thus being unproductive. It suggests that.

Example 2: Identification of intron retention events in gene transcripts by RNAseq using next-generation sequencing for retained introns that have not yet been identified To identify unknown intron retention events herein. Next-generation sequencing was used to perform shotgun sequencing of the entire transcriptome to reveal snapshots of the transcripts produced by the genes described. To this end, polyA + RNA from the nuclear and cytoplasmic fractions of renal epithelial cells was isolated and a cDNA library was constructed using Illumina's TruSeq Stranded mRNA library Prep kit. The library was pair-end sequenced, resulting in a reading of 100 nucleotides mapped to the human genome (February 2009, GRCh37 / hg19 assembly). The mapped reads are (UCSC Genome Infomatics Group (Center for Biomolecule Science & Enginereing, Universal of California, Santa Cruz, 1156 Hig) “The UCSC Genome Browser data: 2015 update,” Nucleic Acids Research 43, Database Issue doi: 10.1093 / nar / gku1177 is described in the UCSC Genome Browser. Can be inferred by the signal. The height of the peak indicates the level of expression given by the reading density in a particular region. The UCSC Genome Browser (under the read signal) provides a schematic diagram (drawn to scale) of the gene so that the peaks can be matched to the exon and intron regions. Based on this display, retained introns are inferred to have high read densities in the nuclear fraction of renal epithelial cells, but very low or no reads in the cytoplasmic fraction of these cells. rice field. This indicates that the intron was retained and that the intron-containing transcript remained in the nucleus, and that these retained RIC pre-mRNA are not excreted in the cytoplasm and are therefore unproductive. It suggests that.

Example 3: Design of ASO Walks Targeting Retained Introns ASO walks were designed to target retained introns using the methods described herein. For example, the region immediately downstream of the intron 5'splice site extending from nucleotides +6 to +69 and the region immediately upstream of the intron 3'splice site extending from nucleotides -16 to -79 of the intron are 2'-O-Me RNA, for example. The PS skeleton was targeted with 18mer ASO shifted at 5 nucleotide intervals. Table 1 lists exemplary ASOs designed and their target sequences.

Example 4: Improved splicing efficiency by ASO targeting of retained introns increases CTNS transcription levels Achieved increased expression of CTNS by improving the splicing efficiency of retained introns using ASO. The methods described herein may be used to determine if this is the case. ARPE-19 cells were independently mock-transfected or transfected with CTNS-targeted ASO or non-targeted ASO controls using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASO for 24 hours (FIGS. 3B and C). The results of Taqman qPCR showed that some targeted ASOs increased CTNS gene transcription levels compared to those sham-transfected. Ct values from CTNS-targeted ASO-transfected cells are normalized to RPL32 and plotted against normalized qPCR products from sham-treated cells. The results of this analysis showed that some CTNS-targeted ASOs increased gene transcription levels. These results indicate that induction of retained intron splicing in genes using ASO results in increased gene expression. Overall, these results indicate that improving the splicing efficiency of rate-determining introns in CTNS genes using ASO increases CTNS gene expression.

Example 5: Improved splicing efficiency by ASO targeting of retained introns increases CYP24A1 transcription levels Achieved increased expression of CYP24A1 by improving the splicing efficiency of retained introns using ASO. The methods described herein may be used to determine if this is the case. ARPE-19 cells were independently mock-transfected using RNAiMAX (Invitrogen) delivery reagent or transfected with CYP24A1 targeted ASO or non-targeted ASO controls. Experiments were performed using 80 nM ASO for 24 hours (FIGS. 4B and C).
The results of Taqman qPCR showed that some targeted ASOs increased CYP24A1 gene transcription levels compared to those sham-transfected. Ct values from CYP24A1 targeted ASO-transfected cells are normalized to RPL32 and plotted against normalized qPCR products from sham-treated cells. The results of this analysis showed that some CYP24A1 targeted ASOs increased gene transcription levels. These results indicate that induction of retained intron splicing in genes using ASO results in increased gene expression. Overall, these results indicate that improving the splicing efficiency of rate-determining introns in the CYP24A1 gene with ASO increases CYP24A1 gene expression.

Example 6: Improved splicing efficiency by ASO targeting of retained introns increases PPARD transcription levels Achieved increased expression of PPARD by improving splicing efficiency of retained introns using ASO. The methods described herein may be used to determine if this is the case. ARPE-19 cells were independently mock-transfected using RNAiMAX (Invitrogen) delivery reagent or transfected with PPARD targeted ASO or non-targeted ASO controls. Experiments were performed using 80 nM ASO for 24 hours (FIGS. 6A and B, FIGS. 6E and F). The results of Taqman qPCR showed that some targeted ASOs increased PPARD gene transcription levels compared to those sham-transfected. Ct values from PPARD-targeted ASO-transfected cells are normalized to RPL32 and plotted against normalized qPCR products from sham-treated cells. The results of this analysis showed that some PPARD-targeted ASOs increased gene transcription levels. These results indicate that induction of retained intron splicing in genes using ASO results in increased gene expression. Overall, these results indicate that improving the splicing efficiency of rate-determining introns in the PPARD gene with ASO increases PPARD gene expression.

Example 7: Improved splicing efficiency by ASO targeting of retained introns increases transcriptional levels To determine if improved intron splicing efficiency in ASO can achieve increased expression of target genes. The method described in the specification was used. ARPE-19 cells, ie the American Type Culture Collection (ATCC), USA, or Huh-7, ie, the human hepatocytoma cell line (NIBIOHN, Japan), or SK-N-AS. That is, the human neuroblastoma cell line (ATCC) is sham-transfected or transfected with the targeted ASOs shown in Figure 3-6 and Table 1. Cells are transfected with Lipofectamine RNAiMax transfection reagent (Thermo Fisher) according to the supplier's specifications. Briefly, ASO is seeded on 96-well tissue culture plates and combined with RNAiMax diluted with Opti-MEM. The cells are stripped with trypsin, resuspended in complete medium and about 25,000 cells are added to the ASO transfection mixture. Transfection experiments are performed in three plate replicas. The final ASO concentration was 80 nM. According to the supplier's specifications, the medium is replaced 6 hours after transfection and the cells are supplemented with DNAse (Thermo Fisher) using Cell-to-Ct lysing reagent and harvested at 24 hours. The cDNA is produced with the Cells-to-Ct RT reagent (Thermo Fisher) according to the supplier's specifications. Quantitative PCR was performed using the Taqman assay on the corresponding exon-exon junction (Thermo Fisher) probes of the retained introns listed in Table 1 to quantify the amount of splicing in the intron of interest. Will be done. The Taqman assay is performed on the QuantStudio 7Flex Real-Time PCR system (Thermo Fisher) according to the supplier's specifications. Target gene assay values are normalized to RPL32 (deltaCt) and plate-matched pseudotransfection samples (delta-delta Ct) to generate fold changes for mock quantification (2 ^-(delta-delta Ct)). Plot mean magnification changes to mock of three plate replicas (FIGS. 3C, 4C, 6B and 6F). Several assays that increase target gene expression have been identified and spliced at their target introns. These results, along with all transcriptom data showing retention of the target intron (Fig. 3-6), confirm that ASO can improve the splicing efficiency of rate-determining introns.

Claims (8)

An antisense oligomer (ASO), the ASO is complementary to the target portion of a retained intron-containing pre-mRNA (RIC pre-mRNA) that encodes the peroxysome growth factor-activated receptor δ, and is a RIC pre-mRNA. The binding of the ASO to the target portion of the RIC pre-mRNA, which contains the body-retained intron, causes the retained intron to be constitutively spliced from the RIC pre-mRNA in cells expressing the RIC pre-mRNA. Increased the level of mature mRNA encoding the peroxysome proliferative factor activated receptor δ in the cell, the target portion of the RIC pre-mRNA is in the retained intron, and the ASO is SEQ ID NO: 59. The ASO comprising a sequence selected from ~ 68, 240, 241, 245 to 248, 809 to 814, 816 to 818, 838 to 840, 843, and 846 . The ASO according to claim 1, wherein the target portion of the RIC mRNA precursor is in the region from +6 to -16 with respect to the 5'splice site of the retained intron. The ASO according to claim 1 or 2, wherein the RIC mRNA precursor comprises a sequence selected from SEQ ID NO: 5-9. The ASO according to any one of claims 1 to 3, wherein the target portion of the RIC mRNA precursor is in the sequence of SEQ ID NO: 10736, 10747, 10739, 10743, 10742, 10737, 10735, or 10744. Peroxisome proliferator-activated receptor δ,
any of claims 1 to 4 , i) produced in a less functional form compared to an equivalent wild-type protein, or ii) in a fully functional form compared to an equivalent wild-type protein. ASO described in. The ASO comprises a skeletal modification or modified sugar moiety or sugar analog comprising phosphorothioate binding or phosphorodiamidate binding, wherein the modified sugar moiety or sugar analog is a phosphorodiamidate morpholino, locked nucleic acid, peptide nucleic acid. The ASO according to any one of claims 1 to 5 , which may contain a 2,'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. The ASO according to any one of claims 1 to 6 , wherein the ASO comprises 18 to 50 nucleobases. A viral vector encoding a polynucleotide comprising the ASO according to any one of claims 1 to 7 .

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