Targeted augmentation of nuclear gene output

• US 9,976,143 B2
• Filed: 10/03/2015
• Issued: 05/22/2018
• Est. Priority Date: 10/03/2014
• Status: Active Grant

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory
• Inventors: Krainer, Adrian, Aznarez, Isabel
• Primary Examiner(s): Whiteman, Brian A
• Application Number:
  US14/874,420
• Publication Number:
  US 20160298121A1
• Time in Patent Office: 962 Days
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/4736 Retinoblastoma protein
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/3341 : 5-Methylcytosine
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2320/11 : for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/33 : Alteration of splicing
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Y 101/01205 : IMP dehydrogenase (1.1.1.205)
• CPC Class Codes: C12Y 304/24087 : ADAMTS13 endopeptidase (3.4.24.87)

Abstract

Provided herein are methods and compositions for increasing production of a target protein or functional RNA by a cell.

Claims

1. A method of treating a subject to increase expression of a target protein or a target functional RNA by cells of the subject, the method comprising contacting the cells of the subject with an antisense oligomer, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′

splice site of the retained intron, and an exon flanking a 3′

splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein or the target functional RNA;

wherein the antisense oligomer binds to a targeted region of the RIC pre-mRNA;

wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region +6 relative to the 5′

splice site of the retained intron to −

16 relative to the 3′

splice site of the retained intron;

whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or the target functional RNA, thereby increasing a level of mRNA encoding the target protein or the target functional RNA and increasing expression of the target protein or the target functional RNA by the cells of the subject;

wherein the cells of the subject produce the target protein or the target functional RNA in a form that is fully-functional compared to a corresponding wild-type protein or wild-type RNA;

wherein the subject has a condition caused by a deficient amount or activity of the target protein or a deficient amount or activity of the target functional RNA; and

wherein the deficient amount or activity of the target protein or the target functional RNA is caused by haploinsufficiency of the target protein or the target functional RNA.

2. The method of claim 1, wherein the targeted region of the RIC pre-mRNA is in the retained intron within:

a region +6 to +100 relative to the 5′

splice site of the retained intron.

3. The method of claim 1, wherein the target protein produced is a full-length protein, or a wild-type protein.

4. The method of claim 1, wherein a total amount of the mRNA encoding the target protein or the target functional RNA produced in the cell contacted with the antisense oligomer is increased at least about 1.1-fold compared to a total amount of the mRNA encoding the target protein or the target functional RNA produced in a control cell.

5. The method of claim 1, wherein a total amount of the target protein produced by the cell contacted with the antisense oligomer is increased at least about 1.1-fold compared to a total amount of the target protein produced by a control cell.

6. The method of claim 1, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

7. The method of claim 1, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′

-O-methyl moiety, a 2′

-Fluoro moiety, or a 2′

-O-methoxyethyl moiety.

8. The method of claim 1, wherein the antisense oligomer comprises a modified sugar moiety.

9. The method of claim 1, wherein the antisense oligomer consists of from 8 to 50 nucleobases.

10. The method of claim 1, wherein the antisense oligomer comprises a sequence with at least 80% complementary to the targeted region of the RIC pre-mRNA that encodes the target protein or the target functional RNA.

11. The method of claim 1, wherein the condition is a disease or disorder.

12. The method of claim 11, wherein the disease or disorder is selected from the group consisting of thrombotic thrombocytopenic purpura, tuberous sclerosis complex, polycystic kidney disease, familial dysautonomia, retinitis pigmentosa type 10, retinitis pigmentosa type 11, cystic fibrosis, retinoblastoma, beta thalassemia, and sickle cell disease.

13. The method of claim 11, wherein the target protein or the target functional RNA and the RIC pre-mRNA are encoded by a gene selected from the group consisting of ADAMTS13, TSC1, PKD1, IKBKAP, IMPDH1, PRPF31, CFTR, RB1, HBG1, HBG2, and HBB.

14. The method of claim 1, wherein the targeted region of the RIC pre-mRNA comprises a sequence selected from the group consisting of SEQ ID NOS:

1-102 and 375-384.

15. The method of claim 1, wherein the subject is a human.

16. The method of claim 1, wherein the subject is a non-human animal.

17. The method of claim 1, wherein the cells are contacted with the antisense oligomer ex vivo.

18. The method of claim 1, wherein the antisense oligomer is administered to the subject by intravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebroventricular injection, intramuscular injection, topical application, or implantation.

19. The method of claim 1, wherein nucleotides that are −

3e to −

1e of the exon flanking the 5′

splice site and +1 to +6 of the retained intron are identical to nucleotides at corresponding positions of a corresponding wild-type sequence.

20. The method of claim 1, wherein nucleotides that are −

15 to −

1 of the retained intron and +1e of the exon flanking the 3′

splice site are identical to nucleotides at corresponding positions of a corresponding wild-type sequence.

21. The method of claim 1, wherein the antisense oligomer comprises a sequence selected from the group consisting of SEQ ID NOs:

103-374 and 385-390.

22. The method of claim 1, wherein the targeted region of the RIC pre-mRNA is in the retained intron within the region −

16 to −

100 relative to the 3′

splice site of the retained intron.

23. A method of treating a subject to increase expression of a target protein or a target functional RNA by cells of the subject, the method comprising contacting the cells of the subject with an antisense oligomer, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′

splice site of the retained intron, and an exon flanking a 3′

splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein or the target functional RNA;

wherein the antisense oligomer binds to a targeted region of the RIC pre-mRNA;

wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region +6 relative to the 5′

splice site of the retained intron to −

16 relative to the 3′

splice site of the retained intron;

whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or the target functional RNA, thereby increasing a level of mRNA encoding the target protein or the target functional RNA and increasing expression of the target protein or the target functional RNA by the cells of the subject;

wherein the cells of the subject produce the target protein or the target functional RNA in a form that is fully-functional compared to a corresponding wild-type protein or wild-type RNA;

wherein the subject has a condition caused by a deficient amount or activity of the target protein or a deficient amount or activity of the target functional RNA;

wherein the condition is a disease or disorder; and

wherein the disease or disorder is selected from the group consisting of thrombotic thrombocytopenic purpura, tuberous sclerosis complex, polycystic kidney disease, familial dysautonomia, retinitis pigmentosa type 10, retinitis pigmentosa type 11, cystic fibrosis, retinoblastoma, beta thalassemia, and sickle cell disease.

27. The method of any one of the claims 23-26, wherein the targeted region of the RIC pre-mRNA is in the retained intron within:

a region +6 to +100 relative to the 5′

splice site of the retained intron.

28. The method of any one of the claims 23-26, wherein the target protein produced is a full-length protein, or a wild-type protein.

29. The method of any one of the claims 23-26, wherein a total amount of the mRNA encoding the target protein or the target functional RNA produced in the cell contacted with the antisense oligomer is increased at least about 1.1-fold compared to a total amount of the mRNA encoding the target protein or the target functional RNA produced in a control cell.

30. The method of any one of the claims 23-26, wherein a total amount of the target protein produced by the cell contacted with the antisense oligomer is increased at least about 1.1-fold compared to a total amount of the target protein produced by a control cell.

31. The method of any one of the claims 23-26, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

32. The method of any one of the claims 23-26, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2′

-O-methyl moiety, a 2′

-Fluoro moiety, or a 2′

-O-methoxyethyl moiety.

33. The method of any one of the claims 23-26, wherein the antisense oligomer comprises a modified sugar moiety.

34. The method of any one of the claims 23-26, wherein the antisense oligomer consists of from 8 to 50 nucleobases.

35. The method of any one of the claims 23-26, wherein the antisense oligomer comprises a sequence with at least 80% complementary to the targeted region of the RIC pre-mRNA that encodes the target protein or the target functional RNA.

36. The method of any one of the claims 23-26, wherein the subject is a human.

37. The method of any one of the claims 23-26, wherein the subject is a non-human animal.

38. The method of any one of the claims 23-26, wherein the cells are contacted with the antisense oligomer ex vivo.

39. The method of any one of the claims 23-26, wherein the antisense oligomer is administered to the subject by intravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebroventricular injection, intramuscular injection, topical application, or implantation.

40. The method of any one of the claims 23-26, wherein nucleotides that are −

3e to −

1e of the exon flanking the 5′

splice site and +1 to +6 of the retained intron are identical to nucleotides at corresponding positions of a corresponding wild-type sequence.

41. The method of any one of the claims 23-26, wherein nucleotides that are −

15 to −

1 of the retained intron and +1e of the exon flanking the 3′

splice site are identical to nucleotides at corresponding positions of a corresponding wild-type sequence.

42. The method of any one of the claims 23-26, wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region −

16 to −

100 relative to the 3′

splice site of the retained intron.

24. A method of treating a subject to increase expression of a target protein or a target functional RNA by cells of the subject, the method comprising contacting the cells of the subject with an antisense oligomer, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′

splice site of the retained intron, and an exon flanking a 3′

splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein or the target functional RNA;

wherein the antisense oligomer binds to a targeted region of the RIC pre-mRNA;

wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region +6 relative to the 5′

splice site of the retained intron to −

16 relative to the 3′

splice site of the retained intron;

whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or the target functional RNA, thereby increasing a level of mRNA encoding the target protein or the target functional RNA and increasing expression of the target protein or the target functional RNA by the cells of the subject;

wherein the cells of the subject produce the target protein or the target functional RNA in a form that is fully-functional compared to a corresponding wild-type protein or wild-type RNA;

wherein the subject has a condition caused by a deficient amount or activity of the target protein or a deficient amount or activity of the target functional RNA;

wherein the condition is a disease or disorder; and

wherein the target protein or the target functional RNA and the RIC pre-mRNA are encoded by a gene selected from the group consisting of ADAMTS13, TSC1, PKD1, IKBKAP, IMPDH1, PRPF31, CFTR, RB1, HBG1, HBG2, and HBB.

25. A method of treating a subject to increase expression of a target protein or a target functional RNA by cells of the subject, the method comprising contacting the cells of the subject with an antisense oligomer, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′

splice site of the retained intron, and an exon flanking a 3′

splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein or the target functional RNA;

wherein the antisense oligomer binds to a targeted region of the RIC pre-mRNA comprising a sequence selected from the group consisting of SEQ ID NOS;

1-102 and 375-384;

wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region +6 relative to the 5′

splice site of the retained intron to −

16 relative to the 3′

splice site of the retained intron; and

whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or the target functional RNA, thereby increasing a level of mRNA encoding the target protein or the target functional RNA and increasing expression of the target protein or the target functional RNA by the cells of the subject;

wherein the cells of the subject produce the target protein or the target functional RNA in a form that is fully-functional compared to a corresponding wild-type protein or wild-type RNA.

26. A method of treating a subject to increase expression of a target protein or a target functional RNA by cells of the subject, the method comprising contacting the cells of the subject with an antisense oligomer, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), wherein the RIC pre-mRNA comprises a retained intron, an exon flanking a 5′

splice site of the retained intron, and an exon flanking a 3′

splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein or the target functional RNA;

wherein the antisense oligomer binds to a targeted region of the RIC pre-mRNA;

wherein the targeted region of the RIC pre-mRNA is in the retained intron within a region +6 relative to the 5′

splice site of the retained intron to −

16 relative to the 3′

splice site of the retained intron;

wherein the antisense oligomer comprises a sequence selected from the group consisting of SEQ ID NOs;

103-374 and 385-390; and

whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or the target functional RNA, thereby increasing a level of mRNA encoding the target protein or the target functional RNA and increasing expression of the target protein or the target functional RNA by the cells of the subject;

wherein the cells of the subject produce the target protein or the target functional RNA in a form that is fully-functional compared to a corresponding wild-type protein or wild-type RNA.