Reducing intron retention

• US 9,714,422 B2
• Filed: 05/06/2016
• Issued: 07/25/2017
• Est. Priority Date: 06/16/2014
• Status: Active Grant

• Current Assignee: University of Southampton
• Original Assignee: University of Southampton
• Inventors: Vorechovsky, Igor, Kralovicova, Jana
• Primary Examiner(s): Bowman, Amy
• Application Number:
  US15/148,303
• Publication Number:
  US 20160244762A1
• Time in Patent Office: 445 Days
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• 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: 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/113 targeting other non-coding nucleic acids, e.g. antagomirs
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/33 Alteration of splicing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material

Abstract

Disclosed herein are methods, compositions, polynucleic acid polymers, assays, and kits for inducing processing of a partially processed mRNA transcript to remove a retained intron to produce a fully processed mRNA transcript that encodes a full-length functional form of a protein. Also described herein are methods and compositions for treating a disease or condition characterized by impaired production of a full-length functional form of a protein or for treating a disease or condition characterized by a defective splicing in a subject.

Claims

1. A method of inducing processing of a partially processed mRNA transcript to facilitate removal of an entire retained intron to produce a fully processed mRNA transcript that encodes a functional form of a protein, the method comprising:

(a) contacting an isolated polynucleic acid polymer to a target cell of a subject, wherein the polynucleic acid polymer is from about 10 to about 50 nucleotides in length;

(b) hybridizing the isolated polynucleic acid polymer to a wild-type target sequence of the partially processed mRNA transcript, wherein the polynucleic acid polymer comprises a sequence that is complementary to at least 10 contiguous bases of the wild-type target sequence, wherein the partially processed mRNA transcript is capable of encoding the functional form of a protein and comprises at least one entire retained intron, optionally interfering with one or more conformational transitions of canonical and noncanonical RNA structures or interacting with trans-acting factors that bind to an intron;

(c) removing the at least one entire retained intron from the partially processed mRNA transcript to produce the fully processed mRNA transcript that encodes the functional form of a protein; and

(d) translating the functional form of a protein from the fully processed mRNA transcript.

2. The method of claim 1, wherein the intron of step (b) is an INS intron.

3. The method of claim 1, wherein the partially processed mRNA transcript comprising the entire retained intron further induces a disease or condition or a predisposition to a disease or condition.

4. The method of claim 3, wherein the disease or condition is diabetes.

5. The method of claim 1, wherein the polynucleic acid polymer comprises an artificial nucleotide.

6. The method of claim 1, wherein the artificial nucleotide is selected from the group consisting of 2′

-O-methyl, 2′

-O-methoxyethyl (2′

-O-MOE), 2′

-O-aminopropyl, 2′

-deoxy, T-deoxy-2′

-fluoro, 2′

-O-aminopropyl (2′

-O-AP), 2′

-O-dimethylaminoethyl (2′

-O-DMAOE), 2′

-O-dimethylaminopropyl (2′

-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′

-O-DMAEOE), 2′

-O—

N-methylacetamido (2′

-O-NMA), a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′

-fluoro N3-P5′

-phosphoramidite.

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

8. The method of claim 1, wherein the target sequence is a binding motif that forms a hairpin structure.

9. The method of claim 1, wherein the target sequence is between two G quadruplexes of a partially processed mRNA transcript.

10. The method of claim 1, wherein the target sequence is within the entire retained intron of the partially processed mRNA transcript.

11. The method of claim 1, wherein the target sequence does not form a G quadruplex.

12. The method of claim 1, wherein the target sequence is an intronic sequence.

13. The method of claim 12, wherein the intronic sequence comprises an intronic splicing regulatory element comprising a first CCC motif or a second CCC motif.

14. The method of claim 1, wherein the polynucleic acid polymer is from about 10 to about 30 nucleotides in length.

15. The method of claim 1, wherein the polynucleic acid polymer comprises a sequence that is at least 60% complementary to the wild-type target sequence of the partially processed mRNA transcript.

16. The method of claim 1, wherein the polynucleic acid polymer comprises a sequence that is complementary to a sequence with:

(i) at least 80% sequence identity to at least 13 contiguous bases of SEQ ID NO;

46;

(ii) at least 10 contiguous bases of SEQ ID NO;

46;

(iii) at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO;

6, SEQ ID NO;

9, SEQ ID NO;

12, SEQ ID NO;

15, SEQ ID NO;

18, SEQ ID NO;

21, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

30, SEQ ID NO;

33, SEQ ID NO;

36, SEQ ID NO;

39, SEQ ID NO;

42, and SEQ ID NO;

45;

or

(iv) at least 60% sequence identity to SEQ ID NO;

3.

17. The method of claim 1, wherein the polynucleic acid polymer is modified at a nucleoside moiety, at a phosphate moiety, at a 5′

terminus, at a 3′

terminus, or a combination thereof.

18. The method of claim 3, wherein the disease or condition is a hereditary disease.

19. The method of claim 1, wherein the polynucleic acid polymer comprises a sequence having at least 80% sequence identity to at least 13 contiguous bases of a sequence selected from the group consisting of SEQ ID NOs:

47-434.

20. The method of any one of claims 1-7, 8-13 and 14-19, wherein the wild-type target sequence does not comprise a mutation-induced aberrant splice site.