Reducing intron retention

• US 9,745,577 B2
• Filed: 06/16/2015
• Issued: 08/29/2017
• 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:
  US14/741,071
• Publication Number:
  US 20160017322A1
• Time in Patent Office: 805 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 pharmaceutical composition comprising:

a polynucleic acid polymer that is from about 10 to about 50 nucleotides in length and hybridizes to a wild-type target sequence of a 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 encodes a protein and comprises an entire retained intron, wherein the polynucleic acid polymer induces splicing out of the entire retained intron from the partially processed mRNA transcript; and

a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable delivery vehicle.

2. The pharmaceutical composition of claim 1, wherein the wild-type target sequence is a binding motif that forms a hairpin structure.

3. The pharmaceutical composition of claim 1, wherein the wild-type target sequence is between two G quadruplexes of a partially processed mRNA transcript.

4. The pharmaceutical composition of claim 1, wherein the wild-type target sequence is within the entire retained intron of the partially processed mRNA transcript.

5. The pharmaceutical composition of claim 1, wherein the wild-type target sequence does not form a G quadruplex.

6. The pharmaceutical composition of claim 1, wherein the wild-type target sequence is an intronic sequence.

7. The pharmaceutical composition of claim 6, wherein the intronic sequence comprises an intronic splicing regulatory element comprising a first CCC motif or a second CCC motif.

8. The pharmaceutical composition of claim 1, wherein the delivery vehicle comprises a cell penetrating peptide or a peptide-based nanoparticle.

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

10. The pharmaceutical composition 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.

11. The pharmaceutical composition 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.

12. The pharmaceutical composition of claim 11, wherein the polynucleic acid polymer comprises an artificial nucleotide.

13. The pharmaceutical composition of claim 12, 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), a 1′

,5′

-anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′

-fluoro N3-P5′

-phosphoramidite.

14. The pharmaceutical composition 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.

15. The pharmaceutical composition 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.

16. The pharmaceutical composition of claim 1, wherein the splicing occurs within a human cell.

17. The pharmaceutical composition of any one of claims 1-8, 9-14, or 15-16, wherein the wild-type target sequence does not comprise a mutation-induced aberrant splice site.