Oligonucleotides matching COL7A1 exon 73 for epidermolysis bullosa therapy

• US 10,563,198 B2
• Filed: 03/11/2016
• Issued: 02/18/2020
• Est. Priority Date: 03/11/2015
• Status: Active Grant

• Current Assignee: Wings Therapeutics, Inc.
• Original Assignee: Wings Therapeutics, Inc.
• Inventors: Haisma, Elisabeth Marlene, Potman, Marko, Beumer, Wouter, Brinks, Vera
• Primary Examiner(s): Bowman, Amy H
• Application Number:
  US15/554,420
• Publication Number:
  US 20180216106A1
• Time in Patent Office: 1,439 Days
• 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 2310/315 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

Abstract

Antisense oligonucleotides capable of preventing or reducing exon 73 inclusion into the human COL7A mRNA are characterized in various ways: (a) the oligonucleotide'"'"'s sequence includes at most two CpG sequences; (b) the oligonucleotide has a length of no more than 24 nucleotides; (c) the oligonucleotide is capable of annealing to the (SRp40/SC35 binding/ESE) element in exon73. These oligonucleotides can usefully be oligoribonucleotides with modified internucleosidic linkages e.g. phosphorothioate linkages.

Claims

1. An antisense oligoribonucleotide capable of preventing or reducing exon 73 inclusion into a human COL7A1 mRNA when the mRNA is produced by splicing from a pre-mRNA in a human cell, wherein the oligoribonucleotide comprises a nucleotide sequence selected from SEQ ID NO:

5, 24, 25, 26, 27, 28, 39, 40, 41, 42, 43, 29 and 35, the oligoribonucleotide has a length of between 16 and 24 nucleotides, and the oligoribonucleotide comprises at least one of (i) a nucleotide residue having a modified base, (ii) a modified backbone, and (iii) a non-natural internucleoside linkage.

2. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide has no more than one CpG sequence.

3. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of between 16 and 23 nucleotides.

4. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of 16, 17, or 18 nucleotides.

5. The antisense oligoribonucleotide of claim 1, wherein the non-natural internucleoside linkage is a phosphorothioate linkage.

6. The antisense oligoribonucleotide of claim 1, wherein the modified base comprises a lower 2′

-O-alkyl modification.

7. The antisense oligoribonucleotide of claim 6, wherein the modification is a 2′

-O-methyl modification.

8. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 70%.

9. The antisense oligoribonucleotide of claim 8, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 80%.

10. The antisense oligoribonucleotide of claim 9, wherein the oligoribonucleotide is capable of reducing exon 73 inclusion in a HeLa cell, or a sample derived therefrom, by more than 90%.

11. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide comprises the nucleotide sequence of SEQ ID NO:

35.

12. The antisense oligoribonucleotide of claim 1, wherein the modified base comprises a 2′

-O-alkyl-O-alkyl modification.

13. The antisense oligoribonucleotide of claim 1, wherein the modified base comprises a 2′

-methoxyethoxy modification.

14. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of 19, 20, or 21 nucleotides.

15. The antisense oligoribonucleotide of claim 1, wherein the oligoribonucleotide has a length of 22, 23 or 24 nucleotides.