Granted Patent B2

US 12,203,072 · App. 18/185,929 · Granted Jan 21, 2025

Inventors: Janne Juha Turunen (Leiden, NL); Antti Aalto (Leiden, NL); Bart Klein (Leiden, NL); Lenka Van Sint Fiet (Leiden, NL); Julien Auguste Germain Boudet (Leiden, NL)

Assignee: ProQR Therapeutics II B.V.

C12N15/113C12N2310/11C12N2310/344C12N2310/533C12N2320/34C12Y305/04004

Abstract

Claims (43)

1. An antisense oligonucleotide (AON) targeting an alpha-1-antitrypsin (A1AT) target RNA molecule comprising a target adenosine in a human cell, wherein the AON comprises a Central Triplet of 3 sequential DNA nucleotides (5′-YXZ-3′) wherein:

(a) Y is a nucleotide comprising a uracil nucleobase;

(b) X is a nucleotide that is directly opposite the target adenosine; and

(c) Z is a nucleotide comprising a hypoxanthine nucleobase;

wherein the AON is capable of forming a double stranded complex with the A1AT target RNA molecule;

wherein the AON can mediate the deamination of the target adenosine in the A1AT target RNA molecule by a human ADAR enzyme naturally present in the human cell;

wherein the target RNA molecule is located within the sequence set forth in SEQ ID NO:1; and

wherein the deamination of the target adenosine treats A1AT deficiency.

2. The AON of claim 1 , wherein X is a modified uridine.

3. The AON of claim 2 , wherein the modified uridine is selected from the group consisting of 4-thio-uridine, 5-methoxyuridine, 5-methyluridine, dihydrouridine, pseudouridine, and thienouridine.

4. The AON of claim 1 , wherein the AON is 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.

5. The AON of claim 4 , wherein the AON is 30 nucleotides in length.

6. The AON of claim 1 , wherein the nucleotides outside of the Central Triplet consist of RNA nucleotides each comprising a 2′ substitution in the ribose.

7. The AON of claim 6 , wherein the nucleotides outside of the Central Triplet comprise a 2′ substitution each independently selected from the group consisting of 2′-OMe, 2′-F, and 2′-MOE.

8. The AON of claim 1 , wherein the AON does not comprise a 5′-terminal 06-benzylguanosine.

9. The AON of claim 1 , wherein the AON does not comprise a portion that is capable of forming an intramolecular stem-loop structure capable of binding a human ADAR enzyme present in the cell.

10. The AON of claim 1 , wherein the AON comprises phosphorothioate linkages between the two, three, four or five most terminal 5′ and/or 3′ nucleotides of the AON.

11. The AON of claim 1 , wherein the AON comprises at least one peptidic internucleotidic linkage.

12. The AON of claim 1 , wherein the AON is a peptidonucleic acid.

13. The AON of claim 1 , wherein the uracil nucleobase is selected from the group consisting of methoxyuracil, 5-methyluracil, dihydrouracil, pseudouracil, thienouracil, 4-thiouracil, and 5-hydroxymethyluracil.

14. The AON of claim 1 , wherein the target adenosine is the c.1096G>A mutation in human SERPINA1.

15. A pharmaceutical composition comprising the AON of claim 1 , and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15 , wherein the AON is formulated for intravenous administration.

17. A method of treating A1AT deficiency in a human subject in need thereof comprising administering the AON of claim 1 or a pharmaceutical composition comprising said AON to the human subject.

18. The method of claim 17 , wherein administering is in vivo or ex vivo.

19. The method of claim 17 , wherein the A1AT deficiency is caused by the c.1096G>A mutation in the SERPINA1 gene.

20. A method for the deamination of at least one specific target adenosine present in an A1AT target RNA molecule in a human cell comprising contacting the human cell with the AON of claim 1 .

21. The method of claim 20 , wherein the human cell is a human liver cell.

22. The method of claim 20 , wherein the human cell is an ex vivo human stem cell.

23. The method of claim 22 , wherein the ex vivo human stem cell is a human embryonic stem cell, a human pluripotent stem cell, a human totipotent stem cell, or a human induced pluripotent stem cell.

24. An antisense oligonucleotide (AON) targeting an alpha-1-antitrypsin (A1AT) target RNA molecule comprising a target adenosine in a human cell, wherein the AON comprises a Central Triplet of 3 sequential DNA nucleotides (5′-YXZ-3′) wherein:

(a) Y is a nucleotide comprising 5-methyluracil;

(b) X is a nucleotide that is directly opposite the target adenosine; and

(c) Z is a nucleotide comprising a hypoxanthine nucleobase;

wherein the AON is capable of forming a double stranded complex with the A1AT target RNA molecule;

wherein the AON can mediate the deamination of the target adenosine in the A1AT target RNA molecule by a human ADAR enzyme naturally present in the human cell;

wherein the target RNA molecule is located within the sequence set forth in SEQ ID NO:1; and

wherein the deamination of the target adenosine treats A1AT deficiency.

25. The AON of claim 24 , wherein X is a modified uridine.

26. The AON of claim 25 , wherein the modified uridine is selected from the group consisting of 4-thio-uridine, 5-methoxyuridine, 5-methyluridine, dihydrouridine, pseudouridine, and thienouridine.

27. A pharmaceutical composition comprising the AON of claim 24 , and a pharmaceutically acceptable carrier.

28. A method of treating A1AT deficiency in a human subject in need thereof comprising administering the AON of claim 24 or a pharmaceutical composition comprising said AON to the human subject.

29. The method of claim 28 , wherein the A1AT deficiency is caused by the c.1096G>A mutation in the SERPINA1 gene.

Assignments (1)

Priority Claims (5)

Continuity (3)

References Cited (390)

Cited By (1)

US 12,655,423