Artificial nucleic acid molecules for improved protein expression

Patent metadata

Field	Value
Publication number	US-11761009-B2
Application number	US-202217713533-A
Country	US
Kind code	B2
Filing date	Apr 5, 2022
Priority date	Dec 12, 2014
Publication date	Sep 19, 2023
Grant date	Sep 19, 2023

Abstract

The invention relates to an artificial nucleic acid molecule or a vector comprising an open reading frame and a 3′-UTR comprising at least two poly(A) sequences. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.

First claim

The invention claimed is:

1. An artificial RNA molecule comprising:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding a protein; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,

wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule comprises at least one nucleotide analogue, which is a modified form of uridine.

2. The artificial RNA molecule of claim 1 , wherein at of the first poly(A) or second poly(A) sequences is located at the 3′ terminus of the RNA molecule.

3. The artificial RNA molecule of claim 2 , wherein the 5′-cap structure is m7GpppN, wherein N is the terminal 5′ nucleotide of the RNA molecule.

4. The artificial RNA molecule of claim 3 , wherein the RNA molecule comprises a heterologous 5′ UTR sequence.

5. The artificial RNA molecule of claim 4 , wherein the protein is an antigen.

6. The artificial RNA molecule of claim 5 , wherein the antigen is from a pathogen associated with an infectious disease.

7. The artificial RNA molecule of claim 5 , wherein the antigen is a tumor antigen.

8. The artificial RNA molecule of claim 5 , wherein the ORF encoding the protein has a G/C content that is increased by at least 7% relative to a corresponding reference ORF encoding the protein.

9. The artificial RNA molecule of claim 8 , wherein the ORF encoding the protein has a G/C content that is increased by at least 15% relative to a corresponding reference ORF encoding the protein.

10. The artificial RNA molecule of claim 5 , wherein the first poly(A) sequence comprises at least 30 adenine nucleotides.

11. A pharmaceutical composition comprising the artificial RNA molecule of claim 10 and one or more pharmaceutically acceptable vehicles.

12. The pharmaceutical composition of claim 11 , wherein the RNA molecule is complexed with a cationic or polycationic compound.

13. The pharmaceutical composition of claim 12 , wherein the cationic or polycationic compound comprises a cationic or polycationic peptide.

14. The pharmaceutical composition of claim 12 , wherein the cationic or polycationic compound comprises a cationic lipid.

15. The artificial RNA molecule of claim 5 , wherein the modified form of uridine is chemically altered by methylation.

16. The artificial RNA molecule of claim 15 , wherein the modified form of uridine is a naturally occurring variant of uridine.

17. The artificial RNA molecule of claim 6 , wherein the antigen is a viral antigen.

18. The artificial RNA molecule of claim 17 , wherein the viral antigen is associated with a virus selected from the group consisting of an influenza virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus, human immunodeficiency virus, dengue virus, cytomegalovirus, hepatitis B virus, rabies virus, coronavirus, and yellow fever virus.

19. The artificial RNA molecule of claim 18 , wherein the viral antigen is an influenza virus antigen.

20. The artificial RNA molecule of claim 19 , wherein the influenza virus antigen is a HA antigen.

21. The artificial RNA molecule of claim 18 , wherein the viral antigen is a coronavirus antigen.

22. An artificial RNA molecule comprising:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding an antigen; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,

wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the ORF encoding the antigen has a G/C content that is increased by at least 7% relative to a corresponding reference ORF encoding the antigen.

23. The artificial RNA molecule of claim 22 , wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation.

24. The artificial RNA molecule of claim 23 , wherein the ORF encoding the antigen has a G/C content that is increased by at least 15% relative to a corresponding reference ORF encoding the antigen.

25. The artificial RNA molecule of claim 24 , wherein the antigen is a viral antigen.

26. The artificial RNA molecule of claim 25 , wherein the viral antigen is associated with a virus selected from the group consisting of an influenza virus, respiratory syncytial virus, herpes simplex virus, human papilloma virus, human immunodeficiency virus, dengue virus, cytomegalovirus, hepatitis B virus, rabies virus, coronavirus, and yellow fever virus.

27. The artificial RNA molecule of claim 26 , wherein the viral antigen is a coronavirus antigen.

28. The artificial RNA molecule of claim 26 , wherein the viral antigen is an influenza virus antigen.

29. The artificial RNA molecule of claim 28 , wherein the influenza virus antigen is a HA antigen.

30. A pharmaceutical composition comprising an artificial RNA molecule and one or more pharmaceutically acceptable vehicles, said artificial RNA comprising:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding an antigen; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,

wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule is complexed with a cationic carrier or a polycationic carrier,
wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation, and
wherein when the RNA molecule is expressed in an organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference RNA comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.

31. The pharmaceutical composition of claim 30 , wherein one of the first poly(A) or second poly(A) sequences of the RNA molecule is located at the 3′ terminus of the RNA molecule.

32. The pharmaceutical composition of claim 31 , wherein the 5′-cap structure is m7GpppN.

33. The pharmaceutical composition of claim 32 , wherein the cationic carrier or polycationic carrier comprises a cationic lipid.

34. The pharmaceutical composition of claim 33 , wherein the ORF encoding the antigen has a G/C content that is increased by at least 7% relative to a corresponding reference ORF encoding the antigen.

35.

Assignees

• CureVac SE

Inventors

• Thess Andreas
• Schlake Thomas
• Grund Stefanie

Classifications

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title

• A61K2039/53

  DNA (RNA) vaccination · CPC title

• A61K2039/54

  characterised by the route of administration · CPC title

• C12N2760/16134

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title