Artificial nucleic acid molecules for improved protein expression
The invention relates to an artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal. 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 as a medicament, as a vaccine or in gene therapy.
1. A method for stimulating an immune response in an organism, the method comprising administering to the organism a RNA molecule comprising:
a) a 5′-cap structure;
b) an open reading frame encoding an antigen; and
c) a heterologous 3′-untranslated region comprising 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 when the RNA molecule is expressed in the organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence; and
wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation.
2. The method of claim 1 , wherein the RNA molecule is administered intramuscularly.
3. The method of claim 1 , wherein the antigen is a tumor antigen.
4. The method of claim 1 , wherein the antigen is a viral antigen.
5. The method of claim 4 , 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, plasmodium, staphylococcus aureus, dengue virus, chlamydia trachomatis, cytomegalovirus, hepatitis B virus, mycobacterium tuberculosis, rabies virus, coronavirus, and yellow fever virus.
6. The method of claim 5 , wherein the viral antigen in an influenza virus antigen.
7. The method of claim 5 , wherein the influenza virus antigen is a HA antigen.
8. The method of claim 5 , wherein the antigen is a coronavirus antigen.
9. The method of claim 8 , wherein the RNA molecule is formulated with a cationic lipid.
10. The method of claim 8 , wherein the RNA molecule is transfected into cells of the organism in a nanoparticle.
11. The method of claim 10 , wherein the first poly(A) sequence comprises at least 30 adenine nucleotides.
12. The method of claim 11 , wherein the nucleotide sequence which separates the first and the second poly(A) sequences consists of 10 nucleotides and has no more than 2 consecutive adenine nucleotides.
13. The method of claim 12 , wherein when administered intramuscularly, the RNA molecule yields an increased immune response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
14. The method of claim 13 , wherein when administered intramuscularly, the RNA molecule yields an increased neutralizing antibody response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
15. The method of claim 1 , wherein the open reading frame has a guanidine/cytosine content that is increased by at least 15% relative to a corresponding reference open reading frame.
16. A method for stimulating an immune response in an organism, the method comprising administering to the organism a RNA molecule comprising:
a) a 5′-cap structure;
b) an open reading frame encoding an antigen; and
c) a heterologous 3′-untranslated region comprising a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 30 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 10 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein when the RNA molecule is expressed in the organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence;
wherein the RNA molecule is formulated with a cationic lipid; and
wherein the RNA molecule is administered intramuscularly.
17. The method of claim 16 , wherein the antigen is a coronavirus antigen.
18. The method of claim 17 , wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation.
19. The method of claim 18 , wherein the open reading frame has a guanidine/cytosine content that is increased by at least 15% relative to a corresponding reference open reading frame.
20. The method of claim 16 , wherein the RNA molecule is transfected into cells of the organism in a nanoparticle.
21. The method of claim 20 , wherein the RNA molecule yields an increased immune response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
22. The method of claim 21 , wherein when administered intramuscularly, the RNA molecule yields an increased neutralizing antibody response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
23. A method for stimulating an immune response in an organism, the method comprising administering to the organism a RNA molecule comprising:
a) a 5′-cap structure;
b) an open reading frame encoding a coronavirus antigen; and
c) a heterologous 3′-untranslated region comprising 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 10 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule is administered intramuscularly;
wherein when the RNA molecule is expressed in the organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence;
wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation; and
wherein the RNA molecule is transfected into cells of the organism in a nanoparticle.
24. The method of claim 23 , wherein the open reading frame has a guanidine/cytosine content that is increased by at least 15% relative to a corresponding reference open reading frame.
25. The method of claim 23 , wherein the RNA molecule yields an increased immune response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
26. The method of claim 25 , wherein when administered intramuscularly, the RNA molecule yields an increased neutralizing antibody response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.
27. A method for stimulating an immune response in an organism, the method comprising administering to the organism a RNA molecule comprising:
a) a 5′-cap structure;
b) an open reading frame encoding a coronavirus antigen; and
c) a heterologous 3′-untranslated region comprising a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 30 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 10 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule is administered intramuscularly;
wherein when administered intramuscularly, the RNA molecule yields an increased neutralizing antibody response in comparison to an intramuscular injection of a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence;
wherein the RNA molecule comprises at least one nucleotide analog comprising a modified form of uridine chemically altered by methylation; and
wherein the RNA molecule is transfected into cells of the organism in a nanoparticle.
28. The method of claim 27 , wherein when the RNA molecule is expressed in the organism, the RNA molecule yields increased expression of the antigen encoded by the open reading frame in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking the second poly(A) sequence.