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 further relates to a vector comprising the artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal, to a cell comprising the artificial nucleic acid molecule or the vector, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition. 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.
1. A method for treating or preventing an infectious disease, the method comprising administering an RNA molecule comprising:
a) at least one open reading frame (ORF) encoding an antigen from a pathogen associated with the infectious disease; and
b) a 3′-untranslated region (3′-UTR) comprising at least two poly(A) sequences, wherein at least one of the poly(A) sequences comprises at least 70 adenine nucleotides, wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides,
wherein the RNA molecule is administered intramuscularly.
2. The method of claim 1 , wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising a poly(C) element and/or a histone stem-loop element.
3. The method of claim 1 , wherein the pathogen is selected from the group consisting of a bacterial, a viral, a fungal, and a protozoan pathogen.
4. The method of claim 1 , wherein at least one of the poly(A) sequences comprises at least 150 adenine nucleotides.
5. The method of claim 1 , wherein at least one of the poly(A) sequences is located at the 3′ terminus of the RNA molecule.
6. The method of claim 1 , wherein the 3′-UTR further comprises at least one 3′-UTR element that is not a poly(A) sequence.
7. The method of claim 1 , wherein the RNA molecule further comprises a 5′-cap structure, a poly(C) sequence, a histone stem-loop, and/or an IRES motif.
8. The method of claim 7 , wherein the histone stem-loop comprises a sequence according to SEQ ID NO: 11.
9. The method of claim 1 , wherein the RNA molecule further comprises a 5′-UTR and/or a promoter containing-sequence.
10. The method of claim 9 , wherein the 5′-UTR comprises at least a portion of a 5′-UTR of a 5′ Terminal Oligopyrimidine Tract (TOP) gene.
11. The method of claim 1 , wherein the open reading frame is at least partially G/C modified, wherein the encoded antigen sequence is not altered.
12. The method of claim 11 , wherein the G/C content of the open reading frame is increased compared to a wild type open reading frame, wherein the encoded antigen sequence is not altered.
13. The method of claim 1 , wherein the open reading frame is at least partially codon-optimized.
14. The method of claim 13 , wherein the RNA molecule comprises at least one nucleotide analogue.
15. The method of claim 14 , wherein the at least one nucleotide analogue is a modified form of uridine.
16. The method of claim 15 , wherein the modified form of uridine is chemically altered by methylation.
17. The method of claim 16 , wherein the modified form of uridine is a naturally occurring variant of uridine.
18. The method of claim 17 , wherein the RNA molecule is associated or complexed with a cationic carrier or a polycationic carrier.
19. The method of claim 1 , wherein the at least two poly(A) sequence elements are separated by a nucleic acid sequence comprising a poly(C) element and a histone stem-loop element.
20. The method of claim 1 , wherein the RNA molecule is associated with or complexed with a cationic or polycationic compound or polymeric carrier.