Artificial nucleic acid molecules for improved protein or peptide expression
The invention relates to an artificial nucleic acid molecule comprising at least one 5′UTR element which is derived from a TOP gene, at least one open reading frame, and preferably at least one histone stem-loop. Optionally the artificial nucleic acid molecule may further comprise, e.g. a poly(A)sequence, a poyladenylation signal, and/or a 3′UTR. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination.
1. An artificial nucleic acid molecule comprising, from 5′ to 3′:
a) at least one 5′-untranslated region element (5′UTR element) which comprises a nucleic acid sequence having an identity of at least 95% to a nucleic acid sequence according to any of SEQ ID NOs: 1368 and 1452-1458, or a corresponding RNA sequence, wherein the 5′UTR element lacks a 5′TOP motif; and
b) at least one open reading frame (ORF),
wherein the ORF is heterologous relative to the 5′UTR element.
2. The artificial nucleic acid molecule according to claim 1 , further comprising:
c) at least one histone stem-loop.
3. The artificial nucleic acid molecule according to claim 1 , further comprising at least one 3′ UTR element of a mammalian gene positioned 3′ relative to the ORF.
4. The artificial nucleic acid molecule according to claim 3 , wherein the 3′ UTR element comprises a 3′ UTR element of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene.
5. The artificial nucleic acid molecule according to claim 4 , wherein the 3′ UTR element comprises a 3′ UTR element of a human albumin gene.
6. The artificial nucleic acid molecule according to claim 3 , further comprising a poly(A) sequence and/or polyadenylation signal wherein the poly(A) sequence and/or polyadenylation signal is located within the 3′ UTR element.
7. The artificial nucleic acid molecule according to claim 6 , wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides.
8. The artificial nucleic acid molecule according to claim 3 , wherein the at least one 3′ UTR element comprises an mRNA-stabilizing element.
9. The artificial nucleic acid molecule according to claim 1 , further comprising a poly(A) sequence and/or a polyadenylation signal.
10. The artificial nucleic acid molecule according to claim 1 , further comprising a poly(C) sequence positioned 3′ relative to the ORF.
11. The artificial nucleic acid molecule according to claim 1 , wherein the molecule is a DNA and further comprises a promoter containing-sequence operably linked to the ORF.
12. The artificial nucleic acid molecule according to claim 1 , wherein the molecule is a RNA.
13. The artificial nucleic acid molecule according to claim 12 , wherein the RNA is a mRNA and comprises a 5′ cap.
14. The artificial nucleic acid molecule according to claim 12 , wherein the RNA comprises at least one nucleotide position that is substituted with an analogue of the nucleotide selected from the group consisting of 2-amino-6-chloropurineriboside-5′triphosphate, 2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodouridine-5′triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazoleriboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,triphosphate, N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-pseudouridine-5′-triphosphate, puromycin-5′-triphosphate andxanthosine-5′-triphosphate.
15. The artificial nucleic acid molecule according to claim 1 , wherein one or more codon of the open reading frame is modified to a codon encoding the same amino acid but having an increased G/C content compared to the wild-type codon, thereby increasing the G/C content of the open reading frame compared to the wild type version of the open reading frame.
16. The artificial nucleic acid molecule according to claim 1 , wherein the ORF encodes a reporter polypeptide, a human polypeptide, a tumour antigen or an infectious disease antigen.
17. The artificial nucleic acid molecule according to claim 1 , wherein the ORF encodes an antibody or a portion thereof.
18. A pharmaceutical composition comprising a RNA molecule in accordance with claim 12 in a pharmaceutically acceptable carrier.
19. A method of treatment comprising administering to a subject a pharmaceutically effective dose of the pharmaceutical composition of claim 18 .
20. A method of expressing a polypeptide in a subject comprising administering an artificial nucleic acid molecule according to claim 1 to the subject, wherein the polypeptide is encoded by the ORF of said artificial nucleic acid molecule.