Artificial nucleic acid molecules comprising a 5′TOP UTR
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 optionally at least one 3′UTR element comprising a nucleic acid sequence which is preferably derived from the 3′UTR of a gene providing a stable mRNA, such as an albumin gene, or from a variant of the 3′UTR of a gene providing a stable mRNA. 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) a 5′-untranslated region element (5′UTR element) of a mammalian TOP gene selected from the group consisting of AIG1, ASAH1, ATP5A1, COX6C, HSD17B4, RPL21, RPL32, and RPL35; and
(b) at least one heterologous open reading frame (ORF); and
(c) at least one 3′UTR element of a human albumin gene with greater than 90% identity to the gene encoded by SEQ ID NO: 1376.
2. The artificial nucleic acid molecule according to claim 1 , further comprising (d) a poly(A) sequence and/or a polyadenylation signal.
3. The artificial nucleic acid molecule according to claim 1 , further comprising a polyadenylation signal wherein the polyadenylation signal is located within the 3′UTR element.
4. The artificial nucleic acid molecule according to claim 2 , wherein the poly(A) sequence has a length of about 20 to about 300 adenine nucleotides.
5. The artificial nucleic acid molecule according to claim 1 , wherein the open reading frame does not code for a GFP protein, a luciferase protein, a globin protein, human growth hormone, or human albumin.
6. The artificial nucleic acid molecule according to claim 1 , further comprising a poly(C) sequence positioned 3′ relative to the ORF.
7. 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.
8. The artificial nucleic acid molecule according to claim 1 , wherein the molecule is a RNA.
9. The artificial nucleic acid molecule according to claim 8 , wherein the RNA is a mRNA and comprises a 5′ cap.
10. The artificial nucleic acid molecule according to claim 8 , 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, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, puromycin-5′-triphosphate and xanthosine-5′-triphosphate.
11. The artificial nucleic acid molecule according to claim 1 , wherein the G/C content of the open reading frame is increased compared to the wild type version of the open reading frame.
12. The artificial nucleic acid molecule according to claim 1 , wherein the ORF encodes a human polypeptide, a tumour antigen or an infectious disease antigen.
13. The artificial nucleic acid molecule according to claim 1 , wherein the ORF encodes an antibody or a portion thereof.
14. A pharmaceutical composition comprising a RNA molecule in accordance with claim 8 in a pharmaceutically acceptable carrier.
15. 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.
16. The artificial nucleic acid molecule according to claim 1 , further comprising a histone stem-loop sequence.
17. The artificial nucleic acid molecule according to claim 1 , wherein the 5′ UTRS element is from RPL32.