RNA vector with an open reading frame, an albumin 3′-UTR, and a histone stem loop
The invention relates to an artificial nucleic acid molecule comprising at least one open reading frame and at least one 3′UTR element comprising a nucleic acid sequence which is derived from the 3′UTR of an albumin gene or from a variant of the 3′UTR of an albumin gene. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination. Furthermore, the invention relates to the use of a 3′UTR element comprising a nucleic acid sequence which is derived from the 3′UTR of an albumin gene or from a variant of the 3′UTR of an albumin gene for the stabilization and/or prolongation of protein expression from a nucleic acid sequence comprising such 3′UTR element.
1. An isolated RNA molecule comprising:
a) at least one open reading frame (ORF);
b) a heterologous 3′-untranslated region element (3′UTR element) comprising a nucleic acid sequence having:
i) at least 90% identity to a mammalian albumin mRNA 3′UTR; or
ii) at least 50 consecutive nucleotides of a mammalian albumin mRNA 3′UTR; and
c) a histone stem-loop.
2. The RNA molecule of claim 1 , wherein:
(i) the ORF does not code for beta-globin, if the 3′UTR element is from the rat albumin 3′UTR; and
(ii) the ORF does not code for human factor IX, if the 3′UTR element is from the human albumin 3′UTR.
3. The RNA molecule of claim 1 , wherein the 3′UTR element comprises a nucleic acid sequence having at least 50, 75 or 100 consecutive nucleotides of a mammalian albumin mRNA 3′UTR.
4. The RNA molecule of claim 1 , wherein the 3′UTR element comprises a nucleic acid sequence having at least 90% or 95% identity to a mammalian albumin mRNA 3′UTR.
5. The RNA molecule of claim 1 , wherein the 3′UTR element comprises a nucleic acid sequence having at least 90% or 95% identity to a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.
6. The RNA molecule of claim 1 , wherein the 3′UTR element comprises a human albumin mRNA 3′UTR.
7. The RNA molecule of claim 1 , wherein the RNA further comprises a 5′-cap, a Poly(C) sequence, an IRES motif and/or a poly(A) sequence.
8. The RNA molecule of claim 1 , wherein the ORF has an increased amount of guanine and/or cytosine residues as compared to a corresponding wild type ORF.
9. The RNA molecule of claim 7 , wherein the RNA further comprises a poly(A) sequence of 20 to 300 nucleotides.
10. The RNA molecule of claim 1 , wherein the at least one ORF encodes a therapeutic polypeptide or an antigen.
11. The RNA molecule of claim 10 , wherein the at least one ORF encodes an antigen selected from the group consisting of an infectious disease antigen, and allergic antigen or a tumour antigen.
12. The RNA molecule of claim 1 , wherein at least one guanosine, uridine, adenosine, or cytidine position of the RNA molecule is substituted with an analogue of these nucleotides selected from 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, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, and xanthosine-5′-triphosphate.
13. A pharmaceutical composition comprising a RNA molecule of claim 1 and a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 13 , further comprising an adjuvant.
15. The pharmaceutical composition of claim 13 , wherein the composition further comprises a cationic or polycationic compound in complex with the nucleic acid molecule.
16. The isolated RNA molecule of claim 1 , comprising, from 5′ to 3′:
a) the at least one open reading frame (ORF);
b) the heterologous 3′-untranslated region element (3′UTR element) comprising a nucleic acid sequence having:
i) at least 90% identity to a mammalian albumin mRNA 3′UTR; or
ii) at least 50 consecutive nucleotides of a mammalian albumin mRNA 3′UTR; and
c) the histone stem-loop.