Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded protein
The present application describes a coding nucleic acid sequence, particularly a messenger RNA (mRNA), comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal and the use thereof for increasing the expression of an encoded protein. It also discloses its use for the preparation of a pharmaceutical composition, especially a vaccine e.g. for the use in the treatment of tumours and cancer diseases, cardiovascular diseases, infectious diseases, autoimmune diseases or genetic diseases, or in gene therapy. The present invention further describes an in vitro transcription method, in vitro methods for increasing the expression of a protein using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal and an ex vivo and in vivo method.
1. An isolated nucleic acid comprising:
a) a coding region encoding a therapeutic polypeptide, wherein the coding region does not code for histone polypeptides, reporter polypeptides selected from EGFP and Luciferase, or marker or selection polypeptides selected from alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT),
b) at least one histone stem-loop, and
c) a poly(A) sequence or a polyadenylation signal.
2. The nucleic acid of claim 1 , wherein the nucleic acid does not contain one of the components of the group consisting of: a sequence encoding a ribozyme, a viral nucleic acid sequence, a histone stem-loop processing signal, a Neo gene, an inactivated promoter sequence, and an inactivated enhancer sequence.
3. The nucleic acid of claim 1 , wherein the nucleic acid does not contain a ribozyme and one of the group consisting of: a Neo gene, an inactivated promotor sequence, an inactivated enhancer sequence, and a histone stem-loop processing signal.
4. The nucleic acid of claim 1 , wherein the nucleic acid is an RNA.
5. The nucleic acid of claim 4 , wherein the RNA comprises a 5′ cap structure and a poly(A) sequence of about 25 to about 400 adenosine nucleotides.
6. The nucleic acid of claim 1 , wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides.
7. The nucleic acid of claim 1 , wherein the polyadenylation signal comprises the consensus sequence NNUANA, AAUAAA, or AUUAAA.
8. The nucleic acid of claim 1 , wherein the nucleic acid is monocistronic or bicistronic.
9. The nucleic acid of claim 1 , wherein the therapeutic polypeptide is selected from the group consisting of a cytokine, lymphokine, monokine, growth factor, receptor, signal transduction molecule, transcription factor, anticoagulant, antithrombins, antiallergic protein, apoptotic factor, therapeutic active enzyme, and a protein connected with an acquired or hereditary disease.
10. The nucleic acid of claim 1 , wherein the therapeutic polypeptide is a therapeutic antibody.
11. The nucleic acid of claim 1 , wherein at least one guanosine, uridine, adenosine, thymidine, or cytidine position of the nucleic acid molecule is substituted with a nucleotide analog 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.
12. The nucleic acid of claim 1 , wherein the nucleic acid further comprises a sequence of at least 10 consecutive cytidines.
13. The nucleic acid of claim 1 , wherein the nucleic acid further comprises a stabilizing sequence.
14. The nucleic acid of claim 13 , wherein the stabilizing sequence comprises a sequence from the alpha globin 3′ UTR, positioned 3′ relative to the polypeptide coding region of the nucleic acid.
15. A pharmaceutical composition comprising the nucleic acid of claim 1 , in complex with a cationic or polycationic compound.
16. The pharmaceutical composition of claim 15 , wherein the cationic or polycationic compound is a cationic or polycationic lipid or polypeptide.
17. A pharmaceutical composition comprising the nucleic acid of claim 1 and a pharmaceutically acceptable carrier.
18. A method of treatment comprising administering the pharmaceutical composition of claim 17 to a subject, and expressing a therapeutic polypeptide from the nucleic acid, thereby treating the subject.
19. The method of claim 18 , wherein the pharmaceutical composition is administered by injection.
20. The method of claim 19 , wherein the pharmaceutical composition is administered by subcutaneous, intravenous, or intramuscular injection.