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 tumors 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. A method of expressing a therapeutic polypeptide in a patient comprising providing the patient with a composition comprising an isolated nucleic acid molecule comprising:
a) a coding region, encoding the therapeutic polypeptide, wherein the coding region does not code for histone polypeptides, reporter polypeptides selected from enhanced green fluorescent protein (EGFP) and Luciferase and 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 method 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 method 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 method of claim 1 , wherein the nucleic acid is an RNA.
5. The method 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 method of claim 1 , wherein the poly(A) sequence comprises a sequence of about 25 to about 400 adenosine nucleotides.
7. The method of claim 1 , wherein the polyadenylation signal comprises the consensus sequence NNUANA, AAUAAA, or AUUAAA.
8. The method of claim 1 , wherein the coding region encodes a therapeutic polypeptide selected from groups consisting 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 disease or a hereditary disease.
9. The method of claim 1 , wherein the coding region encodes a therapeutic antibody.
10. The method of claim 1 , wherein the nucleic acid is monocistronic or bicistronic.
11. The method of claim 1 , wherein the composition comprises a pharmaceutically acceptable carrier.
12. The method of claim 1 , wherein the patient has a disease selected from the group consisting of a hereditary disease, autoimmune disease, allergy, cancer disease, tumour disease, and infectious disease.
13. The method of claim 1 , wherein at least one guanosine, uridine, adenosine, thymidine, or cytidine position of the nucleic acid molecule is substituted with a nucleotide analogue 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, puromycin-5′-triphosphate, and xanthosine-5′-triphosphate.
14. The method of claim 1 , wherein the composition is administered by injection.
15. The method of claim 14 , wherein the composition is administered by subcutaneous, intravenous or intramuscular injection.
16. The method of claim 1 , wherein the nucleic acid molecule further comprises a sequence of at least 10 consecutive cytidines.
17. The method of claim 1 , wherein the nucleic acid molecule further comprises a stabilizing sequence.
18. The method of claim 17 , 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 molecule.
19. The method of claim 1 , wherein the composition further comprises a cationic or polycationic compound in complex with the nucleic acid molecule.
20. The method of claim 19 , wherein the polycationic compound is a polycationic lipid or polypeptide.