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 therapeutic protein
The present disclosure concerns a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. The present disclosure further concerns a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.
1. A method of treating a subject comprising administering an effective amount of a pharmaceutical composition comprising a ribonucleic acid (RNA) encoding:
a) a polypeptide coding region encoding IL-12;
b) at least one histone stem-loop; and
c) a poly(A) sequence or polyadenylation signal,
wherein said RNA does not include a histone downstream element (HDE) to the subject.
2. The method of claim 1 , wherein the polyadenylation sequence comprises a consensus sequence of NN(U/T)ANA, AA(U/T)AAA or A(U/T)(U/T)AAA.
3. The method of claim 1 , wherein the RNA is a mRNA.
4. The method of claim 3 , wherein the mRNA comprises a 5′ cap structure.
5. The method of claim 3 , wherein the mRNA is modified by introduction of a non-native nucleotide compared with a native mRNA sequence or by covalent coupling of the mRNA with a further chemical moiety.
6. The method of claim 5 , wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA.
7. The method of claim 5 , wherein the non-native nucleotide is 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, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, and xanthosine-5′-triphosphate.
8. The method of claim 1 , comprising a poly(A) sequence.
9. The method of claim 8 , wherein the poly(A) sequence comprises about 25 to about 400 adenosine nucleotides.
10. The method of claim 1 , wherein the G/C content of the polypeptide coding region is increased compared with the G/C content of a wild-type nucleic acid encoding IL-12.
11. The method of claim 1 , wherein the nucleic acid molecule additionally comprises a poly(C) sequence of about 10 to about 200 cytosine nucleotides.
12. The method of claim 1 , wherein the RNA further comprises a stabilizing sequence from an alpha globin 3′ UTR.
13. The method of claim 1 , wherein the subject has a cancer.
14. The method of claim 13 wherein the cancer is a sarcoma, melanoma, lung cancer, ovarian cancer, leukemia, lymphoma, brain and central nervous system tumors, testicular cancer, prostate cancer, pancreatic cancer, or breast cancer.
15. The method of claim 13 , wherein the pharmaceutical composition is administered by intralesional injection.
16. The method of claim 1 , wherein the pharmaceutical composition is administered by injection.