RNA constructs and uses thereof
Disclosed herein are RNA polynucleotides comprising a 5′ Cap, a 5′ UTR comprising a cap proximal sequence disclosed herein, and a sequence encoding a payload. Also disclosed herein are compositions and medical preparations comprising the same, and methods of making and using the same.
1. A composition or medical preparation comprising an RNA polynucleotide comprising:
(a) a 5′ cap comprising a Cap1 structure;
(b) a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide;
(c) a 5′ UTR sequence comprising the nucleotide sequence of SEQ ID NO: 27;
(d) a sequence encoding a payload;
(e) a 3′ UTR sequence comprising the nucleotide sequence of SEQ ID NO: 13; and
(f) a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14;
wherein:
(i) the Cap1 structure comprises m7(3′ OMeG)(5′)ppp(5)(2′OMeA 1 )pG 2 , wherein A 1 is position +1 of the RNA polynucleotide, and G2 is position +2 of the RNA polynucleotide;
(ii) the cap proximal sequence comprises A 1 and G 2 of the Cap1 structure, and a sequence comprising: A 3 A 4 U 5 (SEQ ID NO: 5) at positions +3, +4 and +5 respectively of the RNA polynucleotide; and
(iii) the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines.
2. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, the 5′ cap, the cap proximal sequence, the 5′ UTR, the sequence encoding a payload, the 3′ UTR, and the polyA sequence.
3. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises a modified uridine in place of one or more uridines.
4. The composition or medical preparation of claim 3 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
5. The composition or medical preparation of claim 1 , wherein the sequence encoding a payload comprises a sequence encoding a protein replacement polypeptide; a sequence encoding an antibody agent; a sequence encoding a cytokine; a sequence encoding an antigenic polypeptide; a sequence encoding a gene editing component; a sequence encoding a regenerative medicine component, or combinations thereof
6. The composition or medical preparation of claim 5 , wherein the sequence encoding a payload comprises a sequence encoding an antigenic polypeptide.
7. The composition or medical preparation of claim 6 , wherein the antigenic polypeptide comprises a plurality of distinct epitopes from one or more antigens.
8. The composition or medical preparation of claim 6 , wherein the antigenic polypeptide comprises: a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, or an antigenic polypeptide from a pathogen.
9. The composition or medical preparation of claim 8 , wherein the RNA polynucleotide is mRNA.
10. A pharmaceutical composition comprising an RNA polynucleotide formulated in lipid nanoparticles (LNPs) comprising a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-conjugated lipid, the RNA polynucleotide comprising:
(a) a 5′ cap comprising a Cap1 structure;
(b) a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide;
(c) a 5′ UTR sequence comprising the nucleotide sequence of SEQ ID NO: 27;
(d) a sequence encoding a payload;
(e) a 3′ UTR sequence comprising the nucleotide sequence of SEQ ID NO: 13; and
(f) a polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14;
wherein:
(i) the Cap1 structure comprises m7(3′ OMeG)(5′)ppp(5)(2′OMeA 1 )pG 2 , wherein A 1 is position +1 of the RNA polynucleotide, and G2 is position +2 of the RNA polynucleotide;
(ii) the cap proximal sequence comprises A 1 and G 2 of the Cap1 structure, and a sequence comprising: A 3 A 4 U 5 (SEQ ID NO: 5) at positions +3, +4 and +5 respectively of the RNA polynucleotide; and
(iii) the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines.
11. The pharmaceutical composition of claim 10 , which is formulated as a liquid.
12. The pharmaceutical composition of claim 10 , which is formulated as a solid.
13. The composition or medical preparation of claim 6 , wherein the antigenic polypeptide comprises an antigenic polypeptide from an allergen.
14. The composition or medical preparation of claim 6 , wherein the antigenic polypeptide comprises a tumor antigenic polypeptide.
15. The composition or medical preparation of claim 6 , wherein the antigenic polypeptide comprises a self-antigenic polypeptide.
16. The pharmaceutical composition of claim 10 , wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, the 5′ cap, the cap proximal sequence, the 5′ UTR, the sequence encoding a payload, the 3′ UTR, and the polyA sequence.
17. The pharmaceutical composition of claim 10 , wherein the RNA polynucleotide comprises a modified uridine in place of one or more uridines.
18. The pharmaceutical composition of claim 17 , wherein the modified uridine is N1-methyl-pseudouridine (m1 ).
19. The pharmaceutical composition of claim 10 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
20. The pharmaceutical composition of claim 19 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
21. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
22. The composition or medical preparation of claim 21 , wherein the modified nucleoside uridine is N1-methyl-pseudouridine (m1Ψ).