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 sequence encoding a payload, wherein:
the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines; and
the 5′ terminus of the RNA polynucleotide comprises m 7 (3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pA 4 pN 5 , wherein N5 is uridine or a modified uridine.
2. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide further comprises a 3′ UTR sequence optionally comprising a modified uridine in place of one or more uridines.
3. The composition or medical preparation of claim 2 , wherein the 3′ UTR sequence comprises the nucleotide sequence of SEQ ID NO: 13 optionally comprising a modified uridine in place of one or more uridines.
4. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide further comprises a polyA sequence.
5. The composition or medical preparation of claim 4 , wherein the polyA sequence comprises at least 100 nucleotides.
6. The composition or medical preparation of claim 5 , wherein:
(i) the polyA sequence is an interrupted sequence of adenine nucleotides; and/or
(ii) the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
7. The composition or medical preparation of claim 6 , wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 14 optionally comprising a modified uridine in place of one or more uridines.
8. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, the m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pA 4 pN 5 , other 5′ UTR sequences, the sequence encoding a payload, 3′ UTR sequences, and a polyA sequence.
9. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
10. The composition or medical preparation of claim 9 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
11. 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.
12. The composition or medical preparation of claim 11 , wherein the sequence encoding a payload comprises a sequence encoding an antigenic polypeptide.
13. The composition or medical preparation of claim 12 , wherein the antigenic polypeptide comprises an epitope from an antigen.
14. The composition or medical preparation of claim 12 , wherein the antigenic polypeptide comprises a plurality of distinct epitopes from one or more antigens.
15. The composition or medical preparation of claim 12 , wherein the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.
16. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide is mRNA.
17. The composition or medical preparation of claim 1 , wherein the RNA polynucleotide comprises a human alpha globin (hAg) 5′ UTR or a fragment thereof, and wherein the hAg 5′ UTR or a fragment thereof optionally comprise a modified uridine in place of one or more uridines.
18. The composition or medical preparation of claim 17 , wherein the RNA polynucleotide further comprises a Kozak sequence optionally comprising a modified uridine in place of one or more uridines.
19. The composition or medical preparation of claim 18 , wherein the RNA polynucleotide comprises the nucleotide sequence of SEQ ID NO: 27 optionally comprising a modified uridine in place of one or more uridines.
20. A pharmaceutical composition comprising an RNA polynucleotide formulated as a particle, wherein the RNA polynucleotide comprises a sequence encoding a payload, wherein:
the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines; and
the 5′ terminus of the RNA polynucleotide comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pA 4 pN 5 , wherein N 5 is uridine or a modified uridine.
21. The pharmaceutical composition of claim 20 , Wherein the particle is a lipid nanoparticle (LNP) or a lipoplex (LPX) particle.
22. The pharmaceutical composition of claim 21 , wherein the particle is an LNP comprising a cationically ionizable lipid; a sterol; a phospholipid; and a pegylated lipid.
23. The pharmaceutical composition of claim 22 , wherein the concentration of the cationically ionizable lipid in the LNP is within a range of about 40 to about 55 mole percent, the concentration of the sterol in the LNP is within a range of about 30 to about 50 mole percent, the concentration of the phospholipid in the LNP is within a range of about 5 to about 15 mole percent, and the concentration of the pegylated lipid in the LNP is within a range of about 1 to about 10 mole percent.
24. The pharmaceutical composition of claim 20 , which is formulated as a liquid.
25. The pharmaceutical composition of claim 20 , which is formulated as a solid.
26. The pharmaceutical composition of claim 20 , wherein the RNA polynucleotide comprises m 7 (3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pN 4 pN 5 , the 5′ UTR, the sequence encoding a payload, the 3′ UTR, and a polyA sequence.
27. The pharmaceutical composition of claim 20 , wherein the RNA polynucleotide comprises a modified uridine in place of at least one uridine.
28. The pharmaceutical composition of claim 27 , wherein, the modified uridine is N1-methyl-pseudouridine (m1Ψ).
29. The pharmaceutical composition of claim 20 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
30. The pharmaceutical composition of claim 29 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
31. A composition or medical preparation comprising an RNA polynucleotide comprising a sequence encoding a payload, wherein:
the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines; and
the 5′ terminus of the RNA polynucleotide comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pN 4 pN 5 , wherein N 4 and N 5 are each independently selected from A, G, C, U, or a modified uridine.
32. The composition or medical preparation of claim 31 , wherein the RNA polynucleotide comprises the nucleotide sequence of SEQ ID NO: 27 optionally comprising a modified uridine in place of one or more uridines.
33. The composition or medical preparation of claim 31 , wherein the RNA polynucleotide further comprises a 3′ UTR sequence, wherein the 3′ UTR sequence comprises the nucleotide sequence of SEQ ID NO: 13 optionally comprising a modified uridine in place of one or more uridines.
34. The composition or medical preparation of claim 31 , wherein the RNA polynucleotide further comprises a polyA sequence.
35. The composition or medical preparation of claim 34 , wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO: 14 optionally comprising a modified uridine in place of one or more uridines.
36. The composition or medical preparation of claim 31 , wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, the m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pN 4 pN 5 , other 5′ UTR sequences, the sequence encoding a payload, 3′ UTR sequences, and a polyA sequence.
37. The composition or medical preparation of claim 31 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
38. The composition or medical preparation. of claim 37 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
39. The composition or medical preparation of claim 31 , 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.
40. The composition or medical preparation of claim 39 , wherein the sequence encoding a payload comprises a sequence encoding an antigenic polypeptide.
41. The composition or medical preparation of claim 40 , wherein the antigenic polypeptide comprises a plurality of distinct epitopes from one or more antigens.
42. The composition or medical preparation of claim 39 , wherein the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.
43. A pharmaceutical composition comprising an RNA polynucleotide formulated as a particle, wherein the RNA polynucleotide comprises a sequence encoding a payload, wherein:
the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines; and
the 5′ terminus of the RNA polynucleotide comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pN 4 pN 5 , wherein N4 and Ns are each independently selected from A, G, C, U, or a modified uridine.
44. The pharmaceutical composition of claim 43 , wherein the particle is a lipid nanoparticle (LNP) comprising a cationically ionizable lipid; a sterol; a phospholipid; and a pegylated lipid.
45. The pharmaceutical composition of claim 43 , wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, m7(3′OMeG)(5′)ppp(5′)(2′OMeA 1 )pG 2 pA 3 pN 4 pN 5 , other 5′ UTR sequences, the sequence encoding a payload, a 3′UTR, and a polyA sequence.
46. The pharmaceutical composition of claim 43 , wherein the RNA polynucleotide comprises a modified uridine in place of at least one uridine.
47. The pharmaceutical composition of claim 46 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).
48. the pharmaceutical composition of claim 43 , wherein the RNA polynucleotide comprises a modified uridine in place of every uridine.
49. The pharmaceutical composition of claim 48 , wherein the modified uridine is N1-methyl-pseudouridine (m1Ψ).