CORONAVIRUS VACCINE
This disclosure relates to the field of RNA to prevent or treat coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses such as antibody and/or T cell responses. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccine antigen.
1 .- 73 . (canceled)
74 . A pharmaceutical composition comprising:
an RNA comprising a nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to the polypeptide of SEQ ID NO: 7 and includes proline residues at positions 986 and 987 of SEQ ID NO: 7, which nucleotide sequence is at least 90% identical to SEQ ID NO: 9,
wherein the RNA is formulated in lipid nanoparticles comprising a cationically ionizable lipid, a neutral lipid, a sterol, and a lipid conjugate,
wherein the cationically ionizable lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), the neutral lipid is or comprises a phospholipid, the sterol is or comprises cholesterol, and the lipid conjugate is or comprises a polyethylene glycol (PEG)-lipid.
75 . The composition of claim 74 , wherein the nucleotide sequence includes modified uridines in place of all uridines.
76 . The composition of claim 75 , wherein the modified uridines are each N1-methyl-pseudouridine.
77 . The composition of claim 74 , wherein the phospholipid is or comprises distearoylphosphatidylcholine (DSPC).
78 . The composition of claim 74 , wherein the PEG-lipid is or comprises the structure:
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
79 . The composition of claim 78 , wherein the PEG-lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
80 . The composition of claim 74 , wherein the lipid nanoparticles comprise each of:
(a) ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate);
(b) cholesterol;
(c) distearoylphosphatidylcholine (DSPC); and
(d) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
81 . The composition of claim 74 , wherein:
the cationically ionizable lipid is present in a concentration ranging from about 40 to about 50 mol percent of the total lipids;
the neutral lipid is present in a concentration ranging from about 5 to about 15 mol percent of the total lipids;
the sterol is present in a concentration ranging from about 30 to about 50 mol percent of the total lipids; or
the PEG-lipid is present in a concentration ranging from about 1 to about 10 mol percent of the total lipids.
82 . The composition of claim 80 , wherein the lipid nanoparticles comprise from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.
83 . The composition of claim 74 , wherein the RNA comprises: a 5′ cap, a 5′ UTR, a 3′ UTR, and a polyA sequence.
84 . The composition of claim 83 , wherein the RNA comprises at least one of the following features: (i) polyA sequence comprises at least 100 Å nucleotides and/or is an interrupted sequence of A nucleotides; and (ii) the 5 cap′ is or comprises a cap1 structure.
85 . The composition of claim 74 , further comprising at least one salt and/or a cryoprotectant.
86 . The composition of claim 85 , wherein the cryoprotectant is or comprises sucrose.
87 . The composition of claim 74 , wherein the RNA is present in an amount within a range of about 1 μg to about 100 μg per dose in the composition.
88 . The composition of claim 74 , wherein the nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide is codon-optimized for human subjects.
89 . The composition of claim 74 , wherein the nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide is characterized in that its G/C content is increased by about 55% as compared to the G/C content of the nucleotide sequence of SEQ ID NO: 2.
90 . A method of vaccinating by administering a pharmaceutical composition comprising:
an RNA comprising a nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to the polypeptide of SEQ ID NO: 7, and includes proline residues at positions 986 and 987 of SEQ ID NO: 7, which nucleotide sequence is at least 90% identical to SEQ ID NO: 9,
wherein the RNA is formulated in lipid nanoparticles comprising a cationically ionizable lipid, a neutral lipid, a sterol, and a lipid conjugate,
wherein the cationically ionizable lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), the neutral lipid is or comprises a phospholipid, the sterol is or comprises cholesterol, and the lipid conjugate is or comprises a polyethylene glycol (PEG)-lipid.
91 . The method of claim 90 , comprising administering to a subject at least one dose of the composition.
92 . The method of claim 91 , wherein the RNA is administered in an amount within a range of about 1 μg to about 100 μg per dose.
93 . The method of claim 91 , wherein the RNA is administered in an amount within a range of about 10 μg to about 30 μg per dose.
94 . The method of claim 91 , wherein the administration is by intramuscular administration.
95 . The method of claim 91 , comprising administering to the subject at least a second dose a period of time after a first dose.
96 . The method of claim 95 , wherein the second dose is administered about 7 to about 28 days following administration of the first dose.
97 . A pharmaceutical composition comprising:
an RNA comprising a nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to the polypeptide of SEQ ID NO: 7, and includes proline residues at positions 986 and 987 of SEQ ID NO: 7, which nucleotide sequence is at least 95% identical to SEQ ID NO: 9.
98 . The composition of claim 97 , wherein the RNA comprises at least one of the following features:
(i) a 5′-cap that is or comprises m2 7,3′-O Gppp(m 1 2′-O )ApG;
(ii) a polyA sequence comprising 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence;
(iii) a 5′-UTR that is or comprises a modified human alpha-globin 5′-UTR;
(iv) a 3′-UTR that is or comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA; and
(v) a combination of any one of (i)-(iv).
99 . The composition of claim 97 , wherein the RNA is formulated in lipid nanoparticles comprising each of: (i) a cationically ionizable lipid; (ii) a neutral lipid; (iii) a sterol; and (iv) a lipid conjugate.
100 . The composition of claim 99 , wherein the cationically ionizable lipid is within a range of about 40 to about 50 mole percent, the neutral lipid is within a range of about 5 to about 15 mole percent, the sterol is within a range of about 35 to about 45 mole percent, and the lipid conjugate is within a range of about 1 to about 10 mole percent.
101 . A pharmaceutical composition comprising:
an RNA comprising a nucleotide sequence that includes modified uridines and encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to the polypeptide of SEQ ID NO: 7, and includes proline residues at positions 986 and 987 of SEQ ID NO: 7, wherein the RNA comprises a nucleotide sequence that is at least 85% identical to SEQ ID NO: 20.
102 . The composition of claim 101 , wherein the RNA comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20.
103 . The composition of claim 101 , wherein the RNA is formulated in lipid nanoparticles comprising each of:
(a) ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate);
(c) distearoylphosphatidylcholine (DSPC);
(b) cholesterol; and
(d) 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.