RNA constructs and uses thereof

Abstract

To provide RNA polynucleotides that improve the translation efficiency of an RNA encoding a payload, and / or the expression of a polypeptide payload encoded by the RNA, and methods of making and using the same.SOLUTION: Disclosed herein are RNA polynucleotides comprising a 5' Cap, a 5' UTR comprising a cap proximal sequence, 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.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claim This application claims priority under 35 U.S.C. § 119 to each of the following applications, the disclosures of each of which are incorporated herein by reference in their entirety: International Application No. PCT / EP20 / 61239 (filed April 22, 2020); and International Application No. PCT / EP20 / 66968 (filed June 18, 2020). [Background technology]

[0002] The use of RNA polynucleotides as therapeutic agents is an emerging field. Summary of the Invention [Means for solving the problem]

[0003] This disclosure identifies particular challenges that may be associated with RNA therapeutics.

[0004] For example, in some embodiments, the present disclosure identifies sources of particular problems that may be encountered in the expression of polypeptides encoded by RNA therapeutics. Among other things, the present disclosure provides techniques for improving the translation efficiency of RNA encoding a payload and / or the expression of a polypeptide payload encoded by the RNA. In some embodiments, the translation efficiency and / or expression of an RNA-encoded payload is improved by the presence of a cap1 structure (e.g., m2 7,3’-O Gppp(m1 2’-O ) ApG cap); a 5'UTR comprising a cap-proximal sequence disclosed herein, and an RNA polynucleotide comprising a sequence encoding a payload. Without wishing to be bound by theory, the present disclosure suggests that improved translation efficiency and / or polypeptide payload expression may be achieved by using the cap 1 structure disclosed herein of eukaryotic translation initiation factor 4E (eIF4E), e.g., m2, as opposed to IFN-induced proteins having tetratricopeptide repeat-1 (IFIT1). 7,3’-O Gppp(m12’-O It is proposed that this can be achieved through preferential binding to RNAs containing an ApG cap and / or a 5'UTR containing a cap-proximal sequence. For example, in some embodiments, it is proposed that eIF4E can compete with IFIT1 for binding to RNA polynucleotides based on the 5' cap structure. Among other things, the present disclosure provides certain techniques that can prioritize eIF4E binding and / or otherwise enhance translation, at least compared to IFIT1 binding.

[0005] In some embodiments, the present disclosure teaches that the identity of a particular sequence proximal to the 5' cap (e.g., a 5' Cap 1 structure) can affect the translation efficiency of an associated payload. Without wishing to be bound by any particular theory, the present disclosure proposes that eIF4E competes with IFIT1 for binding to an RNA polynucleotide based on the identity of one or more nucleotides downstream of the 5' cap, e.g., the cap-proximal sequences disclosed herein. In some embodiments, the present disclosure demonstrates that an AGAAU or AGCAC sequence downstream of the 5' cap (e.g., a 5' Cap 1 structure) can improve translation. The present disclosure proposes that the presence of such a sequence (e.g., AGAAU or AGCAC) can increase eIF4E binding, at least compared to IFIT1.

[0006] Alternatively, or in addition, the present disclosure identifies certain advantages of avoiding (e.g., ensuring the absence of) self-hybridizing sequences (which may sometimes be referred to as self-complementary sequences) within an RNA polynucleotide encoding a payload. For example, the present disclosure demonstrates that such absence may improve and / or be required for translation (e.g., translation efficiency) of an associated (e.g., RNA-encoded) payload and / or expression of a polypeptide otherwise encoded thereby. While not wishing to be bound by theory, it is believed that self-hybridizing sequences (particularly sequences that hybridize with sequences within or including one or more of a Kozak sequence, a 5' UTR element, and / or a 3' UTR element) interfere with one or more aspects of translation. For example, in some embodiments, it is proposed that such self-hybridization may inhibit the binding of transcription and / or translation factors to an RNA polynucleotide by self-hybridizing to a complementary sequence within the RNA polynucleotide.

[0007] Alternatively, or in addition, in some embodiments, the present disclosure defines particular lipid components, and / or ratios thereof, that may be useful or effective, inter alia, for delivering nucleic acids, particularly RNA (e.g., therapeutic RNA, or other RNA encoding a polypeptide), immediately upon administration (e.g., by injection, e.g., intramuscular or intravenous injection) to a subject. For example, in some embodiments, the present disclosure demonstrates that the lipid ALC-0315, as described herein, is highly and particularly useful for delivery.

[0008] Disclosed herein, inter alia, are compositions or medical preparations comprising: (i) a 5' cap that is or includes a Cap 1 structure, e.g., as disclosed herein; (ii) a 5' UTR sequence that includes a cap-proximal sequence, e.g., as disclosed herein; and (iii) an RNA polynucleotide that includes a sequence encoding a payload. Also disclosed herein are methods for making the same and methods for using the same, e.g., to induce an immune response in a subject.

[0009] 1. A composition or medical preparation comprising an RNA polynucleotide, a 5' cap comprising a Cap 1 structure; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the Cap 1 structure comprises m7G(5')ppp(5')(2'OMeN1)pN2, where N1 is position +1 of the RNA polynucleotide and N2 is position +2 of the RNA polynucleotide, and N1 and N2 are each independently selected from A, C, G, or U; (ii) the cap-proximal sequence is N1 and N2 of the cap 1 structure; and (a) a sequence selected from the group consisting of A3A4X5 (SEQ ID NO: 1); C3A4X5 (SEQ ID NO: 2); A3C4A5 (SEQ ID NO: 3), and A3U4G5 (SEQ ID NO: 4); or (b) a sequence comprising X3Y4X5 (SEQ ID NO: 7); Including, X3 (the nucleotide X at position +3 in SEQ ID NO:7) or X5 (the nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) are each independently selected from A, G, C, or U; Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C; Compositions or medical preparations are provided herein.

[0010] The present disclosure also provides a composition or medical preparation comprising an RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is G * ppp(m1 2’-O ) Cap 1 structure containing N1pN2, N1 is position +1 of the RNA polynucleotide and N2 is position +2 of the RNA polynucleotide, and N1 and N2 are each independently selected from A, C, G, or U; G * has the following structure: [ka] Including, [ka] is G * represents the bond attached to the first phosphorus atom of the ppp group, and R 1 is CH3 and R 2 is OH or O-CH3, and R 3 is O-CH3; (ii) the cap-proximal sequence is N1 and N2 of the cap 1 structure; and (a) a sequence selected from the group consisting of A3A4X5 (SEQ ID NO: 1); C3A4X5 (SEQ ID NO: 2); A3C4A5 (SEQ ID NO: 3), and A3U4G5 (SEQ ID NO: 4); or (b) a sequence comprising X3Y4X5 (SEQ ID NO: 7); Including, X3 (the nucleotide X at position +3 in SEQ ID NO:7) or X5 (the nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) are each independently selected from A, G, C, or U; Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C; A composition or medical preparation is provided.

[0011] a 5' cap comprising a Cap 1 structure; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the Cap 1 structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence comprises A1 and G2 of the cap 1 structure and a sequence comprising A3A4U5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively; Also provided herein are compositions or medical preparations comprising the RNA polynucleotides.

[0012] The present disclosure provides a composition or medical preparation comprising a capped RNA polynucleotide encoding a gene product, wherein the RNA polynucleotide has the formula: [ka] Including, In the formula, R 1 is CH3 and R 2 is OH or O-CH3, and R 3 is O-CH3, B1 is any nucleobase, preferably A; B2 is any nucleobase, preferably G; B3 is any nucleobase, preferably A or C; B4 is any nucleobase; B5 is any nucleobase, The composition or medical preparation is provided such that, when the RNA polynucleotide is administered to a subject, the level of expression of the encoded gene product at about 6 hours after administration and at about 48 hours after administration does not differ by more than 5-fold.

[0013] Provided herein is a pharmaceutical composition comprising the RNA polynucleotides disclosed herein. In some embodiments, the pharmaceutical composition comprises the compositions or medical preparations disclosed herein.

[0014] Also provided herein are methods of producing pharmaceutical compositions comprising the RNA polynucleotides disclosed herein, e.g., by combining an RNA polynucleotide with a lipid to form a lipid nanoparticle that encapsulates the RNA.

[0015] The present disclosure provides a nucleic acid template suitable for producing RNA capped with Cap1, wherein the first five nucleotides transcribed from the template strand of the nucleic acid template comprise the sequence N1pN2pN3pN4pN5, where N1 is any nucleotide, preferably T; N2 is any nucleotide, preferably C; N3 is any nucleotide, preferably T or G; N4 is any nucleotide; and N5 is any nucleotide. In some embodiments, the DNA template comprises a 5' UTR, a sequence encoding a payload, a 3' UTR, and a polyA sequence.

[0016] (i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence disclosed herein; (ii) a polymerase; and (iii) RNA polynucleotide Provided herein is an in vitro transcription reaction comprising:

[0017] Also provided herein are RNA polynucleotides isolated from the provided in vitro transcription reactions.

[0018] The present disclosure provides a method for producing capped RNA, comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure is a G * ppp(m1 2’-O )N1pN2, N1 is complementary to position +1 of the nucleic acid template and N2 is complementary to position +2 of the nucleic acid template, and N1 and N2 are independently selected from A, C, G, or U; position +3 of the nucleic acid template is any nucleotide, preferably T or G; position +4 of the nucleic acid template is any nucleotide; position +5 of the nucleic acid template is any nucleotide; G * has the following structure: [ka] Including, [ka] is G * represents the bond attached to the first phosphorus atom of the ppp group, and R 1 is CH3 and R 2 is OH or O-CH3, and R 3 is O-CH3, a method is provided.

[0019] Also provided herein are compositions comprising DNA polynucleotides comprising sequences complementary to the provided RNA polynucleotide sequences. In some embodiments, the DNA polynucleotides disclosed herein can be used to transcribe the RNA polynucleotides disclosed herein.

[0020] The present disclosure provides methods comprising administering to a subject a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, for example, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein.

[0021] Also provided herein are methods of inducing an immune response in a subject, comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, for example, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein.

[0022] Provided herein are methods of vaccinating a subject, for example, by administering a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein.

[0023] The present disclosure provides a method of reducing the interaction of an RNA polynucleotide comprising a 5' cap and a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide with IFIT1, comprising: providing a variant of an RNA polynucleotide that differs from the parent RNA polynucleotide by substitution of one or more residues within a cap-proximal sequence; determining that the mutant has a reduced interaction with IFIT1 compared to that of the parent RNA polynucleotide; The present invention provides a method comprising:

[0024] providing an RNA polynucleotide comprising a 5' cap, a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload; the RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition comprising the same is administered, higher expression and / or a prolonged duration of expression of the payload is observed compared to a suitable reference comparator; Methods for producing the polypeptides are also provided herein.

[0025] 1. A method for increasing translatability of an RNA polynucleotide comprising a 5′ cap, a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload, comprising: providing a variant of the RNA polynucleotide that differs from the parent RNA polynucleotide by substitution of one or more residues within the cap-proximal sequence; determining that expression of the variant is increased compared to that of the parent RNA polynucleotide; Provided herein is a method comprising:

[0026] Provided herein are therapeutic RNAs comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, where the improvement includes inclusion of one or more of the following residues within the cap-proximal sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide, which have been demonstrated to increase expression of the RNA in an LNP formulation when administered to a subject. In some embodiments, X is selected from A, C, G, or U.

[0027] The present disclosure provides therapeutic RNAs comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, wherein the improvement comprises including one or more of the following residues within the cap-proximal sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide, which have been demonstrated to increase expression of the RNA in an LNP formulation when administered to a subject. In some embodiments, X is selected from A, C, G, or U.

[0028] Also provided herein is a therapeutic RNA comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, wherein the improvement includes inclusion of one or more of the following residues within the cap-proximal sequence: an A at position +1 of the RNA polynucleotide, a G at position +2 of the RNA polynucleotide, an A at position +3 of the RNA polynucleotide, an A at position +4 of the RNA polynucleotide, and a U at position +5 of the RNA polynucleotide, which have been demonstrated to increase expression of the RNA in an LNP formulation when administered to a subject.

[0029] The present disclosure provides methods for increasing translation of an RNA polynucleotide comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues within the cap-proximal sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

[0030] Provided herein are methods for increasing translation of an RNA polynucleotide comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues within the cap-proximal sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

[0031] Also provided herein are methods for increasing translation of an RNA polynucleotide comprising a 5' cap comprising a Cap 1 structure, a cap-proximal sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues within the cap-proximal sequence: an A at position +1 of the RNA polynucleotide, a G at position +2 of the RNA polynucleotide, an A at position +3 of the RNA polynucleotide, an A at position +4 of the RNA polynucleotide, and a U at position +5 of the RNA polynucleotide.

[0032] 1. A method for providing a framework for an RNA polynucleotide comprising a 5′ cap, a cap-proximal sequence, and a payload sequence, comprising: evaluating at least two variants of an RNA polynucleotide, Each variant contains the same 5' cap and payload sequences; The variants differ from each other at one or more specific residues in the cap-proximal sequence; The evaluating step includes determining the expression level and / or duration of expression of the payload sequence; and a process of evaluating; selecting at least one combination of 5' cap and cap-proximal sequence that exhibits higher expression compared to at least one other combination of 5' cap and cap-proximal sequence; Also provided herein are methods comprising: [Brief explanation of the drawings]

[0033] [Figure 1] Figure 1 demonstrates the plasma levels of EPO 6, 24, 48, and 72 hours after intravenous administration of mice with murine EPO (mEPO) mRNA constructs containing or not containing Lig3 in the 3'UTR sequence. Blood was collected 6, 24, 48, and 72 hours after administration, and samples were analyzed for mEPO levels via ELISA. [Figure 2] 2A-C are schematic diagrams of Lig3 sequences self-hybridizing to the 5'UTR (FIGS. 2A-B) or 3'UTR (FIG. 2C). [Figure 3-1] Figures 3A-I show the structures of 5' caps that can be incorporated into mRNA. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 4] Figure 4 demonstrates the plasma levels of mEPO 6, 24, 48, and 72 hours after intravenous administration of mice with mEPO mRNA constructs that differed in the nucleotide at positions +3, +4, or +5. Blood was collected 6, 24, 48, and 72 hours after administration, and samples were analyzed for mEPO levels via ELISA. [Figure 5]Figure 5 demonstrates anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with BNT162a1. BALB / c mice were immunized once IM with 1, 5, or 10 μg of LNP-formulated RBL063.3. Animals were bled 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG), measured via ELISA. Values ​​for a 1:100 serum dilution for days 7, 14, 21, and 28 are included within the graph. Each point in the graph represents one mouse, with duplicate measurements per mouse sample (group size n = 8; mean + SEM for each group is included). [Figure 6] Figure 6 demonstrates anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with BNT162b1. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBP020.3. Animals were bled 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) levels measured via ELISA. Various serum dilutions are included within the graph for days 7 (1:100), 14 (1:300), 21 (1:900), and 28 (1:2700). Each point within the graph represents one mouse, with duplicate measurements per mouse sample (group size n = 8; mean + SEM is included for each group). [Figure 7]Figure 7 demonstrates neutralization of SARS-CoV-2 pseudovirus 14, 21, and 28 days after immunization with BNT162b1. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBP020.3. Animals were bled 14, 21, and 28 days after immunization, and serum was tested for neutralization of SARS-CoV-2 pseudovirus. Graph represents pVN50 serum dilution (50% reduction in infection events compared to the no-serum positive control). Each point in the graph represents one mouse. Two replicates were measured per mouse sample. Group size n=8. Mean + SEM is indicated by horizontal bars with whiskers per group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 8] Figure 8 demonstrates anti-S protein IgG responses 7, 14, and 21 days after immunization with BNT162c1. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.3. Animals were bled 7, 14, and 21 days post-immunization, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG), as measured via ELISA. Various serum dilutions are included within the graph for days 7 (1:100), 14 (1:300), and 21 (1:900). Each point within the graph represents one mouse, with duplicate measurements per mouse sample (group size n = 8; mean + SEM is included for each group). [Figure 9]Figure 9 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162c1. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.3. 14 and 21 days after immunization, animals were bled and serum was tested for neutralization of SARS-CoV-2 pseudovirus. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to the no serum positive control). One point in the graph represents one mouse. Duplicate measurements were performed per mouse sample. Group size n=8. Mean + SEM is indicated by horizontal bars with whiskers per group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 10] Figure 10 demonstrates anti-S protein IgG responses 7, 14, 21, and 28 days after immunization with LNP-formulated RBL063.1. BALB / c mice were immunized once IM with 1, 5, or 10 μg of LNP-formulated RBL063.1. Animals were bled 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG), as measured via ELISA. Various serum dilutions are included within the graph for days 7 (1:100), 14 (1:100), 21 (1:300), and 28 (1:900). Each point within the graph represents one mouse, with duplicate measurements per mouse sample (group size n = 8; mean + SEM is included for each group). [Figure 11]Figure 11 shows the neutralization of SARS-CoV-2 pseudovirus 14, 21, and 28 days after immunization with LNP-formulated RBL063.1. BALB / c mice were immunized once IM with 1, 5, or 10 μg of LNP-formulated RBL063.1. Animals were bled 14, 21, and 28 days after immunization, and serum was tested for neutralization of SARS-CoV-2 pseudovirus. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to the no-serum positive control). Each point in the graph represents one mouse. Duplicate measurements were performed per mouse sample. Group size n=8. The mean + SEM is indicated by a horizontal bar with whiskers per group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 12] Figure 12 shows anti-S protein IgG responses 7, 14, and 21 days after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBP020.1. Animals were bled 7, 14, and 21 days post-immunization, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG), as measured via ELISA. Various serum dilutions were included within the graph for days 7 (1:100), 14 (1:300), and 21 (1:1100). Each point within the graph represents one mouse, with duplicate measurements per mouse sample (group size n=8; mean + SEM is included for each group). [Figure 13]Figure 13 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBP020.1. Animals were bled 14 and 21 days after immunization, and serum was tested for neutralization of SARS-CoV-2 pseudovirus. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to the no-serum positive control). Each point in the graph represents one mouse. Duplicate measurements were performed per mouse sample. Group size n=8. The mean + SEM is indicated by the horizontal bar with whiskers per group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 14] Figure 14 shows anti-S protein IgG responses 7, 14, and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.2. At 7, 14, and 21 days after immunization, animals were bled, and serum samples were analyzed for total anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) levels measured via ELISA. Various serum dilutions are included for days 7 (1:100), 14 (1:300), and 21 (1:900). Each point in the graph represents one mouse, with duplicate measurements per mouse sample (group size n = 8; mean + SEM is included for each group). [Figure 15]Figure 15 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized once IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.2. Animals were bled 14 and 21 days after immunization, and serum was tested for neutralization of SARS-CoV-2 pseudovirus. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to the no-serum positive control). Each point in the graph represents one mouse. Duplicate measurements were performed per mouse sample. Group size n=8. The mean + SEM is indicated by the horizontal bar with whiskers per group. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation. [Figure 16] FIG. 16 shows the activity of ALC-0315 in the screening process. [Figure 17-1] Figure 17 demonstrates that after intramuscular administration in wild-type (WT) or ApoE knockout (ApoE) C57B1 / 6 mice, luciferase expression was monitored in the right (site of injection), dorsal (site of injection), and ventral (liver drainage) sides of the animals in the presence or absence of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject at 4, 24, 72, and 96 hours after administration. [Figure 17-2] Same as above. [Figure 18] Figure 18 shows luciferase activity in wild-type (WT) or ApoE knockout (APOE) C57B1 / 6 mice after intravenous (IV) and intramuscular (IM) administration in the presence (KO+) or absence (KO) of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject 4 hours after administration. DETAILED DESCRIPTION OF THE INVENTION

[0034] Specific Definitions Although the present disclosure will be described in detail below, it should be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0035] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0036] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0037] The elements of the present disclosure are described below. The elements are listed according to specific embodiments. However, it should be understood that the elements can be combined in any manner and in any number to produce additional embodiments. The variously described examples and embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to disclose and encompass embodiments combining the explicitly described embodiment with any number of the disclosed elements. Furthermore, all permutations and combinations of all described elements should be considered disclosed by the description unless the context indicates otherwise. The term "about" means approximately or near, and in the context of a numerical value or range set forth herein, means, in some embodiments, ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0038] As used in the context of describing this disclosure (especially in the context of the claims), the terms "a," "an," and "the," and similar designations, should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0039] Unless expressly stated otherwise, the term "comprising" is used in the context of this document to indicate that additional members may be present in addition to the members of the list introduced by "comprising." However, for certain embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of" or "consisting essentially of."

[0040] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0041] The following provides definitions applicable to all aspects of this disclosure. The following terms have the following meanings unless otherwise specified: Any term not given a definition has its art-recognized meaning.

[0042] Agent: As used herein, the term "agent" may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular configuration and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, polypeptide, nucleic acid, monosaccharide, lipid, metal, or combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is devoid of or substantially free of any polymer or polymer moiety.

[0043] Amino acid: In its broadest sense, the term "amino acid" as used herein refers to a compound and / or substance that can be, is, or is incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-naturally occurring amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the above general structure. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to one containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to one containing an otherwise identical unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

[0044] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity to the reference substance, e.g., shares a core or consensus structure, but differs in certain discrete ways. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by the performance of a synthetic process that is substantially similar to (e.g., shares multiple steps with) that which produces the reference substance. In some embodiments, an analog is produced, or can be produced, by the performance of a synthetic process that is different from that used to produce the reference substance.

[0045] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence contains one or more structural elements recognized by those skilled in the art as a complementarity-determining region (CDR); in some embodiments, an antibody agent is or comprises a polypeptide that contains at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are sequence-identical or contain one to five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR has been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR have been deleted, added, or substituted compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR has been substituted relative to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as corresponding to CDR1, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent is or comprises a polypeptide or set of polypeptides whose amino acid sequence together comprises structural elements recognized by those skilled in the art as corresponding to both heavy chain variable region CDRs and light chain variable region CDRs, e.g., heavy chain CDR1, 2, and / or 3 and light chain CDR1, 2, and / or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may comprise one or more constant region sequences characteristic of a particular organism, e.g., camel, human, mouse, primate, rabbit, or rat; in many embodiments, an antibody agent may comprise one or more constant region sequences characteristic of humans. In some embodiments, an antibody agent may include one or more sequence elements recognized by those skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a standard antibody (e.g., may include two heavy chains and two light chains).In some embodiments, the antibody agent includes, but is not limited to, an intact IgA, IgG, IgE, or IgM antibody; a bi- or multispecific antibody [e.g., Zybodies. (登録商標) antibody fragments, e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide Fc fusions; single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR, or fragments thereof); camelid antibodies; masked antibodies [e.g., Probodies (登録商標) ];Small Modular ImmunoPharmaceuticals (商標) ]; single chain or Tandem diabody [TandAb (登録商標) ];VHH;Anticalins (登録商標) Nanobodies (登録商標) Mini Body; BiTE (登録商標) ankyrin repeat proteins or DARPINs (登録商標) ;Avimers (登録商標) ;DARTs;TCR-like antibodies;Adnectins (登録商標) ;Affilins (登録商標) ;Trans-bodies (登録商標) ;Affibodies (登録商標) ;TrimerX (登録商標) ;MicroProteins;Fynomers (登録商標) , Centyrins (登録商標) ; and KALBITOR (登録商標) In some embodiments, the antibody may lack a covalent modification (e.g., glycan attachment) that it would have if it were naturally produced. In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.)).

[0046] Related: As used herein, the term refers to two events or entities being "associated" with one another if the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence, susceptibility, severity, stage, etc. of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly so as to be physically close to one another and / or remain physically close to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0047] Binding: As used herein, the term "binding" will be understood to typically refer to a non-covalent association between two or more entities. "Direct" binding encompasses physical contact between entities or moieties; indirect binding encompasses physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation or in association with more complex systems (e.g., simultaneously, covalently or otherwise associated with a carrier entity and / or within a biological system or cell). Binding between two entities can be considered "specific" if, under the conditions being assessed, the associated entities are more likely to associate with each other than with other available binding partners.

[0048] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., a tissue, organism, or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or biological fluid. In some embodiments, the biological sample may be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; free-flowing nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural effusion; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washing or lavage, e.g., ductal washing or bronchoalveolar lavage; aspirate; scraping; bone marrow specimen; tissue biopsy specimen; surgical specimen; feces, other body fluids, secretions, and / or excretions; and / or cells derived therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells derived from the individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), such as by filtering using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, isolation and / or purification of specific components, and the like.

[0049] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administration of one or more agents or modalities to a subject receiving other agents or modalities in combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or necessarily simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combination composition or combination compound (e.g., as part of a single chemical complex or covalent entity).

[0050] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, circumstances, sets of conditions, etc., which need not be identical to one another, but which are sufficiently similar to permit comparisons between them, so that one of skill in the art would recognize that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few different characteristics. One of skill in the art would understand the degree of identity required to be considered comparable in any given situation for two or more such agents, entities, circumstances, sets of conditions, etc., in context. For example, one of skill in the art would recognize that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to justify a reasonable conclusion that differences in the results obtained under or with different sets of circumstances, individuals, or populations, or in the phenomena observed, are caused by or indicate changes in the varying characteristics.

[0051] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to indicate the location / identity of a structural element in a compound or composition relative to another compound or composition (e.g., an appropriate reference compound or composition). For example, in some embodiments, a monomer residue within a polymer (e.g., an amino acid residue within a polypeptide, or a nucleic acid residue within a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, one of ordinary skill in the art will recognize that, for simplicity's sake, residues within a polypeptide are often designated based on the relevant reference polypeptide using a standard numbering system, so that an amino acid "corresponding to" a residue at position 190 corresponds to the residue found at 190 in the reference polypeptide, for example, without necessarily being the actual 190th amino acid in a particular amino acid chain; one of ordinary skill in the art will readily recognize how to identify a "corresponding" amino acid. For example, those of skill in the art will be aware of various sequence alignment strategies, including, for example, software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, Parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, that can be utilized to identify "corresponding" residues within polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also recognize that, in some cases, the term "corresponding to" can be used to describe an event or entity that shares relevant similarity with another event or entity (e.g., a suitable reference event or entity).As just one example, a gene or protein in one organism may, in some embodiments, be described as "corresponding to" a gene or protein from another organism to indicate that they play a similar role or perform a similar function, and / or exhibit a particular degree of sequence identity or homology, or share certain characteristic sequence elements.

[0052] Designed: As used herein, the term "designed" refers to (i) an agent whose structure is selected or chosen by the hand of man; (ii) an agent produced by a process requiring the hand of man; and / or (iii) an agent that is distinct from natural substances and other known agents.

[0053] Dosage regimen: Those skilled in the art will recognize that the term "dosage regimen" can be used to refer to a set of unit doses (typically more than one) that are typically separated by time and administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more doses. In some embodiments, a dosing regimen includes multiple doses that are each separated from the other doses by a suitable time. In some embodiments, the individual doses are separated from each other by the same length of time; in some embodiments, a dosing regimen includes multiple doses and at least two different time periods that separate the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dosage in a first dosage, followed by one or more additional doses in a second dosage that is different from the first dosage. In some embodiments, the dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0054] Engineered: Generally, the term "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when it is manipulated by the hand of man so that two or more sequences that are not naturally linked together in that order are directly linked to each other in the engineered polynucleotide, and / or when certain residues within the polynucleotide are caused through the action of the hand of man to be linked to entities or moieties that are not naturally occurring and / or not naturally linked.

[0055] Epitope: As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding entity. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a related three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms and groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0056] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0057] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation.

[0058] Polypeptide: As used herein, polypeptide refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it is designed and / or created through the act of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, such as modified or attached to one or more amino acid side chains at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc. (including combinations thereof). In some embodiments, a polypeptide may be cyclic and / or include cyclic portions. In some embodiments, a polypeptide is not cyclic and / or does not include any cyclic portions. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be attached to the name of a reference polypeptide, activity, or structure; in such instances, the term "polypeptide" is used herein to refer to polypeptides that share a related activity or structure and therefore can be considered members of the same class or family of polypeptides.For each such class, exemplary polypeptides within the class are provided herein and / or known to those of skill in the art, whose amino acid sequence and / or function are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with the reference polypeptide of the class (and in some embodiments, with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at comparable levels or within a specified range) with the reference polypeptide of the class (and in some embodiments, with all polypeptides in the class). For example, in some embodiments, member polypeptides exhibit an overall degree of sequence homology or identity with a reference polypeptide of at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or contain at least one region (e.g., a conserved region, which in some embodiments may be or may include a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least 3-4, and often up to 20 or more, amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of the parent polypeptide.

[0059] Prevention: As used herein, prevention, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predefined period of time.

[0060] Reference: As used herein, reference describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or assessed substantially simultaneously with the test or assessment of interest. In some embodiments, the reference or control is a historical reference or control, which may be embodied in a tangible medium. Typically, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those skilled in the art will recognize when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0061] Risk: "Risk" of a disease, disorder, and / or condition, as understood from the context, refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, up to 100%. In some embodiments, risk is expressed as risk compared to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples is known to be at risk for the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is from an individual comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect, for example, one or more genetic attributes that may (or may not) predispose an individual to developing a particular disease, disorder, and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes, and / or one or more lifestyle or environmental events or attributes.

[0062] Susceptible to: An individual "susceptible to" a disease, disorder, and / or condition is an individual who is at a higher risk of developing the disease, disorder, and / or condition than members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0063] Vaccination: As used herein, the term "vaccination" refers to the administration of a composition intended to generate an immune response, for example, to a disease-associated (e.g., pathogenic) agent. In some embodiments, vaccination can be administered before, during, and / or after exposure to a disease-associated agent, and in certain embodiments, before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination involves multiple administrations of a vaccine composition appropriately spaced apart. In some embodiments, vaccination generates an immune response against an infectious agent. In some embodiments, vaccination generates an immune response against a tumor; in some such embodiments, vaccination is "personalized" in that it is partially or completely directed to epitopes (which may be or include, for example, one or more neoepitopes) determined to be present in a particular individual's tumor.

[0064] Variant: As used herein in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits substantial structural identity with a reference molecule but differs structurally from the reference molecule, e.g., by the presence or absence, or level, of one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be recognized by those skilled in the art, every biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a different molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid as a result of one or more differences in the amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently constituent of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid.In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, except for minor sequence modifications at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the reference. Often, a variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, 4, 3, 2, or 1) of functional residues (i.e., residues involved in a particular biological activity) substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains no more than about 5, 4, 3, 2, or 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, a variant polypeptide or nucleic acid contains less than about 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, 6 additions or deletions compared to the reference, and typically less than about 5, 4, 3, or 2 additions or deletions. In some embodiments, the reference polypeptide or nucleic acid is one found in nature.

[0065] Detailed Description of Specific Embodiments The present disclosure provides, inter alia, an RNA polynucleotide comprising: (i) a 5' cap that is or includes a Cap1 structure, e.g., as disclosed herein; (ii) a 5' UTR sequence that includes a cap-proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also provided herein are compositions and pharmaceutical preparations comprising the same, as well as methods of making and using the same. In some embodiments, the translation efficiency of an RNA encoding a payload and / or the expression of a payload encoded by the RNA is improved by the addition of a 5' cap that includes a Cap1 structure, e.g., m2, as disclosed herein. 7,3’-O Gppp(m1 2’-O ) ApG cap; a 5'UTR comprising a cap-proximal sequence as disclosed herein, and an RNA polynucleotide comprising a sequence encoding a payload. In some embodiments, the absence of self-hybridizing sequences in the RNA polynucleotide encoding a payload can further improve translation efficiency of the RNA encoding the payload and / or expression of the payload encoded by the RNA payload.

[0066] RNA polynucleotides As used herein, the term "polynucleotide" or "nucleic acid" refers to DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0067] In some embodiments, the nucleic acid may be contained within a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including a plasmid vector, a cosmid vector, a phage vector, such as lambda phage, a viral vector, such as a retroviral, adenoviral, or baculoviral vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). In some embodiments, the vector may be an expression vector; alternatively, or in addition, in some embodiments, the vector may be a cloning vector. As will be apparent to those skilled in the art, in some embodiments, the expression vector may be, for example, a plasmid; alternatively, or in addition, in some embodiments, the expression vector may be a viral vector. Typically, an expression vector will contain a desired coding sequence and other appropriate sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., a bacterium, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are commonly used to manipulate and amplify specific desired fragments (typically DNA fragments). It may also lack functional sequences required for expression of the desired fragment.

[0068] In some embodiments, the nucleic acids described and / or utilized herein may be or include recombinant and / or isolated molecules.

[0069] Those skilled in the art who have read this disclosure will understand that the term "RNA" typically refers to a nucleic acid molecule containing ribonucleotide residues. In some embodiments, RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. In some embodiments, RNA may be partially or completely double-stranded RNA; in some embodiments, RNA may comprise two or more different nucleic acid strands (e.g., separate molecules) that are partially or completely hybridized with each other. In many embodiments, RNA is single-stranded, which in some embodiments may self-hybridize or may normally fold into a secondary and / or tertiary structure. In some embodiments, the RNA described and / or utilized herein does not self-hybridize, at least with respect to the specific sequences described herein. In some embodiments, the RNA may be isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and / or modified RNA (wherein the term "modified" is understood to indicate that one or more residues or other structural elements of the RNA differ from naturally occurring RNA; e.g., in some embodiments, modified RNA differs by the addition, deletion, substitution, and / or modification of one or more nucleotides and / or by one or more portions or characteristics of the nucleotides, e.g., of the nucleosides, or of the backbone structure or linkage). In some embodiments, the modification may be or may include the addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA. It is also contemplated herein that nucleotides within an RNA (e.g., within a modified RNA) may be non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For the purposes of the present disclosure, modified RNA is considered an analog of naturally occurring RNA.

[0070] In some embodiments of the present disclosure, the RNA is or includes messenger RNA (mRNA), which refers to an RNA transcript that encodes a polypeptide.

[0071] In some embodiments, the RNA disclosed herein comprises a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region (5' UTR) comprising a cap-proximal sequence, a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3' UTR); and / or a polyadenylation (polyA) sequence.

[0072] In some embodiments, the RNA disclosed herein comprises, from 5' to 3', the following components: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region comprising a cap-proximal sequence (5'UTR), a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'UTR); and a polyA sequence.

[0073] In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. Here, DNA refers to a nucleic acid containing deoxyribonucleotides.

[0074] In some embodiments, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a vector suitable for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0075] In some embodiments, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In some embodiments, the replicon or self-replicating RNA is derived from or includes elements derived from an ssRNA virus, particularly a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and in protein modification) and structural proteins (which form the virus particle). Typically, two open reading frames (ORFs) are present in the genome. Four nonstructural proteins (nsP1-nsP4) are co-encoded by the first ORF, which typically begins near the 5' end of the genome, while the alphavirus structural proteins are co-encoded by the second ORF, which is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA polynucleotides similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly as a messenger RNA for the translation of an open reading frame encoding a nonstructural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be trans-replicated by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be trans-replicated by the replicase must contain specific alphavirus sequence elements that allow recognition and RNA synthesis by the alphavirus replicase.

[0076] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridine.

[0077] The term "uracil" as used herein describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is as follows: [ka]

[0078] As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is as follows: [ka]

[0079] UTP (uridine 5'-triphosphate) has the following structure: [ka]

[0080] Pseudo-UTP (pseudouridine-5'-triphosphate) has the following structure: [ka]

[0081] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring through a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0082] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the following structure: [ka]

[0083] N1-methylpseudouridine-5'-triphosphate (m1ΨTP) has the following structure: [ka]

[0084] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: [ka]

[0085] In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides.In some embodiments, the modified nucleosides are modified uridines.In some embodiments, the RNA comprises modified nucleosides instead of at least one uridine.In some embodiments, the RNA comprises modified nucleosides instead of each uridine.

[0086] In some embodiments, the modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U).

[0087] In some embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0088] In some embodiments, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine is partially or completely, preferably completely, substituted with cytidine in the RNA. In some embodiments, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA contains 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0089] In some embodiments, RNA encoding a payload, e.g., a vaccine antigen, is expressed in the cells of a subject treated to provide the payload, e.g., vaccine antigen. In some embodiments, the RNA is transiently expressed in the cells of the subject. In some embodiments, the RNA is in vitro transcribed RNA. In some embodiments, expression of the payload, e.g., vaccine antigen, occurs on the cell surface. In some embodiments, the payload, e.g., vaccine antigen, is expressed and presented in the context of MHC. In some embodiments, expression of the payload, e.g., vaccine antigen, is in the extracellular space, i.e., the vaccine antigen is secreted.

[0090] The term "transcription" in the context of the present disclosure relates to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0091] According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in a cell-free system in vitro, preferably using a suitable cell extract. Preferably, a cloning vector is used to generate the transcription product. Such a cloning vector is commonly referred to as a transcription vector and, according to the present invention, is encompassed by the term "vector." According to the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a vector suitable for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0092] With respect to RNA, the terms "expression" or "translation" refer to the process by which a chain of mRNA directs the assembly of a series of amino acids to form a peptide or protein in the ribosomes of a cell.

[0093] In some embodiments, after administration of the RNA described herein, for example, formulated as an RNA-lipid particle, at least a portion of the RNA is delivered to the target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the encoded peptide or protein. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, for example, a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or a macrophage. RNA particles, such as the RNA-lipid particles described herein, may be used to deliver RNA to such target cells. Thus, the present disclosure also relates to a method of delivering RNA to a target cell in a subject, comprising administering to the subject the RNA particles described herein. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA. "Code" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0094] In some embodiments, the nucleic acid compositions described herein, e.g., compositions comprising lipid nanoparticle-encapsulated mRNA, are characterized by sustained expression of the encoded polypeptide (e.g., when administered to a subject). For example, in some embodiments, such compositions, when administered to a human, achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some embodiments, are characterized in that such expression persists for at least 36 hours, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

[0095] In some embodiments, the RNA encoding the vaccine antigen to be administered according to the present invention is non-immunogenic. RNA-encoded immunostimulants can be administered according to the present invention to provide an adjuvant effect. RNA-encoded immunostimulants can be standard RNA or non-immunogenic RNA.

[0096] The term "non-immunogenic RNA" as used herein refers to RNA that does not induce a response by the immune system immediately after administration, for example to a mammal, or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., standard RNA (stdRNA). In a preferred embodiment, the non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that suppress RNA-mediated activation of innate immune receptors, thereby eliminating double-stranded RNA (dsRNA).

[0097] To make non-immunogenic RNA non-immunogenic by incorporating modified nucleosides, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises the replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine]. In a particularly preferred embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m 5 U), especially N1-methyl-pseudouridine.

[0098] In some embodiments, the replacement of one or more uridines with nucleosides containing modified nucleobases includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridines. During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, a significant amount of abnormal products, including double-stranded RNA (dsRNA), are produced due to the unconventional activity of the enzyme. dsRNA induces inflammatory cytokines, activates effector enzymes, and causes the inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by, for example, ion-pair reverse-phase HPLC using a non-porous or porous C-18 polystyrene divinylbenzene (PS-DVB) matrix. Alternatively, enzyme-based methods can be used to remove dsRNA contaminants from IVT RNA preparations using E. coli RNase III, which specifically hydrolyzes dsRNA but not ssRNA. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA preparation is contacted with the cellulose material and the ssRNA is separated from the cellulose material under conditions that allow binding of dsRNA but not ssRNA to the cellulose material.

[0099] As used herein, the term "removing" or "removal" refers to the characteristic of a population of a first substance, such as a non-immunogenic RNA, being separated from the vicinity of a population of a second substance, such as a dsRNA, where the population of the first substance is not necessarily free of the second substance, and the population of the second substance is not necessarily free of the first substance. However, the population of the first substance characterized by the removal of the population of the second substance has a significantly lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0100] In some embodiments, removing dsRNA from non-immunogenic RNA comprises removing dsRNA so that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA is free or essentially free of dsRNA.In some embodiments, non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside-modified RNA.For example, in some embodiments, a purified preparation of single-stranded nucleoside-modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, a purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0101] In some embodiments, the non-immunogenic RNA is translated more efficiently in cells than standard RNA of the same sequence. In some embodiments, translation is enhanced by 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced by 3-fold. In some embodiments, translation is enhanced by 4-fold. In some embodiments, translation is enhanced by 5-fold. In some embodiments, translation is enhanced by 6-fold. In some embodiments, translation is enhanced by 7-fold. In some embodiments, translation is enhanced by 8-fold. In some embodiments, translation is enhanced by 9-fold. In some embodiments, translation is enhanced by 10-fold. In some embodiments, translation is enhanced by 15-fold. In some embodiments, translation is enhanced by 20-fold. In some embodiments, translation is enhanced by 50-fold. In some embodiments, translation is enhanced by 100-fold. In some embodiments, translation is enhanced by 200-fold. In some embodiments, translation is enhanced by 500-fold. In some embodiments, translation is enhanced by a factor of 1000-fold. In some embodiments, translation is enhanced by a factor of 2000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0102] In some embodiments, the non-immunogenic RNA exhibits significantly lower natural immunogenicity than standard RNA of the same sequence. In some embodiments, the non-immunogenic RNA exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced by a factor of 3. In some embodiments, the natural immunogenicity is reduced by a factor of 4. In some embodiments, the natural immunogenicity is reduced by a factor of 5. In some embodiments, the natural immunogenicity is reduced by a factor of 6. In some embodiments, the natural immunogenicity is reduced by a factor of 7. In some embodiments, the natural immunogenicity is reduced by a factor of 8. In some embodiments, the natural immunogenicity is reduced by a factor of 9. In some embodiments, the natural immunogenicity is reduced by a factor of 10. In some embodiments, the natural immunogenicity is reduced by a factor of 15. In some embodiments, the natural immunogenicity is reduced by a factor of 20. In some embodiments, the natural immunogenicity is reduced by a factor of 50. In some embodiments, the natural immunogenicity is reduced by a factor of 100. In some embodiments, the natural immunogenicity is reduced by a factor of 200. In some embodiments, the natural immunogenicity is reduced by a factor of 500. In some embodiments, the natural immunogenicity is reduced by a factor of 1000. In some embodiments, the natural immunogenicity is reduced by a factor of 2000.

[0103] The term "exhibiting significantly reduced innate immunogenicity" refers to a detectable reduction in innate immunogenicity. In some embodiments, the term refers to a reduction such that an effective amount of the non-immunogenic RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA. "Immunogenicity" refers to the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively non-specific and immediate component of the immune system. Along with the adaptive immune system, it is one of the two major components of the vertebrate immune system.

[0104] As used herein, "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0105] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0106] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0107] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements, components, or domains.

[0108] Codon optimization In some embodiments, the payloads (e.g., polypeptides) described herein are encoded by coding sequences that are codon-optimized and / or have increased G / C content compared to wild-type coding sequences. In some embodiments, one or more sequence regions of the coding sequence are codon-optimized and / or have increased G / C content compared to corresponding sequence regions of wild-type coding sequences. In some embodiments, codon optimization and / or increased G / C content do not change the sequence of the encoded amino acid sequence.

[0109] The term "codon-optimized" is understood by those skilled in the art to refer to the modification of codons in the coding region of a nucleic acid molecule, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule, so as to reflect the typical codon usage of the host organism.In the context of the present disclosure, the coding region is preferably codon-optimized for optimal expression in the subject to be treated with the RNA polynucleotide described herein.Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of tRNA occurrence in cells.Therefore, the sequence of RNA can be modified so that codons that are available for frequently occurring tRNAs are inserted instead of "rare codons".

[0110] In some embodiments, the guanosine / cytidine (G / C) content of the coding region of the RNA (e.g., payload sequence) is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA encoding the payload, and the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the effective translation of that mRNA. Sequences with an increased G (guanosine) / C (cytidine) content are more stable than sequences with an increased A (adenosine) / U (uridine) content. In conjunction with the fact that several codons encode one and the same amino acid (the so-called degeneracy of the genetic code), the most favorable codon for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to the wild-type sequence. In particular, codons containing A and / or U nucleosides can be modified by replacing the codon with other codons that encode the same amino acids but that do not contain A and / or U or that contain a lower content of A and / or U nucleosides.

[0111] In some embodiments, the G / C content of the coding region of an RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding region of a wild-type RNA.

[0112] 5' Cap RNA capping has been thoroughly studied and described, for example, in Decroly E et al. (2012) Nature Reviews 10: 51-65; and Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which are incorporated herein by reference. 5' caps include cap-0 (also referred to herein as "cap-0"), cap-1 (also referred to herein as "cap-1"), or cap-2 (also referred to herein as "cap-2"). See, for example, Figure 1 in Ramanathan A et al. and Figure 1 in Decroly E et al.

[0113] As used herein, the term 5' cap refers to a structure found on the 5' end of an RNA, e.g., an mRNA, and generally comprises a guanosine nucleotide linked to the RNA, e.g., an mRNA, via a 5'-5' triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, the guanosine nucleoside contained within the 5' cap can be modified, for example, by methylation at one or more positions on the base (guanine) (e.g., at the 7 position) and / or by methylation at one or more positions on the ribose. In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a 3'O methylation at the ribose (3'OMeG). In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7 position of the guanine (m7G). In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7 position of the guanine and a 3'O methylation on the ribose [m7(3'OMeG)].

[0114] In some embodiments, providing an RNA with a 5' cap or 5' cap analog disclosed herein may be accomplished by in vitro transcription, in which the 5' cap is transcriptionally expressed into the RNA strand, or may be attached to the RNA post-transcriptionally using a capping enzyme. In some embodiments, transcription-associated capping with a cap disclosed herein, such as Cap1 or a Cap1 analog, improves the capping efficiency of the RNA compared to transcription-associated capping with an appropriate reference comparator. In some embodiments, improving capping efficiency may increase the translation efficiency and / or rate of the RNA and / or increase expression of the encoded polypeptide.

[0115] In some embodiments, the RNA described herein comprises a 5' cap or a 5' cap analog, e.g., Cap 0, Cap 1, or Cap 2. In some embodiments, the provided RNA does not have an uncapped 5' triphosphate cap. In some embodiments, the RNA may be capped with a 5' cap analog. In some embodiments, the RNA described herein comprises Cap 0. In some embodiments, the RNA described herein comprises Cap 1, e.g., as described herein. In some embodiments, the RNA described herein comprises Cap 2.

[0116] In some embodiments, the Cap 0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G). In some embodiments, the Cap 0 structure is linked to the RNA via a 5'-5' triphosphate linkage, also referred to herein as m7Gppp or m7G(5')ppp(5').

[0117] In some embodiments, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and a first nucleotide (2'OMeN1) that is 2'O-methylated within the RNA. In some embodiments, the Cap 1 structure is linked to the RNA via a 5'-5' triphosphate linkage, also referred to herein as m7Gppp(2'OMeN1) or m7G(5')ppp(5')(2'OMeN1). In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0118] In some embodiments, the m7G(5')ppp(5')(2'OMeN1) Cap 1 structure includes a second nucleotide, N2, which is the cap proximal to the nucleotide at position 2 and is selected from A, G, C, or U (m7G(5')ppp(5')(2'OMeN1)N2). In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0119] In some embodiments, the Cap 1 structure is or includes m7G(5')ppp(5')(2'OMeA1)pG2, where A is the cap proximal to the nucleotide at the +1 position and G is the cap proximal to the nucleotide at the +2 position, and has the following structure: [ka]

[0120] In some embodiments, the Cap 1 structure is or includes m7G(5')ppp(5')(2'OMeA1)pU2, where A is the cap proximal to the nucleotide at position 1 and U is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0121] In some embodiments, the Cap 1 structure is or includes m7G(5')ppp(5')(2'OMeG1)pG2, where G is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0122] In some embodiments, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G), one or more additional modifications, e.g., a methylation on the ribose, and a 2'O-methylated first nucleotide within the RNA. In some embodiments, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine and 3'O-methylated on the ribose [m7(3'OMeG)]; and a 2'O-methylated first nucleotide within the RNA (2'OMeN1). In some embodiments, the Cap 1 structure is linked to the RNA via a 5'-5' triphosphate linkage, also referred to herein as m7(3'OMeG)ppp(2'OMeN1) or m7(3'OMeG)(5')ppp(5')(2'OMeN1). In some embodiments, N1 is selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U.

[0123] In some embodiments, the m7(3'oMeG)(5')ppp(5')(2'OMeN1) Cap 1 structure includes a second nucleotide, N2, which is the cap proximal to the nucleotide at position 2 and is selected from A, G, C, or U [m7(3'OMeG)(5')ppp(5')(2'OMeN1)N2]. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0124] In some embodiments, the Cap 1 structure is or includes m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0125] In some embodiments, the Cap 1 structure is or includes m7(3'OMeG)(5')ppp(5')(2'OMeG1)pG2, where G is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0126] In some embodiments, the second nucleotide in the Cap 1 structure can include one or more modifications, such as methylation. In some embodiments, a Cap 1 structure that includes a second nucleotide that includes a 2'O methylation is a Cap 2 structure.

[0127] In some embodiments, an RNA polynucleotide comprising a Cap 1 structure exhibits increased translation efficiency, increased translation rate, and / or increased expression of the encoded payload relative to a suitable reference comparator. In some embodiments, an RNA polynucleotide comprising a Cap 1 structure having m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2 (where A is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2) exhibits increased translation efficiency compared to an RNA polynucleotide comprising a Cap 1 structure having m7(3'OMeG)(5')ppp(5')(2'OMeG1)pG2, where G1 is the cap proximal to the nucleotide at position 1 and G2 is the cap proximal to the nucleotide at position 2. In some embodiments, increased translation efficiency is assessed by administering the RNA polynucleotide to a cell or organism.

[0128] In some embodiments, the cap analog used in the RNA polynucleotide is m2 7,3’-O Gppp(m1 2’-O )ApG(occasionally, m2 7,3’O G(5')ppp(5')m 2’-O ApG, or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG), which has the following structure: [ka]

[0129] The following is an exemplary cap1 RNA, which is a nucleotide sequence of RNA and m2 7,3’O G(5')ppp(5')m 2’-O Including ApG, [ka]

[0130] The following is another exemplary Cap1 RNA: [ka]

[0131] In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in any one of Figures 3A-I. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3A. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3B. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3C. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3D. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3E. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3F. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3G. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3H. In some embodiments, an RNA polynucleotide disclosed herein comprises a cap shown in Figure 3I.

[0132] 5'UTR and cap-proximal sequences In some embodiments, the RNA disclosed herein comprises a 5' UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5' UTR) and / or 3' (downstream) of an open reading frame (3' UTR). If present, the 5' UTR is located at the 5' end upstream of the start codon of a protein-coding region. The 5' UTR is located downstream of the 5' cap (if present), for example, directly adjacent to the 5' cap.

[0133] In some embodiments, a 5'UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some embodiments, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some embodiments, the cap-proximal sequence comprises nucleotides within positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0134] In some embodiments, the cap structure comprises one or more polynucleotides of a cap-proximal sequence. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide.

[0135] Those skilled in the art will recognize, upon reading this disclosure, that in some embodiments, one or more residues of the cap-proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in the RNA by being included in a cap entity (e.g., a cap 1 structure, etc.); and in other embodiments, at least a portion of the residues in the cap-proximal sequence may be added enzymatically (e.g., by a polymerase such as T7 polymerase). For example, m2 7,3’-O Gppp(m1 2’-O In certain exemplary embodiments where an ApG cap is utilized, +1 and +2 are the (m1 2’-O ) A and G residues, and +3, +4, and +5 are added by a polymerase (e.g., T7 polymerase).

[0136] In some embodiments, the cap-proximal sequence comprises cap structures N1 and N2, where N1 and N2 are any nucleotides, e.g., A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0137] In some embodiments, N1 is A and N2 is A. In some embodiments, N1 is A and N2 is C. In some embodiments, N1 is A and N2 is G. In some embodiments, N1 is A and N2 is U.

[0138] In some embodiments, N1 is C and N2 is A. In some embodiments, N1 is C and N2 is C. In some embodiments, N1 is C and N2 is G. In some embodiments, N1 is C and N2 is U.

[0139] In some embodiments, N1 is G and N2 is A. In some embodiments, N1 is G and N2 is C. In some embodiments, N1 is G and N2 is G. In some embodiments, N1 is G and N2 is U.

[0140] In some embodiments, N1 is U and N2 is A. In some embodiments, N1 is U and N2 is C. In some embodiments, N1 is U and N2 is G. In some embodiments, N1 is U and N2 is U.

[0141] In some embodiments, the cap-proximal sequence includes N1 and N2, and N3, N4, and N5 of the cap structure, where N1-N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0142] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is A. In some embodiments, N5 is A.

[0143] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is C. In some embodiments, N5 is A.

[0144] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is G. In some embodiments, N5 is A.

[0145] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is U. In some embodiments, N5 is A.

[0146] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is A. In some embodiments, N5 is G.

[0147] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is G. In some embodiments, N5 is G.

[0148] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is C. In some embodiments, N5 is G.

[0149] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is U. In some embodiments, N5 is G.

[0150] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is A. In some embodiments, N5 is C.

[0151] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is C. In some embodiments, N5 is C.

[0152] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is G. In some embodiments, N5 is C.

[0153] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is U. In some embodiments, N5 is C.

[0154] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is A. In some embodiments, N5 is U.

[0155] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is C. In some embodiments, N5 is U.

[0156] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is G. In some embodiments, N5 is U.

[0157] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is A. In some embodiments, N4 is U. In some embodiments, N5 is U.

[0158] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is A. In some embodiments, N5 is A.

[0159] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is A.

[0160] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is G. In some embodiments, N5 is A.

[0161] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is U. In some embodiments, N5 is A.

[0162] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is A. In some embodiments, N5 is G.

[0163] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is G. In some embodiments, N5 is G.

[0164] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is G.

[0165] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is U. In some embodiments, N5 is G.

[0166] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is A. In some embodiments, N5 is C.

[0167] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is C.

[0168] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is G. In some embodiments, N5 is C.

[0169] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is U. In some embodiments, N5 is C.

[0170] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is A. In some embodiments, N5 is U.

[0171] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is C. In some embodiments, N5 is U.

[0172] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is G. In some embodiments, N5 is U.

[0173] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is C. In some embodiments, N4 is U. In some embodiments, N5 is U.

[0174] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is A. In some embodiments, N5 is A.

[0175] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is C. In some embodiments, N5 is A.

[0176] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is G. In some embodiments, N5 is A.

[0177] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is U. In some embodiments, N5 is A.

[0178] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is A. In some embodiments, N5 is G.

[0179] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is G. In some embodiments, N5 is G.

[0180] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is C. In some embodiments, N5 is G.

[0181] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is U. In some embodiments, N5 is G.

[0182] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is A. In some embodiments, N5 is C.

[0183] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is C. In some embodiments, N5 is C.

[0184] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is G. In some embodiments, N5 is C.

[0185] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is U. In some embodiments, N5 is C.

[0186] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is A. In some embodiments, N5 is U.

[0187] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is C. In some embodiments, N5 is U.

[0188] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is G. In some embodiments, N5 is U.

[0189] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is G. In some embodiments, N4 is U. In some embodiments, N5 is U.

[0190] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N5 is A.

[0191] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is C. In some embodiments, N5 is A.

[0192] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is G. In some embodiments, N5 is A.

[0193] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is U. In some embodiments, N5 is A.

[0194] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N5 is G.

[0195] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is G. In some embodiments, N5 is G.

[0196] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is C. In some embodiments, N5 is G.

[0197] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is U. In some embodiments, N5 is G.

[0198] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N5 is C.

[0199] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is C. In some embodiments, N5 is C.

[0200] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is G. In some embodiments, N5 is C.

[0201] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is U. In some embodiments, N5 is C.

[0202] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N5 is U.

[0203] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is C. In some embodiments, N5 is U.

[0204] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is G. In some embodiments, N5 is U.

[0205] In some embodiments, N1, N2, N3, N4, or N5 is any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, N3 is U. In some embodiments, N4 is U. In some embodiments, N5 is U.

[0206] In some embodiments, a 5'UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some embodiments, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some embodiments, the cap-proximal sequence comprises nucleotides within positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0207] In some embodiments, the cap structure comprises one or more polynucleotides of a cap-proximal sequence. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide.

[0208] In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0209] In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0210] In some embodiments, N1 is A and N2 is A. In some embodiments, N1 is A and N2 is C. In some embodiments, N1 is A and N2 is G. In some embodiments, N1 is A and N2 is U.

[0211] In some embodiments, N1 is C and N2 is A. In some embodiments, N1 is C and N2 is C. In some embodiments, N1 is C and N2 is G. In some embodiments, N1 is C and N2 is U.

[0212] In some embodiments, N1 is G and N2 is A. In some embodiments, N1 is G and N2 is C. In some embodiments, N1 is G and N2 is G. In some embodiments, N1 is G and N2 is U.

[0213] In some embodiments, N1 is U and N2 is A. In some embodiments, N1 is U and N2 is C. In some embodiments, N1 is U and N2 is G. In some embodiments, N1 is U and N2 is U.

[0214] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3A4X5 (SEQ ID NO: 1). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is selected from A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0215] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising C3A4X5 (SEQ ID NO: 2). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is selected from A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0216] In some embodiments, the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure, and X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X3 and X5 are each independently selected from A, C, G, or U. In some embodiments, X3 and / or X5 are A. In some embodiments, X3 and / or X5 are C. In some embodiments, X3 and / or X5 are G. In some embodiments, X3 and / or X5 are U. In some embodiments, Y4 is not C. In some embodiments, Y4 is A. In some embodiments, Y4 is G. In some embodiments, Y4 is U.

[0217] In some embodiments, the cap-proximal sequence comprises a sequence comprising cap structures N1 and N2 and X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X3 and X5 are each independently selected from A, C, G, or U. In some embodiments, X3 and / or X5 are A. In some embodiments, X3 and / or X5 are C. In some embodiments, X3 and / or X5 are G. In some embodiments, X3 and / or X5 are U. In some embodiments, Y4 is not G. In some embodiments, Y4 is A. In some embodiments, Y4 is C. In some embodiments, Y4 is U.

[0218] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3C4A5 (SEQ ID NO: 3). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0219] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3U4G5 (SEQ ID NO: 4). In some embodiments, N1 and N2 are each independently selected from A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0220] In some embodiments, the cap structure comprises one or more polynucleotides of a cap-proximal sequence. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide.

[0221] In some embodiments, N1 and N2 are any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0222] In some embodiments, N1 and N2 are any nucleotide, for example, A, C, G, or U. In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1 is G. In some embodiments, N1 is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U.

[0223] In some embodiments, N1 is A and N2 is A. In some embodiments, N1 is A and N2 is C. In some embodiments, N1 is A and N2 is G. In some embodiments, N1 is A and N2 is U.

[0224] In some embodiments, N1 is C and N2 is A. In some embodiments, N1 is C and N2 is C. In some embodiments, N1 is C and N2 is G. In some embodiments, N1 is C and N2 is U.

[0225] In some embodiments, N1 is G and N2 is A. In some embodiments, N1 is G and N2 is C. In some embodiments, N1 is G and N2 is G. In some embodiments, N1 is G and N2 is U.

[0226] In some embodiments, N1 is U and N2 is A. In some embodiments, N1 is U and N2 is C. In some embodiments, N1 is U and N2 is G. In some embodiments, N1 is U and N2 is U.

[0227] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3A4X5 (SEQ ID NO: 1). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is selected from A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0228] In some embodiments, the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure and C3A4X5 (SEQ ID NO: 2). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X5 is any nucleotide, e.g., A, C, G, or U. In some embodiments, X5 is A. In some embodiments, X5 is C. In some embodiments, X5 is G. In some embodiments, X5 is U.

[0229] In some embodiments, the cap-proximal sequence comprises a sequence comprising the cap structure N1 and N2, and X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X3 and X5 are any nucleotide, e.g., A, C, G, or U. In some embodiments, X3 and / or X5 are A. In some embodiments, X3 and / or X5 are C. In some embodiments, X3 and / or X5 are G. In some embodiments, X3 and / or X5 are U. In some embodiments, Y4 is not C. In some embodiments, Y4 is A. In some embodiments, Y4 is G. In some embodiments, Y4 is U.

[0230] In some embodiments, the cap-proximal sequence comprises a sequence comprising the cap structure N1 and N2, and X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G. In some embodiments, X3 and X5 are any nucleotide, e.g., A, C, G, or U. In some embodiments, X3 and / or X5 are A. In some embodiments, X3 and / or X5 are C. In some embodiments, X3 and / or X5 are G. In some embodiments, X3 and / or X5 are U. In some embodiments, Y4 is not G. In some embodiments, Y4 is A. In some embodiments, Y4 is C. In some embodiments, Y4 is U.

[0231] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3C4A5 (SEQ ID NO: 3). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0232] In some embodiments, the cap-proximal sequence comprises N1 and N2 of the cap structure and a sequence comprising A3U4G5 (SEQ ID NO: 4). In some embodiments, N1 and N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0233] Exemplary 5'UTRs include human alpha globin (hAg) 5'UTR or a fragment thereof, TEV 5'UTR or a fragment thereof, HSP70 5'UTR or a fragment thereof, or c-Jun 5'UTR or a fragment thereof.

[0234] In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR or a fragment thereof. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 11. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 12. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 12.

[0235] 3'UTR In some embodiments, the RNA disclosed herein comprises a 3'UTR. If present, the 3'UTR is located at the 3' end downstream of the termination codon of the protein-coding region, but the term "3'UTR" preferably does not include a poly(A) sequence. Thus, the 3'UTR is located upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0236] In some embodiments, the RNA disclosed herein comprises a 3'UTR comprising an F element and / or an I element. In some embodiments, the 3'UTR or a sequence proximal thereto comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is a XhoI site.

[0237] In some embodiments, the RNA disclosed herein comprises a 3'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the 3'UTR provided in SEQ ID NO: 13. In some embodiments, the RNA disclosed herein comprises a 3'UTR provided in SEQ ID NO: 13.

[0238] Poly A In some embodiments, the RNAs disclosed herein comprise a polyadenylation (polyA) sequence, e.g., as described herein. In some embodiments, the polyA sequence is located downstream of the 3' UTR, e.g., adjacent to the 3' UTR.

[0239] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and can follow the 3'UTR in the RNAs described herein. Uninterrupted poly(A) sequences are characterized by consecutive adenylate residues. Uninterrupted poly(A) sequences are typical in nature. The RNAs disclosed herein can have poly(A) sequences attached to the free 3' end of the RNA by a non-template-dependent RNA polymerase after transcription, or poly(A) sequences encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0240] It has been demonstrated that poly(A) sequences of approximately 120 A nucleotides have a beneficial effect on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0241] The poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly(A) sequence, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0242] In some embodiments, poly(A) sequences are attached during RNA transcription based on a DNA template containing repetitive dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand, e.g., during preparation of in vitro transcribed RNA. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

[0243] In some embodiments, a poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 A1 can be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5 to 50 nucleotides, exhibit consistent propagation of plasmid DNA in E. coli at the DNA level, yet are associated with beneficial properties at the RNA level related to support of RNA stability and translation efficiency. In some embodiments, the poly(A) sequences contained within the RNA polynucleotides described herein consist essentially of A nucleotides but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0244] In some embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked by or followed by a nucleotide other than A at its 3' end.

[0245] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises approximately 150 nucleotides. In some embodiments, the poly(A) sequence comprises approximately 120 nucleotides.

[0246] In some embodiments, the RNA disclosed herein comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the RNA disclosed herein comprises the poly(A) sequence of SEQ ID NO: 14.

[0247] payload In some embodiments, the RNA polynucleotides disclosed herein comprise a sequence encoding a payload, such as those described herein. In some embodiments, the sequence encoding the payload comprises a promoter sequence. In some embodiments, the sequence encoding the payload comprises a sequence encoding a secretory signal peptide.

[0248] In some embodiments, the payload is selected from a protein replacement polypeptide; an antibody agent; a cytokine; an antigenic polypeptide; a gene editing component; a tissue engineering component, or a combination thereof.

[0249] In some embodiments, the payload is or comprises a protein replacement polypeptide. In some embodiments, the protein replacement polypeptide comprises a polypeptide whose expression is aberrant in a disease or disorder. In some embodiments, the protein replacement polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein. In some embodiments, the protein replacement polypeptide comprises an enzyme.

[0250] In some embodiments, the disease or disorder in which polypeptide expression is aberrant includes, but is not limited to, a rare disease, a metabolic disorder, a muscular dystrophy, a cardiovascular disease, or a monogenic disease.

[0251] In some embodiments, the payload is or comprises an antibody agent. In some embodiments, the antibody agent binds to a polypeptide expressed on a cell. In some embodiments, the antibody agent comprises a CD3 antibody, a claudin 6 antibody, or a combination thereof.

[0252] In some embodiments, the payload is or comprises a cytokine, or a fragment or variant thereof. In some embodiments, the cytokine comprises IL-12, or a fragment, variant, or fusion thereof, IL-15, or a fragment, variant, or fusion thereof, GMCSF, or a fragment or variant thereof; or IFN-alpha, or a fragment or variant thereof.

[0253] In some embodiments, the payload is or comprises an antigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the antigenic polypeptide comprises one epitope derived from an antigen. In some embodiments, the antigenic polypeptide comprises multiple different epitopes derived from an antigen. In some embodiments, the antigenic polypeptide comprising multiple different epitopes derived from an antigen is a polyepitope.

[0254] In some embodiments, the antigenic polypeptide comprises an antigenic polypeptide derived from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide derived from an infectious agent, an antigenic polypeptide derived from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

[0255] In some embodiments, the viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a coronavirus antigenic polypeptide, a rabies antigenic polypeptide, or a Zika virus antigenic polypeptide.

[0256] In some embodiments, the viral antigenic polypeptide is or comprises a coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigen is or comprises a SARS-CoV-2 protein. In some embodiments, the SARS-CoV-2 protein comprises a SARS-CoV-2 spike (S) protein, or an immunogenic variant or immunogenic fragment thereof. In some embodiments, the SARS-CoV-2 protein, or an immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions 986 and 987.

[0257] In some embodiments, the SARS-CoV-2 S polypeptide is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to a SARS-CoV-2 S polypeptide disclosed herein. In some embodiments, the SARS-CoV-2 S polypeptide is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:9.

[0258] In some embodiments, the SARS-CoV-2 S polypeptide is encoded by RNA that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to a SARS-CoV-2 S polynucleotide disclosed herein. In some embodiments, the SARS-CoV-2 S polypeptide is encoded by RNA that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:10.

[0259] In some embodiments, the payload is or comprises a tumor antigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the tumor antigenic polypeptide comprises a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the tumor antigenic polypeptide is p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE -A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE -A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or a combination thereof.

[0260] In some embodiments, the tumor antigenic polypeptide comprises a tumor antigen from a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, or a combination thereof.

[0261] In some embodiments, the tumor antigenic polypeptide comprises a melanoma tumor antigen.

[0262] In some embodiments, the tumor antigenic polypeptide comprises a prostate cancer antigen.

[0263] In some embodiments, the tumor antigenic polypeptide comprises an HPV16-positive head and neck cancer antigen.

[0264] In some embodiments, the tumor antigenic polypeptide comprises a breast cancer antigen.

[0265] In some embodiments, the tumor antigenic polypeptide comprises an ovarian cancer antigen.

[0266] In some embodiments, the tumor antigenic polypeptide comprises a lung cancer antigen.

[0267] In some embodiments, the tumor antigenic polypeptide comprises an NSCLC antigen.

[0268] In some embodiments, the payload is or comprises an autoantigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the autoantigenic polypeptide comprises an antigen that is typically expressed on cells and recognized as an autoantigen by the immune system. In some embodiments, the autoantigenic polypeptide comprises a multiple sclerosis antigenic polypeptide, a rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof.

[0269] Exemplary Polynucleotides In some embodiments, the RNA polynucleotides described herein, or compositions or medical preparations comprising the same, comprise a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence that is at least 80% identical to a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence that encodes a polypeptide that is at least 80% identical to a polypeptide sequence disclosed herein. Exemplary nucleotide and polypeptide sequences are provided, for example, in Table 1, in this section entitled "Exemplary Polynucleotides," or in Example 2.

[0270] In some embodiments, the RNA polynucleotides described herein, or compositions or medical preparations comprising the same, are transcribed using a DNA template. In some embodiments, the DNA template used to transcribe the RNA polynucleotides described herein comprises a sequence complementary to the RNA polynucleotide.

[0271] In some embodiments, the payloads described herein are encoded by an RNA polynucleotide described herein, including a nucleotide sequence disclosed herein, e.g., in Table 1, in this section entitled "Exemplary Polynucleotides," or in Example 2. In some embodiments, the RNA polynucleotide encodes a polypeptide payload that is at least 80% identical to a polypeptide payload sequence disclosed herein. In some embodiments, the payloads described herein are encoded by an RNA polynucleotide that is transcribed by a DNA template that includes a sequence complementary to the RNA polynucleotide.

[0272] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0273] RBL063.1 (SEQ ID NO:28, nucleotide; SEQ ID NO:9, amino acid) Structure: beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variants) SEQ ID NO: 28 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu 60 guuucuugu cugcugccuc uugugucuuc ucagugugu aauuugacaa caagaacaca 120 gcugccacca gcuuauacaa auucuuuuac cagaggagug uauuauccug auaaaguguu 180 uagaucuucu gugcugcaca gcacacagga ccuguuucug ccauuuuuua gcaaugugac 240 augguuucau gcaauucaug ugucuggaac aaauggaaca aaaagauuug auauccugu 300 cguccuuuuu aaugauggag ugaauuuugc uucaacagaa aagucaaaua uuauuagagg 360 auggauuuuu ggaacaacac uggauucuaa aacacagucu cugcugauug ugaauaaugc 420 aacaaaugug gugauuaaag ugugugaauu ucaguuuugu aaugauccuu uucugggagu 480 guauuaucac aaaaauaaua aaucuuggau ggaaucugaa uuuagagugu auuccugc 540 aaauaauugu acauuugaau augugucuca gccuuuuucug auggaucugg aagaaaaca 600 gggcaauuu aaaaaauuga gagaauuuu guuuaaaaau auuuuuuuuuuuuuuuu 660 uuauucuaaa cacacaccaa uuauuuuga gagagaucug ccucagggau uuucugcucu 720 ggaaccucug guggauucugc caauuggcau uauauaca agauuucaga caucuguggc 780 ucugcacaga ucuuacuga caccuggaga uucuucuucu ggauggacag ccggagcugc 840 agcuauuau gugggcuauc ugcagccaag aacauuucug ugaaauua augaaaaugg 900 aacaauuaca gaugcugugg augugcucu ggauccucug ucugaaaca aauguacauu 960 aaaaucuuuu acaguggaaa aaggcauuuua ucagacaucu aauuuuaagag ugcagccaac 1020 agaaucuauu gugagauuuc caaauauuac aaaucugugu ccauuuggag aaguguuuaa 1080 ugcaacaaga uuugcaucug uguaugcaug gaauagaaaa agaauuucua auuguguggc 1140 ugauuauucu gugcuguaua auagugcuuc uuuuccaca uuuaaauguu auggaguguc 1200 uccaacaaaa uuaaaugauu uauguuuuac aaauguguau gcugauucuu uugugaucag 1260 aggugaugaa gugagacaga uugccccccgg acagacagga aaaauugcug auuacaauua 1320 caaacugccu gaugauuuua caggaugugu gauugcuugg aauucuaaua auuuagauuc 1380 uaaaguggga ggaaauuaca auuaucugua cagacuguuu agaaaaucaa aucugaaacc 1440 uuuugaaaga gauauuucaa cagaaauuua ucaggcugga ucaacaccuu guaauggagu 1500 ggaaggauuu aauuguuauu uuccauuaca gagcuaugga uuucagccaa caaoggugu 1560 gggauaucag ccauauagag ugguggugcu gucuuuugaa cugcugcaug caccugcaac 1620 agugugugga ccuaaaaaau cuacaaauuu agugaaaaau aaauguguga auuuuaauuu 1680 uaauggauua acaggaacag gagugcugac agaaucuaau aaaaaauuc ugccuuuuca 1740 gcaguuuuggc agagauauug cagauaccac agaugcagug agagauccuc agacauuaga 1800 aauucuggau auuacaccuu guucuuugg gggugugucu gugauuacac cuggaacaaa 1860 uacaucuaau cagguggcug ugcuguauca ggaugugaau uguacagaag ugccaguggc 1920 aauucaugca gaucagcuga caccaacaug gagaguguau ucuacaggau cuaauguguu 1980 ucagacaaga gcaggauguc ugauuggagc agaacaugug aauaauucuu augaauguga 2040 uauuccaauu ggagcaggca uuugugcauc uuaucagaca cagacaaauu ccccaaggag 2100 agcaagaucu guggcaucuc agucuauuau ugcauacacc augucucugg gagcagaaaa 2160 uucuguggca uauucuaaua auucuauugc uauuccaaca aauuuuacca uuucugugac 2220 aacagaaauu uuaccugugu cuaugacaaa aacaucugug gauuguacca uguacauuug 2280 uggagauucu acagaauguu cuaaucugcu gguccaguau ggaucuuuuu guacacagcu 2340 gaauagagcu uuaacaggaa uugcuguga acaggauaa aauacacagg aaguguuugc 2400 ucaggugaaa cagauuuaca aaacaccacc aauuaaagau uuuggaggau uuaauuuuag 2460 ccagauucug ccugauccu cuaaaccuuc uaaaagaucu uuuauugaag aucugcuguu 2520 uaauaaagug acacuggcag augcaggauu uauuaaacag uauggagaau gccuggguga 2580 uauugcugca agagauucuga uuugugcuca gaauuuuaau ggacugacag ugcugccucc 2640 ucugcugaca gaugaauga uugcucagua cacaucugcu uaacuggcug gaacaauuac 2700 aagcggaugg acauuuggag cuggagcugc ucugcagauu ccuuuugcaa ugcagauggc 2760 uuacagauuu aauggaauug gagugacaca gaaugugua uaugaaaauc agaaacugau 2820 ugcaaaucag uuuaauucug caauuggcaa aauucaggau ucucugucuu cuacagcuuc 2880 ugcucuggga aaacugcagg auguggugaa ucagaaugca caggcacuga aucucuggu 2940 gaacagcug ucuagcaauu uugggcaau uucuucugug cugaaugaua uucugucuag 3000 acuggauccu ccugaagcug aagugcagau ugauaagacug aucaggaa gaucgaguc 3060 ucugcagacu uaugugacac agcagcugau uagagcugcu gaauuagag cuucugcuaa 3120 ucuggcugcu aaaaaaugu cugaaugugu gcugggacag ucaaaaagag uggauuuug 3180 uggaaagga uaucaucuga ugucuuuuucc acagucugcu ccacauggag ugguguuuuu 3240 acaugugaca uaugugccag cacaggaaaa gaauuuuacc acagcaccag caauuuguga 3300 ugauggaaaa gcacauuuuuc caagagaagg aguguuguug ucuaauggaa cacauugguu 3360 ugugacacag agaaauuuuu augaaccuca gauuauuaca acagauaaua cauuuguguc 3420 aggaauugu gaugugguga uggaauugu gaauaauaca guguaugauc caucugcagcc 3480 agaacuggau ucuuuuaaag aagaacugga uaauauuuuu aaaaaucaca caucuccuga 3540 uguggauuua ggagaauuuuu cuggaaucaa ugcauucugug gugaauuc agaaagaaau 3600 ugauagacug aaugaagugg ccaaaaaucu gaaugaaucu cugauugauc ugcaggaacu 3660 uggaaau gaacaguaca uaaauggcc uuggguacauu uggcuuggau uuauugcagg 3720 auuaauugca auugugaugg ugacaauuau guuauguugu augacaucau guuguucuug 3780 uuuaaaagga uguuguucuu guggaaagcug uuguaauu gaugaagaug auucugaacc 3840 ugguuaaaa ggagugaaau ugcauuaac augaugacuc gagcugguac ugcaugcacg 3900 caugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc 3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc 4020 aagcacgcag caugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca 4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu 4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaaa aa 4282

[0274] RBL063.2 (SEQ ID NO:29, nucleotide; SEQ ID NO:9, amino acid) Structure: beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variants) SEQ ID NO: 29 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguucgu 60 guuccuggug cugcugccuc ugguguccag ccagugug aaccugacca ccagaacaca 120 gcugccucca gccuacacca acagcuuuac cagaggcgug uacuacccg acaagguguu 180 cagauccagc gugcugcacu cuacccagga ccuguuccug ccuuucuuca gcaacgugac 240 cugguuccac gccauccacg uguccggcac caauggcacc aagagauucg acaaccccgu 300 gguccccuuc aacgacgggg uguacguugc cagcaccgag aaguccaaa ucaucagagg 360 420 caccaacgug gucaucaaag ugugcgaguu ccaguucugc aacgacccu uccugggcgu 480 cuacuaccac aagaacaaca agagcuggau ggaaagcgag uuccgggugu acagcagcgc 540 caacaacugc accuucgagu acguuccca gccuuuccug auggaccugg aaggcaagca 600 gggcaacuuc aagaaccugc gcgaguucgu guuuaagaac aucgacggcu acuucaagau 660 cuacagcaag cacaccccua ucaaccucgu gcgggauucug ccucagggcu ucucugcucu 720 ggaacccccug guggauucugc ccaucggcau caacaucacc cgguuucaga cacugcuggc 780 840 900 caccaucacc gacgccgugg auugugcucu ggauccucug agcgagacaa agugcacccu 960 gaaguccuuc accguggaaa agggcaucua ccagaccagc aacuuccggg ugcagcccac 1020 cgaauccauc gugcgguucc ccaauaucac caaucugugc cccuucggcg agguguucaa 1080 ugccaccaga uucgccucug uguacgccug gaaccggaag cggaucagca auugcguggc 1140 cgacuacucc gugcuguaca acuccgccag cuucagcacc uucaagugcu acggcguguc 1200 cccuaccaag cugaacgacc ugugcuucac aaacguguac gccgacagcu ucgugauccg 1260 gggagaugaa gugcggcaga uugccccugg acagacaggc aagaucgccg acuacaacua 1320 caagcugccc gacgacuuca ccggcugugu gauugccugg aacagcaaca accuggacuc 1380 caaagucggc ggcaacuaca auuaccugua ccggcuguuc cggaagucca aucugaagcc 1440 cuucgagcgg gacaucucca ccgagaucua ucaggccggc agcaccccuu guaacggcgu 1500 ggaaggcuuc aacugcuacu ucccacugca guccuacggc uuucagccca caaauggcgu 1560 gggcuaucag cccuacagag ugguggugcu gagcuucgaa cugcugcaug ccccugccac 1620 agugugcggc ccuagaaaa gcaccaaucu cgugagaac aaugcguga acuucaucu 1680 caacggccug accggcaccg gcgugcugac agagagcaac aagaaguucc ugccaucca 1740 gcaguuuggc cgggauucg ccgauaccac agacgccguu agagaucccc agacacugga 1800 aauccuggac aucaccccuu gcagcuucgg cggagugucu gugaucuccc cuggcaccaa 1860 caccagcaau cagguggcag ugcuguacca ggacgugaac ugcccgag ugcccguggc 1920 CaucacGcc Gaucaugcuga Caucaug Gcggguac Caucaccggca Gcaugguu 1980 ucagaccaga gccggcuguc ugaucggagc cgagcacgug aacaauagcu acgagugcga 2040 cauccccauc ggcgcuggaa ucugcgccag cuaccagaca caucahaaca gcccucggag 2100 agccagaagc guggccagcc agccaucau ugccucaca augucucugg gcgccgagaa 2160 cagcguggcc uacuccaca acucuuucgc uauccccacc aacuucacca ucagcgugac 2220 cacagagauc cugccugugu cacaugaccaa gaccagcgug gacugcacca uguacaucug 2280 cggcgauucc accgagugcu gcugcaguac ggcagcuucu gcaccagcu 2340 gaauagagcc cugacaggga ucgccgugga acaggacaag aacacccaag agguguucgc 2400 ccaagugaag cagaucuaca agacccccucc uaucaaggac uucggcggcu ucaauuucag 2460 ccagauucug cccgauccua gcaagcccag caagcggagc uucaucgagg accugcuguu 2520 caacaaagug acacuggccg acgccggcuu caucaagcag uauggcgauu gucugggcga 2580 cauugccgcc agggaucuga uuugcgccca gaaguuuaac ggacugacag ugcugccucc 2640 ucugcugacc gaugagauga ucgcccagua cacaucgcc cugcuggccg gcacaaucac 2700 aagcggcugg acauuuggag caggcgccgc ucugcagauc cccuuugcua ugcagauggc 2760 cuaccgguuc aacggcaucg gagugaccca gaaugugcug uacgagaacc agaagcugau 2820 cgccaaccag uucaacagcg ccaucggcaa gauccaggac agccugagca gcacagcaag 2880 cgcccuggga aagcugcagg acguggucaa ccagaaugcc caggcacuga acacccuggu 2940 caagcagcug uccuccaacu ucggcgccau cagcucugug cugaacgaua uccugagcag 3000 acuggacccu ccugaggccg aggugcagau cgacagacug aucacaggca gacugcagag 3060 ccuccagaca uacgugaccc agcagcugau cagagccgcc gagauuagag ccucugccaa 3120 ucuggccgcc accaagaugu cugagugugu gcugggccag agcaagagag uggacuuuug 3180 cggcaagggc uaccaccuga ugagcuuccc ucagucugcc ccucacggcg ugguguuucu 3240 gcacgugaca uaugugcccg cucaagagaa gaauuucacc accgcuccag ccaucugcca 3300 cgacggcaaa gcccacuuuc cuagagaagg cguguucgug uccaacggca cccauugguu 3360 cgugacacag cggaacuucu acgagcccca gaucaucacc accgacaaca ccuucguguc 3420 uggcaacugc gacgucguga ucggcauugu gaacaauacc guguacgacc cucugcagcc 3480 cgagcuggac agcuucaaag aggaacugga caaguacuuu aagaaccaca caagccccga 3540 cguggaccug ggcgauauca gcggaaucaa ugccagcguc gugaacaucc agaaagagau 3600 cgaccggcug aacgaggugg ccaagaaucu gaacgagagc cugaucgacc ugcaagaacu 3660 ggggaaguac gagcaguaca ucaaguggcc cugguacauc uggcugggcu uuaucgccgg 3720 acugauugcc aucgugaugg ucacaaucau gcuguguugc augaccagcu gcuguagcug 3780 ccugaagggc uguuguagcu guggcagcug cugcaaguuc gacgaggacg auucugagcc 3840 cgugcugaag ggcgugaaac ugcacuacac augaugacuc gagcugguac ugcaugcacg 3900 caugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc 3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc 4020 aagcacgcag caugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca 4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu 4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaaa aa 4282

[0275] BNT162a1; RBL063.3 (SEQ ID NO: 30, nucleotide; SEQ ID NO: 21, amino acid) Structure: beta-S-ARCA(D1)-hAg-Kozak-RBD-GS-fibritin-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S protein) [partial sequence, receptor-binding domain (RBD) of S1S2 protein] SEQ ID NO: 30 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu 60 guuucuugug cugcugccuc uugugucuuc ucagugugug gugagauuuc caauauuuac 120 aaaucugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug uguaugcaug 180 240 uuuuuccaca uuuaaauuguu auggaguguc uccaacaaaa uuaaaugauu uuuuuuuac 300 aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga uugccccccgg 360 acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua caggaugugu 420 gauugcuugg aauucuaua auuauagauuc uaaaaguggga ggaaauuaca auuauucagua 480 540 ucaggcugga ucaacaccuu guaauggagu ggaaggauu aauuguuuau uuccauuaca 600 gagcuaugga uuucagccaa ccaauggugu gggauaucag ccaauauagag ugguggugcu 660 gucuuuugaa cugcugcaug caccugcaac aguguguga ccuaaaggcu cccccggcuc 720 cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu acguucguaa 780 agauggcgaa uggguauuac uuucuaccuu uuuaggccgg ucccuggagg ugcuguucca 840 gggccccggc ugaugacucg agcugguacu gcaugcacgc aaugcuagcu gccccuuucc 900 cguccugggu accccgaguc ucccccgacc ucggguccca gguaugcucc caccuccacc 960 ugccccacuc accaccug cuaguuccag acaccuccca agcacgcagc aaugcagcuc 1020 aaaacgcuua gccuagccac acccccacgg gaaacagcag ugauuaaccu uuagcaauaa 1080 acgaaaguuu aacuaagcua uacuaacccc aggguugguc aauuucgugc cagccacacc 1140 cuggagcuag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260 a 1261

[0276] BNT162b2; RBP020.1 (SEQ ID NO:31, nucleotide; SEQ ID NO:9, amino acid) structure m2 7,3’-O Gppp(m1 2’-O )ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variants) Sequence number 31 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugugu gaauuugaca acaagaacac 120 agcugccacc agcuuauaca aauucuuuua ccagaggagu guauuauccu gauaaagugu 180 uuagaucuuc ugugcugcac agcacacagg accuguuucu gccauuuuuu agcaauguga 240 caugguuuca ugcaauucau gugucuggaa caaauggaac aaaaagauuu gauaauccug 300 ugcugccuuu uaaugaugga guguauuuug cuucaacaga aaagucaaau auuauuagag 360 gauggauuuu uggaacaaca cuggauucua aaacacaguc ucugcugauu gugaauaaug 420 caacaaaugu ggugauuaaa gugugugaau uucaguuuug uaaugauccu uuucugggag 480 uguauuauca caaaaauaau aaaucuugga uggaaucuga auuuagagug uauuccucug 540 caaauaauug uacauuugaa uaugugucuc agccuuuucu gauggaucug gaaggaaaac 600 agggcaauuu uaaaaaucug agagaauuug uguuuaaaaa uauugaugga uauuuuaaaa 660 uuuauucuaa acacacacca auuaauuuag ugagagaucu gccucaggga uuuucugcuc 720 uggaaccucu gguggaucug ccaauuggca uaauauauac aagauuucag acacugcugg 780 840 cagcuuauua ugugggcuau cugcagccaa gaacauuucu gcugaaau aaugaaaaug 900 gaacaauuac agaugcugug gauugugcuc uggauuccucu gucugaaaca aaauguacau 960 uaaaaucuuu uacaguggaa aaaggcauuu aucagacauc uaauuuuaaga gugcagccaa 1020 cagaaucuau ugugaguuuu ccaauauua caaaucugug uccauuugga gaaguguuua 1080 augcaacaag auuugcaucu guguaugcau ggaauagaaa aagaauuucu aauugugg 1140 cugauuauuc ugugcuguau aauagugcuu cuuuuuccac auuuaaaugu uauggagugu 1200 cuccaacaaa auuaaaugau uuauguuuua caaugugua ugcugauucu uuugugauca 1260 gaggugauga agugagacag auugccccg gacagacagg aaaaauugcu gauuacaauu 1320 acaaacugcc ugaugauuuu acaggaugug ugauugcuug gaauucuaau aauuuagauu 1380 cuaaaguggg aggaauuac aauuaucugu acagacuguu uagaaaauca aaucugaaac 1440 cuuugaaag agaauuuca acagaauuu aucaggcugg aucacaccu uguaaoggag 1500 uuggaggau uaauuguuau uuuccauac agagcuaugg auuucagcca accaugggg 1560 ugggauauca gccauauaga guggugc ugucuuuuga acugcugcau gcaccugcau 1620 cagugoogg accuaaaaa ucuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaw 1680 uaaaoggau aaaaggaaca ggagugcuga cagaaucuaa uaaaaaauuu cugccuuuuc 1740 agcaguuugg cagagauauu gcagauacca cagaugcagu gagagauccu cagacauuag 1800 aaauucugga uauuacaccu uguucuuuug gggguguguc ugugauuaca ccuggaacaa 1860 auacaucuaa ucagguggcu gugcuguauc aggauguga uguacaga gugccagugg 1920 caaucaugc agaucagcug acacaucaucaucaucaucaucaugue 1980 oucagacaag aggagoug cugauuggag cagaauugu gaauaauucu oauugaougug 2040 auauuccaau uggagcaggc auuugugcau cuaucagac acagacaau ucccaagga 2100 2160 2160 auucuguggc auucucuaau aauucuauug cuauuccaac aaauuuuacc auuucuguga 2220 caacagaaau uuuaccugug ucuaugacaa aaacaucugu ggauuguacc auguacauu 2280 guggagauuc uacagaaugu ucuaaucugc ugcugcagua uggaucuuuu uguacacagc 2340 ugaauagagc uuuaacagga auugcugugg aacaggauaa aaaauacacag gaaguguuuug 2400 cucaggugaa acagauuac aaacaccac caauuaaaga uuuuggagga uuuaauuuua 2460 gccagauucu gccugauccu ucuaaaccuu cuaaaaagauc uuuuuugaa gaucugcugu 2520 uuauaaagu gacacuggca gaugcaggau uuauuaaaca guauggagau ugccugggug 2580 auugcugc aagagaucug auuugugcuc agaaauuuaa uggacugaca gugcugccuc 2640 cugcugac agaugaaaug auugcucagu acacaucugc uuuacuggcu ggaacaauua 2700 caagcggaug gacauuugga gcuggagcug cucugcagau uccuuuugca augcagaugg 2760 cuuacagauu uaauggaauu ggagugacac agaauguguu auugaaaau cagaaacuga 2820 uugcaaauca guuuauaucu gcaauuggca aaauucagga uucucugucu ucuacagcuu 2880 cugcucugggg aaaacugcag gaugugguga aucagaaugc acagcacug aauacucugg 2940 ugaacagcu gucuagcaau uuuggggcau uucucugu gcugaauugau auucugucua 3000 gacuggaacc uccugaagcu gaguagacu gaucaquaga agaquaga 3060 cucugcagac uuaugugaca cagcagcuga uuagagcugc ugaaauuaga gcucugcua 3120 aucuggcugc uacaaaaug ucugaaugug ugcugggaca whaaaaga guggauuuuu 3180 guggaaagg auaucaucug augucuuuuc cacagucugc uccacaugga gugguuuuu 3240 uacaugugac auaugugcca gcacaggaaa agauuuuac cacagcacca gcaauuuguc 3300 augauggaaa acacauuuu ccaagagaag gagugouugu accauugga acacauuggu 3360 uugugacaca gagaaauuuu uuugaaccuc agauuuuac acagauaau acauuugugu 3420 caggaaoug ugauggug auoggauug ugauaauac aguguaugau ccacugcagc 3480 cagaaacugga oocuuuuaaaaaaaaaaaaaaaaaaaaaaacucug 3540 auguggauu aggagaauu ucuggauca auugcaucugu ggugaauau cagaaagaaa 3600 uugauagacu gaaugaagug gccaaaaauc ugaaugaauc ucugauugau cugcaggaac 3660 uuggaaaaua ugaacaguac auuaaauggc cuugguacau uuggcuugga uuuauugcag 3720 gauuaauugc aauugugaug gugacaauua uguuauguug uaugacauca uguuguucuu 3780 guuuaaaagg auguuguucu uguggaagcu guuguaaauu ugaugaagau gauucugaac 3840 cuguguuaaa aggagugaaa uugcauuaca caugaugacu cgagcuggua cugcaugcac 3900 gcaaugcuag cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc 3960 cagguaugcu cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc 4020 caagcacgca gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc 4080 agugauuaac cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg 4140 ucaauuucgu gccagccaca cccuggagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aaagcauaug acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aaa 4283

[0277] RBP020.2 (SEQ ID NO: 10, nucleotide; SEQ ID NO: 9, amino acid) (see Table 1) structure m2 7,3’-O Gppp(m1 2’-O )ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variants)

[0278] BNT162b1; RBP020.3 (SEQ ID NO: 32; SEQ ID NO: 21, amino acid) structure m2 7,3’-O Gppp(m1 2’-O )ApG)-hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) [partial sequence, receptor binding domain (RBD) of S1S2 protein fused to fibritin] SEQ ID NO: 32 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugu gggagauuu ccaaauauua 120 caaaucugu uccauuugga gaaguguuua augcaacaag auuugcaucu guguaugcau 180 ggaauagaaa aagaauuucu aauugugggg cugauuauuc ugugcuguau aauagugcuu 240 cuuuuuccac auuuaaaugu uauggagugu cuccaacaaa auuaaaugau uuauguuuua 300 360 gacagacagg aaaaauugcu gauuacaauu acaaacugcc ugaugauuuu acaggaugug 420 ugauugcuug gaauucuaau aauuuagauu cuaaagugg aggaauuac aauuaucugu 480 acagacuguu uagaaaauca aaucugaaac cuuuugaaag agaauuuuca acagaaauuu 540 aucaggcugg aucaacaccu uguaauggag uggaaggau uaauuguuau uuuccauuac 600 agagcuaugg auuucagcca accaauggug ugggauauca gccaauaga gugguggugc 660 ugucuuuuga acugcugcau gcaccugcaa cagugugugg accuaaaggc ucccccggcu 720 ccggcuccgg aucugguuau auuccugaag cuccaagaga ugggcaagcu uacguucgua 780 aagauggcga auggguauua cuuucuaccu uuuaggccg gucccuggag gugcuguucc 840 agggccccgg cugaugacuc gagcuggac ugcaugcacg caaugcuagc ugccccuuuc 900 ccguccuggg uaccccgagu cucccccgac cucggguccc agguaugcuc ccaccuccac 960 1020 caaaacgcuu agccuagcca caccccacg ggaaacagca gugauuaacc uuuagcaaua 1080 aacgaaaguu uaacuaagcu auacuaaccc caggguuggu caauuucgug ccagccacac 1140 ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagcauauga cuaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260 aa 1262

[0279] RBS004.1 (SEQ ID NO: 33; SEQ ID NO: 9, amino acid) Structure: beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S protein) (S1S2 full-length protein, sequence variants) SEQ ID NO: 33 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 480 caauguccug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 780 accaucuacc acgaaaagag ggacuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcacag augucagugc ggacgacgcg aaaacugc ugguugggcu 1140 auagucguca acggucgcac ccagagaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggcccc aggcauugc uagguggggca aggaaua aggaagauca agagaugaa 1260 aggccacuag gacuacgaga uagacaguua guugggu guguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa caucauca agugacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaca cauggagau cgggcugaga 1440 acaagaauca ggaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag agcuaagug cgcagccgau gaggcuagg agggcgagg agccgagg 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agccucucu ggagccgau 1620 gucgacuuga uguacaaga ggcuggggcc ggcucagugg agaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccaccccuc ucgcugaaca agucauagug 1800 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 The 1980s 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug ccauauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuaa caugaugugc 2520 2580. cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2640. aaucugugac uucggucguc uuuacgacaa aaaaaugaga 2700. acgacgaauc cgaaagagac uagauugug auugacacua ccggcaguac caaaccuaag 2760. snow snow snow snow snow snow snow snow aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau gccguucggu acaaggugaa ugaaauccu cuguacgcac ccaccucaga acaugugaac snow cccgcacgga ggaccgcauc snow cacuagccgg cgacccaugg auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc auagacauga ccacugaaca auggacacu guggauuauu uugaaacgga caaagcucac 3240. ucagcagaga uaguauuga ccaacuaugc gugagguucu uuggacucga ucuggacucc ggucuauuuu cugcacccac uguuccguua uccauuagga auaauacug gguaaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacgu agaccuaug aaugaacac uggacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagaacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu aucacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 cgagauguag ccauauacug caggcaaag aaugggaaa ugacucucaa ggaagcagug 4500 ccuaggagag aagcagugga ggagauaugc auauccgacg aucuucagu gacagaaccu 4560 gaugcagagc uggugagggu caucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860 agaguacagc gccuaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4980. snowflake snowflake snowflake 4980. snowflake ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5100. cggagacuc cggagccauc ggcagagac caauccacag aggggacacc ugaacaacca ccacuuauaa ccgaggauga ccgaggauga agaacgccug agccgaucau caucgaagaa gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5340. uccgacuuug auguggacag uuuauccaua cuugacaccc uggggagc uagcgugacc agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucaggacacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuaggca uauuugaag ccagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 cgaguggaag ccuguaacgc cauguugaaa gagacuuuc cgacuguggc uucuuacuugu 6120 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacuc cuauuuugaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 cgucccacaa aaagaaauug caaugucacg caaugagagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauuugugu ucuggaaacu gacaucgcgu cguuguuaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggagaacu uaggugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 cgagcauuca uuggagauga caauacgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuau uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag accccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaucaaggu augaaaccgu aggaacuucc 7440 7500 ggggccccua uaacucucua cggcuaaccu gauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguuu cuugugcugc ugccuugu gucuucucag ugugugaauu 7620 ugacaacaag aacacagcug ccaccagcuu auacaaauuc uuuaaccaga ggaguguauu 7680 auccugauaa aguguuuaga ucuucugugc ugcacagcac acaggaccug uuucugccau 7740 uuuuuagcaa ugugacaugg uuucaugcaa uucauguguc uggaaacaaau ggaacaaaaa 7800 gauuugauaa uccugugcug ccuuuuaaug auggaguguua uuuugcuuca acagaaaagu 7860 caauauuau uagaggaugg auuuuuggaa caacacugga uucuaaaaca cagucucugc 7920 ugauugugaa uaaugcaaca aaugugguga uuaaagugug ugaauuuucag uuuuguaaug 7980 auccuuuuucu gggaguguau uaucacaaaa auaauaaauc uuggauggaa ucugaauuuua 8040 gaguguauuc cucugcaaau aauuguacau uugaauaugu gucucagccu uuucugaugg 8100 aucuggaagg aaaacagggc aauuuuaaaa aucugagaga auuuguuu aaaaaauauug 8160 auggauauuu uaaaauuuau ucuaaacaca caccaauuaa uuuaugaga gaucugccuc 8220 agggauuuuc ugcucuggaa ccucuggugg aucugccaau uggcauuaau auuacaagau 8280 uucagacacu gcuggcucug cacagaucuu aucugacacc uggagaaucu ucuucuggau 8340 ggacagccgg agcugcagcu uauuaugugg gcuaucugca gccaagaaca uuucugcuga 8400 aauaaauga aaauggaaca auuacagaug cuguggauug ugcucuggau ccucugucug 8460 aaacaaaaug uacauuaaaa ucuuuuacag uggaaaaagg cauuuaucag acaucuauau 8520 uuagagugca gccaacagaa ucuauuguga gauuuccaaa uauuacaaau cuguguccau 8580 uuggagaagu guuuaaugca acaagauuug caucugugua ugcauggaau agaaaaaagaa 8640 uuucuaauug uguggcugau uauucugugc uguauaauag ugcuucuuuu uccacauuua 8700 aauguuaugg agugucucca acaaaauuuaa augauuuaug uuuuacaaau guguaugcug 8760 aucuuuugu gaucagaggu gaugaaguga gacagauugc ccccggacag acaggaaaaa 8820 uugcugauua caauuacaaa cugccugaug auuuuacagg auguugugauu gcuuggaauu 8880 cuauauauuu agauucuaa gugggagaa auuacaauua ucuguacaga cuguuuagaa 8940 aaucaaaucu gaaaccuuuu gaagagaua uuucaacaga aauuuaucag gcuggaucaa 9000 caccuuguaa uggaguggaa ggauuuaauu guuauuuucc auuacagagc uauggauuuc 9060 agccaaccaa ugguguggga uaucagccau auagaguggu ggugcugucu uuugaacugc 9120 ugcaugcacc ugcaacagug ugggaccua aaaaucuac aaauuuagug aaaaauaaau 9180 gugugaauuu uaauuuuaau ggauuaacag gaacaggagu gcugacagaa ucuaauaaaa 9240 aauuucugcc uuuucagcag uuuggcagag auugcaga uaccacagau ccagugagag 9300 auccucagac auuagaaauu cuggauauua caccuuguuc uuuugggggu gugucuguga 9360 uuacaccugg aacaaauaca ucuaaucagg uggcugugcu guaucaggau gugaauugua 9420 cagaagugcc aguggcaauu caugcagauc agcugacacc aacaugga guguauucua 9480 caggauucaa uguguuucag acaagagcag gaugucugau uggagcaga caugugaaua 9540 9600 caaauucccc aaggagca aguaucugg caucucaguc uauuauugca uacaccaugu 9660 cucugggagc agaaaauucu guggcauauu cuauauauuc uauugcuauu ccaacaaauu 9720 uuaccauuuc ugugacaaca gaauuuuac cugugucuau uuaccauuuc ucuguggauu 9780 Guaccaugua Cauuugugga Gauucuacag Auguaugua Ucugcugcug Caguaugau 9840 cuuuuguac acagcugaau agagcuuaa caggaauugc uguggaacag gauaaaaaaaaa 9900 cacaggaagu guogcucag gugaaaaaaaaaaaaaaaaaaaaooug 9960 gaggauuuaa uuuuagccag auucugccug auccuucuaa accuuuuaa agaucuuuua 10020 uugagaucu gcuguuuaau aaagugacac uggcagaugc aggauuuuu aaacaguaug 10080 gagauugccu gggugauauu gcugcaagg aucugauuug ugcucagaa uuuaauggac 10140 ugacagugcu gccuccucug cugacagaug aaugauugc ucaguacaca ucugcuuuac 10200 uggcuggaac auuuacaagc ggauggacau uuggagcugg agcugcucug cagauuccuu 10260 uugcaugca gauggcuuac agauuuaaug gaauuggagu gacagaau guguauauug 10320 aaaaucagaa acugauugca auucaguua auucugcaau uggcaauu caggauucuc 10380 stir stir stir stir neighbors stir stir stir stir stir 10440 cacugaauac ucuggugaaa cagcugucua gcaauuuugg ggcaauuucu ucugugcuga 10500 augauauucu gucuagacug gauccuccug aagcugaagu gcagauugau agacugauca 10560 caggaagacu gcagucucug cagacuuaug ugacacagca gcugauuaga gcugcugaaa 10620 uuagagcuuc ugcuaaucug gcugcuacaa aaaugucuga augugugcug ggacagucaa 10680 aaagagugga uuuuugugga aaaggauauc aucugauguc uuuuccacag ucugcuccac 10740 auggaguggu guuuuuacau gugacauaug ugccagcaca ggaaaagaau uuuaccacag 10800 caccagcaau uugucaugau ggaaaagcac auuuuccaag agaaggagug uuugugucua 10860 auggaacaca uugguuugug acacagagaa auuuuuauga accucagauu auuacaacag 10920 auaauacauu ugugucagga aauugugaug uggugauugg aauugugaau aauacagugu 10980 augauccacu gcagccagaa cuggauucuu uuaaagaaga acuggauaaa uauuuuaaaa 11040 aucacacauc uccugaugug gauuuaggag auauuucugg aaucaaugca ucugugguga 11100 auauucagaa agaaougau agacugaaug agoggccaa aaucugaau gaocucuga 11160 uugaucugca ggaacuugga aaauauugaac aguacauuaa auggccuugg uacauuuggc 11220 Oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo Goodooooo 11280 caucaugoog ooooooooa nooooooooooooooooooooooooooooooooooughe 11340 aagaugauuc ugaaccugug uaaaaggg uuacacauca ugacucgagc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccccucc accucuccua 11520 guuccagaca ccuccaagc acgcagcaau gcagcucaa acgcuuagcc uagccacacc 11580 cccacgggaa acaccaguga uaaccuuua gcauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa auuuuuuauu 11760 uuuuuuuuuuuuuuuuuuuuuuuuuuuaaaaaaaaaaaaaaaaaaaa 11820 aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 11917

[0280] RBS004.2 (SEQ ID NO: 34; SEQ ID NO: 9, amino acid) Structure: beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S protein) (S1S2 full-length protein, sequence variants) SEQ ID NO: 34 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 480 caauguccug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 780 accaucuacc acgaaaagag ggacuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auagucguca acggucgcac ccagagaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggcccc aggcauugc uagguggggca aggaaua aggaagauca agagaugaa 1260 aggccacuag gacuacgaga uagacaguua guugggu guguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa caucauca agugacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaca cauggagau cgggcugaga 1440 acaagaauca ggaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag agcuaagug cgcagccgau gaggcuagg agggcgagg agccgagg 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agccucucu ggagccgau 1620 gucgacuuga uguacaaga ggcuggggcc ggcucagugg agaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccaccccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaa agggcguauau gccguggaac cauaccaugg uaaguaguug 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 The 1980s 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug ccauauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacgu agaccuaug aaugaacac uggacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagaacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu aucacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 cgagauguag ccauauacug caggcaaag aaugggaaa ugacucucaa ggaagcagug 4500 ccuaggagag aagcagugga ggagauaugc auauccgacg aucuucagu gacagaaccu 4560 gaugcagagc uggugagggu caucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860 agaguacagc gccuaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4980. snowflake snowflake snowflake 4980. snowflake ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5100. cggagacuc cggagccauc ggcagagac caauccacag aggggacacc ugaacaacca ccacuuauaa ccgaggauga ccgaggauga agaacgccug agccgaucau caucgaagaa gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5340. uccgacuuug auguggacag uuuauccaua cuugacaccc uggggagc uagcgugacc agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5460. cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucaggacacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuaggca uauuugaag ccagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 cgaguggaag ccuguaacgc cauguugaaa gagacuuuc cgacuguggc uucuuacuugu 6120 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacuc cuauuuugaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 cgucccacaa aaagaaauug caaugucacg caaugagagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauuugugu ucuggaaacu gacaucgcgu cguuguuaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggagaacu uaggugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 cgagcauuca uuggagauga caauacgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuau uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag accccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaucaaggu augaaaccgu aggaacuucc 7440 7500 ggggccccua uaacucucua cggcuaaccu gauggacua cgacauaguc uaguccgcca 7560 agacuaguau guucguguuc cuggugcugc ugccucuggu guccagccag ugugugaacc 7620 ugaccaccag aacacagcug ccuccagccu acaccaacag cuuuaccaga ggcguguacu 7680 accccgacaa gguguucaga uccagcgugc ugcacucuac ccaggaccug uuccugccuu 7740 ucuucagcaa cgugaccugg uuccacgcca uccacguguc cggcaccaau ggcaccaaga 7800 gauucgacaa ccccgugcug cccuucaacg acggggugua cuuugccagc accgagaagu 7860 ccaacaucau cagaggcugg aucuuccggca ccacacugga cagcaagacc cagagccugc 7920 ugaucgugaa caacgccacc aacgugguca ucaaagugug cgaguuccag uucugcaacg 7980 acccccuuccu gggcgucuac uaccacaaga acaacaagag cuggauggaa agcgaguucc 8040 ggggugacag cagcgccaac aacugcaccu ucgaguaggu gucccagccu uuccugaugg 8100 accuggaagg caaccaggc aacuucaaga accugcgcga guucguuu aagaacaucg 8160 8220 agggcuucuc ugcucuggaa ccccuggugg aucugcccau cggcaucaac aucacccggu 8280 uucagacacu gcuggcccug cagaagcu accugacacc uggcgauagc agcagcggau 8340 ggacagcugg ugccgccgcu uacuaugugg gcuaccugca gccuagaacc uuccugcuga 8400 aguacaacga gaacggcacc aucaccgacg ccguggauug ugcucuggau ccucugagcg 8460 agacaaagug cacccugaag uccuucaccg uggaaaaggg caucuaccag accagcaacu 8520 uccgggugca gcccaccgaa uccaucgugc gguuccccaa uaucaccaau cugugccccu 8580 ucggcgaggu guucaaugcc accagauucg ccucugugua cgccuggaac cggaagcgga 8640 ucagcaauug cguggccgac uacuccgugc uguacaacuc cgccagcuuc agcaccuuca 8700 agugcuacgg cguguccccu accaagcuga acgaccugug cuucacaaac guguacgccg 8760 acagcuucgu gauccgggga gaugaagugc ggcagauugc cccuggacag acaggcaaga 8820 ucgccgacua caacuacaag cugcccgacg acuucaccgg cugugugauu gccuggaaca 8880 gcaacaaccu ggacuccaaa gucggcggca acuacaauua ccuguaccgg cuguuccgga 8940 aguccaaucu gaagcccuuc gagcgggaca ucuccaccga gaucuaucag gccggcagca 9000 ccccuuguaa cggcguggaa ggcuucaacu gcuacuuccc acugcagucc uacggcuuuc 9060 agcccaaaa uggcgugggc uaucagcccu acagaguggu ggugcugagc uucgaacugc 9120 ugcaugcccc ugccacagug ugcggcccua agaaaagcac caaucucgug aagaacaaau 9180 gcgugaacuu caacuucaac ggccugaccg gcaccgggu gcugacagag agcaacaaga 9240 aguucgcc auuccagcag uuuggccggg auucgccga uaccacagac gccguuagag 9300 auccccagac acuggaauc cuggacauca ccccuugcag cuucggcgga gugucuguga 9360 ucaccccugg caccaacacc agcaaucagg uggcagugcu guaccaggac gugaacagua 9420 ccgaagugcc cguggccauu cacgccgauc agcugacacc uacauggcgg guguacucca 9480 ccggcagcaa uguguuucag accagagccg gcugucugau cggagccgag cacgugaaca 9540 9600 caaacagccc ucggagagcc agaagcgugg ccagccagag caucauugcc uacacaaugu 9660 cucugggcgc cgagaacagc guggccuacu ccaacaacuc uaucgcuauc cccaccaacu 9720 ucaccaucag cgugaccaca gagauccugc cuguguccau gaccaagacc agcguggacu 9780 9840 gcuucugcac ccagcugaau agagccuga cagggaucgc cguggaacag gacaagaaca 9900 cccaagaggu guucgcccaa gugaagcaga ucuacaagac cccuccuauc aaagcuucg 9960 gcggcuucaa uuucagccag auucugcccg auccuagcaa gcccagcaag cggagcuuca 10020 ucgaggaccu cguguucaac aaagugacac uggccgacgc cggcuucauc aagcaguaug 10080 gcgauugucu gggcgacauu gccgccaggg aucugauuug cgcccagaag uuuaacggac 10140 ugacagugcu gccuccucug cugaccgaug agaugaucgc ccaguacaca ucugcccugc 10200 uggccggcac aaucacaagc ggcuggacau uuggagcagg cgccgcucug cagaucccu 10260 10320 agaaccagaa gcugaucgcc aaccaguuca acagcgccau cggcaagauc caggacagcc 10380 ugagcagcac agcaagcgcc cugggaaagc ugcaggacgu ggucaaccag aaugcccagg 10440 cacugaacac ccuggucaag cagcuguccu ccaacuucgg cgccaucagc ucugugcuga 10500 10560 10620 uuagagccuc ugccaaucug gccgccacca agaugucuga gugugcug ggccagagca 10680 agagugga cuuuugcggc aagggcuacc accugaugag cuucccucag ucugccccuc 10740 acggcgguggu guuucugcac gugacauaug ugcccgcuca agagaagaau uucaccaccg 10800 cuccagccau cugccacgac ggcaaagccc acuuuccuag agaaggcgug uucgugucca 10860 acggcaccca uuggucggag acaggacgga acucuacga gccccagauc aucaccaccg 10920 acaacaccuu cgugucuggc aacugcgacg ucgugaucgg cauugugaac aauaccgugu 10980 acgacccucu gcagcccgag cuggacagcu ucaaagagga acuggacaag uacuuuuaaga 11040 accacacaag ccccgacgug gaccugggcg auaucagcgg aaucaaugcc agcgucguga 11100 acauccagaa agaaucgac cggcugaacg agguggccaa gaucugaac gagagccuga 11160 ucgaccugca agacugggg aaguacgagc aguacauca guggcccugg uacaucuggc 11220 ugggcuuuau cgccggacug auugccaucg ugauggucac auucaugcug uguugcauga 11280 ccagcugcug uagcugccug aagggcuguu guagcugugg cagcugcugc aaguucgacg 11340 aggacgauuc ogagcccgug cugaagggcg weather ogaacugc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccccucc accucuccua 11520 guuccagaca ccuccaagc acgcagcaau gcagcucaa acgcuuagcc uagccacacc 11580 cccacgggaa acaccaguga uaaccuuua gcauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa auuuuuuauu 11760 uuuuuuuuuuuuuuuuuuuuuuuuuuuaaaaaaaaaaaaaaaaaaaa 11820 Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 11917

[0281] BNT162c1; RBS004.3 (SEQ ID NO: 35; SEQ ID NO: 21, amino acid) Structure: beta-S-ARCA(D1)-replicase-RBD-GS-fibritin-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S protein) [partial sequence, receptor-binding domain (RBD) of S1S2 protein] SEQ ID NO: 35 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480 caauguccug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 780 accaucuacc acgaaaagag ggacuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auaagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggcccc aggcauugc uagguggggca aggaaua aggaagauca agagaugaa 1260 aggccacuag gacuacgaga uagacaguua guugggu guguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa caucauca agugacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaca cauggagau cgggcugaga 1440 acaagaauca ggaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag agcuaagug cgcagccgau gaggcuagg agggcgagg agccgagg 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agccucucu ggagccgau 1620 gucgacuuga uguacaaga ggcuggggcc ggcucagugg agaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccaccccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaa agggcguauau gccguggaac cauaccaugg uaaguaguug 1860 gugccagagg gauggcau acccguccag gacuucaug cucugaguga aagugccacc 1920s The 1980s 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug ccauauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 2700. acgacgaauc cgaaagagac uagauugug auugacacua ccggcaguac caaaccuaag 2760. snow snow snow snow snow snow snow snow aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau gccguucggu acaaggugaa ugaaauccu cuguacgcac ccaccucaga acaugugaac snow cccgcacgga ggaccgcauc snow cacuagccgg cgacccaugg auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc auagacauga ccacugaaca auggacacu guggauuauu uugaaacgga caaagcucac 3240. ucagcagaga uaguauuga ccaacuaugc gugagguucu uuggacucga ucuggacucc ggucuauuuu cugcacccac cover uccauuagga auaaucacug ggauaacucc ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac ccacaacugc cucgggcagu ugccacgu agaccuaug aaugaacac uggacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagaacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu aucacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 cgagauguag ccauauacug caggcaaag aaugggaaa ugacucucaa ggaagcagug 4500 ccuaggagag aagcagugga ggagauaugc auauccgacg aucuucagu gacagaaccu 4560 gaugcagagc uggugagggu caucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 4800 4860. 4860. 4860. 4860. 4860. 4860. 4860. 4860 agaguacagc gccuaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4980. snowflake snowflake snowflake 4980. snowflake ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5100. cggagacuc cggagccauc ggcagagac caauccacag aggggacacc ugaacaacca ccacuuauaa ccgaggauga ccgaggauga agaacgccug agccgaucau caucgaagaa gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5340. uccgacuuug auguggacag uuuauccaua cuugacaccc uggggagc uagcgugacc agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5460. cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucaggacacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuaggca uauuugaag ccagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 cgaguggaag ccuguaacgc cauguugaaa gagacuuuc cgacuguggc uucuuacuugu 6120 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacuc cuauuuugaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 cgucccacaa aaagaaauug caaugucacg caaugagagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauuugugu ucuggaaacu gacaucgcgu cguuguuaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggagaacu uaggugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 cgagcauuca uuggagauga caauacgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuau uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag accccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaucaaggu augaaaccgu aggaacuucc 7440 7500 ggggccccua uaacucucua cggcuaaccu gauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguuu cuugugcugc ugccuugu gucuucucag ugugugguga 7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug 7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc 7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa 7800 aauuuuaug uuuuacaaau guguaugcug auuuuuuugu gaucagaggu gaugaaguga 7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug 7920 auuuuacagg augugugauu gcuuggaauu cuauauauuu agauucuaaa guggagaa 7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaagagaua 8040 uuucaacaga aauuuaucag cuggaguacaa caccuuguaa uggaguggaa ggauuuaauu 8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau 8160 8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agaaggggc 8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggccgguccc 8340 uggaggugcu guuccagggc cccggcugau gacucgagcu gguacugcau gcagcaaug 8400 cuagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua 8460 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca 8520 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau 8580 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu 8640 ucgugccagc cacaccgcgg ccgcaugaau acagcagcaa uuggcaagcu gcuuacauag 8700 aacucgcggc gauuggcaug ccgccuuaaa auuuuuauuu uauuuuuuucu uuucuuuucc 8760 gaucggauu uuguuuuuaa uauuucaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcau 8820 augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 8880 aaaaaaaaaa aaaaaa 8896

[0282] RBS004.4 (SEQ ID NO: 36; SEQ ID NO: 37) Structure: beta-S-ARCA(D1)-replicase-RBD-GS-fibritin-TM-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S protein) [partial sequence, receptor-binding domain (RBD) of S1S2 protein] SEQ ID NO: 36 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 120 gagguaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 300 ugugcggaag aucccggacag auuguuaaag uaugcaacua agcugaagaa aaacuguaag 360 gaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 480 caauguccug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 780 accaucuacc acgaaaagag ggacuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auaagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggccc aggcauuuugc uaggugggca aaggaauaua aggagauca agaagaugaa 1260 aggccacuag gacuacgaga uagacaguua gucaugggu guuguugggc uuuuagaagg 1320 caaagauaa caucuauuua uaagcgccccg gauacccaaa ccaucaucaa agugaacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440 acaagaauca ggaaaauguu agaggagcac aaggaggcu caccucucau uaccgccgag 1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1680. 1680. 1680. 1680. 1680. 1680. 1680. 1680. 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc auuguguaca acgaacguga guucguaaac average accauauugc cacacaugga ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2100. the sword of the sword gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga 2220. acacgaccag ccgcuccuua ccaguacca accauagggg uguauggcgu gccaggauca ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag aaagaaaacu gugcagaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2460. auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc 2580. cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2640. aaucugugac uucggucguc uuuacgacaa aaaaaugaga 2700. acgacgaauc cgaaagagac uagauugug auugacacua ccggcaguac caaaccuaag 2760. snow snow snow snow snow snow snow snow aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau gccguucggu acaaggugaa ugaaauccu cuguacgcac ccaccucaga acaugugaac snow cccgcacgga ggaccgcauc snow cacuagccgg cgacccaugg auaaaaacac ugacugccaa guaccuggg aauuucacug ccacgauaga ggaguggcaa 3000 3060 cagaauaagg caaacgug uugggccaag gcuuuagugc cggugcuga gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuuuu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaauacug gguaaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacgu agaccuaug aaugaacac uggacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagaacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu aucacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu 4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100 ccacuuauaa ccgagauga gaccaggacu agaacgccug agccgaucau caucgaagaa 5160 gaagaaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280 uccgacuuug augggacag uuuauccaua cuugacacc uggagggagc uagcgugacc 5340 5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucaggacacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuaggca uauuugaag ccagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 cgaguggaag ccuguaacgc cauguugaaa gagacuuuc cgacuguggc uucuuacuugu 6120 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacuc cuauuuugaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 cgucccacaa aaagaaauug caaugucacg caaugagagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauuugugu ucuggaaacu gacaucgcgu cguuguuaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggagaacu uaggugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 cgagcauuca uuggagauga caauacgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuau uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag accccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaucaaggu augaaaccgu aggaacuucc 7440 7500 ggggccccua uaacucucua cggcuaaccu gauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguuu cuugugcugc ugccuugu gucuucucag ugugugguga 7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug 7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc 7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa 7800 aauuuuaug uuuuacaaau guguaugcug auuuuuuugu gaucagaggu gaugaaguga 7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug 7920 auuuuacagg augugugauu gcuuggaauu cuauauauuu agauucuaaa guggagaa 7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaagagaua 8040 uuucaacaga aauuuaucag cuggaguacaa caccuuguaa uggaguggaa ggauuuaauu 8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau 8160 8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agaaggggc 8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggaagcggca 8340 gcggaucuga acaguacauu aaauggccuu gguacauuug gcuuggauuu auugcaggau 8400 uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu uguucuguu 8460 uaaaaggaug uugucuugu ggaagcuguu guaauuuga ugaagaugau ucugaaccug 8520 uguuaaaagg agugaaauug cauacacau gaugacucga gcugguacug caugcacgca 8580 augcuagcug ccccuuuccc guccugggua ccccgagucu cccccgaccu cgggucccag 8640 guaugcuccc accucccaccu gccccacuca ccaccucugc uaguuccaga caccucccaa 8700 gcacgcagca augcagcuca aaacgcuuag ccuagccaca cccccacggg aaacagcagu 8760 gauuaaccuu uagcaauaaa cgaaaguuua acuaagcuau acuaacccca ggguugguca 8820 auuucgugcc agccacaccg cggccgcaug aauacagcag caauuggcaa gcugcuuaca 8880 uagaacucgc ggcgauuggc augccgccuu aaaauuuuua uuuuauuuuu ucuuuucuuu 8940 uccgaaucgg auuuuguuuu uaauauuuca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 9000 cauaugacua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060 aaaaaaaaaa aaaaaaaaa 9079 SEQ ID NO: 37 [ka] [ka]

[0283] BNT162b3c (SEQ ID NO: 38; SEQ ID NO: 39) structure m2 7,3’-O Gppp(m1 2’-O )ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) [partial sequence, receptor binding domain (RBD) of S1S2 protein fused to fibritin fused to transmembrane domain (TM) of S1S2 protein]; unique S1S2 protein secretory signal peptide (aa 1-19) at the N-terminus of the antigen sequence SEQ ID NO: 38 [ka] [ka] [ka] SEQ ID NO: 39 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugu gaauuugaca gugagauuuc 120 caaauauuac aaaucugugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug 180 uguaugcaug gaauagaaaa agaauuucua auguguggc ugauuauucu gugcuguaua 240 auagugcuuc uuuuuccaca uuuaaauguu auggaguguc uccaacaaaa uuaaaugauu 300 uauguuuuac aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga 360 uugcccccgg acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua 420 caggaugugu gauugcuugg aauucaaua auuuagauuc uaaaguggga ggaaauuaca 480 auuaucugua cagacuguuu agaaaaucaa aucugaaacc uuuugaaaga gauauuucaa 540 cagaaauuua ucaggcugga ucaacaccuu guaauggagu ggaaggauu aauuguuuau 600 uuccauuaca gagcuaugga uuucagccaa ccaauggugu gggauaucag ccauauagag 660 ugguggugcu gucuuuugaa cugcugcaug caccugcaac agugugugga ccuaaaggcu 720 cccccggcuc cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu 780 acguucguaa agauggcgaa uggguauuac uuucuaccuu uuuaggaagc ggcagcggau 840 cugaacagua cauuaaugg ccuugguaca uuuggcuugg auuuauugca ggauuaauug 900 caauugugau ggugacaauu auguuauguu guaugacauc auguuguucu uguuuaaaag 960 gauguuguuc uuguggaagc uguuguuguau gacucgagcu gguacugcau gcacgcaaug 1020 guagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua 1080 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca 1140 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau 1200 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu 1260 ucgugccagc cacacccugg agcuagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca 1320 uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaa 1397

[0284] BNT162b3d (SEQ ID NO: 40; SEQ ID NO: 41) structure m2 7,3’-O Gppp(m1 2’-O )ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encoded antigen: SARS-CoV-2 viral spike protein (S1S2 protein) [partial sequence, receptor binding domain (RBD) of S1S2 protein fused to fibritin fused to transmembrane domain (TM) of S1S2 protein]; immunoglobulin secretory signal peptide (aa 1-22) at the N-terminus of the antigen sequence SEQ ID NO: 40 [ka] [ka] SEQ ID NO: 41 agaauaaacu aguauucuuc uggucccac agacucagag agaacccgcc accauggauu 60 ggauuuggag aauccuguuc cucgugggag ccgcuacagg agcccacucc cagaugcagg 120 ugagauuucc aaauauuaca aaucuguguc cauuuggaga aguguuuaau gcaacaagau 180 uugcaucugu guaugcaugg aauagaaaaa gaauuucuaa uuguguggcu gauuauucug 240 ugcuguauaa uagcuucu uuuuccacau uaaauguua uggagugucu ccaaaaaau 300 uaaougaooo aoooooaaoooooug cugauocuuu ogogaucaga ggugaooag 360 ugagacagau ugcccccgga cagacaggaa aaauugcuga uaacaauuac aaacugccug 420 augauuuuac aggaugougg auugcuugga auuuuuuaa uuuagauucu aaaogggag 480 gaaauuaca uuaucuguac agacuguuua gaaaucaa ucugaaaccu uuugaagag 540 auauuucaac agaauuuau caggcuggau cacaccuug uaauggagug gaggauua 600 auuguuauuu uccauacag agcuauggau ucagccac caauggug ggauaucagc 660 cauauagagu guggugcug ucuuuugaac ugcugcaugc accugcaca guguguggac 720 cuaaaggcuc ccccggcucc ggcuccggau cugguuauu cuccugaagcu ccagagaug 780 ggcaagcuua cguucguaa gauggcgaau ggguauuacu uucuaccuuu uuaggaagcg 840 gcagcggauc ugacaguac auuaaauggc cugguacau uugcuugga uuuauugcag 900 gauuaauugc auugugaug gugacaua uguuauguug uuugacauca uguugucuu 960 guuuaaaagg auguuguucu uguggaagcu guuguugaug acucgagcug guacugcaug 1020 cacgcaaugc uagcugcccc uuucccgucc uggguacccc gagucucccc cgaccucggg 1080 ucccagguau gcucccaccu ccaccugccc cacucaccac cucugcuagu uccagacacc 1140 ucccaagcac gcagcaaugc agcucaaaac gcuuagccua gccacaccccc cacgggaaac 1200 agcagugauu aaccuuuagc aauaaacgaa aguuuaacua agcuauacua accccagggu 1260 uggucaauuu cgugccagcc acacccugga gcuagcaaaa aaaaaaaaaa aaaaaaaaaa 1320 aaaaaagcau augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaaaaa aaaaaa 1406

[0285] Nucleic acid containing particles The nucleic acids described herein, such as RNA encoding a payload, may be formulated and administered as particles.

[0286] In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or molecular complex. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, for example, a micro- or nano-sized compact structure dispersed in a medium. In some embodiments, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

[0287] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to complex formation and spontaneous formation of nucleic acid particles. In some embodiments, the nucleic acid particles are nanoparticles.

[0288] As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

[0289] "Nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionic lipid or lipid-like material, at least one cationic polymer, such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based formulations and lipoplex (LPX)-based formulations.

[0290] Without intending to be bound by any theory, it is believed that cationic or cationically ionic lipids or lipid-like materials, and / or cationic polymers combine with nucleic acids to form aggregates, which aggregates result in colloidally stable particles.

[0291] In some embodiments, the particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionic lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

[0292] In some embodiments, a nucleic acid particle comprises multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules may be similar or different from each other with respect to molar mass or basic structural elements, such as molecular architecture, capping, coding regions, or other features. In some embodiments, the nucleic acid particles described herein may have an average diameter ranging from about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0293] The nucleic acid particles described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3, or about 0.2 to about 0.3.

[0294] For RNA-lipid particles, the N / P ratio indicates the ratio of nitrogen groups in the lipid to the number of phosphate groups in the RNA. This correlates with the charge ratio, since nitrogen atoms (depending on pH) are usually positively charged and phosphate groups are negatively charged. The N / P ratio is determined by pH when charge balance exists. Lipid formulations are frequently formed with N / P ratios greater than 4, up to 12, because positively charged nanoparticles are considered suitable for transfection. In that case, the RNA is considered fully bound to the nanoparticles.

[0295] The nucleic acid particles described herein can be prepared using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationically ionic lipid or lipid-like material, and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain the nucleic acid particles.

[0296] The term "colloid" as used herein refers to a homogeneous mixture of dispersed particles that do not settle. The insoluble particles in the mixture are fine, ranging in size from 1 to 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0297] For the preparation of colloids comprising at least one cationic or cationically ionic lipid or lipid-like material and / or at least one cationic polymer, methods conventionally used and appropriately adapted for preparing liposome-type vesicles are applicable herein. The most commonly used methods for preparing liposome-type vesicles share the following basic steps: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0298] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to produce a liposomal dispersion. An additional downsizing step may also be included.

[0299] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposomal vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A short sonication of this mixture is required for homogenization. Removal of the organic phase under reduced pressure results in a milky gel, which then transforms into a liposomal suspension.

[0300] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action distributes the lipids throughout the solution, promoting lipid structure formation, such as lipid vesicle formation, e.g., liposome formation. Typically, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in some embodiments as follows: an ethanol solution containing lipids, e.g., cationic lipids and additional lipids, is injected into a stirred aqueous solution. In some embodiments, the RNA lipoplex particles described herein can be obtained without an extrusion step.

[0301] The term "extrusion" or "extrusion" refers to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles by forcing them through a filter with defined pores.

[0302] Other methods having organic solvent-free characteristics may also be used in accordance with the present disclosure to prepare colloids.

[0303] LNPs typically contain four components: ionic cationic lipids, neutral lipids, such as phospholipids, steroids, such as cholesterol, and polymer-conjugated lipids, such as polyethylene glycol (PEG) lipids. Each component is responsible for payload protection and enables effective intracellular delivery. LNPs can be prepared by mixing lipids rapidly dissolved in ethanol with nucleic acid in an aqueous buffer.

[0304] The term "average diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) through data analysis using the so-called cumulant algorithm, which results in the so-called Zaverage, which is a linear dimension, and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the terms "average diameter", "diameter", or "size" of particles are used interchangeably with the value of Zaverage.

[0305] The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of "mean diameter", and can be taken as a measure of the size distribution of an ensemble of nanoparticles under certain prerequisites.

[0306] Various types of nucleic acid-containing particles have previously been described as suitable for delivering nucleic acids in the form of microparticles (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0307] The present disclosure describes particles containing nucleic acids, at least one cationic or cationically ionic lipid or lipid-like material, and / or at least one cationic polymer that associate with nucleic acids to form nucleic acid particles, and compositions containing such particles. The nucleic acid particles may contain nucleic acids complexed to the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they are unable to infect cells. Suitable cationic or cationically ionic lipid or lipid-like materials and cationic polymers that form nucleic acid particles are encompassed by the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with nucleic acids to form nucleic acid particles. Such components include any component that can be part of a nucleic acid particle.

[0308] Some embodiments described herein relate to compositions, methods, and uses that include more than one nucleic acid species, e.g., RNA species, such as: a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parent viral protein, wherein amino acid positions within at least the fragment of the parent viral protein have been modified to include amino acids found within corresponding amino acid positions in one or more viral protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parent viral protein, wherein amino acid positions within at least the fragment of the parent viral protein have been modified to include amino acids found within corresponding amino acid positions in one or more viral protein variants.

[0309] In microparticle preparations, it is possible that each nucleic acid species is separately formulated as an individual microparticle preparation.In this case, each individual microparticle preparation will contain one nucleic acid species.Individual microparticle preparations can exist as separate entities, for example, in separate containers.Such preparations can be obtained by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent that allows particles to form.Each particle will exclusively contain the specific nucleic acid species provided when the particle is formed (individual microparticle preparation).

[0310] In some embodiments, a composition, for example, a pharmaceutical composition, comprises a plurality of individual particle formulations. Each pharmaceutical composition is referred to as a mixed microparticle formulation. The mixed microparticle formulation according to the present invention can be obtained by mixing the individual microparticle formulations after forming them separately as described above. By mixing, a formulation comprising a mixed population of nucleic acid-containing particles can be obtained. The individual microparticle populations can be combined in one container to comprise a mixed population of individual microparticle formulations.

[0311] Alternatively, various nucleic acid species can be formulated together as a combined microparticle preparation.This preparation can be obtained by providing the combined preparation (typically a combined solution) of various RNA species together with the particle forming agent that allows particle formation.In contrast to mixed microparticle preparation, combined microparticle preparation typically comprises particles that contain multiple RNA species.In combined microparticle composition, various RNA species typically exist together in a single particle.

[0312] Cationic polymeric materials (e.g., polymers) Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically compress negatively charged nucleic acids into nanoparticles. The positively charged groups often consist of amines that change protonation state within the pH range of 5.5 to 7.5, which is thought to lead to an ionic imbalance that ultimately results in endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been used for nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have been widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

[0313] As used herein, the term "polymeric material" refers to its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some embodiments, all such repeating units may be identical; otherwise, in some cases, there may be more than one type of repeating unit present within the polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties may also be present within the polymeric material, e.g., a targeting moiety such as those described herein.

[0314] Those skilled in the art will recognize that when more than one type of repeat unit is present within a polymer (or polymer portion), the polymer (or polymer portion) is said to be a "copolymer." In some embodiments, a polymer (or polymer portion) utilized in accordance with the present disclosure may be a copolymer. The repeat units forming the copolymer can be arranged in any manner. For example, in some embodiments, the repeat units can be arranged in a random order; alternatively, or in addition, in some embodiments, the repeat units can be arranged in alternating order or as a "block" copolymer (i.e., comprising one or more regions (e.g., a first block) each comprising a first repeat unit, and one or more regions (e.g., a second block) each comprising a second repeat unit, etc.). A block copolymer can have two (a diblock copolymer), three (a triblock copolymer), or more different blocks.

[0315] In certain embodiments, the polymeric materials used in accordance with the present disclosure are biocompatible. Biocompatible materials are those that typically do not cause significant cell death at reasonable concentrations. In certain embodiments, biocompatible materials are biodegradable, i.e., capable of chemically and / or biologically breaking down within a physiological environment, e.g., within the body.

[0316] In certain embodiments, the polymeric material may be or include protamine or a polyalkyleneimine, particularly protamine.

[0317] Those skilled in the art will recognize that the term "protamine" is often used to refer to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) to associate specifically with DNA in place of somatic histones. In particular, the term "protamine" is often used to refer to a protein found in fish sperm that is strongly basic, soluble in water, heat-resistant, and hydrolyzes to yield primarily arginine. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effects of heparin.

[0318] In some embodiments, the term "protamine" as used herein refers to a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and / or multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from a natural or biological source.

[0319] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In some embodiments, a preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75-10 to 10 Da, preferably 1,000 to 10 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0320] Preferred according to certain embodiments of the present disclosure are linear polyalkyleneimines, such as linear polyethyleneimine (PEI).

[0321] Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those that can electrostatically bind to nucleic acids. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric material with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0322] In some embodiments, the particles described herein may include polymers other than cationic polymers, such as non-cationic and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

[0323] Lipids and lipid-like materials The terms "lipid" and "lipid-like material" are used herein to refer to molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are frequently referred to as amphiphiles. Lipids are typically not sufficiently soluble in water. In an aqueous environment, their amphiphilic nature allows them to self-assemble into organized structures and distinct phases. One such phase consists of a lipid bilayer that resides intact within a vesicle, multilamellar / unilamellar liposome, or membrane in an aqueous environment. Hydrophobicity can be imparted by the inclusion of apolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. In some embodiments, the hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other such groups.

[0324] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Additionally, the polar portion may have a formal positive or negative charge. Alternatively, the polar portion may have both a formal positive and negative charge, or may be a zwitterion or an inner salt. For purposes of this disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0325] The terms "lipid-like material," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, the terms include compounds that can form vesicles, multilamellar / unilamellar liposomes, or amphiphilic layers that reside intact within membranes in an aqueous environment, and include surfactants or synthetic compounds that have both hydrophilic and hydrophobic moieties. Generally speaking, the terms refer to molecules that contain hydrophilic and hydrophobic moieties with different structural organization that may or may not resemble that of lipids. The term "lipid," as used herein, should be interpreted to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

[0326] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0327] In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0328] Fatty acids or fatty acid residues are a diverse group of molecules made up of hydrocarbon chains terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbons long, can be saturated or unsaturated and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. The cis double bond allows the fatty acid chain to bend, which is available for compounding with more double bonds within the chain. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0329] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is esterified, typically with a different fatty acid. An additional subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0330] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) containing a glycerol core linked by ester bonds to two fatty acid-derived "tails" and one "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (sphingomyelins are also classified as phospholipids), include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0331] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The predominant phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples include simple glycosphingolipids and complex glycosphingolipids, such as cerebrosides and gangliosides. Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, together with glycerophospholipids and sphingomyelins.

[0332] Glycolipids describe compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with membrane bilayers. In glycolipids, a monosaccharide replaces the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharide in Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-Lipid A, which is a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0333] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes and repetitive, multimodular enzymes that share mechanistic features with fatty acid synthases. Polyketides have great structural diversity, including numerous secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes. In accordance with the present disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist uncharged or in a neutral zwitterionic form at a selected pH.

[0334] Cationic or cationically ionic lipids or lipid-like materials In some embodiments, the nucleic acid particles described and / or utilized according to the present disclosure may comprise at least one cationic or cationically ionic lipid or lipid-like material as a particle-forming agent.The cationic or cationically ionic lipid or lipid-like material intended for use herein includes any cationic or cationically ionic lipid or lipid-like material that can electrostatically bind to nucleic acid.In some embodiments, the cationic or cationically ionic lipid or lipid-like material intended for use herein can associate with nucleic acid, for example, by forming a complex with nucleic acid or by forming a vesicle in which nucleic acid is enclosed or encapsulated.

[0335] As used herein, "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid-like material that has a net positive charge. Cationic lipids or lipid-like materials bind to negatively charged nucleic acids through electrostatic interactions. Cationic lipids usually have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl, or more acyl chains, and the head group of the lipid typically has a positive charge.

[0336] In certain embodiments, cationic lipids or lipid-like materials have a net positive charge only at certain pHs, particularly acidic pHs, but preferably do not have a net positive charge, and preferably have no charge, i.e., are neutral at a different, preferably higher, pH, such as physiological pH. This ionic behavior is thought to enhance efficacy compared to particles that remain cationic at physiological pH by aiding in endosomal escape and reducing toxicity.

[0337] For the purposes of this disclosure, such "cationically ionic" lipids or lipid-like materials are encompassed by the term "cationic lipids or lipid-like materials," unless contradicted by context.

[0338] In some embodiments, the cationic or cationically ionic lipid or lipid-like material comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated.

[0339] Examples of cationic lipids include, but are not limited to, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC- Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethyl Aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-Octadecadienoxy)propane (CpLinDMA), N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleoyl DLin-K-DMA, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetramethylamino)propanol (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis-(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP -DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propanamin-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-Dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy) (oxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propane-1 -amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]amino)-ethyl ...ethylamino [2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200); or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

[0340] In some embodiments, the cationic lipid is or comprises heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In some embodiments, the cationic lipid is or comprises a cationic lipid represented by the following structure: [ka]

[0341] In some embodiments, the cationic lipid is or includes ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also referred to herein as ALC-0315.

[0342] In some embodiments, the cationic lipid may comprise from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, from about 30 mol% to about 100 mol%, from about 40 mol% to about 100 mol%, or from about 50 mol% to about 100 mol% of the total lipid present in the particle.

[0343] In certain embodiments, particles used in accordance with the present disclosure include ALC-0315, for example, in a weight percent range of about 40-55 mol percent of total lipid.

[0344] Additional lipid or lipid-like materials In some embodiments, the particles described herein comprise one or more lipids or lipid-like materials other than cationic or cationically ionic lipids or lipid-like materials, such as non-cationic lipids or lipid-like materials (including non-cationically ionic lipids or lipid-like materials) (e.g., in addition to cationic lipids such as ALC315). Collectively, anionic lipids or lipid-like materials and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In addition to ionic / cationic lipids or lipid-like materials, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties, such as cholesterol and lipids, can enhance particle stability and the effectiveness of nucleic acid delivery.

[0345] Additional lipids or lipid-like materials may be incorporated, which may or may not affect the overall charge of the nucleic acid particle. In certain embodiments, the additional lipids or lipid-like materials are non-cationic lipids or lipid-like materials. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of several lipid species that are uncharged or exist in a neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and derivatives and mixtures thereof.

[0346] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), as well as phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0347] In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol. In certain embodiments, the nucleic acid particle comprises both a cationic lipid and an additional lipid.

[0348] In some embodiments, the particles described herein comprise a polymer-conjugated lipid, such as a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art. In some embodiments, the PEGylated lipid is ALC-0159, also referred to herein as (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

[0349] Without wishing to be bound by theory, the amount of at least one cationic lipid relative to the amount of at least one additional lipid can affect important nucleic acid particle characteristics, such as nucleic acid charge, particle size, stability, tissue selectivity, and biological activity.Therefore, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0350] In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the particle.

[0351] In some embodiments, particles used in accordance with the present disclosure may comprise, for example, ALC-0315, DSPC, CHOL, and ALC-0159, e.g., ALC-0315 is about 40-55 mole percent; DSPC is about 5-15 mole percent; CHOL is about 30-50 mole percent; and ALC-0159 is about 1-10 mole percent.

[0352] Lipoplex particles In certain embodiments of the present disclosure, the RNA may be present in an RNA lipoplex particle.

[0353] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can typically be synthesized using cationic lipids, such as DOTMA, and additional lipids, such as DOPE. In some embodiments, the RNA lipoplex particle is a nanoparticle.

[0354] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0355] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0356] The RNA lipoplex particles described herein, in some embodiments, have an average diameter ranging from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter ranging from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0357] In some embodiments, the RNA lipoplex particles and / or compositions comprising the RNA lipoplex particles described herein are useful for delivering RNA to target tissues after parenteral administration, particularly intravenous administration. In some embodiments, the RNA lipoplex particles can be prepared using liposomes, which can be obtained by injecting an ethanolic solution of lipids into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase contains acetic acid, for example, in an amount of about 5 mM. Liposomes can be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In some embodiments, the liposomes and RNA lipoplex particles contain at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In some embodiments, at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0358] Spleen-targeting RNA lipoplex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been discovered that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, RNA accumulation and / or expression in the spleen occurs after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or expression in the lung and / or liver does not or essentially does not occur after administration of the RNA lipoplex particles. In some embodiments, RNA accumulation and / or expression in antigen-presenting cells, such as professional antigen-presenting cells, occurs after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In some embodiments, the antigen-presenting cells are dendritic cells and / or macrophages.

[0359] Lipid nanoparticles (LNPs) In some embodiments, nucleic acids such as RNA described herein are administered in the form of lipid nanoparticles (LNPs). LNPs can include any lipid capable of forming particles to which one or more nucleic acid molecules can be attached or in which one or more nucleic acid molecules are encapsulated.

[0360] In some embodiments, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and PEGylated lipids.

[0361] In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

[0362] In some embodiments, the LNP comprises 40-55 molar percent, 40-50 molar percent, 41-49 molar percent, 41-48 molar percent, 42-48 molar percent, 43-48 molar percent, 44-48 molar percent, 45-48 molar percent, 46-48 molar percent, 47-48 molar percent, or 47.2-47.8 molar percent cationic lipid. In some embodiments, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 molar percent cationic lipid.

[0363] In some embodiments, the neutral lipid is present at a concentration ranging from 5 to 15 molar percent, 7 to 13 molar percent, or 9 to 11 molar percent, hi some embodiments, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 molar percent.

[0364] In some embodiments, the steroid is present at a concentration ranging from 30 to 50 mole percent, 35 to 45 mole percent, or 38 to 43 mole percent, hi some embodiments, the steroid is present at a concentration of about 40, 41, 42, 43, 44, 45, or 46 mole percent.

[0365] In some embodiments, the LNP comprises between 1 and 10 mole percent, between 1 and 5 mole percent, or between 1 and 2.5 mole percent of polymer-conjugated lipid.

[0366] In some embodiments, the LNP comprises 40-50 mole percent cationic lipid; 5-15 mole percent neutral lipid; 35-45 mole percent steroid; 1-10 mole percent polymer-conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

[0367] In some embodiments, the mole percentage is determined based on the total moles of lipid present in the lipid nanoparticle.

[0368] In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE and SM.In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.In some embodiments, the neutral lipid is DSPC.

[0369] In some embodiments, the steroid is cholesterol.

[0370] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid. In some embodiments, the PEGylated lipid has the following structure: [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R12 and R13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, optionally interrupted by one or more ester bonds; w has an average value ranging from 30 to 60. In some embodiments, R12 and R13 are each independently a linear, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12 and R13 are each independently a linear, saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

[0371] In some embodiments, the pegylated lipid is DMG-PEG 2000, for example, having the following structure: [ka]

[0372] In some embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa-, or -NRaC(=O)O-, or a direct bond; G1 and G2 are each independently an unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R1 and R2 are each independently a C6-C24 alkyl or a C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4, or -NR5C(=O)R4; R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; x is 0, 1, or 2.

[0373] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB): [ka] During the ceremony: A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R6 at each occurrence is independently H, OH, or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0374] In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0375] In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID): [ka] y and z are each independently an integer ranging from 1 to 12.

[0376] In any of the foregoing embodiments of Formula (III), one of L1 or L2 is -O(C=O)-. For example, in some embodiments, L1 and L2 are each -O(C=O)-. In several different embodiments of any of the foregoing, L1 and L2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L1 and L2 are each -(C=O)O-.

[0377] In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF): [ka]

[0378] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): [ka]

[0379] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0380] In some of the other aforementioned embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0381] In some of the foregoing embodiments of Formula (III), R6 is H. In other of the foregoing embodiments, R6 is C1-C24 alkyl. In other embodiments, R6 is OH.

[0382] In some embodiments of Formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is a linear C1-C24 alkylene or a linear C1-C24 alkenylene.

[0383] In some of the other aforementioned embodiments of Formula (III), R1 or R2, or both, are C6-C24 alkenyl. For example, in some embodiments, R1 and R2 each independently have the following structure: [ka] During the ceremony: R7a and R7b at each occurrence are independently H or C1-C12 alkyl; a is an integer from 2 to 12, R7a, R7b, and a are each selected such that R1 and R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0384] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For example, in some embodiments, each occurrence of R7a is H. In other different embodiments of the foregoing, at least one occurrence of R7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0385] In different embodiments of formula (III), R1 or R2, or both, have one of the following structures: [ka]

[0386] In some of the above-mentioned embodiments of formula (III), R3 is OH, CN, -C(=O)OR4, -OC(=O)R4, or -NHC(=O)R4. In some embodiments, R4 is methyl or ethyl. In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the following table.

[0387] Representative compounds of formula (III). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0388] In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

[0389] In some embodiments, the cationic lipid is present in the LNP in an amount of about 40 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LNP in an amount of about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LNP in an amount of about 1 to about 10 mole percent.

[0390] In some embodiments, the LNPs comprise compound III-3 in an amount of about 40 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 10 mole percent.

[0391] In some embodiments, the LNPs comprise compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

[0392] In various different embodiments, the cationic lipid has one of the structures shown in the table below. [Table 3]

[0393] In some embodiments, the LNP comprises a cationic lipid shown in the table above, for example, a cationic lipid of formula (B) or formula (D), particularly a cationic lipid of formula (D), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is DMG-PEG 2000.

[0394] In some embodiments, the LNPs comprise cationic lipids that are ionic lipid-like materials (lipidoids). In some embodiments, the cationic lipids have the following structure: [ka]

[0395] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

[0396] The LNPs described herein, in some embodiments, can range in average diameter from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

[0397] Pharmaceutical Composition In some embodiments, the pharmaceutical composition comprises an RNA polynucleotide disclosed herein formulated as a particle. In some embodiments, the particle is or comprises a lipid nanoparticle (LNP) or lipoplex (LPX) particle.

[0398] In some embodiments, the RNA polynucleotides disclosed herein may be administered in a pharmaceutical composition or medicament, and may be administered in the form of any suitable pharmaceutical composition.

[0399] In some embodiments, the pharmaceutical compositions described herein are immunogenic compositions for inducing an immune response. For example, in some embodiments, the immunogenic composition is a vaccine.

[0400] In some embodiments, the RNA polynucleotides disclosed herein may be administered in a pharmaceutical composition, which may include a pharmaceutically acceptable carrier, and may also include one or more adjuvants, stabilizers, etc. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatment.

[0401] The term "adjuvant" refers to a compound that prolongs, enhances, or promotes an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Cytokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, and LT-α. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils, such as Montanide. (登録商標)ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys. Pharmaceutical compositions according to the present disclosure are typically used in "pharmaceutically effective amounts" and "pharmaceutically acceptable preparations." The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of the pharmaceutical composition. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves the desired response or desired effect, either alone or together with further doses. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, particularly interrupting or halting the course of the disease. The desired response in the treatment of a disease may also be delaying or preventing the onset of the disease or symptoms. The effective amount of the compositions described herein will depend on the individual parameters of the patient, including the symptoms to be treated, the severity of the disease, age, physiological condition, size and weight, the duration of treatment, the type of concomitant treatment (if any), specific administration route and similar factors.Therefore, the administration dose of the compositions described herein can be determined by such various parameters.If the initial dose does not produce an adequate response in the patient, a higher dose (or an effectively higher dose achieved by a different, more localized administration route) can be used.

[0402] In some embodiments, the pharmaceutical compositions disclosed herein may contain salts, buffers, preservatives, and other therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0403] Suitable preservatives for use in pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0404] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents. The term "diluent" refers to a diluent and / or thinning agent. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension, and / or mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0405] The term "carrier" refers to a component, whether natural, synthetic, organic, or inorganic, with which the active ingredient is combined to facilitate, enhance, or enable administration of the pharmaceutical composition. As used herein, a carrier can be one or more suitable solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0406] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0407] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0408] In some embodiments, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, which includes absorption through the digestive tract or parenteral administration. As used herein, "parenteral administration" refers to any mode of administration other than through the digestive tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration (e.g., intravenous administration).

[0409] Characterization In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, elevated expression of the payload is observed compared to a suitable reference comparator.

[0410] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide has been administered, an extended period of expression (e.g., expression over a longer period of time) of the payload is observed compared to a suitable reference comparator.

[0411] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, a decrease in the interaction of the RNA polynucleotide with IFIT1 is observed compared to a suitable reference comparator.

[0412] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, increased translation of the RNA polynucleotide is observed compared to a suitable reference comparator.

[0413] In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide lacking the m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2 cap. In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide lacking a cap proximal to a sequence disclosed herein. In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide having a self-hybridizing sequence.

[0414] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, higher expression of the payload and a prolonged duration of expression (e.g., expression over a longer period of time) is observed compared to a suitable reference comparator.

[0415] In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 24 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 48 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 72 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 96 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0416] In some embodiments, elevated expression is determined about 24-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide, hi some embodiments, elevated expression is determined about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0417] In some embodiments, the increased expression of the payload is at least 2-fold to at least 10-fold. In some embodiments, the increased expression of the payload is at least 2-fold. In some embodiments, the increased expression of the payload is at least 3-fold. In some embodiments, the increased expression of the payload is at least 4-fold. In some embodiments, the increased expression of the payload is at least 6-fold. In some embodiments, the increased expression of the payload is at least 8-fold. In some embodiments, the increased expression of the payload is at least 10-fold.

[0418] In some embodiments, increased expression of the payload is about 2-fold to about 50-fold. In some embodiments, increased expression of the payload is about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold.

[0419] In some embodiments, elevated expression (e.g., extended duration of expression) of the payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression of the payload persists for at least 24 hours after administration. In some embodiments, elevated expression of the payload persists for at least 48 hours after administration. In some embodiments, elevated expression of the payload persists for at least 72 hours after administration. In some embodiments, elevated expression of the payload persists for at least 96 hours after administration. In some embodiments, elevated expression of the payload persists for at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0420] In some embodiments, the elevated expression of the payload persists for about 24-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide, hi some embodiments, the elevated expression persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0421] use Disclosed herein, inter alia, are methods of making and using RNA polynucleotides comprising a 5' cap; a 5' UTR comprising a cap-proximal structure; and a sequence encoding a payload.

[0422] In some embodiments, disclosed herein are in vitro transcription reactions comprising: (i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence disclosed herein; (ii) a polymerase; and (iii) an RNA polynucleotide. In some embodiments, the polymerase is or comprises T7 polymerase. In some embodiments, the reaction further comprises a 5' cap or a 5' cap analog. In some embodiments, the 5' cap analog is or comprises a Cap 1 structure. In some embodiments, the RNA polynucleotide comprises a cap comprising a Cap 1 structure; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload. In some embodiments, the Cap 1 structure comprises m7G(5')ppp(5')(2'OMeN1)pN2, where N1 is position +1 of the RNA polynucleotide and N2 is position +2 of the RNA polynucleotide, and N1 and N2 are each independently selected from A, C, G, or U.

[0423] Also in some embodiments, there is provided a method of producing capped RNA, comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure is a G * ppp(m1 2’-O )N1pN2, N1 is complementary to position +1 of the nucleic acid template and N2 is complementary to position +2 of the nucleic acid template, and N1 and N2 are independently selected from A, C, G, or U; the RNA comprises N3, which is complementary to position +3 of the nucleic acid template and is any nucleotide, preferably A or C; N4, which is complementary to position +4 of the nucleic acid template and is a nucleotide selected from the group consisting of A, G, and U, preferably T; and N5, which is complementary to position +5 of the nucleic acid template and is any nucleotide; G * has the following structure: [ka] Including, [ka] is G * represents the bond attached to the first phosphorus atom of the ppp group, and R 1 is CH3 and R 2 is OH or O-CH3, and R 3 is O—CH3, methods are disclosed herein.

[0424] In some embodiments, disclosed herein are methods for producing a polypeptide, comprising providing an RNA polynucleotide comprising a 5' cap, a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload; wherein the RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition comprising the same is administered, higher expression and / or a prolonged duration of expression of the payload is observed compared to a suitable reference comparator.

[0425] Disclosed herein, in some embodiments, are methods comprising administering to a subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle disclosed herein.

[0426] Disclosed herein, in some embodiments, is a method of inducing an immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle disclosed herein.

[0427] Disclosed herein, in some embodiments, are methods of vaccinating a subject, the methods comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle as disclosed herein....

Claims

**Claim 1** A composition or medicament comprising an RNA polynucleotide comprising an array encoding a payload, wherein the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines, and The 5'-end of the RNA polynucleotide is m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pA ４ pN ５ and here N ５ is selected from A, G, C, U or a modified uridine, a composition or a pharmaceutical preparation. **Claim 2** The composition or medicament according to claim 1, wherein the RNA polynucleotide comprises a human alpha globin (hAg) 5'UTR or a fragment thereof, and the hAg 5'UTR or a fragment thereof optionally comprises a modified uridine in place of one or more uridines. **Claim 3** The composition or medicament according to claim 2, wherein the RNA polynucleotide further comprises a Kozak sequence, optionally comprising a modified uridine in place of one or more uridines. **Claim 4** The composition or medicament according to claim 3, wherein the RNA polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 27, optionally comprising a modified uridine in place of one or more uridines. **Claim 5** The composition or medicament according to claim 1, wherein the RNA polynucleotide further comprises a 3'UTR sequence, optionally comprising a modified uridine in place of one or more uridines. **Claim 6** The composition or medicament according to claim 5, 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. **Claim 7** The composition or medicament according to claim 1, wherein the RNA polynucleotide further comprises a polyA sequence. **Claim 8** The composition or medicament according to claim 7, wherein the polyA sequence comprises at least 100 nucleotides. **Claim 9** (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, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence, the composition or medicament according to claim 8. **Claim 10** The composition or medicament according to claim 9, 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. **Claim 11** The RNA polynucleotide has, in the 5' to 3' direction, m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pA ４ pN ５ , and other 5'UTR sequences, sequences encoding a payload, 3'UTR sequences, and polyA sequences, the composition or pharmaceutical preparation according to claim 1.

12. The composition or pharmaceutical preparation according to claim 1, wherein the RNA polynucleotide contains a modified uridine in place of all uridines.

13. The composition or pharmaceutical preparation according to claim 12, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

14. The composition or pharmaceutical preparation according to claim 1, wherein the sequence encoding the 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 a combination thereof.

15. The composition or pharmaceutical preparation according to claim 14, wherein the sequence encoding the payload comprises a sequence encoding an antigenic polypeptide.

16. The composition or pharmaceutical preparation according to claim 15, wherein the antigenic polypeptide contains one epitope derived from an antigen.

17. The composition or pharmaceutical preparation according to claim 15, wherein the antigenic polypeptide contains a plurality of different epitopes derived from one or more antigens.

18. The composition or pharmaceutical preparation according to claim 15, wherein the antigenic polypeptide contains an antigenic polypeptide derived from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide derived from an infectious agent, an antigenic polypeptide derived from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

19. The composition or pharmaceutical preparation according to claim 1, wherein the RNA polynucleotide is mRNA.

20. A pharmaceutical composition comprising an RNA polynucleotide formulated as a particle, wherein the RNA polynucleotide contains a sequence encoding a payload, wherein the RNA polynucleotide optionally contains a modified uridine in place of one or more uridines, and The 5'-end of the RNA polynucleotide is m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pA ４ pN ５ and here N ５ is selected from A, G, C, U or a modified uridine, a pharmaceutical composition.

21. The pharmaceutical composition according to claim 20, wherein the particle is a lipid nanoparticle (LNP) or a lipoplex (LPX) particle.

22. The pharmaceutical composition according to claim 20, wherein the particle is an LNP containing a cationically ionic lipid; a sterol; a phospholipid; and a pegylated lipid.

23. The pharmaceutical composition according to claim 20, formulated as a liquid.

24. The pharmaceutical composition according to claim 20, formulated as a solid. **Claim 25** A composition or medicament 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'-end of the RNA polynucleotide is m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pN ４ pN ５ and wherein N4 and N ５ are each independently selected from A, G, C, U or a modified uridine, a composition or a pharmaceutical preparation. **Claim 26** The composition or medicament according to claim 25, wherein the RNA polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 27, optionally comprising a modified uridine in place of one or more uridines. **Claim 27** The composition or medicament according to claim 25, 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. **Claim 28** The composition or medicament according to claim 25, wherein the RNA polynucleotide further comprises a polyA sequence. **Claim 29** The composition or medicament according to claim 28, 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. **Claim 30** The RNA polynucleotide is in the 5' to 3' orientation and is m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pN ４ pN ５ The composition or pharmaceutical preparation according to claim 25, comprising other 5'UTR sequences, a sequence encoding a payload, 3'UTR sequences, and polyA sequences. **Claim 31** The composition or medicament according to claim 25, wherein the RNA polynucleotide comprises modified uridine in place of all uridines. **Claim 32** The composition or medicament according to claim 31, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ). **Claim 33** The composition or medicament according to claim 25, wherein the sequence encoding the 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 a combination thereof. **Claim 34** The composition or medicament according to claim 33, wherein the sequence encoding the payload comprises a sequence encoding an antigenic polypeptide. **Claim 35** The composition or medicament according to claim 34, wherein the antigenic polypeptide comprises a plurality of different epitopes derived from one or more antigens. **Claim 36** The composition or pharmaceutical preparation according to claim 33, wherein the antigenic polypeptide comprises an antigenic polypeptide derived from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide derived from an infectious agent, an antigenic polypeptide derived from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

37. The RNA polynucleotide is in the 5' to 3' orientation and contains m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pA ４ pN ５ The pharmaceutical composition according to claim 20, comprising other 5'UTR sequences, a sequence encoding a payload, a 3'UTR sequence, and a polyA sequence.

38. The pharmaceutical composition according to claim 20, wherein the RNA polynucleotide comprises a modified uridine in place of at least one uridine.

39. The pharmaceutical composition according to claim 38, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

40. The pharmaceutical composition according to claim 20, wherein the RNA polynucleotide comprises a modified uridine in place of all uridines.

41. The pharmaceutical composition according to claim 40, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

42. A pharmaceutical composition comprising an RNA polynucleotide formulated as a particle, wherein the RNA polynucleotide comprises a sequence encoding a payload, the RNA polynucleotide optionally comprises a modified uridine in place of one or more uridines, and The 5'-end of the RNA polynucleotide is m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pN ４ pN ５ and contains, where N4 and N ５ are each independently selected from A, G, C, U or a modified uridine, a pharmaceutical composition.

43. The pharmaceutical composition according to claim 42, wherein the particle is a lipid nanoparticle (LNP) comprising a cationically ionic lipid; a sterol; a phospholipid; and a pegylated lipid.

44. The RNA polynucleotide has, in the 5' to 3' direction, m7(3'OMeG)(5')ppp(5')(2'OMeA １ )pG ２ pC ３ pN ４ pN ５ The pharmaceutical composition according to claim 42, comprising other 5'UTR sequences, a sequence encoding a payload, 3'UTR sequences, and polyA sequences.

45. The pharmaceutical composition according to claim 42, wherein the RNA polynucleotide comprises a modified uridine in place of at least one uridine.

46. The pharmaceutical composition according to claim 45, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

47. The pharmaceutical composition according to claim 42, wherein the RNA polynucleotide comprises a modified uridine in place of all uridines.

48. The pharmaceutical composition according to claim 47, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

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