RNA construct and its use

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. SC § 119 over each of the following applications; the disclosures of each of those applications are incorporated herein by reference in their entirety: International Application PCT / EP20 / 61239 (filed April 22, 2020); and International Application PCT / EP20 / 66968 (filed June 18, 2020). [Background technology]

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

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

[0004] For example, in some embodiments, the disclosure identifies the sources of specific problems that may be faced in the expression of polypeptides encoded by RNA therapeutics. In particular, the disclosure provides techniques to improve the translation efficiency of the RNA encoding the payload and / or the expression of the polypeptide payload encoded by the RNA. In some embodiments, the translation efficiency and / or expression of the RNA-encoded payload is affected by the cap 1 structure (e.g., m2 7,3’-O Gppp(m1 2’-O (ApG cap); This can be enhanced by an RNA polynucleotide containing a 5'UTR with a sequence proximal to the cap disclosed herein, and a sequence encoding the payload. While we do not wish to be constrained by theory, this disclosure suggests that the improvement in translation efficiency and / or polypeptide payload expression may be achieved by a cap 1 structure disclosed herein, e.g., m2, of eukaryotic translation initiation factor 4E (eIF4E), rather than an IFN-induced protein having a tetratricopeptide repeat-1 (IFIT1). 7,3’-O Gppp(m12’-O It is presented that this can be achieved via preferential binding to RNA containing an ApG cap and / or a 5' UTR containing a sequence proximal to the cap. For example, in some embodiments, it is presented that eIF4E may compete with IFIT1 for binding to RNA polynucleotides based on the 5' cap structure. In particular, this disclosure provides specific techniques that may preferentially bind eIF4E to at least IFIT1 binding and / or otherwise enhance translation.

[0005] In some embodiments, the disclosure teaches that the identity of a specific sequence proximal to the 5' cap (e.g., the 5' cap 1 structure) may influence the translation efficiency of the associated payload. While not wishing to be constrained by any particular theory, the disclosure suggests that eIF4E competes with IFIT1 for binding to RNA polynucleotides 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 disclosure demonstrates that AGAAU or AGCAC sequences downstream of the 5' cap (e.g., the 5' cap 1 structure) may improve translation. The disclosure suggests that the presence of such sequences (e.g., AGAAU or AGCAC) may increase eIF4E binding, at least compared to IFIT1.

[0006] Alternatively, or in addition, this disclosure reveals certain advantages of avoiding (e.g., ensuring the absence of) self-hybridizing sequences (which may in some cases be referred to as self-complementary sequences) within the RNA polynucleotide encoding the payload. For example, this disclosure demonstrates that such absence may improve (e.g., translation efficiency) the translation (e.g., translation efficiency) of the relevant (e.g., RNA-encoded) payload and / or the expression of the polypeptide otherwise encoded by it, and / or may be required for these. While we do not wish to be constrained by theory, it is thought that self-hybridizing sequences (in particular sequences that are located within or hybridize with sequences containing the Kozak sequence, 5'UTR elements, and / or 3'UTR elements) may interfere with one or more aspects of translation. For example, in some embodiments, it is presented that such self-hybridization may inhibit the binding of transcription factors and / or translation factors to the RNA polynucleotide by self-hybriding to complementary sequences within the RNA polynucleotide.

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

[0008] In particular, compositions or medical preparations comprising an RNA polynucleotide comprising (i) a 5' cap that is or includes a cap 1 structure, for example, as disclosed herein; (ii) a 5' UTR sequence including a sequence proximal to the cap, for example, as disclosed herein; and (iii) a sequence encoding a payload are disclosed herein. Methods for producing the same and methods for using the same to induce an immune response in a subject, for example, are also disclosed herein.

[0009] A composition or medical preparation containing RNA polynucleotides, The sequence includes a 5' cap containing a cap 1 structure; a sequence proximal to the cap containing the +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding the payload. (i) The cap 1 structure contains m7G(5')ppp(5')(2'OMeN1)pN2, where N1 is the +1 position of the RNA polynucleotide and N2 is the +2 position of the RNA polynucleotide, and N1 and N2 are each independently selected from A, C, G, or U; (ii) The sequence proximal to the cap is N1 and N2 of the cap 1 structure, and (a) Sequences selected from the group consisting of A3A4X5 (Sequence ID 1); C3A4X5 (Sequence ID 2); A3C4A5 (Sequence ID 3); and A3U4G5 (Sequence ID 4); or (b) An array containing X3Y4X5 (sequence number 7); Includes, X3 (nucleotide X at position +3 in SEQ ID NO: 7) or X5 (nucleotide X at position +5 in SEQ ID NO: 1 or 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] Also, the present disclosure provides a composition or a pharmaceutical preparation comprising an RNA polynucleotide comprising a 5' cap; a cap proximal sequence comprising the +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap comprises a cap 1 structure comprising G * ppp(m1 2’-O )N1pN2, where N1 is the +1 position of the RNA polynucleotide, N2 is the +2 position of the RNA polynucleotide, and N1 and N2 are each independently selected from A, C, G, or U; G * has the following structure:

Chemical formula

[0012] This disclosure relates to a composition or medical preparation comprising a capped RNA polynucleotide encoding a gene product, wherein the RNA polynucleotide is of formula: [ka] Includes, In the formula, R 1 It is CH3, and R 2 OH or O-CH3, R 3 It is O-CH3, B1 is any nucleic acid base, preferably A; B2 is any nucleic acid base, preferably G; B3 is any nucleic acid base, preferably A or C; B4 is any nucleic acid base; B5 is any nucleic acid base. The present invention provides a composition or medical preparation in which, when RNA polynucleotides are administered to a target, the expression levels of the encoded gene product differ by more than five times at approximately 6 hours and approximately 48 hours after administration.

[0013] Pharmaceutical compositions comprising RNA polynucleotides disclosed herein are provided herein. In some embodiments, the pharmaceutical composition comprises compositions or medical preparations disclosed herein.

[0014] A method for producing a pharmaceutical composition containing the RNA polynucleotide disclosed herein is also provided herein, for example, by combining RNA polynucleotides with lipids to form lipid nanoparticles that encapsulate the RNA.

[0015] This disclosure provides a nucleic acid template suitable for generating capped RNA by cap 1, 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 payload encoding sequence, a 3'UTR, and a poly(A) sequence.

[0016] (i) template DNA comprising a polynucleotide sequence complementary to the RNA polynucleotide sequence disclosed herein; (ii) polymerase; and (iii) RNA polynucleotides An in vitro transcription reaction including is provided herein.

[0017] RNA polynucleotides isolated from the provided in vitro transcription reaction are also provided herein.

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

[0019] Compositions comprising DNA polynucleotides having a sequence complementary to the RNA polynucleotide sequence provided are also provided herein. In some embodiments, the DNA polynucleotides disclosed herein can be used to transcribe the RNA polynucleotides disclosed herein.

[0020] This disclosure provides a method for administering to a subject a pharmaceutical composition comprising an RNA polynucleotide, as disclosed herein, formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles, as disclosed herein.

[0021] Methods for inducing an immune response in a subject are also provided herein, which include administering to the subject a pharmaceutical composition comprising RNA polynucleotides disclosed herein, formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles, as disclosed herein.

[0022] For example, a method for administering a target vaccine is provided herein by administering a pharmaceutical composition comprising an RNA polynucleotide, as disclosed herein, formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles, as disclosed herein.

[0023] This disclosure relates to a method for reducing the interaction of RNA polynucleotides with IFIT1, including the 5' cap and sequences proximal to the cap, including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, A step of preparing a mutant RNA polynucleotide that is different from the parent RNA polynucleotide by substituting one or more residues in the sequence near the cap, A step to determine that the interaction of the mutant with IFIT1 is reduced compared to that of the parental RNA polynucleotide. This provides a method that includes [something].

[0024] The process includes preparing an RNA polynucleotide containing a 5' cap, a sequence near the cap including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload; RNA polynucleotides are characterized by the observation of higher payload expression and / or prolonged expression duration compared to appropriate reference comparators when evaluated in organisms administered with RNA polynucleotides or compositions containing them. Methods for generating polypeptides are also provided herein.

[0025] A method for increasing the translatability of an RNA polynucleotide comprising a 5' cap, a sequence near the cap including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload, A step of preparing a variant of the RNA polynucleotide that is different from the parent RNA polynucleotide by substituting one or more residues in the sequence near the cap; A step to determine whether the expression of the mutant is increased compared to that of the parental RNA polynucleotide. Methods including the above are provided herein.

[0026] A therapeutic RNA comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding a payload is provided herein, and improvements include including one or more of the following residues within the sequence proximal to the cap: 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 has been demonstrated to increase RNA expression in the LNP formulation when administered to a subject. In some embodiments, X is selected from A, C, G, or U.

[0027] This disclosure provides a therapeutic RNA comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding a payload. Improvements include including one or more of the following residues within the sequence proximal to the cap: 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 RNA expression in the LNP formulation when administered to a subject. In some embodiments, X is selected from A, C, G, or U.

[0028] Therapeutic RNAs comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding the payload are also provided herein, and improvements include including one or more of the following residues within the sequence proximal to the cap: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide, which have been demonstrated to increase RNA expression in the LNP formulation when administered to a subject.

[0029] This disclosure provides a method for increasing the translation of an RNA polynucleotide comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding a payload, the improvement comprising including one or more of the following residues in the sequence proximal to the cap: 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] A method for increasing the translation of an RNA polynucleotide comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding a payload is provided herein, the improvement comprising including one or more of the following residues in the sequence proximal to the cap: 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] Methods for increasing the translation of an RNA polynucleotide comprising a 5' cap containing a cap 1 structure, a sequence proximal to the cap, and a sequence encoding a payload are also provided herein, the improvement comprising including one or more of the following residues in the sequence proximal to the cap: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

[0032] A method for providing a framework for RNA polynucleotides comprising a 5' cap, a sequence near the cap, and a payload sequence, A step of evaluating at least two variants of an RNA polynucleotide, Each variant includes the same 5' cap and payload sequence; Mutants differ from each other in one or more specific residues in the sequence near the cap; The evaluation process includes determining the expression level and / or duration of expression of the payload sequence. The evaluation process; The process involves selecting at least one combination that shows high expression compared to at least one other combination of the 5' cap and the sequence proximal to the cap, and Methods including the above are also provided herein. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 demonstrates the plasma levels of EPO 6, 24, 48, and 72 hours after intravenous administration of mouse EPO (mEPO) mRNA constructs with and without Lig3 in the 3'UTR sequence to mice. Blood was collected 6, 24, 48, and 72 hours after administration, and the samples were analyzed for mEPO levels via ELISA. [Figure 2] Figures 2A-C are schematic diagrams of Lig3 sequences that self-hybridize to the 5'UTR (Figures 2A-B) or 3'UTR (Figure 2C). [Figure 3-1] Figures 3A-I show the structure of a 5' cap 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 mEPO mRNA constructs with different nucleotides at positions +3, +4, or +5 to mice. Blood was collected 6, 24, 48, and 72 hours after administration, and the samples were analyzed for mEPO levels via ELISA. [Figure 5]Figure 5 demonstrates the anti-S protein IgG response at 7, 14, 21, and 28 days after immunization with BNT162a1. BALB / c mice were immunized once by IM with 1, 5, or 10 μg of LNP-formulated RBL063.3. Blood samples were collected from the animals at 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Values ​​for 1:100 serum dilutions are included in the graph for days 7, 14, 21, and 28. Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM included for each group). [Figure 6] Figure 6 demonstrates the anti-S protein IgG response at 7, 14, 21, and 28 days after immunization with BNT162b1. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP-formulated RBP020.3. Blood samples were collected from the animals at 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Various serum dilutions are included in the graph for day 7 (1:100), day 14 (1:300), day 21 (1:900), and day 28 (1:2700). Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM included for each group). [Figure 7]Figure 7 demonstrates the neutralization of SARS-CoV-2 pseudovirus 14, 21, and 28 days after immunization with BNT162b1. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP formulation RBP020.3. Serum was collected from the animals 14, 21, and 28 days after immunization and tested for SARS-CoV-2 pseudovirus neutralization. The graph represents pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). Each dot in the graph represents one mouse. Measurements were taken in two replicates for each mouse sample. Group size n=8. Mean + SEM is shown for each group by a bar with whiskers. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. [Figure 8] Figure 8 demonstrates the anti-S protein IgG response at 7, 14, and 21 days after immunization with BNT162c1. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.3. Serum samples were collected from the animals at 7, 14, and 21 days post-immunization and analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Various serum dilutions are included in the graphs for day 7 (1:100), day 14 (1:300), and day 21 (1:900). Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM included for each group). [Figure 9]Figure 9 demonstrates the neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162c1. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP formulation RBS004.3. Serum was collected from the animals 14 and 21 days after immunization and tested for SARS-CoV-2 pseudovirus neutralization. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). Each dot in the graph represents one mouse. Measurements were taken in two replicates for each mouse sample. Group size n=8. Mean + SEM is shown for each group by a bar with whiskers. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. [Figure 10] Figure 10 demonstrates the anti-S protein IgG response at 7, 14, 21, and 28 days after immunization with LNP-formulated RBL063.1. BALB / c mice were immunized once by IM with 1, 5, or 10 μg of LNP-formulated RBL063.1. Blood samples were collected from the animals at 7, 14, 21, and 28 days post-immunization, and serum samples were analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Various serum dilutions are included in the graph for days 7 (1:100), 14 (1:100), 21 (1:300), and 28 (1:900). Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM 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 by IM with 1, 5, or 10 μg of LNP-formulated RBL063.1. Serum was collected from the animals 14, 21, and 28 days after immunization and tested for SARS-CoV-2 pseudovirus neutralization. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). Each dot in the graph represents one mouse. Measurements were taken in two replicates for each mouse sample. Group size n=8. Mean + SEM is shown for each group by a horizontal bar with whiskers. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. [Figure 12] Figure 12 shows the anti-S protein IgG response at 7, 14, and 21 days after immunization with BNT162b2 (LNP formulation RBP020.1). BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP formulation RBP020.1. Blood samples were collected from the animals at 7, 14, and 21 days post-immunization, and serum samples were analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Various serum dilutions are included in the graph for day 7 (1:100), day 14 (1:300), and day 21 (1:1100). Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM included for each group). [Figure 13]Figure 13 demonstrates the neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with BNT162b2 (LNP formulation RBP020.1). BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP formulation RBP020.1. Serum was collected from the animals 14 and 21 days after immunization and tested for SARS-CoV-2 pseudovirus neutralization. The graph represents pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). Each point in the graph represents one mouse. Measurements were taken in two replicates for each mouse sample. Group size n=8. Mean + SEM is shown for each group by a horizontal bar with whiskers. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. [Figure 14] Figure 14 shows the anti-S protein IgG response at 7, 14, and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.2. Blood samples were collected from the animals at 7, 14, and 21 days after immunization, and serum samples were analyzed for the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. Various serum dilutions are included in the graph for day 7 (1:100), day 14 (1:300), and day 21 (1:900). Each point in the graph represents one mouse, and measurements were taken in two replicates for each mouse sample (group size n=8; mean + SEM included for each group). [Figure 15]Figure 15 demonstrates the neutralization of SARS-CoV-2 pseudovirus 14 and 21 days after immunization with LNP-formulated RBS004.2. BALB / c mice were immunized once by IM with 0.2, 1, or 5 μg of LNP-formulated RBS004.2. Serum was collected from the animals 14 and 21 days after immunization and tested for SARS-CoV-2 pseudovirus neutralization. The graph represents the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). Each dot in the graph represents one mouse. Measurements were taken in two replicates for each mouse sample. Group size n=8. Mean + SEM is shown for each group by a bar with whiskers. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification. [Figure 16] Figure 16 shows the ALC-0315 activity during the screening process. [Figure 17-1] Figure 17 demonstrates that luciferase expression was monitored in the presence or absence of ApoE3 after intramuscular administration in wild-type (WT) or ApoE knockout C57Bl / 6 mice, on the right side (injection site), dorsal side (injection site), and ventral side (hepatic drainage). Luciferase expression was detected using Xenolight D-Luciferin Rediject 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 C57Bl / 6 mice after intravenous (IV) and intramuscular (IM) administration in the presence (KO+) or absence (KO) of ApoE3. Luciferase expression was detected 4 hours after administration using Xenolight D-Luciferin Rediject. [Modes for carrying out the invention]

[0034] Specific definition While this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, for they may differ. Furthermore, the technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

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

[0036] Unless otherwise specified, the implementation of this disclosure will use conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature in the art (see, for example, 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 this disclosure are described below. These elements are listed by specific embodiments. However, it should be understood that these elements can be combined in any way and in any number to produce additional embodiments. The examples and embodiments described herein should not be construed as limiting this disclosure to only the embodiments explicitly described. It should be understood that the description discloses and includes embodiments that combine the explicitly described embodiments with any number of disclosed elements. Furthermore, any rearrangement and combination of all described elements should be considered disclosed by the description unless the context indicates otherwise. The term “about” means approximately or nearly, and in the context of numerical values ​​or ranges shown herein, in some embodiments, means ±20%, ±10%, ±5%, or ±3% of the numerical value or range described or claimed.

[0038] In the context describing this disclosure (particularly in the context of the claims), the terms “a,” “an,” and “the,” and similar references, should be construed to cover both singular and plural unless otherwise specifically stated herein or unless the context clearly contradicts this. The descriptions of value ranges herein are intended merely as a means of reference to each distinct value that falls within the range. Unless otherwise indicated herein, individual values ​​are incorporated herein as they are described individually herein. All methods described herein may be performed in any preferred order unless otherwise specifically stated herein or unless the context clearly contradicts this. The use of any examples or exemplary language (e.g., “etc.”) provided herein is intended merely to better illustrate this disclosure and does not impose any limitation on the claims. The language herein should not be construed as indicating any non-claimed element that is essential for the practice of this disclosure.

[0039] Unless expressly otherwise specified, the term “contains” is used in the context of this Document to indicate that additional members may exist in addition to the members of the list introduced by “contains.” However, in certain embodiments of this Disclosure, the term “contains” is intended to include the possibility that no additional members exist; that is, for the purposes of this embodiment, “contains” should be understood to mean “consisting of” or “essentially consisting of.”

[0040] Several documents are referenced throughout this specification. All documents referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, and others) are incorporated herein by reference in their entirety, whether above or below. Nothing in this specification should be construed as an acknowledgment that this disclosure did not have prior rights to such disclosures.

[0041] The following definitions are provided that apply to all aspects of this disclosure. Unless otherwise specified, the following terms have the meanings set forth below. Any term not given a definition has the meaning as it is perceived in the art.

[0042] Drug: As used herein, the term "drug" may refer to a physical entity or phenomenon. In some embodiments, a drug may be characterized by a particular composition and / or effect. In some embodiments, a drug may be a compound, molecule, or entity of any chemical class, such as small molecules, polypeptides, nucleic acids, monosaccharides, lipids, metals, or combinations or complexes thereof. In some embodiments, the term "drug" may refer to a compound, molecule, or entity containing a polymer. In some embodiments, the term may refer to a compound or entity containing one or more polymer portions. In some embodiments, the term "drug" may refer to a compound, molecule, or entity that is substantially devoid of a particular polymer or polymer portion. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially devoid of any polymer or polymer portion.

[0043] Amino Acids: In its broadest sense, the term “amino acid” as used herein refers to compounds and / or substances that can, are, or are incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, amino acids have the general structure H2N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are non-natural amino acids; in some embodiments, amino acids are D-amino acids; in some embodiments, amino acids are L-amino acids. “Standard amino acids” refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acids” refers to any amino acid other than standard amino acids, whether synthetically prepared or obtained from natural sources. In some embodiments, amino acids, including carboxyl-terminated and / or amino-terminated amino acids in a polypeptide, may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified compared to their 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 hydroxyl groups). In some embodiments, such modifications may alter, for example, the cyclic half-life of a polypeptide containing a 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 a modified amino acid compared to one containing an otherwise identical unmodified amino acid. As is evident 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] Analogue: As used herein, the term “analogue” refers to a substance that shares one or more specific structural features, elements, components, or parts with a reference substance. Typically, an “analogue” exhibits significant structural similarity to the reference substance, sharing, for example, a core or consensus structure, but differing in certain discrete ways. In some embodiments, an analogue is a substance that can be produced from a reference substance, for example, by a chemical operation of the reference substance. In some embodiments, an analogue is a substance that can be produced by performing a synthetic process that is substantially similar to (for example, sharing several steps with) the one that produces the reference substance. In some embodiments, an analogue is produced or can be produced by performing a synthetic process different from the one used to produce the reference substance.

[0045] Antibody Agent: As used herein, the term “antibody agent” refers to a drug that specifically binds to a particular antigen. In some embodiments, the term encompasses polypeptides or polypeptide complexes containing sufficient immunoglobulin structural elements to confer specific binding. For example, in some embodiments, the antibody agent is a polypeptide comprising, or including, one or more structural elements whose amino acid sequence is recognized by those skilled in the art as a complementarity-determining region (CDR); in some embodiments, the antibody agent is a polypeptide comprising, or including, 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 either identical in sequence to a reference CDR or substantially identical to a reference CDR in that they contain one to five amino acid substitutions. 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, compared to the reference CDR, at least one amino acid in the included CDR is deleted, added, or substituted, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR has 1 to 5 amino acids deleted, added, or substituted compared to the reference CDR, but otherwise has the same amino acid sequence as the reference CDR, and is substantially identical to the reference CDR.In some embodiments, the included CDR has at least one amino acid substitution compared to the reference CDR, but otherwise has the same amino acid sequence as the reference CDR, and is substantially identical to the reference CDR. In some embodiments, the included CDR has 1 to 5 amino acids deleted, added, or substituted compared to the reference CDR, but otherwise has the same amino acid sequence as the reference CDR, and is substantially identical to the reference CDR. In some embodiments, the antibody agent is a polypeptide comprising, or including, a structural element whose amino acid sequence is recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide comprising structural elements whose amino acid sequences are recognized by those skilled in the art as corresponding to the antibody variable domains CDR1, 2, and 3; in some such embodiments, the antibody agent is a polypeptide or set of polypeptides comprising structural elements whose amino acid sequences together are recognized by those skilled in the art as corresponding to both the heavy chain variable region CDR and the light chain variable region CDR, e.g., heavy chain CDR1, 2, and / or 3, and light chain CDR1, 2, and / or 3. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to the immunoglobulin binding domain. In some embodiments, the antibody agent may be a polyclonal antibody preparation, or may include one. In some embodiments, the antibody agent may be a monoclonal antibody preparation, or may include one. In some embodiments, the antibody agent may include one or more constant region sequences specific to a particular organism, e.g., camel, human, mouse, primate, rabbit, rat; in many embodiments, the antibody agent may include one or more constant region sequences specific to humans. In some embodiments, the antibody agent may contain one or more sequence elements that are recognized by those skilled in the art as humanized sequences, primated sequences, chimeric sequences, or the like. In some embodiments, the antibody agent may be a standard antibody (for example, it may contain two heavy chains and two light chains).In some embodiments, the antibody agent is, but is not limited to, intact IgA, IgG, IgE, or IgM antibodies; or a bispecific or multispecific antibody [e.g., Zybodies]. (登録商標) Others]; Antibody fragments, e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fd' fragment, Fd fragment, 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); Camelidae antibodies; Mask antibodies [e.g., Probodies] (登録商標) ];Small Modular ImmunoPharmaceuticals (商標) ]; Single chain or Tandem diamond body [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) The format may be selected from the following. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachments) that it would have if it were naturally occurring. In some embodiments, the antibody may contain covalent modifications [e.g., glycans, payloads (e.g., detectable portion, therapeutic portion, catalytic portion, etc.), or attachments of other pendant groups (e.g., polyethylene glycol, etc.)].

[0046] Related: The term "related" means that two events or entities are "related" to each other, as used herein, if the existence, level, degree, type, and / or form of one correlate with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or symptom if its existence, level, and / or form correlate with the onset, morbidity, severity, stage, etc., of a disease, disorder, or symptom (e.g., across an entire population). In some embodiments, two or more entities are "associated" with each other if they interact directly or indirectly so that they are physically close to each other and / or remain physically close. In some embodiments, two or more entities that physically associate with each other are covalently linked; in some embodiments, two or more entities that physically associate with each other are not covalently linked but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

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

[0048] Biological specimen: As used herein, the term “biological specimen” typically refers to a specimen obtained from or derived from a biological source of interest (e.g., tissue, organism, or cell culture) as described herein. In some embodiments, the source of interest includes organisms, e.g., animals or humans. In some embodiments, the biological specimen is or includes biological tissue or biological fluid. In some embodiments, the biological specimen may be bone marrow; blood; blood cells; ascites; tissue or microneedle biopsy specimens; cell-containing fluids; free-flowing nucleic acids; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; mouth swabs; nasal swabs; washing or lavage, e.g., tubal lavage fluid or bronchoalveolar lavage fluid; aspirates; exfoliated material; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excrement; and / or cells and other materials derived therefrom, or may include these. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the obtained cells are or include cells derived from the individual from which the sample is obtained. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any appropriate means. For example, in some embodiments, the 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 others. In some embodiments, as will be apparent from the context, the term “sample” refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components thereof and / or by adding one or more agents thereto), for example, by filtration using a semipermeable membrane. Such a “processed sample” may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to mRNA amplification or reverse transcription, isolation and / or purification of specific components, or other techniques.

[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 treatment 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 “dose” of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered within a duplicate-dosing regimen. In some embodiments, “administration” of combination therapy may include the administration of one or more agents or modalities to a subject receiving combination therapy with other agents or modalities. For clarity, combination therapy does not require that individual agents be administered together (or necessarily simultaneously) in a single composition; however, in some embodiments, two or more agents or their active portions may be administered together in a combination composition or in a 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 sets of drugs, entities, situations, conditions, etc., which do not have to be identical to one another but are similar enough to allow comparison between them, so that a person skilled in the art will recognize that conclusions can be reasonably drawn based on the differences or similarities observed. In some embodiments, a comparable set of conditions, situations, individuals, or groups is characterized by several substantially identical features and one or a few diverse features. A person skilled in the art will understand, in any given context, what degree of identity is required for two or more such sets of drugs, entities, situations, conditions, etc., to be considered comparable. For example, a person skilled in the art will recognize that sets of situations, individuals, or groups are comparable to one another if they are characterized by a sufficient number and type of substantially identical features to justify a reasonable conclusion that differences under different sets of situations, individuals, or groups, or the results or phenomena obtained thereby, are caused by or demonstrate changes in changing features.

[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 position / identity of a structural element in a compound or composition compared to another compound or composition (e.g., a suitable reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide, or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in a suitable reference polymer. For example, a person skilled in the art will recognize that, for the sake of simplicity, residues in a polypeptide are often indicated based on the relevant reference polypeptide using a standard numbering system, so the amino acid "corresponding to" the residue at position 190 does not necessarily have to be, for example, the 190th amino acid in a particular amino acid chain, but corresponds to the residue found at 190 in the reference polypeptide; a person skilled in the art will readily recognize how to identify the "corresponding" amino acid. For example, a person skilled in the art will be familiar with various sequence alignment strategies, such as software programs that can be used to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with this disclosure, including, for example, 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. A person skilled in the art will also recognize that, in some cases, the term “corresponding” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., a suitable reference event or entity).For example, a gene or protein within one organism may be described as "corresponding" to a gene or protein from another organism in some embodiments to indicate that it performs a similar role or performs a similar function and / or exhibits a certain degree of sequence identity or homology, or shares certain characteristic sequence elements.

[0052] Designed: As used herein, the term "designed" means (i) a drug whose structure is selected or chosen by human hands; (ii) a drug produced by a process requiring human hands; and / or (iii) a drug distinct from natural substances and other known drugs.

[0053] Dosing regimen: Those skilled in the art will recognize that the term “dosing regimen” can be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically divided over time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may comprise one or more doses. In some embodiments, a dosing regimen comprises multiple doses, each divided from other doses as appropriate. In some embodiments, individual doses are divided from one another over periods of equal length; in some embodiments, a dosing regimen comprises multiple doses and at least two different periods dividing the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose, followed by one or more additional doses in a second dose different from the first dose. In some embodiments, the administration regimen comprises a first dose in a first dosage, followed by one or more additional doses in a second dosage equal to the first dosage. In some embodiments, the administration regimen, when administered across the entire population in question, correlates with a desired or beneficial outcome (i.e., it is a therapeutic administration regimen).

[0054] Manipulated: Generally, the term "manipulated" refers to a state in which something is altered by human hands. For example, a polynucleotide can be considered "manipulated" if two or more sequences that would not normally be linked together in that order are manipulated by human hands so that they are directly linked to each other in a manipulated polynucleotide, and / or if certain residues within a polynucleotide are linked to entities or parts that do not naturally exist and / or would not normally be linked, through human action.

[0055] Epitope: As used herein, the term “epitope” refers to a portion that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or chemical groups on an antigen. In some embodiments, such chemical atoms or chemical groups are surface-exposed if the antigen adopts a relevant three-dimensional structure. In some embodiments, such chemical atoms or chemical groups are physically close to one another in space if the antigen adopts such a higher-order structure. In some embodiments, at least some of such chemical atoms and chemical groups are physically separated from one another if the antigen adopts an alternative higher-order structure (e.g., linearized).

[0056] Expression: As used herein, “expression” of a nucleic acid sequence refers to the generation of any gene product from a nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence includes one or more of the following: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, or otherwise); (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 activator formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the activator is present in a unit dose suitable for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be formulated for parenteral administration, for example, as a sterile solution or suspension, or as a sustained-release formulation, for example, by subcutaneous, intramuscular, intravenous, or epidural injection.

[0058] Polypeptide: As used herein, polypeptide refers to a polymer chain of amino acids. In some embodiments, polypeptides have amino acid sequences that exist in nature. In some embodiments, polypeptides have amino acid sequences that do not exist in nature. In some embodiments, polypeptides have amino acid sequences that are manipulated in the sense that they are designed and / or produced by human action. In some embodiments, polypeptides may contain or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, polypeptides may contain or consist of only natural amino acids or only non-natural amino acids. In some embodiments, polypeptides may contain D-amino acids, L-amino acids, or both. In some embodiments, polypeptides may contain only D-amino acids. In some embodiments, polypeptides may contain only L-amino acids. In some embodiments, polypeptides may include modification or attachment of one or more amino acid side chains by one or more pendant groups or other modifications, e.g., at the N-terminus of polypeptides, at the C-terminus of polypeptides, 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, and others (including combinations thereof). In some embodiments, the polypeptide may be cyclic and / or contain a cyclic portion. In some embodiments, the polypeptide may not be cyclic and / or contain no cyclic portion. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be a staple polypeptide and / or contain a staple polypeptide. In some embodiments, the term “polypeptide” may be attached to the name of a base polypeptide, activity, or structure; in such examples, the term “polypeptide” is used herein to refer to polypeptides that can be considered members of the same class or family of polypeptides because they share a relevant activity or structure.For each such class, this specification provides, and / or is known to those skilled in the art, exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for a class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit considerable sequence homology or identity with the reference polypeptide of the class (and, in some embodiments, with all polypeptides within the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share common activities (in some embodiments, at comparable levels or within specified ranges). For example, in some embodiments, the member polypeptide exhibits a degree of overall sequence homology or identity with the 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%, and / or often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%, and includes at least one region (for example, in some embodiments, which may be a characteristic sequence element, or a conserved region) exhibiting very high sequence identity. Such a conserved region typically comprises at least 3–4, often up to 20 or more amino acids; in some embodiments, the conserved region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more consecutive amino acid sequences. In some embodiments, the related polypeptide may include or be derived from a fragment of the parent polypeptide.

[0059] Prevention: Where used in connection with the development of a disease, disorder, and / or symptom, prevention as used herein means reducing the risk of developing a disease, disorder, and / or symptom, and / or delaying the onset of one or more characteristics or signs of the disease, disorder, or symptom. Prevention may be considered complete if the onset of the disease, disorder, or symptom is delayed for a predetermined period of time.

[0060] Criteria: As used herein, criteria describe the standard or control on which the comparison is performed. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to the drug, animal, individual, population, sample, sequence, or value of the criterion or control. In some embodiments, the criterion or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the criterion or control is a historical criterion or control, which may be illustrated in tangible media. Typically, as understood by those skilled in the art, the criterion 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 with a particular possible criterion or control.

[0061] Risk: As understood in context, the “risk” of a disease, disorder, and / or symptom refers to the likelihood that a particular individual will develop the disease, disorder, and / or symptom. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 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 a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples is known to have a risk of disease, disorder, symptom, and / or event. In some embodiments, the reference sample or group of reference samples is derived from an individual comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater. In some embodiments, risk may reflect one or more genetic attributes that may (or may not) give an individual a predisposition to developing a particular disease, disorder, and / or symptom. In some embodiments, risk may reflect one or more epigenetic events or attributes, and / or one or more lifestyle, environmental events, or attributes.

[0062] Individuals who are "susceptible" to a disease, disorder, and / or symptom are individuals who are at a higher risk of developing a disease, disorder, and / or symptom than members of the general population. In some embodiments, individuals who are susceptible to a disease, disorder, and / or symptom do not have to have been diagnosed with the disease, disorder, and / or symptom. In some embodiments, individuals who are susceptible to a disease, disorder, and / or symptom may exhibit the symptoms of the disease, disorder, and / or symptom. In some embodiments, individuals who are susceptible to a disease, disorder, and / or symptom do not exhibit the symptoms of the disease, disorder, and / or symptom. In some embodiments, individuals who are susceptible to a disease, disorder, and / or symptom develop the disease, disorder, and / or symptom. In some embodiments, individuals who are susceptible to a disease, disorder, and / or symptom do not develop the disease, disorder, and / or symptom.

[0063] Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to induce an immune response, for example, to a disease-related (e.g., pathogenic) factor. In some embodiments, vaccination may be administered before, during, and / or after, exposure to a disease-related factor, and in certain embodiments, before, during, and / or immediately after, exposure to the factor. In some embodiments, vaccination comprises multiple doses of the vaccine composition, appropriately time-intervened. In some embodiments, vaccination induces an immune response to an infectious agent. In some embodiments, vaccination induces an immune response to a tumor; in some such embodiments, vaccination is “personalized” in that it is directed, in part or all, to an epitope (e.g., one or more neoepitopes) determined to be present within the tumor of a particular individual.

[0064] Mutant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “mutant” refers to a molecule that exhibits considerable structural identity with a reference molecule, but structurally differs from the reference molecule, for example, in the presence or absence, or level, of one or more chemical moieties compared to the reference entity. In some embodiments, mutants are also functionally different from their reference molecule. Generally, whether a particular molecule can be appropriately considered a “mutant” of a reference molecule depends on the degree of structural identity with the reference molecule. As recognized by those skilled in the art, every biological or chemical reference molecule has certain characteristic structural elements. A mutant by definition is a different molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. In some embodiments, a mutant polypeptide or nucleic acid may 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 attached to the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the mutant polypeptide or nucleic acid exhibits overall sequence identity with the reference polypeptide or nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the mutant 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 mutant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the mutant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the mutant polypeptide or nucleic acid exhibits a reduction in the 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 a “mutant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference except for a few sequence alterations at specific positions. Typically, compared to the reference, the mutant has about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or less than 2% of residues substituted, inserted, or deleted in the mutant. In some embodiments, the mutant polypeptide or nucleic acid contains about 10, 9, 8, 7, 6, 5, 4, 3, 2, or about 1 substituted residue compared to the reference. Often, the mutant polypeptide or nucleic acid contains very few (e.g., less than 5, 4, 3, 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues involved in specific biological activity) compared to the reference. In some embodiments, the mutant polypeptide or nucleic acid contains about 5, about 4, about 3, about 2, or less than 1 additional or deleted elements compared to the reference polypeptide, and in some embodiments, it contains neither additional nor deleted elements. In some embodiments, the mutant polypeptide or nucleic acid contains about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, less than 6 additional or deleted elements compared to the reference polypeptide, and generally less than 5, about 4, about 3, or less than 2 additional or deleted elements. In some embodiments, the reference polypeptide or nucleic acid is one found in nature.

[0065] Detailed description of a specific embodiment This disclosure provides, in particular, an RNA polynucleotide comprising (i) a 5' cap that is or includes a cap 1 structure, for example, as disclosed herein; (ii) a 5' UTR sequence that includes a sequence proximal to the cap, for example, as disclosed herein; and (iii) a sequence that encodes a payload. Compositions and medical preparations comprising the same, as well as methods for producing and using the same, are also provided herein. In some embodiments, the translation efficiency of the RNA encoding the payload, and / or the expression of the payload encoded by the RNA, is determined by the 5' cap comprising a cap 1 structure, for example, m2 7,3’-O Gppp(m1 2’-O ) ApG cap; can be enhanced by an RNA polynucleotide containing a 5'UTR including a sequence proximal to the cap as disclosed herein, and a sequence encoding the payload. In some embodiments, the absence of a self-hybridized sequence within the RNA polynucleotide encoding the payload can further improve the translation efficiency of the RNA encoding the payload, and / or the expression of the payload encoded by the RNA payload.

[0066] RNA polynucleotide As used herein, the terms “polynucleotide” or “nucleic acid” refer to DNA and RNA, such as genomic DNA, cDNA, mRNA, molecules produced by recombination, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. Examples of RNA include in vitro transcribed RNA (IVT RNA) or synthetic RNA. In accordance with the present invention, polynucleotides are preferably isolated.

[0067] In some embodiments, nucleic acids may be contained within the vector. As used herein, the term “vector” includes all vectors known to those skilled in the art, such as plasmid vectors, cosmid vectors, phage vectors (e.g., lambda phage), viral vectors (e.g., retrovirus, adenovirus, or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). In some embodiments, the vector may be an expression vector; instead, 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; instead, 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 suitable sequences essential for the expression of an operablely linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used to manipulate and amplify a specific desired fragment (typically a DNA fragment). Furthermore, it may lack the functional sequence required for the expression of the desired fragment.

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

[0069] A person skilled in the art who has 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 most 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 partially or completely hybridize with each other. In many embodiments, RNA is single-stranded, which may, in some embodiments, self-hybridize or, normally, fold into secondary and / or tertiary structures. In some embodiments, 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, recombinant RNA, and / or modified RNA (wherein the term “modified” is understood to mean that one or more residues or other structural elements of the RNA differ from naturally occurring RNA; for example, in some embodiments, modified RNA differs by the addition, deletion, substitution, and / or alteration of one or more nucleotides, and / or by one or more parts or features of the nucleotides, e.g., nucleosides, or the skeletal structure or linkages). In some embodiments, the modification may include, or constitutes, the addition of non-nucleotide material to the internal RNA nucleotides or to the ends of the RNA. It is also intended herein that the nucleotides within the RNA (e.g., within modified RNA) may be non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, modified RNA is considered an analogue of naturally occurring RNA.

[0070] In some embodiments of this disclosure, RNA is or includes messenger RNA (mRNA) related to an RNA transcript encoding a polypeptide.

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

[0072] In some embodiments, the RNA disclosed herein comprises the following components in a 5' to 3' orientation: a 5' cap containing the 5' cap disclosed herein; a 5' untranslated region containing a sequence proximal to the cap (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, where DNA refers to nucleic acids 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 nucleic acids, particularly cDNA, and introducing them into a vector suitable for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0075] In some embodiments, the RNA is “replicon RNA” or simply “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In some embodiments, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses, such as alphaviruses. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses is typically 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, and in protein modification) and structural proteins (forming the viral particle). Typically, two open reading frames (ORFs) exist within the genome. Four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF beginning near the 5' end of the genome, while alphaviral structural proteins are found downstream of the first ORF and are encoded together by a second ORF extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In alphavirus-infected cells, only the nucleic acid sequences encoding non-structural proteins are translated from genomic RNA, while the genetic information encoding structural proteins is translatable from subgenome 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., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly as messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for the delivery of exogenous genetic information to target cells or target organisms. In a simple approach, an open reading frame encoding an alphavirus structural protein is replaced by an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two distinct nucleic acid molecules: one nucleic acid molecule encoding a viral replicase, and the other nucleic acid molecule being 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. The nucleic acid molecule that can be trans-replicated by the replicase must contain specific alphavirus sequence elements that enable 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 contains modified nucleosides instead of at least one (e.g., all) uridines.

[0077] As used herein, the term "uracil" refers to one of the nucleic acid bases that can be present in RNA. The structure of uracil is as follows: [ka]

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

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

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

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

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

[0083] N1-methylpseudridine-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 contains modified nucleosides in place of at least one uridine. In some embodiments, the RNA contains modified nucleosides in place of each uridine.

[0086] In some embodiments, the modified nucleoside is independently selected from pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (Ψ). In some embodiments, the modified nucleoside includes N1-methylpseudridine (m1Ψ). In some embodiments, the modified nucleoside includes 5-methyluridine (m5U). In some embodiments, the RNA may contain more than one modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (Ψ) and N1-methylpseudridine (m1Ψ). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises N1-methylpseudridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), and 5-methyluridine (m5U).

[0087] In some embodiments, the modified nucleoside that replaces one or more, for example, all, uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (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-pseudridine, 2-thio-pseudridine, 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-oxyacetate methyl ester (mcmo 5U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethyl-pseudridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudolidine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuduridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m 3ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psuduridine, 4-methoxy-2-thio-psuduridine, N1-methylpsuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (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-pseudridine(ψm), 2-thio-2'-O-methyl-uridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 This may be one or more of the following modified uridines known in the art: 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 for cytidine in the RNA. In some embodiments, the RNA contains 5-methylcytidine, as well as one or more selected from pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA contains 5-methylcytidine and N1-methyl-pseudridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine instead of each cytidine, and N1-methyl-pseudridine (m1ψ) instead of each uridine.

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

[0090] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code within a DNA sequence is transcribed into RNA. RNA can then be translated into peptides or proteins.

[0091] In this invention, the term “transcription” includes “in vitro transcription,” which refers to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used to produce the transcript. Such a cloning vector is usually referred to as a transcription vector and, in this invention, is encompassed by the term “vector.” In this invention, the RNA used 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 this invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning 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] In relation to RNA, the terms "expression" or "translation" refer to the process in which a chain of mRNA instructs a sequence of amino acids to assemble into a peptide or protein within the ribosome of a cell.

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

[0094] In some embodiments, nucleic acid compositions described herein, such as compositions comprising lipid nanoparticle-encapsulated mRNA, are characterized by the sustained expression of the encoded polypeptide (for example, when administered to a subject). For example, in some embodiments, such compositions achieve detectable polypeptide expression in such human-derived biological samples (e.g., serum) when administered to humans, and in some embodiments, such expression is characterized by persisting for at least 36 hours, for example, 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. The RNA-coding immunostimulant, when administered according to the present invention, may produce an adjuvant effect. The RNA-coding immunostimulant may be standard RNA or non-immunogenic RNA.

[0096] As used herein, the term "non-immunogenic RNA" refers to RNA that does not induce an immediate immune response in mammals, for example, or that induces a weaker response than that induced by the same RNA, except that it has not been modified and treated to make it non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In a preferred embodiment, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating a modified nucleoside into the RNA that suppresses RNA-mediated activation of innate immune receptors and by removing double-stranded RNA (dsRNA).

[0097] To render non-immunogenic RNA non-immunogenic by incorporation of modified nucleosides, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress the RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises substitution of one or more uridines with nucleosides containing modified nucleobases. In some embodiments, the modified nucleobase is modified uracil. In some embodiments, the nucleoside containing the 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), methyl ester of uridine 5-oxyacetic acid (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), methyl ester of 5-carboxyhydroxymethyl-uridine (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (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-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudolidine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseuduridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m 3 ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psuduridine, 4-methoxy-2-thio-psuduridine, N1-methylpsuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (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-pseudridine(ψm), 2-thio-2'-O-methyl-uridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(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 are selected from the group. In one particularly preferred embodiment, the nucleoside containing the modified nucleic acid base is pseudouridine (ψ), N1-methyl-pseudridine (m1ψ), or 5-methyl-uridine (m 5 U), particularly N1-methyl-pseuduridine.

[0098] In some embodiments, the substitution of one or more uridines with nucleosides containing modified nucleic acid bases includes substitutions of 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 uridine. During mRNA synthesis by in vitro transcription (IVT) using T7 RNA polymerase, a considerable amount of abnormal products, such as double-stranded RNA (dsRNA), are generated due to the enzyme's atypical activity. dsRNA induces inflammatory cytokines, activating effector enzymes and causing inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix, for example. In addition, an enzyme-based method can be used to remove dsRNA contamination from IVT RNA preparations by using E. coli RNase III, which specifically hydrolyzes dsRNA rather than ssRNA. Furthermore, dsRNA can be separated from ssRNA by using cellulose material. In some embodiments, the RNA preparation comes into contact with the cellulose material, enabling the binding of dsRNA to the cellulose material, but ssRNA is separated from the cellulose material under conditions that do not enable the binding of ssRNA to the cellulose material.

[0099] As used herein, the terms “remove” or “remove” refer to the characteristic of separating a population of a first substance, such as non-immunogenic RNA, from a population of a second substance, such as 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, the removal of dsRNA from non-immunogenic RNA includes the removal of dsRNA such 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, the non-immunogenic RNA is dsRNA-free or essentially dsRNA-free. In some embodiments, the non-immunogenic RNA composition includes a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside-modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, the 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 compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

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

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

[0103] The term “showing significantly low 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 non-immunogenic RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that non-immunogenic RNA can be repeatedly administered without inducing an innate immune response, sufficient to detectably reduce the production of proteins encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that non-immunogenic RNA can be repeatedly administered without inducing an innate immune response, sufficient to eliminate the detectable production of proteins encoded by the non-immunogenic RNA. “Immunogenicity” is the ability of an exogenous substance, such as RNA, to trigger an immune response in the body of a human or other animal. The innate immune system is a relatively non-specific and immediate component of the immune system. It is one of the two main components of the immune system in vertebrates, along with the adaptive immune system.

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

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

[0106] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence.

[0107] As used herein, the terms “linked,” “fused,” and “fused” are interchangeable. These terms 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 a coding sequence that is codon-optimized and / or has an increased G / C content compared to the wild-type coding sequence. In some embodiments, one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content do not alter 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 within the coding region of a nucleic acid molecule to reflect the typical codon usage frequency of a host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, the coding region is preferably codon-optimized for optimal expression in the subject to be treated with the RNA polynucleotides described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of tRNA generation within the cell. Thus, the RNA sequence may be modified so that codons available to frequently occurring tRNAs are inserted in place of “rare codons.”

[0110] In some embodiments, the guanosine / cytidine (G / C) content of the RNA coding region (e.g., the payload sequence) is increased compared to the G / C content of the corresponding coding sequence of the 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 its mRNA. Sequences with increased G (guanosine) / C (cytidine) content are more stable than sequences with increased A (adenosine) / U (uridine) content. In relation to the fact that some codons encode one and the same amino acid (so-called degeneracy of the genetic code), the most preferred codon for stability can be determined (so-called alternative codon use). Depending on the amino acids encoded by the RNA, various possibilities exist for the modification of the RNA sequence compared to the wild-type sequence. In particular, codons containing A and / or U nucleosides can be modified by substituting them with other codons that encode the same amino acids but do not contain A and / or U, or contain lower levels of A and / or U nucleosides.

[0111] In some embodiments, the G / C content of the coding region of the 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 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 these publications are incorporated herein by reference. Examples of 5' caps include cap-0 (also referred herein as "cap-0"), cap-1 (also referred herein as "cap-1"), or cap-2 (also referred 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 RNA, such as mRNA, and generally includes a guanosine nucleotide linked to RNA, such as mRNA, via a 5'-5' triphosphate bond (also referred to as Gppp or G(5')ppp(5')). In some embodiments, the guanosine nucleoside contained within the 5' cap may be modified, for example, by methylation at one or more positions on a base (guanine) (e.g., at position 7) and / or by methylation at one or more positions on ribose. In some embodiments, the guanosine nucleoside contained within the 5' cap includes 3'O methylation at ribose (3'OMeG). In some embodiments, the guanosine nucleoside contained within the 5' cap includes methylation at position 7 of guanine (m7G). In some embodiments, the guanosine nucleoside contained within the 5' cap includes methylation at the 7th position of guanine and 3'O methylation at ribose [m7(3'OMeG)].

[0114] In some embodiments, the provision of a 5' cap or 5' cap analogue to RNA as disclosed herein may be achieved by in vitro transcription, in which the 5' cap is expressed in the RNA chain during transcription, or by post-transcriptional attachment to the RNA using a capping enzyme. In some embodiments, transcriptional capping with the caps disclosed herein, for example, cap 1 or cap 1 analogue, improves the capping efficiency of RNA compared to transcriptional capping with a suitable reference comparator. In some embodiments, improved capping efficiency may increase the translation efficiency and / or translation rate of RNA, and / or increase the expression of the encoded polypeptide.

[0115] In some embodiments, the RNA described herein includes a 5' cap or a 5' cap analogue, 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 analogue. In some embodiments, the RNA described herein includes cap 0. In some embodiments, the RNA described herein includes cap 1, e.g., as described herein. In some embodiments, the RNA described herein includes cap 2.

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

[0117] In some embodiments, the cap 1 structure comprises a guanosine nucleoside (m7G) methylated at the 7th position of guanine, and a first nucleotide (2'OMeN1) methylated at 2'O within the RNA. In some embodiments, the cap 1 structure is linked to the RNA via a 5'-5' triphosphate bond and is also referred herein to 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 a 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 m7G(5')ppp(5')(2'OMeA1)pG2, or comprises the same, where A is a cap proximal to the nucleotide at position +1, G is a cap proximal to the nucleotide at position +2, and has the following structure: [ka]

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

[0121] In some embodiments, the cap 1 structure is m7G(5')ppp(5')(2'OMeG1)pG2 or comprises the same, where G is a cap proximal to the nucleotide at position 1, and G is a 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 position 7 of guanine (m7G), one or more additional modifications, e.g., methylation on ribose, and a 2'O methylated first nucleotide in the RNA. In some embodiments, the cap 1 structure comprises a 7-position methylation of guanine, and a 3'O methylated guanosine nucleoside [m7(3'OMeG)]; and a 2'O methylated first nucleotide in the RNA (2'OMeN1). In some embodiments, the cap 1 structure is linked to the RNA via a 5'-5' triphosphate bond and is 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 a 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 m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, or comprises the same, where A is a cap proximal to the nucleotide at position 1, G is a cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0125] In some embodiments, the cap 1 structure is m7(3'OMeG)(5')ppp(5')(2'OMeG1)pG2, or comprises the same, where G is a cap proximal to the nucleotide at position 1, G is a 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 may include one or more modifications, such as methylation. In some embodiments, the cap 1 structure containing the second nucleotide including 2'O methylation is the cap 2 structure.

[0127] In some embodiments, RNA polynucleotides containing a cap1 structure exhibit increased translation efficiency, increased translation rate, and / or increased expression of the encoded payload compared to a suitable reference comparator. In some embodiments, RNA polynucleotides containing a cap1 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) exhibit increased translation efficiency compared to RNA polynucleotides containing a cap1 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, the increase in translation efficiency is evaluated by administration of the RNA polynucleotide to cells or organisms.

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

[0129] The following is an example of cap 1 RNA, which is RNA and m2 7,3’O G(5')ppp(5')m 2’-O Including ApG, [ka]

[0130] The following is another example of cap 1 RNA: [ka]

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

[0132] Sequences near the 5'UTR and cap In some embodiments, the RNA disclosed herein includes a 5'UTR. The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) may be located at the 5' (upstream) end of the open reading frame (5'UTR) and / or at the 3' (downstream) end of the open reading frame (3'UTR). The 5'UTR, if present, is located at the 5' end upstream of the start codon of the 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, the 5'UTR disclosed herein includes, for example, a sequence proximal to the cap, as disclosed herein. In some embodiments, the sequence proximal to the cap includes a sequence adjacent to the 5' cap. In some embodiments, the sequence proximal to the cap includes nucleotides within the +1, +2, +3, +4, and / or +5 positions of the RNA polynucleotide.

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

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

[0136] In some embodiments, the sequence proximal to the cap includes N1 and N2 of the cap structure, 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as N3, N4, and N5, where N1-N5 correspond to the +1, +2, +3, +4, and / or +5 positions 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, e.g., 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, the 5'UTR disclosed herein includes, for example, a sequence proximal to the cap, as disclosed herein. In some embodiments, the sequence proximal to the cap includes a sequence adjacent to the 5' cap. In some embodiments, the sequence proximal to the cap includes nucleotides within the +1, +2, +3, +4, and / or +5 positions of the RNA polynucleotide.

[0207] In some embodiments, the cap structure includes one or more polynucleotides in the sequence proximal to the cap. In some embodiments, the cap structure includes an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure includes an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure includes 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 sequence proximal to the cap includes the cap structure N1 and N2, as well as the sequence A3A4X5 (Sequence ID 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 sequence proximal to the cap includes the cap structure N1 and N2, as well as the sequence C3A4X5 (Sequence ID 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 sequence proximal to the cap includes the cap structure N1 and N2, and the sequence 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 is A. In some embodiments, X3 and / or X5 is C. In some embodiments, X3 and / or X5 is G. In some embodiments, X3 and / or X5 is 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 sequence proximal to the cap includes the cap structure N1 and N2, and the sequence 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 is A. In some embodiments, X3 and / or X5 is C. In some embodiments, X3 and / or X5 is G. In some embodiments, X3 and / or X5 is 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as the sequence containing 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 sequence proximal to the cap includes the cap structure N1 and N2, as well as the sequence 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 includes one or more polynucleotides in the sequence proximal to the cap. In some embodiments, the cap structure includes an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure includes an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure includes 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 nucleotides, 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 nucleotides, 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as the sequence containing A3A4X5 (SEQ ID NO: 1). In some embodiments, N1 and N2 are any nucleotides, 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as a sequence containing C3A4X5 (SEQ ID NO: 2). In some embodiments, N1 and N2 are any nucleotides, 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as the sequence containing X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are any nucleotides, 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 nucleotides, e.g., A, C, G, or U. In some embodiments, X3 and / or X5 is A. In some embodiments, X3 and / or X5 is C. In some embodiments, X3 and / or X5 is G. In some embodiments, X3 and / or X5 is 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as the sequence containing X3Y4X5 (SEQ ID NO: 7). In some embodiments, N1 and N2 are any nucleotides, 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 nucleotides, e.g., A, C, G, or U. In some embodiments, X3 and / or X5 is A. In some embodiments, X3 and / or X5 is C. In some embodiments, X3 and / or X5 is G. In some embodiments, X3 and / or X5 is 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 sequence proximal to the cap includes N1 and N2 of the cap structure, as well as a sequence containing A3C4A5 (SEQ ID NO: 3). In some embodiments, N1 and N2 are any nucleotides, e.g., A, C, G, or U. In some embodiments, N1 is A and N2 is G.

[0232] In some embodiments, the sequence proximal to the cap includes N1 and N2 of the cap structure, as well as a sequence containing A3U4G5 (SEQ ID NO: 4). In some embodiments, N1 and N2 are any nucleotides, 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 its fragment, TEV 5'UTR or its fragment, HSP70 5'UTR or its fragment, or c-Jun 5'UTR or its fragment.

[0234] In some embodiments, the RNA disclosed herein comprises hAg 5'UTR or a fragment thereof. In some embodiments, the RNA disclosed herein comprises hAg 5'UTR having identity with respect to human alphaglobin 5'UTR provided in SEQ ID NO: 11 of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some embodiments, the RNA disclosed herein comprises hAg 5'UTR having identity with respect to human alphaglobin 5'UTR provided in SEQ ID NO: 12 of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some embodiments, the RNA disclosed herein comprises hAg 5'UTR having identity with respect to human alphaglobin 5'UTR provided in SEQ ID NO: 12 of 99%.

[0235] 3'UTR In some embodiments, the RNA disclosed herein includes a 3'UTR. The 3'UTR, if present, is located at the 3' end downstream of the end 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 includes a 3'UTR containing an F element and / or an I element. In some embodiments, the 3'UTR or a sequence proximal thereto includes a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

[0237] In some embodiments, the RNA disclosed herein includes a 3'UTR having an identity of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% with respect to the 3'UTR provided in SEQ ID NO: 13.

[0238] Poly A In some embodiments, the RNA disclosed herein includes, for example, a polyadenylated (poly-A) sequence as described herein. In some embodiments, the poly-A sequence is located downstream of the 3'UTR, for example, adjacent to the 3'UTR.

[0239] As used herein, the terms “poly(A) sequence” or “polyA tail” typically refer to an uninterrupted or interrupted sequence of adenylate residues located at the 3' end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and may follow the 3'UTR in the RNA described herein. An uninterrupted poly(A) sequence is characterized by a continuous adenylate residue. In nature, uninterrupted poly(A) sequences are typical. The RNA disclosed herein may have a poly(A) sequence attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

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

[0241] The poly(A) sequence may be of any length. In some embodiments, the poly(A) sequence contains, is essentially, 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, “essentially from” means that most of the 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, but the remaining nucleotides may be other nucleotides, such as U nucleotides (uridilates), G nucleotides (guanilates), or C nucleotides (cytidilates). In this context, "consists of" means that all nucleotides in a poly(A) sequence, i.e., 100% of the nucleotides in a poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0242] In some embodiments, the poly(A) sequence is attached during RNA transcription based on a DNA template containing repeating dT nucleotides (deoxythymidilates) in a strand complementary to the coding strand, for example, during the preparation of in vitro transcription RNA. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as the poly(A) cassette.

[0243] In some embodiments, poly(A)cassettes present within the coding strand of DNA essentially consist of dA nucleotides but are interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5–50, 10–30, or 10–20 nucleotides long. Such cassettes are disclosed in International Publication 2016 / 005324A1, which is incorporated herein by reference. Any poly(A)cassette disclosed in International Publication 2016 / 005324A1 may be used in the present invention. Poly(A)cassettes essentially consisting of dA nucleotides but interrupted by a random sequence in which the distribution of four nucleotides (dA, dC, dG, and dT) is equal and the length is, for example, 5–50 nucleotides, exhibit, at the DNA level, consistent proliferation of plasmid DNA in E. coli, and still, at the RNA level, beneficial properties relating to the support of RNA stability and encompass translation efficiency. In some embodiments, the poly(A) sequence contained within the RNA polynucleotide described herein consists essentially of A nucleotides but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides long.

[0244] In some embodiments, nucleotides other than A nucleotides are not located on the side of the poly(A) sequence at its 3' end; that is, the poly(A) sequence is not masked at its 3' end by nucleotides other than A, and is not followed by nucleotides other than A.

[0245] 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 may essentially 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 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 contains at least 100 nucleotides. In some embodiments, the poly(A) sequence contains about 150 nucleotides. In some embodiments, the poly(A) sequence contains about 120 nucleotides.

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

[0247] payload In some embodiments, the RNA polynucleotides disclosed herein include, for example, a payload-coding sequence as described herein. In some embodiments, the payload-coding sequence includes a promoter sequence. In some embodiments, the payload-coding sequence includes a secretion signal peptide-coding sequence.

[0248] In some embodiments, the payload is selected from protein substitution polypeptides; antibody agents; cytokines; antigenic polypeptides; gene editing components; regenerative medicine components; or a combination thereof.

[0249] In some embodiments, the payload is or comprises a protein-substituted polypeptide. In some embodiments, the protein-substituted polypeptide comprises a polypeptide that is abnormally expressed in a disease or disorder. In some embodiments, the protein-substituted polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein. In some embodiments, the protein-substituted polypeptide comprises an enzyme.

[0250] In some embodiments, diseases or disorders characterized by abnormal polypeptide expression include, but are not limited to, rare diseases, metabolic disorders, muscular dystrophy, cardiovascular diseases, or monogenic disorders.

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

[0252] In some embodiments, the payload is or includes a cytokine, or a fragment or variant thereof. In some embodiments, the cytokine includes 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, an immunogenic variant thereof, or an immunogenic fragment thereof. In some embodiments, the antigenic polypeptide comprises one epitope derived from the antigen. In some embodiments, the antigenic polypeptide comprises multiple different epitopes derived from the antigen. In some embodiments, the antigenic polypeptide comprising multiple different epitopes derived from the antigen is a polyepitope.

[0254] In some embodiments, the antigenic polypeptide includes allergen-derived antigenic polypeptide, viral antigenic polypeptide, bacterial antigenic polypeptide, fungal antigenic polypeptide, parasitic antigenic polypeptide, infectious agent-derived antigenic polypeptide, pathogen-derived antigenic polypeptide, tumor antigenic polypeptide, or autoantigenic polypeptide.

[0255] In some embodiments, the viral antigenic polypeptide includes HIV antigenic polypeptide, influenza antigenic polypeptide, coronavirus antigenic polypeptide, rabies antigenic polypeptide, or 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 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with respect to the SARS-CoV-2 S polypeptide disclosed herein. In some embodiments, the SARS-CoV-2 S polypeptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with respect to SEQ ID NO: 9.

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

[0259] In some embodiments, the payload is or comprises a tumor antigenic polypeptide, an immunogenic variant thereof, or an immunogenic 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 This includes minor BCR-abL, Place-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 combinations thereof.

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

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

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

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

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

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

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

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

[0268] In some embodiments, the payload is or comprises an autoantigenic polypeptide, an immunogenic variant thereof, or an immunogenic 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 Sjögren'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 containing them, include the nucleotide sequences disclosed herein. In some embodiments, the RNA polynucleotides include sequences that are at least 80% identical to the nucleotide sequences disclosed herein. In some embodiments, the RNA polynucleotides include sequences encoding polypeptides that are at least 80% identical to the polypeptide sequences disclosed herein. Exemplary nucleotide and polypeptide sequences are provided, for example, in Table 1, in this section titled “Exemplary Polynucleotides,” or in Example 2.

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

[0271] In some embodiments, the payloads described herein are encoded by RNA polynucleotides described herein, including nucleotide sequences disclosed herein, for example in Table 1, in this section titled “Exemplary Polynucleotides,” or in Example 2. In some embodiments, the RNA polynucleotide encodes a polypeptide payload having at least 80% identity to the polypeptide payload sequences disclosed herein. In some embodiments, the payloads described herein are encoded by RNA polynucleotides, which are transcribed by a DNA template containing 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variant). Sequence ID 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 aaaaaaaaa 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variant). Sequence ID 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 agaccccucc 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 ucgccagua 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 aaaaaaaaa 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] Sequence ID 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variant). 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[[ID=二十]] 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) (full-length S1S2 protein, sequence variant).

[0278] BNT162b1;RBP020.3 (SEQ ID NO: 32; SEQ ID NO: 21, amino acids) 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 the S1S2 protein fused to fibrintin] Sequence ID 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 acids) Structure: Beta-S-ARCA(D1)-Replicaze-S1S2-PP-FI-A30L70 The encoded antigen is the SARS-CoV-2 viral spike protein (S protein) (S1S2 full-length protein, sequence variant). Sequence ID 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 gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugugau 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. 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100 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 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 cguugauaa 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 agaaaaagaa 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 acagagaa 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 acids) Structure: Beta-S-ARCA(D1)-Replicaze-S1S2-PP-FI-A30L70 The encoded antigen is the SARS-CoV-2 viral spike protein (S protein) (S1S2 full-length protein, sequence variant). Sequence ID 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 uaaguagug 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 gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugugau 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. 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100 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 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 cguugauaa 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 acids) 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] Sequence ID 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 uauggccuc 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 uaaguagug 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 gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugugau 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. 5100. 5100. 5100. 5100. 5100. 5100. 5100. 5100 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 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 cguugauaa 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 guuuuguuu 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 agaaugggc 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 (Sequence ID 36; Sequence ID 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] Sequence ID 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 gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugugau 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 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 cguugauaa 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 guuuuguuu 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 agaaugggc 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 aaaaaaaa 9079 Sequence ID 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) [partial sequence, receptor-binding domain (RBD) of the S1S2 protein fused to fibrin fused to the transmembrane domain (TM) of the S1S2 protein]; the N-terminus of the antigen sequence contains the intrinsic S1S2 protein secretion signal peptides (aa1-19). Sequence ID 38 [ka] [ka] [ka] Sequence ID 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 cuagcugccc 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 The encoded antigen is the SARS-CoV-2 viral spike protein (S1S2 protein) [partial sequence, receptor-binding domain (RBD) of the S1S2 protein fused to fibrin fused to the transmembrane domain (TM) of the S1S2 protein]; immunoglobulin secretion signal peptides (aa1-22) at the N-terminus of the antigen sequence. Sequence ID 40 [ka] [ka] Sequence ID 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 the payload, may be formulated as particles and administered.

[0286] In the context of this 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, such as a micro or nano-sized compact structure dispersed in a medium. In some embodiments, the particles are nucleic acid-containing particles, such as particles containing DNA, RNA, or mixtures 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 the formation of complexes 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 target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles may 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 acids. Examples of nucleic acid particles include lipid nanoparticle (LNP)-based formulations and lipoplex (LPX)-based formulations.

[0290] While not intended to be constrained by any particular 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, and that this aggregation produces 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 cationic lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

[0292] In some embodiments, the nucleic acid particles comprise multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules may be similar or different from one another in terms of molar mass or basic structural elements, such as molecular architecture, capping, coding region, or other features. In some embodiments, the nucleic acid particles described herein may have average diameters 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] 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 less than or equal to about 0.2. For example, 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 represents the ratio of nitrogen groups in the lipid to the number of phosphate groups in the RNA. This correlates with the charge ratio, because 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 equilibrium is present. Lipid formulations are frequently formed with N / P ratios greater than 4, up to 12, because positively charged nanoparticles are considered suitable for translocation. In this case, the RNA is thought to be completely bound to the nanoparticles.

[0295] The nucleic acid particles described herein can be prepared using a wide range 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 nucleic acid particles.

[0296] As used herein, the term “colloid” refers to a homogeneous mixture of dispersed particles that do not precipitate. The insoluble particles in the mixture are fine, with a particle size of 1 to 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. Occasionally, 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 the preparation of liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolution of the lipid in an organic solvent, (ii) drying of the resulting solution, and (iii) hydration of the dried lipid (using various aqueous media).

[0298] In the membrane hydration method, the lipids are first dissolved in a suitable organic solvent, dried, and a thin film is obtained at the bottom of the flask. The resulting lipid film is then hydrated using a suitable aqueous medium to produce a liposome-type dispersion system. Further, an additional downsizing step may be included.

[0299] Reverse-phase evaporation is an alternative to membrane hydration for preparing liposomal vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. A brief sonication of this mixture is required for homogenization. Removal of the organic phase under reduced pressure results in a milky gel, which subsequently 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 into an aqueous solution through a needle. This action disperses the lipids throughout the solution, promoting lipid structure formation, such as lipid vesicle formation or liposome formation. Typically, the RNA lipoplex particles described herein are obtained by adding RNA to a colloidal liposome dispersion system. Using the ethanol injection technique, such a colloidal liposome dispersion system is formed in some embodiments as follows: an ethanol solution containing lipids, such as cationic lipids and further lipids, is injected into an aqueous solution under agitation. In some embodiments, the RNA lipoplex particles described herein are obtainable without an extrusion step.

[0301] The term "extrude" refers to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles, in which particles are forced to pass through a filter with defined pores.

[0302] Furthermore, other methods having the characteristic of being organic solvent-free may be used in accordance with this disclosure to prepare colloids.

[0303] LNPs typically consist of 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, enabling effective intracellular delivery. LNPs can be prepared by rapidly dissolving lipids in ethanol and mixing them with nucleic acids in an aqueous buffer.

[0304] The term "average diameter" refers to the average hydrodynamic diameter of a particle measured by dynamic laser light scattering (DLS) using data analysis with the so-called cumulant algorithm, which consequently provides the so-called Zaverage in terms of length and a dimensionless multi-dispersion index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, "average diameter," "diameter," or "size" for a particle are used synonymously with the corresponding value of Zaverage.

[0305] The "polydispersion index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as referred to in the definition of "average diameter." This can be used as a measure of the size distribution of an ensemble of nanoparticles under certain preconditions.

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

[0307] This 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, which associate with nucleic acids to form nucleic acid particles, and compositions comprising such particles. Nucleic acid particles may contain nucleic acids that are complexed with particles in various forms by non-covalent interactions. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they cannot infect cells with viruses. Suitable cationic or cationically ionic lipid or lipid-like materials and cationic polymers that form nucleic acid particles are included by the terms “particle-forming components” or “particle-forming agents.” The terms “particle-forming components” or “particle-forming agents” refer to any component that associates with nucleic acids to form nucleic acid particles. Such components include any component that may be part of a nucleic acid particle.

[0308] Some embodiments described herein relate to compositions, methods, and uses comprising more than one, for example, two, three, four, five, six, or even more than one nucleic acid species, such as RNA species, for example, a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parental viral protein (the amino acid positions in at least the fragment of the parental viral protein are modified to include amino acids found in the corresponding amino acid positions of 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 parental viral protein (the amino acid positions in at least the fragment of the parental viral protein are modified to include amino acids found in the corresponding amino acid positions of one or more viral protein variants).

[0309] In particulate formulations, each nucleic acid species can be formulated separately as an individual particulate formulation. In this case, each individual particulate formulation will contain one nucleic acid species. Individual particulate formulations may exist as separate entities, for example, in separate containers. Such formulations are available by providing each nucleic acid species separately (typically in the form of a nucleic acid-containing solution) along with a particle-forming agent that enables particle formation. Each particle will exclusively contain the specific nucleic acid species provided when the particle is formed (individual particulate formulation).

[0310] In some embodiments, a composition, such as a pharmaceutical composition, comprises a plurality of individual particle formulations. Each pharmaceutical composition is referred to as a mixed microparticle formulation. Mixed microparticle formulations according to the present invention can be obtained by a mixing step, after the individual microparticle formulations have been formed separately as described above. The mixing step can yield a formulation containing a mixed population of nucleic acid-containing particles. The individual microparticle populations may come together in a single container to constitute a mixed population of individual microparticle formulations.

[0311] In addition to the above, various nucleic acid species can be formulated together as combined microparticle formulations. Such formulations are available by providing a formulation (typically a combined solution) of various RNA species together with a particle-forming agent that enables particle formation. In contrast to mixed microparticle formulations, combined microparticle formulations typically contain particles containing multiple RNA species. In combined microparticle compositions, various RNA species typically coexist within a single particle.

[0312] Cationic polymer materials (e.g., polymers) Given their high degree of chemical flexibility, polymer materials are commonly used for nanoparticle-based deliveries. Typically, cationic materials are used to electrostatically compress negatively charged nucleic acids into nanoparticles. The positively charged groups often consist of amines that change their protonation state within the pH range of 5.5–7.5, which is thought to lead to an ionic imbalance resulting in endosomal disruption. Polymers, such as poly-L-lysine, polyamidoamines, protamines, and polyethyleneimines, as well as naturally occurring polymers, such as chitosan, have all been used in nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some researchers have synthesized polymer materials specifically for nucleic acid delivery. Poly(β-aminoesters) have been widely used in nucleic acid delivery, particularly 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, “polymer material” refers to a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds, in its ordinary sense. In some embodiments, all such repeating units may be identical; otherwise, there may be more than one type of repeating unit present in the polymer material. In some cases, the polymer material may be of biological origin, such as biopolymers such as proteins. In some cases, additional parts may also be present in the polymer material, such as targeted parts, as described herein.

[0314] Those skilled in the art will know that if a polymer (or polymer portion) contains more than one type of repeating unit, the polymer (or polymer portion) is said to be a “copolymer.” In some embodiments, the polymer (or polymer portion) utilized pursuant to this disclosure may be a copolymer. The repeating units forming the copolymer can be arranged in any manner. For example, in some embodiments, the repeating units may be arranged in a random order; instead, or in addition, in some embodiments, the repeating units may be arranged in an alternating order or as a “block” copolymer (i.e., comprising one or more regions (e.g., a first block), each containing a first repeating unit, and one or more regions (e.g., a second block), etc.). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more different blocks.

[0315] In certain embodiments, the polymer materials used in accordance with this disclosure are biocompatible. Biocompatible materials do not typically cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible materials are biodegradable, i.e., they can be chemically and / or biologically degraded in a physiological environment, for example, within the body.

[0316] In certain embodiments, the polymer material may be a protamine or a polyalkyleneimine, particularly a protamine, or may contain the same.

[0317] Those skilled in the art will know that the term “protamine” is often used to refer to any of the various relatively low molecular weight, strongly basic proteins that are rich in arginine and found to associate with DNA, in particular, in place of somatic histones, in the sperm cells of various animals (such as fish). In particular, the term “protamine” is often used to refer to a strongly basic, water-soluble, non-coagulating protein found in fish sperm, which hydrolyzes primarily to produce arginine. In its purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0318] In some embodiments, the term “protamine” as used herein refers to protamine amino acid sequences obtained from or derived from natural or biological sources [including fragments thereof, and / or polymeric forms of said amino acid sequences or fragments thereof, as well as artificial, specifically designed for a particular purpose, and not isolated from natural or biological sources (synthesized) polypeptides].

[0319] In some embodiments, the polyalkyleneimine includes polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In some embodiments, the preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75·10² to 10⁷ Da, preferably 1000 to 10⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, and even more preferably 20000 to 25000 Da.

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

[0321] Cationic materials intended for use herein (e.g., polymer materials such as polycationic polymers) include those that can electrostatically bond to nucleic acids. In some embodiments, cationic polymer materials intended for use herein include any cationic polymer material to which nucleic acids can associate, for example, by forming a complex with nucleic acids or by forming vesicles in which nucleic acids are encapsulated or contained.

[0322] In some embodiments, the particles described herein may include polymers other than cationic polymers, such as non-cationic polymer materials and / or anionic polymer materials. Collectively, anionic polymer materials and neutral polymer materials are referred to herein as non-cationic polymer 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 hydrophobic and hydrophilic moieties are also frequently referred to as amphiphilic substances. Lipids are typically not fully soluble in water. In an aqueous environment, due to their amphiphilic nature, molecules can self-assemble into organized structures and different phases. One such phase consists of vesicles, multilayer / monolayer liposomes, or lipid bilayers that remain intact within membranes in an aqueous environment. Hydrophobicity can be given by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated aliphatic hydrocarbon groups and long-chain unsaturated aliphatic hydrocarbon groups, as well as groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. In some embodiments, hydrophilic groups may include polar and / or charged groups, such as carbohydrates, phosphate groups, carboxylic acid 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 a polar and a nonpolar portion. In many cases, amphiphilic compounds have a polar head attached to a long, hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble. Additionally, the polar portion may have either a formal positive or formal negative charge. Furthermore, the polar portion may have both formal positive and negative charges, and may be a zwitterion or an intramolecular salt. For the purposes of this disclosure, amphiphilic compounds may be one or more natural or non-natural lipids and lipid-like compounds, but are not limited to those described below.

[0325] The terms “lipid-like materials,” “lipid-like compounds,” or “lipid-like molecules” 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, multilayer / monolayer liposomes, or amphiphilic layers that remain intact within membranes in an aqueous environment, and include surfactants or synthetic compounds having both hydrophilic and hydrophobic parts. Generally speaking, the terms refer to molecules that have hydrophilic and hydrophobic parts with different structural organization, whether similar or different to those of lipids. As used herein, the term “lipid” should be interpreted to cover both lipids and lipid-like materials unless otherwise specified herein or the context clearly indicates otherwise.

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

[0327] In certain embodiments, amphiphilic compounds are lipids. The term “lipid” refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Typically, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenolipids (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 triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0328] Fatty acids, or fatty acid residues, are diverse groups of molecules made up of hydrocarbon chains ending in a carboxylic acid group; this arrangement confers to the molecule polar, hydrophilic ends and nonpolar, hydrophobic ends that are insoluble in water. Typically 4-24 carbon chains 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 a possibility of cis or trans geometric isomerism, which significantly affects the stereochemistry of the molecule. A cis double bond causes the fatty acid chain to bend, which is effective in 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 by 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 amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that have a glycerol core linked to two fatty acid-derived "tails" by ester bonds and to a single "head" group by a phosphate ester bond. Examples of glycerophospholipids commonly referred to as phospholipids (sphingomyelin is also classified as a phospholipid) 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 skeleton. The major sphingoid base in mammals is generally referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives containing amide-linked fatty acids. These fatty acids are typically saturated or monounsaturated and have a chain length of 16–26 carbon atoms. The major phosphosphingolipid in mammals is sphingomyelin (ceramidephosphocholine), while insects primarily contain ceramidephosphoethanolamine, and fungi have phytoceramidephosphoinositol and mannose-containing head groups. Sphingoglycolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples of these include simple and complex sphingoglycolipids, such as cerebrosides and gangliosides. Sterol lipids, such as cholesterol and its derivatives, or tocopherols and their derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelin.

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

[0333] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classical enzymes and repetitive, multimodular enzymes that share mechanical features with fatty acid synthases. Polyketides exhibit great structural diversity, including numerous secondary metabolites and endemic products from animal, plant, bacterial, fungal, and marine sources. Many polyketides are cyclic molecules whose backbone is often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes. According to this disclosure, lipids and lipid-like materials may 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, nucleic acid particles described and / or utilized in accordance with this disclosure may include at least one cationic or cationically ionic lipid or lipid-like material as a particle-forming agent. Cationic or cationically ionic lipid or lipid-like materials intended for use herein include any cationic or cationically ionic lipid or lipid-like material that can electrostatically bind to nucleic acids. In some embodiments, cationic or cationically ionic lipid or lipid-like materials intended for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or contained.

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

[0336] In certain embodiments, cationic lipids or lipid-like materials have a net positive charge only at specific pH levels, particularly acidic pH levels, while preferably having no net positive charge, and more preferably being chargeless, i.e., neutral at different, preferably higher, pH levels, such as physiological pH. This ionic behavior is thought to enhance efficacy compared to particles that remain cationic at physiological pH by assisting endosomal escape and reducing toxicity.

[0337] For the purposes of this disclosure, such “cationically ionic” lipids or lipid-like materials are included by the term “cationic lipids or lipid-like materials” unless otherwise inconsistent with the context.

[0338] In some embodiments, cationic or cationically ionic lipids or lipid-like materials include a head group comprising at least one nitrogen atom (N) that can be positively charged or protonated.

[0339] Examples of cationic lipids include, but are not limited to, the following: ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC- Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; 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-trimethylammoniumpropane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium (propanamium) trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethyl Aminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-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-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl Lyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[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(Te Tradecyloxy)-1-propaneaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis-(cis-9-tetradecenyloxy)-1-propaneaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (GAP -DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-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]propane-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-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyl) Xy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di((Z)-nona-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)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)-nona-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecaneoate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-e [Tyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (Lipidoid C12-200); or heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

[0340] In some embodiments, the cationic lipid is or comprises heptadecan-9-yl8-((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 ((4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), also referred to herein as ALC-0315, or comprises the same.

[0342] In some embodiments, cationic lipids may constitute about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the particles.

[0343] In some specific embodiments, the particles used in accordance with this disclosure include, for example, ALC-0315 in a weight percentage in the range of about 40 to 55 mol percent of total lipids.

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

[0345] Additional lipids or lipid-like materials may be incorporated that may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the additional lipids or lipid-like materials are noncationic lipids or lipid-like materials. Noncationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, “anionic lipids” refers to any lipid that is negatively charged at a selected pH. As used herein, “neutral lipids” 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 lipids include the following neutral lipid components: (1) phospholipids, (2) cholesterol or its derivatives; or (3) a mixture of phospholipids and cholesterol or one of its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol, and their 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, diacylphosphatidylcholine, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), and 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Examples include diether PC, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and phosphatidylethanolamines, particularly 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 lipids are DSPC or DSPC and cholesterol. In certain embodiments, the nucleic acid particles include both cationic lipids and additional lipids.

[0348] In some embodiments, the particles described herein include polymer-conjugated lipids, such as pegylated lipids. The term "pegylated lipid" refers to a molecule containing 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] While we do not wish to be constrained by theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid may 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, noncationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may constitute about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipids present in the particles.

[0351] In some embodiments, the particles used in accordance with this disclosure may include, for example, ALC-0315, DSPC, CHOL, and ALC-0159, for example, ALC-0315 in amounts of about 40 to 55 mole percent; DSPC in amounts of about 5 to 15 mole percent; CHOL in amounts of about 30 to 50 mole percent; and ALC-0159 in amounts of about 1 to 10 mole percent.

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

[0353] In the context of this disclosure, the term “RNA lipoplex particles” refers to particles containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in the complex formation 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 particles are nanoparticles.

[0354] In certain embodiments, RNA lipoplex particles contain both cationic lipids and additional lipids. In exemplary embodiments, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0355] 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. In certain embodiments, the molar ratio may 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 at least one cationic lipid to at least one additional lipid is about 2:1.

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

[0357] In some embodiments, RNA lipoplex particles and / or compositions, including the RNA lipoplex particles described herein, are useful for the delivery of RNA to target tissues after parenteral administration, particularly after intravenous administration. In some embodiments, RNA lipoplex particles may be prepared using liposomes, which may be obtained by injecting an ethanol solution of lipids into water or a suitable aqueous phase. In some embodiments, the aqueous phase is acidic in pH. In some embodiments, the aqueous phase contains acetic acid, for example, in an amount of about 5 mM. Liposomes may be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In some embodiments, the liposomes and RNA lipoplex particles include at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium-propane (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-targeted RNA lipoplex particles are described in WO2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles having 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 RNA expression occurs in the spleen after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, RNA accumulation and / or RNA expression in the lungs and / or liver does not occur, or is essentially absent, after administration of RNA lipoplex particles. In some embodiments, RNA accumulation and / or RNA expression occurs in antigen-presenting cells in the spleen, such as professional antigen-presenting cells, after administration of RNA lipoplex particles. Therefore, the RNA lipoplex particles of this 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 may comprise any lipid capable of forming particles to which one or more nucleic acid molecules are attached or to which one or more nucleic acid molecules are encapsulated.

[0360] In some embodiments, the LNP comprises one or more cationic lipids and one or more stabilized lipids. Examples of stabilized lipids include neutral lipids and pegylated lipids.

[0361] In some embodiments, LNPs include cationic lipids, neutral lipids, steroids, polymer-conjugated lipids; and RNA encapsulated within or associated with lipid nanoparticles.

[0362] In some embodiments, the LNP contains 40–55 mol percent, 40–50 mol percent, 41–49 mol percent, 41–48 mol percent, 42–48 mol percent, 43–48 mol percent, 44–48 mol percent, 45–48 mol percent, 46–48 mol percent, 47–48 mol percent, or 47.2–47.8 mol percent of cationic lipids. In some embodiments, the LNP contains approximately 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol percent of cationic lipids.

[0363] In some embodiments, neutral lipids are present at concentrations ranging from 5–15 mole percent, 7–13 mole percent, or 9–11 mole percent. In some embodiments, neutral lipids are present at concentrations of approximately 9.5, 10, or 10.5 mole percent.

[0364] In some embodiments, the steroid is present at concentrations ranging from 30–50 mole percent, 35–45 mole percent, or 38–43 mole percent. In some embodiments, the steroid is present at concentrations of approximately 40, 41, 42, 43, 44, 45, or 46 mole percent.

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

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

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

[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 conjugate lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure: [ka] or having a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, in the formula: R12 and R13 are each independently linear or branched saturated or unsaturated alkyl chains containing 10 to 30 carbon atoms, the alkyl chains may be interrupted by one or more ester bonds; w has an average value of 30 to 60. In some embodiments, R12 and R13 are each independently linear saturated alkyl chains containing 12 to 16 carbon atoms. In some embodiments, w has an average value of 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12 and R13 are each independently linear saturated alkyl chains 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, having, for example, the following structure: [ka]

[0372] In some embodiments, the cationic lipid component of LNP has the structure of formula (III): [ka] or having a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, in the formula: Either 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 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, independently, unsubstituted C1-C12 alkylenes or C1-C12 alkenylenes; G3 consists of C1-C24 alkylenes, C1-C24 alkenylenes, C3-C8 cycloalkylenes, and C3-C8 cycloalkenylenes; Ra is either H or a C1-C12 alkyl group; R1 and R2 are each independently a C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4, or -NR5C(=O)R4; R4 is a C1-C12 alkyl group; R5 is either H or a C1-C6 alkyl group; x is 0, 1, or 2.

[0373] In some of the aforementioned 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; Each instance of R6 is independently H, OH, or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0374] In some of the aforementioned 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 independent integers ranging from 1 to 12.

[0376] In any of the embodiments of formula (III) described above, one of L1 or L2 is -O(C=O)-. For example, in some embodiments, L1 and L2 are each -O(C=O)-. In any of the several different embodiments described above, 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 several different embodiments of formula (III), the lipid has one of the following structures: (IIIE) or (IIIF): [ka]

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

[0379] In some of the embodiments of formula (III) described above, n is an integer ranging from 2 to 12, for example, 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 embodiments of equation (III) described above, y and z are each an integer ranging from 2 to 10, independently of each other. For example, in some embodiments, y and z are each an integer ranging from 4 to 9 or 4 to 6, independently of each other.

[0381] In some of the embodiments of formula (III) described above, R6 is H. In other embodiments described above, R6 is a C1-C24 alkyl group. In other embodiments, R6 is an OH group.

[0382] In some embodiments of formula (III), G3 is not substituted. 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 embodiments of formula (III) described above, R1 or R2, or both, are C6-C24 alkenyls. For example, in some embodiments, R1 and R2 each independently have the following structure: [ka] During the ceremony: R7a and R7b, each appearing independently, are either H or C1-C12 alkyl groups; a is an integer between 2 and 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 embodiments described above of formula (III), the presence of at least one R7a is H. For example, in some embodiments, R7a is H whenever it appears. In other different embodiments described above, the presence of at least one R7b is a C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

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

[0386] In some of the aforementioned 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] A representative compound of formula (III). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0388] In some embodiments, 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, cationic lipids are present in LNPs in an amount of about 40 to about 50 mole percent. In some embodiments, neutral lipids are present in LNPs in an amount of about 5 to about 15 mole percent. In some embodiments, steroids are present in LNPs in an amount of about 35 to about 45 mole percent. In some embodiments, pegylated lipids are present in LNPs in an amount of about 1 to about 10 mole percent.

[0390] In some embodiments, the LNP contains 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 LNP contains 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 includes cationic lipids shown in the table above, for example, cationic lipids of formula (B) or formula (D), particularly cationic lipid of formula (D), RNA, neutral lipids, steroids, and pegylated lipids. 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 LNP includes a cationic lipid, which is an ionic lipid-like material (lipidoid). In some embodiments, the cationic lipid has 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] In some embodiments, the LNPs described herein may have an average diameter ranging 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 RNA polynucleotides formulated as particles, as disclosed herein. In some embodiments, the particles are or comprise lipid nanoparticles (LNPs) or lipoplex (LPX) particles.

[0398] In some embodiments, the RNA polynucleotides disclosed herein may be administered in the form of a pharmaceutical composition or drug, and may be administered in any suitable form of 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 that includes a pharmaceutically acceptable carrier and may also include one or more adjuvants, stabilizers, and the like. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatment.

[0401] The term "adjuvant" refers to compounds that prolong, enhance, or promote the 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, but not limited to, include LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Cytokines may include 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 is one example. Other suitable adjuvants used in this disclosure include lipopeptides, such as Pam3Cys. Pharmaceutical compositions according to this disclosure are typically used in “pharmaceutically effective amounts” and in “pharmaceutically acceptable preparations.” The term “pharmaceutically acceptable” means non-toxic material that does not interact with the action of the active ingredient of the pharmaceutical composition. The terms “pharmaceutically effective amount” or “therapeutably effective amount” mean an amount that achieves the desired reaction or effect, either alone or in combination with additional doses. In the case of treatment of a particular disease, the desired reaction preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, in particular interrupting or halting the course of the disease. Alternatively, the desired reaction in treatment of a disease may be the delay or prevention of 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 being treated, the severity of the disease, age, physiological condition, build, and weight, as well as the duration of treatment, the type of any accompanying treatments, the specific route of administration, and similar factors. Therefore, the dosage of the compositions described herein may depend on these various parameters. If the patient's response is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may 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 may contain one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

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

[0404] As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical composition of this 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 colorants. The term “diluent” refers to diluents and / or diluting agents. Furthermore, the term “diluent” includes one or more fluids, liquids, or solid suspensions and / or mixed media. Examples of suitable diluents include ethanol, glycerol, and water.

[0405] The term “carrier” refers to a component, which may be natural, synthetic, organic, or inorganic, that, when combined with an active ingredient, facilitates, enhances, or enables the administration of the pharmaceutical composition. As used herein, a carrier may be one or more suitable solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a target. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of this disclosure contain isotonic saline.

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

[0407] The carrier, excipient, or diluent of the pharmaceutical can be selected in relation to the intended route of administration and the standard practice of administering the pharmaceutical.

[0408] In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration may include enteral administration, which encompasses absorption through the gastrointestinal tract or parenteral administration. As used herein, “parenteral administration” refers to any mode of administration other than through the gastrointestinal tract, such as by intravenous injection. In preferred embodiments, the pharmaceutical compositions are formulated for intramuscular administration. In other embodiments, the pharmaceutical compositions are formulated for systemic administration (e.g., intravenous administration).

[0409] Characteristic evaluation In some embodiments, the RNA polynucleotides disclosed herein are characterized by exhibiting higher payload expression compared to appropriate reference comparators when evaluated in organisms administered a composition or medical preparation containing the RNA polynucleotide.

[0410] In some embodiments, the RNA polynucleotides disclosed herein are characterized by the observation of an extended duration of payload expression (e.g., prolonged expression) compared to a suitable reference comparator when evaluated in an organism administered a composition or medical preparation containing the RNA polynucleotide.

[0411] In some embodiments, the RNA polynucleotides disclosed herein are characterized by exhibiting reduced interaction with IFIT1 compared to a suitable reference comparator when evaluated in organisms administered a composition or medical preparation containing the RNA polynucleotide.

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

[0413] In some embodiments, the reference comparator includes organisms administered with an RNA polynucleotide that is otherwise similar but lacks the m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2 cap. In some embodiments, the reference comparator includes organisms administered with an RNA polynucleotide that is otherwise similar but lacks the cap proximal to the sequence disclosed herein. In some embodiments, the reference comparator includes organisms administered with an RNA polynucleotide that is otherwise similar but has a self-hybridized sequence.

[0414] In some embodiments, the RNA polynucleotides disclosed herein are characterized by exhibiting higher payload expression and extended expression duration (e.g., prolonged expression) compared to appropriate reference comparators when evaluated in organisms administered a composition or medical preparation containing the RNA polynucleotide.

[0415] In some embodiments, high 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 the RNA polynucleotide-containing composition or medical preparation.

[0416] In some embodiments, high expression is determined approximately 24 to 120 hours after administration of the RNA polynucleotide-containing composition or medical preparation. In some embodiments, high expression is determined approximately 24 to 110 hours, approximately 24 to 100 hours, approximately 24 to 90 hours, approximately 24 to 80 hours, approximately 24 to 70 hours, approximately 24 to 60 hours, approximately 24 to 50 hours, approximately 24 to 40 hours, approximately 24 to 30 hours, approximately 30 to 120 hours, approximately 40 to 120 hours, approximately 50 to 120 hours, approximately 60 to 120 hours, approximately 70 to 120 hours, approximately 80 to 120 hours, approximately 90 to 120 hours, approximately 100 to 120 hours, or approximately 110 to 120 hours after administration of the RNA polynucleotide-containing composition or medical preparation.

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

[0418] In some embodiments, the high expression of the payload is approximately 2 to 50 times. In some embodiments, the high expression of the payload is approximately 2 to 45 times, approximately 2 to 40 times, approximately 2 to 30 times, approximately 2 to 25 times, approximately 2 to 20 times, approximately 2 to 15 times, approximately 2 to 10 times, approximately 2 to 8 times, approximately 2 to 5 times, approximately 5 to 50 times, approximately 10 to 50 times, approximately 15 to 50 times, approximately 20 to 50 times, approximately 25 to 50 times, approximately 30 to 50 times, approximately 40 to 50 times, or approximately 45 to 50 times.

[0419] In some embodiments, high payload expression (e.g., prolonged expression period) 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 the RNA polynucleotide-containing composition or medical preparation.

[0420] In some embodiments, high payload expression persists for approximately 24 to 120 hours after administration of the RNA polynucleotide-containing composition or medical preparation. In some embodiments, high expression persists for approximately 24 to 110 hours, approximately 24 to 100 hours, approximately 24 to 90 hours, approximately 24 to 80 hours, approximately 24 to 70 hours, approximately 24 to 60 hours, approximately 24 to 50 hours, approximately 24 to 40 hours, approximately 24 to 30 hours, approximately 30 to 120 hours, approximately 40 to 120 hours, approximately 50 to 120 hours, approximately 60 to 120 hours, approximately 70 to 120 hours, approximately 80 to 120 hours, approximately 90 to 120 hours, approximately 100 to 120 hours, or approximately 110 to 120 hours after administration of the RNA polynucleotide-containing composition or medical preparation.

[0421] use In particular, methods for producing RNA polynucleotides comprising a 5' cap; a 5' UTR including a cap-proximal structure; and a payload-coding sequence, as well as methods for using such polynucleotides, are disclosed herein.

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

[0423] Furthermore, in some embodiments, a method for generating capped RNA, comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure is G * ppp(m1 2’-O )Includes N1pN2, N1 is complementary to the +1 position of the nucleic acid template, and N2 is complementary to the +2 position of the nucleic acid template. N1 and N2 are independently selected from A, C, G, or U. RNA comprises N3, which is complementary to the +3 position of the nucleic acid template and is any nucleotide, preferably A or C; N4, which is complementary to the +4 position 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 the +5 position of the nucleic acid template and is any nucleotide. G * The structure is as follows: [ka] Includes, [ka] G * However, this represents a bond that is attached to the first phosphorus atom of the ppp group, R 1 It is CH3, and R 2 is OH or O-CH3, and R 3 A method is disclosed herein in which O-CH3 is used.

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

[0425] In some embodiments, methods are disclosed herein that involve administering to a subject a pharmaceutical composition comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles as disclosed herein.

[0426] In some embodiments, a method for inducing an immune response in a subject is disclosed herein, which includes administering to the subject a pharmaceutical composition comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles as disclosed herein.

[0427] In some embodiments, a method for vaccinating a target is disclosed herein, comprising administering to the target a pharmaceutical composition comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipoplex (LPX) particles as disclosed herein.

[0428] In some embodiments, a method is provided herein for reducing the interaction of an RNA polynucleotide with IFIT1, including the 5' cap and a sequence near the cap containing positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, the method comprising the steps of: preparing a mutant of the RNA polynucleotide different from the parent RNA polynucleotide by substituting one or more residues in the sequence near the cap; and determining that the interaction of the mutant with IFIT1 is reduced compared to that of the parent RNA polynucleotide. In some embodiments, the determination includes administering the RNA polynucleotide or a composition containing the same to a cell or organism.

[0429] In some embodiments, a method is disclosed herein for increasing the translatability of an RNA polynucleotide comprising a 5' cap, a sequence near the cap including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload, the method comprising the steps of: preparing a mutant of the RNA polynucleotide different from the parent RNA polynucleotide by substituting one or more residues in the sequence near the cap; and determining that the expression of the mutant is increased compared to that of the parent RNA polynucleotide. In some embodiments, determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or organism. In some embodiments, the increase in translatability is evaluated by the increase in the expression and / or the persistence of the expression of the payload. In some embodiments, the increase in expression is determined at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments, the increase in expression is at least 2 to 10 times. In some embodiments, the increase in expression is about 2 to 50 times. In some embodiments, the high expres...

Claims

1. A composition or medical preparation comprising an RNA polynucleotide containing a payload encoding sequence, The RNA polynucleotide optionally contains one or more modified uridines instead of 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 This includes, where N ５ A composition or medicinal preparation in which is selected from A, G, C, U or modified uridine.

2. The composition or medical preparation according to claim 1, wherein the RNA polynucleotide comprises human alpha globin (hAg) 5'UTR or a fragment thereof, and the hAg 5'UTR or fragment thereof optionally comprises modified uridine instead of one or more uridines.

3. The composition or medical preparation according to claim 2, wherein the RNA polynucleotide is a Kozak sequence, and further comprises a Kozak sequence optionally containing modified uridines instead of one or more uridines.

4. The composition or medical preparation according to claim 3, wherein the RNA polynucleotide comprises the nucleotide sequence described in Sequence ID No. 27, wherein optionally the nucleotide sequence includes modified uridines instead of one or more uridines.

5. The composition or medical preparation according to claim 1, wherein the RNA polynucleotide further comprises a 3'UTR sequence, optionally comprising a 3'UTR sequence containing modified uridine instead of one or more uridines.

6. The composition or medical preparation according to claim 5, wherein the 3'UTR sequence is the nucleotide sequence of SEQ ID NO: 13, and optionally comprises a nucleotide sequence containing modified uridine instead of one or more uridines.

7. The composition or medical preparation according to claim 1, wherein the RNA polynucleotide further comprises a polyA sequence.

8. The composition or medical preparation according to claim 7, wherein the polyA sequence comprises at least 100 nucleotides.

9. (i) The polyA sequence is a suspended sequence of adenine nucleotides; and / or (ii) The composition or medical preparation according to claim 8, wherein 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.

10. The composition or medical preparation according to claim 9, wherein the polyA sequence is the nucleotide sequence of SEQ ID NO: 14, and optionally comprises a nucleotide sequence containing modified uridines instead of one or more uridines.

11. The RNA polynucleotide has, in the 5' to 3' orientation, 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 medicament according to claim 1.

12. The composition or medical preparation according to claim 1, wherein the RNA polynucleotide comprises modified uridine instead of all uridines.

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

14. The composition or medical preparation according to claim 1, wherein the sequence encoding the payload comprises: a sequence encoding a protein-substituted 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 medical preparation according to claim 14, wherein the payload encoding sequence comprises a sequence encoding an antigenic polypeptide.

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

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

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

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

20. A pharmaceutical composition containing RNA polynucleotides, which is formulated as particles, The RNA polynucleotide contains a sequence that codes for the payload. The RNA polynucleotide optionally contains one or more modified uridines instead of 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 This includes, where N ５ A pharmaceutical composition in which is selected from A, G, C, U, or modified uridine.

21. The pharmaceutical composition according to claim 20, wherein the particles are lipid nanoparticles (LNPs) or lipoplex (LPX) particles.

22. The pharmaceutical composition according to claim 20, wherein the particles are LNPs comprising cationically ionic lipids; sterols; phospholipids; and pegylated lipids.

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

24. The pharmaceutical composition according to claim 20, which is formulated as a solid.

25. The RNA polynucleotides are oriented from 5' to 3', with 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 payload encoding sequence, a 3'UTR sequence, and a polyA sequence.

26. The pharmaceutical composition according to claim 20, wherein the RNA polynucleotide comprises modified uridine instead of at least one uridine molecule.

27. The pharmaceutical composition according to claim 26, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

28. The pharmaceutical composition according to claim 20, wherein the RNA polynucleotide comprises modified uridine instead of all uridine.

29. The pharmaceutical composition according to claim 28, wherein the modified uridine is N1-methyl-pseudouridine (m1ψ).

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