RNA constructs and uses thereof

Abstract

Disclosed herein are RNA polynucleotides comprising a 5' cap, a 5' UTR comprising a cap-proximal sequence as disclosed herein, and a sequence encoding a payload. Also disclosed herein are compositions and medical preparations comprising same, as well as compositions and methods of making and using same.

Description

[Technical Field]

[0001] The use of RNA polynucleotides as therapeutic agents is an emerging field. Summary of the Invention

[0002] This disclosure identifies particular challenges that can be associated with the in vitro production of RNA, e.g., RNA therapeutics.

[0003] For example, in some embodiments, the present disclosure identifies the cause of certain problems that may be encountered in the expression of polypeptides encoded by RNA therapeutics. Among other things, the present disclosure provides techniques for improving capping efficiency (e.g., the proportion of capped transcripts in an in vitro transcription reaction), the quality of an RNA preparation (e.g., an in vitro transcribed RNA, e.g., the amount of short polynucleotide by-products produced), the translation efficiency of an RNA encoding a payload, and / or the expression of a polypeptide payload encoded by the RNA. In some embodiments, the translation efficiency and / or expression of an RNA-encoded payload can be improved by an RNA polynucleotide comprising a 5' cap as defined and described herein, a 5' UTR comprising a cap-proximal sequence as defined and described herein, and a sequence encoding a payload. While not wishing to be bound by theory, the present disclosure proposes that improved RNA transcription, capping efficiency, translation efficiency, and / or polypeptide payload expression, and / or reduced transcription by-product formation can be achieved by using the 5' cap structures described herein in combination with specific transcription start site sequences in template DNA.

[0004] In some embodiments, the present disclosure recognizes that certain transcription start sites, when used with, for example, certain caps, provide improved RNA transcription, capping efficiency, translation efficiency, and / or polypeptide payload expression, and / or reduced by-product formation.

[0005] T7 RNA polymerase most commonly utilizes GGG transcription start sites (e.g., producing RNA in which the first three residues, N1, N2, and N3, are each "G") and has been reported to prefer "G" as the initiating residue (e.g., producing RNA in which the first residue, N1, is "G"). Conrad, et al. (2020) Communications Biology 3:439. A study comparing T7 transcription of templates with different initiating residues reported that the level of transcripts beginning with "A" was only 25% of the level observed for transcripts beginning with "G." Milligan, et al. (1987) Nucleic Acids Research 15:8783-8798.

[0006] The 3' terminus of commonly used dinucleotide caps also contains a "G" (e.g., [ka] ) is utilized. Grudzien-Nogalska, et al. RNA 13:1745-1755. Indeed, certain such caps, such as β-S-ARCA, offer advantages, including greater resistance to human decapping enzymes (Kowalska et al. (2008) RNA 14:1119-1131) and interferon-inducible protein with tetratricopeptide repeats (IFIT), which inhibits Cap0-dependent translation (Diamond et al. (2014) Cytokine & Growth Factor Reviews 25:543-550, and Miedziak et al. (2019) RNA 25:58-68). However, poor capping efficiency can be observed. Without wishing to be bound by any particular theory, the present disclosure proposes that competition with GTP in the transcription reaction may contribute to such poor capping efficiency.

[0007] Furthermore, the present disclosure provides the surprising discovery that DNA template sequence, particularly the sequence of the transcription start site in the DNA template, can affect the usefulness of particular caps (e.g., 3'-terminal G caps) in in vitro transcription reactions such as those described herein. Specifically, for example, the present disclosure shows that DNA templates containing a GGG transcription start sequence can promote the production of undesirable short poly(G) by-products, for example, when a 3'-terminal cap is utilized. Thus, the present disclosure identifies the source of problems with certain in vitro transcription strategies and further provides surprising insights into in vitro transcription, including solutions to such problem(s).

[0008] For example, the present disclosure provides the insight that RNA transcripts containing particular initiation sequences (e.g., those containing a pyrimidine base (C or U) at the +2 position, such as GCG, GUG, or GCA) exhibit particular advantages compared to a purine base (A or G) at the same position. For example, in some embodiments, the present disclosure provides the insight that RNA transcripts with a pyrimidine base (C or U) at the +2 position, compared to a purine base (A or G) at the same position, such as GGG as the initial sequence, exhibit improved transcription and / or translation, higher capping efficiency, less immunogenicity, and / or improved and / or prolonged expression.

[0009] Additionally or alternatively, in some embodiments, the present disclosure recognizes that particular 5' cap structures, when paired with particular transcription start sites, provide improved RNA transcription, translation efficiency, and / or polypeptide payload expression. In some embodiments, the present disclosure recognizes that particular 5' cap structures (e.g., m2 (7,3′O) Gppp (m2′O)In some embodiments, the present disclosure also provides that a specific trinucleotide 5' cap structure (e.g., m2) when paired with a specific transcription start site (e.g., AGN, e.g., AGA) results in higher capping efficiency, less immunogenicity, and significantly improved and prolonged expression compared to transcripts containing other 5' cap structures in combination with other transcription start sequences (e.g., the β-S-ARCA cap used in combination with a GGG transcription start sequence). (7,3′O) Gppp (m2′O) ApG) can be used in combination with a transcription initiation site that is not perfectly complementary to the 5' cap (e.g., in some embodiments, the present disclosure provides (7,3′O) Gppp (m2′O) (Provided that ApG can be used in combination with GGG or GCG transcription start sites.) This can be advantageous because it allows for the incorporation of a 5' cap with certain desired properties (e.g., reduced immunogenicity) without creating a new DNA template that is complementary to the selected 5' cap.

[0010] In some embodiments, the present disclosure provides the insight that RNA generated with a particular ARCA cap structure, when paired with a particular transcription start site other than a GGG start sequence, which is believed to be the preferred start site for the ARCA cap, can produce surprisingly high protein expression compared to RNA generated with the same cap and a GGG start sequence. For example, in some embodiments, the present disclosure demonstrates that RNA generated with a β-S-ARCA D1 cap ("D1 cap") and a GCG start sequence produces surprisingly high protein expression compared to a D1 cap with a GGG start sequence.

[0011] Additionally or alternatively, in some embodiments, the present disclosure recognizes that the identity of particular sequence(s) proximal to the 5' cap can affect the efficiency of RNA transcription and / or translation of the associated payload. Without wishing to be bound by any particular theory, the present disclosure proposes that eIF4E competes with IFIT1 for binding to an RNA polynucleotide based on the identity of one or more nucleotides downstream of the 5' cap, e.g., the cap-proximal sequences disclosed herein.

[0012] Thus, in some embodiments, the present disclosure provides, inter alia, compositions or medical preparations comprising an RNA polynucleotide comprising (i) a 5' cap; (ii) a cap-proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also disclosed herein are methods of making the same and methods of using the same, e.g., to induce an immune response in a subject.

[0013] In some embodiments, the present disclosure recognizes that a GGG transcription start site, when paired with a particular 5' cap as defined and described herein, provides improved RNA transcription, translation efficiency, and / or polypeptide payload expression. For example, in some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is C and N2 is G; (b) N1 is U and N2 is G; or (c) N1 is A and N2 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 and N4 are G and N5 is selected from A, C, G, and U.

[0014] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are each G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is G and each of N4 and N5 is selected from A, C, G, and U.

[0015] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a dinucleotide cap structure N1 and a sequence comprising N2N3N4N5 at positions +2, +3, +4, and +5 of an RNA polynucleotide, respectively, wherein each of N2 and N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0016] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is G; (b) N1 is U, N2 is G, and N3 is G; or (c) N1 is A, N2 is G, and N3 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein N4 is G and N5 is selected from A, C, G, and U.

[0017] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is G, N2 is G, and N3 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0018] In some embodiments, the present disclosure recognizes that a pyrimidine at the +2 position of a transcription start site can improve capping efficiency (e.g., the proportion of capped transcripts in an in vitro transcription reaction), the quality of an RNA preparation (e.g., an in vitro transcribed RNA, e.g., the amount of short polynucleotide by-products produced), the translation efficiency of an RNA encoding a payload, and / or the expression of a polypeptide payload encoded by the RNA. In some embodiments, such technical effects may be observed regardless of the identity of the 5' UTR, the capping method (e.g., enzymatic capping vs. co-transcriptional capping), the cap structure (e.g., Cap0, Cap1, or Cap2), the coding sequence, the type of ribonucleotide (e.g., modified nucleotides vs. unmodified nucleotides), or a combination thereof.

[0019] For example, in some embodiments, the present disclosure recognizes that a GCG transcription start site, when paired with a particular 5' cap as defined and described herein, provides improved RNA transcription, translation efficiency, and / or polypeptide payload expression. For example, in some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is G; (b) N1 is U and N2 is G; (c) N1 is A and N2 is G; or (d) N1 is C and N2 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is C, N4 is G, and N5 is selected from A, C, G, and U.

[0020] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 is G and N2 is C; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is G and each of N4 and N5 is selected from A, C, G, and U.

[0021] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) The composition or medical preparation is provided, wherein the cap-proximal sequence comprises N1 of the dinucleotide cap structure and sequences comprising N2N3N4N5 at positions +2, +3, +4, and +5 of the RNA polynucleotide, respectively, where N2 is a pyrimidine (e.g., C or U) and N3, N4, and N5 are each selected from A, C, G, and U. In some embodiments, N3 is G or A, and N4 and N5 are each separately and independently selected from A, C, G, and U.

[0022] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a dinucleotide cap structure N1 and a sequence comprising N2N3N4N5 at positions +2, +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N2 is C, N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0023] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is C; (b) N1 is U, N2 is G, and N3 is C; or (c) N1 is A, N2 is G, and N3 is C; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein N4 is G and N5 is selected from A, C, G, and U.

[0024] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is G, N2 is C, and N3 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0025] In some embodiments, the present disclosure recognizes that a CGC transcription start site, when paired with a particular 5' cap as defined and described herein, provides improved RNA transcription, translation efficiency, and / or polypeptide payload expression. For example, in some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is C; (b) N1 is U and N2 is C; (c) N1 is A and N2 is C; or (d) N1 is C and N2 is C; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is G, N4 is C, and N5 is selected from A, C, G, and U.

[0026] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, N1 is C, and N2 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is C, and each of N4 and N5 is selected from A, C, G, and U.

[0027] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is G, N2 is C, and N3 is G; (b) N1 is U, N2 is C, and N3 is G; or (c) N1 is A, N2 is C, and N3 is G; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein N4 is C and N5 is selected from A, C, G, and U.

[0028] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is C, N2 is G, and N3 is C; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0029] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, N1 is A, and N2 is U; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of an RNA polynucleotide, respectively, wherein N3 is A and each of N4 and N5 is selected from A, C, G, and U.

[0030] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is A, N2 is U, and N3 is A; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of an RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0031] Additionally or alternatively, in some embodiments, the present disclosure recognizes that certain 5' cap structures (e.g., as defined and described herein) provide improved RNA transcription, translation efficiency, and / or polypeptide payload expression when paired with particular transcription start sites. In some embodiments, the 5' cap is a dinucleotide cap structure (e.g., comprising N1, where N1 is defined and described herein), a trinucleotide cap structure (e.g., comprising N1pN2, where N1 and N2 are as defined and described herein), or a tetranucleotide cap structure (e.g., comprising N1pN2pN3, where N1, N2, and N3 are as defined and described herein). In some embodiments, the 5' cap comprises G*, G* is a structure of formula (I): [ka] or a salt thereof, wherein R 2 , R 3 and X is as defined herein.

[0032] In some embodiments, the present disclosure provides a method for determining G*N 1 a 5' cap having a dinucleotide cap structure comprising: 1

[0013] The present disclosure recognizes that a 5' cap, where G is G, in combination with a GCG transcription start site exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression. In some embodiments, the present disclosure provides a 5' cap, where G*N is G, in combination with a GCG transcription start site. 1 a 5' cap having a dinucleotide cap structure comprising: 1 recognizes that a 5' cap, which is C, in combination with a CGC transcription start site exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0033] In some embodiments, the present disclosure provides a method for determining G*N 1 It is recognized that a 5' cap having a trinucleotide cap structure comprising pN2, wherein N1 and N2 are selected from one of the following combinations: (a) N1 is C and N2 is G; (b) N1 is U and N2 is G; (c) N1 is A and N2 is G; or (d) N1 and N2 are each G, when combined with a GGG transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0034] In some embodiments, the present disclosure provides a method for determining G*N 1 It is recognized that a 5' cap having a trinucleotide cap structure comprising pN2, wherein N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is G; (b) N1 is U and N2 is G; (c) N1 is A and N2 is G; (d) N1 is C and N2 is G; or (e) N1 is G and N2 is C, when combined with a GCG transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0035] In some embodiments, the present disclosure provides a method for determining G*N 1It is recognized that a 5' cap having a trinucleotide cap structure comprising pN2, wherein N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is C; (b) N1 is U and N2 is C; (c) N1 is A and N2 is C; (d) N1 is C and N2 is C; or (e) N1 is C and N2 is G, when combined with a CGC transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0036] In some embodiments, the present disclosure provides a method for determining G*N 1 It is recognized that a 5' cap having a tetranucleotide cap structure comprising pN2pN3, wherein N1 and N2 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is G; (b) N1 is U, N2 is G, and N3 is G; (c) N1 is A, N2 is G, and N3 is G; or (d) N1 is G, N2 is G, and N3 is G; when combined with a GGG transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0037] In some embodiments, the present disclosure provides a method for determining G*N 1 It is recognized that a 5' cap having a tetranucleotide cap structure comprising pN2pN3, wherein N1 and N2 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is C; (b) N1 is U, N2 is G, and N3 is C; (c) N1 is A, N2 is G, and N3 is C; or (d) N1 is G, N2 is C, and N3 is G; when combined with a GCG transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0038] In some embodiments, the present disclosure provides a method for determining G*N 1It is recognized that a 5' cap having a tetranucleotide cap structure comprising pN2pN3, wherein N1 and N2 are selected from one of the following: (a) N1 is G, N2 is C, and N3 is G; (b) N1 is U, N2 is C, and N3 is G; (c) N1 is A, N2 is C, and N3 is G; or (d) N1 is C, N2 is G, and N3 is C, when combined with a CGC transcription start site, exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression.

[0039] In some embodiments, the present disclosure provides a 5' cap having a trinucleotide cap structure comprising G*ApG, e.g., m2 (7,3′O) Gppp (m2′O) It is recognized that ApG in combination with an AGN (e.g., AGA or AGC) transcription start site exhibits improved RNA transcription, translation efficiency, and / or polypeptide payload expression, for example, compared to a GGG transcription start site. For example, in some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a 5' cap of A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, where N3-N5 are selected from A, C, G, and U.

[0040] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a 5' cap comprising A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is A, and N4 and N5 are selected from A, C, G, and U.

[0041] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a 5' cap of A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 and N4 are G and N5 is selected from A, C, G, and U.

[0042] In some embodiments, the present disclosure provides a composition or medical preparation comprising an RNA polynucleotide, a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O)A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides a composition or medical preparation comprising a 5' cap A1 and G2 and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is C, N4 is G, and N5 is selected from A, C, G, and U.

[0043] The present disclosure provides a composition or medical preparation comprising a capped RNA polynucleotide encoding a gene product, wherein the RNA polynucleotide has the formula: [ka] Including, In the formula, R 1 is CH3 and R 2 and R 3 is as defined above and herein; B1 is any nucleobase, preferably A; B2 is any nucleobase, preferably G; B3 is any nucleobase, preferably A or C; B4 is any nucleobase; B5 is any nucleobase, The composition or medical preparation is provided such that, when the RNA polynucleotide is administered to a subject, the level of expression of the encoded gene product at about 6 hours after administration and at about 48 hours after administration does not differ by more than 5-fold.

[0044] Provided herein are pharmaceutical compositions comprising the RNA polynucleotides disclosed herein. In some embodiments, the pharmaceutical compositions comprise the compositions or medical preparations disclosed herein.

[0045] Also provided herein are methods of producing pharmaceutical compositions comprising the RNA polynucleotides disclosed herein, e.g., by combining an RNA polynucleotide with a lipid to form a lipid nanoparticle that encapsulates the RNA.

[0046] The present disclosure provides a nucleic acid template suitable for producing capped RNA, 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 sequence encoding a 5' UTR, a sequence encoding a payload, a sequence encoding a 3' UTR, and a sequence encoding a polyA sequence.

[0047] 1. An in vitro transcription reaction comprising: (i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence disclosed herein; (ii) a polymerase; and (iii) Provided herein are in vitro transcription reactions comprising an RNA polynucleotide.

[0048] Also provided herein are RNA polynucleotides isolated from the provided in vitro transcription reactions.

[0049] Also provided herein are compositions comprising DNA polynucleotides comprising sequences complementary to the provided RNA polynucleotide sequences. In some embodiments, the DNA polynucleotides disclosed herein can be used to transcribe the RNA polynucleotides disclosed herein.

[0050] The present disclosure provides methods that include administering to a subject a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, for example, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein. In some embodiments, the compositions, pharmaceutical preparations, and therapeutic agents provided herein increase expression of the RNA when administered in an LNP formulation.

[0051] Also provided herein are methods of inducing an immune response in a subject, comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, for example, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein.

[0052] Provided herein are methods of vaccinating a subject, for example, by administering a pharmaceutical composition comprising an RNA polynucleotide disclosed herein, formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle, as disclosed herein.

[0053] The present disclosure provides a method for reducing the interaction of an RNA polynucleotide comprising a 5' cap and a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide with IFIT1, the method comprising the steps of preparing a mutant form of the RNA polynucleotide that differs from a parent RNA polynucleotide by substitution of one or more residues within the cap-proximal sequence, and determining that the interaction of the mutant form with IFIT1 is reduced compared to that of the parent RNA polynucleotide.

[0054] Also disclosed herein is a method for producing a polypeptide, comprising providing an RNA polynucleotide comprising a 5' cap, a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload; the RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition comprising the same is administered, higher expression and / or a prolonged duration of expression of the payload is observed compared to a suitable reference comparator.

[0055] Disclosed herein are methods for increasing the translatability of an RNA polynucleotide comprising a 5' cap, a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload, the method comprising: providing a variant of the RNA polynucleotide that differs from the parent RNA polynucleotide by substitution of one or more residues within the cap-proximal sequence; and determining that expression of the variant is increased compared to that of the parent RNA polynucleotide.

[0056] Also provided herein are methods for improving the capping efficiency of RNA transcripts (e.g., the proportion of capped transcripts in an in vitro transcription reaction), the improvement comprising including a pyrimidine at the +2 position of the transcription start site in a DNA template for in vitro transcription. Exemplary pyrimidines include, for example, C or U. In some embodiments, the +1 position of the transcription start site is G. In some embodiments, the +3 position of the transcription start site is a pyrimidine or purine. In some embodiments, the transcription start site can be GCG, GUG, or GCA. In some embodiments, such improvements can be observed regardless of the identity of the 5'UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structure (e.g., Cap0, Cap1, or Cap2), coding sequence, type of ribonucleotide (e.g., modified nucleotides vs. unmodified nucleotides), formulation (e.g., lipoplexes vs. lipid nanoparticles), or a combination thereof.

[0057] Also provided herein are methods for improving the quality of an RNA preparation (e.g., in vitro transcribed RNA, e.g., the amount of short polynucleotide by-products produced), the improvement comprising including a pyrimidine at the +2 position of the transcription start site in a DNA template for in vitro transcription. Exemplary pyrimidines include, for example, C or U. In some embodiments, the +1 position of the transcription start site is G. In some embodiments, the +3 position of the transcription start site is a pyrimidine or purine. In some embodiments, the transcription start site can be GCG, GUG, or GCA. In some embodiments, such improvements can be observed regardless of the identity of the 5'UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structure (e.g., Cap0, Cap1, or Cap2), coding sequence, type of ribonucleotide (e.g., modified nucleotides vs. unmodified nucleotides), formulation (e.g., lipoplexes vs. lipid nanoparticles), or combinations thereof.

[0058] Also provided herein are methods for improving the translation efficiency of an RNA encoding a payload and / or the expression of a polypeptide payload encoded by the RNA, the improvement comprising including a pyrimidine at the +2 position of the transcription start site in a DNA template for in vitro transcription. Exemplary pyrimidines include, for example, C or U. In some embodiments, the +1 position of the transcription start site is G. In some embodiments, the +3 position of the transcription start site is a pyrimidine or a purine. In some embodiments, the transcription start site can be GCG, GUG, or GCA. In some embodiments, such improvements can be observed regardless of the identity of the 5'UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structure (e.g., Cap0, Cap1, or Cap2), coding sequence, type of ribonucleotide (e.g., modified nucleotides vs. unmodified nucleotides), formulation (e.g., lipoplex vs. lipid nanoparticle), or a combination thereof.

[0059] 1. A method for providing a framework for an RNA polynucleotide comprising a 5′ cap, a cap-proximal sequence, and a payload sequence, the method comprising: evaluating at least two variants of an RNA polynucleotide, Each variant contains the same 5' cap and payload sequences; The variants differ from each other at one or more specific residues in the cap-proximal sequence; The evaluating step includes determining the expression level and / or duration of expression of the payload sequence. Also provided herein is a method comprising the steps of: evaluating; and selecting at least one combination that exhibits higher expression compared to at least one other combination of 5' cap and cap-proximal sequences. [Brief explanation of the drawings]

[0060] [Figure 1] Chemical structures of specific functional caps characterized herein. Red circles indicate modifications (-CH3) at the C2' or C3' position of the 7-methylguanosine of each anti-reverse cap analog (ARCA) to prevent reverse orientation. The β-S-ARCA dinucleotide cap (m2 7,2'OGppspG) has a single phosphorothioate moiety at the β position of the 5',5'-triphosphate bridge (blue circle). This cap exists in two diastereomers, designated D1 and D2, according to the fractionation of an HPLC run (Kowalska, et al. (2008) RNA 14:1119-1131). The CleanCap AG 3'OMe trinucleotide cap (CC413-m2 (7,3'O)Gppp(m2'O)ApG) contains another methyl group (-CH3), highlighted by an orange circle, at the 2'OH position of the first ribose sugar of the first nucleotide on the opposite side of the dinucleotide cap. The non-ARCA version of CC413 corresponds to the canonical CleanCap AG (CC113-m2(7)Gppp(m2′O)ApG) with no modification at the C3′ position of the 7-methylguanosine. [Figure 2]Characteristics of in vitro transcribed mRNAs disclosed herein. (A) The yields and corresponding fractions of mRNA encoding mouse erythropoietin (EPO mRNA) or firefly luciferase (LUC mRNA) were analyzed by spectrophotometry and electrophoresis on a 1.4% agarose gel, respectively. All mRNAs contain N1-methylpseudouridine (m1Ψ) nucleoside modifications. To examine the mRNA capping efficiency, a ribozyme assay was performed, followed by homemade urea polyacrylamide gel electrophoresis (Urea PAGE). To quantify the capping reaction, the percentage of capped transcripts in the total pool of capped and uncapped mRNA was determined. (B) Quantification of luminescence signals obtained by LUC mRNA that was uncapped (none) or capped with anti-reverse cap analog (ARCA-G), β-S-ARCA (D1), enzyme cap (Ecap1), and CleanCap AG 3'OMe (CC413) using a rabbit reticulocyte lysate translation system. All data are expressed as the mean ± standard deviation (SEM) of values ​​obtained from quadruplicate data points. RLU = relative light units. G = guanosine; A = adenosine. [Figure 3]Durability and biodistribution of luciferase translated from m1Ψ-modified LUC mRNA in mice. (A) Representative IVIS images of a group of four BALB / c mice intravenously injected with 3.0 μg of TransIT-complexed LUC mRNA containing different 5' cap structures (ARCA-G, D1, Ecap1, and CC413). LUC activity was measured at the indicated time points. Relative luminescence images are shown, and the scale of mean brightness is indicated. (B) Quantification of bioluminescence signals measured in mice 6, 24, and 48 hours after injection of 3.0 μg of TransIT-complexed mRNA encoding firefly luciferase. All data are expressed as the mean ± standard deviation of values ​​obtained from four animals per group. A p-value of 0.05 or less was considered statistically significant (asterisk indicates p<0.05). RLU = relative light units; A = adenosine, G = guanosine. hAg = 5'UTR derived from human α-globin mRNA; ARCA-G = anti-reverse cap analog; D1 = β-S-ARCA; Ecap1 = enzymatic cap; CC413 = CleanCap AG 3'OMe. [Figure 4] Biological activity of mouse EPO-encoding mRNA prepared with different 5'-cap structures. Mice received a single intravenous injection of 3.0 μg of mRNA capped with ARCA-G, β-S-ARCA (D1), enzymatic capping (Ecap1), or CleanCap AG 3'OMe (CC413) complexed with TransIT mRNA Reagent. (A) Plasma EPO levels were determined by ELISA 6, 24, 48, and 72 hours after injection. (B) Hematocrit was measured at the indicated time points using 20 μl of blood. Three animals per group were analyzed. Error bars represent standard error of the mean (SEM); values ​​less than 0.05 were considered statistically significant (asterisks indicate p<0.05). Mock = TransIT Reagent (no RNA sample); A = adenosine; G = guanosine. [Figure 5]Reduced immunogenicity of in vitro transcribed mRNA capped with CleanCap AG. (A) Heatmap showing changes in the levels of symbolized proinflammatory cytokines and chemokines in the Mesoscale Disorder (MSD) of human peripheral blood mononuclear cells (PBMCs) treated for 24 hours with cationic lipid-complexed mRNA (RNA-LPX) carrying various 5' cap structures (anti-reverse cap analog (ARCA), β-S-ARCA (D1) enzyme cap (Ecap1), or CleanCap AG 3'OMe (CC413)). (B) Capped mRNA samples were derived from a (Discovery) immunoassay. Each capped mRNA was used at three different final concentrations, as indicated. Values ​​obtained from PBS-treated cells were used as a baseline for comparison. Results are expressed as the mean ± standard deviation of values ​​from three independent experiments performed in triplicate in three donors. (B) Short abortive by-products generated during in vitro transcription of capped mRNA starting with GGG or AGA were separated by homemade denaturing urea polyacrylamide gel electrophoresis. A single-stranded RNA (ssRNA) ladder was used as a marker for approximating the size of small transcripts. G = guanosine; A = adenosine; TNF-α = tumor necrosis factor alpha; IFN-γ = interferon gamma; IL-6 = interleukin 6; IL-1β = interleukin 1 beta; MIP-1β = macrophage inflammatory protein 1 beta. [Figure 6] Physiological responses to injection of EPO mRNA capped with conventional or anti-reverse cap1 analogs in mice. Plasma EPO levels and hematocrit were determined in mice after intravenous injection of 3.0 μg of CC113 (CleanCap AG) or CC413 (CleanCap AG 3'OMe) EPO mRNA complexed with TransIT on the indicated days. Error bars represent the standard error of the mean for data sets obtained from three mice per group. [Figure 7]Schematic diagram of each mRNA used in Example 1. The in vitro transcribed mRNAs contain a 5' cap (anti-reverse cap analog (ARCA-G), phosphorothioate-containing cap analog (β-S-ARCA), Ecap1 (enzyme cap), or CC413 (CleanCap AG 3'OMe)); GGG and AGA as two different initiation sites (S); the 5' UTR of human alpha globin (hAg) mRNA, the coding sequence (CDS) of mouse erythropoietin (EPO-582 nt) or firefly luciferase (Luc-1,653 nt), an FI element as the 3' UTR, and an encoded poly(A) tail (AAA100, 100 nt) (A30LA70) interrupted by a linker (L, 10 nt). All mRNAs used in this study contain N1-methylpseudouridine (m1Ψ) nucleoside modification. UTR = untranslated region, G = guanosine, A = adenosine [Figure 8] Comparison of cytokine and chemokine levels in human PBMCs transfected with mRNA capped with CleanCap AG 3'OMe, with or without nucleoside modifications. mRNA capped with CleanCap AG 3'OMe (CC413) containing 1-methylpseudouridine (m1Ψ) or uridine (U) was synthesized by in vitro transcription and then purified on cellulose. Human peripheral blood mononuclear cells (PBMCs) were transfected with mRNA complexed with cationic lipids (RNA-LPX) at final concentrations of 0.2, 0.5, and 1.5 μg / ml. Supernatants were collected 24 h posttransfection, and the levels of the indicated proinflammatory cytokines and chemokines were determined by MSD. Data presented in heat maps are from three independent experiments performed in three donors. TNF-α = tumor necrosis factor alpha; IFN-γ = interferon gamma; IL-6 = interleukin 6; IL-1β = interleukin 1 beta; MIP-1β = macrophage inflammatory protein 1 beta. [Figure 9-1]D1-capped mRNA starting with GGA provides improved expression compared to D1-capped mRNA starting with AGA. (A) Quantification of plasma concentrations of mouse EPO (mEPO) in mice 6, 24, 48, and 72 hours after IV injection of 3 μg of TransIT-formulated RNA containing modified nucleotides (m1Ψ) encoding mEPO and containing a cap structure (D1-cap or CC413-cap) with an initiation sequence (e.g., GGA or AGA) and a TEV 5'UTR. (B) Quantification of luciferase expression in mice 6 and 24 hours after injection of 3 μg of TransIT-formulated mRNA transcripts containing modified nucleotides (m1Ψ) encoding firefly luciferase and containing a cap structure (D1-cap or CC413-cap) with an initiation sequence (GGA or AGA) and a TEV 5'UTR. [Figure 9-2] Same as above. [Figure 10] The beneficial effect of a pyrimidine base at the +2 position of IVT mRNA as described herein on the performance of IVT mRNA. (A) Quantification of plasma concentrations of mouse EPO (mEPO) in mice 6, 24, and 48 hours after IV injection of TransIT-formulated mRNA encoding mEPO and containing a modified nucleotide (mΨ) containing a cap structure (D1 cap) with an initiation sequence (GGG, GAG, GGA, GGU, GGC, GUG, GCA, or GCG) and the TEV 5'UTR. In the figure, R represents a purine nucleotide and Y represents a pyrimidine nucleotide. (B) Hematocrit levels in the same mice 0, 7, and 14 days after injection of the RNA. [Figure 11]The effect of the pyrimidine base at the +2 position of IVT mRNA on IVT mRNA performance is independent of the 5' cap. (A) Quantification of mouse EPO (mEPO) in the plasma of mice 6, 24, 48, and 72 hours after injection of 3 μg of TransIT-formulated mRNA encoding mEPO and containing a modified nucleotide (m1Ψ) containing a cap structure (D1 cap, enzymatically incorporated cap (Ecap1), or CC413 cap) with a start sequence (GGG, GGA, GUG, or GCG) and the TEV 5' UTR. mRNA containing a CC413 cap with a start sequence of AGC was used as a control for comparison. (B) Hematocrit levels measured in the same mice 0, 7, and 14 days after injection. [Figure 12] The pyrimidine base effect is independent of nucleoside modifications and / or the 5'UTR of the IVT mRNA. (A) Plasma concentrations of mouse EPO (mEPO) in mice 6, 24, 48, and 72 hours after injection of 3 μg of TransIT-formulated mRNA transcripts encoding mEPO and containing a cap structure (enzymatically incorporated cap0 (Ecap0), enzymatically incorporated cap1 (Ecap1), ARCA-G cap, or D1 cap) with an initiation sequence (GGG or GCG) and the hAg 5'UTR. The mRNA transcripts used in this experiment contained an unmodified uracil residue. mRNA containing a CC413 cap with an initiation sequence of AGA was used as a control for comparison. (B) Hematocrit levels measured in the same mice 0 and 7 days after mRNA injection (each dot represents one dot). [Figure 13-1]The pyrimidine base effect is independent of the coding sequence and / or formulation. (A) Representative IVIS images of mice 24, 48, and 72 hours after injection with 10 μg of F-12 (lipoplex)-formulated mRNA or no mRNA, where the mRNA encodes firefly luciferase and contains an initiation sequence (GGG, GAG, GGA, GGU, GGC, GUG, GCA, or GCG) and a cap structure (D1 cap or CC413 cap) with the hAg 5'UTR. The mRNA transcripts used in this experiment contained unmodified uracil residues. In the figure, R represents a pyrimidine nucleotide and Y represents a purine nucleotide. (B) Quantification of luciferase expression for the mice shown in (A). [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 14] A schematic comparison of DNA templates with GGG or GCG transcription start sites and their resulting RNA transcripts is shown. RNA transcripts synthesized from DNA templates with and without a Lig3 self-hybridization sequence in the 3'UTR are compared. In constructs with a Lig3 self-hybridization sequence in the 3'UTR, the coding strand of the DNA template with a GGG or GCG transcription start site has CG or AA at its +4 and +5 positions, respectively. In constructs without a Lig3 self-hybridization sequence in the 3'UTR, the coding strand of the DNA template with a GGG or GCG transcription start site has the same AT at its +4 and +5 positions. In such constructs, the only difference between the two templates with a GGG or GCG transcription start site is the nucleotide at the second position (+2). [Figure 15]The initiation sequence GGG results in higher capping efficiency for D1-capped mRNA. We compared the capping efficiency of D1-capped mRNAs encoding EPO or firefly luciferase and containing GGG or GCG initiation sequences. After in vitro transcription, the reaction mixture was run on a urea-PAGE gel and an agarose gel, and the capping efficiency was determined by comparing the intensity of the upper band (capped) to the intensity of the lower band (uncapped) in the urea-PAGE gel. [Figure 16] Effect of changing a nucleotide from a purine to a pyrimidine at the second position of a translational RNA construct. (A) Plasma concentrations of mouse EPO (mEPO) in mice 6, 24, 48, and 72 hours after injection of 3 μg of TransIT-formulated m1Ψ-RNA transcripts encoding mEPO and containing a cap structure (ARCA-G, D1, Ecap1, or CC413 cap) with an initiation sequence (GGG or GCG; or AGA for CC413 only) and the hAg 5'UTR. (B) Hematocrit levels measured in the same mice characterized in (A) 0, 7, and 14 days after injection of the RNA. [Figure 17] Changing the nucleotide at the second position of the RNA construct from a purine to a pyrimidine can eliminate short by-products. In vitro transcription reactions were performed to produce m1Ψ-mRNA transcripts encoding mEPO and containing a cap (Ecap1, ARCA-G, D1, or CC413 cap) with an initiation sequence (GGG or GCG; or CC413 only, AGA) and the hAg 5'UTR. After in vitro transcription, the reaction mixtures were run on a urea-PAGE gel. The short by-products correspond to bands toward the bottom of the gel. [Figure 18]Changing the nucleotide at the second position of the RNA construct from a purine to a pyrimidine can result in less immunogenic mRNA. Secretion of various proinflammatory cytokines by human PBMCs was analyzed after incubation with 1.5 or 5.0 μg of D1-capped m1Ψ-mRNA containing a GGG or GCG initiation sequence. TNF-α = tumor necrosis factor alpha; IFN-γ = interferon gamma; IL-6 = interleukin 6; IL-1β = interleukin 1 beta; MIP-1β = macrophage inflammatory protein 1 beta.

[0061] Specific Definitions Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0062] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0063] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology as described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0064] The elements of the present disclosure are described below. The elements are listed according to specific embodiments. However, it should be understood that the elements can be combined in any manner and in any number to produce additional embodiments. The variously described examples and embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to disclose and encompass embodiments combining the explicitly described embodiment with any number of the disclosed elements. Furthermore, all permutations and combinations of all described elements should be considered disclosed by the description unless the context indicates otherwise. The term "about" means approximately or near, and in the context of a numerical value or range set forth herein, in some embodiments, means ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

[0065] As used in the context of describing this disclosure (especially in the context of the claims), the terms "a," "an," and "the," and similar designations, should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0066] Unless expressly stated otherwise, the term "comprising" is used in the context of this document to indicate that additional members may be present in addition to the members of the list introduced by "comprising." However, for certain embodiments of the present disclosure, the term "comprising" is intended to encompass the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of" or "consisting essentially of."

[0067] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0068] The following provides definitions applicable to all aspects of this disclosure. The following terms have the following meanings unless otherwise specified: Any term not given a definition has its art-recognized meaning.

[0069] Agent: As used herein, the term "agent" may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular configuration and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, a polypeptide, a nucleic acid, a monosaccharide, a lipid, a metal, or a combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is devoid of or substantially free of any polymers or polymer moieties.

[0070] Amino acid: In its broadest sense, the term "amino acid" as used herein refers to a compound and / or substance that can be, is, or is incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-naturally occurring amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, may contain structural modifications compared to the above general structure. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid compared to one comprising the otherwise identical amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, the term may be used to refer to an amino acid residue of a polypeptide.

[0071] Analog: As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity to the reference substance, e.g., shares a core or consensus structure, but differs in certain discrete ways. In some embodiments, an analog is a substance that can be produced from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be produced by the performance of a synthetic process that is substantially similar to (e.g., shares multiple steps with) that which produces the reference substance. In some embodiments, an analog is produced, or can be produced, by the performance of a synthetic process that is different from that used to produce the reference substance.

[0072] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. For example, in some embodiments, an antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity-determining region (CDR); in some embodiments, an antibody agent is or includes a polypeptide that includes 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 CDR is substantially identical to the reference CDR in that it is sequence-identical or contains one to five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that one to five amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been substituted relative to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as corresponding to CDR1, 2, and 3 of an antibody variable domain; in some such embodiments, the antibody agent is or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together comprise structural elements recognized by those skilled in the art as corresponding to both heavy chain variable region CDRs and light chain variable region CDRs, e.g., heavy chain CDR1, 2, and / or 3, and light chain CDR1, 2, and / or 3. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent may be or include a polyclonal antibody preparation. In some embodiments, the antibody agent may be or include a monoclonal antibody preparation. In some embodiments, the antibody agent may include one or more constant region sequences unique 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 unique to humans. In some embodiments, the antibody agent may include one or more sequence elements recognized by those skilled in the art as humanized sequences, primatized sequences, chimeric sequences, etc. In some embodiments, the antibody agent may be a standard antibody (e.g., may include two heavy chains and two light chains).In some embodiments, antibody agents include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR, or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); Small Modular The antibody may be in a format selected from ImmunoPharmaceuticals [SMIPs™]; single chain or Tandem diabodies [TandAb®]; VHH; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., glycan attachment, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0073] Associated: Two events or entities are "associated" with one another, as this term is used herein, when the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence, susceptibility, severity, stage, etc. of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly to be in physical proximity and / or remain in close proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, e.g., by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0074] Binding: As used herein, the term "binding" will be understood to typically refer to a non-covalent association between two or more entities. "Direct" binding encompasses physical contact between the entities or moieties; indirect binding encompasses physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation or in association with more complex systems (e.g., covalently or otherwise associated with a carrier entity, and / or in a biological system or cell). Binding between two entities can be considered "specific" if, under the conditions being assessed, the associated entities are more likely to associate with each other than with other available binding partners.

[0075] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or fluid. In some embodiments, the biological sample may be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; suspended nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural effusion; feces; lymph; gynecological body fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces; other body fluids, secretions, and / or excretions; and / or cells derived therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells derived from the individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), for example, by filtration using a semipermeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of specific components, etc.

[0076] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may include administration of one or more agent(s) or modality(s) to a subject receiving the other agent(s) or modality(s) in combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or necessarily simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in a combination composition or combination compound (e.g., as part of a single chemical complex or covalent entity).

[0077] Complementary: As used herein, the term "complementary" refers to oligonucleotide hybridization related by base pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA". Complementarity can be partial or complete. Therefore, any degree of partial complementarity is intended to be included within the scope of the term "complementary", provided that the partial complementarity allows oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to base pairing rules. Complete or complete complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base pairing rules.

[0078] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but that are sufficiently similar to permit a comparison between them where one of skill in the art would understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, an equivalent set of conditions, circumstances, individuals, or populations is characterized by multiple substantially identical characteristics and one or a few different characteristics. One of skill in the art will understand the degree of identity required to be considered comparable in any given situation for two or more such agents, entities, circumstances, sets of conditions, etc., in context. For example, one of skill in the art will understand that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are attributable to or indicative of differences in those different characteristics.

[0079] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to indicate the location / identity of a structural element in a compound or composition relative to another compound or composition (e.g., an appropriate reference compound or composition). For example, in some embodiments, a monomer residue within a polymer (e.g., an amino acid residue within a polypeptide, or a nucleic acid residue within a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, one of ordinary skill in the art will understand that, for simplicity's sake, residues within a polypeptide are often designated based on the relevant reference polypeptide using a standard numbering system, so that an amino acid "corresponding to" a residue at position 190, for example, corresponds to the residue found at 190 in the reference polypeptide, rather than necessarily being the actual 190th amino acid in a particular amino acid chain; one of ordinary skill in the art will readily understand how to identify a "corresponding" amino acid. For example, those of skill in the art will be aware of various sequence alignment strategies, including, for example, software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, Parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, that can be utilized to identify "corresponding" residues within polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also recognize that, in some cases, the term "corresponding to" can be used to describe an event or entity that shares relevant similarity with another event or entity (e.g., a suitable reference event or entity).As just one example, a gene or protein in one organism may, in some embodiments, be described as "corresponding to" a gene or protein from another organism to indicate that they play a similar role or perform a similar function, and / or exhibit a particular degree of sequence identity or homology, or share certain characteristic sequence elements.

[0080] Designed: As used herein, the term "designed" refers to (i) an agent whose structure is selected or chosen by the hand of man; (ii) an agent produced by a process requiring human intervention; and / or (iii) an agent that differs from natural substances and other known agents.

[0081] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically separated by time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by the same length of time; in some embodiments, a dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dosage. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dosing at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen comprises a first dosing at a first dosage amount, followed by one or more additional dosings at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered within a relevant population (i.e., is a therapeutic dosing regimen).

[0082] Encode: As used herein, the terms "encode" or "encoding" refer to the sequence information of a first molecule that directs the production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription, which involves a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by the process of translation). Thus, a gene, cDNA, or single-stranded RNA (e.g., mRNA) encodes a polypeptide if transcription and translation of the mRNA corresponding to the gene produces the polypeptide in a cell or other biological system. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to the coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such target polypeptide agent. In some embodiments, the coding region of a single-stranded RNA encoding a target polypeptide agent refers to the non-coding strand of such target polypeptide agent, which can be used as a template for transcription of the gene or cDNA.

[0083] Engineered: Generally, the term "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when the hand of man manipulates two or more sequences that are not naturally linked together in that order so that they are directly linked to each other in the engineered polynucleotide, and / or when certain residues within the polynucleotide are caused through the action of man to be linked to entities or moieties that are not naturally occurring and / or not naturally linked.

[0084] Epitope: As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding entity. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a related three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0085] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) generation of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0086] Improved, increased, or reduced: As used herein, these terms, or grammatically equivalent comparative terms, refer to values ​​that are relative to a comparable reference measurement. For example, in some embodiments, a value achieved by a subject or system of interest may be "improved" compared to that obtained by a comparable reference drug. Alternatively or additionally, in some embodiments, a value achieved in a subject or system of interest may be "improved" compared to that obtained in the same subject or system under different conditions (e.g., before and after an event such as administration of the subject or system of interest) or in a different comparable subject (e.g., in a different comparable subject or system than the subject or system of interest, in the presence of one or more indicators of a particular disease, disorder, or condition of interest, or prior to exposure to the condition, drug, etc.). In some embodiments, comparative terms refer to a statistically relevant difference (e.g., one of sufficient incidence and / or magnitude to achieve statistical relevance). One of skill in the art will know or be able to readily determine the degree and / or incidence of difference necessary or sufficient to achieve such a statistically significant difference in a given context.

[0087] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism, but in an artificial environment, e.g., in a test tube or reaction vessel (e.g., a bioreactor), in cell culture, etc.

[0088] In vitro transcription: As used herein, the term "in vitro transcription" or "IVT" refers to a process in which transcription occurs in vitro in a non-cellular system to produce synthetic RNA products for use in a variety of applications, including, for example, the production of proteins or polypeptides. Such synthetic RNA products may be translated in vitro or directly introduced into cells and translated therein. Such synthetic RNA products include, for example, but are not limited to, mRNA, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNAs (e.g., for CRISPR), ribosomal RNA, small nuclear RNA, small nucleic acid RNA, and the like. IVT reactions typically utilize a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase as described and / or utilized herein.

[0089] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation.

[0090] Polypeptide: As used herein, polypeptide refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or generated through the act of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from acetylation, amidation, lipidation, methylation, pegylation, etc. (including combinations thereof). In some embodiments, a polypeptide may be cyclic and / or include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share a related activity or structure and can therefore be considered members of the same class or family of polypeptides.For each such class, exemplary polypeptides within the class are provided herein, and / or those of skill in the art will be aware of, 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 polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class (and, in some embodiments, with all polypeptides in the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range) with the reference polypeptide of the class (and, in some embodiments, with all polypeptides in the class). For example, in some embodiments, member polypeptides exhibit an overall degree of sequence homology or identity with a reference polypeptide of at least about 30-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or contain at least one region (e.g., a conserved region, which in some embodiments may be or may include a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least 3-4, and often up to 20 or more, amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0091] Prevent or prophylaxis: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

[0092] Pure or purified: As used herein, an agent or entity is "pure" or "purified" if it is substantially free of other components. For example, a preparation containing greater than about 90% of a particular agent or entity is typically considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure in the preparation.

[0093] Reference: As used herein, reference describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, which may be embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular reference or control considered.

[0094] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can contain one or more modifications, including, but not limited to, (a) terminal modifications, such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3'-terminal modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, such as substitution with a modified base, a stabilized base, a destabilized base, or a base that base pairs with an expanded repertoire of partners, or a conjugated base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) internucleoside linkage modifications, including modifications or replacements of phosphodiester bonds. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0095] Risk: As understood from the context, "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, up to 100%. In some embodiments, risk is expressed as risk compared to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is from individuals comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect, for example, one or more genetic attributes that may (or may not) predispose an individual to developing a particular disease, disorder, and / or condition, hi some embodiments, risk may reflect one or more epigenetic events or attributes, and / or one or more lifestyle or environmental events or attributes.

[0096] Susceptible to: An individual "susceptible to" a disease, disorder, and / or condition is an individual who is at a higher risk of developing the disease, disorder, and / or condition than members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0097] Vaccination: As used herein, the term "vaccination" refers to the administration of a composition intended to generate an immune response, for example, to a disease-associated (e.g., pathogenic) agent. In some embodiments, vaccination can be administered before, during, and / or after exposure to the disease-associated agent, and in certain embodiments, before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination involves multiple administrations of a vaccine composition, appropriately spaced apart. In some embodiments, vaccination generates an immune response against an infectious agent. In some embodiments, vaccination generates an immune response against a tumor; in some such embodiments, vaccination is "personalized" in that it is directed partially or completely to epitope(s) (which may be or include, for example, one or more neo-epitopes) determined to be present in a particular individual's tumor.

[0098] Variant: As used herein, in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or level of one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., to which the polypeptide or nucleic acid backbone is attached). In some embodiments, a variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid.In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, but with minor sequence modifications at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the reference. Often, a variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, 4, 3, 2, or 1) of functional residues (i.e., residues involved in a particular biological activity) substituted, inserted, or deleted compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, 4, 3, 2, or 1 additions or deletions, and in some embodiments no additions or deletions, relative to the reference. In some embodiments, the variant polypeptide or nucleic acid contains less than about 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, 6, and typically less than about 5, 4, 3, or 2 additions or deletions, relative to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature.

[0099] Detailed Description of Specific Embodiments The present disclosure provides, inter alia, an RNA polynucleotide comprising: (i) a 5' cap; (ii) a 5' UTR sequence comprising a cap-proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Compositions and pharmaceutical preparations comprising the same, as well as methods of making and using the same, are also provided herein. In some embodiments, the translation efficiency of an RNA encoding a payload and / or the expression of a payload encoded by the RNA can be improved by an RNA polynucleotide comprising a 5' cap comprising a structure disclosed herein, a 5' UTR comprising a cap-proximal sequence disclosed herein, and a sequence encoding a payload. In some embodiments, the absence of self-hybridizing sequences in the RNA polynucleotide encoding a payload can further improve the translation efficiency of an RNA encoding a payload and / or the expression of a payload encoded by the RNA payload.

[0100] RNA polynucleotides As used herein, the term "polynucleotide" or "nucleic acid" refers to DNA and RNA, e.g., genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0101] In some embodiments, the nucleic acid may be contained within a vector. As used herein, the term "vector" includes any vector known to those of skill in the art, including a plasmid vector, a cosmid vector, a phage vector, e.g., lambda phage, a viral vector, e.g., a retroviral, adenoviral, or baculoviral vector, or an artificial chromosome vector, e.g., a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). In some embodiments, the vector may be an expression vector; alternatively or additionally, in some embodiments, the vector may be a cloning vector. As will be apparent to those of skill in the art, in some embodiments, the expression vector may be, for example, a plasmid; alternatively or additionally, in some embodiments, the expression vector may be a viral vector. Typically, an expression vector will contain a desired coding sequence and other appropriate sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., a bacterium, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are commonly used to manipulate and amplify specific desired fragments (typically DNA fragments). It may also lack functional sequences required for expression of the desired fragment(s).

[0102] In some embodiments, the nucleic acids described and / or utilized herein may be or include recombinant and / or isolated molecules.

[0103] Those skilled in the art who have read this disclosure will understand that the term "RNA" typically refers to a nucleic acid molecule containing ribonucleotide residues. In some embodiments, RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. In some embodiments, RNA may be partially or completely double-stranded; in some embodiments, RNA may comprise two or more different nucleic acid strands (e.g., separate molecules) that are partially or completely hybridized with each other. In many embodiments, RNA is single-stranded, which in some embodiments self-hybridizes or otherwise folds to form secondary and / or tertiary structures. In some embodiments, the RNA described and / or utilized herein does not self-hybridize, at least with respect to the specific sequences described herein. In some embodiments, the RNA may be isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and / or modified RNA (wherein the term "modified" is understood to indicate that one or more residues or other structural elements of the RNA differ from naturally occurring RNA; e.g., in some embodiments, modified RNA differs by the addition, deletion, substitution, and / or modification of one or more nucleotides and / or by one or more portions or characteristics of the nucleotides, e.g., of the nucleosides, or of the backbone structure or linkage). In some embodiments, the modification may be or include the addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that nucleotides within an RNA (e.g., within a modified RNA) may be non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For the purposes of the present disclosure, modified RNA is considered an analog of naturally occurring RNA.

[0104] As will be understood by those skilled in the art, the RNA polynucleotides disclosed herein can comprise or consist of naturally occurring ribonucleotides and / or modified ribonucleotides. Thus, those skilled in the art will understand that throughout the specification described herein, references to A, U, G, or C can refer to the naturally occurring ribonucleotides and / or modified ribonucleotides described herein. For example, in some embodiments, U is uridine. In some embodiments, U is a modified uridine (e.g., pseudouridine, 1-methylpseudouridine).

[0105] In some embodiments of the present disclosure, the RNA is or comprises messenger RNA (mRNA), which refers to an RNA transcript that encodes a polypeptide.

[0106] In some embodiments, the RNA disclosed herein comprises a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region (5' UTR) comprising a cap-proximal sequence, a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3' UTR); and / or a polyadenylation (polyA) sequence.

[0107] In some embodiments, the RNA disclosed herein comprises, from 5' to 3', the following components: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region (5'UTR) comprising a cap-proximal sequence, a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'UTR); and a polyA sequence.

[0108] In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. Here, DNA refers to a nucleic acid containing deoxyribonucleotides.

[0109] In some embodiments, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a vector suitable for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0110] In some embodiments, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In some embodiments, the replicon or self-replicating RNA is derived from or contains elements derived from an ssRNA virus, particularly a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication, and protein modification) and structural proteins (forming viral particles). Typically, two open reading frames (ORFs) are present within the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF beginning near the 5' end of the genome, while the alphavirus structural proteins are encoded together by a second ORF found downstream from the first ORF and extending toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA polynucleotides similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly as a messenger RNA for the translation of an open reading frame encoding a nonstructural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be trans-replicated by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules capable of trans-replication by the replicase must contain specific alphavirus sequence elements that allow recognition and RNA synthesis by the alphavirus replicase.

[0111] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridine.

[0112] The term "uracil" as used herein describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is as follows: [ka]

[0113] As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is as follows: [ka]

[0114] UTP (uridine 5'-triphosphate) has the following structure: [ka]

[0115] Pseudo-UTP (pseudouridine-5'-triphosphate) has the following structure: [ka]

[0116] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring through a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0117] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the following structure: [ka]

[0118] N1-methylpseudouridine-5'-triphosphate (m1ΨTP) has the following structure: [ka]

[0119] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: [ka]

[0120] In some embodiments, one or more uridines in the RNA described herein are replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0121] In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0122] In some embodiments, the modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In some embodiments, modified nucleosides include pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include N1-methylpseudouridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U).

[0123] In some embodiments, the modified nucleoside that replaces one or more, e.g., all, uridines in the RNA is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0124] In some embodiments, the RNA includes 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 includes 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA includes 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA includes 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

[0125] In some embodiments, RNA encoding a payload, e.g., a vaccine antigen, is expressed in cells of a subject treated to provide the payload, e.g., vaccine antigen. In some embodiments, the RNA is transiently expressed in the subject's cells. In some embodiments, the RNA is in vitro transcribed RNA. In some embodiments, expression of the payload, e.g., vaccine antigen, occurs at the cell surface. In some embodiments, the payload, e.g., vaccine antigen, is expressed and presented in the context of MHC. In some embodiments, expression of the payload, e.g., vaccine antigen, is in the extracellular space, i.e., the vaccine antigen is secreted.

[0126] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0127] According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used to generate the transcript. These cloning vectors are commonly referred to as transcription vectors and, according to the present invention, are encompassed by the term "vector." According to the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a vector suitable for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0128] With respect to RNA, the terms "expression" or "translation" refer to the process by which a chain of mRNA directs the assembly of a series of amino acids to form a peptide or protein in the ribosomes of a cell.

[0129] In some embodiments, after administration of the RNA described herein, for example, formulated as an RNA-lipid particle, at least a portion of the RNA is delivered to a target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the encoded peptide or protein. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, for example, a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or a macrophage. RNA particles, such as the RNA-lipid particles described herein, may be used to deliver RNA to such target cells. Thus, the present disclosure also relates to methods of delivering RNA to target cells in a subject, including administering to the subject an RNA particle described herein. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.

[0130] "Encoding" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0131] In some embodiments, nucleic acid compositions described herein, e.g., compositions comprising lipid nanoparticle-encapsulated mRNA, are characterized by sustained expression of the encoded polypeptide (e.g., when administered to a subject). For example, in some embodiments, such compositions, when administered to a human, achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some embodiments, are characterized in that such expression persists for at least 36 hours, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

[0132] In some embodiments, the RNA encoding the payload to be administered in accordance with the present invention is non-immunogenic. RNA-encoded immunostimulants can be administered in accordance with the present invention to provide an adjuvant effect. RNA-encoded immunostimulants can be standard RNA or non-immunogenic RNA.

[0133] As used herein, the term "non-immunogenic RNA" refers to RNA that does not induce a response by the immune system immediately upon administration, e.g., to a mammal, or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and processes that render it non-immunogenic, i.e., standard RNA (stdRNA). In a preferred embodiment, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors, thereby eliminating double-stranded RNA (dsRNA).

[0134] To make non-immunogenic RNA non-immunogenic by incorporating modified nucleosides, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises the replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 In a particularly preferred embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0135] In some embodiments, replacement of one or more uridines with nucleosides comprising modified nucleobases comprises replacement 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 uridines.

[0136] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, a significant amount of aberrant products, including double-stranded RNA (dsRNA), is generated due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines, activates effector enzymes, and causes inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a non-porous or porous C-18 polystyrene divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used to remove dsRNA contaminants from IVT RNA preparations by using Escherichia coli (E. coli) RNase III, which specifically hydrolyzes dsRNA but not ssRNA. Furthermore, dsRNA can be separated from ssRNA by using cellulose materials. In some embodiments, the RNA preparation is contacted with the cellulosic material and the ssRNA is separated from the cellulosic material under conditions that allow binding of the dsRNA to the cellulosic material, but not of the ssRNA to the cellulosic material.

[0137] As used herein, the term "removing" or "removal" refers to the characteristic of a population of a first substance, e.g., non-immunogenic RNA, being separated from a nearby population of a second substance, e.g., 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, a population of a first substance characterized by the removal of a population of a second substance will have a significantly lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0138] In some embodiments, removing dsRNA from non-immunogenic RNA comprises removing 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 free of dsRNA or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises 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, a purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0139] In some embodiments, the non-immunogenic RNA is translated more efficiently in cells than standard RNA of the same sequence. In some embodiments, translation is enhanced by 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced by 3-fold. In some embodiments, translation is enhanced by 4-fold. In some embodiments, translation is enhanced by 5-fold. In some embodiments, translation is enhanced by 6-fold. In some embodiments, translation is enhanced by 7-fold. In some embodiments, translation is enhanced by 8-fold. In some embodiments, translation is enhanced by 9-fold. In some embodiments, translation is enhanced by 10-fold. In some embodiments, translation is enhanced by 15-fold. In some embodiments, translation is enhanced by 20-fold. In some embodiments, translation is enhanced by 50-fold. In some embodiments, translation is enhanced by 100-fold. In some embodiments, translation is enhanced by 200-fold. In some embodiments, translation is enhanced by a factor of 500-fold. In some embodiments, translation is enhanced by a factor of 1000-fold. In some embodiments, translation is enhanced by a factor of 2000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0140] In some embodiments, the non-immunogenic RNA exhibits significantly lower natural immunogenicity than standard RNA of the same sequence. In some embodiments, the non-immunogenic RNA exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced by a factor of 3. In some embodiments, the natural immunogenicity is reduced by a factor of 4. In some embodiments, the natural immunogenicity is reduced by a factor of 5. In some embodiments, the natural immunogenicity is reduced by a factor of 6. In some embodiments, the natural immunogenicity is reduced by a factor of 7. In some embodiments, the natural immunogenicity is reduced by a factor of 8. In some embodiments, the natural immunogenicity is reduced by a factor of 9. In some embodiments, the natural immunogenicity is reduced by a factor of 10. In some embodiments, the natural immunogenicity is reduced by a factor of 15. In some embodiments, the natural immunogenicity is reduced by a factor of 20. In some embodiments, the natural immunogenicity is reduced by a factor of 50. In some embodiments, the natural immunogenicity is reduced by a factor of 100. In some embodiments, the natural immunogenicity is reduced by a factor of 200. In some embodiments, the natural immunogenicity is reduced by a factor of 500. In some embodiments, the natural immunogenicity is reduced by a factor of 1000. In some embodiments, the natural immunogenicity is reduced by a factor of 2000.

[0141] The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of the non-immunogenic RNA can be administered without triggering a detectable natural immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA can be repeatedly administered without eliciting a natural immune response sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA.

[0142] "Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively non-specific and immediate component of the immune system. It is one of the two major components of the vertebrate immune system, along with the adaptive immune system.

[0143] As used herein, "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0144] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0145] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0146] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements, components, or domains.

[0147] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is C and N2 is G; (b) N1 is U and N2 is G; or (c) N1 is A and N2 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 and N4 are G, and N5 is selected from A, C, G, and U.

[0148] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are each G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0149] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a dinucleotide cap structure N1 and a sequence comprising N2N3N4N5 at positions +2, +3, +4, and +5 of the RNA polynucleotide, respectively, wherein each of N2 and N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0150] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is G; (b) N1 is U, N2 is G, and N3 is G; or (c) N1 is A, N2 is G, and N3 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein N4 is G and N5 is selected from A, C, G, and U.

[0151] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is G, N2 is G, and N3 is G; (ii) the cap-proximal sequence is An RNA polynucleotide is provided, comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0152] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is G; (b) N1 is U and N2 is G; (c) N1 is A and N2 is G; or (d) N1 is C and N2 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is C, N4 is G, and N5 is selected from A, C, G, and U.

[0153] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 is G and N2 is C; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0154] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) the RNA polynucleotide comprises an N1 dinucleotide cap structure and sequences comprising N2N3N4N5 at positions +2, +3, +4, and +5 of the RNA polynucleotide, respectively, where N2 is a pyrimidine (e.g., C or U), and N3, N4, and N5 are each selected from A, C, G, and U. In some embodiments, N3 is G or A, and N4 and N5 are each separately and independently selected from A, C, G, and U.

[0155] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a dinucleotide cap structure that includes N1, wherein N1 is at position +1 of the RNA polynucleotide and N1 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a dinucleotide cap structure N1 and a sequence comprising N2N3N4N5 at positions +2, +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N2 is C, N3 is G, and each of N4 and N5 is selected from A, C, G, and U.

[0156] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is C, N2 is G, and N3 is C; (b) N1 is U, N2 is G, and N3 is C; or (c) N1 is A, N2 is G, and N3 is C; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein N4 is G and N5 is selected from A, C, G, and U.

[0157] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is G, N2 is C, and N3 is G; (ii) the cap-proximal sequence is An RNA polynucleotide is provided, comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0158] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide and N2 is at position +2 of the RNA polynucleotide, and N1 and N2 are selected from one of the following combinations: (a) N1 is G and N2 is C; (b) N1 is U and N2 is C; (c) N1 is A and N2 is C; or (d) N1 is C and N2 is C; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is G, N4 is C, and N5 is selected from A, C, G, and U.

[0159] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, N1 is C, and N2 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is C, and each of N4 and N5 is selected from A, C, G, and U.

[0160] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1, N2, and N3 are selected from one of the following combinations: (a) N1 is G, N2 is C, and N3 is G; (b) N1 is U, N2 is C, and N3 is G; or (c) N1 is A, N2 is C, and N3 is G; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a tetranucleotide cap structure N1, N2, and N3, and a sequence comprising N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein N4 is C and N5 is selected from A, C, G, and U.

[0161] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is C, N2 is G, and N3 is C; (ii) the cap-proximal sequence is An RNA polynucleotide is provided, comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0162] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, N1 is A, and N2 is U; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a trinucleotide cap structure N1 and N2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is A and each of N4 and N5 is selected from A, C, G, and U.

[0163] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1 is at position +1 of the RNA polynucleotide, N2 is at position +2 of the RNA polynucleotide, and N3 is at position +3 of the polynucleotide, and N1 is A, N2 is U, and N3 is A; (ii) the cap-proximal sequence is An RNA polynucleotide is provided, comprising a sequence comprising tetranucleotide cap structures N1, N2, and N3, and N4N5 at positions +4 and +5 of the RNA polynucleotide, respectively, wherein each of N4 and N5 is selected from A, C, G, and U.

[0164] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a 5' cap, A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is A, and N4 and N5 are selected from A, C, G, and U.

[0165] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a 5' cap, A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 and N4 are G, and N5 is selected from A, C, G, and U.

[0166] In some embodiments, the present disclosure provides: 1. An RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, (i) The 5' cap is m2 (7,3’O) Gppp (m2’O) A1pG2, where A1 is the +1 position of the RNA polynucleotide and G2 is the +2 position of the RNA polynucleotide; (ii) the cap-proximal sequence is The present invention provides an RNA polynucleotide comprising a 5' cap, A1 and G2, and a sequence comprising N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein N3 is C, N4 is G, and N5 is selected from A, C, G, and U.

[0167] Codon optimization In some embodiments, the payloads (e.g., polypeptides) described herein are encoded by coding sequences that are codon-optimized and / or have an increased G / C content compared to a 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 a corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content does not alter the sequence of the encoded amino acid sequence.

[0168] The term "codon-optimized" is understood by those skilled in the art to refer to the modification of codons in the coding region of a nucleic acid molecule, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule, so as to reflect the typical codon usage of the host organism.In the context of the present disclosure, the coding region is preferably codon-optimized for optimal expression in the subject to be treated with the RNA polynucleotide described herein.Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of tRNA occurrence in cells.Therefore, the sequence of RNA can be modified so that the codons that are available to frequently occurring tRNAs are inserted instead of "rare codons".

[0169] In some embodiments, the guanosine / cytidine (G / C) content of the coding region of the RNA (e.g., the payload sequence) is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA encoding the payload, and the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the effective translation of that mRNA. Sequences with an increased G (guanosine) / C (cytidine) content are more stable than sequences with an increased A (adenosine) / U (uridine) content. In conjunction with the fact that several codons encode one and the same amino acid (the so-called degeneracy of the genetic code), the most favorable codon for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the RNA, there are various possibilities for modifying the RNA sequence compared to the wild-type sequence. In particular, a codon containing A and / or U nucleosides can be modified by replacing the codon with another codon that encodes the same amino acid but does not contain A and / or U or contains a lower content of A and / or U nucleosides.

[0170] 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 the wild-type RNA.

[0171] 5' Cap A structural feature of mRNA is the cap structure at the 5'-prime end. Natural eukaryotic mRNAs contain a 7-methylguanosine cap linked to the mRNA via a 5'-5'-triphosphate bridge, resulting in the cap0 structure (m7GpppN). In most eukaryotic mRNAs and some viral mRNAs, further modifications can occur at the 2'-hydroxyl group (2'-OH) of the first and subsequent nucleotides (e.g., the 2'-hydroxyl group can be methylated to form 2'-O-Me), producing the "cap1" and "cap2" 5'-prime ends, respectively. Diamond et al. (2014) Cytokine & Growth Factor Reviews, 25:543-550 reported that cap0-mRNAs cannot be translated as efficiently as cap1-mRNAs, in which the role of 2'-O-Me at the penultimate position of the mRNA 5' end is crucial. The lack of 2'-O-Me has been shown to trigger innate immunity and activate IFN responses. Daffis, et al. (2010) Nature, 468:452-456; and Zust et al. (2011) Nature Immunology, 12:137-143.

[0172] RNA capping has been thoroughly studied and described, for example, in Decroly E et al. (2012) Nature Reviews 10:51-65; and Ramanathan A. et al., (2016) Nucleic Acids Res;44(16):7511-7526, the entire contents of each of which are incorporated herein by reference. In some embodiments, to mimic the 5' cap structure of native mRNA, in vitro transcribed mRNA (IVT mRNA) can be post-transcriptionally capped using a recombinant Vaccinia virus-derived enzyme (e.g., Kyrieleis, et al. (1993) Structure 22:452-465; and Corbett, et al. (2020) The New England Journal of Medicine 383:1544-1555) or co-transcriptionally capped by immediately adding a cap to the in vitro transcription reaction (see, e.g., Jemielity, et al. (2003) RNA 9:1108-1122; and Kocmik, et al. (2018) Cell Cycle 17:1624-1636). In some embodiments, enzymatic capping can result in cap1-mRNA, but can be time-consuming because it requires additional purification steps and a heating step to improve accessibility of the structured 5' end, thereby further increasing the risk of RNA degradation. Notably, capping by this method is more reproducible and less expensive than enzymatic capping. mRNA produced in the presence of these caps is more resistant to human decapping enzymes (Kowalska et al. (2008) RNA 14:1119-1131) and / or interferon-inducible proteins with tetratricopeptide repeats (IFITs), which inhibit cap0-dependent translation (Diamond et al. (2014) Cytokine & Growth Factor Reviews 25:543-550; and Miedziak, et al. (2019) RNA 26:58-68).However, using this approach, GTP typically competes with the cap during transcription, potentially reducing capping efficiency and translational competence. Certain Cap1 structures can be incorporated into IVT mRNA in the correct orientation to produce cap1-mRNA with high capping efficiency in rapid co-transcription reactions. (Henderson et al., (2021) Current Protocols 1:e39) For example, trinucleotide Cap1 structures require an AG initiator, preventing RNA polymerase slippage on the template DNA strand, as opposed to those containing a G triplet as the transcription start site. (Imburgio, et al., (2000) Biochemistry 39:10419-10430)

[0173] In some embodiments, the 5' cap includes cap-0 (also referred to herein as "Cap0"), cap-1 (also referred to herein as "Cap1"), or cap-2 (also referred to herein as "Cap2"). See, e.g., Figure 1 of Ramanathan A et al. and Figure 1 of Decroly E et al.

[0174] As used herein, the term 5' cap refers to a structure found on the 5' end of an RNA, e.g., an mRNA, and generally comprises a guanosine nucleotide linked to the RNA, e.g., an mRNA, via a 5'-5' triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, the guanosine nucleoside contained within the 5' cap may be modified, for example, by methylation at one or more positions on the base (guanine) (e.g., at the 7 position) and / or by methylation at one or more positions on the ribose. In some embodiments, the guanosine nucleoside contained within the 5' cap is modified by 3' O methylation ("(m 3’-O In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7-position of the guanine (represented as "(m )G" or "3'OMeG"). 7In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7 position of the guanine and a 3' O methylation at the ribose (represented as "(m7G" or "m7G"). 7,3’-O In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a 2'O methylation at the ribose (represented by "(m )G" or "m7(3'OMeG)"). 2’-O In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7-position of the guanine and a 2'O methylation at the ribose (represented as "(m2 7,2’-O )G" or "m7(2'OMeG)". 7,3’-O )G" or "m7(3'OMeG)" is understood to apply to other structures described herein.

[0175] In some embodiments, providing an RNA with a 5' cap or 5' caps disclosed herein can be accomplished by in vitro transcription, where the 5' cap is transcriptionally expressed into the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme. In some embodiments, transcription-associated capping with a cap disclosed herein, e.g., with a Cap0, Cap1, or Cap2 structure, improves the capping efficiency of the RNA compared to transcription-associated capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase the translation efficiency and / or rate of the RNA and / or increase expression of the encoded polypeptide.

[0176] In some embodiments, T7 RNA polymerase prefers a G as the initial site. Accordingly, in some such embodiments, the present disclosure provides caps (e.g., the trinucleotide and tetranucleotide caps described herein) wherein the 3' end of the trinucleotide (e.g., N2) or tetranucleotide cap (e.g., N3) is a G.

[0177] In some embodiments, all compounds or structures (e.g., 5' caps) provided herein contain a suitable counterion (e.g., Na + ), including free base or salt forms (e.g., Na + It will be understood that compounds or structures (e.g., 5' caps) shown as salts also include the free base and a suitable counter ion (e.g., Na + ) is included.

[0178] In some embodiments, the RNA described herein comprises a 5' cap or 5' cap, e.g., Cap0, Cap1, or Cap2. In some embodiments, the RNA provided does not have an uncapped 5' triphosphate cap. In some embodiments, the RNA may be capped with a 5' cap. In some embodiments, the RNA described herein comprises Cap0. In some embodiments, the RNA described herein comprises Cap1, e.g., as described herein. In some embodiments, the RNA described herein comprises Cap2.

[0179] In some embodiments, the Cap0 structure comprises a guanosine nucleoside methylated at the 7 position of guanine (m7G). In some embodiments, the Cap0 structure is linked to the RNA via a 5'-5' triphosphate linkage, also referred to herein as m7Gppp or m7G(5')ppp(5').

[0180] In some embodiments, the Cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and a first nucleotide (2'OMeN1) that is 2'O-methylated within the RNA. In some embodiments, the Cap1 structure is linked to the RNA via a 5'-5' triphosphate linkage and is also referred to herein as m7Gppp(2'OMeN1) or m7G(5')ppp(5')(2'OMeN1), where N1 is as defined and described herein.

[0181] In some embodiments, the m7G(5')ppp(5')(2'OMeN1)Cap1 structure includes a second nucleotide, N2, which is the cap proximal to the nucleotide at position 2 and is selected from A, G, C, or U (m7G(5')ppp(5')(2'OMeN1)N2), where each of N1 and N2 is as defined and described herein.

[0182] In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure comprising N1, where N1 is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G*N1, where N1 is as defined above and described herein. G* is a structure of formula (I), [ka] or a salt thereof, During the ceremony, Each R 2 and R 3 is -OH or -OCH3, X is O or S.

[0183] In some embodiments, R 2 is —OH. In some embodiments, R 2 is —OCH. In some embodiments, R 3 is —OH. In some embodiments, R 3 is —OCH. In some embodiments, R 2 is -OH and R 3 is —OH. In some embodiments, R 2 is -OH and R 3 is -CH3. In some embodiments, R 2 is -CH3 and R 3 is —OH. In some embodiments, R2 is -CH3 and R 3 is -CH3.

[0184] In some embodiments, X is O. In some embodiments, X is S.

[0185] In some embodiments, the 5' cap is a dinucleotide Cap0 structure (e.g., (m 7 )GpppN1, (m2 7,2’-O )GpppN1, (m2 7,3’-O )GpppN1, (m 7 )GppSpN1, (m2 7,2’-O )GppSpN1, or (m2 7,3’-O )GppSpN1, where N1 is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide Cap0 structure (e.g., (m 7 )GpppN1, (m2 7,2’-O )GpppN1, (m2 7,3’-O )GpppN1, (m 7 )GppSpN1, (m2 7,2’-O )GppSpN1, or (m2 7,3’-O )GppSpN1, where N1 is G. In some embodiments, the 5' cap is a dinucleotide Cap0 structure (e.g., (m 7 )GpppN1, (m2 7,2’-O )GpppN1, (m2 7,3’-O )GpppN1, (m 7 )GppSpN1, (m2 7,2’-O )GppSpN1, or (m2 7,3’-O )GppSpN1, where N1 is A, U, or C. In some embodiments, the 5' cap is a dinucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N1, (m2 7,2’-O )Gppp(m 2’-O )N1, (m2 7,3’-O )Gppp(m 2’-O )N1, (m 7 )GppSp(m 2’-O )N1, (m2 7,2’-O)GppSp(m 2’-O )N1, or (m2 7,3’-O )GppSp(m 2’-O )N1, where N1 is as defined and described herein. In some embodiments, the 5' cap is (m 7 )GpppG("Ecap0"), (m 7 )Gppp(m 2’-O )G("Ecap1"), (m2 7,3’-O ) GpppG ("ARCA" or "D1"), and (m2 7,2’-O ) GppSpG (“beta-S-ARCA”). In some embodiments, the 5′ cap has the following structure: 7 )GpppG("Ecap0"): [ka] or a salt thereof.

[0186] In some embodiments, the 5' cap has the following structure: 7 )Gppp(m 2’-O )G("Ecap1"): [ka] or a salt thereof.

[0187] In some embodiments, the 5' cap has the following structure: 7,3’-O )GpppG("ARCA" or "D1"): [ka] or a salt thereof.

[0188] In some embodiments, the 5' cap has the following structure: 7,2’-O ) GppSpG ("beta-S-ARCA"): [ka] or a salt thereof.

[0189] In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure comprising N1pN2, where N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide cap G*N1pN2, where N1 and N2 are as defined and described herein. G* is a structure of formula (I), [ka] or a salt thereof, wherein R 2 , R 3 , and X are as defined and explained herein.

[0190] In some embodiments, the 5' cap is a trinucleotide CapO structure (e.g., (m 7 )GpppN1pN2, (m2 7,2’-O )GpppN1pN2, or (m2 7,3’-O )GpppN1pN2, where N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N1pN2, (m2 7,2’-O )Gppp(m 2’-O )N1pN2, (m2 7,3’-O )Gppp(m 2’-O )N1pN2, where N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N1p(m 2’-O )N2, (m2 7,2’-O )Gppp(m 2’-O )N1p(m 2’-O )N2, (m2 7,3’-O )Gppp(m 2’-O )N1p(m 2’-O)N2, where N1 and N2 are as defined and described herein. In some embodiments, the 5' cap is (m2 7,3’-O )Gppp(m 2’-O )ApG("CleanCap AG", "CC413"), (m2 7,3’-O )Gppp(m 2’-O )GpG("CleanCap GG"), (m 7 )Gppp(m 2’-O )ApG, (m2 7,3’-O )Gppp(m2 6,2’-O ) ApG, and (m 7 )Gppp(m 2’-O In some embodiments, the 5' cap is selected from the group consisting of (m2 7,3’-O )Gppp(m 2’-O )ApG("CleanCap AG", "CC413"), (m2 7,3’-O )Gppp(m 2’-O )GpG("CleanCap GG"), (m 7 )Gppp(m 2’-O )ApG, (m2 7,3’-O )Gppp(m2 6,2’-O )ApG, (m 7 )Gppp(m 2’-O )ApU, and (m2 7,3’-O )Gppp(m 2’-O ) CpG.

[0191] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m 2’-O )ApG("CleanCap AG 3' OMe", "CC413"): [ka] or a salt thereof.

[0192] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m 2’-O )GpG("CleanCap GG"): [ka] or a salt thereof.

[0193] In some embodiments, the 5' cap has the following structure: 7 )Gppp(m 2’-O )ApG: [ka] or a salt thereof.

[0194] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m2 6,2’-O )ApG: [ka] or a salt thereof.

[0195] In some embodiments, the 5' cap has the following structure: 7 )Gppp(m 2’-O )ApU: [ka] or a salt thereof.

[0196] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m 2’-O )CpG: [ka] or a salt thereof.

[0197] In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising N1pN2pN3, where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap G*N1pN2pN3, where N1, N2, and N3 are as defined and described herein. G* is a structure of formula (I), [ka] or a salt thereof, wherein R 2 , R 3 , and X are as defined and explained herein.

[0198] In some embodiments, the 5' cap is a tetranucleotide Cap0 structure (e.g., (m 7 )GpppN1pN2pN3, (m2 7,2’-O )GpppN1pN2pN3, or (m2 7,3’-O )GpppN1N2pN3, where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide Cap1 structure (e.g., (m 7 )Gppp(m 2’-O )N1pN2pN3, (m2 7,2’-O )Gppp(m 2’-O )N1pN2pN3, or (m2 7,3’-O )Gppp(m 2’-O )N1pN2N3, where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2’-O )N1p(m 2’-O )N2pN3, (m2 7,2’-O )Gppp(m 2’-O )N1p(m 2’-O )N2pN3, (m2 7,3’-O )Gppp(m2’-O )N1p(m 2’-O )N2pN3, where N1, N2, and N3 are as defined and described herein. In some embodiments, the 5' cap is (m2 7,3’-O )Gppp(m 2’-O )Ap(m 2’-O )GpG, (m2 7,3’-O )Gppp(m 2’-O )Gp(m 2’-O ) GpC, (m 7 )Gppp(m 2’-O )Ap(m 2’-O )UpA, and (m 7 )Gppp(m 2’-O )Ap(m 2’-O ) GpG.

[0199] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m 2’-O )Ap(m 2’-O )GpG: [ka] or a salt thereof.

[0200] In some embodiments, the 5' cap has the following structure: 7,3’-O )Gppp(m 2’-O )Gp(m 2’-O )GpC: [ka] or a salt thereof.

[0201] In some embodiments, the 5' cap has the following structure: 7 )Gppp(m 2’-O )Ap(m 2’-O )UpA: [ka] or a salt thereof.

[0202] In some embodiments, the 5' cap has the following structure: 7 )Gppp(m 2’-O )Ap(m 2’-O )GpG: [ka] or a salt thereof.

[0203] In some embodiments, the Cap1 structure is or includes m7G(5')ppp(5')(2'OMeA1)pG2, where A is the cap proximal to the +1 nucleotide and G is the cap proximal to the +2 nucleotide, and has the following structure: [ka]

[0204] In some embodiments, the Cap1 structure is or includes m7G(5')ppp(5')(2'OMeA1)pU2, where A is the cap proximal to the nucleotide at position 1 and U is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0205] In some embodiments, the Cap1 structure is or includes m7G(5')ppp(5')(2'OMeG1)pG2, where G is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0206] In some embodiments, the 5' cap is or includes m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0207] In some embodiments, the 5' cap is or includes m7(3'OMeG)(5')ppp(5')(2'OMeG1)pG2, where G is the cap proximal to the nucleotide at position 1 and G is the cap proximal to the nucleotide at position 2, and has the following structure: [ka]

[0208] In some embodiments, the second nucleotide in the Cap1 structure can include one or more modifications, such as methylation. In some embodiments, a Cap1 structure that includes a second nucleotide that includes a 2'O methylation is a Cap2 structure.

[0209] In some embodiments, an RNA polynucleotide comprising a Cap1 structure exhibits increased translation efficiency, increased translation rate, and / or increased expression of the encoded payload relative to a suitable reference comparator. In some embodiments, an RNA polynucleotide comprising a 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, exhibits increased translation efficiency relative to an RNA polynucleotide comprising 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, increased translation efficiency is assessed by administering the RNA polynucleotide to a cell or organism.

[0210] In some embodiments, the cap used in the RNA polynucleotide is m2 7,3’-O Gppp(m1 2’-O )ApG(occasionally, m2 7,3`O G(5')ppp(5')m 2’-O ApG, or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG), which has the following structure: [ka]

[0211] The following is an exemplary Cap1 RNA, which is a nucleotide sequence of RNA and m2 7,3`O G(5')ppp(5')m 2’-O Contains ApG. [ka]

[0212] Below is another exemplary Cap1 RNA. [ka]

[0213] 5'UTR and cap-proximal sequences In some embodiments, the RNAs disclosed herein include a 5' UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA polynucleotide, e.g., an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5' UTR) and / or 3' (downstream) of an open reading frame (3' UTR). If present, the 5' UTR is located at the 5' end upstream of the start codon of a protein-coding region. The 5' UTR is located downstream of the 5' cap (if present), e.g., immediately adjacent to the 5' cap.

[0214] In some embodiments, a 5' UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some embodiments, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some embodiments, the cap-proximal sequence comprises nucleotides within positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0215] In some embodiments, the cap structure comprises one or more polynucleotides of the cap-proximal sequence. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide. In some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1, +2, and +3 (N1, N2, and N3) of the RNA polynucleotide.

[0216] Those of skill in the art will recognize upon reading this disclosure that in some embodiments, one or more residues of the cap-proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in the RNA by being included in a cap entity (e.g., a Cap1 or Cap2 structure); alternatively, in some embodiments, at least a portion of the residues in the cap-proximal sequence may be added enzymatically (e.g., by a polymerase such as T7 polymerase). For example, m2 7,3’-O Gppp(m1 2’-O In certain exemplary embodiments where an ApG cap is utilized, +1 (i.e., N1) and +2 (i.e., N2) are the (m1 2’-O ) A and G residues, and +3, +4, and +5 are added by a polymerase (e.g., T7 polymerase).

[0217] A. The cap-proximal sequence containing the nucleotides of the dinucleotide 5' cap In some embodiments, the 5' cap is a dinucleotide cap structure and the cap-proximal sequence comprises N1 of the 5' cap, where N1 is any nucleotide, e.g., A, C, G, or U. In some embodiments, the 5' cap is a dinucleotide cap structure and the cap-proximal sequence comprises N1 of the 5' cap, where N1 is G.

[0218] B. Cap-proximal sequence including the nucleotide(s) of the trinucleotide 5' cap In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises 5' cap N1 and N2, where N1 and N2 are independently any nucleotide, e.g., A, C, G, or U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises 5' cap N1 and N2, where N1 and N2 are A. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises 5' cap N1 and N2, where N1 and N2 are C. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises 5' cap N1 and N2, where N1 and N2 are G. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 and N2 are U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is A and N2 is C. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is A and N2 is G. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2, where N1 is A and N2 is U.In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is C and N2 is A. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is C and N2 is G. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is C and N2 is U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is G and N2 is A. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is G and N2 is C. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is G and N2 is U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is U and N2 is A. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is U and N2 is C.In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1 and N2 of the 5' cap, where N1 is U and N2 is G.

[0219] In some embodiments, for example, when the 5' cap is a trinucleotide cap structure and the cap-proximal sequence is described in the previous paragraph, N3 is G. In some embodiments, for example, when the 5' cap is a trinucleotide cap structure and the cap-proximal sequence is described in the previous paragraph, N4 is G.

[0220] C. The cap-proximal sequence containing the nucleotide(s) of the tetranucleotide 5' cap In some embodiments, the 5' cap is a tetranucleotide cap structure (a trinucleotide cap structure as described above and herein), and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are any nucleotide, e.g., A, C, G, or U.

[0221] i. Exemplary embodiments where N1 is A In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is A, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is A, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is A, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is C, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is C, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is A, N2 is C, and N3 is G.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is C, and N3 is U. In some embodiments, N1 is A, N2 is G, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is G, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is G, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is G, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is U, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is A, N2 is U, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is A, N2 is U, and N3 is G.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is A, N2 is U, and N3 is U.

[0222] ii. Exemplary Embodiments Where N1 is C In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is A, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is A, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is A, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is A, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is C, and N3 is A. In some embodiments, N1 is C, N2 is C, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is C, N2 is C, and N3 is G.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is C, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is G, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is G, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is G, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is G, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is U, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is C, N2 is U, and N3 is C.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is U, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is C, N2 is U, and N3 is U.

[0223] iii. Exemplary Embodiments Wherein N1 is G In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is A, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is A, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is A, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is A, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is C, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is G, N2 is C, and N3 is C.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is C, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is C, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is G, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is G, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is G, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is G, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is G, N2 is U, and N3 is A.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is U, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is U, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is G, N2 is U, and N3 is U.

[0224] iv. Exemplary Embodiments Where N1 is U In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1, N2, and N3 are U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is A, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is A, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is A, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is A, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is C, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is U, N2 is C, and N3 is C.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is C, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is C, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is G, and N3 is A. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is G, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is G, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is G, and N3 is U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence includes N1, N2, and N3 of the 5' cap, where N1 is U, N2 is U, and N3 is A.In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is U, and N3 is C. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is U, and N3 is G. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure described above and herein) and the cap-proximal sequence comprises N1, N2, and N3 of the 5' cap, where N1 is U, N2 is U, and N3 is U.

[0225] D. Exemplary Cap-Proximal Sequences In some embodiments, for example, the 5' cap is a dinucleotide cap structure and the cap-proximal sequence includes N1, and N2, N3, N4, and N5 of the 5' cap, where N1-N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, for example, the 5' cap is a trinucleotide cap structure and the cap-proximal sequence includes N1 and N2, and N3, N4, and N5 of the 5' cap, where N1-N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, for example, the 5' cap is a tetranucleotide cap structure and the cap-proximal sequence includes N1, N2, and N3, and N4 and N5 of the 5' cap, where N1-N5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0226] i. An exemplary cap-proximal sequence wherein N1 is A and N2 is A. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is A, N4 is U, and N5 is U.

[0227] In some embodiments, N1 is A, N2 is A, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is C, N4 is U, and N5 is U.

[0228] In some embodiments, N1 is A, N2 is A, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is U, and N5 is U.

[0229] In some embodiments, N1 is A, N2 is A, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is U, and N5 is U.

[0230] ii. An exemplary cap-proximal sequence wherein N1 is A and N2 is C. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is A, N4 is U, and N5 is U.

[0231] In some embodiments, N1 is A, N2 is C, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is C, N4 is U, and N5 is U.

[0232] In some embodiments, N1 is A, N2 is C, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is G, N4 is U, and N5 is U.

[0233] In some embodiments, N1 is A, N2 is C, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is C, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is U, N4 is U, and N5 is U.

[0234] iii. An exemplary cap-proximal sequence wherein N1 is A and N2 is G. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is A, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is A, N4 is U, and N5 is U.

[0235] In some embodiments, N1 is A, N2 is G, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is C, N4 is U, and N5 is U.

[0236] In some embodiments, N1 is A, N2 is G, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is G, N4 is U, and N5 is U.

[0237] In some embodiments, N1 is A, N2 is G, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is G, N3 is U, N4 is U, and N5 is U.

[0238] iv. An exemplary cap-proximal sequence where N1 is A and N2 is U. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is A, N4 is U, and N5 is U.

[0239] In some embodiments, N1 is A, N2 is U, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is C, N4 is U, and N5 is U.

[0240] In some embodiments, N1 is A, N2 is U, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is G, N4 is U, and N5 is U.

[0241] In some embodiments, N1 is A, N2 is U, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is A, N2 is U, N3 is U, N4 is U, and N5 is U.

[0242] v. An exemplary cap-proximal sequence where N1 is C and N2 is A. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is A, N4 is U, and N5 is U.

[0243] In some embodiments, N1 is C, N2 is A, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is C, N4 is U, and N5 is U.

[0244] In some embodiments, N1 is C, N2 is A, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is G, N4 is U, and N5 is U.

[0245] In some embodiments, N1 is C, N2 is A, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is U, and N5 is U.

[0246] vi. An exemplary cap-proximal sequence wherein N1 is C and N2 is C. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is A, N4 is U, and N5 is U.

[0247] In some embodiments, N1 is C, N2 is C, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is C, N4 is U, and N5 is U.

[0248] In some embodiments, N1 is C, N2 is C, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is G, N4 is U, and N5 is U.

[0249] In some embodiments, N1 is C, N2 is C, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is C, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is A, N3 is U, N4 is U, and N5 is U.

[0250] vii. An exemplary cap-proximal sequence wherein N1 is C and N2 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is A, N4 is U, and N5 is U.

[0251] In some embodiments, N1 is C, N2 is G, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is C, N4 is U, and N5 is U.

[0252] In some embodiments, N1 is C, N2 is G, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is G, N4 is U, and N5 is U.

[0253] In some embodiments, N1 is C, N2 is G, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is G, N3 is U, N4 is U, and N5 is U.

[0254] viii. An exemplary cap-proximal sequence wherein N1 is C and N2 is U. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is A, N4 is U, and N5 is U.

[0255] In some embodiments, N1 is C, N2 is U, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is C, N4 is U, and N5 is U.

[0256] In some embodiments, N1 is C, N2 is U, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is G, N4 is U, and N5 is U.

[0257] In some embodiments, N1 is C, N2 is U, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is C, N2 is U, N3 is U, N4 is U, and N5 is U.

[0258] ix. An exemplary cap-proximal sequence wherein N1 is G and N2 is A. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is A, N4 is U, and N5 is U.

[0259] In some embodiments, N1 is G, N2 is A, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is C, N4 is U, and N5 is U.

[0260] In some embodiments, N1 is G, N2 is A, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is G, N4 is U, and N5 is U.

[0261] In some embodiments, N1 is G, N2 is A, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is U, and N5 is U.

[0262] x. An exemplary cap-proximal sequence wherein N1 is G and N2 is C. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is A, N4 is U, and N5 is U.

[0263] In some embodiments, N1 is G, N2 is C, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is C, N4 is U, and N5 is U.

[0264] In some embodiments, N1 is G, N2 is C, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is G, N4 is U, and N5 is U.

[0265] In some embodiments, N1 is G, N2 is C, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is C, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is A, N3 is U, N4 is U, and N5 is U.

[0266] xi. An exemplary cap-proximal sequence wherein N1 is G and N2 is G. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is A, N4 is U, and N5 is U.

[0267] In some embodiments, N1 is G, N2 is G, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is C, N4 is U, and N5 is U.

[0268] In some embodiments, N1 is G, N2 is G, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is G, N4 is U, and N5 is U.

[0269] In some embodiments, N1 is G, N2 is G, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is G, N3 is U, N4 is U, and N5 is U.

[0270] xii. An exemplary cap-proximal sequence wherein N1 is G and N2 is U. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is A, N4 is U, and N5 is U.

[0271] In some embodiments, N1 is G, N2 is U, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is C, N4 is U, and N5 is U.

[0272] In some embodiments, N1 is G, N2 is U, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is G, N4 is U, and N5 is U.

[0273] In some embodiments, N1 is G, N2 is U, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is G, N2 is U, N3 is U, N4 is U, and N5 is U.

[0274] xiii. An exemplary cap-proximal sequence wherein N1 is U and N2 is A. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is A, N4 is U, and N5 is U.

[0275] In some embodiments, N1 is U, N2 is A, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is C, N4 is U, and N5 is U.

[0276] In some embodiments, N1 is U, N2 is A, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is G, N4 is U, and N5 is U.

[0277] In some embodiments, N1 is U, N2 is A, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is U, and N5 is U.

[0278] xiv. An exemplary cap-proximal sequence wherein N1 is U and N2 is C. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is A, N4 is U, and N5 is U.

[0279] In some embodiments, N1 is U, N2 is C, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is C, N4 is U, and N5 is U.

[0280] In some embodiments, N1 is U, N2 is C, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is G, N4 is U, and N5 is U.

[0281] In some embodiments, N1 is U, N2 is C, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is C, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is A, N3 is U, N4 is U, and N5 is U.

[0282] xv. An exemplary cap-proximal sequence wherein N1 is U and N2 is G. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is A, N4 is U, and N5 is U.

[0283] In some embodiments, N1 is U, N2 is G, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is C, N4 is U, and N5 is U.

[0284] In some embodiments, N1 is U, N2 is G, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is G, N4 is U, and N5 is U.

[0285] In some embodiments, N1 is U, N2 is G, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is G, N3 is U, N4 is U, and N5 is U.

[0286] xvi. An exemplary cap-proximal sequence wherein N1 is U and N2 is U. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is A, N4 is U, and N5 is U.

[0287] In some embodiments, N1 is U, N2 is U, N3 is C, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is C, N4 is U, and N5 is U.

[0288] In some embodiments, N1 is U, N2 is U, N3 is G, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is G, N4 is U, and N5 is U.

[0289] In some embodiments, N1 is U, N2 is U, N3 is U, N4 is A, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is A, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is A, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is A, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is C, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is C, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is C, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is C, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is G, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is G, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is G, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is G, and N5 is U. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is U, and N5 is A. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is U, and N5 is C. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is U, and N5 is G. In some embodiments, N1 is U, N2 is U, N3 is U, N4 is U, and N5 is U.

[0290] It will be understood that embodiments for variables described above and herein (e.g., Sections Di-D-xvi) may be combined with other embodiments for other variables described above and herein (e.g., 5' cap).

[0291] Exemplary 5'UTRs include human alpha globin (hAg) 5'UTR or a fragment thereof, TEV 5'UTR or a fragment thereof, HSP70 5'UTR or a fragment thereof, or c-Jun 5'UTR or a fragment thereof.

[0292] In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR or a fragment thereof. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 11. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 12. In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the human alpha globin 5'UTR provided in SEQ ID NO: 12.

[0293] 3'UTR In some embodiments, the RNAs disclosed herein include a 3'UTR. If present, the 3'UTR is located at the 3' end downstream of the termination codon of the protein-coding region, although 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.

[0294] In some embodiments, the RNAs disclosed herein comprise a 3'UTR comprising a sequence element derived from an "amino-terminal enhancer of split" (AES) mRNA and / or a sequence element derived from mitochondrially encoded 12S ribosomal RNA (MT-RNR1). In some embodiments, the RNAs disclosed herein comprise a 3'UTR comprising an AES 3'UTR, or a fragment or variant thereof. In some embodiments, the RNAs disclosed herein comprise a 3'UTR comprising an MT-RNR1 non-coding RNA, or a fragment or variant thereof. In some embodiments, the RNAs disclosed herein comprise a 3'UTR comprising a combination of (i) an AES 3'UTR, or a fragment or variant thereof, and (ii) an MT-RNR1 non-coding RNA, or a fragment or variant thereof. Such and additional 3'UTR sequences were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see, e.g., WO2017 / 060314, the entire contents of which are incorporated herein by reference for purposes described herein). In some embodiments, the RNA disclosed herein comprises a 3'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the 3'UTR provided in SEQ ID NO: 13. In some embodiments, the RNA disclosed herein comprises a 3'UTR provided in SEQ ID NO: 13.

[0295] In some embodiments, the RNA disclosed herein comprises a 3'UTR comprising two copies of the 3'UTR of a heterologous gene. In some embodiments, the RNA disclosed herein comprises a 3'UTR comprising two copies of the 3'UTR of a human globulin mRNA, in some embodiments, the 3'UTR of a human beta-globulin mRNA. For example, see WO2007 / 036366, the entire contents of which are incorporated herein by reference for the purposes described herein.

[0296] In some embodiments, the 3'UTR or a sequence proximal thereto comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is a XhoI site.

[0297] Poly A In some embodiments, the RNAs disclosed herein comprise a polyadenylation (polyA) sequence, e.g., as described herein. In some embodiments, the polyA sequence is located downstream of the 3' UTR, e.g., adjacent to the 3' UTR.

[0298] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and can follow the 3'UTR in the RNAs described herein. Uninterrupted poly(A) sequences are characterized by consecutive adenylate residues. Uninterrupted poly(A) sequences are typical in nature. The RNAs disclosed herein can have poly(A) sequences attached to the free 3' end of the RNA by a non-template-dependent RNA polymerase after transcription, or poly(A) sequences encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0299] In some embodiments, poly(A) sequences of approximately 120 A nucleotides have been demonstrated to have a beneficial effect on the levels of RNA in transfected eukaryotic cells and on the levels of protein translated from open reading frames located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0300] Poly(A) sequences can be of any length. In some embodiments, poly(A) sequences comprise, consist essentially of, or 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 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly(A) sequence, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% of the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0301] In some embodiments, poly(A) sequences are attached during RNA transcription based on a DNA template containing repetitive dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand, e.g., during preparation of in vitro transcribed RNA. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as a poly(A) cassette.

[0302] In some embodiments, a poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 can be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5 to 50 nucleotides, exhibit consistent propagation of plasmid DNA in E. coli at the DNA level, yet are associated with beneficial properties at the RNA level related to support of RNA stability and translation efficiency. In some embodiments, the poly(A) sequences contained within the RNA polynucleotides described herein consist essentially of A nucleotides but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. In some embodiments, interrupted poly(A) sequences according to the present disclosure are described in WO2016 / 005324, the entire contents of which are incorporated herein by reference for purposes described herein.

[0303] In some embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed by a nucleotide other than A at its 3' end.

[0304] In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0305] In some embodiments, the RNA disclosed herein comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the RNA disclosed herein comprises the poly(A) sequence of SEQ ID NO: 14.

[0306] payload In some embodiments, the RNA polynucleotides disclosed herein comprise a sequence encoding a payload, e.g., as described herein. In some embodiments, the sequence encoding the payload comprises a promoter sequence. In some embodiments, the sequence encoding the payload comprises a sequence encoding a secretory signal peptide.

[0307] In some embodiments, the payload is selected from a protein replacement polypeptide; an antibody agent; a cytokine; an antigenic polypeptide; a gene editing component; a tissue engineering component, or a combination thereof.

[0308] In some embodiments, the payload is or comprises a protein replacement polypeptide. In some embodiments, the protein replacement polypeptide comprises a polypeptide whose expression is aberrant in a disease or disorder. In some embodiments, the protein replacement polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein. In some embodiments, the protein replacement polypeptide comprises an enzyme.

[0309] In some embodiments, the disease or disorder in which polypeptide expression is aberrant includes, but is not limited to, a rare disease, a metabolic disorder, a muscular dystrophy, a cardiovascular disease, or a monogenic disease.

[0310] In some embodiments, the payload is or comprises an antibody agent. In some embodiments, the antibody agent binds to a polypeptide expressed on a cell. In some embodiments, the antibody agent comprises a CD3 antibody, a claudin 6 antibody, or a combination thereof.

[0311] In some embodiments, the payload is or comprises a cytokine, or a fragment or variant thereof, hi some embodiments, the cytokine comprises IL-12, or a fragment, variant, or fusion thereof, IL-15, or a fragment, variant, or fusion thereof, GM-CSF, or a fragment or variant thereof; or IFN-alpha, or a fragment or variant thereof.

[0312] In some embodiments, the payload is or comprises an antigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the antigenic polypeptide comprises one epitope from an antigen. In some embodiments, the antigenic polypeptide comprises multiple different epitopes from an antigen. In some embodiments, the antigenic polypeptide comprises multiple different epitopes from at least two or more antigens. In some embodiments, an antigenic polypeptide comprising multiple different epitopes from one or more antigens is a polyepitope.

[0313] In some embodiments, the antigenic polypeptide comprises an antigenic polypeptide derived from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide derived from an infectious agent, an antigenic polypeptide derived from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

[0314] In some embodiments, the antigenic polypeptide comprises one or more antigenic polypeptides from influenza virus, Pneumoviridae (e.g., parainfluenza (PIV3), henipavirus), Paramyxoviridae (e.g., respiratory syncytial virus (RSV)), metapneumovirus (e.g., hMPV), coronavirus, herpes simplex virus (HSV) type 1 and / or type 2, Staphylococcus aureus, tuberculosis, Ebola / alphavirus, malaria, varicella-zoster virus, cytomegalovirus (CMV), norovirus, Zika virus, shingles, monkeypox virus, hepatitis C virus, or human immunodeficiency virus (HIV), or a combination thereof.

[0315] In some embodiments, the parasitic antigenic polypeptide comprises a malaria antigenic polypeptide.

[0316] In some embodiments, the viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a coronavirus antigenic polypeptide, a rabies antigenic polypeptide, a varicella-zoster virus antigenic polypeptide, a cytomegalovirus (CMV) antigenic polypeptide, a norovirus antigenic polypeptide, or a Zika virus antigenic polypeptide. In some embodiments, the viral antigenic polypeptide comprises an antigen from a virus associated with a zoonotic disease. In some embodiments, the viral antigenic polypeptide comprises a monkeypox virus antigenic polypeptide.

[0317] In some embodiments, the viral antigenic polypeptide is or comprises a coronavirus antigenic polypeptide. In some embodiments, the viral antigenic polypeptide is or comprises an alpha-coronavirus antigenic polypeptide. In some embodiments, the viral antigenic polypeptide is or comprises a beta-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 comprises at least two proline substitutions (e.g., at least three, at least four, at least five, at least six proline substitutions). In some embodiments, the SARS-CoV-2 protein, or an immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions corresponding to positions 986 and 987 of the SARS-CoV-2 S protein from the Wuhan strain. Additionally or alternatively, in some embodiments, the SARS-CoV-2 protein, or immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions corresponding to positions 817, 892, 899, and 942 of the SARS-CoV-2 S protein from the Wuhan strain. See, e.g., WO2021 / 243122, the entire contents of which are incorporated herein by reference for purposes described herein.

[0318] In some embodiments, the SARS-CoV-2 S polypeptide is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to a SARS-CoV-2 S polypeptide disclosed herein. In some embodiments, the SARS-CoV-2 S polypeptide is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:9.

[0319] In some embodiments, the SARS-CoV-2 S polypeptide is encoded by RNA that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to a SARS-CoV-2 S polynucleotide disclosed herein. In some embodiments, the SARS-CoV-2 S polypeptide is encoded by RNA that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to SEQ ID NO:10.

[0320] In some embodiments, the SARS-CoV-2 S polypeptide comprises one or more mutations characteristic of a SARS-CoV-2 variant, e.g., a SARS-CoV-2 variant predicted to be endemic and / or rapidly spreading in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided at the GISAID initiative database: https: / / www.gisaid.org, and / or data provided by the World Health Organization (WHO) (e.g., provided at https: / / www.who.int / activities / tracking-SARS-CoV-2-variants). Mutations characteristic of SARS-CoV-2 variants are known in the art. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences, and particularly their modifications compared to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., compared to SEQ ID NO: 9, may be useful in accordance with the present disclosure:

[0321] B.1.1.7 ("Variants of Concern 202012 / 01" (VOC-202012 / 01) B.1.1.7 is a SARS-CoV-2 variant first detected in October 2020 during the COVID-19 pandemic in the UK in samples collected the previous month and quickly began spreading by mid-December. This correlated with a significant increase in COVID-19 infection rates in the UK, an increase thought to be at least in part due to an N501Y mutation in the receptor-binding domain of the spike glycoprotein, which is required for binding to ACE2 on human cells. The B.1.1.7 variant is defined by 23 mutations: 13 nonsynonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., 17 protein-altering mutations and 6 non-altering mutations). Spike protein alterations in B.1.1.7 include deletions 69-70, 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0322] B.1.351(501.V2) The B.1.351 lineage, colloquially known as the South African COVID-19 variant, is a variant of SARS-CoV-2. Preliminary results indicate that this variant may have increased transmissibility. The B.1.351 variant is defined by multiple spike protein changes, including L18F, D80A, D215G, deletions 242-244, R246I, K417N, E484K, N501Y, D614G, and A701V. The spike region of the B.1.351 genome contains three mutations of particular interest: K417N, E484K, and N501Y.

[0323] B.1.1.298 (Cluster 5) B.1.1.298 was discovered in North Jutland, Denmark, and is believed to have been transmitted from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been identified. Specific mutations include deletions 69-70, Y453F, D614G, I692V, M1229I, and optionally S1147L.

[0324] P.1(B.1.1.248) The lineage B.1.1.248, also known as the Brazil (Brazilian) variant, is one of the SARS-CoV-2 variants designated the P.1 lineage. P.1 has several S protein modifications [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F] and is similar to the South African variant B.1.351 at certain critical RBD positions (K417, E484, N501).

[0325] B.1.427 / B.1.429(CAL.20C) Lineage B.1.427 / B.1.429, also known as CAL.20C, is defined by the following modifications in the S-protein: S13I, W152C, L452R, and D614G (of which the L452R modification is of particular concern). The CDC lists B.1.427 / B.1.429 as a "variant of concern."

[0326] B.1.525 B.1.525 has the same E484K modification found in the P.1 and B.1.351 variants and the same ΔH69 / ΔV70 deletion found in B.1.1.7 and B.1.1.298. It also has the modifications D614G, Q677H, and F888L.

[0327] B.1.526 B.1.526 was detected as an emerging lineage of virus isolates in the New York area that shares mutations with previously reported variants. The most common set of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

[0328] B.1.1.529 B.1.1.529 was first detected in South Africa in November 2021. Omicron grows approximately 70 times faster than the delta variant and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, several Omicron sublineages have emerged. The current Omicron variants of concern, along with certain characteristic mutations associated with each S protein, are listed below. The BA.4 and BA.5 S proteins have the same set of characteristic mutations, which is why the table below has a single column for "BA.4 or BA.5" and why, in some embodiments, the present disclosure refers to the "BA.4 / 5" S protein. [Table 1-1] [Table 1-2]

[0329] In addition to the Omicron variants described above, additional variants of BA.5 have been observed that contain one or more of the following mutations in the S protein (positions shown relative to SEQ ID NO: 9): R346X, K444X, V445X, N450D, and S:N460X (e.g., such variants include BF.7, BF.14, and BQ.1).

[0330] In one embodiment, the vaccine antigen described herein comprises, consists essentially of, or consists of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof. In some embodiments, the RNA described herein comprises a nucleotide sequence encoding a SARS-CoV-2 S protein containing one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron variant. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein containing one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations listed in Table 2. In some such embodiments, the one or more mutations may be derived from two or more variants as listed in Table 2. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations identified in Table 2 as being characteristic of a particular Omicron variant (e.g., in some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations listed in Table 2 as being characteristic of the Omicron BA.1, BA.2, BA.2.12.1, BA.4 / 5, BA.2.75, BA.2.75.1, BA.4.6, or XBB variant).

[0331] In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes a subset of mutations listed in Table 2. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes a mutation listed in Table 2 that is most prevalent for a particular variant (e.g., a mutation detected in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequences collected on the day of sequencing for a given variant). Mutation prevalence can be determined, for example, based on published sequences (e.g., sequences collected and made publicly available by GISAID).

[0332] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4 / 5 variant.

[0333] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant.

[0334] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75.2 variant.

[0335] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 variant.

[0336] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron XBB variant.

[0337] In some embodiments, the payload is or comprises a tumor antigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the tumor antigenic polypeptide comprises a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the tumor antigenic polypeptide is p53, ART-4, BAGE, ss-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1. , MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, WT-1, or a combination thereof.

[0338] In some embodiments, the tumor antigenic polypeptide comprises a tumor antigen from a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, or a combination thereof. In some embodiments, the tumor antigenic polypeptide comprises a melanoma tumor antigen. In some embodiments, the tumor antigenic polypeptide comprises a prostate cancer antigen. In some embodiments, the tumor antigenic polypeptide comprises an HPV16-positive head and neck cancer antigen. In some embodiments, the tumor antigenic polypeptide comprises a breast cancer antigen. In some embodiments, the tumor antigenic polypeptide comprises an ovarian cancer antigen. In some embodiments, the tumor antigenic polypeptide comprises a lung cancer antigen. In some embodiments, the tumor antigenic polypeptide comprises an NSCLC antigen.

[0339] In some embodiments, the payload is or comprises an autoantigenic polypeptide, or an immunogenic variant or fragment thereof. In some embodiments, the autoantigenic polypeptide comprises an antigen that is typically expressed on cells and recognized as an autoantigen by the immune system. In some embodiments, the autoantigenic polypeptide comprises a multiple sclerosis antigenic polypeptide, a rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof.

[0340] In vitro synthesis of RNA polynucleotides Generally, an in vitro transcription reaction includes a double-stranded DNA template consisting of a template strand (also known as a non-coding strand) and a coding strand. As RNA synthesis proceeds in the 5' to 3' direction, RNA polymerase reads the template strand in the 3' to 5' direction. Thus, those skilled in the art will understand that when a template strand is described in this disclosure as including a sequence containing positions +1, +2, +3, ... +N, these positions are read in the 3' to 5' direction. Similarly, those skilled in the art will understand that when an RNA transcript is described in this disclosure as including a sequence containing positions +1, +2, +3, ... +N, such positions are read in the 5' to 3' direction.

[0341] Those skilled in the art will understand that a "transcription start site" sequence, when presented as a single-stranded (SS) sequence, is typically relative to the coding strand sequence and reflects the canonical location where the associated RNA polymerase will initiate transcription. Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, a cap (e.g., a co-transcriptional cap) can include one or more residues corresponding to the location of such a "transcription start site sequence," such that the first residue added by the RNA polymerase can actually represent the second (or later) residue of the canonical transcription start site.

[0342] In some embodiments, the DNA template is a linear DNA molecule. In some embodiments, the DNA template is a circular DNA molecule. The DNA can be obtained or generated using methods known in the art, including, for example, gene synthesis, recombinant DNA technology, or a combination thereof. In some embodiments, the DNA template includes a nucleotide sequence encoding a transcribed region of interest (e.g., encoding an RNA described herein) and a promoter sequence recognized by an RNA polymerase selected for use in in vitro transcription. Various RNA polymerases are known in the art, including, for example, DNA-dependent RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, N4 virion RNA polymerase, or variants or functional domains thereof). Those skilled in the art will readily understand that the RNA polymerase utilized herein can be a recombinant RNA polymerase and / or a purified RNA polymerase, i.e., not as part of a cell extract but containing other components in addition to the RNA polymerase. Those skilled in the art will recognize appropriate promoter sequences for a selected RNA polymerase. In some embodiments, the DNA template comprises a promoter sequence for T7 RNA polymerase.

[0343] In some embodiments, the present disclosure provides the insight that double-stranded DNA templates containing a pyrimidine base (e.g., C or U) at the +2 position of the transcription start site from an RNA polymerase promoter (e.g., a T7 promoter) can be useful for improving capping efficiency (e.g., the proportion of capped transcripts in an in vitro transcription reaction), the quality of an RNA preparation (e.g., an in vitro transcribed RNA, e.g., the amount of short polynucleotide by-products produced), the translation efficiency of an RNA encoding a payload, and / or the expression of a polypeptide payload encoded by the RNA. In some embodiments, such improvements can be observed regardless of the identity of the 5' UTR, capping method (e.g., enzymatic capping vs. co-transcriptional capping), cap structure (e.g., Cap0, Cap1, or Cap2), coding sequence, type of ribonucleotide (e.g., modified nucleotides vs. unmodified nucleotides), formulation (e.g., lipoplexes vs. lipid nanoparticles), or a combination thereof. In certain embodiments, the double-stranded DNA template comprises a pyrimidine base (e.g., C or U) at position +2 of the transcription start site and a G at position +1 of the transcription start site. A pyrimidine base (e.g., C or U) or a purine base (e.g., G or A) can be present at position +3 of the transcription start site of the double-stranded DNA template, and in certain embodiments, such a double-stranded DNA template comprises a G at position +3 of the transcription start site.

[0344] As will be understood by those skilled in the art, the 3' end of the cap structure can be extended by RNA polymerase using naturally occurring and / or modified ribonucleotides. Thus, those skilled in the art will understand that throughout the specification described herein, references to A, U, G, or C can refer to the naturally occurring and / or modified ribonucleotides described herein. For example, in some embodiments, U is uridine. In some embodiments, U is a modified uridine (e.g., pseudouridine, 1-methylpseudouridine).

[0345] In some embodiments, the provided RNA polynucleotides are produced by in vitro transcription reactions described herein, e.g., using different combinations of cap structures and transcription start sites (e.g., as described herein).

[0346] AGA transcription start site In some embodiments, a transcription start site that may be useful according to the present disclosure is AGA. In some embodiments, an in vitro transcription reaction includes: (i) a template DNA strand comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence described herein, wherein the template DNA strand comprises a sequence complementary to an AGA transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide ...

Claims

1. 1. A composition or medical preparation comprising an RNA polynucleotide, said RNA polynucleotide comprising: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of said RNA polynucleotide; and a sequence encoding a payload; The cap-proximal sequence is N １ , N ２ , N ３ , N ４ , and N ５ Including N １ ~N ５ corresponds to positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; the 5' cap and a portion of the cap-proximal sequence form a cap structure comprising at least N 1 , wherein N １ is G and N ２ is C or U, and each of N 3 , N 4 and N 5 is selected from A, C, G and U, preferably N 3 is G or A.

2. The cap structure has the structure: G*N １ pN ２ and G* is a compound having the structure of formula (I): 【Chemistry 1】 or a salt thereof, During the ceremony, Each R ２ and R ３ is —OH or —OCH ３ and 2. The composition or medical preparation of claim 1, wherein X is O or S, preferably X is O.

3. R ２ The composition or medical preparation of claim 2, wherein is -OH or R 2 is -OCH 3 .

4. R ３ The composition or medical preparation of claim 2, wherein is -OH or R 3 is -OCH 3 .

5. A composition or medical preparation as described in claim 1, wherein the cap structure comprises a Cap0 or Cap1 structure.

6. N ４ 2. The composition or medical preparation of claim 1, wherein N4 is A, or N4 is C, or N4 is G, or N4 is U.

7. N ５ 2. The composition or medical preparation of claim 1, wherein N is A, or N 5 is C, or N 5 is G, or N 5 is U. (i) a template DNA strand comprising a polynucleotide sequence complementary to the RNA polynucleotide sequence of claim 1; (ii) a polymerase; (iii) ribonucleotides, and (iv) N １ pN ２ a 5' cap structure comprising 1. An in vitro transcription reaction mixture comprising:

9. A method for producing a capped RNA polynucleotide, comprising producing the capped RNA polynucleotide using the in vitro transcription reaction mixture described in claim 8.

10. 1. A method of making a capped RNA polynucleotide, wherein the RNA polynucleotide comprises: a 5' cap; a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload; The cap-proximal sequence is N １ , N ２ , N ３ , N ４ , and N ５ Including N １ ~N ５ correspond to positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; N １ is G and N ２ is U or C, and N ３ , N ４ , and N ５ are each independently selected from A, C, G, and U; The method comprises transcribing, in the presence of the 5' cap and an RNA polymerase, a template DNA strand comprising a polynucleotide sequence complementary to the RNA polynucleotide sequence.

11. 11. The method of claim 9 or 10, wherein the +4 and +5 positions of the template DNA strand are each independently A, C, G, or T.

12. 11. The method of claim 9 or 10, further comprising purifying the capped RNA polynucleotide.

13. A capped RNA polynucleotide produced by the method of claim 9 or 10.

14. A method for preparing a pharmaceutical composition, comprising combining a preparation comprising an RNA polynucleotide according to any one of claims 1 to 7, or a capped RNA polynucleotide produced by the method of claim 9 or 10, with a preparation comprising a lipid.

15. A pharmaceutical composition for pharmaceutical use comprising an RNA polynucleotide described in any one of claims 1 to 7, or a capped RNA polynucleotide produced by the method described in claim 9 or 10.

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