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 the same, as well as compositions and methods of making and using the 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 sources of particular problems that may be encountered in the expression of polypeptides encoded by RNA therapeutics. Among other things, the present disclosure provides techniques for improving 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 caps provide improved RNA transcription, capping efficiency, translation efficiency, and / or polypeptide payload expression, and / or reduced by-product formation. In some embodiments, the present disclosure recognizes that certain caps, when utilized with specific transcription start sites, 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 initiation sites (e.g., the first three residues are each "G," producing RNA), and has been reported to prefer "G" as the initiation residue (e.g., the first residue is "G," producing RNA). Conrad, et al. (2020) Communications Biology 3:439. A study comparing T7 transcription of templates with different initiation 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] Commonly used dinucleotide cap analogs also contain a "G" at the 3' end (e.g., m2 7,2’-OGppSpG ("β-S-ARCA" or "D1") 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] In some embodiments, cap1 analogs (including, for example, commercially available ones) can be incorporated into synthetic RNA (e.g., RNA produced by in vitro transcription (IVT)) in the correct orientation to produce cap1 RNA with high capping efficiency, all in a rapid co-transcription reaction. For example, the cap analog for synthetic self-amplifying RNA (saRNA) can be or include CleanCap AU, TriLink (#N7-114). For example, the cap analog for synthetic mRNA can be or include CleanCap AG, Trilink, #N7-413. See Henderson, JM, et al. (2021) Current protocols, 1, e39. An attractive feature of these trinucleotide cap1 analogs is that they require an A initiator, which, in contrast to those containing a G triplet as the transcription start site, may avoid potential slippage of RNA polymerase on the DNA template strand. See Imburgio, et al. (2000) Biochemistry, 39, 10419-10430.

[0008] Furthermore, mRNA capped with an anti-reverse cap analog (ARCA) may have higher translation efficiency compared to conventional cap analogs. See Stepinski, J., et al. (2001) RNA (New York, NY), 7, 1486-1495, and Kuhn, A. N., et al. (2010) Gene therapy, 17, 961-971. For example, a variant of CleanCap AG with a modification at the C3' position of 7-methylguanosine (CleanCap AG 3' OMe) may play an important role in the development of immunotherapeutic vaccination strategies against SARS-CoV-2. See Sahin, U. et al. (2021) Nature, 595, 572-577. In some embodiments, without wishing to be bound by theory, the present disclosure provides the recognition that ARCA cap1 analogs may exhibit better translation efficiency and / or biological activity compared to those capped with their non-ARCA versions (see Figure 1). Additionally or alternatively, cap analogs paired with specific initiation sequences, e.g., WO2021 / 214204A1, have been described that attempt to address one or more of these problems.

[0009] Additionally or alternatively, incorporation of nucleoside modifications (e.g., modified uridines (e.g., N1-methylpseudouridine (m1ψ)) and / or modified adenosines (e.g., N6-methyladenine (m6A)) into synthetic RNA (e.g., in some embodiments, IVT mRNA) may increase the biological stability and thereby improve the durability of the encoded protein compared to unmodified RNA. See Kariko, K., et al. (2008) Molecular therapy: the journal of the American Society of Gene Therapy, 16, 1833-1840, and Gao, Y., et al. (2020) Immunity, 52, 1007-1021.e8. However, without wishing to be bound by theory, because certain saRNAs cannot contain modified nucleosides, the use of such modified nucleosides has been primarily limited to prophylactic vaccines against infectious diseases, as opposed to non-replicating mRNAs. Bloom, K., et al. See, e.g., J. et al. (2021) Gene therapy, 28, 117-129. Additionally or alternatively, non-replicating mRNAs also have great potential in research areas such as gene editing and protein replacement therapy, where reducing and eliminating immune regulation is important to reach appropriate therapeutic goals.

[0010] Although the potential benefits of using various modified nucleosides are contemplated, the impact of cap analogs containing modified nucleosides on the quality, translation efficiency, and biological activity or immunogenicity of mRNAs encoding potential therapeutic proteins is not understood. In some embodiments, the present disclosure also provides the recognition that a 5' cap comprising modified nucleoside(s) may be a promising alternative to current capping strategies in mRNA vaccines, particularly RNA-based therapeutics.

[0011] In some embodiments, the present disclosure recognizes that certain 5' cap structures (e.g., a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof and N2 is U or an analog thereof), when paired with, for example, a particular transcription start site (e.g., an AUN such as AUA), provide improved RNA transcription, improved translation efficiency, and / or improved and / or elongated polypeptide payload expression compared to transcripts comprising other 5' cap structures (e.g., the use of CC114 or CC413 caps). Additionally or alternatively, in some embodiments, the present disclosure recognizes that certain 5' cap structures (e.g., a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof and N2 is U or an analog thereof), when paired with, for example, a particular transcription start site (e.g., an AUN such as AUA), result in higher capping efficiency, reduced amounts of short contaminants, and reduced toxicity due to cytokine / chemokine secretion compared to transcripts comprising other 5' cap structures (e.g., the use of CC114 or CC413 caps). Additionally or alternatively, in some embodiments, the present disclosure recognizes that certain 5' cap structures (e.g., a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof and N2 is U or an analog thereof) can be accommodated in not only replicating but also non-replicating mRNAs, for example, when paired with a particular transcription start site (e.g., AUN, such as AUA).

[0012] Additionally or alternatively, in some embodiments, the present disclosure provides compounds in which N2 is a modified U (e.g., pseudouridine, i.e., Ψ), and 1-methylpseudouridine (m 1The disclosed 5' cap structures, wherein N2 is a modified U (e.g., a pseudouridine, i.e., Ψ), and its analogs such as (m 1 The disclosed 5' cap structures, in which N2 is a modified U (e.g., pseudouridine, i.e., Ψ, and its analogs such as (m 1 ) and analogs thereof, such as Ψ) display, it is recognized that the demonstrated efficacy of the disclosed 5' cap structures can be adapted to non-replicating as well as replicating mRNAs.

[0013] Thus, in some embodiments, the present disclosure provides compositions or medical preparations comprising, inter alia, an RNA polynucleotide comprising (i) a 5' cap, e.g., as disclosed herein; (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.

[0014] In some embodiments, the present disclosure also provides a trinucleotide cap G*N1pN2, or a salt thereof, G* comprises the structure of formula I'; [ka] During the ceremony, Each R 2 and R3 are independently -OH or -OCH3; X is OH or SH, N1 is A or an analog thereof; N2 is U or an analog thereof; p is a phosphate (e.g., -P(=O)(OH)- or -P(=O)(O - )-) or thiophosphates (e.g., -P(=S)(OH)- or -P(=S)(O - )-).

[0015] It will be appreciated that in some embodiments, a trinucleotide cap having the structure of Formula I' (e.g., a trinucleotide cap comprising an N2 nucleotide of Formula II'' or Formula II''') exhibits surprising advantages, such as, for example, improved translation efficiency, as discussed in more detail herein. [Brief explanation of the drawings]

[0016] [Figure 1] Comparison of mouse EPO and hematocrit levels is shown. CC113 corresponds to (m7)Gppp(m2'-O)ApG, and CC413 corresponds to (m27,3'-O)Gppp(m2'-O)ApG. The translation efficiency and biological activity of EPO mRNA capped with CC413 are significantly better than those of CC113. [Figure 2] A shows a comparison of RNA quality after in vitro transcription using the (m2 7,3'-O)Gppp(m2'-O)ApU cap (i.e., compound I'-1) and various start sites. The highest yield was observed with the AUAGU start site. B shows a comparison of capping efficiency by 21% urea-PAGE. High yields were observed when the (m2 7,3'-O)Gppp(m2'-O)ApU cap (i.e., compound I'-1) was used at concentrations ranging from 3 to 6 mM. Regardless of the concentration used, capping efficiency was close to 100%. [Figure 3]The capping efficiencies of compounds I'-1 and I'-6 were compared using 21% urea-PAGE. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. The capping efficiencies of compounds I'-1 and I'-6 were close to 100%, comparable to those of CC114 and CC413. [Figure 4] A comparison of the amount of short contaminants at specific caps and initiation sites is shown. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. Minimal amounts of short contaminants were observed for compound I'-6 and CC413 mRNA, but significant amounts were observed for the other unmodified mRNAs tested, regardless of CAP. [Figure 5] Figure 1 shows a comparison of XTT assay results for viable PMBCs at 24 hours. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. No toxic effects on PMBC cell viability were observed with up to 1 μg / well of mRNA from compound I'-1 or compound I'-6. Transfection of unmodified mRNA resulted in a decrease in cell viability starting at a dose of 0.333 μg / well, but this effect was independent of the cap used and dependent on the mRNA modification. [Figure 6]Panels A, B, C, D, E, F, and G show a comparison of cytokine / chemokine secretion in human PBMCs. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. Compound I'-6 is comparable to CC413 in terms of the amount of cytokine / chemokine secreted by human PBMCs after transfection with m1Ψ-modified mRNA. In terms of unmodified mRNA cytokines / chemokines, compound I'-1 produces cytokines / chemokines comparable to CC114 and significantly higher than CC413. [Figure 7] This figure shows a comparison of EPO secretion in human hepatocytes on day 1 after transfection with 0.1 μg / well of TransIT-EPO mRNA (IV187). Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. Compounds I'-1 and I'-6 show higher translation compared to CC413 in human hepatocytes at 24 hours. [Figure 8A]Comparison of plasma EPO levels in mice IV-injected with 3 μg of TransIT-formulated somEPO mRNA (JR81) is shown. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. EPO mRNA capped with compound I'-6 showed 2-3 times greater translation at later time points compared to that capped with CC413, demonstrating that compound I'-6 has a strong beneficial effect on mRNA translation capacity and biological activity. [Figure 8B] Figure 1 shows hematocrit levels in mice injected IV with 3 μg of TransIT-complexed somEPO mRNA (hAg) capped with specific caps. Cap1 corresponds to compound I'-1 ((m2 7,3'-O)Gppp(m2'-O)ApU), Cap2 corresponds to compound I'-6 ((m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ), CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. Hemoatocrit values ​​in mice injected with EPO mRNA capped with compound I'-6 were very high and further increased 14 days after injection. [Figure 9]Figure 1 shows a comparison of plasma EPO levels in mice IV injected with 3 μg of TransIT-formulated somEPO mRNA capped with the cap analog of formula I'. CC114 corresponds to (m7)Gppp(m2'-O)ApU, and CC413 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApG. I'-1 corresponds to (m2 7,3'-O)Gppp(m2'-O)ApU. I'-2 corresponds to (m2 7,2'-O)Gppp(m2'-O)ApU. I'-13 corresponds to m7Gppp(m2'-O)Ap(m1)Ψ. I'-5 corresponds to (m2 7,2'-O)Gppp(m2'-O)Ap(m1)Ψ. I'-6 corresponds to (m2 7,3'-O)Gppp(m2'-O)Ap(m1)Ψ. ARCA analogs I'-1 and I'-6 were significantly better translated than the non-ARCA-capped CC114 and I'-13, regardless of RNA modification. m1Ψ-mRNA capped with the non-ARCA cap I'-13 containing the m1Ψ-modified RNA was translated 8- and 25-fold more than U-mRNA capped with the non-ARCA CC114 without nucleoside modifications at 6 and 24 h postinjection, respectively. [Figure 10] Comparison of EPO levels in mice injected with 3 μg of TransIT formulated with U-containing mRNA capped with I'-1 and mΨ-modified mRNA capped with I'-6 is shown. I'-6 (i.e., mRNA with the mΨ-mΨ combination) performed best at each time point after administration, translating 2-3 times more than I'-1. U-containing mRNA capped with I'-1 showed significantly lower translation capacity at each time point than that observed for the mΨ modification present in both the cap analog (I'-6) and the mRNA. [Figure 11]Figure 1 shows a comparison of EPO levels in mice injected with 3 μg of TransIT-formulated mΨ-modified mRNA with caps containing unmodified uridine (I'-1) and unmodified pseudouridine (I'-3) and modified uridine (U) or pseudouridine (Ψ) (N5-methyluridine (I'-9), N5-methoxyuridine (I'-12), N1-methylpseudouridine (I'-6), and N1-propargylpseudouridine (I'-16). At 48 and 72 hours post-injection, EPO levels in mice injected with mRNA capped with the Ψ-containing cap analog (I'-3-(m2 7,3'-O)G(5')ppp(5')(m2'-O)ApΨ) were significantly higher than mΨ (I'-6-(m2 The potency of the N-methylpseudouridine (1-methyl-Ψ)-containing cap (I'-6) was equal to or slightly lower than that of the N-methylpseudouridine (1-methyl-Ψ)-containing cap (I'-6) (Figure 11). Neither uridine (U) nor its derivatives (5-methylU, 5-methoxyU) nor the pseudouridine derivative 1-propargylΨ could improve the potency of the N-methylpseudouridine (1-methyl-Ψ)-containing cap (I'-6). [Figure 12] Figure 1 shows the effects on cytokine and chemokine levels after application of EPO mRNA formulated with lipoplex (LPX). A shows the effect of CC413 and I'-6 on IL-6 levels. B shows the effect of CC413 and I'-6 on TNF-α levels. C shows the effect of CC413 and I'-6 on IL-1β levels. D shows the effect of CC413 and I'-6 on IFN-γ levels. E shows the effect of CC413 and I'-6 on MIP-1β levels. I'-6 showed a smaller increase in proinflammatory cytokine and chemokine levels compared to CC413 across the concentrations tested (i.e., I'-6 was less immunogenic). [Figure 13]Comparison of EPO levels in primary human hepatocytes transfected with 0.1 μg / well of TransIT-formulated somEPO mRNA. EPO levels were measured from supernatants transfected with uRNA capped with I'-6 or CC114. Increased EPO secretion in primary human cells was detected at all three test time points: 24 h, 48 h, and 144 h. These results suggest that cap1 analogs, such as I'-6, are suitable for translation of the encoded protein and can be used to synthesize non-replicating functional mRNA. [Figure 14] Figures A-D show a comparison of EPO uRNA capped with I'-6 and I'-1. In this case, both mRNAs had the same TAGT 5' end. I'-6 demonstrated benefits leading to significantly lower cytokines (IL-6 (Figure 14A), TNF-α (Figure 14B), IL-1β (Figure 14C), and IFN-γ (Figure 14D)) 24 hours after application to human PBMCs. Therefore, I'-6 results in lower immunogenicity. [Figure 15] Figure 1 shows EPO secretion after application of EPO-encoding Ψ-mRNA capped with uridine (U) or pseudouridine (Ψ) derivatives CC413 and I'-3, respectively. EPO levels were higher with I'-3 compared to CC413 at 24 and 48 hours. [Figure 16] A comparison of EPO-encoding mRNAs capped with cap1 analogs containing N5-methyluridine (I'-9), N5-methoxyuridine (I'-12), N1-methylpseudouridine (I'-6), and N1-propargylpseudouridine (I'-16) is shown. I'-6 showed increased levels of secreted EPO at 24 hours compared with the other caps. In addition, mRNAs with modified caps resulted in increased EPO secretion at 24 and 48 hours compared with unmodified I'-1.

[0017] 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.

[0018] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0019] 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).

[0020] The compounds of the present disclosure include those generally described above, and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. Additionally, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0021] Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that remain substantially unchanged when subjected to conditions that enable their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0022] The recitation of a list of chemical groups within any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0023] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydriodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Examples of the salts include phonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0024] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0025] Unless otherwise stated, structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, e.g., R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformer isomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure. Unless otherwise stated, all tautomeric forms are within the scope of the disclosure. Additionally, unless otherwise stated, the disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms. For example, replacement of hydrogen by deuterium or tritium, or 13 C or 14 Compounds having this structure, including the replacement of a carbon with a C-enriched carbon, are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as therapeutic agents according to this disclosure. In some embodiments, compounds of this disclosure include one or more deuterium atoms.

[0026] 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.

[0027] 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 for 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.

[0028] 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."

[0029] 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.

[0030] 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.

[0031] Agent: As used herein, the term "agent" may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular configuration and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class, including, for example, a small molecule, polypeptide, nucleic acid, monosaccharide, lipid, metal, or combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is devoid of or substantially free of any polymers or polymer moieties.

[0032] Aliphatic or aliphatic group: As used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocycle," "carbocyclic," "cycloaliphatic," or "cycloalkyl") and has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0033] Unsaturated: As used herein, means that a moiety has one or more units of unsaturation.

[0034] Partially unsaturated: As used herein, refers to a ring moiety that contains at least one double or triple bond. As used herein, the term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0035] Amino acid: In its broadest sense, as used herein, the term "amino acid" 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.

[0036] 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 generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated by performance of a synthetic process that is substantially similar to (e.g., shares multiple steps with) that which generates the reference substance. In some embodiments, an analog is generated or can be generated by performance of a synthetic process that is different from that used to generate the reference substance.

[0037] 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.).

[0038] 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 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, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] 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, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few different characteristics. One of skill in the art 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.

[0044] 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).By way of example only, 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 it plays a similar role or performs a similar function, and / or that it exhibits a particular degree of sequence identity or homology or shares particular characteristic sequence elements.

[0045] 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.

[0046] 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).

[0047] 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.

[0048] 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.

[0049] 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).

[0050] 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.

[0051] 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 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 recognize or be able to readily determine the degree of difference and / or number of patients necessary or sufficient to achieve such statistical significance in a given context.

[0052] 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.

[0053] 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 various applications, including, for example, the production of proteins or polypeptides. Such synthetic RNA products may be translated in vitro or may be 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, etc. 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.

[0054] 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.

[0055] 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 the parent polypeptide.

[0056] 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.

[0057] 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.

[0058] 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 linkage, etc.), 3'-terminal modifications (e.g., conjugation, inverted linkage, 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 replacement of phosphodiester bonds. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the 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. DETAILED DESCRIPTION OF THE INVENTION

[0063] 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 having 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.

[0064] RNA polynucleotides As used herein, the terms "polynucleotide" or "nucleic acid" refer 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 synthetic RNA. In some embodiments, synthetic RNA is or comprises in vitro transcribed RNA (IVT RNA). According to the present invention, polynucleotides are preferably isolated.

[0065] 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, such as lambda phage, a viral vector, such as a retroviral, adenoviral, or baculoviral vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). In some embodiments, the vector may be an expression vector; alternatively or 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).

[0066] In some embodiments, the nucleic acids described and / or utilized herein may be or include recombinant and / or isolated molecules.

[0067] Those skilled in the art who 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 may self-hybridize or otherwise fold into 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.

[0068] 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).

[0069] 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.

[0070] In some embodiments, the RNA disclosed herein comprises a 5' cap as 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.

[0071] In some embodiments, the RNAs disclosed herein comprise, in a 5' to 3' direction, the following components: a 5' cap as 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.

[0072] 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.

[0073] 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.

[0074] In some embodiments, the RNA is a "replicon RNA" or simply a "replicon," particularly a "self-replicating RNA" or "self-amplifying RNA." In some embodiments, the replicon or self-replicating RNA is derived from or includes elements derived from an ssRNA virus, particularly a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are a typical example of a positive-stranded RNA virus. 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, as well as protein modification) and structural proteins (which form the virus particle). Typically, two open reading frames (ORFs) are present in the genome. Four nonstructural proteins (nsP1-nsP4) are typically co-encoded by a first ORF beginning near the 5' end of the genome, while the alphavirus structural proteins are co-encoded by a second ORF found downstream of 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 encoding the viral replicase and the other capable of being replicated by the replicase in trans (hence the term trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a particular host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must contain specific alphavirus sequence elements to enable recognition by the alphavirus replicase and RNA synthesis.

[0075] 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.

[0076] As used herein, the term "uracil" describes one of the nucleobases that can occur in RNA nucleic acids. The structure of uracil is: [ka]

[0077] As used herein, the term "uridine" describes one of the nucleosides that can occur in RNA. The structure of uridine is: [ka]

[0078] UTP (uridine 5'-triphosphate) has the following structure: [ka]

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

[0080] "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.

[0081] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the following structure: [ka]

[0082] N1-methylpseudouridine-5'-triphosphate (m1ΨTP) has the following structure: [ka]

[0083] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: [ka]

[0084] In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines. In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0085] 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).

[0086] In some embodiments, the 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, and 5-[3-(1-E-propenylamino)uridine]uridine, or any other modified uridine known in the art.

[0087] In some embodiments, the RNA includes other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in some embodiments of the RNA, 5-methylcytidine is partially or completely, preferably completely, substituted with cytidine. In some embodiments, the RNA includes 5-methylcytidine and one or more nucleosides 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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 a cell's ribosomes.

[0092] 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. "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.

[0093] 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 or longer, 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.

[0094] 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.

[0095] 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 the immunogenic RNA 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).

[0096] To render 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 (mnm 5se 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 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2 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.

[0097] In some embodiments, the replacement of one or more uridines with nucleosides containing modified nucleobases includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridines. During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, a significant amount of abnormal products, including double-stranded RNA (dsRNA), are produced due to the unconventional activity of the enzyme. dsRNA induces inflammatory cytokines, activates effector enzymes, and causes the inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by, for example, ion-pair reverse-phase HPLC using a non-porous or porous C-18 polystyrene divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method can be used to remove dsRNA contaminants from IVT RNA preparations by using E. coli RNase III, which specifically hydrolyzes dsRNA but not ssRNA. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA preparation is contacted with the cellulose material, and the ssRNA is separated from the cellulose material under conditions that allow binding of dsRNA but not ssRNA to the cellulose material.

[0098] 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 the first substance characterized by the removal of the population of the second substance will have a significantly lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0099] 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.).

[0100] 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.

[0101] 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.

[0102] The term "exhibiting significantly reduced innate immunogenicity" refers to a detectable reduction in innate 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 innate immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response, sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response, sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA. "Immunogenicity" refers to the ability of a foreign substance, such as RNA, to elicit an immune response in humans or other animals. The innate immune system is a relatively non-specific and immediate component of the immune system. Along with the adaptive immune system, it is one of the two major components of the vertebrate immune system.

[0103] As used herein, "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0104] As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0105] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence.

[0106] 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.

[0107] In some embodiments, the present disclosure provides: 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 and N2 is at position +2 of the RNA polynucleotide; N1 is A or an analog thereof; N2 is U or an analog thereof; (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, N4, and N5 are selected from A, C, G, and U.

[0108] Codon optimization In some embodiments, the payloads (e.g., polypeptides) described herein are encoded by coding sequences that are codon-optimized and / or have 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.

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

[0110] In some embodiments, the guanosine / cytidine (G / C) content of the coding region of the RNA (e.g., 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, codons containing A and / or U nucleosides can be modified by replacing these codons with other codons that encode the same amino acids but that do not contain A and / or U or that contain fewer A and / or U nucleosides.

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

[0112] 5' Cap A structural feature of mRNA is the cap structure at the 5'-prime (5') end. Natural eukaryotic mRNAs contain a 7-methylguanosine cap linked to the mRNA via a 5'-to-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 Zuest et al. (2011) Nature Immunology, 12:137-143.

[0113] 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 (see, 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 analog 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, co-transcriptional capping can be more reproducible and less expensive than enzymatic capping. mRNA produced in the presence of cap analogs can be resistant to human decapping enzymes (see, e.g., Kowalska et al. (2008) RNA 14:1119-1131) and / or interferon-inducible proteins with tetratricopeptide repeats (IFITs), which inhibit cap0-dependent translation (see, e.g., Diamond et al. (2014) Cytokine & Growth Factor Reviews 25:543-550 and Miedziak, et al. (2019) RNA 26:58-68).However, in co-transcriptional capping, GTP typically competes with cap analogs during transcription, potentially resulting in reduced capping efficiency and reduced translational capacity. Certain Cap1 structures can be incorporated into IVT mRNA in the correct orientation to produce cap1-mRNA with high capping efficiency in rapid co-transcriptional reactions. See, for example, Henderson et al. (2021) Current Protocols 1:e39. For example, trinucleotide Cap1 structures containing an AG initiator can reduce RNA polymerase slippage on the DNA template strand (compared to, for example, a DNA template containing a G triplet as the transcription start site). See, for example, Imburgio et al. (2000) Biochemistry 39:10419-10430.

[0114] In some embodiments, the 5' cap includes a Cap-0 structure (also referred to herein as "Cap0"), a Cap-1 structure (also referred to herein as "Cap1"), or a Cap-2 structure (also referred to herein as "Cap2"). See, e.g., Figure 1 of Ramanathan A et al. and Figure 1 of Decroly E et al.

[0115] As used herein, the term "5' cap" refers to the 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'-to-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 has a 3'O methylation ("(m 3’-OIn 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"). 7 In some embodiments, the guanosine nucleoside contained within the 5' cap comprises a methylation at the 7 position of the guanine and a 3' O methylation 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.

[0116] In some embodiments, providing an RNA with a 5'-cap or 5'-cap analog disclosed herein may be achieved by in vitro transcription, in which case the 5'-cap is co-transcriptionally incorporated into the RNA strand. In some embodiments, the 5'-cap may 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 RNA capping efficiency compared to transcription-associated capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can improve RNA translation efficiency and / or translation rate and / or increase expression of the encoded polypeptide.

[0117] In some embodiments, the RNA described herein comprises a 5'-cap or a 5'-cap analog, e.g., a 5'-cap, including a Cap0, Cap1, or Cap2 structure. In some embodiments, the provided RNA does not have an uncapped 5'-triphosphate cap. In some embodiments, the RNA can be capped with a 5'-cap analog. In some embodiments, the RNA described herein comprises a Cap0 structure. In some embodiments, the RNA described herein comprises a Cap1 structure, e.g., as described herein. In some embodiments, the RNA described herein comprises a Cap2 structure.

[0118] In some embodiments, the Cap0 structure comprises a guanosine nucleoside methylated at the 7 position of the guanine (m7G). In some embodiments, the Cap0 structure is linked to the RNA via a 5'- to 5'-triphosphate linkage, also referred to herein as m7Gppp or m7G(5')ppp(5').

[0119] In some embodiments, the Cap1 structure comprises a guanosine nucleoside (m7G) methylated at the 7-position of the guanine 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'-to-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. In some embodiments, the m7G(5')ppp(5')(2'OMeN1)Cap1 structure comprises a second nucleotide, N2, which is the cap proximal to the nucleotide at position 2, (m7G(5')ppp(5')(2'OMeN1)N2), where each of N1 and N2 is as defined and described herein.

[0120] 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, During the ceremony, Each R 2 and R 3 is -OH or -OCH3, X is OH or SH.

[0121] Each nucleotide, e.g., N1 and N2, contains a phosphate group "p" (e.g., -P(=O)(OH)-, or a salt thereof, e.g., -P(=O)(OH)-). - It will be understood that the link is via .

[0122] 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, R 2 is -CH3 and R 3 is -CH3. In some embodiments, R 2 is -OH and R3 is —OCH. In some embodiments, R 2 is -OCH3, and R 3 is —OH. In some embodiments, R 2 is -OCH3, and R 3 is -OCH3.

[0123] X being OH or SH may be a salt thereof, e.g., O - or S - In some embodiments, X is OH. In some embodiments, X is SH. In some embodiments, X is O - In some embodiments, X is S - is.

[0124] 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 explained herein.

[0125] In some embodiments, N1 is A or an analog thereof. In some embodiments, N1 is adenosine. In some embodiments, N1 is 6-methyladenosine. In some embodiments, N1 is [ka] and where % represents the point of attachment to G*.

[0126] In some embodiments, N2 is U or an analog thereof. In some embodiments, N2 is a modified U. In some embodiments, N2 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 2U), 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 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 5U), 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, and 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art. In some embodiments, N2 is 5-methyluridine (m 5 In some embodiments, N2 is 1-methyl-pseudouridine (m 1 In some embodiments, N2 is pseudouridine (ψ). In some embodiments, N2 is 1-(2,2,2-trifluoroethyl)pseudouridine (tfet 1 In some embodiments, N2 is 1-propargylpseudouridine (ppg). 1 In some embodiments, N2 is 1-benzylpseudouridine (bn 1 In some embodiments, N2 is 1-(cyclopropylmethyl)pseudouridine (cpm 1 In some embodiments, N2 is 1-(pyridin-4-ylmethyl)pseudouridine ((4-pm) 1 ψ).

[0127] In some embodiments, N2 is a group represented by Formula II [ka] or a salt thereof, wherein [ka] are each independently a single bond or a double bond as allowed by valence; Y 1 is O or S, Y 2 is N, C, or CH; Y 3 But N, NR a1 , C.R. a1 , or CHR a1 and Y 4 But NR a2 or CHR a2 and R a1 or R a2 each independently represents hydrogen or C 1-6 is aliphatic, R 4 is -OH or -OMe, # represents the attachment point of N1p to p.

[0128] In some embodiments, Y 1 is O. In some embodiments, Y 1 is S.

[0129] In some embodiments, Y 2 is N. In some embodiments, Y 2 is C or CH. In some embodiments, Y 2 is C. In some embodiments, Y 2 is CH.

[0130] In some embodiments, Y 3 is N or CR a1In some embodiments, Y 3 is N. In some embodiments, Y 3 is CR a1 In some embodiments, Y 3 is CH or C(CH). In some embodiments, Y 3 is CH. In some embodiments, Y 3 is C(CH). In some embodiments, Y 3 is NR a1 or CHR a1 In some embodiments, Y 3 is NH or N(CH). In some embodiments, Y 3 is NH. In some embodiments, Y 3 is N(CH). In some embodiments, Y 3 is CH2 or CH(CH3). In some embodiments, Y 3 is CH. In some embodiments, Y 3 is CH(CH3).

[0131] In some embodiments, Y 4 is NR a2 In some embodiments, Y 4 is NH or NCH. In some embodiments, Y 4 is NH. In some embodiments, Y 4 is NCH3. In some embodiments, Y 4 is CHR a2 In some embodiments, Y 4 is CH2 or CH(CH3). In some embodiments, Y 4 is CH. In some embodiments, Y 4 is CH(CH3).

[0132] In some embodiments, R a1 is hydrogen. In some embodiments, R a1 is C 1-6 In some embodiments, Ra1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R a1 is methyl.

[0133] In some embodiments, R a2 is hydrogen. In some embodiments, R a2 is C 1-6 In some embodiments, R a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R a2 is methyl.

[0134] In some embodiments, R 4 is —OH. In some embodiments, R 4 is -OMe.

[0135] In some embodiments, N2 is a group represented by formula IIa [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is as defined above and described herein.

[0136] In some embodiments of Formula IIa, Y 1 is O. In some embodiments of Formula IIa, Y 3 is CR a1 In some such embodiments, R a1 is hydrogen, C 1-6 Aliphatic or -O(C 1-4 In some embodiments of Formula IIa, R a1 is hydrogen, C 1-3 Aliphatic or -O(C 1-2 In some embodiments of Formula IIa, R a1 is -CH3 or -OCH3.

[0137] In some embodiments, N2 is a group of formula IIb [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is as defined above and described herein.

[0138] In some embodiments of Formula IIb, Y 3 is CR a1 In some embodiments of Formula IIb, R a1 is hydrogen. In some embodiments, R a1 is C 1-6 In some embodiments of Formula IIb, R a1 is C 1-3 In some embodiments of Formula IIb, R a1 In some embodiments of Formula IIb, R a1 In some such embodiments, R is —CHR. In some such embodiments, R is —CHR. 1-4 In some embodiments of Formula IIb, R a1 is -CHR, where R is a C substituted with halogen 1-2 In some embodiments of Formula IIb, R a1 is -CHR, where R is -CF. In some embodiments of Formula IIb, R a1 is —CHR, where R is phenyl. In some embodiments of Formula IIb, R a1 is —CHR, where R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of Formula IIb, R a1 is —CHR, where R is a 3-4 membered saturated carbocyclic ring. In some embodiments of Formula IIb, R a1 is —CHR, where R is a 3-membered saturated carbocyclic ring. In some embodiments of Formula IIb, R a1is —CHR, where R is a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of Formula IIb, R a1 is —CHR, where R is a 6-membered heteroaryl ring having 1 to 3 nitrogen atoms. In some embodiments of Formula IIb, R a1 is —CHR, where R is a 6-membered heteroaryl ring having one nitrogen atom.

[0139] In some embodiments, N2 is a group of formula II'' [ka] or a salt thereof, wherein [ka] are each independently a single or double bond as allowed by valence; Y 1 is O or S, Y 2 is N, C, or CH; Y 3 But N, NR a1 , C.R. a1 , or CHR a1 and Y 4 But NR a2 or CHR a2 and R a1 or R a2 each independently being hydrogen, C 1-6 Aliphatic, -CHR, or -O(C 1-4 alkyl), R is substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 1-4 is aliphatic, R 4 is -OH or -OMe, # represents the attachment point of N1p to p.

[0140] In some embodiments of Formula II″, Y 1 is O. In some embodiments of Formula II'', Y 1 is S.

[0141] In some embodiments of Formula II″, Y 2 is N. In some embodiments of Formula II'', Y 2 is C or CH. In some embodiments of Formula II'', Y 2 is C. In some embodiments of Formula II'', Y 2 is CH.

[0142] In some embodiments of Formula II″, Y 3 is N or CR a1 In some embodiments of Formula II'', Y 3 is N. In some embodiments of Formula II'', Y 3 is CR a1 In some embodiments of Formula II'', Y 3 is NR a1 or CHR a1 In some embodiments of Formula II'', Y 3 is NR a1 In some embodiments of Formula II'', Y 3 is CHR a1 is.

[0143] In some embodiments of Formula II″, Y 4 is NR a2 In some embodiments of Formula II'', Y 4 is CHR a2 is.

[0144] In some embodiments of Formula II″, R a1 is hydrogen. In some embodiments of Formula II'', R a1 is C 1-6 In some embodiments of Formula II'', R a1 is C1-3 In some embodiments of Formula II'', R a1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of Formula II'', R a1 is methyl. In some embodiments of Formula II'', R a1 is ethyl. In some embodiments of Formula II'', R a1 is -CH=CH2. In some embodiments of Formula II'', R a1 is n-propyl. In some embodiments of Formula II'', R a1 is isopropyl. In some embodiments of Formula II'', R a1 is —CHC≡CH. In some embodiments of Formula II″, R a1 is -CHCH=CH. In some embodiments of Formula II'', R a1 is —CHR. In some embodiments of Formula II″, R a1 is -O(C 1-4 In some embodiments of Formula II'', R a1 is -OMe.

[0145] In some embodiments of Formula II″, R a2 is hydrogen. In some embodiments of Formula II'', R a2 is C 1-6 In some embodiments of Formula II'', R a2 is C 1-3 In some embodiments of Formula II'', R a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of Formula II'', R a2 is methyl. In some embodiments of Formula II'', R a2 is ethyl. In some embodiments of Formula II'', R a2 is -CH=CH2. In some embodiments of Formula II'', R a2 is n-propyl. In some embodiments of Formula II'', R a2is isopropyl. In some embodiments of Formula II'', R a2 is —CHC≡CH. In some embodiments of Formula II″, R a2 is -CHCH=CH. In some embodiments of Formula II'', R a2 is —CHR. In some embodiments of Formula II″, R a2 is -O(C 1-4 In some embodiments of Formula II'', R a2 is -OMe.

[0146] In some embodiments of Formula II″, R is C substituted with halogen. 1-4 In some embodiments of Formula II", R is a C substituted with a halogen. 1-2 In some embodiments of Formula II", R is -CF. Thus, in some embodiments of Formula II", R a1 or R a2 is -CH2CF3.

[0147] In some embodiments of Formula II", R is phenyl. Thus, in some embodiments of Formula II", R a1 or R a2 is benzyl (i.e., [ka] )

[0148] In some embodiments of Formula II", R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of Formula II", R is a 3- to 4-membered saturated carbocyclic ring. In some embodiments of Formula II", R is a 3-membered saturated carbocyclic ring. Thus, in some embodiments of Formula II", R a1 or R a2 teeth, [ka] is.

[0149] In some embodiments of Formula II", R is a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of Formula II", R is a 6 membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments of Formula II", R is a 6 membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments of Formula II", R is a 6 membered heteroaryl ring having 1 nitrogen atom. In some embodiments of Formula II", R is 4-pyridyl. Thus, in some embodiments of Formula II", R a1 or R a2 teeth, [ka] is.

[0150] In some embodiments of Formula II″, R 4 is —OH. In some embodiments of Formula II″, R 4 is -OMe.

[0151] In some embodiments, N2 is a group of formula IIa'' [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is defined above and as described herein for Formula II″.

[0152] In some embodiments, N2 is a group of formula IIb" [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is defined above and as described herein for Formula II″.

[0153] In some embodiments, N2 is a group represented by Formula II''' [ka] or a salt thereof, wherein [ka] are each independently a single or double bond as allowed by valence; Y 1 is O or S, Y 2 is N, C, or CH; Y 3 But N, NR a1 , C.R. a1 , or CHR a1 and Y 4 But NR a2 or CHR a2 and Y 5 But, CR a3 and R a1 , R a2 , or R a3 each independently being hydrogen, C 1-6 Aliphatic, -CHR, or -O(C 1-4 alkyl), R is substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 1-4 is aliphatic, R 4 is -OH or -OMe, # represents the attachment point of N1p to p.

[0154] In some embodiments of Formula II''', Y 1 is O. In some embodiments of Formula II''', Y 1 is S.

[0155] In some embodiments of Formula II''', Y2 is N. In some embodiments of Formula II''', Y 2 is C or CH. In some embodiments of Formula II''', Y 2 is C. In some embodiments of Formula II''', Y 2 is CH.

[0156] In some embodiments of Formula II''', Y 3 is N or CR a1 In some embodiments of Formula II''', Y 3 is N. In some embodiments of Formula II''', Y 3 is CR a1 In some embodiments of Formula II''', Y 3 is NR a1 or CHR a1 In some embodiments of Formula II''', Y 3 is NR a1 In some embodiments of Formula II''', Y 3 is CHR a1 is.

[0157] In some embodiments of Formula II''', Y 4 is NR a2 In some embodiments of Formula II''', Y 4 is CHR a2 is.

[0158] In some embodiments of Formula II''', R a1 is hydrogen. In some embodiments of Formula II''', R a1 is C 1-6 Aliphatic, -CHR, or -O(C 1-4 In some embodiments of Formula II''', R a1 is C 1-6 In some embodiments of Formula II''', R a1 is C 1-3 In some embodiments of Formula II''', R a1is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of Formula II''', R a1 is methyl. In some embodiments of Formula II''', R a1 is ethyl. In some embodiments of Formula II''', R a1 is -CH=CH2. In some embodiments of Formula II''', R a1 is n-propyl. In some embodiments of Formula II''', R a1 is isopropyl. In some embodiments of Formula II''', R a1 is —CHC≡CH. In some embodiments of Formula II′″, R a1 is -CHCH=CH. In some embodiments of Formula II''', R a1 is —CHR. In some embodiments of Formula II′″, R a1 is -O(C 1-4 In some embodiments of Formula II''', R a1 is -OMe.

[0159] In some embodiments of Formula II''', R a2 is hydrogen. In some embodiments of Formula II''', R a2 is C 1-6 Aliphatic, -CHR, or -O(C 1-4 In some embodiments of Formula II''', R a2 is C 1-6 In some embodiments of Formula II''', R a2 is C 1-3 In some embodiments of Formula II''', R a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of Formula II''', R a2 is methyl. In some embodiments of Formula II''', R a2 is ethyl. In some embodiments of Formula II''', R a2 is -CH=CH2. In some embodiments of Formula II''', R a2is n-propyl. In some embodiments of Formula II''', R a2 is isopropyl. In some embodiments of Formula II''', R a2 is —CHC≡CH. In some embodiments of Formula II′″, R a2 is -CHCH=CH. In some embodiments of Formula II''', R a2 is —CHR. In some embodiments of Formula II′″, R a2 is -O(C 1-4 In some embodiments of Formula II''', R a2 is -OMe.

[0160] In some embodiments of Formula II''', R a3 is hydrogen. In some embodiments of Formula II''', R a3 is C 1-6 Aliphatic, -CHR, or -O(C 1-4 In some embodiments of Formula II''', R a3 is C 1-6 In some embodiments of Formula II''', R a3 is C 1-3 In some embodiments of Formula II''', R a3 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments of Formula II''', R a3 is methyl. In some embodiments of Formula II''', R a3 is ethyl. In some embodiments of Formula II''', R a3 is n-propyl. In some embodiments of Formula II''', R a3 is isopropyl.

[0161] In some embodiments of Formula II''', R is C substituted with halogen. 1-4 In some embodiments of Formula II''', R is a C substituted with a halogen. 1-2In some embodiments of Formula II''', R is -CF3. Thus, in some embodiments of Formula II''', R a1 , R a2 , or R a3 is -CH2CF3.

[0162] In some embodiments of Formula II''', R is phenyl. Thus, in some embodiments of Formula II''', R a1 , R a2 , or R a3 is benzyl (i.e., [ka] )

[0163] In some embodiments of Formula II'", R is a 3- to 6-membered saturated carbocyclic ring. In some embodiments of Formula II'", R is a 3- to 4-membered saturated carbocyclic ring. In some embodiments of Formula II'", R is a 3-membered saturated carbocyclic ring. Thus, in some embodiments of Formula II'", R a1 , R a2 , or R a3 teeth, [ka] is.

[0164] In some embodiments of Formula II'", R is a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments of Formula II'", R is a 6 membered heteroaryl ring having 1-3 nitrogen atoms. In some embodiments of Formula II'", R is a 6 membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments of Formula II'", R is a 6 membered heteroaryl ring having 1 nitrogen atom. In some embodiments of Formula II'", R is 4-pyridyl. Thus, in some embodiments of Formula II'", R a1 , R a2 , or R a3 teeth, [ka] is.

[0165] In some embodiments of Formula II''', R 4 is —OH. In some embodiments of Formula II′″, R 4 is -OMe.

[0166] In some embodiments of Formula II" or Formula II'", Y 3 is NR a1 and Y 4 is NH, where R a1 is C 1-6 In some embodiments of Formula II" or Formula II'", Y is aliphatic or -CHR. 3 is NH and Y 4 is NR a2 where R a2 is C 1-6 In some embodiments of Formula II" or Formula II'", Y is aliphatic or -CHR. 3 is NR a1 and Y 4 is NR a2 where R a1 and R a2 Each of the 1-6 It is aliphatic or -CH2R.

[0167] In some embodiments, N2 is uridine, 1-methylpseudouridine, 2-thio-uridine, or 5-methyluridine.

[0168] In some embodiments, N2 is [ka] Or or a salt thereof, wherein # represents the point of attachment of N1p to p.

[0169] In some embodiments, N2 is [ka] [ka] Or or a salt thereof, wherein # represents the point of attachment of N1p to p.

[0170] In some embodiments, N2 is [ka] Or or a salt thereof, wherein # represents the point of attachment of N1p to p.

[0171] In some embodiments, N2 is [ka] [ka] Or or a salt thereof, wherein # represents the point of attachment of N1p to p.

[0172] In some embodiments, p is —P(═O)(OH)—, or a salt thereof.

[0173] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 2’-O )A1pU2, (m 7,3’-O )Gppp(m 2’-O )A1pU2, (m 7,2’-O )Gppp(m 2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,2’-O)Gppp(m 2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 2’-O )A1p(m 5 )U2.

[0174] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 6,2’-O )A1pU2, (m 7,3’-O )Gppp(m 6,2’-O )A1pU2, (m 7,2’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 5 )U2.

[0175] In some embodiments, the 5' cap is (m 7,2’-O )GpppA1(m 2’-O )pU2, (m 7,3’-O )GpppA1(m 2’-O )pU2, (m 7,2’-O )GpppA1(m 2’-O )pΨ2, (m 7,3’-O)GpppA1(m 2’-O )pΨ2, (m 7,2’-O )GpppA1(m 2’-O )p(m 1 )Ψ2, (m 7,3’-O )GpppA1(m 2’-O )p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 2’-O )A1(m 2’-O )pS 2 U2, (m 7,3’-O )GpppA1(m 2’-O )pS 2 U2, (m 7,2’-O )GpppA1(m 2’-O )p(m 5 )U2, or (m 7,3’-O )GpppA1(m 2’-O )p(m 5 )U2.

[0176] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 6,2’-O )A1pU2, (m 7,3’-O )Gppp(m 6,2’-O )A1pU2, (m 7,2’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 5 )U2.

[0177] In some embodiments, the 5' cap is 7 G( 3’-OMe )pppA1( 2’-OMe )pm 3 U2, m 7 G( 3’-OMe )pppA1( 2’-OMe )pmo 5 U2, m 7 GpppA1( 2’-OMe )pm 1 Ψ2, m 7 G( 3’-OMe )pppA1( 2’-OMe )pm 3 Ψ2, m 7 G( 3’-OMe )pppA1( 2’-OMe )PTFE 1 Ψ2, m 7 G( 3’-OMe )pppA1( 2’-OMe )p(ppg) 1 Ψ2, m 7 G( 3’-OMe )pppA1( 2’-OMe )pbn 1 Ψ2, m 7 G( 3’-OMe )pppA1( 2’-OMe )pcpm 1 Ψ2, or m 7 G( 3’-OMe )pppA1( 2’-OMe )p(4-pm) 1 Ψ2.

[0178] In some embodiments, the 5' cap provided herein is selected from those in Table 1, or [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] or a salt thereof.

[0179] In some embodiments, the 5' cap has the following structure: 7,2’-O )Gppp(m 2’-O )A1pU2: [ka] or a salt thereof.

[0180] In some embodiments, the 5' cap is (m 7,3’-O )Gppp(m 2’-O )A1pU2, [ka] or a salt thereof.

[0181] In some embodiments, the 5' cap is (m 7,3’-O )Gppp(m 2’-O )A1pΨ2, [ka] or a salt thereof.

[0182] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 2’-O )A1pΨ2, [ka] or a salt thereof.

[0183] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, [ka] or a salt thereof.

[0184] In some embodiments, the 5' cap is (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, [ka] or a salt thereof.

[0185] In some embodiments, the 5' cap is (m 7,3’-O )Gppp(m 2’-O )A1pS 2 U2, [ka] or a salt thereof.

[0186] In some embodiments, the 5' cap is (m 7,2’-O )Gppp(m 2’-O )A1pS 2 U2, [ka] or a salt thereof.

[0187] In some embodiments, the 5' cap is (m 7,3’-O )Gppp(m 2’-O )A1p(m 5 )U2, [ka] or a salt thereof.

[0188] In some embodiments, the 5' cap is (m7,2’-O )Gppp(m 2’-O )A1p(m 5 )U2, [ka] or a salt thereof.

[0189] It will be understood that in some embodiments, the disclosure of the 5' cap above and herein includes the 5' cap by itself or as part of a larger molecule (e.g., RNA). For example, the structure shown above includes a 3' ether linkage to the next nucleotide or as a free -OH.

[0190] In some embodiments, the present disclosure provides a compound of formula G*N1pN2, wherein: G* is a compound of formula I' [ka] or a salt thereof, 2 , R 3 , X, N1, p, and N2 are as defined above and described herein.

[0191] In some embodiments, N2 is of formula II' [ka] or a salt thereof, [ka] Y 1 , Y 2 , Y 3 , Y 4 , R a1 , R a2 , R 4 Each of , and # is as defined above and described herein.

[0192] In some embodiments, N2 is of formula IIa' [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is as defined above and described herein.

[0193] In some embodiments, N2 is of formula IIb' [ka] or a salt thereof, wherein Y 1 , Y 3 , R 4 Each of , and # is as defined above and described herein.

[0194] In some embodiments, N2 is of formula IIa′ or IIb′, wherein Y 1 , Y 3 , and R 4 Each of is as defined above for formula II″.

[0195] In some embodiments, N2 is [ka] Or or a salt thereof, In the formula, # represents the point of attachment of N1p to p.

[0196] In some embodiments, N2 is [ka] [ka] Or or a salt thereof, In the formula, # represents the point of attachment of N1p to p.

[0197] In some embodiments, N2 is [ka] [ka] Or or a salt thereof, In the formula, # represents the point of attachment of N1p to p.

[0198] In some embodiments, N1 is [ka] and or a salt thereof.

[0199] In some embodiments, p is —P(═O)(OH)—, or a salt thereof, such as —P(═O)(O - )-.

[0200] In some embodiments, the present disclosure provides a compound having the following structure: 7,2’-O )Gppp(m 2’-O )A1pU2: [ka] or a salt thereof.

[0201] In some embodiments, the present disclosure provides a compound (m 7,3’-O )Gppp(m 2’-O )A1pU2, [ka] or a salt thereof.

[0202] In some embodiments, the present disclosure provides a compound (m 7,3’-O )Gppp(m 2’-O )A1pΨ2, [ka] or a salt thereof.

[0203] In some embodiments, the present disclosure provides a compound (m 7,2’-O )Gppp(m 2’-O )A1pΨ2, [ka] or a salt thereof.

[0204] In some embodiments, the present disclosure provides a compound (m 7,2’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, [ka] or a salt thereof.

[0205] In some embodiments, the present disclosure provides a compound (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, [ka] or a salt thereof.

[0206] In some embodiments, the present disclosure provides a compound (m 7,3’-O )Gppp(m 2’-O )A1pS 2 U2, [ka] or a salt thereof.

[0207] In some embodiments, the present disclosure provides a compound (m 7,2’-O )Gppp(m 2’-O )A1pS 2 U2, [ka] or a salt thereof.

[0208] In some embodiments, the present disclosure provides a compound (m7,3’-O )Gppp(m 2’-O )A1p(m 5 )U2, [ka] or a salt thereof.

[0209] In some embodiments, the present disclosure provides a compound (m 7,2’-O )Gppp(m 2’-O )A1p(m 5 )U2, [ka] or a salt thereof.

[0210] In some embodiments, provided compounds are salts. In some embodiments, provided compounds are pharmaceutically acceptable salts.

[0211] 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 of an RNA upstream of the start codon of the protein-coding region. The 5'-UTR can be located downstream of the 5'-cap (if present), e.g., immediately adjacent to the 5'-cap.

[0212] 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 sequence adjacent to the 5' cap (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides immediately adjacent to the 5' cap). In some embodiments, the cap-proximal sequence comprises a nucleotide at position +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0213] In some embodiments, the 5'UTR comprises a Kozak sequence (e.g., GCCACC). In some embodiments, the Kozak sequence is immediately adjacent to the payload sequence (e.g., immediately upstream of the start codon).

[0214] In some embodiments, the cap structure comprises one or more polynucleotides of a cap-proximal sequence, hi some embodiments, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 and N2) of the RNA polynucleotide.

[0215] 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 ApU cap is utilized, +1 (i.e., N1) and +2 (i.e., N2) are the (m1 2’-O ) A and U residues, and +3, +4, and +5 are added by the polymerase (e.g., T7 polymerase).

[0216] 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 as defined above and described herein, and N2 is as defined above and described herein.

[0217] 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, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U.

[0218] In some embodiments, N3 is A, N4 is A, and N5 is A. In some embodiments, N3 is A, N4 is A, and N5 is C. In some embodiments, N3 is A, N4 is A, and N5 is G. In some embodiments, N3 is A, N4 is A, and N5 is U. In some embodiments, N3 is A, N4 is C, and N5 is C. In some embodiments, N3 is A, N4 is C, and N5 is G. In some embodiments, N3 is A, N4 is C, and N5 is U. In some embodiments, N3 is A, N4 is G, and N5 is C. In some embodiments, N3 is A, N4 is G, and N5 is G. In some embodiments, N3 is A, N4 is G, and N5 is U. In some embodiments, N3 is A, N4 is U, and N5 is C. In some embodiments, N3 is A, N4 is U, and N5 is G. In some embodiments, N3 is A, N4 is U, and N5 is U.

[0219] In some embodiments, N3 is C, N4 is A, and N5 is A. In some embodiments, N3 is C, N4 is A, and N5 is C. In some embodiments, N3 is C, N4 is A, and N5 is G. In some embodiments, N3 is C, N4 is A, and N5 is U. In some embodiments, N3 is C, N4 is C, and N5 is C. In some embodiments, N3 is C, N4 is C, and N5 is G. In some embodiments, N3 is C, N4 is C, and N5 is U. In some embodiments, N3 is C, N4 is G, and N5 is C. In some embodiments, N3 is C, N4 is G, and N5 is G. In some embodiments, N3 is C, N4 is G, and N5 is U. In some embodiments, N3 is C, N4 is U, and N5 is C. In some embodiments, N3 is C, N4 is U, and N5 is G. In some embodiments, N3 is C, N4 is U, and N5 is U.

[0220] In some embodiments, N3 is G, N4 is A, and N5 is A. In some embodiments, N3 is G, N4 is A, and N5 is C. In some embodiments, N3 is G, N4 is A, and N5 is G. In some embodiments, N3 is G, N4 is A, and N5 is U. In some embodiments, N3 is G, N4 is C, and N5 is C. In some embodiments, N3 is G, N4 is C, and N5 is G. In some embodiments, N3 is G, N4 is C, and N5 is U. In some embodiments, N3 is G, N4 is G, and N5 is C. In some embodiments, N3 is G, N4 is G, and N5 is G. In some embodiments, N3 is G, N4 is G, and N5 is U. In some embodiments, N3 is G, N4 is U, and N5 is C. In some embodiments, N3 is G, N4 is U, and N5 is G. In some embodiments, N3 is G, N4 is U, and N5 is U.

[0221] In some embodiments, N3 is U, N4 is A, and N5 is A. In some embodiments, N3 is U, N4 is A, and N5 is C. In some embodiments, N3 is U, N4 is A, and N5 is G. In some embodiments, N3 is U, N4 is A, and N5 is U. In some embodiments, N3 is U, N4 is C, and N5 is C. In some embodiments, N3 is U, N4 is C, and N5 is G. In some embodiments, N3 is U, N4 is C, and N5 is U. In some embodiments, N3 is U, N4 is G, and N5 is C. In some embodiments, N3 is U, N4 is G, and N5 is G. In some embodiments, N3 is U, N4 is G, and N5 is U. In some embodiments, N3 is U, N4 is U, and N5 is C. In some embodiments, N3 is U, N4 is U, and N5 is G. In some embodiments, N3 is U, N4 is U, and N5 is U.

[0222] Exemplary 5'UTRs include a human alpha globin (hAg) 5'UTR or fragment thereof, a TEV 5'UTR or fragment thereof, an HSP70 5'UTR or fragment thereof, or a c-Jun 5'UTR or fragment thereof.

[0223] In some embodiments, the RNA disclosed herein comprises an hAg 5'UTR sequence or a fragment thereof. In some embodiments, the RNA disclosed herein comprises a 5'UTR comprising an AUAGU cap-proximal sequence and an hAg 5'UTR sequence (e.g., a 5'UTR that is at least 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 a 5'UTR that is at least 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 provided in SEQ ID NO: 12.

[0224] 3'UTR In some embodiments, the RNAs disclosed herein include a 3'UTR. If present, the 3'-UTR is located at the 3' end of the RNA, 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 upstream of the poly(A) sequence (if present), for example, directly adjacent to the upstream side of the poly(A) sequence.

[0225] In some embodiments, the RNAs disclosed herein comprise a 3' UTR that includes a first sequence ("F element") from the amino-terminal enhancer of a split (AES) messenger RNA and / or a second sequence ("I element") from a mitochondrially encoded 12S ribosomal RNA. 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.

[0226] In some embodiments, the RNA disclosed herein comprises a 3'UTR that is at least 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.

[0227] 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.

[0228] As used herein, the term "poly(A) sequence" or "poly(A) sequence" or "poly(A) tail" typically refers to an uninterrupted or interrupted sequence of adenylic acid residues located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and may follow the 3'-UTR in the RNAs described herein. A continuous poly(A) sequence is characterized by consecutive adenylic acid residues. Uninterrupted poly(A) sequences are typical in nature. The RNAs disclosed herein may have a poly(A) sequence attached to the free 3'-end of the RNA by a non-template-dependent RNA polymerase after transcription, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0229] It has been demonstrated that poly(A) sequences of approximately 120 A nucleotides have a beneficial effect on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0230] The poly(A) sequence may be of any length. In some embodiments, the poly(A) sequence comprises at least 20, at least 30, at least 40, at least 80, or at least 100 and no more than 500, 400, 300, 200, or 150 A nucleotides, particularly about 120 A nucleotides; consists essentially of at least 20, at least 30, at least 40, at least 80, or at least 100 and no more than 500, 400, 300, 200, or 150 A nucleotides, particularly about 120 A nucleotides; or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and no more than 500, 400, 300, 200, or 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, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). 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.

[0231] 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.

[0232] In some embodiments, the poly(A) cassette present in the coding strand of the DNA template consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1 (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.

[0233] In some embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at the 3' end (i.e., the poly(A) sequence is not masked at the 3' end by nucleotides other than A and is not followed at the 3' end by nucleotides other than A).

[0234] In some embodiments, the poly(A) sequence is at least 20, at least 30, at least 40, at least 80, or at least 100, and may contain no more than 500, 400, 300, 200, or 150 nucleotides. In some embodiments, the poly(A) sequence is at least 20, at least 30, at least 40, at least 80, or at least 100, and may consist essentially of no more than 500, 400, 300, 200, or 150 nucleotides. In some embodiments, the poly(A) sequence is at least 20, at least 30, at least 40, at least 80, or at least 100, and may consist of no more than 500, 400, 300, 200, or 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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, an antigenic polypeptide comprising multiple different epitopes from an antigen is a polyepitope.

[0243] 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.

[0244] In some embodiments, the viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a respiratory syncytial virus antigenic polypeptide, a coronavirus antigenic polypeptide, a rabies antigenic polypeptide, or a Zika virus antigenic polypeptide. In some embodiments, the viral antigenic polypeptide comprises an antigenic polypeptide of a virus associated with a respiratory infection.

[0245] In some embodiments, the viral antigenic polypeptide is or comprises a coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigen is or comprises a SARS-CoV-2 protein. In some embodiments, the SARS-CoV-2 protein comprises a SARS-CoV-2 spike (S) protein, or an immunogenic variant or immunogenic fragment thereof. In some embodiments, the SARS-CoV-2 protein, or an immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions 986 and 987.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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. 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 at which an associated RNA polymerase will initiate transcription. Upon reading this disclosure, those skilled in the art will understand that in some embodiments, a cap (e.g., a co-transcriptional cap) can include one or more residues corresponding to the position of such a "transcription start site sequence," such that the first residue added by an RNA polymerase can actually represent the second (or subsequent) residue of the canonical transcription start site.

[0252] 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.

[0253] In some embodiments, the present disclosure provides the insight that double-stranded DNA templates containing A and U at positions +1 and +2, respectively, 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 the RNA preparation (e.g., in vitro transcribed RNA, e.g., the amount of short polynucleotide by-products produced), the translation efficiency of the RNA encoding the payload, and / or the expression of a polypeptide payload encoded by the RNA.

[0254] 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. In some particular embodiments, the double-stranded DNA template comprises an A and a U at the +1 and +2 positions, respectively, of the transcription start site. In some embodiments, a pyrimidine base (e.g., C or U) or a purine base (e.g., G or A) can independently be present at the +3, +4, or +5 positions of the transcription start site of the double-stranded DNA template. In some particular embodiments, such a double-stranded DNA template comprises an A at the +3 position of the transcription start site.

[0255] 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).

[0256] 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).

[0257] AUA transcription start site In some embodiments, a transcription start site that can be useful according to the present disclosure is AUA. 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, where the template DNA strand comprises a sequence complementary to the AUA transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein), and the sequence in the template DNA strand that is complementary to AUA is the start site of an RNA polymerase promoter. One of skill in the art reading this disclosure will understand that when an AUA transcription start site is referenced in relation to a double-stranded DNA template, the coding strand of the double-stranded DNA template contains the AUA start sequence, while the template DNA strand of the double-stranded DNA template contains the TAT, which is the start site for the RNA polymerase promoter.

[0258] In some embodiments, such an in vitro transcription reaction can produce 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, wherein (i) N1 is the +1 position of the RNA polynucleotide, (ii) N2 is the +2 position of the RNA polynucleotide, and N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein), and (iii) the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure, and N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein each of N3, N4, and N5 is independently selected from A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5' cap and a cap-proximal sequence comprising A1U2A3N4N5. In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein.

[0259] AUC transcription start site In some embodiments, a transcription start site that can be useful according to the present disclosure is AUA. 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 the AUC transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N1pN2, wherein N1 is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein), and the sequence in the template DNA strand that is complementary to AUC is the start site of an RNA polymerase promoter. One of skill in the art reading this disclosure will understand that when an AUC transcription start site is referenced in relation to a double-stranded DNA template, the coding strand of the double-stranded DNA template contains the AUC start sequence, while the template DNA strand of the double-stranded DNA template contains the TAG, which is the start site for the RNA polymerase promoter.

[0260] In some embodiments, such an in vitro transcription reaction can produce 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, wherein (i) N1 is the +1 position of the RNA polynucleotide, (ii) N2 is the +2 position of the RNA polynucleotide, and N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein), and (iii) the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure, and N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein each of N3, N4, and N5 is independently selected from A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5' cap and a cap-proximal sequence comprising A1U2C3N4N5. In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein.

[0261] AUG transcription start site In some embodiments, a transcription start site that can be useful according to the present disclosure is AUA. 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, where the template DNA strand comprises a sequence complementary to an AUG transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein), and the sequence in the template DNA strand that is complementary to AUG is the start site of an RNA polymerase promoter. One of skill in the art reading this disclosure will understand that when an AUG transcription start site is referenced in relation to a double-stranded DNA template, the coding strand of the double-stranded DNA template contains the AUG start sequence, while the template DNA strand of the double-stranded DNA template contains the TAC, which is the start site for the RNA polymerase promoter.

[0262] In some embodiments, such an in vitro transcription reaction can produce 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, wherein (i) N1 is the +1 position of the RNA polynucleotide, (ii) N2 is the +2 position of the RNA polynucleotide, and N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein), and (iii) the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure, and N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein each of N3, N4, and N5 is independently selected from A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5' cap and a cap-proximal sequence comprising A1U2G3N4N5. In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein.

[0263] AUU transcription start site In some embodiments, a transcription start site that can be useful according to the present disclosure is AUA. 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, where the template DNA strand comprises a sequence complementary to an AUU transcription start site; (ii) a polymerase (e.g., an RNA polymerase such as T7 polymerase); (iii) ribonucleotides; and (iv) a trinucleotide cap comprising N1pN2, where N1 is A or an analog thereof (e.g., as described above and herein) and N2 is U or an analog thereof (e.g., as described above and herein), and the sequence in the template DNA strand that is complementary to AUG is the start site of an RNA polymerase promoter. One of skill in the art reading this disclosure will understand that when an AUU transcription start site is referenced in relation to a double-stranded DNA template, the coding strand of the double-stranded DNA template contains the AUU start sequence, while the template DNA strand of the double-stranded DNA template contains TAA, the start site for the RNA polymerase promoter.

[0264] In some embodiments, such an in vitro transcription reaction can produce 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, wherein (i) N1 is the +1 position of the RNA polynucleotide, (ii) N2 is the +2 position of the RNA polynucleotide, and N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein), and (iii) the cap-proximal sequence comprises a sequence comprising N1 and N2 of the cap structure, and N3N4N5 at positions +3, +4, and +5 of the RNA polynucleotide, respectively, wherein each of N3, N4, and N5 is independently selected from A, C, G, and U (e.g., as described above and herein). By way of example only, in some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction comprises a 5' cap and a cap-proximal sequence comprising A1U2U3N4N5. In some embodiments, an RNA polynucleotide resulting from such an in vitro transcription reaction can be an RNA polynucleotide described herein.

[0265] Complex In certain aspects, provided herein are complexes formed during the in vitro transcription reactions described herein, e.g., using different combinations of caps (e.g., as described herein) and transcription start sites (e.g., as described herein).

[0266] In some embodiments, the present disclosure provides a complex comprising a DNA template strand and a 5' cap analog, wherein the DNA template strand comprises a sequence complementary to an RNA polymerase promoter sequence and a transcription start site, the 5' cap analog comprises the structure N1pN2, where N1 is A or an analog thereof (e.g., as described above and herein), and N2 is U or an analog thereof (e.g., as described above and herein), where N1 interacts with position +1 of the DNA template strand (corresponding to the first nucleotide of the transcription start site) and N2 interacts with position +2 of the DNA template strand (corresponding to the second nucleotide of the transcription start site), and the sequence in the template strand that is complementary to the transcription start site is the start site of the RNA polymerase promoter. In some embodiments, N1 is A, N2 is U, and positions +1 and +2 of the DNA template strand are T and A, respectively.

[0267] In various aspects described herein, one or more nucleotides of the cap (e.g., those described herein) interact with one or more nucleotides of the template DNA strand of the RNA polymerase initiation site via standard Watson-Crick base pairing. In some embodiments, the provided complex comprises a DNA template strand comprising an RNA polymerase promoter sequence, which may be or may comprise a T7 RNA polymerase promoter sequence. In some embodiments, the complexes disclosed herein further comprise an RNA polymerase (e.g., T7 RNA polymerase).

[0268] Exemplary Polynucleotides In some embodiments, the RNA polynucleotides described herein, or compositions or medical preparations comprising same, comprise a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence that is at least 80% identical to a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence that encodes a polypeptide that is at least 80% identical to a polypeptide sequence disclosed herein. Exemplary nucleotide and polypeptide sequences are provided, for example, in Table 2, in this section entitled "Exemplary Polynucleotides," or in Examples 1 or 2.

[0269] In some embodiments, the RNA polynucleotides described herein, or compositions or medical preparations comprising the same, are transcribed using a DNA template. In some embodiments, the DNA template used to transcribe the RNA polynucleotides described herein comprises a sequence complementary to the RNA polynucleotide.

[0270] In some embodiments, the payloads described herein are encoded by an RNA polynucleotide described herein, including a nucleotide sequence disclosed herein, e.g., in Table 2, in this section entitled "Exemplary Polynucleotides," or in Examples 1 or 2. In some embodiments, the RNA polynucleotide encodes a polypeptide payload that is at least 80% identical to a polypeptide payload sequence disclosed herein. In some embodiments, the payloads described herein are encoded by an RNA polynucleotide that is transcribed by a DNA template that includes a sequence complementary to the RNA polynucleotide. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

[0271] RBL063.1 (SEQ ID NO:28, nucleotide; SEQ ID NO:9, amino acid) [Table 3] SEQ ID NO: 28 [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0272] RBL063.2 (SEQ ID NO:29, nucleotide; SEQ ID NO:9, amino acid) [Table 9] SEQ ID NO: 29 [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0273] BNT162a1; RBL063.3 (SEQ ID NO: 30, nucleotide; SEQ ID NO: 21, amino acid) [Table 15] SEQ ID NO: 30 [Table 16] [Table 17]

[0274] BNT162b2; RBP020.1 (SEQ ID NO:31, nucleotide; SEQ ID NO:9, amino acid) [Table 18] SEQ ID NO: 31 [Table 19] [Table 20] [Table 21] [Table 22] [Table 23]

[0275] RBP020.2 (SEQ ID NO: 10, nucleotide; SEQ ID NO: 9, amino acid) (see Table 2) [Table 24]

[0276] BNT162b1; RBP020.3 (SEQ ID NO: 32; SEQ ID NO: 21, amino acid) [Table 25] SEQ ID NO: 32 [Table 26] [Table 27]

[0277] RBS004.1 (SEQ ID NO: 33; SEQ ID NO: 9, amino acid) [Table 28] SEQ ID NO: 33 [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39]

[0278] RBS004.2 (SEQ ID NO: 34; SEQ ID NO: 9, amino acid) [Table 40] SEQ ID NO: 34 [Table 41] [Table 42] [Table 43] [Table 44] [Table 45] [Table 46] [Table 47] [Table 48] [Table 49] [Table 50] [Table 51] [Table 52]

[0279] BNT162c1; RBS004.3 (SEQ ID NO: 35; SEQ ID NO: 21, amino acid) [Table 53] SEQ ID NO: 35 [Table 54] [Table 55] [Table 56] [Table 57] [Table 58] [Table 59] [Table 60] [Table 61] [Table 62]

[0280] RBS004.4 (SEQ ID NO: 36; SEQ ID NO: 37) [Table 63] SEQ ID NO: 36 [Table 64] [Table 65] [Table 66] [Table 67] [Table 68] [Table 69] [Table 70] [Table 71] [Table 72] SEQ ID NO: 37 [Table 73] [Table 74] [Table 75]

[0281] BNT162b3c (SEQ ID NO: 38; SEQ ID NO: 39) [Table 76] SEQ ID NO: 38 [Table 77] [Table 78] [Table 79] SEQ ID NO: 39 [Table 80] [Table 81]

[0282] BNT162b3d (SEQ ID NO: 40; SEQ ID NO: 41) [Table 82] SEQ ID NO: 40 [Table 83] [Table 84] SEQ ID NO: 41 [Table 85] [Table 86] [Table 87]

[0283] Nucleic acid containing particles The nucleic acids described herein, for example, RNA encoding a payload, may be formulated and administered as particles.In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or molecular complex.In some embodiments, the term "particle" refers to a micro- or nano-sized structure, for example, a micro- or nano-sized compact structure dispersed in a medium.In some embodiments, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

[0284] Electrostatic interactions between positively charged molecules, such as polymers and lipids, and negatively charged nucleic acids are responsible for particle formation, leading to complexation and spontaneous formation of nucleic acid particles. In some embodiments, the nucleic acid particles are nanoparticles.

[0285] As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

[0286] "Nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., a cell, tissue, organ, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionic lipid or lipid-like material, at least one cationic polymer, such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0287] Without intending to be bound by any theory, it is believed that cationic or cationically ionic lipids or lipid-like materials, and / or cationic polymers combine with nucleic acids to form aggregates, which aggregate to give rise to colloidally stable particles.

[0288] In some embodiments, the particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionic lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

[0289] In some embodiments, nucleic acid particles comprise multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules may be similar or different from each other with respect to molar mass or basic structural elements, such as molecular architecture, capping, coding regions, or other features. The nucleic acid particles described herein may, in some embodiments, have an average diameter ranging from about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0290] The nucleic acid particles described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index in the range of about 0.1 to about 0.3, or about 0.2 to about 0.3.

[0291] For RNA-lipid particles, the N / P ratio indicates the ratio of nitrogen groups in the lipid to the number of phosphate groups in the RNA. This correlates with the charge ratio, since nitrogen atoms (depending on pH) are usually positively charged and phosphate groups are negatively charged. The N / P ratio is determined by pH when charge balance exists. Lipid formulations are frequently formed with N / P ratios greater than 4, up to 12, because positively charged nanoparticles are considered suitable for transfection. In that case, the RNA is considered fully bound to the nanoparticles.

[0292] The nucleic acid particles described herein can be prepared using a wide variety of methods, which can include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain the nucleic acid particles.

[0293] As used herein, the term "colloid" refers to a homogeneous mixture of dispersed particles that do not settle. The insoluble particles in the mixture are fine, ranging in size from 1 to 1000 nanometers. This mixture is sometimes called a colloid or colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture, rather than the entire suspension.

[0294] For the preparation of colloids comprising at least one cationic or cationically ionic lipid or lipid-like material and / or at least one cationic polymer, methods conventionally used and appropriately adapted for preparing liposome-type vesicles are applicable herein. The most commonly used methods for preparing liposome-type vesicles share the following basic steps: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0295] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to produce a liposomal dispersion. An additional downsizing step may also be included.

[0296] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposomal vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A short sonication of this mixture is required for homogenization. Removal of the organic phase under reduced pressure results in a milky gel, which then transforms into a liposomal suspension.

[0297] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action distributes the lipids throughout the solution, promoting lipid structure formation, e.g., lipid vesicle formation, e.g., liposome formation. Typically, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed, in some embodiments, as follows: an ethanol solution containing lipids, e.g., cationic lipids and additional lipids, is injected into a stirred aqueous solution. In some embodiments, the RNA lipoplex particles described herein can be obtained without an extrusion step.

[0298] The term "extrusion" or "extrusion" refers to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles by forcing them through a filter with defined pores.

[0299] Other methods that have organic solvent-free characteristics may also be used in accordance with the present disclosure to prepare colloids.

[0300] LNPs typically contain four components: ionic cationic lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer-conjugated lipids such as polyethylene glycol (PEG) lipids. Each component is responsible for payload protection and enables effective intracellular delivery. LNPs can be prepared by mixing lipids rapidly dissolved in ethanol with nucleic acid in an aqueous buffer.

[0301] The term "average diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) through data analysis using the so-called cumulant algorithm, which results in the so-called Zaverage, which is a linear dimension, and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the terms "average diameter," "diameter," or "size" of particles are used interchangeably with the value of Zaverage.

[0302] The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, which is mentioned in the definition of "mean diameter". It can be taken as a measure of the size distribution of an ensemble of nanoparticles under certain prerequisites.

[0303] Various types of nucleic acid-containing particles have previously been described as suitable for delivering nucleic acids in the form of microparticles (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral nucleic acid delivery vehicles, encapsulation of nucleic acids in nanoparticles can physically protect the nucleic acid from degradation and, depending on its inherent chemical properties, can aid in cellular uptake and endosomal escape.

[0304] The present disclosure describes particles containing nucleic acid, at least one cationic or cationically ionic lipid or lipid-like material, and / or at least one cationic polymer that associate with nucleic acid to form nucleic acid particles, and compositions containing such particles.Nucleic acid particles can contain nucleic acid that is complexed to the particles in various forms through non-covalent interactions.The particles described herein are not virus particles, particularly infectious virus particles, i.e., they cannot infect cells with the virus.

[0305] Suitable cationic or cationically ionic lipids or lipid-like materials and cationic polymers form nucleic acid particles and are encompassed by the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with nucleic acid to form a nucleic acid particle. Such components include any component that can be part of a nucleic acid particle.

[0306] Some embodiments described herein relate to compositions, methods, and uses that include more than one nucleic acid species, e.g., RNA species, such as: a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parent viral protein, wherein amino acid positions within at least the fragment of the parent viral protein have been modified to include amino acids found within corresponding amino acid positions in one or more viral protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parent viral protein, wherein amino acid positions within at least the fragment of the parent viral protein have been modified to include amino acids found within corresponding amino acid positions in one or more viral protein variants.

[0307] In microparticle preparations, it is possible that each nucleic acid species is separately formulated as an individual microparticle preparation.In this case, each individual microparticle preparation will contain one nucleic acid species.Individual microparticle preparations can exist as separate entities, for example, in separate containers.Such preparations can be obtained by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent that allows particles to form.Each particle will exclusively contain the specific nucleic acid species provided when the particle is formed (individual microparticle preparation).

[0308] In some embodiments, a composition, such as a pharmaceutical composition, comprises a plurality of individual particle formulations. Each pharmaceutical composition is referred to as a mixed microparticle formulation. A mixed microparticle formulation according to the present invention can be obtained by mixing the individual microparticle formulations after forming them separately as described above. By mixing, a formulation comprising a mixed population of nucleic acid-containing particles can be obtained. The individual microparticle populations can be combined in one container to comprise a mixed population of individual microparticle formulations.

[0309] Alternatively, various nucleic acid species can be formulated together as a combined microparticle preparation.This preparation can be obtained by providing the combined preparation (typically combined solution) of various RNA species together with particle forming agent that allows particle formation.In contrast to mixed microparticle preparation, combined microparticle preparation will typically comprise particles that contain multiple RNA species.In combined microparticle composition, various RNA species typically exist together in a single particle.

[0310] Cationic polymeric materials (e.g., polymers) Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically compact negatively charged nucleic acids into nanoparticles. The positively charged groups often consist of amines that change protonation state within the pH range of 5.5 to 7.5, which is thought to lead to an ionic imbalance that ultimately results in endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been used for nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have been widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

[0311] As used herein, "polymeric material" refers to its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some embodiments, all such repeating units may be identical; alternatively, in some cases, there may be more than one type of repeating unit present within the polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties, e.g., targeting moieties as described herein, may also be present in the polymeric material.

[0312] Those skilled in the art will recognize that when more than one type of repeat unit is present in a polymer (or polymer portion), the polymer (or polymer portion) is said to be a "copolymer." In some embodiments, a polymer (or polymer portion) utilized in accordance with the present disclosure may be a copolymer. The repeat units forming the copolymer can be arranged in any manner. For example, in some embodiments, the repeat units can be arranged in random order; alternatively or additionally, in some embodiments, the repeat units can be arranged in alternating order or as a "block" copolymer (i.e., comprising one or more regions (e.g., first blocks) each comprising a first repeat unit, and one or more regions (e.g., second blocks) each comprising a second repeat unit, etc.). A block copolymer can have two (diblock copolymer), three (triblock copolymer), or more different blocks.

[0313] In certain embodiments, polymeric materials for use in accordance with the present disclosure are biocompatible. Biocompatible materials are those that typically do not cause significant cell death at reasonable concentrations. In certain embodiments, biocompatible materials are biodegradable, i.e., capable of chemically and / or biologically breaking down within a physiological environment, e.g., within the body.

[0314] In certain embodiments, the polymeric material may be or include protamine or a polyalkyleneimine, particularly protamine.

[0315] Those skilled in the art will recognize that the term "protamine" is often used to refer to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (e.g., fish) to associate specifically with DNA in place of somatic histones. In particular, the term "protamine" is often used to refer to a protein found in fish sperm that is strongly basic, soluble in water, heat-resistant, and hydrolyzes to yield primarily arginine. In purified form, they are also used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

[0316] In some embodiments, the term "protamine," as used herein, refers to a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and / or multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from a natural or biological source.

[0317] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. In some embodiments, the preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75-10 to 10 Da, preferably 1,000 to 10 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0318] Preferred according to certain embodiments of the present disclosure are linear polyalkyleneimines, such as linear polyethyleneimine (PEI).

[0319] Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those that can electrostatically bind to nucleic acids. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric material with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0320] In some embodiments, the particles described herein can include polymers other than cationic polymers, such as non-cationic and / or anionic polymeric materials, anionic and neutral polymeric materials being collectively referred to herein as non-cationic polymeric materials.

[0321] Lipids and lipid-like materials The terms "lipid" and "lipid-like material" are used herein to refer to molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and distinct phases. One such phase consists of lipid bilayers, such as those found in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by the inclusion of non-polar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic group(s). In some embodiments, hydrophilic groups may include polar and / or charged groups, which may include carbohydrate groups, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0322] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have a formal positive or negative charge. Alternatively, the polar portion may have both a formal positive and negative charge, or may be a zwitterion or an inner salt. For purposes of this disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipid and lipid-like compounds.

[0323] The terms "lipid-like material," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, the terms include compounds that can form vesicles, multilamellar / unilamellar liposomes, or amphiphilic layers that reside intact within membranes in an aqueous environment, and include surfactants or synthetic compounds that have both hydrophilic and hydrophobic portions. Generally speaking, the terms refer to molecules that contain hydrophilic and hydrophobic portions with different structural organizations that may or may not resemble those of lipids. As used herein, the term "lipid" should be interpreted to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

[0324] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0325] In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including tri-, di-, and monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0326] Fatty acids or fatty acid residues are a diverse group of molecules made up of hydrocarbon chains terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbons long, can be saturated or unsaturated and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. The cis double bond causes the fatty acid chain to bend, which is available for compounding with more double bonds within the chain. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

[0327] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is esterified, typically with a different fatty acid. An additional subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0328] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) containing a glycerol core linked by ester bonds to two fatty acid-derived "tails" and one "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (sphingomyelins are also classified as phospholipids), include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0329] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The predominant phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples include simple glycosphingolipids and complex glycosphingolipids, such as cerebrosides and gangliosides.

[0330] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, together with glycerophospholipids and sphingomyelins.

[0331] Glycolipids describe compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with membrane bilayers. In glycolipids, a monosaccharide replaces the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, which is a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0332] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes and by iterative, multimodular enzymes that share mechanistic features with fatty acid synthases. Polyketides possess great structural diversity, including numerous secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0333] According to the present disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials are uncharged or exist in a neutral zwitterionic form at a selected pH.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0370] In some embodiments, the polymer-conjugated lipid is a PEGylated lipid. In some embodiments, the PEGylated lipid has the following structure: [ka] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein: R 12 and R 13 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester linkages; and w has an average value ranging from 30 to 60. In some embodiments, R 12 and R 13 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w ranges from an average of 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R 12 and R 13 is each independently a linear saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

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

[0372] In some embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka] or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: L 1 or L 2 One of the groups is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a- , -OC(=O)NR a -, or -NR a C(=O)O-, and L 1 or L 2 -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a- , -OC(=O)NR a - or -NR a C(=O)O-, or a direct bond, G 1 and G 2 are each independently unsubstituted C1-C 12 Alkylene or C1-C 12 is alkenylene, G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1~C 12 is alkyl, R 1 and R2 are each independently C6 to C 24 Alkyl or C6-C 24 is alkenyl, R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 and R 4 is C1~C 12 is alkyl, R 5 is H or C1-C6 alkyl, x is 0, 1 or 2.

[0373] In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB): [ka] During the ceremony: A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is independently H, OH, or C1-C 24 is alkyl, n is an integer ranging from 1 to 15.

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

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

[0376] In any of the foregoing embodiments of formula (III), L 1or L 2 One of the groups is —O(C═O)—. For example, in some embodiments, L 1 and L 2 In some different embodiments of any of the foregoing, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 are each —(C═O)O—.

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

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

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

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

[0381] In some of the foregoing embodiments of formula (III), R 6is H. In other of the foregoing embodiments, R 6 is C1~C 24 In another embodiment, R 6 is OH.

[0382] In some embodiments of Formula (III), G 3 is unsubstituted. In another embodiment, G 3 is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0383] In some of the other aforementioned embodiments of formula (III), R 1 Or R 2 , or both are C6~C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka] During the ceremony: R 7a and R 7b Each occurrence of is independently H or C1-C 12 is alkyl, a is an integer from 2 to 12, R 7a , R 7b , and a are R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0384] In some of the foregoing embodiments of formula (III), R 7a At least one occurrence of is H. For example, in some embodiments, R 7a Each occurrence of is H. In another different embodiment of the foregoing, R 7bis C1-C8 alkyl. For example, in some embodiments, the C1-C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0385] In different embodiments of formula (III), R 1 Or R 2 , or both have one of the following structures: [ka]

[0386] In some of the foregoing embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NHC(=O)R 4 In some embodiments, R 4 is methyl or ethyl.

[0387] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

[0388] Representative compounds of formula (III). [Table 88] [Table 89] [Table 90] [Table 91] [Table 92] [Table 93] [Table 94]

[0389] In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

[0390] In some embodiments, the cationic lipid is present in the LNP in an amount of about 40 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LNP in an amount of about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LNP in an amount of about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LNP in an amount of about 1 to about 10 mole percent.

[0391] In some embodiments, the LNPs comprise compound III-3 in an amount of about 40 to about 50 mole percent, DSPC in an amount of about 5 to about 15 mole percent, cholesterol in an amount of about 35 to about 45 mole percent, and ALC-0159 in an amount of about 1 to about 10 mole percent.

[0392] In some embodiments, the LNPs comprise compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

[0393] In various different embodiments, the cationic lipid has one of the structures shown in the table below. [Table 95]

[0394] In some embodiments, the LNP comprises a cationic lipid shown in the table above, e.g., a cationic lipid of Formula (B) or Formula (D), particularly a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000.

[0395] In some embodiments, the LNPs comprise cationic lipids that are ionic lipid-like materials (lipidoids). In some embodiments, the cationic lipids have the following structure: [ka] The N / P value is preferably at least about 4. In some embodiments, the N / P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N / P value is about 6.

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

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

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

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

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

[0401] The term "adjuvant" refers to a compound that prolongs, enhances, or promotes an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Cytokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, and LT-α. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oils, such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

[0402] Pharmaceutical compositions according to the present disclosure are typically used in "pharmaceutically effective amounts" and in "pharmaceutically acceptable preparations."

[0403] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0404] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves the desired response or desired effect, either alone or together with further doses. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, particularly interrupting or halting the course of the disease. The desired response in the treatment of a disease may also be delaying or preventing the onset of the disease or symptoms. The effective amount of the compositions described herein will depend on the condition to be treated, the severity of the disease, individual parameters of the patient, including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors.

[0405] Thus, the dosage of the compositions described herein may depend on such various parameters: if the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0406] In some embodiments, the pharmaceutical compositions disclosed herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0407] Suitable preservatives for use in pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0408] As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0409] The term "diluent" refers to a diluent and / or thinning agent. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension, and / or mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0410] The term "carrier" refers to a component, whether natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier can be one or more suitable solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and particularly biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0411] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art; see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0412] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0413] In some embodiments, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, which includes absorption through the digestive tract or parenteral administration. As used herein, "parenteral administration" refers to any mode of administration other than through the digestive tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration (e.g., intravenous administration).

[0414] Characterization In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, elevated expression of the payload is observed compared to a suitable reference comparator.

[0415] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide has been administered, a prolonged period of expression (e.g., expression over a longer period of time) of the payload is observed compared to a suitable reference comparator.

[0416] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, a decrease in the interaction of the RNA polynucleotide with IFIT1 is observed compared to a suitable reference comparator.

[0417] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, increased translation of the RNA polynucleotide is observed compared to a suitable reference comparator.

[0418] In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a cap as described herein. In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a cap proximal to the sequence disclosed herein. In some embodiments, the reference comparator comprises an organism administered an otherwise similar RNA polynucleotide with a self-hybridizing sequence.

[0419] In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in an organism to which a composition or medical preparation comprising the RNA polynucleotide is administered, higher expression of the payload and a prolonged duration of expression (e.g., expression over a longer period of time) are observed compared to a suitable reference comparator.

[0420] In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 24 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 48 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 72 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 96 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression is determined at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0421] In some embodiments, elevated expression is determined about 24-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide, hi some embodiments, elevated expression is determined about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.

[0422] In some embodiments, the increased expression of the payload is at least 2-fold to at least 10-fold. In some embodiments, the increased expression of the payload is at least 2-fold. In some embodiments, the increased expression of the payload is at least 3-fold. In some embodiments, the increased expression of the payload is at least 4-fold. In some embodiments, the increased expression of the payload is at least 6-fold. In some embodiments, the increased expression of the payload is at least 8-fold. In some embodiments, the increased expression of the payload is at least 10-fold.

[0423] In some embodiments, increased expression of the payload is about 2-fold to about 50-fold. In some embodiments, increased expression of the payload is about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold.

[0424] In some embodiments, elevated expression (e.g., extended duration of expression) of the payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide. In some embodiments, elevated expression of the payload persists for at least 24 hours after administration. In some embodiments, elevated expression of the payload persists for at least 48 hours after administration. In some embodiments, elevated expression of the payload persists for at least 72 hours after administration. In some embodiments, elevated expression of the payload persists for at least 96 hours after administration. In some embodiments, elevated expression of the payload persists for at least 120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0425] In some embodiments, high expression of the payload persists for about 24-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide, hi some embodiments, high expression persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising the RNA polynucleotide.

[0426] use Disclosed herein, inter alia, are methods of making and using RNA polynucleotides comprising a sequence encoding a 5' cap; a 5' UTR comprising a cap-proximal structure; and a payload.

[0427] In some embodiments, disclosed herein are methods for producing a polypeptide, comprising providing an RNA polynucleotide comprising a 5' cap (e.g., as described herein), a cap-proximal sequence including positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload; wherein the RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition comprising the same is administered, higher expression and / or a prolonged duration of expression of the payload is observed compared to a suitable reference comparator.

[0428] In some embodiments, methods are disclosed herein that include administering to a subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle as disclosed herein.

[0429] Disclosed herein, in some embodiments, is a method of inducing an immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle disclosed herein.

[0430] Disclosed herein, in some embodiments, are methods of vaccinating a subject, the methods comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or lipoplex (LPX) particle as disclosed herein.

[0431] In some embodiments, there is provided a method of reducing interaction of an RNA polynucleotide comprising a 5' cap and a cap-proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide with IFIT1, comprising: providing a variant of 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 the mutant has a reduced interaction with IFIT1 compared to that of the parent RNA polynucleotide. In some embodiments, the determining step comprises administering the RNA polynucleotide or a composition comprising the same to a cell or organism.

[0432] Disclosed herein, in some embodiments, are methods for increasing the translatability of 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, the methods comprising: providing a variant 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 expression of the variant is increased relative to that of the parent RNA polynucleotide. In some embodiments, the determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or organism. In some embodiments, the increased translatability is assessed by increased expression and / or persistence of expression of the payload. In some embodiments, the increased expression is determined at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments, the increased expression is at least 2-fold to 10-fold. In some embodiments, the increased expression is about 2-fold to 50-fold. In some embodiments, the elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration.

[0433] In some embodiments of any of the methods disclosed herein, an immune response is induced in the subject. In some embodiments of any of the methods disclosed herein, the immune response is a prophylactic or therapeutic immune response.

[0434] In some embodiments of any of the methods disclosed herein, the subject is a mammal.

[0435] In some embodiments of any of the methods disclosed herein, the subject is a human.

[0436] In some embodiments of any of the methods disclosed herein, the subject has a disease or disorder disclosed herein.

[0437] In some embodiments of any of the methods disclosed herein, the vaccination generates an immune response to the agent. In some embodiments, the immune response is a prophylactic immune response.

[0438] In some embodiments of any of the methods disclosed herein, the subject has a disease or disorder disclosed herein.

[0439] In some embodiments of any of the methods disclosed herein, a single dose of the pharmaceutical composition is administered.

[0440] In some embodiments of any of the methods disclosed herein, multiple doses of the pharmaceutical composition are administered.

[0441] In some embodiments of any of the methods disclosed herein, the method further comprises administration of one or more therapeutic agents, hi some embodiments, the one or more therapeutic agents are administered before, after, or simultaneously with the administration of the pharmaceutical composition comprising the RNA polynucleotide.

[0442] 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), comprising including A or its analog, and U or its analog, at positions +1 and +2, respectively, of the transcription start site in the coding strand of a double-stranded DNA template for in vitro transcription. In some embodiments, the transcription start site can be AUA, AUC, AUG, or AUU. 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.

[0443] Also provided in some embodiments is a method of 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. Also provided herein is a method comprising 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.

[0444] In some embodiments, the evaluating comprises administering the RNA construct or a composition comprising the same to a cell or organism.

[0445] In some embodiments, increased expression of the payload is detected at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments, increased expression is at least 2-fold to 10-fold. In some embodiments, increased expression is about 2-fold to about 50-fold.

[0446] In some embodiments, high expression of the payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration.

[0447] In some embodiments of any of the methods disclosed herein, the RNA polynucleotide comprises one or more features of the RNA polynucleotides provided herein.

[0448] In some embodiments of any of the methods disclosed herein, the composition comprising the RNA polynucleotide comprises a pharmaceutical composition provided herein.

[0449] Enumeration of Embodiments 1. 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 said 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 and N2 is at position +2 of the RNA polynucleotide; N1 is A or an analog thereof; N2 is U or an analog thereof; (ii) the cap-proximal sequence is The composition or medical preparation comprises 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, N4, and N5 are selected from A, C, G, and U.

[0450] 2. The composition or medical preparation of embodiment 1, wherein N3 is A.

[0451] 3. A composition or medical preparation according to embodiment 1 or 2, wherein N5 is U.

[0452] 4. A composition or medical preparation according to any one of embodiments 1 to 3, wherein N4 is A.

[0453] 5. A composition or medical preparation according to any one of embodiments 1 to 3, wherein N4 is C.

[0454] 6. A composition or medical preparation according to any one of embodiments 1 to 3, wherein N4 is G.

[0455] 7. A composition or medical preparation according to any one of embodiments 1 to 3, wherein N4 is U.

[0456] 8. The trinucleotide cap structure has the structure: G*N1pN2, wherein: 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 OH or SH (e.g., O - or S - 8. The composition or medical preparation of any one of embodiments 1 to 7, wherein

[0457] 9.R 2The composition or medical preparation of embodiment 8, wherein is —OH.

[0458] 10.R 2 The composition or medical preparation of embodiment 8, wherein is —OCH3.

[0459] 11.R 3 A composition or medical preparation according to any one of embodiments 8 to 10, wherein is —OH.

[0460] 12.R 3 A composition or medical preparation according to any one of embodiments 8 to 10, wherein is —OCH3.

[0461] 13. X is OH (e.g., O - 13. The composition or medical preparation of any one of embodiments 8 to 12, wherein

[0462] 14. A composition or medical preparation according to any one of embodiments 1 to 13, wherein the trinucleotide cap structure comprises a Cap1 structure.

[0463] 15. N2 is a compound of formula II [ka] or a salt thereof, wherein [ka] are each independently a single or double bond as allowed by valence; Y 1 is O or S, Y 2 is N, C, or CH; Y 3 But N, NR a1 , C.R. a1 , or CHR a1 and Y 4 But NR a2 or CHR a2 and R a1 or R a2 each independently represents hydrogen or C 1-6 is aliphatic, R 4 is -OH or -OMe, 15. A composition or medical preparation according to any one of embodiments 1 to 14, wherein # represents the point of attachment of N1p to p.

[0464] 16. N2 is of formula IIa, [ka] or a salt thereof.

[0465] 17. N2 is of formula IIb, [ka] or a salt thereof.

[0466] 18.Y 1 A composition or medical preparation according to any one of embodiments 15 to 17, wherein is O.

[0467] 19.Y 1 A composition or medical preparation according to any one of embodiments 15 to 17, wherein is S.

[0468] 20.Y 3 The composition or medical preparation of embodiment 15 or 16, wherein is N.

[0469] 21.Y 3 But, CR a1 17. The composition or medical preparation of embodiment 15 or 16, wherein

[0470] 22.Y 3 But NR a1 18. The composition or medical preparation of embodiment 15 or 17, wherein

[0471] 23.Y 3 But CHR a1 18. The composition or medical preparation of embodiment 15 or 17, wherein

[0472] 24.Each R a1 or R a2 24. The composition or medical preparation of any one of embodiments 15 to 23, wherein is independently hydrogen or methyl.

[0473] 25.R 4 A composition or medical preparation according to any one of embodiments 15 to 24, wherein is —OH.

[0474] 26.R 4 A composition or medical preparation according to any one of embodiments 15 to 24, wherein is -OMe.

[0475] 27. A composition or medical preparation according to any one of embodiments 1 to 14, wherein N2 is uridine or a modified uridine (eg m1ψ, 2-thio-uridine, or 5-methyluridine).

[0476] 28.N2 is [ka] or a salt thereof, 15. A composition or medical preparation according to any one of embodiments 1 to 14, wherein # represents the point of attachment of N1p to p.

[0477] 29. A composition or medical preparation according to any one of embodiments 1 to 28, wherein N1 is adenosine or a modified adenosine (for example 6-methyladenosine).

[0478] 30. The 5' cap is 7,2’-O )Gppp(m 2’-O )A1pU2, (m 7,3’-O )Gppp(m 2’-O )A1pU2, (m 7,2’-O)Gppp(m 2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 2’-O )A1p(m 5 ) U2.

[0479] 31. The 5' cap is 7,2’-O )Gppp(m 6,2’-O )A1pU2, (m 7,3’-O )Gppp(m 6,2’-O )A1pU2, (m 7,2’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 6,2’-O )A1p(m5 ) U2.

[0480] 32. An in vitro transcription reaction, comprising: (i) a template DNA strand comprising a polynucleotide sequence complementary to the RNA polynucleotide sequence of any one of embodiments 1 to 31, wherein the template DNA strand comprises a sequence complementary to an AUA, AUC, AUG, or AUU transcription start site; (ii) a polymerase; (iii) ribonucleotides, and (iv) a 5' cap comprising N1pN2; N1 is A or an analog thereof and N2 is U or an analog thereof; the in vitro transcription reaction, wherein the sequence in the template strand that is complementary to AUA, AUC, AUG, or AUU is the start site of an RNA polymerase promoter.

[0481] 33. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence complementary to a transcription start site comprising AUA.

[0482] 34. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence complementary to a transcription start site comprising AUC.

[0483] 35. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence complementary to a transcription start site comprising an AUG.

[0484] 36. The in vitro transcription reaction of embodiment 32, wherein the template DNA strand comprises a sequence complementary to a transcription start site comprising AUU.

[0485] 37. An in vitro transcription reaction according to any one of embodiments 32 to 36, wherein the template DNA strand 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.

[0486] 38. The in vitro transcription reaction of any one of embodiments 32-37, wherein N2 is uridine, or a modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine).

[0487] 39. An RNA polynucleotide produced from an in vitro transcription reaction according to any one of embodiments 32 to 38.

[0488] 40. A method for making a capped RNA polynucleotide, comprising: a 5' cap comprising N1pN2; 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 comprises N1 and N2, and N3, N4, and N5 of the 5' cap, where N1 through N5 correspond to positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, N1 is A or an analog thereof, N2 is U or an analog thereof, and N3, N4, and N5 are each independently selected from A, C, G, and U; The method includes transcribing a template DNA strand in the presence of the 5' cap and an RNA polymerase, wherein the template DNA strand comprises an RNA polymerase promoter sequence and a sequence complementary to a transcription start site, and the sequence complementary to the transcription start site is a start site of the RNA polymerase promoter.

[0489] 41. The method of embodiment 39, wherein N1 is complementary to the +1 position of the template DNA strand (corresponding to the first nucleotide of the transcription start site) and N2 is complementary to the +2 position of the template DNA strand (corresponding to the second nucleotide of the transcription start site).

[0490] 42. The method of embodiment 39 or 40, wherein the RNA polymerase is T7 RNA polymerase.

[0491] 43. The method of any one of embodiments 39-41, wherein N2 is uridine or a modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine).

[0492] 44. A method for producing a capped RNA polynucleotide, comprising: transcribing a DNA template strand in the presence of a 5' cap, wherein the 5' cap comprises the structure N1pG2; the DNA template strand comprises a sequence complementary to an RNA polymerase promoter sequence and an AUA, AUC, AUG, or AUU transcription start site; The method, wherein N1 is A or an analog thereof, and N2 is U or an analog thereof.

[0493] 45. The method of embodiment 44, wherein N2 is uridine or a modified uridine (e.g., m1ψ, 2-thio-uridine, or 5-methyluridine).

[0494] 46. ​​A complex comprising a DNA template strand and a 5' cap analog comprising the structure N1pN2, wherein the DNA template strand comprises a sequence complementary to an RNA polymerase promoter sequence and a transcription start site; N1 is A or an analog thereof and N2 is U or an analog thereof; N1 interacts with the +1 position of the DNA template strand (corresponding to the first nucleotide of the transcription start site), and N2 interacts with the +2 position of the DNA template strand (corresponding to the second nucleotide of the transcription start site), the complex, wherein the sequence in the template strand that is complementary to the transcription start site is an RNA polymerase promoter start site.

[0495] 47. The conjugate of embodiment 46, wherein the +1 and +2 positions of the DNA template strand are T and A, respectively.

[0496] 48. The complex of embodiment 46 or 47, wherein the nucleotide of the cap interacts with the nucleotide of the template DNA strand via standard Watson-Crick base pairing.

[0497] 49. The complex of any one of embodiments 46 to 48, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence.

[0498] 50. The complex of any one of embodiments 46 to 49, wherein the complex further comprises an RNA polymerase (e.g., T7 RNA polymerase).

[0499] 51. The conjugate of any one of embodiments 46-50, wherein N2 is uridine or a modified uridine (eg m1ψ, 2-thio-uridine, or 5-methyluridine).

[0500] 52. A method for preparing a pharmaceutical composition, comprising combining a preparation comprising an RNA polynucleotide according to any one of embodiments 1 to 31 with a preparation comprising a lipid.

[0501] 53. The method of embodiment 52, wherein the method comprises combining the preparation comprising an RNA polynucleotide with the preparation comprising a lipid to form lipid nanoparticles that encapsulate the RNA polynucleotide.

[0502] 54. The method of embodiment 52, wherein the method comprises combining the preparation comprising an RNA polynucleotide with the preparation comprising a lipid to form an RNA lipoplex.

[0503] 55. A compound of the formula G*N1pN2, wherein: G* is a compound of formula I' [ka] or a salt thereof, wherein Each R 2 and R3 is -OH or -OCH3, X is OH or SH (e.g., O - or S - ) and p is a phosphate linker; N1 is A or an analog thereof; The compound wherein N2 is U or an analog thereof.

[0504] 56.R 2 is —OH.

[0505] 57.R 2 is —OCH 3 .

[0506] 58.R 3 The compound of any one of embodiments 55-57, wherein is —OH.

[0507] 59.R 3 The compound of any one of embodiments 55-57, wherein is —OCH 3 .

[0508] 60.X is OH (e.g., O - 60. The compound of any one of embodiments 55-59, wherein

[0509] 61. N2 is a compound of formula II' [ka] or a salt thereof, wherein [ka] are each independently a single or double bond as allowed by valence; Y 1 is O or S, Y 2 is N, C, or CH; Y 3 But N, NR a1 , C.R. a1, or CHR a1 and Y 4 But NR a2 or CHR a2 and R a1 or R a2 each independently represents hydrogen or C 1-6 is aliphatic, R 4 is -OH or -OMe, The compound of any one of embodiments 55-60, wherein # represents the point of attachment of N1p to p.

[0510] 62.N 2 Formula IIa [ka] or a salt thereof.

[0511] 63.N 2 Formula IIb [ka] or a salt thereof.

[0512] 64.Y 1 is O.

[0513] 65.Y 1 is S.

[0514] 66.Y 3 The compound of embodiment 61 or 62, wherein is N.

[0515] 67.Y 3 But, CR a1 63. The compound of embodiment 61 or 62, wherein

[0516] 68.Y 3 But NRa1 64. The compound of embodiment 61 or 63, wherein

[0517] 69.Y 3 But CHR a1 64. The compound of embodiment 61 or 63, wherein

[0518] 70.Each R a1 or R a2 The compound of any one of embodiments 61-69, wherein is independently hydrogen or methyl.

[0519] 71.R 4 The compound of any one of embodiments 61-70, wherein is —OH.

[0520] 72.R 4 The compound of any one of embodiments 61-70, wherein is -OMe.

[0521] 73.N2 is [ka] or a salt thereof, The compound of any one of embodiments 55-60, wherein # represents the point of attachment of N1p to p.

[0522] 74. The compound according to any one of embodiments 55-73, wherein N1 is adenosine or 6-methyladenosine.

[0523] 75.N1 is, [ka] or a salt thereof.

[0524] 76. The compound of any one of embodiments 55-75, wherein p is -P(=O)(OH)-, or a salt thereof.

[0525] 77. The compound is (m 7,2’-O)Gppp(m 2’-O )A1pU2, (m 7,3’-O )Gppp(m 2’-O )A1pU2, (m 7,2’-O )Gppp(m 2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m 2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 2’-O )A1p(m 5 ) U2, or a salt thereof.

[0526] 78. The compound is (m 7,2’-O )Gppp(m 6,2’-O )A1pU2, (m 7,3’-O )Gppp(m 6,2’-O )A1pU2, (m 7,2’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,3’-O )Gppp(m 6,2’-O )A1pΨ2, (m 7,2’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 1 )Ψ2, (m 7,2’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,3’-O )Gppp(m 6,2’-O )A1pS 2 U2, (m 7,2’-O )Gppp(m6,2’-O )A1p(m 5 )U2, or (m 7,3’-O )Gppp(m 6,2’-O )A1p(m 5 ) U2, or a salt thereof.

[0527] 79.Y 3 The composition or medical preparation of embodiment 15 or 17, wherein is N.

[0528] 80. A method for producing a capped RNA polynucleotide, comprising: transcribing a DNA template strand in the presence of a 5' cap, wherein the 5' cap comprises the structure N1pN2; the DNA template strand comprises a sequence complementary to an RNA polymerase promoter sequence and an AUA, AUC, AUG, or AUU transcription start site; The method, wherein N1 is A or an analog thereof, and N2 is U or an analog thereof. [Example]

[0529] Example 1-(m2 7,3’-O )Gppp(m 2’-O )ApU and (m 7,3’-O )Gppp(m 2’-O )A1p(m 1 ) Evaluation of Ψ2, A linearized plasmid encoding codon-optimized mouse erythropoietin (EPO) was used as a template. mRNAs beginning with AUAAU, AUACU, AUAGU, or AUAUU were designed to contain the 5' untranslated region (5' UTR) sequence of human α-globin (hAg) mRNA, an FI element as the 3' UTR, and an interrupted 100-nt 3' poly(A) tail adjacent to the coding sequence. A MEGAscript T7 transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for transcription, and UTP was either retained or replaced with N1-methylpseudouridine (m1ψ) triphosphate (TriLink, San Diego, CA, USA). Capping of in vitro transcribed mRNA was performed by co-transcription using a trinucleotide cap analog (Cap1 is compound I'-1 ((m2 7,3’-O )Gppp(m 2’-O )ApU), and Cap2 corresponds to compound I'-6 ((m2 7,3’-O )Gppp(m 2’-O )Ap(m 1 )Ψ)), and CC114 corresponds to (m 7 )Gppp(m 2’-O )ApU (TriLink, USA) and CC413 (m2 7,3’-O )Gppp(m 2’-O) corresponded to ApG (TriLink, USA). To obtain the desired transcripts with cap analogs, the initial GTP and UTP or mΨTP concentrations in the transcription reaction were reduced from 7.5 mM to 1.5 mM, and the 1.5 mL tubes were incubated in a hybridization chamber at 37°C for 30 min. Sequential addition of 1.5 mM GTP and UTP or mΨTP was required to complement the reaction at 30, 60, 90, and 120 min, followed by an additional 30 min of incubation at 37°C. To remove template DNA, Turbo DNase (Thermo Fisher Scientific, USA) was added to the reaction mixture after the transcription reaction was completed and incubated at 37°C for 15 min. The synthesized mRNA was precipitated by adding half the volume of 8 M LiCl solution (Merck, Darmstadt, Germany) to the reaction mixture and then pelleted by centrifugation. After dissolution in nuclease-free water, the mRNA was purified with cellulose to remove double-stranded RNA contaminants as described in Baiersdoerfer, M., et al. (2019) Molecular therapy. Nucleic acids, 15, 26-35. The concentration and quality of the mRNA were measured using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, USA). Small aliquots of the mRNA samples were stored at -20°C in siliconized tubes. These findings were matched to the AUAGU transcription start site (m2). 7,3’-O )Gppp(m 2’-O ) demonstrated that ApU produced EPO-encoding mRNA (EPO mRNA) with the highest RNA yield (64 μg / unit) (Figure 2A).

[0530] To determine the mRNA capping efficiency, in vitro transcription reactions followed by ribozyme assays were performed in a cap analog concentration-dependent manner (1, 3, 6, 9, and 12 mM). Ribozyme cleavage reactions contained 0.45 μM mRNA. A 3-fold molar excess of ribozyme over the mRNA substrate was added in an aqueous solution containing 30 mM HEPES and 150 mM NaCl. Ribozyme cleavage reactions were performed on a PCR machine using the following program: 95°C for 2 min, ramping at 0.1°C / s, and 37°C for 5 min. The mixture was cooled to a maximum of 37°C. After adding 30 mM MgCl2 solution to each sample, the mixture was maintained at 37°C for 60 min, followed by annealing at 80°C for 2 min and transferring to ice for 5 min. Short and long RNA fragments were then separated using an RNA Clean & Concentrator-5 kit (Zymo Research Europe, Freiburg, Germany) according to the manufacturer's instructions. In this study, the following custom-designed hammerhead ribozyme specific for the hAg 5'UTR was used: 5'-UGU GGG CUG AUG AGG CCG UGA GGC CGA AAC CAG AAG AAU-3' (SEQ ID NO: 44) (synthesized by Metabion International AG, Planegg, Germany). To detect short fragments, samples (30 ng) were resolved on a 21% (vol / vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). Prior to loading, samples were denatured by incubation at 75°C for 5 min in the presence of 2x RNA loading buffer (New England Biolabs, Germany). The gel was pre-run at 180V for 60 min. After the pre-run, the pockets were rinsed with 1x TBE buffer. Immediately after loading, the samples were loaded, and the gel was run continuously at 200V until the dye front reached the end of the gel.To identify short cleavage products, gels were incubated in 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA), and fluorescent signals were captured using a Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). High yields were observed (m2). 7,3’-O )Gppp(m 2’-O ) was observed when ApU was used in the concentration range of 3 to 6 mM (Figure 2B), and therefore, in further tests, a concentration of 4 mM was applied. Regardless of the concentration used, (m 7,3’-O )Gppp(m 2’-O ) The capping efficiency of ApU is close to 100% (Figure 2B).

[0531] Using optimized conditions, in vitro transcribed (IVT) EPO mRNA containing U or mΨ was produced with the trinucleotide cap analogs CC114, CC413, compound I'-1, or compound I'-6. High yields and capping efficiencies were observed for each mRNA tested, regardless of the cap analog. Ribozyme assay analysis showed that the capping efficiencies of compound I'-1 and compound I'-6 were close to 100%, comparable to those of the commercially available CC114 and CC413 cap analogs (Figure 3).

[0532] For detection of short by-products, IVT mRNA (1.5–2 μg) was resolved on a 21% (vol / vol) 19:1 acrylamide:bisacrylamide denaturing gel supplemented with 8 M urea (Merck, Germany). For both pre-runs, the pockets were rinsed with 1x TBE buffer. Immediately after pre-run, the sample was applied, and the gel was run continuously at 180 V until the dye front reached the end of the gel. To identify short by-products, the gel was incubated in 1x TBE buffer containing 0.01% SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA), and the fluorescent signal was captured using a Gel Doc EZ Imager (Bio-Rad, USA). Denaturing urea polyacrylamide gel electrophoresis showed minimal amounts of short contaminants in EPO m1Ψ-mRNA for compounds I'-6 and CC413, but significant amounts were observed for the unmodified version, independent of the cap analog (Figure 4).

[0533] Human buffy coats from healthy individuals were obtained from the Faculty of Medicine at Johannes Gutenberg University, Mainz, and used to isolate peripheral blood mononuclear cells (PBMCs) on a Ficoll-Paque™ PLUS (Cytiva, Marlborough, MA, USA) density gradient. In preparation for mRNA transfection, cryopreserved PBMCs were thawed and plated at 5 × 10 cells per well in 190 μL of RPMI medium supplemented with 1% non-essential amino acids (NEAA), 1% sodium pyruvate, and 10% fetal bovine serum (Merck, Germany). 5Cells were seeded into 96-well plates at a density of 1 / 3 cells / well. Cells were maintained at 37°C with 5% CO2 until transfection with 0.5, 1.5, 5.0, and 15 μg / ml lipoplex-formulated EPO mRNA (LPX-RNA) as described in Kranz, LM, et al. (2016) Nature, 534, 396-401. Complexed RNA (10 μl per well) was added in triplicate, and supernatants were collected 24 hours posttransfection for single-cell cytotoxicity assays and cytokine / chemokine profiles. To determine cell viability and production of selected cytokines / chemokines, supernatants from LPX-RNA-transfected human PBMCs were subjected to XTT cytotoxicity testing using the Cell Proliferation Kit II (Sigma-Aldrich) and cytokine / chemokine profile analysis using the Meso Scale Discovery V-PLEX Custom Human Biomarkers Proinflammatory and Chemokine Panel (Meso Scale Diagnostics-MSD, Rockville, MD, USA) according to the manufacturer's instructions. For cytokine / chemokine profile analysis, a 1:5 sample dilution (supernatant:MSD diluent) was used in each experiment. The levels of IL-6 (interleukin 6), TNF-α (tumor necrosis factor alpha), IL-1β (interleukin 1 beta), IFN-γ (interferon gamma), MCP-1 (monocyte-inducible protein-1), MIP-1β (macrophage inflammatory protein-1 beta), and IL-10 (interleukin 10) were quantified 24 hours after mRNA transfection. These results indicated that no toxic effects on cell viability due to compounds I'-1 and I'-6 were observed (Figure 5). For m1Ψ-modified EPO mRNA, there was no toxic effect on PBMCs up to 1 μg / well mRNA (Figure 5). Transfection of unmodified mRNA resulted in a decrease in cell viability starting at a dose of 1.5 μg / ml, but this effect was independent of the cap used and dependent on the mRNA modification (Figure 5).The levels of each cytokine / chemokine showed a zero to moderate increase in each measured sample (Figure 6). Compound I'-6 was comparable to CC413 in terms of the amount of cytokine / chemokine secreted by human PBMCs after transfection with EPO m1Ψ-mRNA (Figure 6). This low immunogenicity can be directly attributed to the nucleoside modifications incorporated into the mRNA. To illustrate this, cells treated with U-containing mRNA complexed with lipid capped compounds I'-1, CC114, and CC413 induced a much higher inflammatory cytokine / chemokine response compared to m1Ψ-modified mRNA, even at their lowest doses (0.5 μg / ml) (Figure 6). With regard to cytokine / chemokine levels from unmodified mRNA, compound I'-1 produced cytokines / chemokines comparable to CC114 and significantly higher than CC413 (Figure 6).

[0534] To measure the translation efficiency of EPO mRNA capped with Compound I'-1, Compound I'-6, CC114, and CC413 in vitro, human primary hepatocytes were cultured at 2.5 x 10 per well. 4 Cells were seeded into 96-well plates at a density of 1 / 3 of a cell population and transfected with mRNA samples (0.1 μg) complexed with TransIT Reagent (Mirus Bio, Madison, WI, USA) in a final volume of 200 μL of InVitroGRO CP medium (Sigma-Aldrich) supplemented with Thorpedo Antibiotic Mix (Sigma-Aldrich). To quantify EPO levels, supernatants were collected 1, 2, 3, 4, 5, and 6 days after transfection, and EPO levels were analyzed using a mouse erythropoietin DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Compound I'-1 resulted in higher translation compared to CC413 at each time point after transfection in the context of unmodified RNA (Figure 7). However, compound I'-6 in the context of m1Ψ-mRNA results in lower translation at later time points but near-equivalent translation to the standard CC413 cap analog in human primary hepatocytes.

[0535] To confirm the in vitro data, 8-10 week-old female BALB / c mice from Jackson Laboratory (Bar Harbor, ME, USA) were used for in vivo experiments in accordance with federal policy on animal research (ethics approval number: G18-12-027). Mice (n = 3 / group) were intravenously injected with 3 μg of TransIT-complexed EPO mRNA (Mirus Bio) in a final volume of 200 μL of Dulbecco's modified Eagle's medium (DMEM). Mice used as controls were injected with TransIT reagent diluted in DMEM but without RNA. Mice were injected with TransIT reagent-complexed EPO mRNA, and plasma EPO levels were measured using ELISA (Figure 8A). Hematocrit was measured from 18 μl of blood collected at the indicated times using centrifugation in Drummond microcap glass capillary tubes (20 μl volume, Merck, Germany) as described (20) (Figure 8B). After hematocrit determination, the capillary tubes were snapped open, plasma collected, and EPO levels were measured and analyzed using a mouse erythropoietin DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. These results again demonstrated that, regardless of the cap initiator, mRNA capped with an ARCA cap analog was translated much better than mRNA capped with a non-ARCA version (Figure 1, Figures 8A and 8B). EPO RNA containing mΨ was translated twice as much as U-containing mRNA at each time point (Figure 8A). Both compound I'-1 and compound I'-6 were suitable for translation of the encoded protein, and each cap analog can be used to synthesize non-replicating functional mRNA (Figures 8A and 8B). Interestingly, compound I'-6 had a beneficial effect on mRNA translation efficiency, meaning that it showed 1.5- to 3-fold more translation compared to mRNA capped with CC413 at 48 and 72 h after injection, respectively (Figure 8A). This is confirmed by the fact that the 24-hour EPO values ​​were higher than the 6-hour values, which was surprising after IV injection and has never been experienced before (Figure 8A).The positive effect of compound I'-6 on mRNA translational activity was also reflected in the biological activity of mRNA, as hematocrit remained at a very high level in mice injected with EPO mRNA capped with compound I'-6 even 21 days after injection (Figure 8B). Hematocrit began to decrease on day 14 after injection of U-containing mRNA capped with CC114 and CC413, but this was not observed for mRNA capped with compound I'-1 (Figure 8B). After 21 days of administration, hematocrit in mice injected with EPO mRNA capped with compound I'-6 was at the same level as that in mice injected with CC413-capped mRNA on days 7 and 14 (Figure 8B).

[0536] In conclusion, the presented data demonstrated that mΨ-containing cap analogs can be adapted for the synthesis of nonreplicative mRNAs. These studies evaluated a unique, nucleoside-modified, anti-reverse trinucleotide cap analog, compound I'-6, which was used to generate functional mRNAs with a 5' cap structure that results in long-term maintenance of the encoded protein. These data demonstrated that the appropriate combination of initial sequences with compound I'-6 resulted in mRNAs that significantly exceeded the translational capacity and biological activity of IVT mRNAs capped with commonly used trinucleotide cap analogs, which have already been successfully used, for example, in mRNA-based vaccines against SARS-CoV-2.

[0537] Example 2. Synthesis of 5' cap. General trinucleotide cap1 structure: [ka] During the ceremony, R2 / R3: OH / OMe N1 and N2: A, U, G, C, Ψ, m 6 A, modified U, modified Ψ, any natural / unnatural nucleoside, any modified nucleoside

[0538] A general trinucleotide cap1 structure containing a 2'-OMe-Ψ analog at N1 and another nucleoside at N2: [ka] During the ceremony, R2 / R3: OH / OMe R 3* , R 4* , R 5* : H, Me, any alkyl, aryl, benzyl, naphthyl, vinyl, allyl, propargyl, carbocyclic, heterocyclic N2: A, G, m 6 A

[0539] A general trinucleotide cap1 structure containing 2'-OMe-A at N1 and a U / Ψ analog at N2: [ka] During the ceremony, R2 / R3: OH / OMe N2:U, Ψ, modified U, modified Ψ

[0540] General synthetic route to different cap analogues [ka]

[0541] A general synthetic route for different cap analogs containing 2'-OMe-A at N1 and various Ψ derivatives at N2 [ka]

[0542] Different m 7 General synthetic route to GDP derivatives (1-3) [ka]

[0543] 7-methyl-guanosine 5'-diphosphate derivative (m7 Synthesis of GDP derivatives, 1-3) m 7 The synthesis of GDP derivatives was carried out according to modified published procedures (Patent US2003 / 0194759A1; Patent WO2008 / 016473A2; Patent WO2017053297A1; Bioorg. Med. Chem. Lett. 2007, 17, 5295.).

[0544] 7-methylguanosine 5'-diphosphate (m 7 GDP, 1): [ka] 1 H NMR(300 MHz,D2O):δ 6.04(d,J=3.4 Hz,1H),4.66(dd,J=4.8,3.4 Hz,1H),4.50(t,J=5.2 Hz,1H),4.42-4.28(m,2H),4.20(ddd,J=11.9,5.3,2.1 Hz,1H),4.10(s,3H),3.19(q,J=7.3 Hz,22H),1.26(t,J=7.3 Hz,33H).

[0545] 31 P NMR(121 MHz,D2O): δ-10.40(d,J=20.9 Hz),-11.38(d,J=20.7 Hz).

[0546] MS(ESI - ):C 11 H 16 N5O 11 P2[MH] - Calculated exact mass: 456.03. Measured value: 456.01.

[0547] 7-methyl-2'-O-methylguanosine 5'-diphosphate (m 7 GDP(2'-OMe), 2): [ka] 1H NMR (300 MHz, D2O): δ 6.21 (d, J = 3.0 Hz, 1H), 4.69 (dd, J = 6.1, 4.9 Hz,1H),4.43-4.34(m,3H),4.31-4.22(m,1H),4.17(s,3H),3.65(s,3H),3.25(q,J=7.4 Hz,9H,Et3NH + ),1.33(t,J=7.3 Hz,14H,Et3NH + ).

[0548] 31 P NMR (121 MHz, D2O): δ-8.98 (d, J=21.7 Hz), -11.03--11.36 (m).

[0549] MS (ESI - ):C 12 H 18 N5O 11 P2[MH] - についてcalculatedされたcorrectなquality, 470.05. The measured value is 470.02.

[0550] 7-メチル-3'-O-メチルグアノシン5'-ジホスフェート(m 7 GDP(3'-OMe), 3):

change

[0551] 31 P NMR (121 MHz, D2O): δ-8.95(d,J=21.5 Hz),-11.20(dq,J=21.2,4.3 Hz).

[0552] MS(ESI - ):C 12 H 18 N5O 11 P2[MH] - Calculated exact mass: 470.05. Measured mass: 470.09.

[0553] General scheme for the synthesis of different cap analogues [ka]

[0554] General Procedure (I) The multi-step cap synthesis was performed according to a modified published procedure (patents WO2017053297A1, WO2021162567A1, Nucleic Acids Res. 2020, 48, 1607).

[0555] Step I: Coupling + Oxidation The 2',3'-N-protected nucleoside derivative (SM-1, 5 mmol), 5-ethylthio-1H-tetrazole (activator, 25 mmol), and 2'-OMe phosphoramidite derivative (SM-2, 5 mmol) were placed in a 100 mL Schlenk flask and 50 mL of dry ACN was added. The solution was stirred at room temperature for 30 min. Then, tert-butyl hydroperoxide solution (25 mmol to oxidize P(III) to the P(V) state) was added dropwise, turning the solution yellow. The solution was stirred at room temperature for another 30 min. The solvent was evaporated, and the residue was redissolved in DCM (300 mL) and extracted with water (3 × 100 mL) and brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated in vacuo to give a yellow sticky solid. The crude residue was purified by flash chromatography (0-5% MeOH / DCM) to give the coupled product (Int-1) as a pale yellow solid foam (84-92% yield).

[0556] Step II: Detritylation The coupling product (Int-1, 4 mmol) was placed in a 250 mL Schlenk flask and 150 mL of dry DCM was added to form a colorless solution. Dichloroacetic acid (40 mmol) was then added dropwise over 5 min at room temperature, turning the solution deep red. The reaction was stirred at room temperature for 15 min. TLC confirmed complete deprotection of the DMT group. MeOH was then added until a faint red color persisted. The solvent was evaporated, and the residue was dissolved in 300 mL of DCM. MeOH was added again until only a faint red color persisted. The solution was extracted with saturated aqueous NaHCO3 (3 × 100 mL) followed by brine (100 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was evaporated in vacuo to give a yellow foam. The crude residue was purified by flash chromatography (0–10% MeOH / DCM) to give compound (Int-2) as a pale yellow solid foam (86–91% yield).

[0557] Step III: Coupling + Oxidation Compound Int-2 (3 mmol) and 5-ethylthio-1H-tetrazole (activator, 15 mmol) were placed in a 100 mL Schlenk flask and 30 mL of dry ACN was added. 3-({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)propanenitrile (4.5 mmol) was then added dropwise. The solution was stirred at room temperature for 30 minutes. tert-Butyl hydroperoxide solution (15 mmol) was then added dropwise, turning the solution yellow. The solution was stirred at room temperature for another 30 minutes. The solvent was evaporated, and the residue was redissolved in DCM (200 mL) and extracted with water (3 × 50 mL) and brine (50 mL). The organic phase was dried over anhydrous NaSO, filtered, and the solvent was evaporated in vacuo to give a yellow sticky residue. The crude residue was purified by flash chromatography (0-10% MeOH / DCM) to give compound (Int-3) as a pale yellow solid foam (72-80% yield).

[0558] Step IV: Deprotection Compound (Int-3, 2 mmol) was placed in an autoclave and 20 mL of absolute EtOH was added, followed by 60 mL of aqueous NH3 (32%). The reaction was stirred at 60 °C for 6 h, and LCMS studies showed complete deprotection of the substrate. The solution was concentrated under reduced pressure, redissolved in water / absolute ethanol (1:1, 50 mL), and evaporated in vacuo to give a yellow residue.

[0559] When R* = Bz, the residue was directly purified by anion exchange chromatography followed by reverse-phase chromatography to give the compound (pN1(2'-OMe)pN2, Int-4) as a triethylammonium salt as a white solid foam (77-89% yield).

[0560] When R* = TBS, the residue was further treated with 1 M TBAF in THF solution. The reaction was stirred at 40 °C for 18 h. Deprotection of the TBS group was monitored by LCMS studies. After complete deprotection, 100 mM TEAB (pH = 7.5) was added, and the product was directly purified by anion exchange chromatography and reverse-phase chromatography. Compound (pN1(2'-OMe)pN2, Int-4) was obtained as a triethylammonium salt as a white solid foam (65-72% yield).

[0561] Anion exchange chromatography: XK 50 / 30 column packed with DEAE Sephadex A-25 (approximately 75 g of resin, column volume approximately 550 mL, Cytiva. Detection wavelength: 220, 260 nm; solvent system: Buffer A: 100 mM TEAB (pH = 7.5); Buffer B: 1.0 M TEAB (pH = 7.5); flow rate: 30 ml / min; gradient: 0% B for 30 min, 0–80% B for 160 min, 80–100% B for 5 min, 100% B for 25 min). The product obtained after anion exchange chromatography was further purified by reversed-phase chromatography.

[0562] Reverse-phase chromatography: Column: FP Select C18 330 g; Detection wavelength: 220 nm, 260 nm; Solvent system: Solvent A: 100 mM TEAB (pH = 7.5), Solvent B: acetonitrile; Flow rate: 60 mL / min; Gradient: 5% B for 6 min, 5–20% B for 24 min, 20–50% B for 5 min, 50% B for 5 min). Product-containing fractions were collected, the solvent evaporated, and lyophilized.

[0563] Step V: Phosphorimidazolide Formation Compound (pN1(2'-OMe)pN2, Int-4, 1 mmol) was placed in a 100 mL RB flask and coevaporated with dry DMF (2 × 15 mL). 35 mL of dry DMF was then added. In a separate dry 50 mL Schlenk flask, triphenylphosphine (2 mmol), 2,2'-dipyridyl disulfide (2 mmol), and imidazole (2.5 mmol) were dissolved in 20 mL of dry DMF and EtN (10 mmol). The solution was then added dropwise to the dinucleotide derivative solution, resulting in a yellow solution. The reaction was stirred at room temperature for 18 hours. The solution was then poured into 1 L of 2% anhydrous NaClO4 in dry acetone (w / v), and the formation of a white precipitate was observed. Filtration was performed, and the residue was washed with 1 L of dry acetone. The product was dried over anhydrous PO5 under reduced pressure. The dinucleotide phosphorimidazolide (Im-pN1(2'-OMe)pN2, Int-5) was obtained as the disodium salt (91-98%).

[0564] Step VI: Final Coupling 7-methyl-guanosine 5'-diphosphate derivative (m 7GDP derivative (0.75 mmol) and dinucleotide phosphorimidazolide (Im-pN1(2'-OMe)pN2, Int-5, 0.9 mmol) were taken in ...

Claims

1. Trinucleotide Cap G*N １ pN ２ or a salt thereof, G* comprises the structure of formula I'; 【Chemical 1】 During the ceremony, Each R ２ and R ３ are independently —OH or —OCH ３ and X is OH or SH; N １ is A or an analog thereof, N ２ is U or an analog thereof; p is a phosphate (e.g., —P(═O)(OH)— or —P(═O)(O － )-) or thiophosphates (e.g., -P(=S)(OH)- or -P(=S)(O － )-).

2. R ２ is —OH, and R ３ But, -OCH ３ The trinucleotide cap of claim 1, wherein

3. R ２ But, -OCH ３ and R ３ The trinucleotide cap of claim 1, wherein is -OH.

4. X is OH or O － The trinucleotide cap according to any one of claims 1 to 3,

5. X is SH or S － The trinucleotide cap according to any one of claims 1 to 3,

6. N １ The trinucleotide cap of any one of claims 1 to 5, wherein is adenosine.

7. N １ The trinucleotide cap of any one of claims 1 to 5, wherein is 6-methyladenosine.

8. N １ but, 【Chemistry 2】 and 6. The trinucleotide cap of any one of claims 1 to 5, wherein % represents the point of attachment to G*.

9. N ２ is a modified U.

10. N ２ is represented by formula II''' 【Chemistry 3】 or a salt thereof, wherein 【Chemistry 4】 are each independently a single bond or a double bond as allowed by valence; Y １ is O or S, Y ２ is N, C, or CH; Y ３ But N, NR ａ１ , C.R. ａ１ , or CHR ａ１ and Y ４ But NR ａ２ or CHR ａ２ and Y ５ But, CR ａ３ and R ａ１ , R ａ２ , or R ａ３ each independently being hydrogen, C １－６ Aliphatic, -CH ２ R, or -O(C １－４ alkyl), R is substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. １－４ is aliphatic, R ４ is —OH or —OMe, # is N １ 9. The trinucleotide cap of any one of claims 1 to 8, which represents the point of attachment of p to p.

11. N ２ is of formula II″, 【Chemistry 5】 During the ceremony, 【Chemistry 6】 are each independently a single bond or a double bond as allowed by valence; Y １ is O or S, Y ２ is N, C, or CH; Y ３ But N, NR ａ１ , C.R. ａ１ , or CHR ａ１ and Y ４ But NR ａ２ or CHR ａ２ and R ａ１ or R ａ２ each independently being hydrogen, C １－６ Aliphatic, -CH ２ R, or -O(C １－４ alkyl), R is substituted with halogen, phenyl, a 3- to 6-membered saturated carbocyclic ring, or a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. １－４ is aliphatic, R ４ is —OH or —OMe, # is N １ 9. The trinucleotide cap of any one of claims 1 to 8, which represents the point of attachment of p to p.

12. N ２ The trinucleotide cap of claim 11, wherein is of formula IIa″. 【Chemistry 7】

13. N ２ The trinucleotide cap of claim 11, wherein is of formula IIb''. 【Chemistry 8】

14. Y １ The trinucleotide cap of any one of claims 10 to 13, wherein is O.

15. Y １ The trinucleotide cap of any one of claims 10 to 13, wherein is S.

16. Y ３ But, CR ａ１ The trinucleotide cap of claim 12, wherein:

17. Y ３ But NR １ａ 14. The trinucleotide cap of claim 13, wherein:

18. R ａ１ is hydrogen.

19. R ａ１ But C １－６ 18. The trinucleotide cap of claim 16 or claim 17, which is aliphatic.

20. R ａ１ is methyl, ethyl, n-propyl, or isopropyl.

21. R ａ１ is methyl.

22. R ａ１ But -CH ２ 18. The trinucleotide cap of claim 16 or claim 17, wherein C≡CH.

23. R ａ１ But -O(C １－４ 17. The trinucleotide cap of claim 16, wherein the trinucleotide cap is aryl, ...

24. R ａ１ The trinucleotide cap of claim 23, wherein is -OMe.

25. R ａ１ But -CH ２ 18. The trinucleotide cap of claim 16 or claim 17, wherein R is R.

26. R is C substituted with halogen １－４ 26. The trinucleotide cap of claim 25, which is aliphatic.

27. R is C substituted with halogen １－２ 27. The trinucleotide cap of claim 26, which is aliphatic.

28. R is -CF ３ 28. The trinucleotide cap of claim 27, wherein:

29. 26. The trinucleotide cap of claim 25, wherein R is phenyl.

30. 26. The trinucleotide cap of claim 25, wherein R is a 3- to 6-membered saturated carbocyclic ring.

31. 31. The trinucleotide cap of claim 30, wherein R is a 3- to 4-membered saturated carbocyclic ring.

32. 32. The trinucleotide cap of claim 31, wherein R is a three-membered saturated carbocyclic ring.

33. 26. The trinucleotide cap of claim 25, wherein R is a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

34. 34. The trinucleotide cap of claim 33, wherein R is 4-pyridyl.

35. R ４ The trinucleotide cap of any one of claims 10 to 34, wherein is -OH.

36. R ４ The trinucleotide cap of any one of claims 10 to 34, wherein is -OMe.

37. N ２ 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-oxyacetic 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-thio-uridine (mcm ５ s ２ U), 5-aminomethyl-2-thio-uridine (nm ５ s ２ U), 5-methylaminomethyl-uridine (mnm ５ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ５ s ２ U), 5-methylaminomethyl-2-seleno-uridine (mnm ５ se ２ U), 5-carbamoylmethyl-uridine (ncm ５ U), 5-carboxymethylaminomethyl-uridine (cmnm ５ U), 5-carboxymethylaminomethyl-2-thio-uridine (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-thio-uridine (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, 5-methyluridine (m ５ U), 1-methyl-pseudouridine (m １ ψ), pseudouridine (ψ), 1-(2,2,2-trifluoroethyl)pseudouridine (tfet １ ψ), 1-propargylpseudouridine (ppg) １ ψ), 1-benzylpseudouridine (bn １ ψ), 1-(cyclopropylmethyl)pseudouridine (cpm １ ψ), and 1-(pyridin-4-ylmethyl)pseudouridine ((4-pm) １ The trinucleotide cap of any one of claims 1 to 8, selected from the group consisting of:

38. a trinucleotide cap selected from: 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 Or its salt.

39. A composition or medical preparation comprising the trinucleotide cap or salt thereof according to any one of claims 1 to 38.

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