Methods and modifications for reducing the innate immune response to RNA

Abstract

Compositions and methods are provided for reducing RNA interaction with cellular RNA sensors, including but not limited to, Toll-like receptors (TLRs), RIG-I, and MDA-5, by cloaking RNA using selective 2'-hydroxyl post-synthetic modifications after transcription. The post-synthetic modification of the RNA's 2'-hydroxyl group (referred to as "cloaking") can optionally be spontaneously reversed within the cell, restoring biologically functional RNA within the cell. This RNA modification reduces the natural immunogenicity of the RNA while preserving other biological activities, such as translation.

Description

[Background technology]

[0001] RNA has several uses as a therapeutic agent. For example, mRNA vaccines are being used or are under development for immunization against viruses, cancer, etc. The recent development and rapid distribution of mRNA-based SARS-COVID-19 vaccines demonstrates the potential of mRNA for vaccination purposes. RNA is also being developed for use in siRNA, circRNA, miRNA sponges, CRISPR technology, and base editing technology.

[0002] However, RNA delivered to cells can also overstimulate the human innate immune system by activating cellular RNA sensors, triggering cellular antiviral defense mechanisms that result in the inhibition of mRNA translation and systemic inflammation in humans. For example, RNA can be recognized in human cells by TLR3, TLR7, TLR8, RIG-I, and MDA-5 in a sequence- and structure-dependent manner. The resulting activation of the innate immune system is cell- and tissue-specific. Unmodified mRNA synthesized by in vitro transcription has been found to be a potent inducer of type I interferon, as well as proinflammatory cytokines and chemokines, mediated by TLR3, TLR7, TLR8, and RIG-I, which can inhibit the translation efficiency of encoded antigen proteins and lead to other undesirable physiological effects, such as pain.

[0003] Multiple approaches have been developed to minimize the innate immune response to mRNA, including RNA sequence optimization, advanced purification and RNA delivery methods, and the incorporation of natural and modified nucleobases. For example, modified nucleobases (e.g., m5C, pseudouridine, 1-methylpseudouridine) can reduce such immunogenic activity by disrupting RNA interactions with cellular receptors / RNA sensors.

[0004] Chemically modifying nucleosides at multiple 2'-OH groups of RNA via post-synthetic modification (termed "cloaking") for therapeutic indications is of interest, especially if the modification can be reversed to provide a biologically active RNA molecule. The present disclosure provides such protection and methods for modulating the effects of RNA on innate immunity. Summary of the Invention

[0005] As disclosed herein, compositions and methods are provided for reducing RNA interaction with cellular RNA sensors, including but not limited to, Toll-like receptors (TLRs), RIG-I, and MDA-5, by post-transcriptionally selectively cloaking RNA through reaction with cloaking reagents such as water-soluble acylimidazoles, sulfonylimidazoles, sulfonyltriazoles, and related activated acyl reagents. The 2'-acylation can optionally be spontaneously reversed in cells, restoring biologically functional RNA to the cell. This RNA modification reduces the natural immunogenicity of the RNA while preserving other biological activities, such as translation.

[0006] The cloaked RNA can be mRNA, tRNA, rRNA, circRNA, RNA sponge, long non-coding RNA, viral RNA, synthetic RNA such as chemically synthesized or in vitro transcribed forms, or any other form of RNA such as hnRNA and viroid RNA. In some embodiments, the RNA is in vitro transcribed mRNA. In some embodiments, the mRNA encodes a protein, such as an antigen, a therapeutic protein, a growth factor, a structural protein, etc. The antigen can be, for example, a pathogen antigen or an autoantigen, such as a tumor-associated antigen. The cloaked RNA can be formulated for delivery to cells in vivo, for example, as a vaccine, as gene therapy, for cell reprogramming, etc. In such formulations, the RNA can be coated or complexed with lipid nanoparticles, liposomes, lipoplexes, etc.

[0007] The RNA may be a mixture of different types of RNA and may be in single- or double-stranded form. The RNA may be synthetic or naturally occurring. The mRNA may or may not have a cap and / or poly-A tail. The RNA may or may not contain non-naturally occurring modified nucleobases. The RNA may be at least 12 nt in length, at least about 15, at least about 20, at least about 25, about 100 nt, 500 nt, 750 nt, 1 kb, 1.5 kb, 2 kb, or more. In some embodiments, the RNA is greater than about 500 nt, greater than about 750 nt, or greater than about 1 kb in length. The RNA acylated by the methods disclosed herein may contain at least about 10% acylated 2'-OH, at least about 20% acylated 2'-OH, at least about 30% acylated 2'-OH, at least about 50% acylated 2'-OH, or more, up to substantially the entire RNA. In a population of RNAs, there may be at least about 20% acylated 2'-OH, at least about 30% acylated 2'-OH, at least about 50% acylated 2'-OH, or more, up to substantially the entire population. Alternatively, folded RNA acylated by the methods disclosed herein may contain at least about 10% acylated 2'-OH, at least about 20% acylated 2'-OH, at least about 30% acylated 2'-OH, at least about 50% acylated 2'-OH, or more, of nucleotides in unpaired loops and regions.

[0008] In some embodiments, RNA formulations for therapeutic in vivo delivery to cells are provided, wherein the RNA is cloaked by 2'-acylation of at least a portion of the 2'-hydroxyl groups. The RNA in the formulation may be complexed with a carrier, such as a lipid nanoparticle, liposome, lipoplex, or the like. The formulation may be provided in a pharmaceutically acceptable excipient. The formulation may be provided in a unit dose of, for example, about 1 mg to about 500 mg of RNA, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 250 mg, or about 500 mg.

[0009] Cloaking agents used for selective 2' cloaking of RNA include agents having the following general structure:

[0010] [ka]

[0011] Z is selected from imidazole, 1,2,3-triazole, 1,2,4-triazole, azide, cyanide, anhydride, fluoride, NHS ester, etc. In some embodiments, Z is imidazole.

[0012] Z1 may be selected from imidazole and triazole, such as 1,2,3-triazole or 1,2,4-triazole.

[0013] R1 or R2, which may be generally referred to herein as an "R" group, is a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl or heteroaryl group, or a substituted or unsubstituted cycloalkyl group. In some embodiments, R contains 1 to 10 carbons and, optionally, 1 to 4 heteroatoms, particularly N or O. Suitable acyl groups are water-soluble, and the ester products are relatively water-stable yet sufficiently electrophilic to allow for easy inversion by a nucleophilic organic catalyst.

[0014] Suitable R groups include, for example:

[0015] [ka]

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] [ka]

[0021] [ka]

[0022] It has been shown herein that the selection of R groups influences the effect of cloaked RNAs on cellular RNA sensors, and that specific patterns of innate immune mediator expression are associated with exposure to different R groups. For example, exemplary R groups 1, 4, 11, 13, and 19 are shown to provide distinct expression profiles of immune response mediators, including type I interferons, CCL5, IL-8, IL-3, and VEGF, as shown, for example, in Figures 9 and 10. In some embodiments, RNAs are cloaked with selected R groups to achieve a desired pattern of expression of innate immune system mediators in cells contacted with the RNA. For example, R groups can be selected to reduce undesired proinflammatory responses.

[0023] In some embodiments, a method for tuning a cellular innate immune response with an exogenous RNA composition includes cloaking an RNA by 2'-hydroxyl acylation with one or more, typically two or more, three or more, or four or more different RNA cloaking reagents, e.g., using reagents containing an R group selected from R1-R33 disclosed herein; contacting a cell, e.g., a relevant human cell, with the cloaked RNA; and measuring the release of an innate immune mediator by the cell. The level of acylation may vary, e.g., from about 5% to about 95%. The relevant cell may be a cell type targeted by a therapeutic RNA agent, e.g., an antigen-presenting cell, a cancer cell, a blood cell, or the like. The innate immune mediator is optionally selected from TNFα, IL-1β, IFNα, IFNβ, IL-6, CCL5, CXCL8, CXCL10, IL-8, IL-3, VEGF, and PDGFAA. R groups that provide a desired pattern of innate immune response can be selected for use in cloaking RNA, for example, for therapeutic formulations.

[0024] In some embodiments, R is [ka] [ka] [ka] [ka] [ka] [Brief explanation of the drawings]

[0025] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures:

[0026] [Figure 1] N,N-Dimethylglycine acylimidazole (DMG-Im, R group 13) acylates RNA 2'-OH groups in high yield. [Figure 2A] Luminex human 48-plex cytokine analysis showed that cloaking by DMG-Im potently suppressed the expression of several pro-inflammatory cytokines and immune activation markers. [Figure 2B] Luminex human 48-plex cytokine analysis showed that cloaking by DMG-Im potently suppressed the expression of several pro-inflammatory cytokines and immune activation markers. [Figure 2C] Luminex human 48-plex cytokine analysis showed that cloaking by DMG-Im potently suppressed the expression of several pro-inflammatory cytokines and immune activation markers. [Figure 3A] Luminex human 48-plex cytokine analysis showed that cloaking by DMG-Im strongly modulated the expression of several pro-inflammatory cytokines and immune activation markers over time. [Figure 3B] Luminex human 48-plex cytokine analysis showed that cloaking by DMG-Im strongly modulated the expression of several pro-inflammatory cytokines and immune activation markers over time. [Figure 4A] DMG-Im cloaking differentially regulates certain innate immune response pathways over time. (A) VEGF. [Figure 4B] DMG-Im cloaking differentially regulates certain innate immune response pathways over time. (B) CCL5. [Figure 4C]DMG-Im cloaking differentially regulates certain innate immune response pathways over time. (C) IL-6. [Figure 4D] DMG-Im cloaking differentially regulates certain innate immune response pathways over time. (D) IL-8. [Figure 4E] DMG-Im cloaking differentially regulates certain innate immune response pathways over time. (E) IL4. [Figure 5] Cloaking with DMG-Im suppressed ISG15 expression. [Figure 6A] Spontaneous restoration of mRNA encoding eGFP-mRNA in HeLa, HEK293 and SW480 cells cloaked with DMG-Im (a) and destabilized GFP (b). [Figure 6B] Spontaneous restoration of mRNA encoding eGFP-mRNA in HeLa, HEK293 and SW480 cells cloaked with DMG-Im (a) and destabilized GFP (b). [Figure 7] Chemical structures of R groups for immunomodulatory acylimidazole reagents. [Figure 8] Spontaneous restoration of d2GFP-mRNA translation with selected acylimidazole reagents in HEK293 cells. [Figure 9A] Luminex human 48-plex cytokine analysis shows that eGFP-mRNA aliquots cloaked by acylimidazoles with diverse chemical structures differentially regulate the expression of several proinflammatory cytokines and immune activation markers. [Figure 9B] Luminex human 48-plex cytokine analysis shows that eGFP-mRNA aliquots cloaked by acylimidazoles with diverse chemical structures differentially regulate the expression of several proinflammatory cytokines and immune activation markers. [Figure 9C]Luminex human 48-plex cytokine analysis shows that eGFP-mRNA aliquots cloaked by acylimidazoles with diverse chemical structures differentially regulate the expression of several proinflammatory cytokines and immune activation markers. [Figure 9D] Luminex human 48-plex cytokine analysis shows that eGFP-mRNA aliquots cloaked by acylimidazoles with diverse chemical structures differentially regulate the expression of several proinflammatory cytokines and immune activation markers. [Figure 10A] Luminex human 48-plex cytokine analysis comparing R groups 1, 4, 11, and 19 with DMG for IL-8 expression showing a chemical structure-dependent reduction in the pro-inflammatory immune response. [Figure 10B] Luminex human 48-plex cytokine analysis comparing R groups 1, 4, 11, and 19 with DMG for CCL5 expression showing a chemical structure-dependent reduction in pro-inflammatory immune responses. [Figure 10C] Luminex human 48-plex cytokine analysis comparing R groups 1, 4, 11, and 19 with DMG for VEGF expression showing a chemical structure-dependent reduction in pro-inflammatory immune responses. DETAILED DESCRIPTION OF THE INVENTION

[0027] Before the methods and compositions of the present disclosure are described, it is to be understood that this invention is not limited to the particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0028] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either limit is included in the smaller range, neither limit is included in the smaller range, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0029] 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 to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0030] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and a reference to "the peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as polypeptides.

[0031] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0032] As used herein, "commercially available" compounds include those from Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire UK), BDH Inc. (Toronto, Canada), Bionet (Cornwall, UK), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall UK), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall UK), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem, and Argonaut Technology.

[0033] As used herein, "methods known to those skilled in the art" can be identified through various reference books and databases. Suitable reference books and articles that detail the synthesis of reactants useful for preparing the compounds of the present invention or provide references to articles describing their preparation include, for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S.R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H.O. House, "Modern Synthetic Reactions", 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif., 1972; T.L.G. Ilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-Interscience, New York, 1992. Specific and similar reactants can also be identified through indexes of known chemicals prepared by the American Chemical Society's Chemical Abstract Service, available in most public and university libraries, as well as online databases (the American Chemical Society, Washington, DC, can be contacted for more information). Chemicals that are known but not commercially available in catalogs can be prepared by custom chemical synthesis companies, and many of the standard chemical supply companies (e.g., those listed above) offer custom synthesis services.

[0034] The term "alkyl" refers to a C1-C6 alkyl group that may be straight-chain, branched, or cyclic. 20 "Lower alkyl" as in "lower alkyl" or "substituted lower alkyl" refers to a C1-C 10The term "alkyl," "lower alkyl," or "cycloalkyl" refers to any of methyl, ethyl, isopropyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, C6-C8 12 These include spirocycle, cyclopropylethyl, cyclobutylethyl, decalinyl, bicyclo-[1.1.1]-pentyl, norboranyl, bicyclo-[2.2.2]-octyl, cubyl, adamantonyl, and related cage hydrocarbon moieties. In certain embodiments, alkyl is C1-C 20 In certain embodiments, the alkyl group is polydeuterated.

[0035] "Substituted alkyl" typically includes heterocycloalkyl, aryl, substituted aryl, heteroaryl, nitro, cyano (also referred to herein as nitrile), azido, halo, -OR, -SR, -SF, -CHO, -COR, -C(O)OR, -C(O)-NR, -OC(O)R, -OC(O)NR 2、 -OC(O)OR 、 --P(O)(OR)2, -OP(O)(OR)2, -NR2, -N + R3 (counter ion may be present), -CONR2, -NRCOR, -NHC(O)OR, -NHC(O)NR2, -NHC(NH)NR 2、 SO3 -, -SO2OR, -OSO2R, -SO2NR2, or -NRSO2R, where each R is independently hydrogen, lower alkyl, R'-substituted lower alkyl, aryl, R'-substituted aryl, heteroaryl, heteroaryl(alkyl), R'-substituted aryl(alkyl), or aryl(alkyl), and where each R' is independently hydroxy, halo, alkyloxy, cyano, thio, SF5, nitro, alkyl, halo-alkyl, or amino. Of particular interest are substituted alkyls substituted with one to three of the following substituents: alkynyl, cyano, halo, alkyloxy, thio, nitro, amino, or hydroxy.

[0036] The term "aryl" refers to an aromatic ring having (4n+2) pi electrons, which may contain 6 to 20 ring carbon atoms and may be composed of a single ring (e.g., phenyl), or two or more fused rings, such as two to three fused rings (e.g., naphthyl), or two or more aromatic rings, such as two to three aromatic rings linked by a single bond (e.g., biphenylyl). In certain cases, aryl refers to an aromatic ring having 4n+2 pi electrons, such as C6 to C8. 16 or C6~C 14 In certain embodiments, an alkyl group has one or more hydrogen atoms replaced with deuterium.

[0037] Heteroaryl refers to an aromatic ring system containing (4n+2) pi electrons, consisting of 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, having a single ring (e.g., thiophene, pyridine, pyrazine, imidazole, oxazole, tetrazole, etc.) or two or more fused rings, for example, two to three fused rings (e.g., indole, benzimidazole, quinolone, quinoxaline, phenothiazine, etc.), or two or more aromatic rings, such as two to three aromatic rings linked by a single bond (e.g., bipyridyl). Optionally, heteroaryl refers to a C1-C 16 and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O.

[0038] The terms "heterocycloalkyl," "heterocycle," "heterocyclic group," or "heterocyclyl" refer to saturated or unsaturated non-aromatic ring systems containing 1 to 10 ring carbon atoms and 1 to 5 heteroatoms selected from O, N, S, and Se, having a single ring (e.g., tetrahydrofuran, aziridine, azetidine, pyrrolidine, piperidine, tetrathiopyran, hexamethylene oxide, oxazepane, etc.) or two or more fused rings, such as two to three fused rings (e.g., indoline, tetrahydrobenzodiazapine, etc.), including fused, bridged, and spiro ring systems having 3 to 15 ring atoms and containing 1 to 4 heteroatoms. In certain instances, heterocycloalkyl refers to C1-C 16 and a selection of 1 to 5 heteroatoms consisting of S, Se, N, and O. In fused ring systems, one or more of the rings can be cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide the N-oxide, -S(O)-, or -SO2- moieties.

[0039] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, benzimidazole, pyrazole, benzopyrazole, tetrazole, 1,2,3-triazole, benzotriazole, 1,2,4-triazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, benzoisothi ... Examples include azole, phenazine, isoxazole, benzisoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, benzothiazole, thiazolidine, furan, benzofuran, thiophene, benzothiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and benzotetrahydrofuranyl.

[0040] The substituted heterocycloalkyl, aryl, and heteroaryl are optionally selected from hydrogen, 1-3 alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, substituted aryl, aryl(alkyl), -SO2NR 5 R 5 , -PO3H2, -NR 5 SO2R 6 , or -NR 5 C(=O)R 6 wherein R 5 and R 6 are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl(alkyl), aryl, optionally substituted heterocycloalkyl, aryloxy, heteroaryl, heteroaryl(alkyl), or R 5 and R6 Both are -(CH2) 3-6 -or-(CH2) 0-3 X(CH2) 0-3 -(where X=NR, O, S, SO 2、 Substituted aryl(alkyl), halo(alkyl), SF5, NR 5 3 + , azide, cyano (also referred to herein as nitrile), -OR 5 , -SR 5 , -NR 5 R 6 , halogen, nitro, SCH3, OCF3, SO2CH3, SCF3, SO2CF3, CF3, -SO2OR 5 , -OSO2R 5 , CCl3, -C(=O)R 5 , -C(=O)OR 5 , -C(=O)NR 5 R 6 , -OC(=O)R 5 is.

[0041] "Substituted," as in "substituted alkyl," "substituted aryl," etc., means that at least one hydrogen atom bonded to a carbon (or other) atom in a hydrocarbyl, alkyl, aryl, or other moiety, as referred to in some of the above definitions, is replaced with one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to, functional groups and the hydrocarbon moieties C1-C24 alkyl (including C1-C18 alkyl, further including C1-C12 alkyl, further including C1-C6 alkyl), C2-C24 alkenyl (including C2-C18 alkenyl, further including C2-C12 alkenyl, further including C2-C6 alkenyl), C2-C24 alkynyl (including C2-C18 alkynyl, further including C2-C12 alkynyl, further including C2-C6 alkynyl), C5-C30 aryl (including C5-C20 aryl, further including C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, further including C6-C12 aralkyl). The hydrocarbyl moieties described above may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically listed. Unless otherwise indicated, any group described herein is intended to include substituted and / or heteroatom-containing moieties in addition to unsubstituted groups.

[0042] The term "water-soluble group" refers to a functional group that is sufficiently solvated in an aqueous environment and confers improved water solubility to the compound to which it is attached. Water-soluble groups of interest include, but are not limited to, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary, or quaternary amines and polyamines, sulfate groups, sulfonate groups, sulfinate groups, carboxylate groups, phosphate groups, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including polyethylene glycols (PEGs) and modified PEGs, and polyethers. In some cases, the water-soluble group may be a primary, secondary, tertiary, or quaternary amine, carboxylate, phosphonate, phosphate, sulfonate, sulfate, -N(H) 0-1 (CH2CH2OH) 1-2 , -NHCH2CH2N(CH3) 2-3, -NHCH2CH2SO3H, -NHCH2CH2PO3H2 and -NHCH2CH2CO2H, --(CH2CH2O) yy CH2CH2XR yy , --(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yy CH2CH2--, glycol, oligoethylene glycol, and polyethylene glycol, where yy is selected from 1 to 1000, and X is O, S, and NR ZZ Selected from R ZZ and R YY is independently selected from H and C1-3 alkyl.

[0043] The term "carboxyisostere" refers to standard pharmaceutical bioisosteric substituents of carboxylic acids, amides, and esters, including, but not limited to, acylcyanamides, tetrazoles, hydroxychromium, 3-hydroxy-1,2,4-triazoles, 1-hydroxypyrazoles, 2,4-dihydroxyimidazoles, 1-hydroxyimidazoles, 1-hydroxy1,2,3-triazoles, alkylsulfonylcarboxamides, hydroxyisoxazoles, 5-hydroxy1,2,4-oxadiazoles, thiazoles, 1,2,4-oxadiazoles, 1,2,4-oxadiazolones, oxazoles, triazoles, thiazoles, other hydroxamic acids, sulfonimides, acylsulfonamides, sulfonylureas, oxadiazolones, thiazolidinediones, oxadiazoles, thiadiazoles, isothiazoles, difluorophenols, tetramic acids, tetronic acids, squaric acids, hydroxyquinolin-ones, hydroxyquinolin-2-ones, boronic acids, and phosphonic acids.

[0044] As used herein, the term "PEG" refers to polyethylene glycol or modified polyethylene glycol. Modified polyethylene glycol polymers include methoxypolyethylene glycol and polymers that are unsubstituted or substituted at one end with an alkyl, substituted alkyl, or substituent group (e.g., as described herein).

[0045] The term "functional group" includes halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C20 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (wherein X is halo, -(C O)-X), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), mono-substituted C1-C24 alkylcarbamoyl (-(CO)-NH(C1-C24 alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-substituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2) , carbamide (-NH-(CO)-NH2), cyano (-C≡N), isocyano (-N≡C-), cyanato (-OC≡N), isocyanato (-O-N≡C-), isothiocyanato (-SC≡N), azido (-N=N≡N-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24 alkyl)-substituted amino, mono- and di-(C5-C20 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C5-C20 arylamido (-NH-(CO) -aryl), imino (wherein R=hydrogen, -CR=NH, C1-C24 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), alkylimino (wherein R=hydrogen, alkyl, aryl, alkaryl, etc., -CR=N(alkyl)), arylimino (wherein R=hydrogen, alkyl, aryl, alkaryl, etc., -CR=N(aryl)), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl,"Ci-C alkylsulfinyl" refers to chemical groups such as mono- and di-(Ci-C alkyl)-substituted phosphinos, mono- and di-(Ci-C aryl)-substituted phosphines, mono- and di-(Ci-C aryl)-substituted phosphines, and the like. In addition, the foregoing functional groups, if permitted by the particular group, may be further substituted with one or more additional functional groups, or one or more hydrocarbyl moieties, such as those specifically listed above.

[0046] When the term "substituted" appears before a list of possible substituents, it is intended that the term apply to every member of that group. For example, the phrase "substituted alkyl and aryl" shall be interpreted as "substituted alkyl and substituted aryl."

[0047] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can also mean that one or more hydrogen atoms of the particular group or radical are replaced, each independently of the other, with the same or different substituents as defined below.

[0048] In addition to the groups disclosed for each individual term herein, one or more hydrogens on a saturated carbon atom in a particular group or radical (any two hydrogens on a single carbon may be substituted, e.g., ═O, ═NR 70 , =N-OR 70 , =N2 or =S) is substituted with -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + , -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 are independently 70 or alternatively, two R 80’ and together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycloalkyl, optionally containing 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, where the N may have —H or C1-C3 alkyl substitution; + is a counterion with a net single positive charge. + are independently, e.g., K + , Na + , Li + Alkaline ions such as + N(R 60 )4, or ammonium ions such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5 (The "subscript 0.5" means that one of the counterions to such divalent alkaline earth ions may be the ionized form of a compound of the present invention and the other may be a typical counterion such as chloride, or a two-ionized compound disclosed herein may serve as the counterion to such divalent alkaline earth ions, or a doubly-ionized compound of the present invention may serve as the counterion to such divalent alkaline earth ions.) A specific example is -NR 80 R 80 is -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, N-morpholinyl, -N(H) 0-1 (CH2CH2OH) 1-2, -NHCH2CH2N(CH3) 2-3 , —NHCH2CH2SO3H, —NHCH2CH2PO3H2, and —NHCH2CH2CO2H.

[0049] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom in a "substituted" alkene, alkyne, aryl, and heteroaryl group is, unless otherwise specified, -R 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 , and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + provided that it is not.

[0050] In addition to the groups disclosed for each individual term herein, the substituents of the hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70, -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 and M + is as previously defined.

[0051] Salts include, but are not limited to: Na, K, Ca, Mg, ammonium, tetraalkylammonium, aryl and alkyl sulfonates, phosphates, carboxylates, sulfates, Cl, Br, and guanidinium.

[0052] Unless otherwise specified, a reference to an atom is meant to include isotopes of that atom. For example, a reference to H is 1 H, 2 H (i.e., D), and 3 H (i.e., T) and reference to C 12 C and all isotopes of carbon ( 13 C, etc.)

[0053] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0054] Unless otherwise indicated, the naming of substituents not explicitly defined herein is arrived at by naming the adjacent functionality following the terminal portion of the functionality, toward the point of attachment. For example, the substituent "heterocycloalkyl(alkyl)" refers to the group (heterocycloalkyl)-(alkyl)-.

[0055] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0056] In certain embodiments, substituents may contribute to the optical isomerism and / or stereoisomerism of the compound. Salts, solvates, hydrates, and prodrug forms of the compound are also of interest. Polymorphic, pseudopolymorphic, amorphous, and co-crystalline forms of the compound are also of interest. All such forms are encompassed by the present disclosure. Thus, the compounds described herein include their salts, solvates, hydrates, prodrugs, and isomeric forms, including pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and isomers thereof. In certain embodiments, the compounds may be metabolized to pharmaceutically active derivatives.

[0057] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are commercially available. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, and wetting agents, are commercially available. Any compound useful in the methods and compositions of the present invention can be provided as a pharmaceutically acceptable base addition salt. A "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0058] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecydimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum alcohols; These formulations may contain a protein such as globulin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, or other carbohydrate including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0059] The term "sample" with respect to a patient includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washing, or enrichment for certain cell populations, such as diseased cells. This definition also includes samples that have been enriched for specific types of molecules, e.g., nucleic acids, polypeptides, etc. The term "biological sample" encompasses clinical samples, including tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, etc. "Biological sample" includes samples obtained from diseased cells of a patient, e.g., samples containing polynucleotides and / or polypeptides obtained from diseased cells of a patient (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides), as well as samples containing diseased cells from a patient. Biological samples containing diseased cells from a patient can also contain non-diseased cells.

[0060] Innate immune response. The presence and localization of infectious microorganisms are detected in mammalian cells by pattern recognition receptors (PRRs). These receptors are ligand-specific sensors that can recognize both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to orchestrate early host defense against infection or injury. Exogenous nucleic acids are one class of PAMPs, and their molecular features, such as length, double- or single-stranded configuration, nucleoside modifications, and sequence motifs, play important roles in immune recognition.

[0061] DAMPs are endogenous host-derived danger signals released into the extracellular or intracellular space by damaged or dying cells or during cellular stress, promoting inflammation to clear tissue from debris for subsequent regeneration. The most studied DAMPs include mono- and polysaccharides (glycans), high-mobility group box 1 (HMGB1), nucleic acids, and secreted ATP.

[0062] There are several types of PRRs that sense nucleic acids in mammalian cells, located in different cellular compartments (on the plasma membrane, in endosomes, and in the cytoplasm), allowing the sensors to detect both PAMP and DAMP nucleic acids. The first type is the endosomal subfamily of TLRs (TLR3, 7, 8, and 9). Other types include cytoplasmic RNA-binding proteins such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and laboratory genetics and physiological protein 2 (LGP2). Another type of sensor recently described is the cytoplasmic DNA sensor, a DNA-dependent activator of IRF (DAI).

[0063] Each endosomal TLR can recognize a specific type of nucleic acid: dsRNA activates TLR3, non-self ssRNA induces TLR7 and TLR8, and CpG DNA induces TLR9. TLR3 is expressed in myeloid dendritic cells, thus connecting the innate and adaptive immune systems. Other endosomal TLRs are expressed in a wide variety of immune cells, including pDCs, macrophages, monocytes, and lymphocytes.

[0064] Cytoplasmic RNA-binding proteins or RIG-I-like receptors (RLRs) include three members: RIG-I, MDA5, and LGP2. RLRs belong to the SF2 helicase superfamily, which is mostly found in the cytoplasm, while a certain amount of RIG-I is located in the nucleus. RIG-I and MDA5 are signaling proteins, while LGP2 has a regulatory role. These distinct functions result from their structural differences. RIG-I and MDA5 share a similar structure, with a helicase domain and a carboxy-terminal domain (CTD) in the middle. Both of these domains can detect and bind RNA. Furthermore, they both contain a caspase activation and recruitment domain (CARD) that mediates signal transduction and leads to type I IFN gene expression. Although these proteins share structural similarities and conserved downstream signaling pathways, they are activated by different RNA species. RIG-I prefers to bind short dsRNA that is triphosphorylated at the 5' end. Furthermore, RIG-I can distinguish between 5'-diphosphate and 5'-triphosphate dsRNA. These energy differences between binding monophosphate, diphosphate, or triphosphate allow RIG-I to distinguish between endogenous and viral RNA. Conversely, MDA5 is activated by long dsRNA, as confirmed by its activation by poly(I:C), a synthetic mimic of long dsRNA.

[0065] Binding of ligands to TLRs stimulates specific intracellular downstream signaling cascades that initiate host defense responses, leading to the production of proinflammatory cytokines and type 1 interferons. TLR signaling depends on the nature of the stimulus, the activated TLR, and downstream adaptor molecules. Different types of signaling adaptor proteins can be recruited by the TIR domain, including myeloid differentiation primary response protein 88 (MyD88), which is essential for TLRs 2, 4, 5, 7, 8, and 9, leading to the production of proinflammatory cytokines. The TIR domain-containing adaptor protein (TRIF) induces IFN-β. TRIF acts independently of MyD88 in signaling following activation of TLR3 and TLR4, leading to the production of type 1 interferons. Released innate immune mediators (which may be proinflammatory) include, but are not limited to, TNFα, IL-1β, IFNα, IFNβ, IL-6, CCL5, CXCL8, CXCL10, IL-8, IL-3, VEGF, and PDGFAA.

[0066] RNA cloaking method RNA is cloaked to reduce elicitation of an innate immune response by (i) contacting the RNA with a cloaking reagent in aqueous solution and (ii) reacting the RNA with the reagent to produce modified RNA containing acylated 2'-OH ribose. The acylation may be spontaneously reversed when the RNA is inside a cell.

[0067] Cloaking reagents useful in the methods disclosed herein may have the following general structure:

[0068] [ka]

[0069] wherein R1 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl or heteroaryl, or a substituted or unsubstituted cycloalkyl. In some embodiments, R contains 1 to 10 carbons and, optionally, 1 to 4 heteroatoms, particularly N or O.

[0070] Z is selected from imidazole, 1,2,3-triazole, 1,2,4-triazole, azide, cyanide, anhydride, fluoride, NHS ester, etc. In some embodiments, Z is imidazole.

[0071] In other embodiments, the cloaking reagent has the following general structure:

[0072] [ka]

[0073] Z1 may be selected from imidazole and triazole, such as 1,2,3-triazole or 1,2,4-triazole.

[0074] Suitable R1 or R2 groups (collectively R groups) for Formula I or II include, for example:

[0075] [ka]

[0076] [ka]

[0077] [ka]

[0078] [ka]

[0079] [ka]

[0080] [ka]

[0081] [ka]

[0082] In some embodiments, a target region of RNA is cloaked, and a region of the RNA, e.g., one or more of the 5'-UTR, all or a portion of the open reading frame, and the 3'-UTR of an mRNA, ranging in length from about 18 nt to about 120 nt, is hybridized with a complementary DNA oligo. The non-hybridized region of the mRNA-DNA hybrid is then selectively modified with a cloaking reagent of Formula I or II. Subsequent removal of the complementary DNA oligo with DNase produces an mRNA with the selective 2' modification. In some embodiments, selective acylation is achieved on largely unfolded RNA, e.g., RNA in water without added cations, or at elevated temperatures, or with the addition of denaturants. In some embodiments, the modification primarily targets the unpaired region of an otherwise folded RNA.

[0083] Bioorthogonal methods are provided for the optional reversal of 2'-OH RNA acylation using water-soluble organocatalysts that are strong nucleophiles and weak bases, and are carried out in aqueous solution at a neutral pH, e.g., a pH of about 7 to about 8, including pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, etc. In some embodiments, the organocatalyst is tris(tris(hydroxymethyl)aminomethane). In some embodiments, the organocatalyst is DABCO (1,4-diazabicyclo[2.2.2]octane).

[0084] Buffers for reversing acylation include, but are not limited to, Tris (tris(hydroxymethyl)aminomethane), DABCO (1,4-diazabicyclo[2.2.2]octane), NaCN, etc. The buffer may be present at concentrations of about 1 mM, about 5 mM, about 10 mM, about 25 mM, about 50 mM, about 100 mM, and up to about 250 mM. The reaction is carried out at temperatures between room temperature and 37°C for a period of about 1 minute to about 24 hours, or about 30 minutes to about 12 hours.

[0085] Upon reversal of acylation, less than about 75%, less than about 50%, or less than about 25% of the RNA may contain acylated 2'-OH. The deacylated RNA is biologically active and can be used in reactions such as hybridization, translation, reverse transcription, Cas9-mediated gene editing, etc. In other embodiments, cloaking is not reversed, for example, if the acylation is outside of the coding region or if the acylation is in other regions of the sequence that do not substantially reduce translation.

[0086] It has also been shown that, for example, after cloaked mRNA is delivered to a cell for expression, there can be spontaneous reversal of acylation in the intracellular environment.

[0087] Therapeutic preparations Provided is a composition comprising the RNA cloaked by acylated 2'-OH ribose, and the RNA modification is carried out according to the method disclosed herein.In some embodiments, the composition is formulated with a pharmaceutically acceptable excipient.In some embodiments, the acylated RNA is formulated for delivery to mammalian cells, for example, as a vaccine, gene therapy, delivery of biologically active antisense oligonucleotides, delivery of sequences encoding therapeutic proteins, delivery of reprogramming factors, etc.

[0088] Therapeutic modified RNA can be mRNA, antisense mRNA, RNAi, synthetic RNA such as chemically synthesized or in vitro transcribed, or any other form of RNA. In some specific embodiments, the RNA is mRNA. The RNA population acylated by the methods disclosed herein can contain at least about 10% acylated 2'-OH, at least about 20% acylated 2'-OH, at least about 30% acylated 2'-OH, at least about 50% acylated 2'-OH, or more. When the modification is preferentially present in the poly(A) tail, the RNA 5'-UTR, open reading frame, and 3'-UTR of the mRNA can be substantially free of acylated 2'-OH, and the poly(A) tail can contain at least about 30% acylated 2'-OH, at least about 50% acylated 2'-OH, at least about 75% acylated 2'-OH, at least about 90% acylated 2'-OH, or more. In some embodiments, the R groups and levels of acylation are selected to provide a desired profile of innate immune response.

[0089] In some embodiments, the therapeutic formulation comprises RNA formulated with a carrier, the term "carrier" referring to a natural or synthetic organic or inorganic component with which the RNA is combined to facilitate administration. In some embodiments, the formulation comprises at least one RNA (e.g., mRNA) polynucleotide species having an open reading frame encoding an antigen. In some embodiments, the carrier is a lipid carrier such as a lipid nanoparticle (LNP), polymeric nanoparticle, lipidoid, liposome, lipoplex, peptide carrier, nanoparticle mimic, nanotube, or conjugate.

[0090] In some embodiments, the RNA formulation is an RNA vaccine formulation. When the formulation is a vaccine, the vaccine can be a cancer vaccine, a pathogen vaccine, or the like. For example, a cancer vaccine is a vaccine that includes a cancer antigen known to be found in general or in a specific type of cancer or tumor. Antigens within or expressed by tumor cells are referred to as "tumor-associated antigens." A specific tumor-associated antigen may or may not be expressed in non-cancer cells. Many tumor mutations are known in the art. A personalized cancer vaccine may include RNA encoding one or more known tumor-specific cancer antigens or cancer antigens specific to each subject, referred to as neoepitopes, patient-specific epitopes, or antigens. A "patient-specific cancer antigen" is an antigen identified to be expressed in a specific patient's tumor. Patient-specific cancer antigens may or may not typically be present in tumor samples. Tumor-associated antigens that are not expressed, are rarely expressed, or whose expression in non-cancerous cells is sufficiently reduced compared to that in cancerous cells to elicit an immune response upon vaccination are called neoepitopes.

[0091] The vaccine formulation may, for example, comprise mRNA encoding an antigen of interest. An mRNA vaccine may comprise one or more antigens. In some embodiments, an mRNA vaccine comprises three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more antigens. In one embodiment, the antigen is derived from a human pathogen. In another embodiment, the antigen is a tumor-associated antigen, e.g., a cancer neoantigen.

[0092] In some embodiments, the mRNA comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof in addition to an acylated 2'-OH ribose, in embodiments, the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4'-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, N6-methyladenine, and any combination thereof.

[0093] The present invention also encompasses infectious disease vaccines in which the mRNA encodes a viral or bacterial antigen. In some embodiments, the infectious agent is selected from the group consisting of coronavirus (e.g., SARS, CARS-CoV2, etc.), adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, E. The strain of virus is selected from the group consisting of hepatitis virus; human immunodeficiency virus (HIV); influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; measles virus; mumps virus; parainfluenza virus; respiratory syncytial virus; human metapneumovirus; Hendra virus; Nipah virus; rabies virus; hepatitis D; rotavirus; Orbi virus; Colti virus; Banna virus; human enterovirus; hantavirus; West Nile virus; Middle East respiratory syndrome coronavirus; Japanese encephalitis virus; swine vesicular ecchymos virus; and eastern equine encephalitis.

[0094] In other embodiments, the virus is an influenza A or influenza B strain, or a combination thereof. In some embodiments, the influenza A or influenza B strain is associated with an avian, swine, horse, dog, human, or non-human primate. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. In some embodiments, the hemagglutinin protein does not include the head domain (HA1). In some embodiments, the hemagglutinin protein comprises a portion of the head domain (HA1). In some embodiments, the virus is selected from the group consisting of H1N1, H3N2, H7N9, and H10N8.

[0095] In some embodiments, the infectious agent is a strain of bacteria selected from Mycobacterium tuberculosis, clindamycin-resistant Clostridium difficile, fluoroquinolone-resistant Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Enterococcus faecalis, multidrug-resistant Enterococcus faecium, multidrug-resistant Pseudomonas aeruginosa, multidrug-resistant Acinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus (VRSA). In some embodiments, the bacterium is Clostridium difficile.

[0096] In some embodiments, RNA, e.g., RNA vaccines, are formulated in lipid nanoparticles (LNPs). The use of LNPs enables effective delivery of RNA. In one embodiment, the lipid nanoparticles comprise an ionizable lipid (e.g., an ionizable cationic lipid), a structural lipid, a phospholipid, and a lipid, including an acylated RNA. Each of the LNPs described herein can be used as a formulation for the RNA described herein. In one embodiment, the lipid nanoparticles comprise an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNPs comprise an ionizable lipid, a PEG-modified lipid, a phospholipid, and a structural lipid. In some embodiments, the LNPs have a molar ratio of about 20-60% ionizable lipid, about 5-25% phospholipid, about 25-55% structural lipid, and about 0.5-15% PEG-modified lipid. In some embodiments, the LNPs comprise a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid, and about 10% phospholipid. In some embodiments, the LNPs comprise a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid, and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid, the phospholipid is a neutral lipid, and the structural lipid is cholesterol. In some embodiments, the LNPs have a molar ratio of ionizable lipid:cholesterol:DSPC:PEG2000-DMG of 50:38.5:10:1.5.

[0097] The ionizable lipids were 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). , 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), (13 Z,165Z)-N,N-Dimethyl-3-nonidocosa-13-16-dien-1-amine (L608), 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), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl The ionizable amino lipid may be selected from the non-limiting group consisting of methyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)-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(2S)). In addition, the ionizable amino lipid may also be a lipid containing a cyclic amine group.

[0098] The lipid composition of the pharmaceutical compositions disclosed herein can include one or more phospholipids, such as one or more saturated or (poly)unsaturated phospholipids, or a combination thereof. Generally, a phospholipid comprises a phospholipid moiety and one or more fatty acid moieties.

[0099] The phospholipid moiety can be selected from the non-limiting group consisting of, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0100] The fatty acid moiety can be selected from the non-limiting group consisting of, for example, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0101] Certain phospholipids can promote membrane fusion. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of a phospholipid to a membrane can allow one or more components (e.g., a therapeutic agent) of a lipid-containing composition (e.g., an LNP) to pass through the membrane, enabling, for example, delivery of the one or more components to a target tissue.

[0102] Non-natural phospholipid species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds) or crosslinked to one or more alkynes. Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to promote membrane penetration or cell recognition, or for conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0103] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidiglycerol, and phosphatidic acid. Phospholipids also include sphingophospholipids such as sphingomyelin. In certain embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC. In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, phospholipids with a modified head group are DSPC or analogs thereof with a modified quaternary amine. In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified tail. In certain embodiments, phospholipids useful or potentially useful in the present invention are DSPC or analogs thereof with a modified tail. As described herein, a "modified tail" can be a tail with a shorter or longer aliphatic chain, an aliphatic chain with introduced branching, an aliphatic chain with introduced substituents, an aliphatic chain in which one or more methylenes are replaced by a cyclic or heteroatom group, or any combination thereof. In certain embodiments, alternative lipids are used in place of the phospholipids of the present invention.

[0104] The LNPs disclosed herein can include one or more structured lipids. As used herein, the term "structured lipid" refers to a sterol and also to a lipid containing a sterol moiety. The incorporation of a structured lipid within a lipid nanoparticle can help reduce aggregation of other lipids within the particle. The structured lipid can be selected from the group including, but not limited to, cholesterol, festerol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol.

[0105] In one embodiment, the amount of structured lipid (e.g., sterol, such as cholesterol) in the lipid composition of the pharmaceutical composition disclosed herein ranges from about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 50 mol%, or about 35 mol% to about 45 mol%. In one embodiment, the amount of structured lipid (e.g., sterol, such as cholesterol) in the lipid composition disclosed herein ranges from about 25 mol% to about 30 mol%, about 30 mol% to about 35 mol%, or about 35 mol% to about 40 mol%. In one embodiment, the amount of structured lipid (e.g., sterol, such as cholesterol) in the lipid composition disclosed herein is about 24 mol%, about 29 mol%, about 34 mol%, or about 39 mol%. In some embodiments, the amount of structured lipid (e.g., a sterol such as cholesterol) in the lipid compositions disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%.

[0106] The lipid composition of the pharmaceutical composition disclosed herein can contain one or more polyethylene glycol (PEG) lipids. As used herein, the term "PEG-lipid" refers to a polyethylene glycol (PEG)-modified lipid. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0107] In some embodiments, the PEG lipid includes, but is not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA). In one embodiment, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the lipid moiety of the PEG-lipid comprises a lipid moiety having a length of about C14 to about C22, preferably about C14 to about C16. In some embodiments, the PEG moiety, e.g., mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons. In one embodiment, the PEG-lipid is PEG2k-DMG. In one embodiment, the lipid nanoparticles described herein can comprise a PEG-lipid that is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. PEG-lipids are described, for example, in U.S. Pat. No. 8,158,601 and WO 2015 / 130584 A2, which are incorporated herein by reference in their entireties.

[0108] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in WO 2012 / 099755, the contents of which are incorporated herein by reference in their entirety. Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxy-PEGylated lipid includes an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0109] In one embodiment, the amount of PEG lipid in the lipid composition of the pharmaceutical composition disclosed herein is from about 0.1 mol% to about 5 mol%, from about 0.5 mol% to about 5 mol%, from about 1 mol% to about 5 mol%, from about 1.5 mol% to about 5 mol%, from about 2 mol% to about 5 mol%, from about 0.1 mol% to about 4 mol%, from about 0.5 mol% to about 4 mol%, from about 1 mol% to about 4 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 4 mol%, from about 0.1 mol% to about 3 mol%, about 0.5 mol% to about 3 mol%, about 1 mol% to about 3 mol%, about 1.5 mol% to about 3 mol%, about 2 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 1.5 mol% to about 2 mol%, about 0.1 mol% to about 1.5 mol%, about 0.5 mol% to about 1.5 mol%, or about 1 mol% to about 1.5 mol%. In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0110] The lipid composition of the pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers (e.g., surfactants), or other components. For example, the permeability enhancer molecule can be a molecule described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0111] A polymer can be included and / or used to encapsulate or partially encapsulate the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions in lipid nanoparticle form). The polymer can be biodegradable and / or biocompatible. The polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0112] The ratio of lipid composition to RNA ranges from about 10:1 to about 60:1 (wt / wt). In some embodiments, the ratio of lipid composition to acylated RNA is about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1 , 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, or 60:1 (weight / weight). In some embodiments, the weight / weight ratio of lipid composition to polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1. In one embodiment, the lipid nanoparticles described herein can comprise polynucleotides at a concentration of approximately 0.1 mg / ml to 2 mg / ml, including, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, or greater than 2.0 mg / ml.

[0113] Nanoparticle compositions are typically submicrometer in size and can contain a lipid bilayer. Nanoparticle compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions can be liposomes with a lipid bilayer having a diameter of 500 nm or less.

[0114] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0115] In one embodiment, the RNA encoding the antigen polypeptide has a size of about 10 to about 100 nm, for example, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm The lipid nanoparticles may be formulated with lipid nanoparticles having a diameter of about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and / or about 90 to about 100 nm.

[0116] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle compositions disclosed herein can be about 0.10 to about 0.20.

[0117] In addition to LNPs, acylated RNAs can be formulated in other carriers such as liposomes, lipoids and liporex, particulate or polymeric nanoparticles, peptide carriers, nanoparticle mimics, nanotubes, conjugates, or emulsion delivery systems such as cationic submicron oil-in-water emulsions.

[0118] Liposomes are amphipathic lipids that can form bilayers in an aqueous environment to encapsulate an RNA-containing aqueous core. These lipids can have anionic, cationic, or amphoteric hydrophilic head groups. Liposomes can be formed from a single lipid or a mixture of lipids. The mixture can include (i) a mixture of anionic lipids, (ii) a mixture of cationic lipids, (iii) a mixture of zwitterionic lipids, (iv) a mixture of anionic and cationic lipids, (v) a mixture of anionic and zwitterionic lipids, (vi) a mixture of zwitterionic and cationic lipids, or (vii) a mixture of anionic, cationic, and zwitterionic lipids. Similarly, the mixture can include both saturated and unsaturated lipids. Exemplary phospholipids include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. Cationic lipids include, but are not limited to, dioleoyltrimethylammoniumpropane (DOTAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids are DPPC, DOPC, and dodecylphosphocholine. The lipids can be saturated or unsaturated.

[0119] Polymeric microparticles or nanoparticles can also be used to encapsulate or adsorb acylated RNA. The particles can be substantially non-toxic and biodegradable. Particles useful for delivering RNA can have an optimal size and zeta potential. For example, microparticles can have diameters ranging from 0.02 μm to 8 μm. When a composition has a population of microparticles or nanoparticles with different diameters, ideally, at least 80%, 85%, 90%, or 95% of the particles have diameters ranging from 0.03 to 7 μm. The particles can also have a zeta potential of 40 to 100 mV to provide maximum adsorption of RNA to the particles.

[0120] Non-toxic and biodegradable polymers include, but are not limited to, poly(hydroxy acids), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyolthioesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidinone, or polyesteramides, and combinations thereof. In some embodiments, particles are formed from poly(hydroxy acids), such as poly(lactide) ("PLA"), copolymers of lactide and glycolide, such as poly(D,L-lactide-co-glycolide) ("PLG"), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having a lactide / glycolide molar ratio ranging from 20:80 to 80:20, e.g., 25:75, 40:60, 45:55, 55:45, 60:40, or 75:25. Useful PLG polymers include, for example, those having a molecular weight between 5,000 and 200,000 Da, for example, 10,000 and 100,000, 20,000 and 70,000, or 40,000 and 50,000 Da.

[0121] Oil-in-water emulsion can also be used to deliver acylated RNA to a subject.Examples of oils useful for making emulsion include animal oils (e.g., fish oil) or vegetable oils (e.g., nuts, seeds and grains).Oils can be biodegradable (metabolizable) and biocompatible.Some exemplary oils include tocopherol and squalene, shark liver oil, which is a branched unsaturated terpenoid, and combinations thereof.Terpenoids are branched-chain oils that are biochemically synthesized from 5-carbon isoprene units.

[0122] The aqueous component of the emulsion can be water or water to which additional components have been added. For example, the aqueous component can include a salt to form a buffer, such as a citrate or phosphate salt, such as a sodium salt. Exemplary buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer.

[0123] The oil-in-water emulsion ideally contains one or more cationic molecules. For example, cationic lipids can be included in the emulsion to provide a positively charged droplet surface to which negatively charged mRNA can adhere. Useful cationic lipids include, but are not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA, e.g., bromide), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), dipalmitoyl (C16:0) trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP). Other useful cationic lipids are benzalkonium chloride (BAK), benzethonium chloride, cetramide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of dedecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide), cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyltrimethylammonium chloride, benzotrimethylammonium bromide ... Dimethylhexadecyl-ammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkylammonium chloride, methyltrioctylammonium chloride, N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemethanaminium chloride (DEBDA), dialkyldimethylammonium salt, [1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (acyl group = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (acyl group = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester, cholesteryl (4'-trimethylammonio)butanoate), N-alkylpyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts ammonium salts, dicationic boraform electrolytes (C12Me6; C12BU6), dialkylglycetylphosphorylcholine, lysolecithin, L-alpha dioleylphosphatidylethanolamine, cholesterol hemiscurate choline ester, lipopolyamines (including but not limited to dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES), lipopoly-L(or D)-lysine (LPLL, LPDL), poly(L(or D)-lysine) conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino groups (C GluPhCnN), ditetradecyl glutamate esters with pendant amino groups (C14GluCnN+), cationic derivatives of cholesterol (including, but not limited to, cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamidoethylene-dimethylamine, cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt, and cholesteryl-3β-carboxyamidoethylenedimethylamine).

[0124] In addition to the oil and cationic lipid, the emulsion can contain nonionic and / or zwitterionic surfactants, including, but not limited to, polyoxyethylene sorbitan ester surfactants, particularly polysorbate 20 and polysorbate 80, copolymers of ethylene oxide, propylene oxide, and / or butylene oxide, linear block copolymers, octoxynol, (octylphenoxy)polyethoxyethanol, phospholipids such as phosphatidylcholine, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols, polyoxyethylene-9-lauryl ether, and sorbitan esters.

[0125] The formulations may be provided in unit dosage form, the term "unit dosage form" referring to a physically discrete unit suitable as a unitary administration dose for a subject, each unit containing a predetermined amount of active agent in an amount calculated to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle sufficient to produce the desired effect. The specifications for the unit dosage forms of the present invention depend on the particular RNA and formulation used, the effect to be achieved, and the pharmacodynamics associated with each formulation in the host. In some embodiments, the unit dose is an effective amount to achieve the desired effect, e.g., expression of the protein encoded by the modified mRNA, e.g., about 1 mg to about 500 mg of RNA, e.g., about 1 mg to about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 250 mg, or about 500 mg. Alternatively, depending on the use and route of administration, the modified RNA may be present in a unit dose ranging from about 100 ng to 1 μg, 10 μg, 100 μg, 1 mg, up to about 10 mg, up to about 100 mg, up to about 1 g, up to about 10 g, up to about 100 g, etc. Dosage amounts are adjusted appropriately for the desired use.

[0126] The modified RNA and carrier can be formulated with a pharmaceutically acceptable excipient. Suitable carriers include sterile saline, although other aqueous and non-aqueous isotonic sterile solutions and suspensions known to be pharmaceutically acceptable are known to those skilled in the art.

[0127] Depending on the desired use, the formulation may contain a pharmaceutically acceptable non-toxic excipient or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers. In pharmaceutical dosage forms, the modified RNA may be provided in the form of a pharmaceutically acceptable salt.

[0128] The RNA can be used in combination with suitable additives, for example, conventional additives such as lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrating agents such as corn starch, potato locust starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if necessary, diluents, buffers, moisturizers, preservatives, and flavoring agents, to form tablets, powders, granules, or capsules.

[0129] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are commercially available. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, and wetting agents, are commercially available. Any compound useful in the methods and compositions of the present invention can be provided as a pharmaceutically acceptable base addition salt. A "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0130] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecydimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum alcohols; These formulations may contain a protein such as globulin, gelatin, or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, histidine, arginine, or lysine; a monosaccharide, a disaccharide, or other carbohydrate including glucose, mannose, or dextrin; a chelating agent such as EDTA; a sugar such as sucrose, mannitol, trehalose, or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0131] The compositions can be prepared as injectables, either as liquid solutions or suspensions, or as solid forms suitable for solution or suspension in liquid vehicles prior to injection. The preparations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactides, polyglycolides, or copolymers, for enhanced adjuvant effect, as described above. See Langer, Science 249:1527, 1990, and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of the present invention can be administered in the form of depot injections or implants, which can be formulated in a manner that allows sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all U.S. Food and Drug Administration Good Manufacturing Practice (GMP) regulations.

[0132] Toxicity of an active agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to further optimize and / or define therapeutic and / or subtherapeutic dosage ranges (e.g., for use in humans). The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0133] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for and / or treated for therapy. In some embodiments, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, an individual with a disease. A subject may be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, such as mice and rats. As used herein, the terms "treatment," "treating," and the like refer to administering an agent or performing a procedure to or in a subject, individual, or patient with the intent of achieving an effect in the subject, individual, or patient. Treating can refer to any indication of successful treatment, amelioration, or prevention of a disease, including any objective or subjective parameter, such as reduction, remission, or reduction of symptoms, or making the disease state more tolerable to the patient, slowing the rate of degeneration or decline, or making the end point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physician's examination.

[0134] kit A kit may be provided. The kit may include reagents suitable for modifying RNA, such as reagents for modifying the 2'-OH group of RNA with the cloaking reagents disclosed herein, such as acylimidazole, sulfonylimidazole, sulfonyltriazole, etc. The components may be packaged separately in two or more containers suitable for use in the methods disclosed herein. The kit may also include reagents for packaging RNA for in vivo delivery. The kit may also include tubes, buffers, etc., and instructions for use.

[0135] experiment The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0136] Example 1 Methods and modifications for suppressing the innate immune response to RNA RNA is emerging as a novel modality for therapy and vaccination. We have shown that 2'-OH acylation ("cloaking") of RNA with the reagents disclosed herein can block RNA interactions with cellular proteins and nucleic acids. Although little structural information is available on these receptor-nucleotide complexes, we hypothesize that RNA 2' modification with acylation reagents may inhibit recognition by RNA receptors and suppress innate immune responses. Furthermore, we hypothesize that variation in the chemical structure of acylation reagents may fine-tune RNA immunogenicity through differential modulation of innate immune response pathways.

[0137] Because high levels of 2' modifications (e.g., 2'-F, 2'-OMe) cannot be efficiently incorporated into RNAs longer than approximately 150 nt in synthetically accessible yields, there have been no reports yet demonstrating how 2'-OH acylation affects the immunogenicity of long RNAs (>600 nt). To test this, we first prepared a 2'-polyacylated ("cloaked") model mRNA by reacting the widely used eGFP-mRNA (996 nt) with N,N-dimethylglycine acylimidazole (DMG-Im), an acylimidazole reagent that selectively acylates 2'-hydroxyl groups in high yield (Figure 1). This resulted in approximately 50% cloaking of the unpaired, accessible regions of eGFP-mRNA. We focused on 2'-acylation with acylimidazoles due to their structural simplicity, ease of preparation, high solubility in aqueous solution, and high-yield acylation of 2'-hydroxyl groups. In addition, acylimidazoles can selectively react with 2'-hydroxyls rather than nucleobases, which can be reversed by design, providing an option for restoring unmodified 2'-hydroxyls and RNA functionality.

[0138] As an initial assessment of how mRNA cloaking affects innate immune responses, we measured the release of cytokines and immune activation markers from HEK293 cells lipofected with eGFP-mRNA, with or without cloaking. HEK293 cells were transfected with 2 μg of mRNA using Lipofectamine MessengerMax. Six hours after transfection, analysis of released immune molecules using the Luminex Human 48-plex kit demonstrated a strong cloaking-dependent reduction in the proinflammatory immune response (Figure 2). For example, transfection with unmodified eGFP-mRNA resulted in substantially enhanced release of proinflammatory chemokines and cytokines, including VEGF, RANTES / CCL5, PDGFAA, and IL-8. In comparison, HEK293 cells transfected with intermediate cloaking of eGFP-mRNA (approximately 50% unpaired 2'-hydroxyls) significantly reduced the release and activation of the above proinflammatory cytokines by up to approximately 90%. Because eGFP-mRNA translation was not significant at this time point (6 h posttransfection), the release of these immunomodulatory cytokines / chemokines was primarily due to cells sensing the mRNA. In addition, the expression of many mRNA-stimulated cytokines was not significantly modulated by cloaking, suggesting that cloaking may selectively regulate specific innate immune response pathways.

[0139] Transfection of in vitro transcribed RNA can induce a potent type I innate immune response in HEK293 cells (PMID: 30011268). Next, we investigated whether cloaking could regulate such responses in HEK293 cells. To do this, HEK293 cells were lipofected with eGFP-mRNA with or without cloaking by DMG-Im. We measured the mRNA expression level of ISG15, a downstream activation marker of type I innate immune responses via the RIG-I pathway. RT-qPCR showed that unmodified eGFP-mRNA moderately enhanced ISG15 expression, whereas cloaking completely suppressed this activation (Figure 3).

[0140] We further evaluated the effect of 2'-acylation on long-term immune responses using a Luminex human 48-plex assay in HEK293 cells transfected with modified eGFP-mRNA (Figures 3 and 4). We found that the release of proinflammatory chemokine ligand 5 (CCL5) and the cytokine granulocyte-macrophage colony-stimulating factor (GMCSF) were increasingly suppressed over time. In contrast, the secretion of the proinflammatory cytokine vascular endothelial growth factor (VEGF) was increasingly suppressed over time, suggesting that cloaking may selectively regulate certain innate immune response pathways in time and space.

[0141] Transfection of in vitro transcribed RNA can induce a potent type I innate immune response in HEK293 cells (PMID: 30011268). Next, we investigated whether cloaking could regulate such responses in HEK293 cells. To do this, HEK293 cells were lipofected with eGFP-mRNA with or without cloaking by DMG-Im. We measured the mRNA expression level of ISG15, a downstream activation marker of type I innate immune responses via the RIG-I pathway. RT-qPCR showed that unmodified eGFP-mRNA moderately enhanced ISG15 expression, whereas cloaking almost completely suppressed this activation (Figure 5).

[0142] Recent studies have shown that 2'-acylation with some acyl groups can terminate translation when introduced into the coding region of mRNA. Therefore, we investigated whether N,N-dimethylglycine acylimidazole adducts can be spontaneously released in cells. To investigate this, we used cloaked mRNA encoding green fluorescent protein (GFP) (Figure 6). Our data showed that translation of GFP-mRNA bearing N,N-dimethylglycine adducts can be spontaneously restored in normal cells (e.g., HEK293) and cancer cells (e.g., HeLa, SW480). Furthermore, cloaking with DMG-Im extended the translational lifetime of mRNA encoding destabilized green fluorescent protein d2GFP by 31% in HeLa cells.

[0143] After confirming that the modified mRNA was translatable, we investigated whether varying the chemical structure of the acylation reagent could fine-tune RNA immunogenicity by differentially modulating innate immune response pathways. To this end, we assembled a panel of 28 acylation reagents containing structurally diverse substituents (Figure 7). These reagents can be easily prepared by one-step activation of the corresponding low-cost carboxylic acids with 1,1'-carbonyldiimidazole (CDI). Care was taken to install acyl groups with various electrophilicities and sizes, which could subsequently affect RNA interactions with cellular receptors / RNA sensors. Desired characteristics of acyl adducts include sufficient chemical stability during RNA delivery while being sufficiently unstable to efficiently restore mRNA translation. These structural features include aromaticity in reagents 1–3. Additionally, we installed alpha-carbon heteroatoms (N or O) at the carboxyl centers of reagents 4–28, which possess various steric bulk and charge states that could potentially modulate RNA interactions with cellular receptors and RNA sensors.

[0144] We screened these acylimidazole reagents for spontaneous restoration of mRNA translation in HEK293 cells by transfection with d2GFP-mRNA aliquots acylated with these acylimidazole reagents at various levels of 2'-modification. Green fluorescent signal in HEK293 cells was monitored over a 3-day period. Representative translation kinetics data are shown in Figure 8 for d2GFP-mRNA cloaked with 1-28, demonstrating that a group of acylimidazole reagents enabled spontaneous RNA uncloaking and restored mRNA translation in human cells (Figure 8). The chemical structure of the acylimidazole reagent profoundly influences spontaneous RNA uncloaking in human cells, as the shape and electrophilicity of the acyl group appear to regulate translation efficiency, translation duration, and total protein output. Thus, we demonstrated that acylation reagents can be chemically tuned to alter their properties. The data also indicate that acyl groups added to RNA to reduce unwanted immune activation do not necessarily reduce translation but can potentially enhance it.

[0145] Next, we investigated whether the chemical structure of the acylation reagent could differentially regulate certain innate immune response pathways. Among the reagents that enabled spontaneous restoration of mRNA translation, we further evaluated how four structurally diverse reagents, R1, R4, R11, and R19, affected the innate immune response (Figure 9). Because the maximum level of cloaking varied among these reagents, we proceeded with equimolar cloaking at approximately 50% of the unpaired, accessible 2'-hydroxyls of eGFP-mRNA with similar levels of modification. We measured the release of cytokines and immune activation markers from HEK293 cells lipofected with modified eGFP-mRNA, with or without cloaking. HEK293 cells were transfected with 2 μg mRNA aliquots using Lipofectamine MessengerMax. We surprisingly observed that the acylation reagents demonstrated a chemical structure-dependent reduction in the proinflammatory immune response (Figure 9). For example, acylation reagents with aromatic acyl groups (R1) most significantly reduced the release of the proinflammatory cytokines IL8, CCL5, and VEGF. In contrast, acylation reagents with alpha-alkoxy substituents (R4) only significantly suppressed the release of CCL5, but not IL8 and VEGF, strongly suggesting structure-dependent regulation of certain innate immune response pathways (Figure 10). Structural features such as a positive charge at the beta atom to the carboxyl group and additional steric bulk at the alpha carbon did not appear to significantly affect the immunogenicity of the underlying eGFP-mRNA. Thus, acylation reagents with diverse chemical structures can differentially regulate certain innate immune response pathways.

[0146] The foregoing merely illustrates the principles of the present invention. Those skilled in the art will recognize that, although not explicitly described or shown herein, they can devise various arrangements which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and concepts contributed by the inventors to further the art, and should be construed without limitation to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, regardless of structure, i.e., any elements developed to perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

[0147] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 443,651, filed February 6, 2023, the contents of which are incorporated herein by reference in their entirety.

[0148] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under contract GM145357 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

Claims

1. 1. A method for reducing an innate immune response by a cell in response to the introduction of exogenous RNA, comprising: A method comprising cloaking an RNA by acylating at least a portion of the riboses in the RNA at the 2'OH position with a 2' RNA cloaking reagent.

2. The cloaking reagent has the structure: 【Chemistry 1】 In the formula, R １ is a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl or heteroaryl, a substituted or unsubstituted cycloalkyl, 2. The method of claim 1, wherein Z is selected from imidazole, 1,2,3-triazole, 1,2,4-triazole, azide, cyanide, anhydride, fluoride, and NHS ester.

3. 3. The method of claim 2, wherein Z is imidazole.

4. R １ is selected from the following: 【Chemistry 2】 wherein X is Cl or Br, and CH ３ The method according to claim 2 or 3, wherein

5. R1 is, 【Transformation 3】 The method according to any one of claims 2 to 4, wherein the compound is selected from the group consisting of:

6. The cloaking reagent has the structure: 【Chemistry 4】 In the formula, R ２ is a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl or heteroaryl, a substituted or unsubstituted cycloalkyl, Z １ 2. The method of claim 1, wherein is imidazole, 1,2,3-triazole, or 1,2,4-triazole.

7. R ２ is selected from the following: 【Transformation 5】 wherein X is Cl or Br, and CH ３ The method of claim 6, wherein

8. The method according to any one of claims 1 to 7, wherein the RNA is mRNA.

9. The method of claim 8 , wherein the mRNA encodes a protein antigen.

10. The method of claim 8 , wherein the antigen is a pathogen antigen or a tumor-associated antigen.

11. 9. The method of claim 8, wherein the mRNA encodes a therapeutic protein.

12. 12. The method of any one of claims 1 to 11, wherein the RNA further comprises a non-natural modified nucleobase.

13. The method of any one of claims 1 to 12, wherein after cloaking the RNA, the RNA comprises at least 10% acylated 2'-OH.

14. 14. The method of any one of claims 1 to 13, wherein the RNA is formulated with a carrier for in vivo delivery to a cell.

15. 15. The method of claim 14, wherein the carrier is a lipid carrier such as a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipidoid, a liposome, a lipoplex, a peptide carrier, a nanoparticle mimic, a nanotube, or a conjugate.

16. The method of any one of claims 1 to 15, wherein the RNA is formulated in a unit dosage form.

17. 1. An RNA composition formulated for delivery to a cell, comprising: An RNA composition comprising RNA acylated at least in part at the 2'OH position of the ribose, wherein the acylated RNA reduces the innate immune response by cells in response to introduction of the RNA compared to non-acylated RNA.

18. The acylation adduct is selected from: 【Transformation 6】 wherein X is Cl or Br, and CH ３ 18. The RNA composition of claim 17, wherein:

19. R1 is, 【Transformation 7】 19. The RNA composition of claim 17 or 18, wherein the RNA composition is selected from the following:

20. The composition of any one of claims 17 to 19, wherein the RNA is mRNA.

21. The composition of any one of claims 17 to 20, wherein the mRNA encodes a protein antigen.

22. 22. The composition of claim 21, wherein the antigen is a pathogen antigen or a tumor-associated antigen.

23. 22. The composition of claim 21, wherein the mRNA encodes a therapeutic protein.

24. 24. The composition of any one of claims 17 to 23, wherein the RNA further comprises a non-natural modified nucleobase.

25. The composition of any one of claims 17 to 24, comprising at least 10% acylated 2'-OH after cloaking the RNA.

26. 26. The composition of any one of claims 17 to 25, wherein the RNA is formulated with a carrier for in vivo delivery to a cell.

27. 27. The composition of claim 26, wherein the carrier is a lipid carrier such as a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipidoid, a liposome, a lipoplex, a peptide carrier, a nanoparticle mimic, a nanotube, or a conjugate.

28. 28. The composition of any one of claims 17 to 27, wherein the RNA is formulated in a unit dosage form.

29. 1. A method for tuning a cellular innate immune response to an exogenous RNA composition, comprising: cloaking the RNA by 2'-hydroxyl acylation with one or more, typically two or more, three or more, four or more different 2'-OH cloaking reagents; contacting a cell with the acylated RNA; and measuring the release of an innate immune mediator by said cells in response to said acylated RNA.

30. The acylation adduct is selected from: 【Transformation 8】 wherein X is Cl or Br, and CH ３ 30. The method of claim 29, wherein:

31. The acylated adduct is 【Chemistry 9】 31. The method of claim 29 or 30, wherein the

32. 32. The method of any one of claims 29 to 31, wherein the cell is of a cell type in which expression of the RNA is targeted.

33. 33. The method of any one of claims 29 to 32, wherein the innate immune mediators comprise one or more of TNFα, IL-1b, IFNα, IFNβ, IL-6, CCL5, CXCL8, CXCL10, IL-8, IL-3, VEGF, and PDGFAA.

34. 34. The method of any one of claims 29 to 33, wherein the acylated adduct is selected for use in cloaking based on the pattern of innate immune mediator release.

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