Covalent polymer-nucleic acid conjugates for modulation of the RIG-i pathway

Abstract

Disclosed herein are conjugates that can effectively deliver nucleic acid agonists to biological environments. An example conjugate includes a block copolymer including a first block that is hydrophilic, a second block that is hydrophobic, and a linking unit that covalently attaches a nucleic acid agonist to the block copolymer. Also disclosed are particles including self-assembled conjugates, methods of treating diseases, and methods of modulating RIG-I activity in a cell.

Description

Attorney Docket No.093386-0030-WO01 COVALENT POLYMER-NUCLEIC ACID CONJUGATES FOR MODULATION OF THE RIG-I PATHWAY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 579,876 filed on August 31, 2023, which is incorporated fully herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Federal Grant no. EB033822 awarded by the National Institutes of Health and Federal Grant no. W81XWH-20-1-0624 awarded by the Department of Defense. The Federal Government has certain rights to this invention. TECHNICAL FIELD

[0003] This disclosure relates to polymeric carriers that can effectively deliver nucleic acid RIG I agonists for biomedical applications, such as drug delivery. INTRODUCTION

[0004] Engineering of polymeric nanocarriers holds potential for overcoming critical delivery barriers limiting the development of RNA therapeutics, such as 3pRNA therapeutics, and their translation into the clinic. However, there has been relatively little investigation into the development of polymeric delivery systems for 2pRNA and / or 3pRNA, an oligonucleotide therapeutic whose distinctive structure and properties impose unique carrier design requirements. SUMMARY

[0005] In one aspect, disclosed are conjugates, or a pharmaceutically acceptable salt thereof, comprising a block copolymer comprising a first block (A), the first block comprising a recurring hydrophilic unit, and a second block (B), the second block comprising a copolymer, wherein the copolymer comprises a recurring hydrophobic unit and a recurring protonatable unit, wherein the first block, the second block, or both include at least one nucleic acid linking unit; and a nucleic acid retinoic acid-inducible gene I (RIG-I) agonist covalently attached to the nucleic acid linking unit through a linker.Attorney Docket No.093386-0030-WO01

[0006] In another aspect, disclosed are compositions comprising a plurality of conjugates as disclosed herein, or a pharmaceutically acceptable salt thereof, self-assembled into a particle, the particle comprising a corona and a core, wherein the corona comprises the first block and the core comprises the second block.

[0007] In another aspect, disclosed are methods of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition as disclosed herein.

[0008] In another aspect, disclosed are methods of modulating RIG-I activity in a cell, the method comprising administering to the cell an effective amount of a composition as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0010] FIG.1 is a schematic showing design concepts for example polymer nanocarriers of 3pRNA. a) Polymeric nanoparticles (NPs) are capable of loading 3pRNA and promoting endosomal escape to facilitate delivery to the cytosol, resulting in activation of RIG-I signaling. b-d) Illustrations depicting the loading strategies employed by b) polymer 1 (electrostatic complexation to core of NP), c) polymer 2 (conjugation of 3pRNA to NP corona), and d) polymer 3 (conjugation of 3pRNA to the core-forming block of the NP). e) Cutout of two covalent attachment chemistries. f) Illustration highlighting two opposing orientations of polymer-3pRNA conjugates (distal and proximal).

[0011] FIG.2A shows structures of example polymers: a) polymer 1, b) polymer 2, and c) polymer 3.

[0012] FIG.2B is a graph showing diameters of example polymer nanoparticles as measured by dynamic light scattering (DLS) at indicated pH.

[0013] FIG.2C is a graph showing polydispersity indexes (PDIs) of the particle diameter measurements at pH 7.4 in FIG.2B.

[0014] FIG.2D is a graph showing representative histograms of the DLS measurements at pH 7.4 illustrated in FIG.2B and FIG.2C; pol: polymer.Attorney Docket No.093386-0030-WO01

[0015] FIG.2E is a graph showing viability of A549 cells treated with example polymers. Dotted vertical lines depict calculated half-maximal inhibitory concentrations. Data representative of three independent experiments.

[0016] FIG.2F is a schematic depicting the mechanism of the Gal8-YFP recruitment assay for measuring endosomal escape.

[0017] FIG.2G is a graph showing enumeration of fluorescent puncta per cell of Gal8-YFP engineered MDA-MB-231 cells treated with example polymers. Data representative of two independent experiments.

[0018] FIG.2H is a graph showing 22.2 μg mL-1data near the IC50of polymer 2. Comparisons for FIG.2 by ordinary one-way ANOVA with Tukey’s multiple comparisons test. All data shown are mean + / - SD. * P < 0.05. *** P < 0.001. **** P < 0.0001.

[0019] FIG.3A is a schematic depicting 3pRNA loading strategies for polymers 1-3, with polymer 1 loading 3pRNA electrostatically and polymers 2 and 3 loading 3pRNA covalently to the corona- and core-forming blocks, respectively.

[0020] FIG.3B is a representative image of electrophoretic mobility shift assay of indicated polymer + 3pRNA formulations (0.50 ^g 3pRNA per lane) with and without inclusion of 12.5 units of heparin and / or 10 mM TCEP as shown; pol: polymer. TCEP: tris(2- carboxyethyl)phosphine.

[0021] FIG.3C is a graph showing percentage of 3pRNA complexed with polymer 1 as a function of polymer to RNA (pol:RNA) ratio as measured by fluorometric RiboGreen exclusion assay.

[0022] FIG.3D is a graph showing Percentage of 3pRNA conjugated to polymers 2 and 3 at depicted polymer:3pRNA molar ratios as measured by densitometry of heparin+TCEP- bands in Fiji.

[0023] FIG.3E is a graph showing diameter of 3pRNA / polymer NPs formulated with example polymers as measured by dynamic light scattering at depicted polymer:3pRNA molar ratios. Comparisons made by two-way ANOVA with Dunnett’s multiple comparisons test. Comparisons are made between each ratio and pol:3pRNA = 2.5 as a control with each polymer constituting one family. Asterisks (*) indicate comparisons between pol:3pRNA ratios of polymer 2. Hashes (#) indicate comparisons between pol:3pRNA ratios of polymer 3. *,#P < 0.05. *** P < 0.001.

[0024] FIG.3F is a graph showing polydispersity indices (PDIs) of the measurements in FIG. 3E. Comparisons made by two-way ANOVA with Dunnett’s multiple comparisons test. Comparisons are made between each ratio and empty NPs (data shown in FIG.2E) with eachAttorney Docket No.093386-0030-WO01 polymer constituting one family. Asterisks (*) indicate comparisons of polymer 2. * P < 0.05. ** P < 0.01. *** P < 0.001. All data shown are mean + / -SD.

[0025] FIG.4A is a graph showing a dose-response curve for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when complexed to polymer 1. n = 4 for each point. Data shown are representative of three independent experiments. IRF: Interferon Regulatory Factor.

[0026] FIG.4B is a graph showing dose-response curve for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when conjugated to polymer 2. n = 4 for each point. Data shown are representative of three independent experiments. IRF: Interferon Regulatory Factor.

[0027] FIG.4C is a graph showing a dose-response curve for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when conjugated to polymer 3. n = 4 for each point. Data shown are representative of three independent experiments. IRF: Interferon Regulatory Factor.

[0028] FIG.4D is a graph showing calculated half-maximal effective concentrations (EC50) of each formulation shown in FIG.4A-4C as a function of pol:3pRNA ratio. Comparisons made by 2-way ANOVA with Dunnett’s multiple comparisons test. Polymer 1 served as the control and each dose constitutes one family. n = 3 for all points. Asterisks (*) indicate comparisons between polymer 1 and polymer 2. Hashes (#) indicate comparisons between polymer 1 and polymer 3. *,#P < 0.05. ** P < 0.01. ****,####P < 0.0001.

[0029] FIG.4E is a graph showing enumerated fluorescent puncta per cell of Gal8-YFP engineered MDA-231 cells treated with indicated polymer / 5’-AlexaFluor-647 (AF647) RNA formulations at pol:RNA = 10. *** P = 0.0008 and **** P < 0.0001. All comparisons shown are between polymers 2 and 3. n = 12 for all points.

[0030] FIG.4F is a graph showing percentage of cells positive for AF647 in the experiment shown in FIG.4E. Asterisks (*) indicate comparisons between polymers 1 and 3. Hashes (#) indicate comparisons between polymers 1 and 2.##P = 0.008 and ****,####P < 0.0001.

[0031] FIG.4G is a set of representative merged images from the experiment shown in FIG. 4E and 4F at an RNA dose of 3.70 nM (approx.2 ^g mL-1polymer). Hoechst: nuclear stain. YFP: yellow fluorescent protein. AF647: AlexaFluor 647.

[0032] FIG.4H is a graph showing flow cytometric analysis of median fluorescence intensity (MFI) of cells treated with 5’-Cy5 RNA formulated with example polymer (pol:RNA=10) after incubation for indicated duration.

[0033] FIG.4I is a graph showing percentage of cells positive for Cy5 (% Cy5+) in FIG.4H.Attorney Docket No.093386-0030-WO01

[0034] FIG.4J is a graph showing MFI of the Cy5+subset of cells as shown in FIG.4I. For FIG.4, addition signs (+) indicate comparisons between polymer 1 and polymer 2, asterisks (*) indicate comparisons between polymer 1 and polymer 3, and hashes (#) indicate comparisons between polymer 2 and polymer 3.+,#, * P < 0.05. **,##P < 0.01. ***,###P < 0.001. ****,####P < 0.0001. Comparisons shown are by 2-way ANOVA with Tukey’s multiple comparisons test. All data shown are mean + / - SD.

[0035] FIG.5A is representative image of an electrophoretic mobility shift assay of indicated 3pRNA formulations following RNase A challenge.

[0036] FIG.5B is a graph showing densitometric quantification of remaining RNA bands in FIG.5A as shown for example polymer / RNA formulations with polymer:3pRNA ratio of 10. n = 3 independent formulations each. Comparisons by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

[0037] FIG.5C is a graph showing dose-response curves for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when complexed to polymer 1. n = 4 for each point. Data shown are representative of two independent experiments. IRF: Interferon Regulatory Factor. All data shown are mean + / - SD.

[0038] FIG.5D is a graph showing dose-response curves for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when conjugated to polymer 2. n = 4 for each point. Data shown are representative of two independent experiments. IRF: Interferon Regulatory Factor. All data shown are mean + / - SD.

[0039] FIG.5E is a graph showing dose-response curves for induction of IRF signaling in A549-Dual reporter cells at indicated 3pRNA doses when conjugated to polymer 3. n = 4 for each point. Data shown are representative of two independent experiments. IRF: Interferon Regulatory Factor. All data shown are mean + / - SD.

[0040] FIG.6A shows structures of polymers 3 and 4. a) Polymer 3 includes PDS groups that undergo thiol exchange reactions to form a cleavable disulfide bond with thiol-modified RNA while, b) polymer 4 includes maleimide groups that react with thiol-modified RNA to form a stable thioether bond.

[0041] FIG.6B is a representative image of electrophoretic mobility shift assay demonstrating conjugation of 3pRNA to polymer 4.

[0042] FIG.6C is a graph showing dose-response curves for induction of IRF response following treatment of A549-Dual reporter cells at indicated 3pRNA doses with indicated polymers of distal conjugates. n = 4 for each point. Data are representative of three independent experiments.Attorney Docket No.093386-0030-WO01

[0043] FIG.6D is a graph showing dose-response curves for induction of IRF response following treatment of A549-Dual reporter cells at indicated 3pRNA doses with indicated polymers of proximal conjugates. n = 4 for each point. Data are representative of three independent experiments.

[0044] FIG.6E is a graph showing dose-response curves following treatment of A549-Dual reporter cells at indicated 3pRNA doses loaded into polymer 3 with indicated oligonucleotide orientation. n = 4 for each data point. All data shown are mean + / - SD.

[0045] FIG.7A is a graph showing plasma concentrations of cytokines IFN-Į and IFN-ȕ 4 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1body mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD.

[0046] FIG.7B is a graph showing plasma concentrations of cytokines IFN-Ȗ and CXCL104 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1body mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD.

[0047] FIG.7C is a graph showing plasma concentrations of cytokines CCL2 and CCL54 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1body mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD.

[0048] FIG.7D is a graph showing plasma concentrations of cytokines TNF-Į and IL-64 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1body mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD.

[0049] FIG.7E is a graph showing plasma concentrations of cytokines CXCL1 and IL-1ȕ 4 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1body mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD.

[0050] FIG.7F is a graph showing plasma concentrations of cytokines IL-12p70 and IL-104 h after intravenous administration of polymer / 3pRNA nanoparticles at 1.2 mg 3pRNA kg-1bodyAttorney Docket No.093386-0030-WO01 mass formulated with indicated polymer. Comparisons made by one-way ANOVA with Tukey’s multiple comparisons test. n = 6 (vehicle), n = 5 (polymer 1), n = 7 (polymer 3), n = 8 (polymer 4). * P < 0.05. ** P < 0.01. *** P < 0.001. **** P < 0.0001. All data shown are mean + / - SD. DETAILED DESCRIPTION 1. Definitions

[0051] 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. In case of conflict, the present document, including definitions, will control. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in practice or testing of the disclosed technology. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0052] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0053] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0054] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, theAttorney Docket No.093386-0030-WO01 number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are contemplated, and for the range 1.5-2, the numbers 1.5, 1.6, 1.7, 1.8, 1.9, and 2 are contemplated.

[0055] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0056] The term “alkyl,” as used herein, refers to a straight or branched, saturated hydrocarbon chain containing from 1 to 30 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and n-dodecyl. The alkyl group may be optionally substituted with one or more substituents.

[0057] The term “alkenyl” as used herein, means a straight or branched hydrocarbon chain containing from 2 to 30 carbon atoms with at least one carbon-carbon double bond. The alkenyl group may be optionally substituted with one or more substituents.

[0058] The term “alkynyl,” as used herein, refers to straight or branched hydrocarbon chain containing from 2 to 30 carbon atoms with at least one carbon-carbon triple bond. The alkynyl group may be optionally substituted with one or more substituents.

[0059] The term “alkylene,” as used herein, refers to a divalent alkyl group, examples of which include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and - CH2CH2CH2CH2CH2-. An alkylene group may be optionally substituted with one or more substituents.

[0060] The term “alkenylene,” as used herein, refers to a divalent alkenyl group, examples of which include, but are not limited to -CH=CH-, -CH=CH-CH2-, -CH=CH-CH2-CH2- and -CH2- CH=CH-CH2-. An alkenylene group may be optionally substituted with one or more substituents.Attorney Docket No.093386-0030-WO01

[0061] The term “alkynylene,” refers to a divalent alkynyl group, examples of which include, but are not limited to -CŁC-, -CŁC-CH2-, -CŁC-CH2-CH2- and -CH2-CŁC-CH2-. An alkynylene group may be optionally substituted with one or more substituents.

[0062] The term “aryl,” as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e.g., the aryl may be benzo[d][1,3]dioxol-5-yl). The term “phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6- membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).

[0063] The term “alkyl acrylate,” as used herein, refers to , where alkyl is defined herein. Representative examples of alkyl acrylates include, but are not limited to, methyl arylate, butyl acrylate, N-butyl acrylate, ethyl acrylate, tert-butyl acrylate, and 2-octyl acrylate.

[0064] The term “alkyl methacrylate,” as used herein refers to, where alkyl is defined herein. Representative examples of alkyl methacrylates include, but are not limited to, methyl methacrylate, decyl methacrylate, and dodecyl methacrylate.

[0065] The terms “complement” or “complementary,” as used herein, can mean Watson- Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[0066] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control or Control group” as used herein can refer to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. A control may be a subject or cell without a conjugate or composition thereof as detailed herein. A control may be a subject or cell with a conjugate or composition thereof as detailed herein but with the conjugate lacking a nucleic acid RIG-I agonist. A control may be a subject, or a sample therefrom, whose disease state is known. The subject, or sample therefrom, may beAttorney Docket No.093386-0030-WO01 healthy, diseased, diseased prior to treatment, diseased during treatment, or diseased after treatment, or a combination thereof.

[0067] The term “effective dosage” or “therapeutic dosage” or “therapeutically effective amount” or “effective amount,” as used herein, refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results, to modulate a biological process, and / or treat a disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. A therapeutically effective amount is also one in which any toxic or detrimental effects of substance are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In reference to treating an infection, an effective or therapeutically effective amount can include an amount sufficient to, among other things, induce the RIG-I pathway.

[0068] The term “cycloalkyl” or “cycloalkane,” as used herein, refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term “cycloalkyl” is used herein to refer to a cycloalkane when present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e.g., decahydronaphthalenyl), or a bridged cycloalkyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptanyl). Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[1.1.1]pentanyl.

[0069] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0070] The term “haloalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0071] The term “heteroalkyl,” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, O, P and N. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides. A heteroalkyl group may be optionally substituted with one or more substituents.

[0072] The term “heteroalkylene” as used herein, means an alkylene group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, Si, O, P and N. Examples include, but are not limited to, -CH2OCH2-, -CH2NHCH2-,Attorney Docket No.093386-0030-WO01 polyethylene glycol groups (e.g., -(CH2CH2O)n-), polyethyleneimine groups (e.g., -(CH2CH2NH)n- ), and the like. A heteroalkylene group may be optionally substituted with one or more substituents.

[0073] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. The nucleic acid can be natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including, for example, uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. In some embodiments, the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Nucleic acids can be purchased through commercial vendors or obtained by chemical synthesis methods or by recombinant methods.

[0074] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described devices or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgous monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject has a specific genetic marker. The subject may be undergoing other forms of treatment.

[0075] “Substantially identical” means that a first and second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical over aAttorney Docket No.093386-0030-WO01 region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 nucleic acids. This can also be referred to as X% sequence identity, where a first and second nucleic acid sequence are at least X% identical over a region of nucleic acids as listed above.

[0076] The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =O (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, – COOH, ketone, amide, carbamate, and acyl.

[0077] Terms such as "alkyl," "cycloalkyl," "alkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., "C1-4alkyl," "C3-6cycloalkyl," "C1-4alkylene"). These designations are used as generally understood by those skilled in the art. For example, the representation "C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, "C3alkyl" is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in "C1-4," the members of the group that follows may have any number of carbon atoms falling within the recited range. A "C1-4alkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0078] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. 2. Conjugates

[0079] Disclosed herein are conjugates that can be used to effectively deliver nucleic acids, such as nucleic acid retinoic acid-inducible gene I (RIG-I) agonists, within a biological environment. The conjugate includes a nucleic acid RIG-I agonist covalently attached to a block copolymer. The block copolymer, through its structure, can advantageously form a particle with the nucleic acid RIG-I agonist attached thereto and can deliver the nucleic acid RIG-I agonist to specific locations within the cell (e.g., cytosol). The block copolymer can also protect theAttorney Docket No.093386-0030-WO01 attached nucleic acid RIG-I agonist from, e.g., nucleases, serum proteins, and other molecules in a biological environment that may limit therapeutic effects.

[0080] The conjugate can include the block copolymer and the nucleic acid RIG-I agonist at varying molar ratios. For example, the conjugate can include the block copolymer and nucleic acid RIG-I agonist at a molar ratio of about 0.5:1 (block copolymer:nucleic acid RIG-I agonist) to about 20:1, such as about 0.5:1 to about 15:1, about 0.5:1 to about 12:1, about 1:1 to about 12:1, about 2:1 to about 20:1, about 5:1 to about 12:1, or about 8:1 to about 15:1. In some embodiments, the conjugate includes the block copolymer and nucleic acid RIG-I agonist at a molar ratio of about 10:1 (block copolymer:nucleic acid RIG-I agonist).

[0081] The conjugates may exist as salts, such as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the conjugates which are water or oil-soluble or dispersible, suitable for administration to a subject (e.g., treatment of disorders) without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the conjugates or separately by reacting an amino group of the conjugates with a suitable acid. For example, the conjugate may be dissolved in a suitable solvent and treated with at least one equivalent of an acid, like hydrochloric acid. The resulting salt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. Amino groups of the conjugates may also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0082] Basic addition salts may be prepared during the final isolation and purification of the disclosed conjugates by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine,Attorney Docket No.093386-0030-WO01 N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine and N,N’-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like. A. Block Copolymers

[0083] The block copolymer can include two different polymer blocks – the first block can be referred to as the A block and the second block can be referred to as the B block. The two different blocks can differ in hydrophilicity and / or hydrophobicity, which can allow the block copolymer and conjugate thereof to self-assemble into different particulate structures.

[0084] The block copolymer can be synthesized in a variety of different structures. For example, the block copolymer can be an A-B diblock copolymer, an A-B-A triblock copolymer, a B-A-B triblock copolymer, or a star multiblock copolymer. In some embodiments, the block copolymer is an A-B diblock copolymer. Different copolymer structures can facilitate the formation of different particulate structures.

[0085] The block copolymer can have a varying molecular weight. For example, the block copolymer can have a number average molecular weight of about 16 kilodaltons (kDa) to about 200 kDa, such as about 20 kDa to about 200 kDa, about 30 kDa to about 150 kDa, about 35 kDa to about 100 kDa, about 25 kDa to about 100 kDa, about 20 kDa to about 100 kDa, about 40 kDa to about 80 kDa, about 45 kDa to about 75 kDa, or about 50 kDa to about 70 kDa. In some embodiments, the block copolymer has a number average molecular weight of greater than 15 kDa, greater than 16 kDa, greater than 20 kDa, greater than 30 kDa, greater than 40 kDa, greater than 50 kDa, greater than 60 kDa, greater than 70 kDa, greater than 80 kDa, greater than 100 kDa, or greater than 150 kDa. In some embodiments, the block copolymer has a number average molecular weight of less than 200 kDa, less than 175 kDa, less than 150 kDa, less than 125 kDa, less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, or less than 40 kDa.

[0086] The term “molecular weight” in relation to the polymer refers to number average molecular weight (Mn) unless noted otherwise. Molecular weight of the block copolymer and the individual blocks can be measured by techniques used within the art, such as size exclusion chromatography (SEC), SEC combined with multi-angle light scattering, gel permeation chromatography, intrinsic viscosity, nuclear magnetic resonance (NMR) and the like. In some embodiments, the molecular weight is measured by gel permeation chromatography.

[0087] The block copolymer can have a varying structure associated with a varying molecular weight. For example, the block copolymer can have a structure of A1kDa-100kDa-B15kDa-Attorney Docket No.093386-0030-WO01100kDaor A1kDa-75kDa-B15kDa-75kDa.The foregoing are example structures and any combination of molecular weight and block alignment as disclosed herein can be used in the block copolymers.

[0088] The block copolymer can be synthesized via polymerization techniques known within the art, such as reversible additionífragmentation chain-transfer (RAFT) polymerization. Monomers and their resultant recurring units, as well as example polymerizations are described herein.

[0089] The general procedure for polymer synthesis by RAFT polymerization of the polymers described herein is as follows. Polymerization inhibitors can be first removed from commercially obtained monomers by simple gravity chromatography through a column of basic activated alumina immediately before use. Monomer(s), chain transfer agent (CTA), and radical initiator can next be dissolved in a solvent such as toluene, and the polymerization mixture can be purged with nitrogen gas before placing the reaction mixture in an oil bath at indicated temperature for approximately 18 h. The initial CTA:initiator ratio can be about 20:1 for reactions. After polymerization, the reactions can be terminated by briefly purging the crude mixtures with air. Polymers can then be isolated by precipitation into a solvent such as pentane and subsequent redissolution into a solvent such as acetone and reprecipitation into fresh pentane for a total of five precipitations. Polymers can then be dried in vacuo for a sufficient period of time (e.g., about 3 d). i. First Block (A)

[0090] The first block of the block copolymer is a hydrophilic block. Accordingly, the first block can include a recurring hydrophilic unit. The first block may act as a hydrophilic corona block (e.g., in particulate form). The first block may also instill stealth properties (e.g., evading a subject’s immune system and / or clearance from the blood stream) to particles formed by the block copolymer.

[0091] Any suitable hydrophilic monomer can be used to provide the recurring hydrophilic unit. For example, the hydrophilic unit can be derived from an ethylenically unsaturated monomer (e.g., acrylate, methacrylate, acrylamide, methacrylamide, etc.) that also includes a hydrophilic moiety, such as an amine, a hydroxyl, or combinations hereof. Example monomers from which the hydrophilic unit can be derived from include, but are not limited to, dimethylacrylamide, oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) acrylate, N-vinyl pyrrolidone, N-isopropylacrylamide, 2-hydroxyethyl methacrylate methacrylate, acrylamide, methacrylamide, dimethylaminoethyl methacrylate, N- (2-hydroxypropyl) methacrylamide, itaconic acid, acrylic amide, methacrylamide, vinyl alcohol,Attorney Docket No.093386-0030-WO01 3-(methacryloylamino)propyl trimethoxysilane, and combinations thereof. In some embodiments, the hydrophilic unit is derived from dimethylacrylamide, oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) acrylate, or a combination thereof. In some embodiments, the hydrophilic unit is derived from dimethylacrylamide, oligo(ethylene glycol) methyl ether methacrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) acrylate, or a combination thereof. In some embodiments, the first block includes more than one type of hydrophilic unit. In other words, the first block may include hydrophilic units derived from at least two different hydrophilic monomers.

[0092] In some embodiments, the hydrophilic unit is of formula (I)(I), wherein Y1is H or CH3, G1is NH, O, or bond, R1is -NRxRy, -alkylene-NRxRy, -heterolkylene, or - heterolkylene-NRxRy, and Rxand Ryare each independently hydrogen or alkyl.

[0093] In some embodiments, R1is -NRxRy, -C1-8alkylene-NRxRy, -C1-8heterolkylene, or -C1-8heterolkylene-NRxRy.

[0094] In some embodiments, R1is -NRxRy, -C1-6alkylene-NRxRy, -C1-6heterolkylene, or -C1-6heterolkylene-NRxRy, and Rxand Ryare each independently hydrogen or C1-6alkyl.

[0095] In some embodiments, R1is -NRxRyor -C1-8heterolkylene wherein at least 1 carbon of the heteroalkylene is replaced by O.

[0096] In some embodiments, G1is bond and R1is -NRxRy, and Rxand Ryare each independently hydrogen or C1-4alkyl.

[0097] In some embodiments, Rxand Ryare each independently hydrogen or C1-4alkyl.

[0098] The first block can have a varying molecular weight. For example, the first block can have a number average molecular weight of about 1 kDa to about 100 kDa, such as about 5 kDa to about 90 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 50 kDa, about 50 kDa to about 100 kDa, about 60 kDa to about 100 kDa, about 1 kDa to about 50 kDa, or about 5 kDa to about 50 kDa. In some embodiments, the first block has a number average molecular weight of greater than 1 kDa, greater than 5 kDa, greater than 10 kDa, greater than 20 kDa, greater than 30 kDa, greater than 40 kDa, or greater than 50 kDa. In some embodiments, theAttorney Docket No.093386-0030-WO01 first block has a number average molecular weight of less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, or less than 40 kDa.

[0099] The block copolymer can include varying amounts of the first block. For example, the block copolymer can include the first block at about 10% to about 90% by weight of the block copolymer, about 20% to about 80% by weight of the block copolymer, about 30% to about 70% by weight of the block copolymer, about 40% to about 60% by weight of the block copolymer, about 10% to about 60% by weight of the block copolymer, about 10% to about 50% by weight of the block copolymer, about 10% to about 40% by weight of the block copolymer, about 40% to about 90% by weight of the block copolymer, about 50% to about 90% by weight of the block copolymer, or about 60% to about 90% by weight of the block copolymer.

[0100] In some embodiments, the block copolymer includes the first block at greater than 10% by weight of the block copolymer, greater than 20% by weight of the block copolymer, greater than 30% by weight of the block copolymer, greater than 40% by weight of the block copolymer, greater than 50% by weight of the block copolymer, or greater than 60% by weight of the block copolymer. In some embodiments, the block copolymer includes the first block at less than 90% by weight of the block copolymer, less than 80% by weight of the block copolymer, less than 70% by weight of the block copolymer, less than 60% by weight of the block copolymer, less than 50% by weight of the block copolymer, or less than 40% by weight of the block copolymer. ii. Second Block (B)

[0101] The second block is more hydrophobic than the first block. The second block may act as a hydrophobic core block (e.g., in particulate form). The second block can include a copolymer. The copolymer can include at least two different recurring units. For example, the copolymer can include a recurring hydrophobic unit and a recurring protonatable unit. The recurring hydrophobic unit and the recurring protonatable unit can be included in the copolymer randomly or as blocks. The combination of the recurring hydrophobic unit and the recurring protonatable unit can aid in both endosomal escape of the nucleic acid RIG-I agonist and / or conjugate thereof and forming the core of a particle including the conjugate.

[0102] The copolymer can include any suitable polymerized hydrophobic monomer that can be used to provide the recurring hydrophobic unit. For example, the hydrophobic unit can be derived from an ethylenically unsaturated monomer (e.g., acrylate, methacrylate, etc.) that also includes a hydrophobic moiety, such as an alkyl, a cycloalkyl, an aryl, or combinations hereof. Example monomers from which the hydrophobic unit can be derived from include, but are not limited to, an alkyl acrylate, an alkyl methacrylate, styrene, isobornyl acrylate, cyclohexylAttorney Docket No.093386-0030-WO01 methacrylate, phenyl methacrylate, fluorinated methacrylate, allyl methacrylate, vinyl decanoate, dibutyl maleate, N-phenyl maleimide, norbornene, 2-hydroxyethyl methacrylate, 3,4-dihydro-2H- pyran, or a combination thereof. In some embodiments, the hydrophobic unit is derived from an alkyl acrylate, an alkyl methacrylate, an isobornyl acrylate, a cyclohexyl methacrylate, a phenyl methacrylate, or a combination thereof. In some embodiments, the hydrophobic unit is derived from an alkyl acrylate, an alkyl methacrylate, or a combination thereof. In some embodiments, the copolymer includes more than one type of hydrophobic unit. In other words, the copolymer may include hydrophobic units derived from at least two different hydrophobic monomers.

[0103] In some embodiments, the hydrophobic unit is of formula (II)(II), wherein Y2is H or CH3, and R2is C1-12alkyl.

[0104] In some embodiments, R2is C1-10alkyl. In some embodiments, R2is C1-8alkyl. In some embodiments, R2is C1-6alkyl. In some embodiments, R2is C1-4alkyl.

[0105] The copolymer can include the hydrophobic unit at about 25% to about 75% by weight of the copolymer, such as about 30% to about 65% by weight of the copolymer, about 40% to about 60% by weight of the copolymer, about 25% to about 50% by weight of the copolymer, about 35% to about 75% by weight of the copolymer, or about 50% to about 75% by weight of the copolymer. In some embodiments, the copolymer includes the hydrophobic unit at greater than 25% by weight of the copolymer, greater than 30% by weight of the copolymer, greater than 40% by weight of the copolymer, or greater than 50% by weight of the copolymer. In some embodiments, the copolymer includes the hydrophobic unit at less than 75% by weight of the copolymer, less than 70% by weight of the copolymer, less than 60% by weight of the copolymer, or less than 50% by weight of the copolymer.

[0106] The copolymer can include any suitable polymerized protonatable monomer that can be used to provide the recurring protonatable unit. For example, the protonatable unit can be derived from an ethylenically unsaturated monomer (e.g., acrylate, methacrylate, acrylamide, methacrylamide, etc.) that also includes a protonatable moiety, such as an amine, a hydroxyl, a carboxylic acid, or combinations hereof. Example monomers from which the protonatable unit can be derived from include, but are not limited to, 2-(dimethylamino)ethyl methacrylate, 2- (dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, N,N-dimethylaminoethylAttorney Docket No.093386-0030-WO01 acrylate, N,N-diethylaminoethyl acrylate, N-vinyl pyrrolidone, N-vinyl imidazole, 3- (dimethylamino)propyl methacrylate, 4-(dimethylamino)butyl methacrylate, 3-aminopropyl methacrylate, 4-aminobutyl methacrylate, N-(3-aminopropyl) methacrylamide, N-(4-aminobutyl) methacrylamide, 2-aminoethyl methacrylate, N-(2-aminoethyl) acrylamide, N-(3-aminopropyl) acrylamide, N-(4-aminobutyl) acrylamide, 4-(dimethylamino)butyl acrylate, aminoethyl vinyl ether, and combinations thereof.

[0107] In some embodiments, the protonatable unit is derived from 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, N,N- dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, 3-(dimethylamino)propyl methacrylate, 4-(dimethylamino)butyl methacrylate, 3-aminopropyl methacrylate, 4-aminobutyl methacrylate, N-(3-aminopropyl) methacrylamide, N-(4-aminobutyl) methacrylamide, 2- aminoethyl methacrylate, N-(2-aminoethyl) acrylamide, N-(3-aminopropyl) acrylamide, N-(4- aminobutyl) acrylamide, 4-(dimethylamino)butyl acrylate, or a combination thereof.

[0108] In some embodiments, the protonatable unit is derived from 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, N,N- dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, or a combination thereof. In some embodiments, the copolymer includes more than one type of protonatable unit. In other words, the copolymer may include protonatable units derived from at least two different protonatable monomers.

[0109] In some embodiments, the protonatable unit is of formula (III)wherein Y3is H or CH3, G3is NH or O, X is alkylene, heteroalkylene, alkenylene, or alkynylene, R3is -NRxRy, and Rxand Ryare each independently hydrogen or alkyl.

[0110] In some embodiments, X is C1-10alkylene, C1-10heteroalkylene, C1-10alkenylene, or C1-10lkynylene. In some embodiments, X is C1-8alkylene, C1-8heteroalkylene, C1-8alkenylene, or C1-8lkynylene. In some embodiments, X is C1-4alkylene, C1-4heteroalkylene, C1-4alkenylene, or C1-4alkynylene.

[0111] In some embodiments, X is alkylene. In some embodiments, X is C1-10alkylene. In some embodiments, X is C1-4alkylene.Attorney Docket No.093386-0030-WO01

[0112] In some embodiments, X is C1-10alkylene, and Rxand Ryare each independently hydrogen or C1-4alkyl.

[0113] In some embodiments, G is O, X is C1-10alkylene, and Rxand Ryare each independently hydrogen or C1-4alkyl.

[0114] The copolymer can include the protonatable unit at about 25% to about 100% by weight of the copolymer, such as about 30% to about 90% by weight of the copolymer, about 40% to about 80% by weight of the copolymer, about 50% to about 70% by weight of the copolymer, about 30% to about 100% by weight of the copolymer, about 50% to about 95% by weight of the copolymer, about 60% to about 95% by weight of the copolymer, about 25% to about 60% by weight of the copolymer, about 25% to about 50% by weight of the copolymer, or about 25% to about 40% by weight of the copolymer. In some embodiments, the copolymer includes the protonatable unit at greater than 25% by weight of the copolymer, greater than 30% by weight of the copolymer, greater than 40% by weight of the copolymer, greater than 50% by weight of the copolymer, or greater than 60% by weight of the copolymer. In some embodiments, the copolymer includes the protonatable unit at less than 100% by weight of the copolymer, less than 90% by weight of the copolymer, less than 80% by weight of the copolymer, less than 70% by weight of the copolymer, or less than 60% by weight of the copolymer.

[0115] The second block can have a varying molecular weight. For example, the second block can have a number average molecular weight of about 15 kDa to about 100 kDa, such as about 15 kDa to about 90 kDa, about 20 kDa to about 80 kDa, about 25 kDa to about 50 kDa, about 30 kDa to about 100 kDa, about 50 kDa to about 100 kDa, about 15 kDa to about 50 kDa, or about 15 kDa to about 40 kDa. In some embodiments, the second block has a number average molecular weight of greater than 15 kDa, greater than 20 kDa, greater than 25 kDa, greater than 30 kDa, greater than 35 kDa, greater than 40 kDa, or greater than 50 kDa. In some embodiments, the second block has a number average molecular weight of less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, or less than 40 kDa.

[0116] The block copolymer can include varying amounts of the second block. For example, the block copolymer can include the second block at about 10% to about 90% by weight of the block copolymer, about 20% to about 80% by weight of the block copolymer, about 30% to about 70% by weight of the block copolymer, about 40% to about 60% by weight of the block copolymer, about 10% to about 60% by weight of the block copolymer, about 10% to about 50% by weight of the block copolymer, about 10% to about 40% by weight of the block copolymer,Attorney Docket No.093386-0030-WO01 about 40% to about 90% by weight of the block copolymer, about 50% to about 90% by weight of the block copolymer, or about 60% to about 90% by weight of the block copolymer.

[0117] In some embodiments, the block copolymer includes the second block at greater than 10% by weight of the block copolymer, greater than 20% by weight of the block copolymer, greater than 30% by weight of the block copolymer, greater than 40% by weight of the block copolymer, greater than 50% by weight of the block copolymer, or greater than 60% by weight of the block copolymer. In some embodiments, the block copolymer includes the second block at less than 90% by weight of the block copolymer, less than 80% by weight of the block copolymer, less than 70% by weight of the block copolymer, less than 60% by weight of the block copolymer, less than 50% by weight of the block copolymer, or less than 40% by weight of the block copolymer. iii. Nucleic Acid Linking Unit

[0118] The nucleic acid linking unit can be used to attach (e.g., covalently) the nucleic acid RIG-I agonist to the block copolymer. The nucleic acid linking unit can be part of the first block, the second block, or both. In some embodiments, the nucleic acid linking unit is present only in the second block.

[0119] Any suitable monomer (e.g., RAFT polymerizable monomer) used for conjugating biomolecules known in the art can be used to provide the recurring nucleic acid linking unit. For example, the nucleic acid linking unit can be derived from an ethylenically unsaturated monomer (e.g., acrylate, methacrylate, acrylamide, methacrylamide, etc.) that also includes a linking moiety. Example linking moieties include, but are not limited to, a pyridyl disulfide, an azide, a thiol, an amine, a maleimide, and combinations thereof.

[0120] The linking moiety can be a biorthogonal functional group. As used herein, “biorthogonal chemistry” refers to a chemical reaction that can occur inside of living systems without interfering with native biochemical processes. Bioorthogonal reactions proceed in high yield under physiological conditions and result in covalent bonds between reactants that are otherwise stable in these settings. Bioorthogonal reactions are reactions of materials with each other, wherein each material has limited or substantially no reactivity with functional groups found in vivo. For example, the efficient reaction between an azide and a terminal alkyne, i.e., the most widely studied example of “click” chemistry, is known as a useful example of a bioorthogonal reaction. Click chemistry refers to a group of reactions that generate substances quickly and reliably and provide high product yields. In some embodiments, the biorthogonal functional group includes an azide, an alkyne, a maleimide, an iodoacetamide, a thiol, a disulfide, a NHS ester, a tetrazine, a trans-cyclooctene, a ketone / aldehyde, a hydrazine, aAttorney Docket No.093386-0030-WO01 hydrazide, or a thioacid. In some embodiments, the biorthogonal functional group includes an azide or an alkyne.

[0121] In some embodiments, the nucleic acid linking unit is derived from an acrylate monomer, a methacrylate monomer, an acrylamide monomer, or a methylacrylamide monomer, wherein each of the foregoing monomers have at least one linking moiety. In some embodiments, the linking moiety comprises a biorthogonal functional group.

[0122] In some embodiments, the nucleic acid linking unit is of formula (IV)(IV), wherein Y4is H or CH3, G4is NH or O, L is a linker, and R4is a nucleic acid RIG-I agonist.

[0123] In some embodiments, L is a cleavable linker. In some embodiments, L is a non- cleavable linker. In some embodiments, the linker comprises a disulfide moiety or a succinimide moiety. L can be any of the linkers as described herein.

[0124] The nucleic acid RIG-I agonist can be attached to the nucleic acid linking unit and block copolymer through a linker. The linker can be a cleavable linker or a non-cleavable linker. Cleavable linkers are linkers that can be degraded by or in a biological environment in a biologically relevant time scale. For example, cleavable linkers include, but are not limited to, hypoxia sensitive linkers, reactive oxygen species (ROS) sensitive linkers, pH sensitive linkers, redox sensitive linkers, enzyme sensitive linkers, light sensitive linkers, and those degraded by hydrolysis. In contrast, non-cleavable linkers are linkers that are not degraded by or in a biological environment in a biologically relevant time scale, such as amide-based linkers, succinimide-based linkers (e.g., product of maleimide-thiol reaction), and carbamate-based linkers.

[0125] In some embodiments, the linker comprises a hypoxia sensitive linker, a reactive oxygen species (ROS) sensitive linker, a pH sensitive linker, a redox sensitive linker, an enzyme sensitive linker, a light sensitive linker, or a combination thereof.

[0126] In some embodiments, the enzyme sensitive linker is a matrix metalloproteinase sensitive linker, a p-aminobenzyl alcohol system linker, a cathepsin sensitive linker, a beta- glucuronidase sensitive linker, an esterase sensitive linker, or a combination thereof.Attorney Docket No.093386-0030-WO01

[0127] In some embodiments, the redox sensitive linker is a glutathione sensitive linker, a nitroreductase / NADH sensitive linker, or a combination thereof. In some embodiments, the redox sensitive linker comprises a disulfide moiety.

[0128] In some embodiments, the pH sensitive linker is a hydrazone, a silyl ether, a low pH sensitive linker, or a combination thereof.

[0129] In some embodiments, the linker is non-cleavable. It has been found that using a non- cleavable linker to covalently attach the nucleic acid RIG-I agonist to the block copolymer resulted in surprising efficacy.

[0130] The block copolymer can include the nucleic acid linking unit at about 0.5% to about 100% by weight of the block copolymer, such as about 1% to about 100% by weight of the block copolymer, about 10% to about 80% by weight of the block copolymer, about 20% to about 60% by weight of the block copolymer, about 30% to about 50% by weight of the block copolymer, about 1% to about 50% by weight of the block copolymer, about 1% to about 20% by weight of the block copolymer, about 1% to about 10% by weight of the block copolymer, about 0.5% to about 10% by weight of the block copolymer, about 50% to about 100% by weight of the block copolymer, or about 70% to about 100% by weight of the block copolymer.

[0131] In some embodiments, the block copolymer includes the nucleic acid linking unit at greater than 0.5% by weight of the block copolymer, greater than 1% by weight of the block copolymer, greater than 10% by weight of the block copolymer, greater than 20% by weight of the block copolymer, or greater than 50% by weight of the block copolymer. In some embodiments, the block copolymer includes the nucleic acid linking unit at less than 100% by weight of the block copolymer, less than 75% by weight of the block copolymer, less than 50% by weight of the block copolymer, less than 20% by weight of the block copolymer, less than 10% by weight of the block copolymer, or less than 5% by weight of the block copolymer.

[0132] Each of the recurring units of the block copolymer (e.g., recurring units of formula (I), (II), (III), and (IV)) can be repeated a number of times, which can be dependent on the molecular weight of the monomer used for each recurring unit. For example, a recurring unit of the block copolymer can be repeated 2 to 500 times, such as 5 to 500 times, 5 to 400 times, 5 to 350 times, 10 to 300 times, 15 to 350 times, 5 to 200 times, 10 to 150 times, 5 to 400 times, 5 to 275 times, 2 to 100 times, 2 to 50 times, or 2 to 20 times. In some embodiments, a recurring unit of the block copolymer can be repeated greater than 1 time, greater than 5 times, greater than 6 times, greater than 7 times, greater than 8 times, greater than 9 times, greater than 10 times, greater than 15 times, or greater than 20 times. In some embodiments, a recurring unit of the block copolymer can be repeated less than 500 times, less than 450 times, less than 400Attorney Docket No.093386-0030-WO01 times, less than 350 times, less than 325 times, less than 315 times, less than 300 times, less than 250 times, less than 100 times, less than 75 times, less than 50 times, or less than 20 times. The number of repeats of a recurring unit can also be expressed as a subscript “n” associated with a recurring unit as typically done in the art with polymers. For example, formula (I) can be denoted as followswherein n can be as described above, e.g., 5 to 500. All of the disclosed recurring units of the block copolymer can be denoted in the same manner. B. Nucleic Acid RIG-I Agonists

[0133] Pattern recognition receptors (PRRs) can recognize specific molecular patterns associated with pathogen invasion to trigger an inflammatory response that is key to mounting an appropriate immune response to clear the infection. A diversity of nucleic acid PRR agonists (e.g., CpG ODN, poly(I:C)) have been widely explored to activate innate immunity for applications in cancer immunotherapy and vaccine adjuvants. An important PRR involved in the detection of viruses is retinoic acid-inducible gene I (RIG-I, also known as DDX58), which resides in the cytosol and recognizes short, double-stranded RNA that contains a triphosphate group at the 5ƍ end (pppRNA). Activation of RIG-I triggers a multifaceted anti-viral innate immune response that shares significant homology with responses that are associated with productive anti-tumor immunity (e.g., type I interferons, T cell chemokines). Additionally, activation of RIG-I in cancer cells has been shown to increase their immunogenicity as well as induce immunogenic cell death. Hence, agonists of the RIG-I pathway have recently emerged as a promising class of antiviral agents, vaccine adjuvants, and cancer immunotherapeutics,

[0134] The conjugate includes a nucleic acid RIG-I agonist (e.g., a PRR agonist). The nucleic acid RIG-I agonist can include a phosphorylated nucleic acid duplex. As used herein, the term “phosphorylated nucleic acid duplex” refers to any molecule including ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA) base pairing between complementary nucleotide sequences in the same or separate nucleic acid strands, and at least two phosphoryl groups (e.g., diphosphate or triphosphate) at a 5ƍ end of at least one strand. Various PRRs, such as, but not limited to, retinoic acid-inducible gene I (RIG-I), recognize the phosphoryl groups attached to these phosphorylated nucleic acid duplexes, resulting in binding thereto.Attorney Docket No.093386-0030-WO01

[0135] The nucleic acid duplex (of the nucleic acid RIG-I agonist) can include a double- stranded nucleic acid molecule with base pairing between separate, complementary strands. In some embodiments, the nucleic acid duplex includes a double-stranded RNA molecule, which may be comprised entirely of RNA bases or a combination of RNA bases and at least one DNA base. The RNA and / or DNA bases may be modified, such as, for example, by including a phosphothioate linkage. The nucleic acid duplex can include two termini, each terminus including the 5ƍ end of one strand and the 3ƍ end of the other, complementary strand. In such embodiments, the 5ƍ end of at least one strand is phosphorylated with a diphosphate or triphosphate group.

[0136] In some embodiments, the nucleic acid duplex includes a single-stranded nucleic acid molecule with base pairing between complementary regions within the same strand. These single-stranded nucleic acid duplexes have a single terminus including both the 5ƍ and 3ƍ end of the single strand. In such embodiments, the 5ƍ end of the single strand is phosphorylated with a diphosphate or triphosphate group. The single-stranded nucleic acid duplex can include, but is not limited to, a stem-loop or hairpin loop RNA. The stem-loop RNA can include an RNA tetraloop opposite the single terminus. This RNA tetraloop can stabilize the stem-loop RNA molecule and / or block protein binding. Although discussed above in terms of an RNA molecule, as will be appreciated by those skilled in the art, these molecules are not limited solely to RNA bases and may include at least one DNA base.

[0137] The nucleic acid RIG-I agonist can include 10 to 1000 base pairs, such as 10 to 500 base pairs, 10 to 100 base pairs, or 10 to 50 base pairs. In some embodiments, the nucleic acid RIG-I agonist includes greater than 8 base pairs, greater than 10 base pairs, greater than 20 base pairs, greater than 30 base pairs, greater than 50 base pairs, or greater than 100 base pairs. In some embodiments, the nucleic acid RIG-I agonist includes less than 1,000 pairs, less than 500 base pairs, less than 100 base pairs, less than 75 base pairs, less than 50 base pairs, or less than 20 base pairs. The foregoing description of base pairs can also be applied to the nucleic acid duplex of the nucleic acid RIG-I agonist.

[0138] The nucleic acid RIG-I agonist can include any number of different sequences and still function as a RIG-I agonist. In other words, the nucleic acid RIG-I agonist is sequence agnostic. Example sequences include, but are not limited to, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. In some embodiments, the nucleic acid RIG-I agonist includes a nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or aAttorney Docket No.093386-0030-WO01 combination thereof. In some embodiments, the nucleic acid RIG-I agonist includes a nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or a combination thereof. In some embodiments, the nucleic acid RIG-I agonist includes a nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a combination thereof. In some embodiments, the nucleic acid RIG-I agonist includes a nucleic acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. The nucleic acid RIG-I agonists may also include sequences substantially identical to those listed herein. The foregoing description of sequences can also be applied to the nucleic acid duplex of the nucleic acid RIG-I agonist.

[0139] The nucleic acid RIG-I agonist can be covalently attached to the block copolymer (e.g., through the nucleic acid linking unit) in relation to its phosphoryl groups. For example, the nucleic acid RIG-I agonist can be covalently attached to the nucleic acid linking unit through a terminal end of the nucleic acid RIG-I agonist that is either distal or proximal to the at least two phosphoryl groups. In some embodiments, the nucleic acid RIG-I agonist is covalently attached to the nucleic acid linking unit through a terminal end of the nucleic acid RIG-I agonist that is distal from the at least two phosphoryl groups. The foregoing description of attachment can also be applied to the nucleic acid duplex of the nucleic acid RIG-I agonist. C. Synthesis of Conjugates

[0140] Example conjugate synthetic details can be found in the Examples herein. Generally, oligonucleotides can be modified on the 3’ or 5’ end with a reactive group and can be mixed with an ethanolic stock of the block copolymer in a buffer (e.g.,10 mM citrate buffer (pH 4.2) or HEPES buffered glucose at pH 7.4) at a block copolymer:nucleic acid molar ratio as described herein. Mixing can be done at room temperature for a period of time (e.g., about 45 minutes. For reactions that occur in a citrate buffer, phosphate buffer can then be added to neutralize the pH prior to use. D. Particles

[0141] The composition and structure of the block copolymer can afford the block copolymers and conjugates thereof the ability to self-assemble into particulate structures. For example, the block copolymer and conjugate thereof can have a critical micelle concentration (CMC) and at a concentration above the CMC, the block copolymers and conjugates thereof can self-assemble, e.g., with other block copolymers or conjugates thereof into a particulate structure. Accordingly, also disclosed herein are particles that can include a plurality of self- assembled block copolymers or conjugates thereof. Example structures include, but are not limited to, micelles, vesicles, inverted micelles, spherical polymersomes, tubular polymersomes,Attorney Docket No.093386-0030-WO01 and nanofibers. Micelles can be a variety of shapes with varied aspect ratios. For example, the particle can be a filomicelle, a rodlike micelle, a cylindrical micelle, and / or a wormlike micelle. In some embodiments, the particle is a micelle.

[0142] The particle can have a structure that is dependent on the different blocks of the block copolymer. For example, the particle can have a corona and a core, where each of the corona and the core correspond to a block of the block copolymer. In some embodiments, the corona includes the first block. In some embodiments, the core includes the second block. The nucleic acid RIG-I agonist can be covalently attached to the corona, the core, or both. In some embodiments, the nucleic acid RIG-I agonist is covalently attached to the core (e.g., to the second block). In some embodiments, the nucleic acid RIG-I agonist is covalently attached only to the core. It has been found that covalently attaching the nucleic acid RIG-I agonist to the core of the particle can improve therapeutic efficacy.

[0143] The particle can have a varying diameter. For example, the particle can have a diameter of about 10 nm to about 1 μm, such as about 10 nm to about 700 nm, about 10 nm to about 500 nm, about 15 nm to about 400 nm, about 20 nm to about 300 nm, about 25 nm to about 200 nm, about 10 nm to about 100 nm, about 50 nm to about 500 nm, about 10 nm to about 200 nm, or about 10 nm to about 150 nm. In some embodiments, the particle has a diameter of greater than 10 nm, greater than 20 nm, greater than 50 nm, greater than 80 nm, greater than 100 nm, or greater than 500 nm. In some embodiments, the particle has a diameter of less than 1 μm, less than 800 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 200 nm, or less than 100 nm. Particle size can be measured by techniques known within the art, such as dynamic light scattering and electron microscopy (e.g., TEM). 3. Uses of the Conjugates A. Compositions

[0144] Also disclosed herein are compositions that include a plurality of conjugates self- assembled into a particle as described above. Accordingly, the description of the conjugates, the block copolymers, the first block, the second block, the nucleic acid linking unit, the nucleic acid RIG-I agonist, and the particles above can be applied to the disclosed compositions.

[0145] The compositions can also include one or more pharmaceutically acceptable excipients, where such compositions can also be referred to as a pharmaceutical composition. Examples of pharmaceutically acceptable excipients include, but are not limited to, buffering agents (e.g., phosphate buffered saline), carbohydrates (e.g., glucose, trehalose, starch, etc.) solubilizers, solvents, antimicrobial preservatives, antioxidants, suspension agents, or aAttorney Docket No.093386-0030-WO01 combination thereof. The compositions and pharmaceutical compositions can be used in the methods disclosed herein. B. Administration

[0146] The composition can be administered prophylactically or therapeutically. In prophylactic administration, the composition can be administered in an amount sufficient to induce a response. In therapeutic applications, the composition can be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. Amounts effective for this use will depend on, e.g., the particular composition of the conjugate regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0147] The compositions can be administered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, the composition is administered intravenously, subcutaneously, intradermally, intramuscularly, or intraperitoneally. In some embodiments, the composition is administered intravenously.

[0148] The composition may be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub- dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

[0149] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, the severity of the affliction, and subjects treated, the particular conjugates and / or compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods, for example, human clinical trials, in vivo studies and in vitro studies.

[0150] Dosage amount and interval may be adjusted individually to provide plasma levels of the biologically active agent which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC). The MEC will vary for each agent but can be estimated from in vivo and / or in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, assays well known to those in the art can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value. Pharmaceutical compositions can be administered using a regimen which maintainsAttorney Docket No.093386-0030-WO01 plasma levels above the MEC for 10-90% of the time, such as between 30-90% or between 50- 90%. In cases of local administration or selective uptake, the effective local concentration of the conjugate may not be related to plasma concentration.

[0151] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the symptoms to be treated and the route of administration. Further, the dose, and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine. C. Methods of Treating a Disease or Disorder

[0152] Disclosed herein are methods of treating a disease or disorder in a subject (e.g., in need thereof). The method can include administering to the subject an effective amount of the disclosed composition. The composition can further include a pharmaceutically acceptable excipient. The disease or disorder can be any that would benefit from a RIG-I agonist. Example diseases or disorders include, but are not limited to, cancer, viral infection, and bacterial infection. Example cancers include, but are not limited to, melanoma, breast cancer, renal cell carcinoma, colorectal cancer, glioblastoma, pancreatic cancer, lung cancer, bladder cancer, neuroblastoma, cervical cancer, head and neck cancer, liver cancer, prostate cancer, ovarian cancer, multiple myeloma, lymphoma, esophageal cancer, and glioma. In some embodiments, the subject is human.

[0153] The conjugate and composition thereof may also be used as a vaccine adjuvant for treating diseases and disorders as detailed herein. D. Methods of Modulating RIG-I Activity

[0154] Further disclosed herein are methods of modulating (e.g., increasing) RIG-I activity in a cell. The method can include administering to the cell an effective amount of the disclosed composition. The composition can further include a pharmaceutically acceptable excipient.

[0155] Modulating includes increasing or enhancing RIG-I activity. RIG-I activity may be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold, relative to a control. RIG-I activity may be increased by less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, relative to a control.Attorney Docket No.093386-0030-WO01 RIG-I activity may be increased by about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, or about 1.5-fold to about 10-fold, relative to a control.

[0156] The methods are not generally limited to specific cell types and can be used in any nucleated cell type. The cell can be in an in vitro environment, such as cell culture. Alternatively, the cell can be in a subject. In some embodiments, the subject is human.

[0157] The disclosed technology has multiple aspects, illustrated by the following non-limiting examples. 4. Examples Materials and Methods for Example 1

[0158] Materials: Oligonucleotide synthesis reagents were purchased from BioAutomation (Irving, TX) and Glen Research (Sterling, VA).2ƍ-pivaloyloxymethyl amidites were purchased from ChemGenes (Washington, MA). Ethanethiol, sodium hydride (60% in oil), carbon disulfide, molecular iodine, 4,4’-azobis(4-cyanovaleric acid), butyl methacrylate (BMA), 2- (dimethylamino)ethyl methacrylate (DMAEMA), N,N-dimethylacrylamide (DMA), cysteamine hydrochloride, (meth)acryloyl chloride, azobisisobutyronitrile (AIBN), organic solvents, D- glucose (BioXtra), tris(2-carboxyethyl)phosphine (TCEP), heparin sodium salt (from porcine intestinal mucosa, ^180 USP units / mg), Triton-X 100, and oligonucleotide triphosphorylation and deprotection reagents were obtained from Sigma-Aldrich (St. Louis, MO).2,2- Dithiodipyridine and β-mercaptoethanol were purchased from Oakwood Chemical (Estill, SC). V-70 azo initiator was procured from FUJIFILM Wako Chemicals (Richmond, VA). Fetal bovine serum (FBS), heat-inactivated fetal bovine serum (HI-FBS), phosphate-buffered saline (PBS, 1x), penicillin / streptomycin solution, and Dulbecco’s Modified Eagle Medium (DMEM) and opti- MEM reduced serum media were procured from Gibco (Grand Island, NY).0.5 M disodium N,N,N’,N’-ethylenediamine tetraacetic acid (EDTA) solution, 1.0 M (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES) solution, and molecular biology-grade water were purchased from Corning (Corning, NY). Lipofectamine 2000 was acquired from Invitrogen (Carlsbad, CA). Chromatography-grade glacial acetic acid was obtained from Fisher Scientific (Hampton, NH).3’-and 5’-disulfide-modified antisense strand RNA (sequence: 5’ – AUA GGC GUA UUA UAC GCG AUU AAC G – 3’) (SEQ ID NO:1), as well as 5’-non-phosphorylated sense strand, 5’-AlexaFluor 647-tagged sense strand (sequence: 5’ – UAU CCG CAU AAU AUG CGC UAA UUG C – 3’) (SEQ ID NO:2), and unmodified antisense strand RNAs wereAttorney Docket No.093386-0030-WO01 purchased from Integrated DNA Technologies (Coralville, IA). Monomers 2-(pyridin-2- yldisulfaneyl)ethyl methacrylate (PDSEMA) and N-(2-(pyridin-2-yldisulfaneyl)ethyl)acrylamide (PDSEAm) were synthesized as described in Li et al., Chem. Commun.2015, 51, 1425 and Jackson et al., Macromolecules 2012, 45, 2699, both of which are incorporated by reference herein in their entirety. All other reagents were of analytical grade from Sigma-Aldrich.

[0159] Synthesis and purification of 5’ triphosphate (3pRNA): 3pRNA (seq: 5ƍ-ppp-CGU UAA UCG CGU AUA AUA CGC CUA U-3ƍ) (SEQ ID NO:3) was synthesized on a MerMade 12 DNA- RNA synthesizer (BioAutomation) as described in Jiang et al., J. Exp. Med.2019, 216, 2854; Zlatev et al., Curr. Protoc. Nucleic Acid Chem.2012, 50, 1.28.1; and Thillie et al., RNA 2012, 18, 856 – each of which are incorporated by reference herein in their entirety. The 3pRNA was deprotected using ammonium and purified by polyacrylamide gel electrophoresis before further purity analysis by mass spectrometry (Novatia, LLC).

[0160] Synthesis of 5’Cy5 RNA: RNA (seq: Cy5*C*G*U UAA UCG CGU AUA AUA CGC CU*A*U (SEQ ID NO:6), asterisks indicating phosphorothioate bonds) was synthesized at 1 μmole scale on a MerMade 12 DNA synthesizer.5-Ethylthio-1H-Tetrazole (0.25 M in acetonitrile) was used as activator, 0.02M Iodine in Tetrahydrofuran / Pyridine / Water (70:20:10) was used as the oxidizer, 0.05M Sulfurizing Reagent II in Pyridine / Acetonitrile was used as the sulfurizing agent, and a solution of 3% α-dichloroacetic acid in methylene chloride (DCM) was used as the deblock solution. Oligonucleotides were grown on 1000 Å CPG functionalized with Unylinker (~42 μmol / g).2ƍ-TBDMS and 5’ Cy5 phosphoramidite bases were dissolved in acetonitrile at a concentration of 0.15 M with a coupling time of 11 min for each base. The nucleobase protecting groups were removed with concentrated aqueous ammonium hydroxide for 48 h at room temperature. Deprotection of the TBDMS group was achieved with DMSO:NEt3•3 HF (4:1) solution (200 μL) at 65 °C for 3 h. The RNA oligonucleotide was then immediately purified on a Glen-Pak RNA cartridge (Glen Research) according to the manufacturer’s instructions. The purified oligo was lyophilized and resuspended in RNase-free water containing 1 mM EDTA. Molecular weight and purity were confirmed using Liquid Chromatography-Mass Spectrometry (LC-MS) analysis on a ThermoFisher LTQ Orbitrap XL Linear Ion Trap Mass Spectrometer. Chromatography was performed using a Waters XBridge Oligonucleotide BEH C18 Column using a linear gradient from 85% A (16.3 mM triethylamine – 400 mM hexafluoroisopropanol) to 100%B (methanol) at 45 °C.

[0161] Synthesis of chain transfer agent (CTA) 4-cyano-4-(((ethylthio) carbonothioyl)thio) pentanoic acid (ECT): ECT was synthesized as previously described in Moad et al., Polymer 2005, 46, 8458, which is incorporated by reference herein in its entirety, with minorAttorney Docket No.093386-0030-WO01 modifications. Initially, ethanethiol (6.3 g, 100 mmol) was added dropwise to a suspension of sodium hydride (60% in oil, 4.2 g, 105 mmol) in 150 mL diethyl ether with vigorous stirring at room temperature, which was continued until the evolution of gas was no longer observed. After the evolution of gas bubbles ceased, carbon disulfide (8.0 g, 105 mmol) was added to the reaction and allowed to stir for 30 minutes to yield sodium S-ethyl trithiocarbonate as a yellow precipitate, which was collected by filtration, washed with 50 mL diethyl ether, and resuspended in 100 mL of fresh diethyl ether. Then, molecular iodine (13.2 g, 52 mmol) was added portionwise to this resuspension, which was then stirred for 1 hour at room temperature. Sodium iodide byproduct was removed by filtration and the remaining solution was washed three times with equal volumes of saturated aqueous sodium thiosulfate, once with water, and once with brine. This washed solution was then dried over anhydrous sodium sulfate. Solvent was then removed by rotary evaporation to yield bis-(ethylsulfanylthiocarbonyl) disulfide as a dark yellow- orange oil (13.7 g, 50 mmol, quantitative).

[0162] Bis-(ethylsulfanylthiocarbonyl) disulfide (5.3 g, 19.3 mmol) and 4,4’-azobis(4- cyanovaleric acid) (8.1 g, 28.9 mmol, 1.5 eq.) were dissolved in 200 mL of ethyl acetate and were refluxed vigorously for 18 h under a nitrogen atmosphere. The solvent was then removed by rotary evaporation and the resulting crude oil was purified by silica gel chromatography (1:1 hexane:ethyl acetate (v:v) supplemented with + 0.1% glacial acetic acid (v:v), ECT Rf= 0.2, Rf= 0.9 is bis-(ethylsulfanylthiocarbonyl) disulfide). The fractions containing ECT were pooled, and the elution solvent was removed by rotary evaporation, using a few additions of 10 mL of toluene near the end of the evaporation process to co-evaporate residual acetic acid. The remaining material was dried in vacuo for 1 week and ground to afford the title product as a bright yellow powder (3.9 g, 77%).

[0163] Synthesis and characterization of polymers 1-3: The general procedure for polymer synthesis by reversible addition-fragmentation chain-transfer (RAFT) polymerization of the polymers is described as follows. Polymerization inhibitors were removed from commercially obtained monomers by simple gravity chromatography through a column of basic activated alumina (Brockmann type I) immediately before use. Monomer(s), CTA, and radical initiator were dissolved in toluene, an aliquot was taken as a reference for1H NMR analysis (Bruker AV 400), and the polymerization mixture was purged with nitrogen gas for 30 minutes before placing the reaction mixture in an oil bath at indicated temperature for 18 h. The initial CTA:initiator ratio was 20:1 for all reactions. M0:(macro-)CTA ratios, initial monomer compositions, choice of (macro-)CTA, choice of initiator, mass fraction of toluene, and reaction temperature for all polymerizations are summarized in Table 1.Attorney Docket No.093386-0030-WO01 Table 1. Summary of polymerization conditions. M0: initial monomer. CTA: chain transfer agent. DMAEMA: 2-(dimethylamino)ethyl methacrylate. BMA: Butyl methacrylate. PDSEMA: PDS-ethyl methacrylate. HEMA: 2-hydroxyethyl methacrylate. DMA: N,N-dimethylacrylamide. PDSEAm: PDS-ethyl acrylamide.

[0164] After polymerization, the reactions were terminated by briefly purging the crude mixtures with air, and monomer conversion was determined by comparing the proportional peak intensities of vinyl peaks and ester peaks from1H-NMR before and after polymerization. Polymers were isolated by precipitation into pentane and subsequent redissolution into acetone and reprecipitation into fresh pentane for a total of five precipitations. Polymers were then dried in vacuo for 3 d. Polymer compositions were also determined by1H NMR spectroscopy of the pure products. Gel permeation chromatography was performed using 10 mM LiBr in dimethylformamide as a mobile phase to elute through two TSKGel Alpha columns in series (2500 and 3000, Tosoh Biosciences) and comparing to pMMA standards.

[0165] An example synthesis using polymer 2 as reference is as follows. After filtering monomers through simple alumina columns, ECT (26.6 mg, 101 ^mol), DMAEMA (1.99 g, 12.6 mmol), and BMA (1.80 g, 12.6 mmol) were combined in a 10 mL flask with 3.66 g toluene, 1.56 mg AIBN (as 156 mg of 1.0 wt% solution in toluene, 5.05 ^mol), and a magnetic stirrer. TheAttorney Docket No.093386-0030-WO01 mixture was stirred until ECT was fully dissolved, and then the flask was sealed, gently purged with nitrogen gas for 30 min, and held at 75ºC for 18 h. The resulting mixture was precipitated five times into hexane and dried to yield polymer mCTA 2. In a new flask, 716 mg dry mCTA 2 (20.3 ^mol) was combined with 439 mg filtered DMA, 65.6 mg PDSEAm, 1.77 g toluene, and a magnetic stirrer. The flask was sealed and briefly placed in an unheated ultrasonic bath with periodic mixing to assist dissolution of mCTA 2 in the toluenic monomer mixture. When everything was completely in solution, 310 ^g V-70 (as 62 mg of 0.5 wt% solution in toluene, 1.0 ^mol) was added and the mixture was thoroughly mixed before resealing and again gently purging with nitrogen gas for 30 min before holding at 40 ºC for 18 h. Chain extension of DMAEMA-containing mCTAs with PDS-containing monomers (either already copolymerized into the mCTA or included as a chain extension monomer) tend to induce PDS cross-linking during the polymerization process at higher temperatures, necessitating an initiator with a lower temperature elevation requirement for chain extension. The resulting mixture was purified by the same method as mCTA 2 to yield polymer 2 as a yellow solid.

[0166] Synthesis of maleimide-modified polymer 2: Polymer 2 was suspended in ethanol at 1 mM and mixed at a 50:50 ratio with 5mM TCEP for one hour at room temperature. Samples were run through a 7k Zeba column to remove excess TCEP, then reacted with 5 mM N- ethylmaleimide overnight. The following day capped polymer was precipitated in ice cold pentane for five cycles spinning at 200 x g for 5 minutes each. The polymer was then dried for 48 hours prior to use in vitro.

[0167] Synthesis of polymer 4: To synthesize maleimide-functionalized polymer 4, precursor polymer 4-OH was synthesized as a version of polymer 3 with 2-hydroxyethyl methacrylate (HEMA) in place of PDSEMA using otherwise identical polymerization and purification conditions. A flask that had been freshly baked overnight at 120 ºC was charged with a magnetic stir bar, polymer 4-OH (990 mg, 20 ^mol, 244 ^mol HEMA), 6-maleimidohexanoic acid (771 mg, 3.65 mmol, 15 eq with respect to HEMA), dicyclohexyl carbodiimide (902 mg, 4.37 mmol, 18 eq), and a catalytic amount of N,N-dimethylaminopyridine (1.5 mg, 0.05 eq). The solids were dissolved in 50 mL anhydrous DCM by gentle mixing and unheated sonication, and then the flask was sealed, the solution was gently purged with nitrogen gas for 30 min, and the mixture was stirred overnight at room temperature. The following day, insoluble dicyclohexylurea byproduct was removed by filtration of the reaction mixture through celite before removal of the solvent by rotary evaporation. The remaining mixture was redissolved in DCM (~30 wt% solids), and the polymer was selectively precipitated by first diluting the polymer solution with diethyl ether (12.5 mL per 2 mL polymer + DCM solution) then the subsequentAttorney Docket No.093386-0030-WO01 addition of hexane (3 mL per 5 mL ether) precipitated the desired product. The precipitation mixture was vortexed, the polymer was pelleted by centrifugation, and the supernatant was removed by decantation. The polymer was redissolved with minimal DCM and the precipitation process was repeated for 4 additional precipitations. The polymer pellet was then dried in vacuo for 3 days to yield polymer 4 as a light brown, crumbly solid.

[0168] Formulation of and characterization of polymer micelles: Aqueous solutions of self- assembled diblock copolymer micelles were prepared by initially dissolving polymers in ethanol at 100 mg mL-1and diluting by portionwise addition of a sterile-filtered HEPES-buffered glucose solution (HBG, 50 mM HEPES, 5 mM EDTA, 5% D-glucose (w / v), pH 7.4) with gentle mixing until concentration of 10 mg mL-1was reached. These 10 mg mL-1aqueous solutions were diluted to 50 μg mL-1in 1x PBS or 10 mM citrate buffer (pH 4.2) for sizing measurements by dynamic light scattering (DLS) using a Malvern Zetasizer (Malvern, USA).

[0169] Preparation of Thiolated RNAs: Before formulation of polymer / RNA complexes or polymer-RNA conjugates, 5’-triphosphate, 5’-hydroxyl, or 5’-fluorophore sense strand RNA and 5’- (distal) or 3’-thiolated (proximal) antisense RNA (as shipped in protected disulfide form) were mixed in equimolar amounts and supplemented with DTT (100 mM). This mixture was allowed to anneal by incubating at 95 ºC for five minutes and slowly cooling to 4 ºC over the course of 45 min. After completion, the annealed and deprotected thiol-RNA was passed through two Zeba™ Spin Desalting Columns (7 kDa MWCO, Thermo Scientific, Waltham, MA) in series that were pre-equilibrated with 1 mM EDTA. Any remaining DTT was then removed by five consecutive rounds of centrifugal diafiltration (Amicon Ultra centrifugal filters, 10 kDa MWCO, MilliporeSigma, MA). The concentration of the resulting RNA was determined by absorbance at 260 nm using a Synergy H1 microplate reader (BioTek, Winooski, VT). Each batch of 5’ thiol RNA was tested using the fluorometric Measure-IT Thiol Assay kit (Invitrogen) and they were consistently found to be ~80% thiolated. The 3’ thiol RNA was tested using the same kit and was found to be > 95% thiolated.

[0170] Preparation of polymer 1 / RNA complexes, polymer 3-RNA conjugates, and polymer 4-RNA conjugates: Thiolated oligonucleotide was diluted to 1 mg mL-1in a sterile-filtered 10 mM citrate + 1 mM EDTA buffer (CE, pH 4.2) and polymers were diluted from their 100 mg mL-1ethanolic stock solution directly to 25 mg mL-1with the same CE buffer. These oligonucleotide and polymer solutions were mixed at ratios appropriate to achieve the indicated polymer:RNA molar ratios and incubated at room temperature for 45 minutes, at which point a volume of 100 mM phosphate buffer (supplemented with 1 mM EDTA, pH 8.0) equal to 3 volumes of the RNAAttorney Docket No.093386-0030-WO01 plus polymer mixture was added and incubated for 15 min at room temperature to raise the pH to neutrality before dilution to final desired concentration.

[0171] Preparation of polymer 2-RNA conjugates: Polymer 2 was dissolved to 50 mg mL-1in ethanol and diluted to 5 mg mL-1with HBG as described above. The annealed and concentrated oligonucleotide was diluted to 1 mg mL-1in HBG and was mixed with the 5 mg mL-1aqueous solution of polymer 2 in HBG at ratios appropriate to achieve the indicated polymer:RNA molar ratios and incubated for 1 h at room temperature to form the desired conjugates.

[0172] Characterization of polymer + RNA nanoparticle (NP) properties: For particle diameter and surface charge evaluation, polymer / 3pRNA formulations were made as described above, but substituting 100 mM HEPES (pH 8.0) for phosphate buffer and substituting 1 mM EDTA for HBG as a diluent, as buffers containing phosphate, glucose, and / or chloride caused electrode corrosion. The hydrodynamic size and zeta potential of the resultant loaded NPs were measured by DLS and zeta potential analysis using a Malvern Zetasizer at concentrations of 250 μg mL-1of total 3pRNA and polymer material for zeta potential measurements in a disposable folded capillary. Encapsulation of RNA in polymer / RNA complexes was measured by an intercalating fluorescent dye exclusion assay using the Quant-it™ RiboGreen RNA assay kit in triplicate at indicated polymer:RNA molar ratios according to the manufacturer’s instructions (Invitrogen). Conjugation efficiency of RNA in polymer-RNA conjugates was measured by incubating 0.50 ^g of conjugated RNA with 12.5 USP units of heparin sulfate for 15 min before subjecting the samples to an agarose gel electrophoresis mobility shift assay in triplicate (2% agarose gel pre-stained with SYBR-Safe (Invitrogen)) and performing densitometry measurements using Fiji on the resultant gel images (Bio Rad Gel Doc EZ Imager) by subtracting background and linearly interpolating the intensity of the unconjugated band between zero and the intensity of an unconjugated control band containing no polymer. Reductive disulfide bond cleavage was demonstrated with the same electrophoretic mobility shift assay, but additionally including 10 mM TCEP during the heparin incubation step and extending the incubation to 30 min.

[0173] RNase A protection assay: The ribonuclease protection assay was performed by incubating 1 ^g of conjugated or complexed RNA with 5 units of RNase A (Thermo Scientific) for 1 h at 37 ºC in optiMEM. These RNase reactions were terminated by supplementing each reaction mixture with 50 USP units of heparin sulfate, a final concentration of 10 mM TECP, and a final concentration of 0.1% Triton-X 100 for 1 h at 75 ºC before quantifying remaining RNA by the electrophoretic mobility shift and densitometry assays described above, linearly interpolating band intensity between two control lanes of 1 ^g free RNA with or without 5 units of RNase A.Attorney Docket No.093386-0030-WO01

[0174] Cell Culture: A549 lung carcinoma cells, the IRF-reporting A549-Dual cell line (Invivogen), and engineered Gal8-MDA-MB-231 cells were cultured in DMEM supplemented with 4.5 g / L D-glucose, 10% HI-FBS, 2 mM L-glutamine, 100 U mL-1penicillin, 100 ^g mL-1each of streptomycin, and 25 mM HEPES at 37º C in a humidified atmosphere of 5% CO2. A549- Duals were also supplemented with 100 ^g mL-1Normocin™ (Invivogen). Generally, 104cells were plated into each well of 96-well cell culture microplates (Grenier) and allowed to adhere overnight before being transfected in quadruplicate or sextuplicate with indicated doses and polymers. Dose-response sweeps with 3pRNA NPs consisted of serial 2-fold dilutions of polymer or polymer / RNA formulations described above with a typical maximum 3pRNA dose of 100-500 nM after dilution in optiMEM. Treated cells were allowed to incubate for 24 h before measurements. In the case of serum challenge, formulations were diluted 1:1 with either FBS or 1X PBS and incubated at 37 ºC for 4 h before dilution to the desired final concentration in optiMEM.

[0175] Gal8 recruitment assays: Gal8 recruitment assay was performed as previously described with modifications. Treatments included either self-assembled polymer NPs or polymers loaded with 5’ Alexa Fluor 647 RNA (Integrated DNA Technologies). After incubation for 18 h following treatment, the media was aspirated and replaced with FluoroBrite DMEM supplemented with 10% HI-FBS, 2 mM L-glutamine, 100 U mL-1penicillin, 100 ^g mL-1of streptomycin, 25 mM HEPES, and 4 ^M Hoechst 33342 (ThermoFisher Scientific). Cells were imaged on an ImageXpress Micro XLS Widefield High-Content Analysis System outfitted with a 20x objective lens. Four replicate images were taken per well and each were analyzed using the Transfluor Application module of the MetaXpress software package to identify and quantify the fluorescent puncta in each image, which were subsequently normalized to the number of nuclei per image. Replicate images were also analyzed using the Multi Wavelength Cell Scoring module of the MetaXpress software to identify and quantify the cytosolic AF647 signal associated with each cell. These values were then used to establish fluorescent puncta per cell and mean cell-average integrated intensity values, respectively. Each image was treated as an independent replicate, and a threshold cytosolic AF647 fluorescence intensity was established to create AF647+and AF647- categories for each cell.

[0176] In vitro evaluation of polymer and polymer / 3pRNA formulations: Luminescent reporter assays using A549-Duals were performed 24 h after treatment with indicated polymer / 3pRNA formulation at indicated doses using QUANTI-Luc luciferase detection reagent (Invivogen) according to the manufacturer’s instructions. Cell viability assays were performed using CellTiter-Glo reagent (Promega) according to the manufacturer’s instructions. MeasurementsAttorney Docket No.093386-0030-WO01 were taken using a Synergy H1 microplate reader (BioTek, Winooski, VT). All measurements within each experiment were taken with identical gain to normalize signal between treatment groups. All relative measurements were normalized by linear interpolation between the maximum mean value and untreated control. Curve fitting was performed in GraphPad Prism 9. Cell viability curves were fitted using the 4-parameter variable slope logistic curve fitting as [Inhibitor] vs. response. Dose-response curves were fitted using either the 4-parameter variable slope logistic curve fitting as [Agonist] vs. response for sigmoid-type responses (including all tested pol:3pRNA ratios formulated with polymer 1 and pol:3pRNA < 10 with polymer 3) or the bell-shaped curve fitting for bell curve-type responses (including all tested pol:3pRNA ratios formulated with polymer 2 and pol:3pRNA = 12.5, 15 with polymer 3).

[0177] Flow Cytometry: 2.5 x 105A549 cells were plated into 12-well cell culture plates (Grenier) and allowed to adhere overnight before the following treatment schedule. At indicated timepoints, three wells were each aspirated of media and washed with 1x PBS before addition of 5’Cy5 RNA formulated with indicated polymer freshly diluted to 20 nM RNA with complete cell culture medium (1 mL per well). Each well was treated with an independent formulation of polymer and 5’Cy5 RNA. Following treatment, cells were suspended in 50 ^L media containing Fc block for 15 minutes at 4 °C.50 ^L of media containing fixable viability dye (APC-Cy7; ThermoFisher) was added for 30 minutes at 4 °C. Cells were centrifuged at 380 x g for 5 minutes and rinsed in flow buffer (2% FBS in PBS + 0.5% Sodium Azide) and centrifuged again at the same conditions. Cells were then fixed in 2% paraformaldehyde for 10 minutes at room temperature in the dark. Cells were centrifuged at 650 x g for 5 minutes, rinsed twice in flow buffer, then centrifuged at 650 x g for 5 minutes. Samples were then measured using an Amnis CellStream (Cytek Biosciences) and analyzed using FlowJo (BD Biosciences).

[0178] Animals: 6- to 8-week-old Female C57BL / 6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All animals were housed and treated in compliance with the rules and regulations set forth by the Vanderbilt University Institutional Animal Care and Use Committee.

[0179] Preparation of (3p)RNA NPs for in vivo injection: Thiolated 5’OH- or 5’3pRNA was concentrated to 10 mg mL-1in CE buffer before addition to polymer 3 (in the case of 5’OH-RNA) or to polymer 1, 3, or 4 (in the case of 3pRNA) pre-diluted to 25 mg mL-1in CE with rapid mixing. The polymer to RNA molar ratio was 10 in all cases. Polymer / RNA formulations were allowed to incubate at room temperature for 1 hour and a reference aliquot was taken and stored at 4 ºC before dialysis in small-volume dialysis cassettes (Slide-A-Lyzer, 3000 MWCO, ThermoFisher Scientific), once against a 1500x volumetric excess of sterile-filtered 5% D-Attorney Docket No.093386-0030-WO01 glucose (w / v) supplemented with 10 mM HEPES and adjusted to pH 7.4 with iso-osmolar 150 mM KOH for 4 h, and once more against a 1000x volumetric excess of unbuffered sterile-filtered 5% D-glucose overnight. The concentration of 3pRNA was determined by quantification using RiboGreen RNA quantification reagent using the supplied TE buffer as a diluent for both the reference aliquot (as a standard) and sample, and TE + 2% Triton-X 100 as the reagent diluent.

[0180] In vivo activity of 3pRNA + polymer NPs: For the experiment comparing 5’OH- and 5’3pRNA NPs formulated with polymer 3, the NP solutions were diluted to 150 ^g 3pRNA mL-1in sterile 5% D-glucose and administered to pre-weighed 8-week-old female C57BL / 6 mice by retro-orbital injection at 0.75 mg kg-13pRNA. For the experiment examining 3pRNA formulated with polymers 1, 3, and 4, the NP solutions were diluted to 240 ^g 3pRNA mL-1in sterile 5% D- glucose and administered to pre-weighed 8-week-old female C57BL / 6 mice by retro-orbital injection at 1.2 mg kg-13pRNA. Mice were treated with 10 ^L of injection volume per 2 g body weight in both cases.4 h post injection, mice were euthanized, and whole blood was collected by cardiac puncture into K2EDTA-coated tubes (BD Biosciences), and the plasma was separated and collected by centrifugation at 2,000 x g for 15 min at 4 ºC. In the experiment comparing NPs with 5’OH- or 5’3pRNA NPs, blood plasma levels of IFN-Į and IFN-ȕ were measured using LumiKine Xpress mIFN-Į 2.0 and LumiKine Xpress mIFN-ȕ 2.0 ELISA kits (Invivogen) using 2% bovine serum albumin (Research Products International) + 0.01% Tween- 20 (v / v, Sigma Aldritch) as a reagent diluent but otherwise according to the manufacturer’s instructions. In the experiment comparing polymers 1, 3, and 4 (Figure 7), collected plasma was evaluated for concentrations of indicated cytokines using the LEGENDplex™ Mouse Anti-Virus Response Panel (BioLegend) according to the manufacturer’s instructions (v-bottom plate method), and the data were collected using the flow cytometer listed above. Cytokine concentrations were interpolated from standard curves generated using asymmetric sigmoidal 5-paramater logistic curve fits in GraphPad Prism 9. Outliers were identified using the ROUT method (Q = 1%) and pruned from all datasets before any further analysis. GM-CSF measurements are not shown, as there were no notable elevations above zero observed in any test group.

[0181] Statistics: Statistical comparisons were performed using GraphPad Prism 9 software according to the methods indicated in the figure captions for each respective experiment with P < 0.05 set as the threshold for statistical significance. Example 1 Polymer Nanocarriers for 3pRNA DeliveryAttorney Docket No.093386-0030-WO01 Synthesis and characterization of endosomolytic polymers for delivery of 3pRNA

[0182] Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize polymers containing a hydrophilic polyDMA block that imparts aqueous solubility following polymerization from a macro-chain transfer agent (mCTA) comprising a protonatable and hydrophobic 4-50 block to facilitate 3pRNA complexation and NP self-assembly. Thiol- reactive PDS-containing monomers were incorporated at 5 mol% in one of the blocks or withheld from both, enabling the synthesis of polymers for the electrostatic complexation of 3pRNA in addition to those for conjugation of 3pRNA to the “corona” and “core” of the polymer nanocarrier (polymers 1, 2, and 3, respectively, skeleton structures depicted in FIG.2A (a, b, and c, respectively). PDS groups undergo thiol-exchange with free thiols to form disulfides that become reduced in the cytosol, allowing for release of 3pRNA. A cleavable disulfide linker chemistry was selected for the initial carrier designs. A full summary of polymer properties can be found in Table 2. Table 2. Summary of polymer properties. DP1: core-forming block degree of polymerization (DP). DP2: corona-forming block DP. Mn,calc.: estimated number-average MW (Mn) as calculated by conversion1H NMR. Mn,GPC: estimated Mnfrom gel-permeation chromatography (GPC) measurements as compared to poly(methyl methacrylate) standards. Conjugation sites denotes count of PDS groups, except in the case of polymer 4, where it denotes count of maleimide groups.

[0183] Briefly, the endosomolytic blocks of each polymer had a degree of polymerization (DP) of ~220, composed of either 0 or ~10 incorporated PDS-ethyl methacrylate (PDSEMA) groups for thiol-mediated conjugation with the balance of the block comprising an equimolar ratio of protonatable DMAEMA and hydrophobic BMA groups (i.e., 4-50). Using these products as mCTAs, a second, hydrophilic block of dimethylacrylamide (DMA) containing 0 or ~10 incorporated PDS-ethyl acrylamide (PDSEAm) was chain-extended from the first blocks to form second blocks with a DP of ~190. The number-average MWs (Mn) as estimated by1H-NMR agree with Mnestimates from gel permeation chromatography (GPC) measurements forAttorney Docket No.093386-0030-WO01 polymers 1 and 2 as well. This yielded three initial polymers: polymer 1, containing no conjugable PDS groups and intended to solely form electrostatic complexes, polymer 2, including PDS groups in the hydrophilic DMA block and intended to form “corona” conjugates of 3pRNA, and polymer 3, including PDS groups in the endosomolytic 4-50 block, and intended to form “core” conjugates of 3pRNA. Dynamic light scattering (DLS) measurements of self- assembled polymer micelles show consistently sized, low dispersity NPs (PDI < 0.2) at physiological pH (7.4) that transition into smaller species at the late-endosomal pH of 5.8, indicative of pH-responsive micelle disassembly driven by protonation of the DMAEMA groups and subsequent solubilization of the second block (FIG 2B, and FIG.2C). Representative DLS histograms of polymers 1-3 are shown in FIG.2D.

[0184] Polymer cytotoxicity was next evaluated in A549 lung carcinoma epithelial cells. Interestingly, it was found that polymer 2 exhibited significantly more toxicity than polymers 1 or 3, resulting in a significantly lower half-maximal inhibitory concentration (IC50= 18.00 ^g mL-1, 325 nM) in comparison to those treated with polymers 1 (IC50= 386.1 ^g mL-1, 7.35 ^M) and 3 (IC50= 309.0 ^g mL-1, 5.99 ^M) (FIG.2E). It is hypothesized, without being bound by a particular theory, that this is due to the presence of reactive PDS groups on the NP surface that are capable of reacting with free thiols on cell surface proteins, resulting in increased uptake of polymer. To investigate this, a variant of polymer 2 was synthesized, in which the surface PDS groups were reduced to thiols and subsequently capped with N-ethyl maleimide, resulting in a polymer that would not be able to react with cell surface thiols. The cytotoxicity of polymer 2 was compared with this maleimide-capped variant and it was found that replacing the PDS groups resulted in significantly less cytotoxicity, which supports this possible mechanism. A Galectin 8 (Gal8) recruitment assay was also employed in an engineered MDA-MB-231 breast carcinoma cell line to assess the endosomal escape capacity of polymers 1, 2, and 3 at a dose near the IC50of polymer 2. Upon disruption of the endosome, the peptidoglycans of the endosomal lumen are exposed to the cytosolic milieu, including Gal8 fused with yellow fluorescent protein (YFP). Binding of Gal8 to endosomal peptidoglycans results in a redistribution of YFP from diffuse within the cytosol to concentrated as discrete puncta that can be enumerated as a metric of endosomal escape (FIG.2F). All three polymers promoted endosomal disruption compared to PBS control as evidenced by an increased number of observed fluorescent puncta (FIG.2G and FIG.2H). In contrast to polymer 2, which exhibited endosomolysis at all tested doses, polymers 1 and 3 exhibit dose-dependent endosomal disruption. It is hypothesized, without being bound by a particular theory, that the increased activity of polymer 2 may be due to increased cellular internalization mediated by binding to cell surface thiols, which may alsoAttorney Docket No.093386-0030-WO01 contribute to the increased cytotoxicity of polymer 2 as mentioned above. At a dose near the IC50of polymer 2 (22.2 ^g mL-1polymer), treatment with polymer 3 results in significantly less endosomal disruption compared to polymers 1 and 2 but significantly more than background (PBS vehicle control). This could be due to altered polarity of the pH-responsive 4-50 block that includes PDS groups, whose constituent aryl disulfide groups are transformed into thiols upon bioreduction. This may reflect the delicate balance of hydrophobicity and composition of incorporated monomers. Together, these data indicate that these polymers self-assemble into consistently sized, pH-responsive nanoparticles that facilitate endosomal escape. Synthesis and characterization of 3pRNA / polymer complexes and 3pRNA-polymer conjugates

[0185] The capacity of the polymers to electrostatically complex (polymer 1), corona conjugate (polymer 2), and core conjugate (polymer 3) immunostimulatory 3pRNA containing a distal thiol modifier (i.e., on the opposite end of the 5’ triphosphate group, the 5’ position of the complementary strand) was next assessed. Briefly, electrostatic complexes or core conjugates with polymers 1 or 3, respectively, were created by combining polymer and thiolated 3pRNA at pH 4 before raising to physiological pH, and corona conjugates were synthesized by addition of thiolated 3pRNA to pre-formed NPs of polymer 2. These 3pRNA loading mechanisms for each polymer are illustrated in FIG.3A. Upon addition of thiolated 3pRNA to polymers 2 and 3, the mixture immediately turned yellow, which is indicative of the release of pyridine 2-thione, the byproduct of thiol-disulfide conjugation. Initially, polymer / 3pRNA formulations were subjected to an electrophoretic mobility shift assay to assess the nature and degree of oligonucleotide loading for each polymer (FIG.3B). Polymer and 3pRNA formulations containing 0.50 ^g 3pRNA and prepared at a 10:1 polymer:RNA molar ratio were incubated with or without 12.5 USP units of heparin sulfate, a negatively charged biopolymer capable of competitively displacing 3pRNA from the cationic polymer, and / or 10 mM tris(2-carboxyethyl)phosphine (TCEP), a reducing agent that cleaves disulfide bonds between the 3pRNA and the polymer. For polymers 2 and 3, which contain thiol-reactive PDS groups, both heparin and TCEP were required to liberate 3pRNA from the polymer formulation, whereas polymer 1 released 3pRNA cargo while only in the presence of heparin. This result demonstrates that thiol-modified 3pRNA can be covalently conjugated to both the corona-forming (e.g., polymer 2) or core-forming (e.g., polymer 3) blocks of the polymer carrier. Notably, despite conjugation to a pre-formed micelle, heparin is still required for full displacement of 3pRNA from polymer 2, indicating that the cationic moieties of pre-formed micelles are sufficiently accessible to electrostatically interact with 3pRNA. Next, it was sought to determine the minimum number of polymer chains perAttorney Docket No.093386-0030-WO01 3pRNA molecule required to achieve maximum loading (FIG.3C and FIG.3D). To do this, 3pRNA complexation was quantified by polymer 1 complexes using a RiboGreen exclusion assay and found that a minimum required polymer:3pRNA molar ratio (pol:3pRNA) of ~5:1 is required to achieve maximum 3pRNA loading (FIG.3C). For 3pRNA-polymer conjugates, degree of conjugation was measured using agarose gel electrophoresis and densitometric quantification of the free, unbound 3pRNA following incubation with heparin (but not TCEP) at varying pol:RNA ratios and found that a minimum pol:RNA ratio between ~0.5 and 1 is required to achieve maximum conjugation (FIG.3D). Notably, maximum conjugation appears to be ~80%, which is consistent with the measured number of thiols per oligo (Measure-IT Thiol Assay kit, Invitrogen) and the predicted value based on oligo sequence length and the estimated coupling efficiency for the oligonucleotide synthesis process. Additionally, the size, polydispersity, and zeta potential of polymer / 3pRNA formulations were tested at varying pol:3pRNA ratios and all formulations were found to have mean diameters ranging from 25-75 nm, with minimum dispersities (PDI<0.4) observed in the pol:RNA window of 5-10, and a near- neutral zeta potential (FIG.3E and FIG.3F). NP size decreased modestly with increasing pol:3pRNA ratio for all polymers, suggesting that the number of 3pRNA molecules per NP can influence how tightly polymer chains can pack together during the NP self-assembly process, potentially by allowing more hydrophobic moieties in the core-forming blocks to interact with each other (FIG.3E). Further, polymers 1 and 3 showed consistently low PDI (<0.3) at all tested pol:RNA ratios, unlike polymer 2, which had a significantly higher PDI at most tested ratios (FIG. 3F). This is likely due to minor degrees of crosslinking between polymer 2 NPs by spontaneous disulfide bonding, or perhaps by electrostatic attractions between corona conjugated 3pRNA and NP cores that draw particles together. The zeta potential for all three polymer / 3pRNA formulations was near zero for all tested pol:3pRNA ratios. Collectively, these data indicate that covalent conjugation of 3pRNA to the polymer backbone minimizes the required quantity of carrier to fully load 3pRNA while yielding nanoparticles with desirable physicochemical properties for in vivo administration. In particular, charge-neutral particles below ~100 nm in diameter are ideal candidates for increasing RNA half-life, exploiting the enhanced permeation and retention effect for cancer therapy, and accessing draining lymph nodes following vaccine administration. Covalent conjugation enhances 3pRNA activity and uptake in vitro

[0186] Next, the immunostimulatory activity was tested of polymer / 3pRNA formulations at varying pol:RNA ratios in vitro in an A549-Dual reporter cell line that expresses a secretedAttorney Docket No.093386-0030-WO01 luciferase under control of a minimal ISG promoter (FIG.4A, FIG.4B, FIG.4C, and FIG.4D). By establishing dose-response curves and determining half-maximal effective concentration (EC50) values, the relative potency of each formulation for inducing an IFN-I response can be determined. All three polymers were effective at stimulating an IFN-I response via delivery of 3pRNA, with increased activity observed at higher pol:3pRNA ratios which is consistent with previous reports using other cationic materials and nucleic acid cargo. Moreover, higher pol:3pRNA ratios increase the dose of polymer relative to the dose of 3pRNA, which serves to further increase the dose-dependent endosomal escape efficiency observed with these polymers. As cytosolic delivery of 3pRNA cargo is a direct consequence of endosomal escape, it follows that higher pol:3pRNA ratios are more effective at stimulating IFN-I response. In contrast to 3pRNA NPs formulated with polymers 1 and 3, the cells treated with 3pRNA-polymer 2 conjugates show appreciable dose- and pol:3pRNA ratio-dependent cytotoxicity, which is consistent with the cytotoxicity data shown earlier and the decrease in measured IFN-I response at higher doses as shown in FIG.4B. This dose- and pol:3pRNA ratio-dependent cytotoxicity effect was also observed in cells treated with 3pRNA-polymer 3 conjugates, although to a much lesser extent. Collectively, these data support the possibility that residual PDS groups on the surface of polymer 2 NPs may facilitate cytotoxic levels of polymer uptake into cells up by binding to cell surface thiols. The EC50values for IFN-I activation of all polymer / 3pRNA formulations are plotted in FIG.4D, where it is evident that formulations prepared using polymers 2 and 3, in which 3pRNA is covalently conjugated to polymer, are more potent than those assembled via only electrostatic interactions with polymer 1 at pol:RNA ratios of 10 and below. While the maximum measured IFN-I response of cells treated with 3pRNA conjugates of polymers 2 and 3 show a significant decrease with increasing pol:3pRNA ratio when compared to the IFN-I response of those treated with 3pRNA formulated with polymer 1, the magnitude of this effect is modest. Furthermore, the increased cytotoxicity of 3pRNA formulated with polymers 2 and 3 may explain the decrease in the maximum IFN-I response and IFN-I activity at higher doses, as non-viable cells are unable to contribute to an IFN-I response. These data indicate that covalent conjugation achieves higher or equivalent 3pRNA-mediated RIG-I activation potency using lower quantities of carrier material with no or minimal sacrifice in maximum efficacy. For polymers 2 and 3, increasing the pol:RNA ratio beyond ~10:1 resulted in minimal gains in potency, motivating the selection of a 10:1 pol:3pRNA ratio for all subsequent investigations.

[0187] After selecting a common pol:RNA ratio at which to compare all three polymers, it was next sought out to further characterize the differences in activity by examining the intracellularAttorney Docket No.093386-0030-WO01 delivery of RNA cargo. To investigate this, polymers were loaded with 5’-AlexaFluor647 (AF647) RNA, in which the 5’ppp moiety was replaced with AF647, to simultaneously evaluate intracellular RNA uptake in concert with the Gal8 recruitment assay used to assess endosomal escape (FIG.4E, FIG.4F, and FIG.4G). Endosomolytic events (i.e., Gal8 puncta per cell) scaled with polymer / RNA dose for all polymers, with polymer 2 causing the most endosomolysis and polymers 1 and 3 displaying similar levels of activity (FIG.4E). The increased endosomolysis of cells treated with conjugates formulated with polymer 2 is consistent with its elevated cytotoxicity, as endosomal disruption has been found to promote cell death. Surprisingly, the fraction of AF647+cells following incubation with the polymer 1 formulation was nearly zero at all tested doses, whereas those of the polymer 2 and 3 formulations were increased relative to polymer 1 at nearly all doses. To investigate this further, the mean cell- average integrated intensity was investigated of AF647 in each replicate and detected a small subset of cells treated with formulations of AF647-RNA and polymer 1 with an MFI comparable to cells treated with polymer 2 and 3 formulations, but the majority of cells still showed no uptake. Representative images of merged signals from this experiment at an RNA concentration of 3.70 nM (approx.2 ^g mL-1polymer) are displayed in FIG.4G. The low uptake of AF647- labeled RNA measured in the MDA-MB-231-derived cell line was in direct contrast with the dose-dependent immunostimulatory activity observed with 3pRNA / polymer 1 NPs in the A549- Dual reporter cells, potentially suggesting that the extent to which covalent conjugation of RNA increases cellular uptake may be cell-type dependent.

[0188] To further interrogate the influence of covalent conjugation on RNA uptake, polymers were loaded with 5’Cy5-labeled RNA and incubated at 20 nM RNA with A549 lung carcinoma cells for different durations and the relative degree of uptake was analyzed by flow cytometry. A time-dependent increase in the measured median fluorescence intensity (MFI, FIG.4H) and percentage of Cy5+cells (FIG.4I) was observed for all three polymers, with polymer 3 NPs promoting significantly more RNA uptake than NPs of polymer 1 or polymer 2, which had very similar uptake profiles. Interestingly, only polymer 3 resulted in a continuous increase in the MFI of Cy5+cells over the course of 24h (FIG.4J), suggesting that conjugation to the core-forming block promotes continued delivery of RNA payload to cells that have already endocytosed NPs (i.e., are already Cy5+). Collectively, these data demonstrate an interplay between endosomal escape efficiency and the magnitude and / or kinetics of cellular uptake, as well as suggest that covalent conjugation to the core-forming block can have benefits over conjugation to the corona-forming block.Attorney Docket No.093386-0030-WO01 Covalent conjugation of 3pRNA to polymer nanocarrier confers nuclease resistance and serum stability

[0189] It is hypothesized, without being bound by a particular theory, that the discrepancies between the endosomolytic activities and delivery efficiencies of the polymer / 3pRNA formulations could be at least partially explained by differences in the nuclease or serum resistances of the resultant NPs. To investigate this, polymer / 3pRNA formulations were incubated with an excess of RNase A at physiological pH and temperature before being subjected to the electrophoretic mobility shift assay employed previously (FIG.5A). Interestingly, formulations with polymers 2 and 3 show undegraded 3pRNA fractions comparable to the fraction of 3pRNA conjugated to the polymer (FIG.5B and FIG.3D, respectively). This contrasts with electrostatic complexes prepared with polymer 1, where over half of RNA cargo is degraded despite a comparable degree of loading (FIG.3C). These data indicate that covalent conjugation of 3pRNA cargo to the polymer nanocarrier is important for improving nuclease resistance. Surprisingly, the choice of conjugation to the corona of a micelle or the core of an electrostatic complex did not have a significant effect on nuclease resistance.

[0190] To interrogate further, a serum stability assay, was employed in which polymer / 3pRNA NP formulations were incubated 50% in fetal bovine serum (FBS) or PBS and incubated at 37 ºC for 4 h before testing immunostimulatory activity in vitro. The formulation made with polymer 1 (electrostatic only) lost nearly all activity (FIG.5C), while the EC50of the formulation made with polymer 2 (corona conjugate) increased by a factor of about 3 after serum exposure relative to incubation in PBS (EC50’s = 15.48 nM and 5.438 nM, respectively, FIG.5D). Strikingly, and in contrast to polymers 1 and 2, the activity of 3pRNA formulated with polymer 3 was not affected by incubation in 50% serum (FIG.5E). This demonstrates that despite being not particularly important for imparting nuclease resistance, the chosen site of conjugation on the nanocarrier is important for maintaining serum stability. It is hypothesized, without being bound by a particular theory, that 3pRNA conjugated to the core of a nanocarrier may be less accessible to serum components than 3pRNA conjugated to the corona, and / or that core conjugates harness both electrostatic and covalent interactions to increase particle stability in serum. Additionally, it cannot be discounted that residual PDS groups on the surface of NPs prepared with polymer 2 could react with free thiols present on serum proteins, possibly resulting in reduced stability. Taken together, these data indicate that covalent loading of 3pRNA into polymer nanocarrier confers additional durability to the resulting formulation in situ, with conjugation to the core-forming block being particularly advantageous. Furthermore, the reduced RNase resistance of 3pRNA loaded into polymer 1 and reduced serum stability of RNAAttorney Docket No.093386-0030-WO01 loaded into polymers 1 and 2 could potentially explain the poor cellular uptake of RNA observed when formulated with these polymers and the continuously increasing uptake of RNA formulated to polymer 3. Immunostimulatory activity of 3pRNA is site-specific and linker chemistry-dependent with respect to thiol attachment chemistry

[0191] Based on its potent immunostimulatory activity and resistance to nuclease and serum degradation, polymer 3 was selected as the best candidate to further probe the dependence on thiol placement and linker chemistry, which was identified as potentially important variables.Here, thiol placement at the 5’ end of the strand complement to that with the 5’ppp moiety is referred as ‘distal’, which is the site of conjugation used for all studies described above. Thiol placement instead at the 3’ end of the complement strand is referred to herein as ‘proximal’. These contrasting orientations of attachment chemistry are illustrated in FIG.1. Additionally, to evaluate the importance of the disulfide bond cleavability, polymer 4 was synthesized as a cleavage-resistant analog of polymer 3, substituting pendant PDS groups for pendant thiol- reactive maleimide groups such that the bioreducible disulfide bond was replaced with a stable thioether bond (FIG.6A and FIG.6B). When subjected to the denaturing electrophoretic mobility shift assay described above, conjugates of polymer 4 were resistant to TCEP-mediated 3pRNA liberation (FIG.6C), which is in direct contrast to the reductive cleavability of 3pRNA conjugates of polymers 2 and 3.

[0192] The immunostimulatory activity was compared of proximal and distal 3pRNA conjugates prepared using polymers 3 and 4 in A549-Dual reporter cells at a pol:3pRNA ratio of 10 (FIG.6D and FIG.6E). Surprisingly, distal conjugates of polymer 4 were nearly identical in activity and uptake to polymer 3, suggesting that 3pRNA may not have to be released from the polymer if a blunt ended dsRNA with a 5’ triphosphate moiety is accessible to bind RIG-I. This finding is somewhat unexpected given the approximately ten-times higher MW of the polymers previously used. Based on this finding, it is possible that other covalent attachment chemistries can be leveraged for distal ligation, such as sulfonyl fluoride exchange, azide-alkyne cycloaddition, or trans-cyclooctene tetrazine inverse-electron-demand Diels-Alder ligation. Proximal conjugates of polymer 3, but not polymer 4, were capable of stimulating RIG-I. In another independent experiment, the activity of proximal and distal disulfide conjugates of polymer 3 were compared, it was found that the proximal conjugates had reduced activity despite an equivalent activity between unthiolated 3pRNA and proximally thiolated 3pRNA (FIG. 6). It is hypothesized, without being bound by a particular theory, that this might be related to anAttorney Docket No.093386-0030-WO01 incomplete release of proximally ligated 3pRNA, which would be required for RIG-I activation. While further investigation of this behavior is necessary, these data support the possibility that the magnitude and kinetics of RIG-I activation may be modulated via control of proximal site linker chemistry and cleavage rate. Distal conjugation of 3pRNA to polymer nanocarrier improves RIG-I activation in vivo

[0193] The ability of 3pRNA complexed to polymer 1 or distally conjugated to polymers 3 or 4 was next evaluated to activate RIG-I in vivo. It was elected to exclude polymer 2 due to the reduced benefits of corona conjugation and the increased cytotoxicity associated with this carrier. First, while it has been previously demonstrated that replacing the 3p moiety with an OH group eliminates RIG-I binding, nonetheless the in vivo activity of polymer 3 conjugates was compared prepared using 3pRNA or negative control OH-RNA, demonstrating and confirming that the 3p group was essential for activation of an IFN-I response in mice. Next, 3pRNA was formulated with polymers 1, 3, and 4 at a pol:3pRNA ratio of 10:1 as described above. Polymer / 3pRNA formulations were buffer exchanged into sterile 5% D-glucose and injected into healthy, 8-week-old female mice by retro-orbital injection at a dose corresponding to 1.2 mg 3pRNA kg-1body mass.4 h later, the mice were euthanized, and their blood was collected for quantification of plasma IFN-Is and other antiviral cytokines and chemokines. It was found that all three polymer / 3pRNA treatments exhibited in vivo activity as indicated by elevation of one or more of the antiviral cytokines / chemokines assayed. Importantly, polymer / 3pRNA core conjugates formulated using polymers 3 and 4 resulted in significantly increased plasma concentrations of IFN-α and IFN-β — the ‘signature’ cytokines associated with RIG-I activation with important antiviral and antitumor functions — relative to vehicle control and 3pRNA electrostatically complexed with polymer 1 (FIG.7A). Additionally, polymers 3 and 4 elevated the plasma concentrations of IFN-γ, C-X-C motif chemokine ligand 10 (CXCL10), C-C motif chemokine ligand 2 (CCL2), and CCL5 (FIG.7B and FIG.7C). While the response to 3pRNA conjugates prepared with polymer 3 and 4 was generally equivalent for measured cytokines, polymer 3 resulted in significantly elevated levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6, FIG.7D) relative to polymer 4, suggesting that the linker chemistry used to covalently load 3pRNA can potentially affect the nature or kinetics of the downstream immune response following activation of RIG-I. Interestingly, while administration of 3pRNA loaded into polymer 1 by electrostatic complexation increased the plasma concentration of CXCL10 and TNF-α to a level comparable to conjugates made with polymer 3 and 4, this was also associated with a significant increase in CXCL1, IL-1β, IL-12p70, and IL-10 (FIG.7E and FIG.7F), whichAttorney Docket No.093386-0030-WO01 were not significantly elevated by 3pRNA covalently loaded into polymers 3 or 4. Overall, 3pRNA loaded covalently into polymers 3 and 4 performed fairly consistently with each other despite differing attachment chemistry, and, importantly, both conjugates drastically outperformed 3pRNA loaded electrostatically into polymer 1 in terms of the magnitude and specificity of the antiviral immune response.

[0194] Engineering of polymeric nanocarriers holds potential for overcoming critical delivery barriers limiting the development of 3pRNA therapeutics and their translation into the clinic. However, there has been relatively little investigation into the development of polymeric delivery systems for 3pRNA, an oligonucleotide therapeutic whose distinctive structure and properties impose unique carrier design requirements. Using conjugatable and non-conjugatable analogs of a polymeric nanocarrier, it has been demonstrated that the loading efficiency and immunostimulatory activity of 3pRNA can be substantially enhanced if covalently conjugated to the polymer backbone. An overall design concept of this Example can be seen in FIG.1. Importantly, it was also found that covalent conjugation of 3pRNA protected against nuclease degradation and serum destabilization, particularly when the 3pRNA was ligated to the core- forming, endosomolytic block. As a result, systemic administration of nanoparticles formed with core conjugated 3pRNA enhanced type-I IFN production to a greater extent than those assembled using only electrostatics. Notably, core conjugation still enhanced activity despite employing a polymer with reduced endosomolytic activity, reflecting a balance of nucleic acid stability, cellular uptake, and endosomal escape conferred by core conjugation that may also benefit other oligonucleotide therapeutics (e.g., siRNA, miRNA).

[0195] Further, an interplay between the cleavability of the 3pRNA-polymer bond and the site-specificity of the thiol modifier was observed, as conjugation to the distal end of 3pRNA is permissive to stable ligation via a thioether bond, whereas proximal ligation requires a cleavable linkage that allows liberation of 3pRNA from the carrier. These findings have potential implications for the engineering of carrier platforms that enable spatiotemporal control of RIG-I activation through design of environmentally responsive ligation chemistries and / or bond cleavage kinetics. Overall, these studies constitute the first description of covalent 3pRNA- nanocarrier conjugates and highlight how rational integration of covalent 3pRNA conjugation to polymer backbones is a promising strategy for the optimization of future nanocarrier formulations of 3pRNA with potential clinical applications.

[0196] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the technology.Attorney Docket No.093386-0030-WO01

[0197] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use, may be made without departing from the spirit and scope thereof.

[0198] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0199] Clause 1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: a block copolymer comprising a first block (A), the first block comprising a recurring hydrophilic unit, and a second block (B), the second block comprising a copolymer, wherein the copolymer comprises a recurring hydrophobic unit and a recurring protonatable unit, wherein the first block, the second block, or both include at least one nucleic acid linking unit; and a nucleic acid retinoic acid-inducible gene I (RIG-I) agonist covalently attached to the nucleic acid linking unit through a linker.

[0200] Clause 2. The conjugate of clause 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is derived from dimethylacrylamide, oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) acrylate, N-vinyl pyrrolidone (NVP), N- isopropylacrylamide, 2-hydroxyethyl methacrylate methacrylate, acrylamide, methacrylamide, dimethylaminoethyl methacrylate, N-(2-hydroxypropyl) methacrylamide, itaconic acid, acrylic amide, methacrylamide, vinyl alcohol, 3-(methacryloylamino)propyl trimethoxysilane, or a combination thereof.

[0201] Clause 3. The conjugate of clause 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is of formula (I)wherein Y1is H or CH3, G1is NH, O, or bond, R1is -NRxRy, -alkylene-NRxRy, -heterolkylene, or - heterolkylene-NRxRy, and Rxand Ryare each independently hydrogen or alkyl.

[0202] Clause 4. The conjugate of any one of clauses 1-3, or a pharmaceutically acceptable salt thereof, wherein the hydrophobic unit is derived from an alkyl acrylate, an alkyl methacrylate, styrene, isobornyl acrylate, cyclohexyl methacrylate, phenyl methacrylate,Attorney Docket No.093386-0030-WO01 fluorinated methacrylate, allyl, methacrylate, vinyl decanoate, dibutyl maleate, N-phenyl maleimide, norbornene, 2-hydroxyethyl methacrylate, 2-octyl acrylate, 3,4-dihydro-2H-pyran, or a combination thereof.

[0203] Clause 5. The conjugate of any one of clauses 1-4, or a pharmaceutically acceptable salt thereof, wherein the hydrophobic unit is of formula (II)(II), wherein Y2is H or CH3, and R2is C1-12alkyl.

[0204] Clause 6. The conjugate of any one of clauses 1-5, or a pharmaceutically acceptable salt thereof, wherein the protonatable unit is derived from 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, N,N- dimethylaminoethyl acrylate, N,N-diethylaminoethyl acrylate, N-vinyl pyrrolidone, N-vinyl imidazole, 3-(dimethylamino)propyl methacrylate, 4-(dimethylamino)butyl methacrylate, 3- aminopropyl methacrylate, 4-aminobutyl methacrylate, N-(3-aminopropyl) methacrylamide, N-(4- aminobutyl) methacrylamide, 2-aminoethyl methacrylate, N-(2-aminoethyl) acrylamide, N-(3- aminopropyl) acrylamide, N-(4-aminobutyl) acrylamide, 4-(dimethylamino)butyl acrylate, aminoethyl vinyl ether, or a combination thereof.

[0205] Clause 7. The conjugate of any one of clauses 1-6, or a pharmaceutically acceptable salt thereof, wherein the protonatable unit is of formula (III)wherein Y3is H or CH3, G3is NH or O, X is alkylene, heteroalkylene, alkenylene, or alkynylene, R3is -NRxRy, and Rxand Ryare each independently hydrogen or alkyl.

[0206] Clause 8. The conjugate of any one of clauses 1-7, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is derived from a monomer comprising an ethylenically unsaturated group and a linking moiety.Attorney Docket No.093386-0030-WO01

[0207] Clause 9. The conjugate of clause 8, or a pharmaceutically acceptable salt thereof, wherein the linking moiety comprises a pyridyl disulfide, an azide, a thiol, an amine, a maleimide, or a combination thereof.

[0208] Clause 10. The conjugate of any one of clauses 1-9, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is of formula (IV)(IV), wherein Y4is H or CH3, G4is NH or O, L is a linker, and R4is a nucleic acid RIG-I agonist.

[0209] Clause 11. The conjugate of any one of clauses 1-10, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is only present in the second block.

[0210] Clause 12. The conjugate of any one of clauses 1-11, or a pharmaceutically acceptable salt thereof, wherein the linker comprises a hypoxia sensitive linker, a reactive oxygen species (ROS) sensitive linker, a pH sensitive linker, a redox sensitive linker, an enzyme sensitive linker, a light sensitive linker, or a combination thereof.

[0211] Clause 13. The conjugate of clause 12, or a pharmaceutically acceptable salt thereof, wherein the enzyme sensitive linker is a matrix metalloproteinase sensitive linker, a p- aminobenzyl alcohol system linker, a cathepsin sensitive linker, a beta-glucuronidase sensitive linker, an esterase sensitive linker, or a combination thereof.

[0212] Clause 14. The conjugate of clause 12, or a pharmaceutically acceptable salt thereof, wherein the redox sensitive linker is a glutathione sensitive linker, a nitroreductase / NADH sensitive linker, or a combination thereof.

[0213] Clause 15. The conjugate of clause 12, or a pharmaceutically acceptable salt thereof, wherein the pH sensitive linker is a hydrazone, a silyl ether, a low pH sensitive linker, or a combination thereof.

[0214] Clause 16. The conjugate of any one of clauses 1-15 or a pharmaceutically acceptable salt thereof, wherein the linker is non-cleavable.

[0215] Clause 17. The conjugate of any one of clauses 1-16, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist comprises a nucleic acid duplex having at least two phosphoryl groups attached to a 5ƍ end thereof.Attorney Docket No.093386-0030-WO01

[0216] Clause 18. The conjugate of clause 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a double stranded nucleic acid molecule.

[0217] Clause 19. The conjugate of clause 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a single-stranded nucleic acid molecule.

[0218] Clause 20. The conjugate of clause 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a hairpin loop RNA molecule.

[0219] Clause 21. The conjugate of any one of clauses 17-20, or a pharmaceutically acceptable salt thereof, wherein the at least two phosphoryl groups are selected from the group consisting of a diphosphate group and a triphosphate group.

[0220] Clause 22. The conjugate of any one of clauses 17-21, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist is covalently attached to the nucleic acid linking unit through a terminal end of the nucleic acid RIG-I agonist that is distal from the at least two phosphoryl groups.

[0221] Clause 23. The conjugate of any one of clauses 1-22, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist comprises 10 to 100 base pairs.

[0222] Clause 24. The conjugate of any one of clauses 1-23, or a pharmaceutically acceptable salt thereof, wherein the block copolymer comprises the first block at about 10% to about 90% by weight of the block copolymer.

[0223] Clause 25. The conjugate of any one of clauses 1-24, or a pharmaceutically acceptable salt thereof, wherein the block copolymer comprises the second block at about 10% to about 90% by weight of the block copolymer.

[0224] Clause 26. The conjugate of any one of clauses 1-25, or a pharmaceutically acceptable salt thereof, wherein the first block has a number average molecular weight of about 1 kilodalton (kDa) to about 100 kDa as measured by gel permeation chromatography.

[0225] Clause 27. The conjugate of any one of clauses 1-26, or a pharmaceutically acceptable salt thereof, wherein the second block has a number average molecular weight of about 15 kDa to about 100 kDa as measured by gel permeation chromatography.

[0226] Clause 28. The conjugate of any one of clauses 1-27, or a pharmaceutically acceptable salt thereof, wherein the block copolymer is an A-B diblock copolymer.

[0227] Clause 29. The conjugate of any one of clauses 1-28, or a pharmaceutically acceptable salt thereof, wherein the block copolymer has a number average molecular weight of about 16 kDa to about 200 kDa as measured by gel permeation chromatography.

[0228] Clause 30. A composition comprising: a plurality of conjugates of any one of clauses 1-29, or a pharmaceutically acceptable salt thereof, self-assembled into a particle, the particleAttorney Docket No.093386-0030-WO01 comprising a corona and a core, wherein the corona comprises the first block and the core comprises the second block.

[0229] Clause 31. The composition of clause 30, wherein the particle has a diameter of about 10 nm to about 1 μm.

[0230] Clause 32. The composition of clause 30 or 31, further comprising a pharmaceutically acceptable excipient.

[0231] Clause 33. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of clauses 30-32.

[0232] Clause 34. The method of clause 33, wherein the disease or disorder is a cancer, a viral infection, or a bacterial infection.

[0233] Clause 35. A method of modulating RIG-I activity in a cell, the method comprising administering to the cell an effective amount of the composition of any one of clauses 30-32.

[0234] Clause 36. The method of clause 35, wherein the cell is in a subject.

[0235] Clause 37. The method of clause 33 or 35, wherein the subject is human.Attorney Docket No.093386-0030-WO01 Sequences antisense strand RNA (sequence: 5’ – AUA GGC GUA UUA UAC GCG AUU AAC G – 3’) (SEQ ID NO:1), sense strand, 5’-AlexaFluor 647-tagged sense strand (sequence: 5’ – UAU CCG CAU AAU AUG CGC UAA UUG C – 3’) (SEQ ID NO:2) sense strand RNA with phosphorylation (3p) at 5’ end (5ƍ-ppp-CGU UAA UCG CGU AUA AUA CGC CUA U-3ƍ) (SEQ ID NO:3) sense strand with phosphorylation (2p) at 5’ end (5ƍ-pp-CGU UAA UCG CGU AUA AUA CGC CUA U-3ƍ) (SEQ ID NO:4) antisense strand RNA (5ƍ-GCA AUU AGC GCA UAU UAU GCG GAU A-3ƍ) (SEQ ID NO:5) RNA (seq: Cy5*C*G*U UAA UCG CGU AUA AUA CGC CU*A*U (SEQ ID NO:6), asterisks indicating phosphorothioate bonds and Cy5: fluorescent dye) SLR14 (3x P): 5ƍ-ppp-GGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC-3 (SEQ ID NO:7) SLR14 (2x P): 5ƍ-pp-GGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC-3 (SEQ ID NO:8) SLR20 (3x P): 5ƍ-ppp-GGAUCGAUCGAUCGAUCGGCUUCGGCCGAUCGAUCGAUCGAUCC- 3ƍ (SEQ ID NO:9) SLR20 (2x P): 5ƍ-pp-GGAUCGAUCGAUCGAUCGGCUUCGGCCGAUCGAUCGAUCGAUCC-3ƍ (SEQ ID NO:10)

Claims

Attorney Docket No.093386-0030-WO01 CLAIMS What is claimed is:

1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: a block copolymer comprising a first block (A), the first block comprising a recurring hydrophilic unit, and a second block (B), the second block comprising a copolymer, wherein the copolymer comprises a recurring hydrophobic unit and a recurring protonatable unit, wherein the first block, the second block, or both include at least one nucleic acid linking unit; and a nucleic acid retinoic acid-inducible gene I (RIG-I) agonist covalently attached to the nucleic acid linking unit through a linker.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is derived from dimethylacrylamide, oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, hydroxyethyl acrylate, hydroxypropyl acrylate, poly(ethylene glycol) methacrylate, poly(ethylene glycol) acrylate, N-vinyl pyrrolidone (NVP), N- isopropylacrylamide, 2-hydroxyethyl methacrylate methacrylate, acrylamide, methacrylamide, dimethylaminoethyl methacrylate, N-(2-hydroxypropyl) methacrylamide, itaconic acid, acrylic amide, methacrylamide, vinyl alcohol, 3-(methacryloylamino)propyl trimethoxysilane, or a combination thereof.

3. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is of formula (I)wherein Y1is H or CH3, G1is NH, O, or bond, R1is -NRxRy, -alkylene-NRxRy, -heterolkylene, or -heterolkylene-NRxRy, andAttorney Docket No.093386-0030-WO01 Rxand Ryare each independently hydrogen or alkyl.

4. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophobic unit is derived from an alkyl acrylate, an alkyl methacrylate, styrene, isobornyl acrylate, cyclohexyl methacrylate, phenyl methacrylate, fluorinated methacrylate, allyl, methacrylate, vinyl decanoate, dibutyl maleate, N-phenyl maleimide, norbornene, 2- hydroxyethyl methacrylate, 2-octyl acrylate, 3,4-dihydro-2H-pyran, or a combination thereof.

5. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophobic unit is of formula (II)(II), wherein Y2is H or CH3, and R2is C1-12alkyl.

6. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the protonatable unit is derived from 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, N,N-dimethylaminoethyl acrylate, N,N- diethylaminoethyl acrylate, N-vinyl pyrrolidone, N-vinyl imidazole, 3-(dimethylamino)propyl methacrylate, 4-(dimethylamino)butyl methacrylate, 3-aminopropyl methacrylate, 4-aminobutyl methacrylate, N-(3-aminopropyl) methacrylamide, N-(4-aminobutyl) methacrylamide, 2- aminoethyl methacrylate, N-(2-aminoethyl) acrylamide, N-(3-aminopropyl) acrylamide, N-(4- aminobutyl) acrylamide, 4-(dimethylamino)butyl acrylate, aminoethyl vinyl ether, or a combination thereof.

7. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the protonatable unit is of formula (III)Attorney Docket No.093386-0030-WO01(III), wherein Y3is H or CH3, G3is NH or O, X is alkylene, heteroalkylene, alkenylene, or alkynylene, R3is -NRxRy, and Rxand Ryare each independently hydrogen or alkyl.

8. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is derived from a monomer comprising an ethylenically unsaturated group and a linking moiety.

9. The conjugate of claim 8, or a pharmaceutically acceptable salt thereof, wherein the linking moiety comprises a pyridyl disulfide, an azide, a thiol, an amine, a maleimide, or a combination thereof.

10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is of formula (IV)wherein Y4is H or CH3, G4is NH or O, L is a linker, and R4is a nucleic acid RIG-I agonist.Attorney Docket No.093386-0030-WO01 11. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid linking unit is only present in the second block.

12. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker comprises a hypoxia sensitive linker, a reactive oxygen species (ROS) sensitive linker, a pH sensitive linker, a redox sensitive linker, an enzyme sensitive linker, a light sensitive linker, or a combination thereof.

13. The conjugate of claim 12, or a pharmaceutically acceptable salt thereof, wherein the enzyme sensitive linker is a matrix metalloproteinase sensitive linker, a p-aminobenzyl alcohol system linker, a cathepsin sensitive linker, a beta-glucuronidase sensitive linker, an esterase sensitive linker, or a combination thereof.

14. The conjugate of claim 12, or a pharmaceutically acceptable salt thereof, wherein the redox sensitive linker is a glutathione sensitive linker, a nitroreductase / NADH sensitive linker, or a combination thereof.

15. The conjugate of claim 12, or a pharmaceutically acceptable salt thereof, wherein the pH sensitive linker is a hydrazone, a silyl ether, a low pH sensitive linker, or a combination thereof.

16. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker is non-cleavable.

17. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist comprises a nucleic acid duplex having at least two phosphoryl groups attached to a 5ƍ end thereof.

18. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a double stranded nucleic acid molecule.

19. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a single-stranded nucleic acid molecule.Attorney Docket No.093386-0030-WO01 20. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid duplex comprises a hairpin loop RNA molecule.

21. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the at least two phosphoryl groups are selected from the group consisting of a diphosphate group and a triphosphate group.

22. The conjugate of claim 17, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist is covalently attached to the nucleic acid linking unit through a terminal end of the nucleic acid RIG-I agonist that is distal from the at least two phosphoryl groups.

23. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid RIG-I agonist comprises 10 to 100 base pairs.

24. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the block copolymer comprises the first block at about 10% to about 90% by weight of the block copolymer.

25. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the block copolymer comprises the second block at about 10% to about 90% by weight of the block copolymer.

26. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the first block has a number average molecular weight of about 1 kilodalton (kDa) to about 100 kDa as measured by gel permeation chromatography.

27. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the second block has a number average molecular weight of about 15 kDa to about 100 kDa as measured by gel permeation chromatography.

28. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the block copolymer is an A-B diblock copolymer.Attorney Docket No.093386-0030-WO01 29. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the block copolymer has a number average molecular weight of about 16 kDa to about 200 kDa as measured by gel permeation chromatography.

30. A composition comprising: a plurality of conjugates according to claim 1, or a pharmaceutically acceptable salt thereof, self-assembled into a particle, the particle comprising a corona and a core, wherein the corona comprises the first block and the core comprises the second block.

31. The composition of claim 30, wherein the particle has a diameter of about 10 nm to about 1 μm.

32. The composition of claim 30, further comprising a pharmaceutically acceptable excipient.

33. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of claim 30.

34. The method of claim 33, wherein the disease or disorder is a cancer, a viral infection, or a bacterial infection.

35. A method of modulating RIG-I activity in a cell, the method comprising administering to the cell an effective amount of the composition of claim 30.

36. The method of claim 35, wherein the cell is in a subject.

37. The method of claim 33, wherein the subject is human.

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