Modulation of RIG-i using RNA nanostructures

Abstract

The present disclosure relates to nanostructures capable of regulating intracellular immunomodulatory signaling and methods of using the nanostructures.

Description

[0001] MODULATION OF RIG-I USING RNA NANOSTRUCTURES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 762,420, filed February 24, 2025, which is hereby incorporated by reference in its entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under 1AY2AX000031 awarded by Advanced Research Projects Agency for Health (ARPA-H). The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (H049870848WO00-SEQ-HJD.xml; Size: 161,883 bytes; and Date of Creation: February 19, 2026) are herein incorporated by reference in their entirety.

[0008] BACKGROUND

[0009] Modulation of intracellular signaling presents an opportunity to control gene expression and protein production within a cell. Many diseases and disorders, including cancer and viral infections, rely on complex intracellular signaling pathways that can be targeted by synthetic intervention. Identification of compositions and methods to modulate efficiently and potently intracellular signaling remains laborious and cost prohibitive.

[0010] SUMMARY

[0011] Aspects of the technology relate to a nanostructure comprising multiple double- stranded ribonucleic acid (dsRNA) arms, wherein the nanostructure regulates (e.g., activates or inhibits) intracellular immunomodulatory signaling.

[0012] In some embodiments, the nanostructure comprises RNA. For example, the nanostructure can include only RNA or a combination of RNA and DNA.

[0013] In some embodiments, the nanostructure is a nanostar comprising at least three, at least four, or at least five dsRNA arms. The dsRNA “arms” of a nanostar (e.g., RNA nanostar) include doublestranded RNA domains of the nanostar (e.g., no longer than 100 nucleotide base pairs). Thus, each “arm” of a nanostar can be considered a “dsRNA domain” of a nanostar. A single- stranded (ss) RNA domain binds to another ssRNA domain to form a dsRNA domain. In some embodiments, a nanostructure is a nanostar comprising at least three dsRNA arms (i.e., three dsRNA domains). Anexample of a nanostructure that includes three dsRNA arms is depicted in FIG. 7. In some embodiments, a nanostructure is a nanostar comprising at least four dsRNA arms (i.e., or four dsRNA domains). In some embodiments, a nanostructure is a nanostar comprising at least five dsRNA arms (i.e., or five dsRNA domains). Nanostars with six or more dsRNA arms (dsRNA domains) are also contemplated herein.

[0014] In some embodiments, a three-arm RNA nanostar comprises: a first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; and a third RNA strand comprising a fifth domain and a sixth domain, wherein the first domain of the first RNA strand is bound to the sixth domain of the third RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm.

[0015] In some embodiments, a four-arm RNA nanostar comprises: a first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; a third RNA strand comprising a fifth domain and a sixth domain; and a fourth RNA strand comprising a seventh domain and an eighth domain, wherein the first domain of the first RNA strand is bound to the eighth domain of the fourth RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm, the sixth domain of the third strand is bound to the seventh domain of the fourth strand to form a fourth dsRNA arm.

[0016] In some embodiments, a five-arm RNA nanostar comprises: a first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; a third RNA strand comprising a fifth domain and a sixth domain; a fourth RNA strand comprising a seventh domain and an eighth domain; and a fifth RNA strand comprising a ninth domain and a tenth domain, wherein the first domain of the first RNA strand is bound to the tenth domain of the fifth RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm, the sixth domain of the third strand is bound to the seventh domain of the fourth strand to form a fourth dsRNA arm, and the eight domain of the fourth strand is bound to the ninth domain of the fifth strand to form a fifth dsRNA arm.

[0017] In some embodiments, the nanostructure comprises DNA. For example, the nanostructure can include only DNA (e.g., a DNA nanocube or other shape) and further include dsRNA arms linked to the DNA.In some embodiments, the nanostructure is a DNA origami nanostructure. DNA origami refers to a technique that uses the self-assembly properties of DNA to create precise, nanoscale structures. It generally involves designing a long single- stranded DNA scaffold (e.g., from the M13 bacteriophage) and using shorter “staple” strands to fold it into a desired shape through complementary base pairing.

[0018] In some embodiments, at least one of the dsRNA arms comprises a modification, relative to a naturally occurring (non-modified) RNA arm.

[0019] In some embodiments, the modification is at a 5’ end of at least one of the dsRNA arms. In some embodiments, the modification is at a 3’ end of at least one of the dsRNA arms. In some embodiments, the modification is between a a5’ end and a 3’ end of at least one of the dsRNA arms.

[0020] In some embodiments, the modification is selected from sugar modifications, base modifications, and phosphate (backbone) modifications. In some embodiments, the modification is a sugar modification. In some embodiments, the modification is a base modification. In some embodiments, the modification is a phosphate (backbone) modification.

[0021] In some embodiments, the modification is selected from: an unpaired nucleotide, for example selected from unpaired adenine (A) and unpaired uracil (U); a non-canonical nucleotide, for example, selected from Inosine (I), 5 -methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), queuosine (Q), azidothymidine (AZT), 2'-fluoro-modified nucleotides (e.g., 2'-F-uridine), and locked nucleic acids (LNAs); and a chemically modified nucleotide, for example, selected from inosine (I), 5 -methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), azidothymidine (AZT), and 2'-fluoro-modified nucleotides, optionally 2'-F-uridine. In some embodiments, the modification is an unpaired nucleotide. In some embodiments, the modification is an unpaired A. In some embodiments, the modification is an unpaired U. In some embodiments, the modification is an unpaired C. In some embodiments, the modification is an unpaired G. In some embodiments, the modification is a non-canonical nucleotide. In some embodiments, the modification is a chemically modified nucleotide.

[0022] In some embodiments, the modification is a base modification selected from 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), 2-thiouridine (s2U), and inosine (I). In some embodiments, the modification is a sugar modification selected from 2'-O-methyl (2'-0Me), 2'-fluoro (2'-F), locked nucleic acid (LNA), and peptide nucleic acid (PNA). In some embodiments, the modification is a phosphate modification selected from phosphorothioate (PS), cyclization, and a-thio-triphosphates.

[0023] In some embodiments, at least two of the dsRNA arms are linked to each other via Watson-Crick base pairing.In some embodiments, at least one of the dsRNA arms comprises a 5'-terminal monophosphate. In some embodiments, at least one of the dsRNA arms comprises a 5'-terminal diphosphate. In some embodiments, at least one of the dsRNA arms comprises a 5'-terminal triphosphate. In some embodiments, at least one of the dsRNA arms comprises a 5'-terminal hydroxyl group.

[0024] In some embodiments, at least one of the dsRNA arms has a length of about 10-60 base pairs, optionally 11-52 base pairs.

[0025] In some embodiments, each of the multiple dsRNA arms independently comprises a nucleotide sequence of any one of SEQ ID NOs: 1-167.

[0026] In some embodiments, the nanostructure is immunostimulatory. In some embodiments, the dsRNA arms bind to RIG-I. In some embodiments, the dsRNA arms activate a RIG-I-IRF3 pathway, thereby inducing interferon (IFN) production. In some embodiments, the IFN is a type I, type II, or type III IFN.

[0027] In some embodiments, the nanostructure is immunosuppressive. In some embodiments, at least one of the dsRNA arms comprises a nick, for example, at a 5’ end or a 3’ end of a dsRNA arm. In some embodiments, the dsRNA arms bind to RIG-I. In some embodiments, the dsRNA arms (e.g., having a nick) inhibit activation of a RIG-I- IRF3 pathway.

[0028] In some embodiments, at least one of the dsRNA arms comprises a linker, for example, at a 5’ end or a 3’ end of a dsRNA arm. In some embodiments, the linker is selected from: (i) unpaired nucleotides, optionally unpaired A and U; (ii) chemically modified nucleotides, (iii) polyethylene glycol (PEG) spacers, (iv) nucleotide spacers, and (v) amino acid spacers. In some embodiments, the linker comprises an unpaired nucleotide. In some embodiments, the linker comprises a chemically modified nucleotide. In some embodiments, the linker comprises PEG. In some embodiments, the linker comprises a nucleotide spacer.

[0029] Other aspects of the technology relate to a ribonucleic acid (RNA) nanostar comprising three dsRNA arms (arms) described herein. In some embodiments, the RNA nanostar comprises four dsRNA arms. In some embodiments, the RNA nanostar comprises five dsRNA arms. In some embodiments, the RNA nanostar comprises six dsRNA arms.

[0030] Yet other aspects of the technology relate to a scaffold comprising multiple dsRNA arms described herein. A scaffold includes a structured framework that provides stability, organization, and / or spatial control for nucleic acids and other arms. A scaffold can be made from DNA, RNA, or synthetic materials, such as a polymer. In some embodiments, a scaffold comprises DNA. In some embodiments, a scaffold comprises RNA. In some embodiments, a scaffold comprises a protein. In some embodiments, a scaffold comprises a polymer or other synthetic material.Still other aspects of the technology relate to a pharmaceutical composition comprising a nanostructure described herein and a pharmaceutically acceptable excipient.

[0031] Some aspects of the technology relate to a nanoparticle comprising a nanostructure of described herein.

[0032] Other aspects of the technology relate to a method comprising contacting a cell (e.g., a mammalian cell, such as a human cell) with a nanostructure described herein.

[0033] Still other aspects of the technology relate to a method of regulating intracellular immunomodulatory pathway signaling in a subject, the method comprising administering to the subject (e.g., a mammalian subject, such as a human subject) a nanostructure, pharmaceutical composition, or nanoparticle described herein. In some embodiments, the subject has or is suspected of having (i) an immune disorder, optionally an autoimmune disorder, (ii) cancer, or (iii) a viral infection. In some embodiments, the subject has an immune disorder. In some embodiments, the subject has an autoimmune disorder. In some embodiments, the subject has cancer. In some embodiments, the subject has a viral infection.

[0034] Some aspects relate to a DNA nanocube comprising: one or more DNA molecule(s) assembled through complementary base pairing into a nanocube; and dsRNA molecules, optionally about 25 to about 75 dsRNA molecules, covalently linked to the one or more DNA molecule(s).

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this disclosure. The accompanying drawings are not intended to be drawn to scale.

[0037] FIG. 1 shows the mechanism by which a duplex RNA induces interferon (IFN) production via RIG-I activation.

[0038] FIG. 2 provides an examples of a RIG-I molecule binding to a double-stranded RNA (dsRNA) molecule.

[0039] FIG. 3 provides an example of four RIG-I molecules binding a dsRNA molecule (left) and an AlphaFold3 model of four RIG-I molecules binding a dsRNA molecule (right).

[0040] FIG. 4 shows interferon activity as measured by luciferase activation following induction by 52-base pair dsRNA molecules.

[0041] FIG. 5 provides AlphaFold3 models of four RIG-I molecules binding dsRNA molecules of varying lengths (52 base pairs, 44 base pairs, and 36 base pairs).

[0042] FIG. 6 shows interferon activity as measured by luciferase activation following induction by dsRNA molecules of varying lengths (52 base pairs, 44 base pairs, and 36 base pairs).FIG. 7 provides an example of four RIG-I molecules binding an RNA nanostructure (left) and an AlphaFold3 model of four RIG-I molecules binding an RNA nanostructure (right).

[0043] FIG. 8 shows interferon activity as measured by luciferase activation following induction by a dsRNA monomer (RNA-1), a full-length dsRNA duplex (RNA-111), and a 3-arm RNA nanostructure (RNA-113).

[0044] FIG. 9 shows A549 assay data measuring interferon and NF-KB activity following induction by RNA nanostructures with dsRNA arms of varying lengths (26 base pairs, 24 base pairs, 22 base pairs, 20 base pairs, 18 base pairs, 16 base pairs, and 14 base pairs) with or without the presence of RIG-I.

[0045] FIG. 10 shows A549 assay data measuring interferon and NF-KB activity following induction by RNA nanostructures with dsRNA arms of varying lengths (e.g., half-length and full-length) with or without the presence of RIG-I.

[0046] FIG. 11 shows A549 assay data measuring interferon and NF-KB activity following induction by RNA nanostructures with dsRNA arms with or without linkers and with or without the presence of RIG-I.

[0047] FIG. 12 shows A549 assay data measuring interferon and NF-KB activity following induction by RNA nanostructures with dsRNA arms with or without linkers, with or without nicks, and with or without the presence of RIG-I.

[0048] FIG. 13 shows A549 assay data measuring interferon and NF-KB activity following induction by RNA nanostructures with dsRNA arms with or without scrambled sequences and with or without the presence of RIG-I.

[0049] FIG. 14 shows a schematic of encapsulating dsRNA or RNA nanostructures in liquid nanoparticles (LNPs) (left) and hydrodynamic and zeta potential data for the LNPs containing dsRNA or RNA nanostructures (right).

[0050] FIG. 15 shows an experimental outline for delivering LNPs containing dsRNA or RNA nanostructures to mice (top) and in vivo data showing interferon-a induction by dsRNA or RNA nanostructures (bottom).

[0051] FIG. 16 shows impact of 5’ triphosphorylation on IFN-luciferase activity in A549 dual reporter cells for RNA-1 (top left), RNA-111 (top right), and RNA-113 (bottom left). Molar concentrations are reported as concentration of two dsRNA ends to facilitate comparison between duplex RNA and nanostars (bottom right).

[0052] FIG. 17 shows impact of inclusion of phosphorothioate linkages on IFN-luciferase activity in A549 dual reporter cells for RNA-111 (left) and RNA-113 (right). Molar concentrations are reported as concentration of 2 dsRNA ends to facilitate comparison between duplex RNA and nanostars.FIG. 18 shows impact of 3' sugar modifications on IFN-luciferase activity in A549 dual reporter cells for RNA-111.

[0053] FIG. 19 shows comparison of the immuno stimulatory activities of different RNA nanostars. IFN luciferase reporter activity of A549-Dual cells transfected with indicated RNAs formulated in TransIT-X2 for 48 h.

[0054] FIGs. 20A-20C shows biodistribution of organ-tropic LNPs. FIG. 20A shows RNA-lCy5 encapsulated in either lung- or liver-tropic LNPs administered systemically into C57BL / 6 mice. Four hours post dosing major organs were dissected and imaged by IVIS imaging system to detect labeled RNA-1. FIG. 20B shows representative IVIS images showing Cy5 fluorescence signal distribution within the liver, lungs, spleen, kidneys, and heart post-dissection. FIG. 20C shows quantitative analysis of IVIS images presenting the total radiance efficiency signal from the organs (left). Plasma IFNa levels collected 2 h post injection to correlate biodistribution with RNA-1 innate induction activity (right).

[0055] FIG. 21 shows physical characterization of dsRNA analogs full length and nanostar (RNA-111 and RNA-113) formulated in liver-tropic- or lung-tropic-LNPs (by the addition of the DOTAP lipid) (left). Size was measured by DLS (middle), and surface charge was measured by Zeta potential (right).

[0056] FIGs. 22A-22B show in vitro activity of dsRNA analogs. FIG. 22A shows RIG-I activation in A549 dual reporter cells mediated by full length dsRNA and nanostars (RNA-111 and RNA-113) encapsulated in lung- or liver- tropic LNPs compared to previous lead analog RNA-1. Note the table presenting the EC50 of each analog and formulation. FIG. 22B shows cell viability measured by the MTS assay.

[0057] FIG. 23 shows RIG-I activation in A549 dual reporter cells in response to DNA nanocubes loaded with a 52 bp dsRNA analog formulated in TransIT-X2 subsequent to transfection.

[0058] FIG. 24 shows IFN-luciferase reporter activity in A549 dual reporter cells following transfection with RNA-233 (an IVT-synthesized RNA nanostar), RNA-236 (a chemically synthesized 5’-OH version of RNA-233), RNA-113 (a chemically synthesized 3-arm nanostar), and RNA-194 (a chemically synthesized 5’-ppp version of RNA-113), demonstrating that IVT-produced RNA-233 exhibits comparable potency to chemically synthesized 5’-ppp-RNA-113.

[0059] FIGs. 25A-25C show plasma levels of interferon-alpha (IFN-a), interferon-beta (IFN-P), and tumor necrosis factor-alpha (TNF-a) at 2 hours following intravenous administration of dendrimer formulations and poly(beta-amino ester) (pBAE) formulations complexed with RNA-113 in C57BL / 6 mice, demonstrating that both dendrimer and pBAE formulations induce interferon and TNF-a responses.FIG. 26 shows kinetics of interferon-alpha (IFN-a), interferon-beta (IFN-P), interferongamma (IFN-y), and tumor necrosis factor-alpha (TNF-a) in plasma over 24 hours following intravenous administration of dendrimer formulations, pBAE formulations, and liver-tropic lipid nanoparticles (LNPs) complexed with RNA- 113, demonstrating peak cytokine levels at 2 hours postinjection with return to baseline by 24 hours.

[0060] FIGs. 27A-27B show tumor growth inhibition in a subcutaneous Bl 6-F 10 melanoma model following systemic administration of dendrimer formulations (dendrimer-RNA complex and dendrimer 3’ conjugate) and pBAE formulations (linear and branched pBAE) complexed with RNA-113 compared to liver-tropic LNP formulation and PBS control, demonstrating therapeutic efficacy of dendrimer and pBAE formulations in reducing tumor volume.

[0061] FIGs. 28A-28B show body weight measurements over the treatment period for mice treated with dendrimer formulations and pBAE formulations complexed with RNA-113 compared to livertropic LNP formulation and untreated controls in the Bl 6-F 10 melanoma model, demonstrating that dendrimer and pBAE formulations were well tolerated with minimal body weight loss.

[0062] DETAILED DESCRIPTION

[0063] Intracellular signaling is the process by which cells respond to their environment and extracellular cues. It involves a network of signaling pathways that transmit signals from the cell surface to the cellular nucleus, where they influence cellular processes. Prokaryotic and eukaryotic cells have evolved to incorporate various forms of intracellular signaling into a network of signaling pathways, including immunomodulatory intracellular signaling in which intracellular signaling induces or inhibits production of immune molecules. One such form of immunomodulatory intracellular signaling involves the binding of proteins to double- stranded RNA (dsRNA) molecules and dsRNA-like molecules. Proteins that bind to dsRNA molecules and dsRNA-like molecules (e.g., binding proteins) can undergo, for example, conformational changes that induce further downstream signaling or protein multimerization. Protein multimerization is the process by which multiple protein molecules join together through covalent or non-covalent bonds to form large biomacromolecules. Formation of such biomacromolecules can then result in a robust downstream signaling pathway.

[0064] For multiple protein molecules (e.g., proteins) to join together to form large biomacromolecules, the proteins are often in close proximity to one another. Without being bound by theory, one such mechanism by which multiple proteins become close enough for multimerization to occur is through the binding of each protein molecule to a single “core” molecule, for example, a double- stranded RNA (dsRNA) molecule. Once multiple proteins bind to a single dsRNA molecule, the proteins, including “multimerizing binding proteins,” can multimerize and propagate downstreamimmunomodulatory intracellular signaling. The data provided herein demonstrates, surprisingly, that the intensity of downstream signaling can be increased by bringing together multiple dsRNA arms, for example, to form a nanostructure (see, e.g., FIG. 7). This enables efficient binding of multimerizing binding proteins to the RNA nanostructure and a robust downstream signal.

[0065] Accordingly, aspects of the present disclosure provide RNA nanostructures that can regulate intracellular immunomodulatory signaling. Thus, aspects of the present disclosure provide RNA nanostructures that are immunomodulatory. In some embodiments, the RNA nanostructures described herein induce a more robust downstream signaling response than a single dsRNA molecule. The present disclosure also provides, in some aspects, compositions comprising RNA nanostructures and methods of delivering compositions comprising RNA nanostructures to subjects in need of treatment.

[0066] Immunomodulatory Intracellular Signaling

[0067] The technology described herein is based, at least in part, on data demonstrating the immuno stimulatory nature of bringing together double- stranded RNA (dsRNA) molecules to form a nanostructure. The intensity of downstream signaling in immunomodulatory pathways, such as the RIG-I pathway, can be increased by bringing together, for example, at least three or at least four dsRNA arms to form a nanostar (see, e.g., FIG. 7), which enables efficient binding of multimerizing binding proteins, such as RIG-I proteins, to the RNA nanostar and a robust downstream signal. Surprisingly, these RNA nanostars exhibited potent activation of interferon in vivo, demonstrating the ability of multi-arm RNA nanostars to activate immunomodulatory signaling pathways in vivo to induce a downstream signaling response.

[0068] Immunomodulatory intracellular signaling is a process that allows cells to respond to their environment and extracellular cues by modifying their gene expression, protein expression, and protein modifications to increase or decrease the production of immune molecules.

[0069] Immunomodulatory intracellular signaling can be induced by activation of extracellular receptors or cues within a cell. The term “immunomodulatory,” as used herein, includes any agent that changes (e.g., increases or decreases; activates or inhibits) the production of immune molecules. The term “intracellular,” as used herein, includes anything that occurs within a cell. The term “intracellular signaling,” as used herein, includes a series of biochemical reactions that occur within a cell in response to a signal. A series of biochemical reactions that occur within a cell can also be referred to as an “intracellular signaling pathway.”

[0070] One such immunomodulatory intracellular pathway is the RIG-I- IRF3 signaling pathway that, when activated, increases expression of interferon. Interferons (IFN or IFNs) are a class of pleiotropic cytokines that are produced and released by immune cells as a part of the innate immuneresponse to infections. IFNs have been used as a therapeutic in the treatment of autoimmune diseases (e.g., multiple sclerosis and lupus), many types of cancer, and viral infections. In this pathway, RIG-I, a multimerizing binding protein, multimerizes and activates mitochondrial antiviral- signaling protein (MAVS), which in turn induces expression of IFN (FIG. 1). The term “RIG-I,” as used herein, includes retinoic acid-inducible gene I, a cytosolic pattern recognition receptor that induces interferon production. The phrase “multimerizing binding protein,” as used herein, includes proteins that form multimers, or large biomacromolecules, by connecting multiple proteins with covalent or non-covalent bonds. In some embodiments, multimerization of multimerizing binding proteins is required to induce a downstream signaling pathway. The phrase “induces interferon production” or “increases interferon production,” as used herein, encompasses interferon production is increased by at least three-fold following administration of an RNA nanostructure as described herein or following contacting of a cell, population of cells, tissue or organism with such RNA nanostructure. In some embodiments, an increase in interferon production can be at least four-fold, at least fivefold, at least 10-fold, at least 15 -fold, at least 20-fold or more. Interferon production can be measured, for example, by immunoassay (e.g., ELISA, immunoprecipitation, etc.), biological reporter assay or other assays as known in the art.

[0071] Immunomodulatory effects of IFNs are exerted on a wide range of cell types expressing receptors for the IFN polypeptide. Downstream effects of IFN allow for the regulation of the immune system by activating signal transducer and activator of transcription (STAT) complexes and other signaling molecules, for example. STATs are a family of transcription factors that regulate the expression of a number of immune system genes. Interferon signaling pathways are known in the art. See, e.g., Muller U, et al. Science (1994); Honda et al, Immunity, 25, 349-360 (2006); and Marchetti M, et al. Mol Biol Cell (2006); Lee and Ashkar, Front. Immunol., 2018; and Platanias LC. Nat Rev Immunol. (2005) 5:375-86.

[0072] The induction of IFN production plays a role in human immune responses by ‘interfering’ with viral replication. Induction of IFN gene expression can lead to increased cellular resistance to infection, including but not limited to viral infection, by activating immune cells, (e.g., natural killer cells and macrophages), and increasing host defenses by upregulating antigen presentation by virtue of increasing the expression of major histocompatibility complex (MHC) antigens. There are many types of IFN genes and proteins, which are typically divided among three classes in humans: Type I IFN (IFN-a, IFN-b, IFN-e, IFN-k and IFN-co), Type II IFN (IFN-g), and Type III IFN. These IFNs participate in fighting infection and regulating the immune system.

[0073] The regulation of IFN expression is complex and tightly controlled by interferon regulatory factors (IRFs). IRFs are a family of transcription factors that are involved in many aspects of the immune response, including development and differentiation of immune cells and regulatingresponses to pathogens. The functional role and signaling pathways of IRFs are known in the art, see e.g., Jefferries, Front. Immunol., 2019; and Bustamante et al. Clinical Immunology, 5th ed. (2019). One such IRF, IRF3, is a positive regulator of type I interferon gene induction. IRF3 is an intracellular polypeptide that is activated downstream of the pattern recognition receptor, RIG-I, an intracellular RNA sensor. In particular, IRF3 can directly induce the expression of cytokines, such as IFN-b and in addition to type I IFNs, CXCL10, RANTES, ISG56, IL-12p35, IL-23, and IL-15, whilst inhibiting IE- 12b and TGF-b.

[0074] The interferon pathways are involved in many diseases, including pathogenic infections caused by viruses, bacteria, fungi and parasites, as well as cancers, and autoimmune diseases. In many instances, an increase in interferon production is part of the natural response to infection, such that treatments that further promote such production can assist in fighting the infection. In other instances, notably some viral infections, including infection with the SARS-CoV-2 coronavirus, among others, the body’s interferon response is not activated or is suppressed relative to that seen with other viruses or pathogens, such that a treatment that promotes interferon production can assist in fighting the infection. Therefore, the RNA nanostructures described herein can be used to prevent, mitigate, and / or treat diseases that benefit from or are treatable with agents that include interferons or that promote interferon production.

[0075] Accordingly, aspects of the present disclosure relate to induction of IFN production in a cell by delivering to a cell any nanostructure (e.g., RNA nanostructure) described herein. In some embodiments, a nanostructure comprising multiple double- stranded ribonucleic acid (dsRNA) arms binds the multimerizing binding protein RIG-I, which in turn activates the IFN signaling pathway. In other embodiments, a nanostructure comprising multiple dsRNA arms binds RIG-I and inhibits RIG-I multimerization, thus inhibiting the IFN signaling pathway. The dsRNA of such nanostructures, in some embodiments are nicked.

[0076] A non-limiting example of a RIG-I amino acid sequence is provided as UniProt Accession No. Q6Q899 and SEQ ID NO: 168. Amino acid residues 1-172 (the CARD 1 / 2 domains) are responsible for downstream signaling activation, and the remainder of the protein is involved in RNA binding.

[0077] MTAEQRQNLQAFRDYIKKILDPTYILSYMSSWLEDEEVQYIQAEKNNKGPMEAASLFLQYLLKLQSEGWFQAF LDALYHAGYCGLCEAIESWDFQKIEKLEEHRLLLRRLEPEFKATVDPNDILSELSECLINQECEEIRQIRDTK GRMAGAEKMAECLIRSDKENWPKVLQLALEKDNSKFSELWIVDKGFKRAESKADEDDGAEASSIQIFIQEEPE CQNLSQNPGPPSEASSNNLHSPLKPRNYQLELALPAKKGKNTIICAPTGCGKTFVSLLICEHHLKKFPCGQKG KWFFANQIPVYEQQATVFSRYFERLGYNIASISGATSDSVSVQHIIEDNDIIILTPQILVNNLNNGAIPSLS VFTLMIFDECHNTSKNHPYNQIMFRYLDHKLGESRDPLPQVVGLTASVGVGDAKTAEEAMQHICKLCAALDAS VIATVRDNVAELEQVVYKPQKISRKVASRTSNTFKCI ISQLMKETEKLAKDVSEELGKLFQIQNREFGTQKYE QWIVGVHKACSVFQMADKEEESRVCKALFLYTSHLRKYNDALI ISEDAQMTDALNYLKAFFHDVREAAFDETE RELTRRFEEKLEELEKVSRDPSNENPKLRDLYLVLQEEYHLKPETKTILFVKTRALVDALKKWIEENPALSFL KPGILTGRGRTNRATGMTLPAQKCVLEAFRASGDNNILIATSVADEGIDIAECNLVILYEYVGNVIKMIQTRG RGRARDSKCFLLTSSADVIEKEKANMIKEKIMNESILRLQTWDEMKFGKTVHRIQVNEKLLRDSQHKPQPVPDKENKKLLCGKCKNFACYTADIRVVETSHYTVLGDAFKERFVCKPHPKPKIYDNFEKKAKIFCAKQNCSHDWGI FVRYKTFEIPVIKIESFWEDIVSGVQNRHSKWKDFHFERIQFDPAEMSV (SEQ ID NO: 168) Another example of an immunomodulatory intracellular signaling pathway is the NF-KB signaling pathway. The NF-KB signaling pathway regulates cellular responses and belongs to the category of “rapid- acting” primary transcription factors, i.e., transcription factors that are present in cells in an inactive state and do not require new protein synthesis in order to become activated. This allows NF-KB to be a first responder to harmful cellular stimuli. Accordingly, in some embodiments, an RNA nanostructure described herein is used to activate the NF-KB signaling pathway. In some embodiments, an RNA nanostructure described herein is used to reduce activation of the NF-KB signaling pathway.

[0078] Aspects of the present disclosure relate, at least in part, to the use of the RNA nanostructures described herein to modulate any intracellular signaling that can be induced by double- stranded RNA (dsRNA).

[0079] Nanostructures With dsRNA Arms

[0080] As discussed elsewhere herein multi “arm” RNA nanostructures (e.g., three-arm and / or four-arm RNA nanostars) are capable of regulating intracellular immunomodulatory signaling - for example, increasing RIG-I signaling to in turn increase interferon production. Aspects of the technology relate to nanostructures comprising multiple double- stranded ribonucleic acid (dsRNA) arms, wherein the nanostructure regulates (e.g., activates or inhibits) intracellular immunomodulatory signaling. Nanostructures include at least one dimension in the nanometer scale, typically ranging from 1 to 100 nanometers (nm), for example. Nuclei acid nanostructures include “self-assembled” structures made from DNA, RNA, and / or synthetic nucleic acids designed at the nanometer scale. These structures leverage the predictable base-pairing rules of nucleotides (A-T, G-C for DNA; A-U, G-C for RNA) to form precise and programmable nanoscale shapes. Non-limiting examples of nucleic acid nanostructures include DNA nanostructures (e.g., DNA origami, DNA tiles and lattices, etc.), RNA nanostructures, and hybrid DNA-RNA nanostructures.

[0081] The present disclosure provides, in some aspects, RNA nanostars that are useful for regulating intracellular immunomodulatory signaling. The term “RNA nanostar,” as used herein, includes multiple double- stranded RNA (dsRNA) domains (referred to as “arms”) linked to each other. In some embodiments, an RNA nanostar comprises multiple RNA stands bound to each other to form multiple dsRNA domains (see, e.g., FIG. 7). A “domain” is simply a shorter region of nucleotide (e.g., contiguous nucleotides) within a longer nucleic acid molecule. Herein, a singlestranded nucleic acid can have one domain that binds to another domain of another single- stranded nucleic acid to form a double- stranded domain within a longer double- stranded nucleic acidmolecule. A dsRNA arm includes a dsRNA domain that has a “free” 5’ or 3’ end - either a blunt end or an end with overhanging nucleic acids (e.g., 1-5 nucleotide overhang). FIG. 7 depicts a nanostar with three arms, each having a free end (terminus). A domain, such as a dsRNA domain (e.g., arm of a nanostar), in some embodiments, has a length of about 10 to about 100 nucleotides. For example, a dsRNA domain (e.g., arm of a nanostar) can have a length of about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, or about 10 to about 50 nucleotides. In some embodiments, domain, such as a dsRNA domain (e.g., arm of a nanostar), has a length of shorter than 100 nucleotides, shorter than 90 nucleotides, shorter than 80 nucleotides, shorter than 70 nucleotides, or shorter than 60 nucleotides. “Regulating,” or “regulation of,” intracellular immunomodulatory signaling includes increasing / activating or decreasing / inhibiting intracellular immunomodulatory signaling. In some embodiments, RNA nanostars can activate RIG-I signaling or increase RIG-1 signaling. In other embodiments, RNA nanostars (e.g., nicked designs) can inhibit RIG-I signaling or decrease RIG-1 signaling.

[0082] In some embodiments, an RNA nanostar is a 3-arm nanostar. In some embodiments, an RNA nanostar comprises: a first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; and a third RNA strand comprising a fifth domain and a sixth domain, wherein the first domain of the first RNA strand is bound to the sixth domain of the third RNA strand, the second domain of the first RNA strand is bound to the third domain of the second RNA strand, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand.

[0083] In some embodiments, an RNA nanostar is a 4-arm nanostar. In some embodiments, an RNA nanostar comprises: first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; a third RNA strand comprising a fifth domain and a sixth domain; and a fourth RNA strand comprising a seventh domain and an eighth domain, wherein the first domain of the first RNA strand is bound to the eighth domain of the fourth RNA strand, the second domain of the first RNA strand is bound to the third domain of the second RNA strand, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand, the sixth domain of the third strand is bound to the seventh domain of the fourth strand.

[0084] In some embodiments, an RNA nanostar is a 4-arm nanostar. In some embodiments, an RNA nanostar comprises: a first RNA strand comprising a first domain and a second domain; a second RNA strand comprising a third domain and a fourth domain; a third RNA strand comprising a fifth domain and a sixth domain; a fourth RNA strand comprising a seventh domain and an eighth domain; and a fifth RNA strand comprising a ninth domain and a tenth domain, wherein the first domain of the first RNA strand is bound to the tenth domain of the fifth RNA strand, the second domain of the first RNA strand is bound to the third domain of the second RNA strand, the fourthdomain of the second RNA strand is bound to the fifth domain of the third RNA strand, the sixth domain of the third strand is bound to the seventh domain of the fourth strand, and the eight domain of the fourth strand is bound to the ninth domain of the fifth strand.

[0085] A nanostructure (e.g., a DNA or RNA nanostructure) herein comprises multiple (at least two) dsRNA arms. In some embodiments, a nanostructure comprises two dsRNA arms. In some embodiments, a nanostructure comprises three dsRNA arms. In some embodiments, a nanostructure comprises four dsRNA arms. In some embodiments, a nanostructure comprises five dsRNA arms. In some embodiments, a nanostructure comprises at least two, at least three, at least four, or at least five dsRNA arms.

[0086] In some embodiments, each arm of the RNA nanostructure independently comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 1-167. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 1. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 2. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 3. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 4. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 5. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 6. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 7. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 8. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 9. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 10. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 11. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 12. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 13. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 14. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 15. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 16. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 17. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 18. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 19. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 20. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 21. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 22. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 23. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 24. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 25. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 26. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 27. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 28. In some embodiments, a dsRNA arm comprises the sequence of SEQID NO: 29. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 30. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 31. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 32. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 33. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 34. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 35. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 36. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 37. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 38. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 39. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 40. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 41. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 42. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 43. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 44. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 45. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 46. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 47. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 48. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 49. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 50. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 51. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 52. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 53. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 54. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 55. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 56. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 57. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 58. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 59. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 60. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 61. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 62. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 63. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 64. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 65. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 66. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 67. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 68. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 69. In some embodiments, a dsRNA arm comprises thesequence of SEQ ID NO: 70. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 71. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 72. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 73. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 74. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 75. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 76. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 77. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 78. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 79. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 80. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 81. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 82. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 83. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 84. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 85. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 86. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 87. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 88. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 89. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 90. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 91. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 92. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 93. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 94. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 95. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 96. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 97. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 98. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 99. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 100. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 101. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 102. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 103. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 104. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 105. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 106. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 107. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 108. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 109. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 110. In some embodiments, a dsRNA armcomprises the sequence of SEQ ID NO: 111. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 112. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 113. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 114. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 115. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 116. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 117. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 118. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 119. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 120. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 121. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 122. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 123. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 124. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 125. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 126. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 127. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 128. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 129. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 130. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 131. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 132. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 133. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 134. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 135. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 136. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 137. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 138. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 139. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 140. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 141. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 142. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 143. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 144. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 145. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 146. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 147. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 148. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 149. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 150. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 151. In some embodiments, adsRNA arm comprises the sequence of SEQ ID NO: 152. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 153. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 154. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 155. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 156. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 157. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 158. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 159. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 160. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 161. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 162. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 163. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 164. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 165. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 166. In some embodiments, a dsRNA arm comprises the sequence of SEQ ID NO: 167.

[0087] In some embodiments, a dsRNA arm has a length of longer than 10 nucleotide base pairs. In some embodiments, a dsRNA arm has a length of shorter than 100 nucleotide base pairs. In some embodiments, a dsRNA arm has a length of about 10 base pairs to about 100 nucleotide base pairs. In some embodiments, a dsRNA arm has a length of about 10 base pairs to about 75 nucleotide base pairs. In some embodiments, a dsRNA arm has a length of about 10 base pairs to about 50 nucleotide base pairs.

[0088] In some embodiments, a dsRNA arm has a length of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, or at least 55 base pairs. In some embodiments, a dsRNA arm has a length of 10-55 base pairs. In some embodiments, a dsRNA arm has a length of 10-52 base pairs. In some embodiments, a dsRNA arm has a length of 11-52 base pairs. In some embodiments, a dsRNA arm has a length of 12-51 base pairs. In some embodiments, a dsRNA arm has a length of 13-50 base pairs. In some embodiments, a dsRNA arm has a length of 14-49 base pairs. In some embodiments, a dsRNA arm has a length of 15-48 base pairs. In some embodiments, a dsRNA arm has a length of 16-47 base pairs. In some embodiments, a dsRNA arm has a length of 17-46 base pairs. In some embodiments, a dsRNA arm has a length of 18-45 base pairs. In some embodiments, a dsRNA arm has a length of 19-44 base pairs. In some embodiments, a dsRNA arm has a length of 20-43 base pairs. In some embodiments, a dsRNA armhas a length of 21-42 base pairs. In some embodiments, a dsRNA arm has a length of 22-41 base pairs. In some embodiments, a dsRNA arm has a length of 23-40 base pairs. In some embodiments, a dsRNA arm has a length of 24-39 base pairs. In some embodiments, a dsRNA arm has a length of 25-38 base pairs. In some embodiments, a dsRNA arm has a length of 26-37 base pairs. In some embodiments, a dsRNA arm has a length of 27-36 base pairs. In some embodiments, a dsRNA arm has a length of 28-35 base pairs. In some embodiments, a dsRNA arm has a length of 29-34 base pairs. In some embodiments, a dsRNA arm has a length of 30-33 base pairs. In some embodiments, a dsRNA arm has a length of 31-32 base pairs.

[0089] In some embodiments, each dsRNA arm in a nanostructure has the same length (in nucleotide base pairs) relative to one another. In some embodiments, each dsRNA arm in a nanostructure has a different length relative to one another (e.g., at least one longer than at least one other). In some embodiments, a first subset of dsRNA arms in an RNA nanostructure has the same length relative to one another and a second subset of dsRNA arms in an RNA nanostructure has different lengths relative to one another.

[0090] Table 1 provides exemplary RNA nanostructure designs. In some embodiments, an RNA nanostructure of the present disclosure is designed based on one of the design descriptions provided in Table 1. In some embodiments, an RNA nanostructure comprises a combination of oligonucleotides described in Table 1 and is designed based on one of the design descriptions provided in Table 1. Table 2 provides examples of oligonucleotide sequences that can be used to produce an RNA nanostructure of the present disclosure. In some embodiments, an RNA nanostructure of the present disclosure comprises two or more of the oligonucleotide sequences provided in Table 2. Table 3 provides examples of dsRNA molecules that can be used as dsRNA arms. In some embodiments, an RNA nanostructure of the present disclosure comprises two or more of the dsRNA molecules provided in Table 3.

[0091] Table 1. Exemplary RNA Nanostructure Designs

[0092]

[0093]

[0094]

[0095]

[0096] Table 2. RNA Nanostructure Sequences

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Table 3. Double-Stranded RNAs

[0105] >

[0106]

[0107] *GG represents 3'-GG overhanging dinucleotides on each dsRNA arm.

[0108] In some embodiments, an RNA nanostructure is designed based on a design description provided in Table 1 and using two or more of the oligonucleotide sequences provided in Table 2. In some embodiments, an RNA nanostructure comprises a first dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153 and a second dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153. In some embodiments, an RNA nanostructure of the present disclosure comprises a first dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a second dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, and a third dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153. In some embodiments, an RNA nanostructure of the present disclosure comprises a first dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a second dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, athird dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, and a fourth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153. In some embodiments, an RNA nanostructure of the present disclosure comprises a first dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a second dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a third dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a fourth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, and a fifth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153. In some embodiments, an RNA nanostructure of the present disclosure comprises a first dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a second dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a third dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a fourth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, a fifth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153, and a sixth dsRNA arm comprising an oligonucleotide sequence set forth in any one of SEQ ID NOs: 1-153.

[0109] In some embodiments, an RNA nanostructure comprises a first double- stranded RNA arm and a second double- stranded RNA arm. In some embodiments, an RNA nanostructure comprises a first- double- stranded RNA arm, a second double- stranded RNA arm, and a third double- stranded RNA arm. In some embodiments, an RNA nanostructure comprises a first-double-stranded RNA arm, a second double- stranded RNA arm, a third double- stranded RNA arm, and a fourth doublestranded RNA arm. In some embodiments, a first double- stranded RNA arm is RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, or RNA-7, as provided in Table 3. In some embodiments, a second double- stranded RNA arm is RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, or RNA-7, as provided in Table 3. In some embodiments, a third double- stranded RNA arm is RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, or RNA-7, as provided in Table 3. In some embodiments, a fourth double- stranded RNA arm is RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, or RNA-7, as provided in Table 3. In some embodiments, an RNA nanostructure comprises RNA-1 as a first double- stranded RNA arm, RNA-5 as a second double- stranded RNA arm, and RNA-7 as a third double- stranded RNA arm.

[0110] In some embodiments, a dsRNA arm is RNA-1, comprising SEQ ID NOs: 154 and 155. In some embodiments, a dsRNA arm is RNA-2, comprising SEQ ID NOs: 156 and 157. In some embodiments, a dsRNA arm is RNA-3, comprising SEQ ID NOs: 158 and 159. In some embodiments, a dsRNA arm is RNA-4, comprising SEQ ID NOs: 160 and 161. In some embodiments, a dsRNA arm is RNA-5, comprising SEQ ID NOs: 162 and 163. In someembodiments, a dsRNA arm is RNA-6, comprising SEQ ID NOs: 164 and 165. In some embodiments, a dsRNA arm is RNA-7, comprising SEQ ID NOs: 166 and 167. A three-arm nanostar can include any one or more of RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, and RNA-7. Similarly, a four-arm nanostar can include any one or more of RNA-1, RNA-1, RNA-3, RNA-4, RNA-5, RNA-6, and RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-3.

[0111] In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-4. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-4. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-4. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-6.In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-4, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-4, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-4, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-5, RNA-6, RNA-7. In should be understood that, in some embodiments, a three-arm nanostar has two or three arms, each comprising the same sequence, for example, selected from RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, and RNA-7.

[0112] In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-3, RNA-4. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-3, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-3, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-3, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-4, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-2, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-4, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-3, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-4, RNA-6, RNA-7. In someembodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-1, RNA-5, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-4, RNA-5. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-4, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-4, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-3, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-4, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-2, RNA-5, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-5, RNA-6. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-5, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-4, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-3, RNA-5, RNA-6, RNA-7. In some embodiments, a three-arm nanostar comprises the nucleotide sequences of RNA-4, RNA-5, RNA-6, RNA-7. In should also be understood that, in some embodiments, a four-arm nanostar has two, three, or four arms, each comprising the same sequence, for example, selected from RNA-1, RNA-2, RNA-3, RNA-4, RNA-5, RNA-6, and RNA-7.

[0113] An example of a three-arm RNA nanostructure bound by a multimerizing protein is provided in FIG. 7.

[0114] In some embodiments, dsRNA arms are joined together by Watson-Crick base pairing. In other embodiments, dsRNA arms are joined together by Hoogsteen base pairing. In contrast to Watson-Crick base pairing, the phrase “Hoogsteen base pairing,” as used herein, refers to base pairing that occurs when a purine base rotates 180° relative to the helix axis and adopts a syn confirmation.

[0115] In some embodiments, a dsRNA arm comprises one or more modifications. RNA modifications include chemical changes made to RNA molecules that can influence their stability, localization, and / or function, for example. In some embodiments, a modification is on the 5’ end of one or both strands. In some embodiments, a modification is on the 3’ end of one or both strands. In some embodiments, a modification is on the 5’ end and 3’ end of one or more strands. In some embodiments, a modification is between the 5’ end and 3’ end of one or more strands.In some embodiments, a modification is an unnatural nucleic acid. In some embodiments, an unnatural nucleic acid is a locked nucleic acid (LNA). In some embodiments, an unnatural nucleic acid is peptide nucleic acid (PNA). In some embodiments, a modification is the inclusion of a non-nucleic acid polymer. In some embodiments, a non-nucleic acid polymer is a spiroligomer. In some embodiments, a dsRNA arm comprises one or more non-canonical nucleotides. In some embodiments, a dsRNA arm comprises one or more chemically modified nucleotides. In some embodiments, a dsRNA arm comprises a sugar modification, a sugar / backbone modification, a backbone modification, and / or a base modification. In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, a modification is the inclusion of one or more (e.g., 1, 2, 3, or more) phosphate groups. In some embodiments, a modification is the inclusion of one or more functional groups. In some embodiments, the functional group is O-azido-methyl-ether (AOME). In some embodiments, the functional group is O-allyl (Oallyl). In some embodiments, a dsRNA molecule comprises a 2’ -ribose modification, which can be natural or synthetic. Non-limiting examples of natural 2’-ribose modifications include 2'-O-Methyl (2'-0Me, 2'-0-Me), 2'-0-Ribosyl Phosphate (2'-0-ribosyl-P04), 2'-O- Acetyl (2'-O-Ac), and 2'-O-Sulfate (2'-O-SO4) modifications. Non-limiting examples of synthetic 2'-ribose modifications include 2'-Fluoro (2'-F), 2'-Amino (2'-NH2), 2'-0-Methoxyethyl (2'-0-M0E), 2'-0-Propargyl (2'-O-Pr), 2'-O- Allyl (2'-O- Allyl), 2'-O-Benzyl (2'-O-Bn), 2'-Deoxy-2'-fluoro-P-D-arabinonucleic acid (FANA), 2'-O-(2-methoxyethyl) (2'-0-M0E), and 2'-Eocked Nucleic Acid (2'-ENA).

[0116] RNA nanostructures comprising two or more dsRNA arms can form a core structure between the dsRNA arms. In some embodiments, an RNA nanostructure comprises a single unpaired nucleotide (e.g., U or A) in between each dsRNA arm. In some embodiments, one or more of the dsRNA arms within an RNA nanostructure is nicked. The term “nick,” as used herein, refers to a discontinuity in a dsRNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand. Nicks allow dsRNA molecules to untwist, thus affecting the conformation of the dsRNA molecule.

[0117] In some embodiments, a dsRNA comprises a linker or spacer. For example, two dsRNA arms may be linked to each other via a linker. The presence of a linker or spacer on a dsRNA arm within an RNA nanostructure can affect the conformation of the RNA nanostructure and thus impact the potency of the RNA nanostructure in modulating immunomodulatory intracellular signaling. In some embodiments, the linker or spacer can be selected from the group consisting of: photocleavable linkers, hydrolyzable linkers, redox cleavable linkers, phosphate -based cleavable linkers, acid cleavable linkers, ester-based cleavable linkers, peptide-based cleavable linkers, and any combinations thereof. In some embodiments, the cleavable linker can comprise a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a nitroindoline group, abromo hydroxycoumarin group, a bromo hydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin group, or a combination thereof. In some embodiments, a linker is a polyethylene glycol (PEG) linker. In some embodiments, a linker is a nucleotide spacer. In some embodiments, a linker is an amino acid spacer. In some embodiments, each single-stranded RNA of a dsRNA arm comprises one or more unpaired nucleotides. In some embodiments, an unpaired nucleotide is in the middle of a single- stranded RNA.

[0118] In some embodiments, a double- stranded RNA arm comprises a 5'-terminal monophosphate, diphosphate, triphosphate, or hydroxyl group. In some embodiments, a double- stranded RNA arm comprises a 5'-terminal hydroxyl group.

[0119] In some embodiments, an RNA nanostructure is presented on a scaffold to enhance stability and modulation of downstream signaling. In some embodiments, a scaffold is a linear DNA strand, a DNA nanostructure, a DNA nanostar, a DNA nanocube, a DNA origami, or a polymer.

[0120] Aspects of the present disclosure relate, at least in part, to nanostructures (e.g., RNA nanostars) that can regulate intracellular immunomodulatory signaling. dsRNA molecules have been shown to facilitate multimerization of multimerizing binding proteins by providing a landing site for multiple multimerizing binding proteins. Once multiple multimerizing binding proteins bind to a single dsRNA molecule, the multimerizing binding proteins multimerize due to their close proximity. The inventors of the present disclosure made the surprising discovery that RNA nanostructures, which are comprises of multiple dsRNA molecules, can facilitate efficient multimerization of multimerizing binding proteins and increase the increase the production of products of downstream signaling pathways. Binding of multimerizing binding proteins is dependent on access to the ends of dsRNA molecules, so nanostructures providing multiple ends of dsRNA molecules facilitates efficient binding of multimerizing binding proteins and thus increase production of products of downstream signaling pathways.

[0121] Aspects of the present disclosure relate, at least in part, to nanostructures (e.g., RNA nanostars) that can regulate intracellular immunomodulatory signaling by inducing conformational changes of binding proteins. In some embodiments, a binding protein binds to a nanostructure (e.g., RNA nanostars) and undergoes a conformational change to propagate downstream signaling.

[0122] In some embodiments, nanostructures comprising multiple dsRNA arms enhance the activity of any immunomodulatory intracellular signaling pathway that involves binding proteins binding to dsRNA or dsRNA-like molecules. In some embodiments, nanostructures (e.g., RNA nanostars) of the present disclosure enhance the activity of an immunomodulatory intracellular signaling pathway by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold. In some embodiments, nanostructures (e.g., RNA nanostars) comprising multiple dsRNA arms enhance the activity of an immunomodulatory intracellular signaling pathway by 1-foldto 50-fold, 2-fold to 40-fold. 3-fold, to 30-fold, 4-fold, to 20-fold, 5-fold, to 10-fold, 6-fold to 9-fold, or 7-fold to 8-fold. In some embodiments, the intracellular signaling pathway is an immune pathway and a nanostructure (e.g., RNA nanostar) is immunostimulatory.

[0123] The inventors of the present disclosure made the additional surprising discovery that disrupting the conformation of a nanostructure comprising multiple dsRNA arms (e.g., RNA nanostars) does not block binding of binding proteins but it does block multimerization of the binding proteins or conformational changes to the binding proteins. In some embodiments, a nanostructure comprising multiple dsRNA arms comprises modifications that affect immunomodulatory intracellular signaling. As described in Example 2, sequence nicks or the addition of linkers or spacers can decrease the potency of the RNA nanostructure in inducing immunomodulatory intracellular signaling. Accordingly, aspects of the present disclosure relate to nanostructures (e.g., RNA nanostars) comprising sequence nicks or linkers or spacers for use as inhibitors of immunomodulatory intracellular signaling. In some embodiments, nicking one or more of the dsRNA arms of a nanostructure inhibits an immunomodulatory intracellular signaling pathway by inhibiting multimerization of multimerizing binding proteins. In some embodiments, adding a linker to one or more of the dsRNA arms of a nanostructure inhibits an immunomodulatory intracellular signaling pathway by inhibiting multimerization of multimerizing binding proteins. Accordingly, aspects of the present disclosure relate to nanostructures as inhibitors of immunomodulatory intracellular signaling pathways. In some embodiments, the immunomodulatory intracellular signaling pathway is an immune pathway and a nanostructure (e.g., RNA nanostar) is immunoinhibitory.

[0124] The terms “increase,” “enhance,” or “activate” are all used herein to mean an increase by a reproducible statistically significant amount. In some embodiments, the terms “increase,” “enhance,” or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least a 2-fold, or at least a 3 -fold, or at least a 4-fold, or at least a 5 -fold or at least a 10-fold increase, a 20 fold increase, a 30 fold increase, a 40 fold increase, a 50 fold increase, a 6 fold increase, a 75 fold increase, a 100 fold increase, etc. or any increase between 2-fold and 10-fold or greater as compared to an appropriate control. In the context of a marker, an “increase” is a reproducible statistically significant increase in such level.

[0125] The term “decrease,” “reduced,” “reduction,” or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “decrease,” “reduced,” “reduction,” or “inhibit” typically means a decrease by at least 10% as compared to an appropriate control (e.g. the absence of a given treatment) and can include, for example, a decrease by at least10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to an appropriate control.

[0126] The term “reference level”, as used herein, includes a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject, or a biological sample obtained from the subject at a prior time point, e.g., a biological sample obtained from a patient prior to being diagnosed with interferon-mediated disease, or a biological sample that has not been contacted with a composition disclosed herein).

[0127] The term, “appropriate control,” as used herein, includes an untreated, otherwise identical cell or population (e.g., a patient who was not administered an agent described herein, or was administered by only a subset of compositions described herein, as compared to a non-control cell).

[0128] Exemplary Nanostructure Compositions

[0129] The data provided herein demonstrates that RNA nanostars (e.g., three- and four- arm nanostar, such as the three-arm nanostar comprising the nucleotide sequence of RNA- 113) are immunostimulatory. In particular, the data provided herein demonstrates, surprisingly, that the intensity of downstream signaling can be increased by bringing together at least three dsRNA arms to form a nanostar (see, e.g., FIG. 7), which enables efficient binding of multimerizing binding proteins to the RNA nanostar and a robust downstream signal. Surprisingly, RNA-113, for example, exhibited potent activation of interferon in vivo, demonstrating the ability of multi-arm RNA nanostars, such as three- and four-arm RNA nanostars, to activate RIG-I in vivo to induce an interferon signaling response. Thus, in some embodiments, a three-arm RNA nanostar binds to RIG-I and activates a RIG-I-IRF3 pathway, thereby inducing interferon (IFN) production (e.g., type I, type II, or type III IFN). In other embodiments, a four-arm RNA nanostar binds to RIG-I and activates a RIG-I-IRF3 pathway, thereby inducing interferon (IFN) production (e.g., type I, type II, or type III IFN). Other nanostars with additional arms (e.g., more than 4) are contemplated herein.

[0130] Three-Arm Nanostar

[0131] In some embodiments, a nanostructure comprises three dsRNA arms (also referred to as dsRNA domains), also referred to herein as a “three-arm RNA nanostar,” wherein the nanostructure regulates intracellular immunomodulatory signaling. In some embodiments, a three-arm ribonucleic acid (RNA) nanostar comprises a first RNA strand comprising a first domain and a second domain, a second RNA strand comprising a third domain and a fourth domain, and a third RNA strand comprising a fifth domain and a sixth domain, wherein the first domain of the first RNA strand isbound to the sixth domain of the third RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm. In some embodiments, the dsRNA arms comprise the nucleotide sequence of RNA-113 (3-arm nanostar with RNA-1 (SEQ ID NO: 154 and 155), RNA-5 (SEQ ID NO: 162 and 163), and RNA-7 (SEQ ID NO: 166 and 167)).

[0132] In some embodiments, the three-arm nanostar (e.g., comprising the nucleotide sequence of RNA-113) comprises a modification, relative to a naturally occurring RNA molecule. For example, the three-arm nanostar can include a modification at a 5’ end of at least one of the dsRNA arms. As another example, the three-arm nanostar can include a modification at a 3’ end of at least one of the dsRNA arms. In some embodiments, a three-arm nanostar comprises a sugar modification (e.g., 2'-O-methyl (2'-0Me), 2'-fluoro (2'-F), locked nucleic acid (LNA), or peptide nucleic acid (PNA)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a three-arm nanostar comprises a base modification (e.g., 5 -methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), 2-thiouridine (s2U), or inosine (I)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a three-arm nanostar comprises a phosphate (backbone) modification (e.g., phosphorothioate (PS), cyclization, or a-thio-triphosphate), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, the phosphate modification is a triphosphorylation (PPP). In some embodiments, the phosphate modification is phosphorothioate (PS). In some embodiments, a three-arm nanostar comprises an unpaired nucleotide (e.g., unpaired adenine (A) or unpaired uracil (U)), for example, for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a three-arm nanostar comprises a non-canonical nucleotide (e.g., inosine (I), 5 -methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), queuosine (Q), azidothymidine (AZT), 2'-fluoro-modified nucleotides (e.g., 2'-F-uridine), or locked nucleic acids (LNAs)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a three-arm nanostar comprises a chemically modified nucleotide (e.g., from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), azidothymidine (AZT), and 2'-fluoro-modified nucleotides, optionally 2'-F-uridine), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, the three-arm nanostar comprises a 5'-terminal monophosphate, diphosphate, triphosphate, or hydroxyl group in at least one of the multiple dsRNA arms. In some embodiments, the three-arm nanostar comprises a linker, for example, at a 5’ end or a 3’ end of a dsRNA molecule. Examples of such linkers include unpaired nucleotides (e.g., unpaired A and U), chemically modified nucleotides, polyethylene glycol (PEG) spacers, nucleotide spacers, and amino acid spacers.In some embodiments, a three-arm nanostar includes a nicked site in one or more of its dsRNA arms. The data herein shows unexpectedly that disrupting the conformation of a nanostructure comprising multiple dsRNA arms (e.g., RNA nanostars) does not block binding of binding proteins but it does block multimerization of the binding proteins or conformational changes to the binding proteins, thereby rendering the nanostructure immunosuppressive. Thus, in some embodiments, a three-arm RNA nanostar binds to RIG-I and inhibits activation a RIG-I-IRF3 pathway.

[0133] Also contemplated herein are three-arm nanostructures (e.g., 3-arm nanostar with RNA-1 (SEQ ID NO: 154 and 155), RNA-5 (SEQ ID NO: 162 and 163), and RNA-7 (SEQ ID NO: 166 and 167)) formulated in a lipid nanoparticle (LNP), for example, comprising an SM-102 lipid, DOPE / DOTAP, PEG-DMG, and cholesterol. In some embodiments, such nanostars include a modification at a 5’ end or a 3’ end of at least one of the dsRNA arms. These nanoparticles, in some embodiments, are formulated as pharmaceutically acceptable composition, for example, for use in regulating intracellular immunomodulatory pathway signaling in a subject or for use is treating a disease or disorder in a subject.

[0134] Four-Arm Nanostar

[0135] In some embodiments, a nanostructure comprises four dsRNA arms (also referred to as dsRNA domains), also referred to herein as a “four-arm RNA nanostar,” wherein the nanostructure regulates intracellular immunomodulatory signaling. In some embodiments, a four-arm ribonucleic acid (RNA) nanostar comprises a first RNA strand comprising a first domain and a second domain, a second RNA strand comprising a third domain and a fourth domain, a third RNA strand comprising a fifth domain and a sixth domain, and a fourth RNA strand comprising a seventh domain and an eighth domain, wherein the first domain of the first RNA strand is bound to the eighth domain of the fourth RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm, the sixth domain of the third strand is bound to the seventh domain of the fourth strand to form a fourth dsRNA arm. In some embodiments, the dsRNA arms comprise the nucleotide sequence of RNA-92 (4-arm nanostar with RNA-1 (SEQ ID NO: 154 and 155), RNA-5 (SEQ ID NO: 162 and 163), RNA-7 (SEQ ID NO: 166 and 167), and RNA-2 (SEQ ID NO: 156 and 157)).

[0136] In some embodiments, the four-arm nanostar (e.g., comprising the nucleotide sequence of RNA-113) comprises a modification, relative to a naturally occurring RNA molecule. For example, the four- arm nanostar can include a modification at a 5’ end of at least one of the dsRNA arms. As another example, the four- arm nanostar can include a modification at a 3’ end of at least one of the dsRNA arms. In some embodiments, a four-arm nanostar comprises a sugar modification (e.g., 2'-O-methyl (2'-0Me), 2'-fluoro (2'-F), locked nucleic acid (LNA), or peptide nucleic acid (PNA)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a four- arm nanostar comprises a base modification (e.g., 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), 2-thiouridine (s2U), or inosine (I)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a four-arm nanostar comprises a phosphate (backbone) modification (e.g., phosphorothioate (PS), cyclization, or a-thio-triphosphate), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, the phosphate modification is a triphosphorylation (PPP). In some embodiments, the phosphate modification is phosphorothioate (PS). In some embodiments, a four-arm nanostar comprises an unpaired nucleotide (e.g., unpaired adenine (A) or unpaired uracil (U)), for example, for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a four-arm nanostar comprises a non-canonical nucleotide (e.g., inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), queuosine (Q), azidothymidine (AZT), 2'-fluoro-modified nucleotides (e.g., 2'-F-uridine), or locked nucleic acids (LNAs)), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, a four- arm nanostar comprises a chemically modified nucleotide (e.g., from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), azidothymidine (AZT), and 2'-fluoro-modified nucleotides, optionally 2'-F-uridine), for example, at a 5’ end or a 3’ end of at least one of the dsRNA arms. In some embodiments, the four-arm nanostar comprises a 5'-terminal monophosphate, diphosphate, triphosphate, or hydroxyl group in at least one of the multiple dsRNA arms. In some embodiments, the four-arm nanostar comprises a linker, for example, at a 5’ end or a 3’ end of a dsRNA molecule. Examples of such linkers include unpaired nucleotides (e.g., unpaired A and U), chemically modified nucleotides, polyethylene glycol (PEG) spacers, nucleotide spacers, and amino acid spacers.

[0137] In some embodiments, a four-arm nanostar includes a nicked site in one or more of its dsRNA arms. The data herein shows unexpectedly that disrupting the conformation of a nanostructure comprising multiple dsRNA arms (e.g., RNA nanostars) does not block binding of binding proteins but it does block multimerization of the binding proteins or conformational changes to the binding proteins, thereby rendering the nanostructure immunosuppressive. Thus, in some embodiments, a four-arm RNA nanostar binds to RIG-I and inhibits activation a RIG-I-IRF3 pathway.

[0138] Also contemplated herein are four-arm nanostructures (4-arm nanostar with RNA-1 (SEQ ID NO: 154 and 155), RNA-5 (SEQ ID NO: 162 and 163), RNA-7 (SEQ ID NO: 166 and 167), and RNA-2 (SEQ ID NO: 156 and 157)) formulated in a lipid nanoparticle (LNP), for example, comprising an SM-102 lipid, DOPE / DOTAP, PEG-DMG, and cholesterol. In some embodiments,such nanostars include a modification at a 5’ end or a 3’ end of at least one of the dsRNA arms. These nanoparticles, in some embodiments, are formulated as pharmaceutically acceptable composition, for example, for use in regulating intracellular immunomodulatory pathway signaling in a subject or for use is treating a disease or disorder in a subject.

[0139] Methods of Preparing RNA Nanostructures

[0140] The RNA nanostructures described herein can be prepared by synthetic methods known in the art including, but not limited to, chemical synthesis, including but not limited to a nucleoside phosphoramidite approach, or in vitro transcription, among others. Methods for chemical synthesis to include modified nucleotides are also known in the art.

[0141] In in vitro transcription, polymerases can be used including, but not limited to, bacteriophage polymerase such as T7 polymerase, T3 polymerase and SP6 polymerase, viral polymerases, and E. coli RNA polymerase.

[0142] Oligonucleotide strands can be isolated from a sample using RNA extraction and purification methods know in the art. These methods include but are not limited to column purification, ethanol precipitation, phenol-chloroform extraction, or acid guanidinium thiocyanate-phenol chloroform extraction (AGPC). Following isolation of a single stranded oligonucleotide, hybridizing, and / or annealing the top and bottom strands can be performed to form the duplex secondary structure.

[0143] The terms “hybridizing,” “hybridize,” “hybridization,” “annealing,” or “anneal,” as used herein, are used interchangeably in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. In other words, the term “hybridization” includes the process in which two or more single -stranded polynucleotides bind non-covalently to form a doublestranded polynucleotide. The resulting double -stranded polynucleotide is a “hybrid” or “duplex.” Conditions for forming hybridized or duplexed sequences are known to those of skill in the art and generally include salt concentration and temperature at or near normal physiological conditions, e.g., intracellular conditions. Generally, hybridization to form duplexes as described herein can be performed with each strand present in substantially equimolar concentrations.

[0144] Following synthesis, hybridization and, optionally, removal of non-duplexed strands, the RNA nanostructures can be characterized by any method known in the art, e.g., liquid chromatography, mass spectrometry, next generation sequencing, polymerase chain reaction (PCR), gel electrophoresis, or any other method of identifying nucleoside sequences, secondary structures, chemical composition, expression, thermodynamics, binding, or function.

[0145] For further characterization of the RNA nanostructures described herein, the 5'-hydroxyl group can be detected, for example, by a splinted ligation assay. See e.g., Shoenberg et al, Nat ChemBiol 3(9) (2007) and Celesnik H et al. Mol Cell. 2007; 27:79-90. By carefully designing reaction conditions and comparing ligated with non-ligated RNA this assay yields quantitative data of the amount of RNA with a 5'-hydroxyl group.

[0146] For some therapeutic purposes, nanostructures comprising multiple dsRNA arms can have a degree of stability in serum to allow distribution and cellular uptake. The prolonged maintenance of therapeutic levels of the oligonucleotides in serum will have a significant effect on the distribution and cellular uptake and unlike conjugate groups that target specific cellular receptors, the increased serum stability will affect all cells.

[0147] Nanostructures (e.g., RNA nanostars) described herein can also have chemical modifications. Chemical modifications include the addition of one or more linkers, ligands, antigens, and / or other molecules selected from antibodies (e.g., nanobodies or antibody fragments), peptides, and other protein structures. For example, the ligand can improve stability, hybridization thermodynamics with a target nucleic acid, or targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or independent mechanism. Oligonucleotides bearing peptide e.g., antigen) conjugates can be prepared using procedures known in the art. See Trufert et al., Tetrahedron 1996, 52, 3005; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.

[0148] Delivery Modalities

[0149] The nanostructures described herein can be delivered to subjects or cells through various modalities known or developed in the art for nucleic acid delivery. Delivery modalities encompass the methods, formulations, and vehicles used to transport RNA nanostructures to target cells, tissues, or organs while, in some instances, maintaining stability, enhancing cellular uptake, and / or promoting biological activity. Selection of an appropriate delivery modality may depend on factors including, for example, the intended route of administration, target tissue, desired pharmacokinetics, and / or therapeutic application. The RNA nanostructures of the present disclosure can be formulated, in some embodiments, with one or more delivery vehicles, including but not limited to nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles), conjugates, and other carriers that facilitate cellular entry and intracellular trafficking. In some embodiments, the delivery modality is designed to achieve organ- specific and / or tissue-specific distribution, such as liver-tropic or lung-tropic delivery. The following sections describe non-limiting, exemplary delivery modalities suitable for the nanostructures described herein.Nanoparticles for Nanostructure Delivery

[0150] Also provided herein are nanoparticles comprising a nanostructure that includes multiple dsRNA arms, such as an RNA nanostar. Various types of nanoparticles may be utilized for the efficient delivery of nucleic acids. These nanoparticles serve to protect the nucleic acid cargo from degradation, enhance cellular uptake, and promote targeted delivery to specific tissues or cell types. In some embodiments, a nanoparticle for nanostructure delivery is a lipid-based nanoparticle.

[0151] Lipid-based nanoparticles, including liposomes and lipid nanoparticles (LNPs), are widely used for nucleic acid delivery. LNPs are composed, in some embodiments, of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-conjugated lipids, which facilitate encapsulation, stability, and intracellular release.

[0152] In some embodiments, an LNP comprises DOPE:

[0153]

[0154] In some embodiments, an LNP comprises DOTAP:

[0155]

[0156] In some embodiment, an LNP comprises SM-102 lipid:

[0157]

[0158] In some embodiments, an LNP comprises PEG-DMG lipid. In some embodiments, an LNP comprises cholesterol. In some embodiments, an LNP comprises DOPE / DOTAP, cholesterol, PEG-DMG, and SM-102. Other lipid nanoparticle formulations can be used to formulate nucleic acidnanostructures for delivery in vivo (see, e.g., Cullis PR & Feigner PL Nature Reviews Drug Discovery 2024: 23; 709-722; and Saber N et al. Hum Gene Ther. 2024; 35(17-18): 617-627).

[0159] In some embodiments, a nanoparticle for nanostructure delivery is a polymeric nanoparticle. Polymeric nanoparticles, in some embodiments, are composed of biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI), chitosan, or poly(beta-amino esters). These polymers provide structural stability and can be functionalized with targeting moieties or surface modifications to enhance cellular uptake and biodistribution. In some embodiments, a nanoparticle for nanostructure delivery is an inorganic nanoparticle. Inorganic nanoparticles include, for example gold nanoparticles (AuNPs), silica nanoparticles, and magnetic nanoparticles. Gold nanoparticles, in some embodiments, can be functionalized with thiol-modified nucleic acids, enabling stable conjugation and controlled release. Magnetic nanoparticles, such as iron oxide nanoparticles, can be used for magnetically guided nucleic acid delivery, in some embodiments.

[0160] In some embodiments, a nanoparticle for nanostructure delivery is a protein-based nanoparticle. Protein-based nanoparticles, in some embodiments, are derived from albumin, ferritin, or virus-like particles. These can provide biocompatible and biodegradable platforms for nucleic acid delivery. These nanoparticles are engineered, in some embodiments, to optimize loading capacity, stability, and targeted cellular uptake. Virus-like particles mimic the natural viral delivery mechanisms and, in some embodiments, are used to enhance nucleic acid delivery efficiency. In some embodiments, a nanoparticle for nanostructure delivery is a hybrid nanoparticle. Hybrid nanoparticles combine different materials to optimize nucleic acid delivery efficiency. For example, lipid-polymer hybrid nanoparticles integrate the stability of polymeric nanoparticles with the enhanced transfection efficiency of lipid-based carriers. Similarly, inorganic-lipid hybrids may incorporate gold or silica cores coated with lipid bilayers to achieve controlled release and biocompatibility.

[0161] Dendrimers for Nanostructure Delivery

[0162] In some embodiments, a nanoparticle for nanostructure delivery is a dendrimer. Dendrimers include highly branched, nanoscale polymers that can encapsulate nucleic acids via electrostatic interactions or covalent attachment.

[0163] Electrostatic interactions between cationic dendrimers and anionic nucleic acids result in the formation of dendrimer-nucleic acid complexes (also referred to as "dendriplexes"). The positively charged surface groups of dendrimers (such as primary amines) interact with the negatively charged phosphate backbone of nucleic acids through coulombic forces, resulting in spontaneous complex formation. The stoichiometry of dendrimer to nucleic acid can be modified, for example, by adjusting the nitrogen-to-phosphate (N / P) ratio to achieve stable complexes with appropriate size andsurface charge for cellular uptake. Alternatively, nucleic acids can be covalently attached to dendrimers through various chemistries. For example, nucleic acids bearing terminal reactive groups (such as thiol, amine, azide, or alkyne groups) can be conjugated to dendrimers functionalized with complementary reactive groups (such as maleimide, N-hydroxysuccinimide ester, alkyne, or azide groups, respectively). Covalent conjugation can occur at the 3' terminus, 5' terminus, or internal positions of the nucleic acid strands. Cleavable linkers, such as disulfide bonds, hydrazone bonds, or ester bonds, are incorporated, in some embodiments, to enable intracellular release of the nucleic acid cargo following cellular uptake.

[0164] Poly(amidoamine) (PAMAM) dendrimers are particularly effective due to their well-defined structure, multivalent surface functionalities, and ability to promote endosomal escape. PAMAM dendrimers can be synthesized through iterative reaction sequences, resulting in precisely controlled generations (GO to GIO or higher) with exponentially increasing numbers of surface amine groups. The “proton sponge” effect of PAMAM dendrimers, arising from the buffering capacity of their tertiary amine groups, facilitates endosomal disruption and cytoplasmic delivery of nucleic acid cargo. Other suitable dendrimer types include polypropylene imine) (PPI) dendrimers, poly(L-lysine) (PLL) dendrimers, triazine dendrimers, phosphorus-containing dendrimers, carbosilane dendrimers, and peptide dendrimers. Each dendrimer type can offer distinct advantages in terms of biocompatibility, surface functionality, and degradation properties.

[0165] Dendrimers can be further modified with targeting ligands to enhance specificity for particular cell types. Targeting ligands are conjugated to the dendrimer surface, in some embodiments, through reactive surface groups or can be incorporated during dendrimer synthesis. Non-limiting examples of targeting ligands include: (i) peptides, such as RGD peptides for targeting integrin receptors on tumor cells and endothelial cells, cell-penetrating peptides (CPPs) such as TAT peptide or penetratin for enhanced cellular uptake, and tumor-homing peptides such as iRGD or F3 peptide; (ii) antibodies or antibody fragments, such as anti-HER2, anti-EGFR, or anti-PD-Ll antibodies for targeting tumor cells, and anti-CD3 or anti-CD19 antibodies for targeting immune cells; (iii) small molecules, such as folic acid for targeting folate receptors overexpressed on many tumor cells, galactose or N-acetylgalactosamine (GalNAc) for targeting asialoglycoprotein receptors on hepatocytes, and mannose for targeting mannose receptors on antigen-presenting cells; (iv) aptamers, which are single- stranded oligonucleotides that bind specific cell surface markers; and (v) glycans or polysaccharides for targeting lectin receptors. The choice of targeting ligand can depend on the desired tissue distribution, cell type specificity, and therapeutic application, for example.Pharmaceutical Compositions and Methods of Use

[0166] The methods and RNA nanostructure compositions described herein can further comprise formulating the RNA nanostructure described herein with a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable" includes those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0167] In some embodiments, the compositions described herein further comprise a nanoparticle delivery vehicle, such as a lipid nanoparticle, described elsewhere herein.

[0168] The nanostructures described herein can be used, in some embodiments, for inducing interferon (IFN) production (e.g., IFN, Type I IFN, IFN-a, IFN-b, IFN-8, IFN-K and IFN-co, Type II IFN (IFN-g), and Type III IFN) in a cell or subject in need thereof. In some embodiments, administering the RNA nanostructure to a subject is sufficient to increase the levels or activity of IFN.

[0169] The therapeutic formulations to be used for in vivo administration in the methods described herein can be sterile, which is readily accomplished by filtration through sterile filtration membranes, or other methods known to those of skill in the art.

[0170] The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting.

[0171] EXAMPLES

[0172] In order that the invention described in the present application may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided in this disclosure and are not to be construed in any way as limiting their scope.

[0173] Example 1. Identification of RNA Nanostructures with Improved Potency

[0174] This Example relates to the identification of RNA nanostructures as potent activators of retinoic acid-inducible gene I (RIG-I). RIG-I is a cytosolic pattern recognition receptor that can mediate induction of a type-I interferon (IFNI) response. RIG-I induces IFNI by recognizing double- stranded RNA (dsRNA), triggering activation of mitochondrial antiviral- signaling protein (MAVS, which leads to an increase in interferon levels, which in turn leads to molecular outcomes such as interference with viral replication, enhanced antigen presentation, and tumor growth suppression (FIG. 1). While the mechanism of RIG-I activation remains unclear, one possible explanation is that RIG-I binds to dsRNA ends, resulting in a RIG-I conformational change causingRIG-I to translocate down the dsRNA (FIG. 2). When four RIG-I molecules bind to a dsRNA in this manner, their caspase activation and recruitment domains (CARDs) multimerize and trigger MAVS filamentation, resulting in downstream signaling (FIG. 3).

[0175] Given this proposed mechanism of interferon signaling, the inventors of the present disclosure sought to identify RNA molecules that can increase interferon signaling. RIG-I uses approximately 12 base pairs of RNA for binding, so a dsRNA of adequate length enables four RIG-I molecules to bind. To test this, dsRNA molecules with a length of 52 base pairs were generated and screened using the A549 lung carcinoma reporter cell line measuring interferon activity (FIG. 4). Each of the 52-base pair dsRNA molecules were more active than a 25-base pair dsRNA molecule, provided here as RNA-1. In fact, two representative sequences exhibited an approximately five-fold improvement in potency over RNA-1.

[0176] Next, it was sought to determine whether a dsRNA fewer than 52 base pairs in length could be used with the same potency as the 52-base pair dsRNA molecules (FIG. 5). Indeed, it was found that a dsRNA with 44 base pairs can be used without a significant loss in potency, while a dsRNA with 36 base pairs results in loss of potency (FIG. 6).

[0177] After identifying that shorter dsRNA molecules can be used without a significant loss in potency, it was considered whether increasing the number of dsRNA ends from two to three in the form of an RNA nanostructure could further increase potency (FIG. 7). RNA nanostructures were considered because the organization of RIG-I molecules on the RNA nanostructure could result in more efficient CARD multimerization. Indeed, while using a dsRNA longer than RNA-1 (RNA-111) results in a five-fold improvement in potency, using an RNA nanostructure (RNA-113) resulted in an additional order of magnitude in potency (FIG. 8). This was an especially surprising discovery because RNA nanostructures are completely different than any dsRNA molecule that RIG-I could have evolved to recognize.

[0178] Example 2. RNA Nanostructure Experiments

[0179] This Example relates to the development of the RNA nanostructures identified in Example 1.

[0180] First, the length of the arms of the RNA nanostructures was varied from 14 base pairs to 26 base pairs and immune signaling was measured (FIG. 9). Specifically, dual cells or A549-Dual / RIG-I knock-out cells were transfected with the RNA structures formulated in TransIT-X2 and then activation of the IFN pathway and NF-KB pathway was measured by quantifying luciferase reporter activity or alkaline phosphatase activity, respectively. The best activity was identified for RNA nanostructures with arms ranging from 22 to 26 base pairs in length. RNA nanostructure arm length was further modulated with the default being an RNA nanostructure with 26-base pair arms (RNA-112) (FIG. 10). Shortening one arm to 13 base pairs led to some reduction in potency, shorteningtwo arms to 13 base pairs led to an even larger reduction in potency, and shortening all three arms to 13 base pairs nearly eliminated potency. The minimal length of an RNA nanostructure for full IFN induction was determined to be 22-24 bases (FIG. 19 and Table 4). An RNA nanostructure with 24-base pair arms (RNA- 113) consistently exhibited high potency.

[0181] Table 4. Comparison of the immunostimulatory activities of different nanostar analogs.

[0182] > >

[0183] > >

[0184] > >

[0185] > >

[0186] > >

[0187] > >

[0188] > >

[0189] > >

[0190] > >

[0191] > >

[0192] > >

[0193] > >

[0194]

[0195] > >

[0196] > >

[0197] > >

[0198] > >

[0199] > >

[0200] > >

[0201] > >

[0202] > >

[0203] > >

[0204] > >

[0205] > >

[0206] > >

[0207] > >

[0208] > >

[0209] > >

[0210] > >

[0211] > >

[0212] > >

[0213] > >

[0214] > >

[0215] > >

[0216]

[0217] > >

[0218]

[0219] Following assessment of RNA nanostructure arm length, it was determined whether changing linkers in the core of RNA- 113 would affect potency. Changing linkers in the core of RNA- 113 did not appear to affect potency (FIG. 11). Next, it was determined whether adding nicks to the strands in the core of RNA- 113 would affect potency. In fact, adding nicks slightly reduced potency (FIG.

[0220] 12). Finally, it was determined whether changing the structure of the sequences that make up RNA-113 would affect potency. This was determined by scrambling the sequences that make up RNA- 113. Sequence did not appear to affect RNA- 113 potency and RNA- 113 still performed better than a completely new sequence (RNA-143) (FIG. 13). These data demonstrate that RNA-113 is a highly potent RNA nanostructure regardless of the sequences that make up RNA-113. These data also demonstrate that changing linkers in the core of RNA-113 or adding nicks to the core negatively impacts potency.

[0221] Example 3. Development of Nanoparticles for Delivery of RNA Nanostructures

[0222] This Example relates to formulations of nanoparticles for delivery of RNA nanostructures. Given the impressive activity of the RNA nanostructures identified in Example 1 and further development in Example 2, lung-tropic nanoparticles were formulated to deliver RNA- 111 and RNA-113. RNA-111 and RNA-113 were encapsulated in liquid nanoparticles (LNPs) and evaluated for encapsulation efficiency (FIG. 14). LNPs were synthesized by mixing an aqueous phase (10 mM Sodium acetate buffer, pH 5.2, Sigma) containing dsRNA of interest with an organic phase (ethanol, Thermo Fisher Scientific) containing five or four lipid components (SM-102, / Cholesterol (Sigma) / DOPE l,2-dioleoyl-sn-glycero-3-phosphoethanolamine(Avanti Polar Lipids Inc) / polyethylene glycol (PEG)-DMG (1,2-dimyristoyl-rac-glycerol) , l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) in a lipid molar ratio 25:19:5.25:0.75:50 , 20:1 wt:wt (total lipids: nucleic acid) and aqueous to organic volume ratio 3:1. Following assembly, LNPs were then transferred into a dialysis tube (3500 MwCO) against lx PBS (pH 7.4) to completely remove the ethanol. To reach the desired injected dose, LNPs were concentrated using Amicon filters (10,000 MWCO). The composition of the LNP formulation is provided in Table 5. The encapsulation efficiency was over 90%, and it was found that the LNPS have nanoscale size distributions and a roughly neutral charge as measured by zeta potential.Table 5. LNP formulation composition.

[0223]

[0224] Next, using LNPs, RNA-111 and RNA-113 were delivered to mice in an in vivo tolerability study (FIG. 15). Surprisingly, both RNA-111 and RNA-113 exhibited potent activation of interferon in vivo, with RNA-113 being slightly more potent at a lower dose (15 pg). These data demonstrate the ability of RNA nanostructures to activate RIG-I in vivo to induce an interferon signaling response.

[0225] To evaluate the organ-tropic delivery of lipid nanoparticles (LNPs), the biodistribution of both lung- and liver-tropic LNPs containing RNA-1 was assessed. To achieve this, RNA-1 was labeled with the Cy5 fluorescent dye (RNA-lCy5) and encapsulated within both types of particles. The biodistribution of the LNPs was then tracked across major organs using an in vivo imaging system (IVIS), which is capable of detecting the fluorescent signal emitted by the labeled RNA-1 within each organ. LNPs loaded with RNA-1 were administered systemically, and blood samples and major organs were collected at 2 and 4 h post dosing (FIG. 20A). As shown in FIG. 20B and quantitatively analyzed in FIG. 20C, liver- tropic LNPs containing RNA-1 predominantly accumulated in the liver and spleen, whereas lung-tropic LNPs containing RNA- 1 were distributed to the lungs in addition to the liver and spleen. Quantitative analysis revealed no significant difference in the extent of signal in the liver and spleen across both formulations. This suggests that while RNA-1 delivery to the spleen and liver occurs to a similar extent with both lung- and liver-tropic LNPs, the observed in vivo PD effect is only evident when the lungs are targeted using lung-tropic LNPs.

[0226] Next, analogs RNA-111 and RNA-113 encapsulated into LNPs were characterized for their hydrodynamic size (via Dynamic Light Scattering), surface charge (Zeta potential), and encapsulation efficiency (FIG. 21). The nanosized LNPs formulated with RNA-111 measured 103 nm and 105 nm for lung- and liver-tropic formulations, respectively, while those encapsulating RNA-113 were 96 nm and 90 nm for lung- and liver- tropic formulations, respectively. Zeta potential measurements ranged between -3 to +6 mV, consistent with neutrally charged nanoparticles. All formulations demonstrated high encapsulation efficiency (>90%), indicating efficient encapsulation of the RNA cargos. This data suggests that the liver- and lung-tropic LNP formulations are welloptimized for RNA analog delivery into cells and further suggests that any differential activation of RIG-I observed in vivo (i.e. tissue tropism) would be driven by differences in the chemical composition of the nanoparticles.

[0227] Finally, the potential of LNP-encapsulated dsRNA analogs was evaluated for their potential to enhance the potency of RIG-I activation in a dose-response manner. Cells were treated with liver-or lung-tropic LNPs encapsulating the dsRNA analogs, and following a 48-hour incubation period, IRF3-driven luciferase reporter activity was measured. The activity of the new analogs was compared to dsRNA- 1. Both RNA-111 and RNA- 113 demonstrated significantly improved potency compared to dsRNA-1. RNA-113 exhibited the highest RIG-I activation, with EC50 values of 0.002 nM and 0.003 nM for liver- and lung-tropic nanoparticles, respectively. RNA-111 also demonstrated strong activity, with EC50 values of 0.66 nM and 0.75 nM for liver- and lung-tropic nanoparticles, respectively (FIG.22A and Table 6). Cell viability assays confirmed that the reduced activity and cellular toxicity was only observed at higher doses, further validating the observed EC50 values (FIG. 22B). The improved potency of these analogs is likely attributed to their stable RNA structures, which differentiate them from dsRNA-1. In contrast to dsRNA-1, which is hypothesized to require dimerization via G-G overhang Hoogsteen base pairing to initiate RIG-I activation, RNA-111 and RNA-113 already possess stable full-length structures that do not depend on dimerization. RNA-111 achieves this in a one-dimensional configuration, while RNA-113, as a nanostar structure, provides a two-dimensional configuration, further enhancing its ability to activate RIG-I Table 6. Summary and comparison of the immunostimulatory activities of different duplex RNA analogs formulated in lung- and liver- tropic LNPs or TransIT-X2 and transfected into A549-Dual cells.

[0228]

[0229] Example 4. Impact of Phosphorylation.

[0230] Canonically, RIG-I agonists are thought to require di / triphosphorylation (PPP) on their 5' end. To see how potency changes with the inclusion of PPP, phosphorylated analogs RNA-1, duplexRNA-111, nanostar RNA-113, and literature reference SLR14 were evaluated using the A549 dual reporter cell line. A549-Dual cells or A549-Dual / RIG-I knock-out cells were transfected with indicated duplex RNAs formulated in TransIT-X2 and then activation of the IFN pathway and NF-kB pathway was measured by quantifying luciferase reporter activity or alkaline phosphatase activity, respectively (Table 7). 5’ phosphorylation resulted in a ~100x increase in IFN-luciferase reporter activity for RNA-1, ~20x increase for RNA-111, and ~4x for RNA-113. All analogs were significantly more potent than the literature reference SLR14 (FIG. 16).

[0231] Table 7. Comparison of the immunostimulatory activities of different dsRNA analogs with or without phosphorylation.

[0232] >

[0233] > >

[0234] > >

[0235] > >

[0236] > >

[0237] > >

[0238] > >

[0239] > >

[0240]

[0241] The inclusion of phosphorothioate (PS) linkages on the terminal two bases of all strands resulted in a ~4-7x increase in potency for RNA-113 and RNA-111 respectively (FIG. 17 and Table 8). For both PPP and PS modifications, modifying only one of the RNA-111 strands resulted in a more modest potency increase, demonstrating the benefit of having both strands modified. The inclusion of the 3' sugar modifications on RNA-111 did not significantly impact the ability of RNA-111 to activate RIG-I (FIG. 18 and Table 7).Table 8. Comparison of the immunostimulatory activities of different dsRNA analogs with or without chemical modification.

[0242] > >

[0243] >

[0244] > >

[0245] > >

[0246] > >

[0247] > >

[0248] > >

[0249] >

[0250]

[0251] Example 5. In vitro activity of polyvalent DNA nanoparticles (DNA nanocubes).

[0252] This Example relates to the identification of DNA nanoparticles (DNA nanocubes) as potent inducers of the interferon pathway. The in vitro A549 reporter activity of polyvalent DNA nanocubes loaded with a 52 bp dsRNA was evaluated. It was found that polyvalent presentation leads to more potent induction of the interferon pathway (FIG 23 and Table 9). It was confirmed that this activity was not due to the DNA nanocube itself, as mixtures of the dsRNA with nanocubes that are unable to bind the dsRNA did not result in a similar shift of the dose-response curve, and the nanocubes alone were inactive.Table 9. RIG-I activation in A549 dual reporter cells in response to DNA nanocubes loaded with a 52 bp dsRNA analog formulated in TransIT-X2 subsequent to transfection.

[0253]

[0254] Example 6. Development of I VT- Synthesized RNA Nanostars

[0255] This Example relates to the development of RNA nanostructures that are developed for in vitro transcription (IVT) synthesis and designed to avoid unintended sequence homology with endogenous transcripts.

[0256] Design of RNA-233

[0257] RNA-233 was designed as a three-arm nanostar with the following features:

[0258] (1) Enhanced IVT yield: Each strand was designed with a 5’-GGA sequence to increase transcription yield by approximately 10-fold compared to sequences lacking this motif. The 5’-GGA sequence improves T7 polymerase initiation efficiency.

[0259] (2) No BLAST hits: The three RNA strands comprising RNA-233 were systematically screened to ensure that none of the individual strand sequences produced significant matches when searched against nucleotide databases using BLAST (Basic Local Alignment Search Tool). This design minimizes the risk of unintended hybridization with cellular RNAs or triggering of sequencespecific innate immune responses beyond RIG-I activation.

[0260] (3) Validated nanostar folding: The designed sequences were computationally validated to confirm proper three-arm nanostar assembly and structural stability.

[0261] The sequences of the three strands comprising RNA-233 are provided in Table 2 as SEQ ID NO: 167, 170, and 171. RNA-236 was designed as a control, representing the chemically synthesized 5’-OH (hydroxyl) version of RNA-233, with identical sequences to RNA-233 but lacking the 5’-triphosphate groups that result from IVT synthesis.

[0262] In Vitro Activity Assessment

[0263] To evaluate the immuno stimulatory activity of RNA-233, A549 dual reporter cells were transfected with the following RNA constructs formulated in TransIT-X2 transfection reagent:

[0264] • RNA- 113: chemically synthesized 3-arm nanostar with 5 ’-OH termini

[0265] • RNA-194: chemically synthesized version of RNA-113 with 5 ’-triphosphate (5’-ppp) on all three strands

[0266] • RNA-233: IVT-synthesized nanostar with 5’-ppp and no BLAST hits

[0267] • RNA-236: chemically synthesized 5’-OH version of RNA-233Forty-eight hours after transfection, activation of the interferon (IFN) pathway was measured by quantifying luciferase reporter activity (FIG. 24). Concentrations are reported as the concentration of 2 dsRNA ends to facilitate comparison between different constructs.

[0268] Results and Analysis

[0269] The results demonstrated that:

[0270] (1) 5’-OH controls showed similar potency: RNA-236 (the 5’-OH version of RNA-233) exhibited comparable activity to RNA-113, with EC50 values of 0.055 nM and 0.047 nM, respectively. This confirmed that the sequence scrambling in RNA-233 did not adversely affect nanostar activity when the constructs lack 5 ’-phosphorylation.

[0271] (2) 5’-triphosphorylation enhanced potency: Both 5 ’-ppp-containing constructs showed increased activity compared to their 5’-OH counterparts. RNA-233 (IVT-synthesized with 5’-ppp) exhibited an EC50 of 0.015 nM, representing approximately a 4-fold improvement over RNA-236 (EC50 = 0.055 nM). Similarly, chemically synthesized 5’-ppp-RNA-113 (RNA-194) showed an EC50 of 0.020 nM, representing approximately a 2-fold improvement over unphosphorylated RNA- 113 (EC50 = 0.047 nM). This is consistent with the known enhancement of RIG-I recognition by 5’- triphosphate groups.

[0272] (3) IVT-synthesized RNA-233 demonstrated potency comparable to chemically synthesized 5’-ppp-RNA-113: RNA-233 (EC50 = 0.015 nM) and RNA-194 (EC50 = 0.020 nM) exhibited similar activities, demonstrating that IVT-synthesized RNA nanostars can achieve potencies equivalent to chemically synthesized constructs when both contain 5 ’-triphosphate groups.

[0273] (4) Activity is RIG-I dependent: None of the constructs showed significant activity in A549 cells with RIG-I knockout, confirming that the observed interferon induction is specifically mediated by RIG-I activation (data not shown).

[0274] The EC50 values for all tested constructs are summarized in Table 10 below:

[0275]

[0276] This Example demonstrates that RNA nanostars can be successfully designed for IVT synthesis with the following advantages:

[0277] • Enhanced synthesis efficiency: The incorporation of 5’-GGA sequences enables approximately 10-fold higher IVT yields compared to sequences lacking this motif, potentially reducing manufacturing costs and improving scalability.• Sequence specificity: Systematic screening ensures that the nanostar strands do not share significant homology with endogenous transcripts, minimizing potential off-target effects while maintaining robust RIG-I activation.

[0278] • Comparable potency: IVT-synthesized RNA-233 (containing 5 ’-triphosphate groups) demonstrated interferon induction potency comparable to chemically synthesized 5’-ppp- RNA-113, with EC50 values of 0.015 nM and 0.020 nM, respectively.

[0279] • RIG-I specificity: The lack of activity in RIG-I knockout cells confirms that interferon induction is specifically mediated through RIG-I activation, consistent with the mechanism of action for RNA nanostars described throughout this disclosure.

[0280] These results support the use of IVT synthesis as a scalable manufacturing approach for RNA nanostars that maintain the high potency and RIG-I specificity observed with chemically synthesized constructs, while offering the additional benefit of sequence designs that avoid potential homology with endogenous cellular RNAs.

[0281] Example 7. In Vivo Evaluation of Additional Delivery Formulations: Dendrimers and Poly(Beta-Amino Esters)

[0282] This Example relates to the evaluation of additional delivery formulations for RNA nanostars, specifically dendrimer-based and poly(beta-amino ester) (pBAE)-based delivery systems. While lipid nanoparticles (LNPs) have demonstrated effective delivery of RNA nanostars (as described in Examples 3 and 4), other formulation strategies can offer advantages in terms of manufacturing scalability, tissue targeting, or pharmacokinetic properties. This Example evaluates the pharmacodynamic properties and therapeutic efficacy of dendrimer and pBAE formulations delivering RNA- 113 in comparison to an exemplary LNP formulation.

[0283] Formulation Design

[0284] Dendrimer Formulations

[0285] Dendrimers are highly branched, nanoscale polymeric structures that can deliver nucleic acids through electrostatic interactions or covalent conjugation. Three dendrimer-based delivery approaches were evaluated:

[0286] (1) Dendrimer-RNA complex: RNA- 113 was complexed with dendrimers through non-covalent electrostatic interactions, forming polyplexes where multiple RNA molecules associate with dendrimer structures.

[0287] (2) Dendrimer 3’ conjugate: RNA- 113 strands were covalently conjugated to dendrimers at their 3’ termini, creating stable dendrimer- RNA conjugates.(3) Dendrimer 3’ cleavable conjugate: RNA-113 strands were conjugated to dendrimers via a cleavable linker (e.g., disulfide bond) at the 3’ terminus, designed to release free RNA following cellular uptake and exposure to the reducing intracellular environment.

[0288] (4) Dendrimer 5’ conjugate: RNA-113 strands were covalently conjugated to dendrimers at their 5’ termini.

[0289] Poly(Beta-Amino Ester) (pBAE) Formulations

[0290] Poly(beta-amino esters) are biodegradable cationic polymers that can complex with nucleic acids through electrostatic interactions and facilitate endosomal escape through their pH-buffering capacity. Several pBAE variants were evaluated:

[0291] (1) Linear pBAE with peptide end groups: Linear pBAE polymers were synthesized with cationic peptide end groups to enhance RNA binding and cellular uptake. Three variants were tested with different peptide sequences:

[0292] • C6CR3: pBAE terminated with a hexamethylene linker and three arginine residues (CRRR)

[0293] • C6CK3: pBAE terminated with a hexamethylene linker and three lysine residues (CKKK)

[0294] • C6CH3: pBAE terminated with a hexamethylene linker and three histidine residues (CHHH)

[0295] (2) Branched pBAE C6CR3: A branched pBAE architecture with C6CR3 end groups was synthesized to increase the number of cationic sites for RNA binding.

[0296] All formulations used RNA- 113 (the three-arm RNA nanostar with 24 base pair arms described in Example 2) with 5’-hydroxyl (5’-OH) termini. For comparison, RNA-113 was also formulated in lipid nanoparticles (LNPs) as described in Example 3.

[0297] Pharmacodynamic Study Design

[0298] To evaluate the pharmacodynamic properties of dendrimer and pBAE formulations, female C57BL / 6 mice (8-10 weeks old) were administered test formulations by intravenous injection (100 pL, weight-based dosing). The study included the following groups (n=3 mice per group):

[0299] Group 1: PBS (negative control)

[0300] Group 2: Liver LNP / 5’-OH RNA-113 (35 pg RNA per mouse)

[0301] Group 3: Dendrimer-RNA complex / 5’-OH RNA-113 (45 pg RNA per mouse)

[0302] Group 4: Dendrimer 3’ RNA conjugate / 5’-OH RNA-113 (45 pg RNA per mouse) Group 5: Dendrimer 3’ cleavable RNA conjugate / 5’-OH RNA-113 (45 pg RNA per mouse)Group 6: Dendrimer 5’ RNA conjugate / 5’-OH RNA-113 (45 pg RNA per mouse) Group 7: Linear pBAE C6CR3 / 5’-OH RNA- 113 (45 pg RNA per mouse)

[0303] Group 8: Linear pBAE C6CK3 / 5’-OH RNA- 113 (45 pg RNA per mouse)

[0304] Group 9: Linear pBAE C6CH3 / 5’-OH RNA-113 (45 pg RNA per mouse)

[0305] Group 10: Linear pBAE C6CR3 / RNA-223 (freshly prepared, 45 pg RNA per mouse) Note that Group 10 used RNA-223 (Example 6), to evaluate whether the additional formulations are compatible with IVT-produced RNA.

[0306] Mice were monitored for clinical observations post-injection. Blood samples were collected at 2, 6, and 24 hours post-injection for measurement of plasma interferon-alpha (IFN-a), interferonbeta (IFN-P), interferon-gamma (IFN-y), interferon-lambda (IFN-1), and tumor necrosis factor-alpha (TNF-a) levels by enzyme-linked immunosorbent assay (ELISA).

[0307] Pharmacodynamic Results

[0308] Interferon and TNF-a Induction at 2 Hours

[0309] All dendrimer and pBAE formulations induced substantial increases in plasma IFN-a (FIG.

[0310] 25A), IFN-P (FIG. 25B), and TNF-a (FIG. 25C) at the 2-hour timepoint. The dendrimer 3' RNA conjugate formulation induced IFN-a levels of approximately 70,000 pg / mL, substantially higher than the liver LNP formulation (approximately 10,000 pg / mL). The dendrimer-RNA complex and dendrimer 3' cleavable conjugate formulations induced intermediate IFN-a levels of approximately 45,000 and 20,000 pg / mL, respectively. All formulations induced IFN-P levels in the range of 1,000-10,000 pg / mL, with the liver LNP and dendrimer-RNA complex showing the highest IFN-P induction (approximately 6,000-11,000 pg / mL). The dendrimer 3' conjugate formulation induced the highest TNF-a levels (approximately 550 pg / mL), followed by the liver LNP and dendrimer-RNA complex formulations (approximately 400-550 pg / mL). Lower TNF-a levels (approximately ISO-275 pg / mL) were observed with the pBAE formulations and dendrimer 3' cleavable conjugate. The three linear pBAE variants (CeCRs, CeCKs, and CeCHs) induced similar levels of IFN-a (data not shown on FIG. 25), with the CeCRs variant selected for further characterization.

[0311] Temporal Kinetics Over 24 Hours

[0312] The kinetics of IFN-a, IFN-P, IFN-y, and TNF-a were assessed over 24 hours for selected formulations (FIG. 26). All formulations showed maximal IFN-a, IFN-P, IFN-y, and TNF-a levels at the 2-hour timepoint, consistent with rapid RIG-I activation following systemic administration. By 6 hours post-injection, IFN-a and TNF-a levels had decreased substantially for all formulations, approaching baseline levels. By 24 hours, all cytokine levels had returned to near-baseline values, indicating transient immune activation. IFN-y (a Type II interferon) was induced to much lowerlevels (approximately 3-5 pg / mL at peak) compared to IFN-a and IFN-P, consistent with RIG-I primarily activating Type I interferon responses. These kinetic profiles are consistent with the transient nature of innate immune activation by RNA nanostars and suggest that the formulations do not cause sustained inflammatory responses.

[0313] Therapeutic Efficacy Study Design

[0314] To evaluate the therapeutic efficacy of dendrimer and pBAE formulations, a subcutaneous B16-F10 melanoma model was used. B16-F10 is a murine melanoma cell line that forms rapidly growing tumors in syngeneic C57BL / 6 mice. Female C57BL / 6 mice (6-8 weeks old) were inoculated subcutaneously with B16-F10 cells (Day 0). When tumors reached palpable size (approximately 50-100 mm3), mice were randomized into treatment groups (n=3 per group) and received three intravenous doses of test formulations on Days 7, 11, and 15 post-tumor inoculation (100 pL per dose).

[0315] Treatment groups:

[0316] Group 1: PBS (negative control)

[0317] Group 2: Liver LNP / 5’-OH RNA-113 (35 pg RNA per mouse per dose)

[0318] Group 3: Dendrimer-RNA complex / 5’-OH RNA-113 (45 pg RNA per mouse per dose) Group 4: Dendrimer 3’ conjugate / 5’-OH RNA-113 (45 pg RNA per mouse per dose) Group 5: Linear pBAE C6CR3 / 5’-OH RNA-113 (45 pg RNA per mouse per dose) Group 6: Branched pBAE C6CR3 / 5’-OH RNA-113 (45 pg RNA per mouse per dose) Tumor volumes were measured by caliper measurements every 2-3 days, and body weights were monitored throughout the study as a measure of tolerability.

[0319] Therapeutic Efficacy Results

[0320] Tumor Growth Inhibition

[0321] Both dendrimer and pBAE formulations demonstrated significant tumor growth inhibition compared to PBS-treated controls (FIGs. 27A-27B). Both the dendrimer-RNA complex and dendrimer 3' conjugate formulations significantly reduced tumor growth compared to PBS controls. By Day 11 (approximately 4 days after the third dose), untreated tumors reached volumes of approximately 550-600 mm3, while dendrimer-treated tumors were maintained at approximately 200-250 mm3, representing approximately 60-65% tumor growth inhibition. Both linear and branched pBAE CeCRs formulations showed tumor growth inhibition similar to dendrimer formulations, with tumor volumes of approximately 250-350 mm3at Day 11, representing approximately 40-60% tumor growth inhibition relative to PBS controls. The liver LNP formulation showed similar tumor growthinhibition to the dendrimer and pBAE formulations, with tumor volumes of approximately 380-400 mm3at Day 11, representing approximately 30-35% tumor growth inhibition. Tumor growth inhibition was evident after the first dose and was maintained through multiple doses, suggesting that the formulations do not lose efficacy with repeated administration.

[0322] Tolerability Assessment

[0323] Body weight measurements indicated that all formulations were well tolerated (FIGs. 28A-28B). All treatment groups maintained relatively stable body weights throughout the study period, with no significant body weight loss (>10% of baseline) observed in any group, indicating good tolerability. Dendrimer, pBAE, and LNP formulations showed similar body weight profiles, with no formulation showing substantially worse tolerability than others. Clinical observations during and after dosing revealed no signs of acute toxicity, distress, or abnormal behavior in any treatment group, further confirming the favorable safety profile of these formulations.

[0324] This Example demonstrates that dendrimer-based and pBAE-based delivery systems represent viable alternatives to lipid nanoparticles for systemic delivery of RNA nanostars.

[0325] Both dendrimer and pBAE formulations induce robust interferon responses. The dendrimer 3’ conjugate formulation induced particularly strong IFN-a responses (approximately 70,000 pg / mL at 2 hours), substantially higher than liver-tropic LNP formulations (noting, however, that dendrimers and pBAE groups were dosed at higher RNA- 113 concentrations in this study). This suggests that covalent conjugation of RNA to dendrimers may enhance cellular uptake, endosomal escape, or intracellular trafficking to optimize RIG-I activation.

[0326] Alternative formulations demonstrate therapeutic efficacy comparable to LNPs. In the Bl 6-F10 melanoma model, dendrimer and pBAE formulations achieved tumor growth inhibition of 40-65%, comparable to or exceeding that observed with liver-tropic LNP formulations (30-35%). This demonstrates that these alternative delivery systems can effectively deliver RNA nanostars to achieve anti-tumor responses in vivo.

[0327] The formulations are well tolerated with transient immune activation. The rapid rise and fall of cytokine levels (peak at 2 hours, return to baseline by 24 hours) indicates transient immune activation without sustained inflammation. The absence of significant body weight loss or clinical signs of toxicity further supports the safety profile of these formulations.

[0328] Polymer architecture and chemistry influence activity. Among the dendrimer formulations, the 3’ conjugate showed the highest activity. Among the pBAE variants, the linear C6CR3 formulation (with arginine-rich end groups) performed well, consistent with the known cellpenetrating properties of arginine-rich peptides.The alternative formulations may offer manufacturing and targeting advantages. While LNPs have demonstrated excellent performance, dendrimer and pBAE formulations may offer advantages in terms of manufacturing scalability (particularly for chemically synthesized dendrimers), stability during storage, or the ability to incorporate targeting ligands for tissue-specific delivery. The comparable efficacy observed here suggests that these platforms warrant further development for clinical applications.

[0329] These results expand the delivery platform options for RNA nanostars and demonstrate that multiple formulation approaches can achieve effective RIG-I activation and therapeutic responses in vivo. The selection of optimal formulation for clinical development may depend on factors such as manufacturing complexity, cost, stability, tissue targeting requirements, and compatibility with specific RNA nanostar designs (including IVT-synthesized constructs as demonstrated with RNA-223 in Group 10 of the pharmacodynamic study).

[0330] EQUIVALENTS

[0331] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described in the present application. Such equivalents are intended to be encompassed by the following claims.

[0332] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0333] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0334] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0335] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semiclosed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0336] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0337] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

CLAIMSWhat is claimed is:

1. A nanostructure comprising multiple double- stranded ribonucleic acid (dsRNA) arms, wherein the nanostructure regulates intracellular immunomodulatory signaling.

2. The nanostructure of claim 1, wherein the nanostructure comprises RNA.

3. The nanostructure of claim 2, wherein the nanostructure is a nanostar comprising at least 3 dsRNA arms.

4. The nanostructure of claim 1, wherein the nanostructure comprises DNA.

5. The nanostructure of claim 4, wherein the nanostructure is a DNA origami nanostructure.

6. The nanostructure of any preceding claim, wherein at least one of the dsRNA arms comprises a modification, relative to a naturally occurring RNA molecule.

7. The nanostructure of claim 6, wherein the modification is at a 5’ end of at least one of the dsRNA arms or at a 3’ end of at least one of the dsRNA arms.

8. The nanostructure of claim 6, wherein the modification is selected from sugar modifications, base modifications, and phosphate (backbone) modifications.

9. The nanostructure of any of claims 6-8, wherein the modification is selected from:an unpaired nucleotide, optionally selected from unpaired adenine (A) and unpaired uracil (U);a non-canonical nucleotide, optionally selected from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), queuosine (Q), azidothymidine (AZT), 2'-fluoro-modified nucleotides (e.g., 2'-F-uridine), and locked nucleic acids (LNAs); anda chemically modified nucleotide, optionally selected from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), azidothymidine (AZT), and 2'-fluoro-modified nucleotides, optionally 2'-F-uridine.

10. The nanostructure of any of claims 8-9, wherein the modification is selected from:a base modification selected from 5 -methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), 2-thiouridine (s2U), and inosine (I);a sugar modification selected from 2'-O-methyl (2'-0Me), 2'-fluoro (2'-F), locked nucleic acid (ENA), and peptide nucleic acid (PNA); anda phosphate modification selected from phosphorothioate (PS), cyclization, and a-thio-triphosphates.

11. The nanostructure of any preceding claim, wherein at least two of the dsRNA arms are linked to each other via Watson-Crick base pairing.

12. The nanostructure of any preceding claim, wherein at least one of the multiple dsRNA arms comprises a 5'-terminal monophosphate, diphosphate, triphosphate, or hydroxyl group.

13. The nanostructure of any preceding claim, wherein at least one of the dsRNA arms has a length of about 10-60 base pairs, optionally 11-52 base pairs.

14. The nanostructure of any preceding claim, wherein each of the multiple dsRNA arms independently comprises a nucleotide sequence of any one of SEQ ID NOs: 1-167.

15. The nanostructure of any preceding claim, wherein the nanostructure is immunostimulatory.

16. The nanostructure of claim 15, wherein the dsRNA arms bind to RIG-I, optionally activates a RIG-I-IRF3 pathway, thereby inducing interferon (IFN) production, and optionally wherein the IFN is a type I, type II, or type III IFN.

17. The nanostructure of any preceding claim, wherein the nanostructure is immunosuppressive.

18. The nanostructure of claim 17, wherein at least one of the dsRNA arms comprises a nick, optionally at a 5’ end or a 3’ end of a dsRNA molecule, and optionally wherein the RNA nanostar does not block binding of binding proteins but does block multimerization of the binding proteins or conformational changes to the binding proteins.

19. The nanostructure of claim 17 or 18, wherein the dsRNA arms bind to RIG-I, and optionally inhibits activation of a RIG-I- IRF3 pathway.

20. The nanostructure of any preceding claim, wherein at least one of the dsRNA arms comprises a linker, optionally at a 5’ end or a 3’ end of a dsRNA molecule.

21. The nanostructure of claim 20, wherein the linker is selected from: (i) unpaired nucleotides, optionally unpaired A and U; (ii) chemically modified nucleotides, (iii) polyethylene glycol (PEG) spacers, (iv) nucleotide spacers, and (v) amino acid spacers.

22. A ribonucleic acid (RNA) nanostar comprising three double- stranded RNA arms of any preceding claim.

23. A scaffold comprising multiple dsRNA arms of any preceding claim.

24. The scaffold of claim 23, wherein the scaffold comprises DNA, RNA, protein, or a polymer.

25. A pharmaceutical composition comprising the nanostructure of any preceding claim and a pharmaceutically acceptable excipient.

26. A delivery modality, optionally a lipid nanoparticle, a dendrimer, or a poly(beta-amino ester) formulation, comprising the nanostructure of any preceding claim.

27. A method comprising contacting a cell with the nanostructure of any preceding claim.

28. A method of regulating intracellular immunomodulatory pathway signaling in a subject, the method comprising administering to the subject the nanostructure, pharmaceutical composition, or nanoparticle of any preceding claim.

29. The method of claim 28, wherein the subject has or is suspected of having (i) an immune disorder, optionally an autoimmune disorder, (ii) cancer, or (iii) a viral infection.

30. A three-arm ribonucleic acid (RNA) nanostar comprising:a first RNA strand comprising a first domain and a second domain;a second RNA strand comprising a third domain and a fourth domain; anda third RNA strand comprising a fifth domain and a sixth domain,wherein the first domain of the first RNA strand is bound to the sixth domain of the third RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to thethird domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm.

31. A four-arm ribonucleic acid (RNA) nanostar comprising:a first RNA strand comprising a first domain and a second domain;a second RNA strand comprising a third domain and a fourth domain;a third RNA strand comprising a fifth domain and a sixth domain; anda fourth RNA strand comprising a seventh domain and an eighth domain,wherein the first domain of the first RNA strand is bound to the eighth domain of the fourth RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm, the sixth domain of the third strand is bound to the seventh domain of the fourth strand to form a fourth dsRNA arm.

32. A five-arm ribonucleic acid (RNA) nanostar comprising:a first RNA strand comprising a first domain and a second domain;a second RNA strand comprising a third domain and a fourth domain;a third RNA strand comprising a fifth domain and a sixth domain;a fourth RNA strand comprising a seventh domain and an eighth domain; anda fifth RNA strand comprising a ninth domain and a tenth domain,wherein the first domain of the first RNA strand is bound to the tenth domain of the fifth RNA strand to form a first dsRNA arm, the second domain of the first RNA strand is bound to the third domain of the second RNA strand to form a second dsRNA arm, the fourth domain of the second RNA strand is bound to the fifth domain of the third RNA strand to form a third dsRNA arm, the sixth domain of the third strand is bound to the seventh domain of the fourth strand to form a fourth dsRNA arm, and the eight domain of the fourth strand is bound to the ninth domain of the fifth strand to form a fifth dsRNA arm.

33. The RNA nanostar of any one of claims 30-32, wherein at least one of the dsRNA arms comprises a modification, relative to a naturally occurring RNA molecule.

34. The RNA nanostar of any one of claims 30-33, wherein the modification is at a 5’ end of at least one of the dsRNA arms or at a 3’ end of at least one of the dsRNA arms.

35. The RNA nanostar of claim 34, wherein the modification is selected from sugar modifications, base modifications, and phosphate (backbone) modifications.

36. The RNA nanostar of any of claims 33-35, wherein the modification is selected from:an unpaired nucleotide, optionally selected from unpaired adenine (A) and unpaired uracil (U);a non-canonical nucleotide, optionally selected from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), queuosine (Q), azidothymidine (AZT), 2'-fluoro-modified nucleotides (e.g., 2'-F-uridine), and locked nucleic acids (LNAs); anda chemically modified nucleotide, optionally selected from inosine (I), 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), azidothymidine (AZT), and 2'-fluoro-modified nucleotides, optionally 2'-F-uridine.

37. The RNA nanostar of any of claims 33-35, wherein the modification is selected from:a base modification selected from 5-methylcytosine (5mC), N6-methyladenosine (m6A), pseudouridine (T), 2-thiouridine (s2U), and inosine (I);a sugar modification selected from 2'-O-methyl (2'-0Me), 2'-fluoro (2'-F), locked nucleic acid (LNA), and peptide nucleic acid (PNA); anda phosphate modification selected from phosphorothioate (PS), cyclization, and a-thio-triphosphates.

38. The RNA nanostar of any of claims 30-37, wherein at least one of the multiple dsRNA arms comprises a 5'-terminal monophosphate, diphosphate, triphosphate, or hydroxyl group.

39. The RNA nanostar of any of claims 30-38, wherein at least one of the dsRNA arms has a length of about 10-60 base pairs, optionally 11-52 base pairs.

40. The RNA nanostar of any of claims 30-39, wherein each of the multiple dsRNA arms independently comprises a nucleotide sequence of any one of SEQ ID NOs: 1-167.

41. The RNA nanostar of any of claims 30-40, wherein the RNA nanostar is immunostimulatory.

42. The RNA nanostar of claim 41, wherein the dsRNA arms bind to RIG-I, optionally activates a RIG-I-IRF3 pathway, thereby inducing interferon (IFN) production, and optionally wherein the IFN is a type I, type II, or type III IFN.

43. The RNA nanostar of any of claims 30-42, wherein the nanostructure is immunosuppressive.

44. The RNA nanostar of claim 43, wherein at least one of the dsRNA arms comprises a nick, optionally at a 5’ end or a 3’ end of a dsRNA molecule, and optionally wherein the RNA nanostar does not block binding of binding proteins but does block multimerization of the binding proteins or conformational changes to the binding proteins.

45. The RNA nanostar of claim 43 or 44, wherein the dsRNA arms bind to RIG-I, and optionally inhibits activation of a RIG-I- IRF3 pathway.

46. The RNA nanostar of any of claims 30-44, wherein at least one of the dsRNA arms comprises a linker, optionally at a 5’ end or a 3’ end of a dsRNA molecule.

47. The RNA nanostar of claim 46, wherein the linker is selected from: (i) unpaired nucleotides, optionally unpaired A and U; (ii) chemically modified nucleotides, (iii) polyethylene glycol (PEG) spacers, (iv) nucleotide spacers, and (v) amino acid spacers.

48. A pharmaceutical composition comprising the RNA nanostar of any of claims 30-37 and a pharmaceutically acceptable excipient.

49. A nanoparticle, optionally a lipid nanoparticle, comprising the RNA nanostar of any of claims 30-37.

50. A method comprising contacting a cell with the RNA nanostar of any of claims 30-37.

51. A method of regulating intracellular immunomodulatory pathway signaling in a subject, the method comprising administering to the subject the RNA nanostar of any of claims 30-37, pharmaceutical composition of claim 48, or nanoparticle of claim 49.

52. The method of claim 51, wherein the subject has or is suspected of having (i) an immune disorder, optionally an autoimmune disorder, (ii) cancer, or (iii) a viral infection.

53. The RNA nanostar of any of claims 30-47, wherein the RNA nanostar facilitates efficient multimerization of multimerizing binding proteins and increases interferon production.

54. The RNA nanostar of any of claims 30-47, wherein the RNA nanostar activates RIG-I in vivo to induce an interferon signaling.

55. A DNA nanocube comprising:one or more DNA molecule(s) assembled through complementary base pairing into a nanocube; anddsRNA molecules, optionally about 25 to about 75 dsRNA molecules, covalently linked to the one or more DNA molecule(s).

56. A lipid nanoparticle comprising the nanostructure or the nanostar, optionally the 3-arm, 4-arm, or 5-arm RNA nanostar, of any preceding claim.

57. A dendrimer conjugated to the nanostructure or the nanostar, optionally the 3-arm, 4-arm, or 5-arm RNA nanostar, of any preceding claim.

58. A poly(beta-amino ester) formulation comprising the nanostructure or the nanostar, optionally the 3-arm, 4-arm, or 5-arm RNA nanostar, of any preceding claim.

Images