One-component delivery systems for nucleic acids

Abstract

The present invention relates to amphiphilic Janus dendrimers capable of forming nanoparticles. The present invention also relates to a method for inducing an adaptive immune response in a subject, comprising administering to the subject an effective amount of a composition comprising at least one nucleoside modified RNA encoding at least one antigen and at least one amphiphilic Janus dendrimer, as well as a method for delivering a drug to a subject in need thereof, comprising delivering to the subject a composition comprising the drug encapsulated by nanoparticles.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 192,236, filed May 24, 2021, U.S. Provisional Application No. 63 / 253,348, filed October 7, 2021, and U.S. Provisional Application No. 63 / 316,794, filed March 4, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants 1720530, 1807127, 2104554 awarded by the National Science Foundation, and TR002776 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] The delivery of exogenously produced nucleic acids to cells and / or their nuclei to alter protein expression via viral and non-viral vectors represents one of the most fundamental concepts of nanomedicine. Both viral and non-viral delivery systems have advantages and disadvantages. Viral vectors have high transfection efficiency (95%) and higher specificity for cellular targeting than non-native cells. Some disadvantages of viral gene delivery include immunogenicity, cytotoxicity, difficulty in assembly, inflammatory responses to repeated administration, and the possibility of insertional mutagenesis. Non-viral delivery is biologically safe and exhibits lower toxicity and immunogenicity, but results in lower transfection efficiency (1-2%) and vectors that are less stable than viral vectors. Covalent dendrimers and supramolecular dendrimers complexed with nucleic acids via cationic peripheral groups have been used as non-viral vectors for DNA cell transfection.

[0004] Four-component lipid nanoparticles (LNPs), containing ionic lipids, phospholipids, cholesterol for improved mechanical properties, and poly(ethylene glycol) (PEG)-conjugated lipids for stability, are currently the primary nonviral vectors for mRNA delivery. The shortcomings of LNP fabrication and stability are demonstrated by the microfluidic devices required for their assembly and the need for storage at cryogenic temperatures (-70 °C). Their design, synthesis, and assembly were inspired by stealth liposomes, which were developed to deliver low-molar-mass drugs.

[0005] RNA is less stable than DNA and must be protected by encapsulation before it can be released into the cell. a At acidic pH (pH 3-5), LNPs can encapsulate large amounts of mRNA when the pH is less than 7. At physiological pH (7.4), LNPs have a nearly neutral surface charge, whereas at endosomal pH they have a high positive charge. It has been suggested that electrostatic interactions between cationically charged LNPs and naturally occurring anionic lipids in the endosomal membrane are responsible for RNA release. One of the major limitations of four-component vectors is the unknown distribution of their four components in LNPs. The separation of neutral ionic lipids as an oil phase in the core of LNPs is thought to be the cause of their very low transfection efficiency (1-2%). A second drawback of LNPs is caused by PEG-conjugated lipids, known as the "PEG dilemma." PEG conjugated to LNPs increases circulation time in the blood after intravenous injection. However, the same PEG has been shown to reduce gene expression by up to four orders of magnitude by reducing cellular uptake and intracellular trafficking following endosomal escape.

[0006] Charge-altered release transporters (CARTs) have also been demonstrated for mRNA delivery. This delivery concept is independent of the viral and non-viral LNP-based methodologies discussed above. Artificial and synthetic vesicles, such as liposomes and polymersomes, have been created for drug delivery and as mimics of natural cells. Dendrimersomes (DSs) assembled from amphiphilic Janus dendrimers (JDs) have been shown to exhibit excellent mechanical properties and stability, including in serum. Janus glycodendrimers (JGDs), amphiphilic JDs with sugars conjugated to their hydrophilic moieties, self-assemble into glycodendrimersomes (GDSs) that mimic the glycans of biological membranes and bind to sugar-binding proteins. Both JDs and JGDs self-assemble into monodisperse DSs and GDSs with mono- or multilayer structures by simple injection rather than by microfluidic techniques, and their dimensions can be predicted. Sequence-defined JGDs self-assemble upon injection into GDSs. They demonstrated that lower sugar density in a defined sequence induces higher biological activity for sugar-binding proteins.

[0007] Thus, there is a need in the art for compositions and methods for delivering mRNA. The present disclosure fulfills this unmet need. Summary of the Invention

[0008] In one aspect, the present invention relates, in part, to an ionic amphiphilic Janus dendrimer having the structure of Formula (I): [ka]

[0009] In some embodiments, A is [ka] or any combination thereof. In some embodiments, A is a polyvalent group comprising at least one selected from [ka] or a polyvalent group containing at least one selected from any combination thereof.

[0010] In some embodiments, the dashed line represents a binding site for one of X, Y, or Z.

[0011] In some embodiments, X is a hydrophilic group comprising at least one amine.

[0012] In some embodiments, Y is at least one C-C 30 In some embodiments, Y is a lipophilic group comprising an alkyl chain. In some embodiments, Y is at least two C-C alkyl groups having different numbers of carbon atoms. 30 - is a lipophilic group containing an alkyl chain.

[0013] In some embodiments, Z comprises at least one selected from ethylene glycol, diethylene glycol, triethylene glycol, or a polyethylene glycol chain.

[0014] In some embodiments, R A and R B are independently hydrogen, halogen-substituted, hydroxy, C1-C 30 -Alkyl, C1-C 30 -Alkyl halides, C1-C 30 -Alkoxy, C1-C 30 -halogenated alkoxy, or any combination thereof.

[0015] In some embodiments, s is an integer from 0 to 5. In some embodiments, s is an integer from 1 to 5.

[0016] In some embodiments, t is an integer from 0 to 5. In some embodiments, t is an integer from 1 to 5.

[0017] In some embodiments, u is an integer from 0 to 4.

[0018] In some embodiments, the sum of s, t, and u is equal to the valence of A.

[0019] For example, in some embodiments, A is [ka] where s and t are each 2.

[0020] In some embodiments, A is [ka] where t is 2, s is 1, and u is 0 or 1.

[0021] In some embodiments, A is [ka] where s and t are each 1.

[0022] In some embodiments, the ionic amphiphilic Janus dendrimer having the structure of formula (I) is an ionic amphiphilic Janus dendrimer having the structure of formula (II): [ka]

[0023] In some embodiments, each occurrence of X is independently [ka] Or any combination thereof.

[0024] In some embodiments, the dashed line indicates a connection to A.

[0025] In some embodiments, each occurrence of m, n, and o is independently an integer from 1 to 20. In some embodiments, each occurrence of m, n, and o is independently an integer from 1 to 10. In some embodiments, each occurrence of m, n, and o is independently an integer from 1 to 5.

[0026] In some embodiments, each occurrence of W is independently selected from C=O, C(R W )(R W ), NR W , O, or S. In some embodiments, R W Each occurrence of is independently selected from hydrogen, halogen-substituted, hydroxy, alkyl, halogenated alkyl, aryl, halogenated aryl, alkoxy, halogenated alkoxy, or any combination thereof.

[0027] In some embodiments, L 1 , L 2 , L 3 , and L 4 Each occurrence is independently a covalent bond or a divalent linking group selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof.

[0028] In some embodiments, R 11 , R 12 , R 13 , and R 14 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. 11 , R 12 , R 13 , or R 14 At least one occurrence of -C(O)(CH2)m -N(R 1 )(R 2 In some embodiments, each occurrence of m is independently an integer from 1 to 10. In some embodiments, R 1 and R 2 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. 1 and R 2 may be taken together to form a ring. In some embodiments, R 11 , R 12 , R 13 , and R 14 Each entity of [ka] or any combination thereof.

[0029] In some embodiments, X comprises at least two tertiary amines.

[0030] In some embodiments, each occurrence of X is independently [ka] Or any combination thereof.

[0031] In some embodiments, R 3 Each occurrence of is independently hydrogen, (CH2) n , (CH2) n -OH, or any combination thereof.

[0032] In some embodiments, each occurrence of X is [ka] or any combination thereof.

[0033] In some embodiments, each occurrence of Y is [ka] or any combination thereof.

[0034] In some embodiments, R 21 , R 22 , R 23 , and R 24 each occurrence is independently a straight or branched chain C-C 50 In some embodiments, R 21 , R 22 , R 23 , and R 24 Each occurrence of is independently C1-C 30 -Contains alkyl.

[0035] In some embodiments, each occurrence of Y is independently [ka] or any combination thereof. In some embodiments, n is an integer from 1 to 30.

[0036] In some embodiments, each occurrence of Y is independently [ka] Or any combination thereof.

[0037] Some embodiments include -(CH) n CH3, -O(CH2) n - and -O(CH2) n Each occurrence of CH3 is independently straight or branched chain.

[0038] In some embodiments, the ionic amphiphilic Janus dendrimer comprises a first Y and a second Y. In some embodiments, the first Y comprises an alkyl chain having an even number of carbon atoms and the second Y comprises an alkyl chain having an odd number of carbon atoms. In some embodiments, the ratio between the carbon atoms in the first Y and the carbon atoms in the second Y is greater than or equal to 3 and less than 7.

[0039] In some embodiments, u is 1 or 2.

[0040] In some embodiments, each occurrence of Z is independently [ka] Or any combination thereof.

[0041] In some embodiments, R 31 , R 32 , R 33 , and R 34 In some embodiments, each occurrence of Z is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. [ka] In some embodiments, each occurrence of n is independently an integer from 1 to 100. In some embodiments, each occurrence of p is independently an integer from 1 to 10. In some embodiments, R 4 Each occurrence of is independently selected from hydrogen, deuterium, alkyl, aryl, or any combination thereof.

[0042] In some embodiments, the ionic amphiphilic Janus dendrimers comprise homochiral, racemic, or achiral branding points.

[0043] In some embodiments, the ionic amphiphilic Janus dendrimer is a homochiral ionic amphiphilic Janus dendrimer, a racemic ionic amphiphilic Janus dendrimer, or an achiral ionic amphiphilic Janus dendrimer.

[0044] In some embodiments, the ionic amphiphilic Janus dendrimer is an ionic amphiphilic Janus dendrimer having a structure selected from the group consisting of at least one structure in Figure 12, at least one structure in Figure 13, at least one structure in Figure 14, at least one structure in Figure 47, at least one structure in Figure 60, at least one structure in Figure 80, and any combination thereof.

[0045] In one aspect, the present invention relates, in part, to nanoparticles comprising at least one ionic amphiphilic Janus dendrimer of the present invention.

[0046] In some embodiments, the nanoparticles comprise a first ionic amphiphilic Janus dendrimer and a second ionic amphiphilic Janus dendrimer, hi some embodiments, the first ionic amphiphilic Janus dendrimer has a different structure than the second ionic amphiphilic Janus dendrimer.

[0047] In some embodiments, the nanoparticles comprise homochiral ionic amphiphilic Janus dendrimers, achiral ionic amphiphilic Janus dendrimers, or any combination thereof.

[0048] In some embodiments, the nanoparticles are single-layered nanoparticles or onion-multilayered nanoparticles. In some embodiments, the nanoparticles comprise racemic ionic amphiphilic Janus dendrimers. In some embodiments, the nanoparticles are multilayered nanoparticles.

[0049] In some embodiments, the nanoparticles further comprise at least one agent. In some embodiments, the at least one agent comprises a diagnostic agent, a detectable agent, a therapeutic agent, a nucleic acid molecule, or any combination thereof. In some embodiments, the at least one agent is selected from mRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, a small molecule, a protein, an antibody, a peptide, a protein, or any combination thereof.

[0050] In some embodiments, at least one agent comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is selected from cDNA, cRNA, cirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, target nucleic acid, or any combination thereof. In some embodiments, the nucleic acid molecule encodes at least one selected from an antigen, an antibody, a gene editing molecule, a chimeric antigen receptor (CAR), or any combination thereof. In one embodiment, the nucleoside-modified RNA comprises pseudouridine. In one embodiment, the nucleoside-modified RNA comprises pseudouridine + 5-methyl-cytosine. In one embodiment, the nucleoside-modified RNA comprises 5-methyl-uridine. In one embodiment, the nucleoside-modified RNA comprises 1-methyl-pseudouridine.

[0051] In one aspect, the present invention relates to a composition comprising at least one dendrimersome nanoparticle described herein.

[0052] In one aspect, the invention relates, in part, to a composition comprising at least one ionic amphiphilic Janus dendrimer of the invention and / or at least one nanoparticle of the invention.

[0053] In one embodiment, the composition further comprises an adjuvant.

[0054] In a preferred embodiment, the composition is a pharmaceutical composition.

[0055] In one embodiment, the composition is a vaccine.

[0056] In one aspect, the invention relates, in part, to a method of delivering a pharmaceutical agent to a subject in need thereof using at least one nanoparticle or a composition of the invention comprising the same.

[0057] In some embodiments, the method further comprises delivering the agent to the liver of the subject.

[0058] In some embodiments, the method further comprises delivering the agent to the spleen of the subject.

[0059] In some embodiments, the method further comprises delivering the agent to the lungs of the subject.

[0060] In some embodiments, the method treats or prevents at least one condition selected from a viral infection, a bacterial infection, a fungal infection, a parasitic infection, cancer, a cancer-related disease or disorder, an autoimmune disease or disorder, or any combination thereof.

[0061] In some embodiments, the agent is encapsulated within the nanoparticle. In one embodiment, the agent is any agent described herein. For example, in one embodiment, the agent is a composition for protein replacement therapy. In one embodiment, the agent is a composition for gene editing. In one embodiment, the agent is a vaccine.

[0062] In one aspect, the invention relates, in part, to a method of preventing or treating a disease or disorder in a subject in need thereof using at least one nanoparticle or a composition of the invention comprising same, hi some embodiments, the disease or disorder is selected from a viral infection, a bacterial infection, a fungal infection, a parasitic infection, cancer, a cancer-related disease or disorder, an autoimmune disease or disorder, or any combination thereof.

[0063] In one aspect, the invention relates, in part, to a method of inducing an immune response in a subject in need thereof using at least one nanoparticle or a composition of the invention comprising same.

[0064] In one aspect, the present invention relates to a method of delivering a drug to a subject in need thereof, comprising administering to the subject at least one dendrimersome nanoparticle described herein or a composition comprising same. [Brief explanation of the drawings]

[0065] The following detailed description of the embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings, it being understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0066] [Figure 1] FIG. 1 shows a schematic diagram illustrating an exemplary one-component nanoparticle (DNP) for mRNA delivery.

[0067] [Figure 2] A schematic diagram illustrating a four-component lipid nanoparticle (LNP) system for mRNA delivery is shown.

[0068] [Figure 3] 1 shows a schematic diagram of the hydrophilic acid, hydrophobic acid, and linker used in an exemplary DNP.

[0069] [Figure 4] A schematic diagram of six libraries containing 52 ionic amphiphilic Janus dendrimers (IAJDs) is shown.

[0070] [Figure 5] Representative luciferase expression in HEK293T cells with luciferase-mRNA encapsulated DNPs is shown.

[0071] [Figure 6]Representative assay results of in vivo transfection of one-component DNP are shown. The diameter and polydispersity of DNP (both in black) and the pKa value of IAJD (in blue) are shown below the number of the IAJD molecule. All of this data is printed above each mouse image. Luminescence values ​​are also shown.

[0072] [Figure 7] Representative results are shown in which luciferase signals from in vivo images were quantified.

[0073] [Figure 8] Representative results comparing the concentration and sequence of IA of the IAJD for in vitro and in vivo activity are shown. On the right is a schematic diagram of the IAJD used.

[0074] [Figure 9] Representative in vivo images of organs are shown.

[0075] [Figure 10] Representative images of mRNA delivery to different organs by one-component DNP are shown.

[0076] [Figure 11] Representative results are shown demonstrating examples of the excellent stability of DNPs assembled from IAJD9, IAJD22, IAJD33, IAJD34, IAJD32, IAJD33 + IAJD32 (2%), IAJD46, and IAJD47.

[0077] [Figure 12-1] Exemplary compounds from single-single IAJD libraries 1-4 are shown. Dark yellow means that the molecule is active both in vitro and in vivo, with the exception of IAJD19, 23, and 45, which only exhibit in vivo activity and not in vitro activity. Light yellow means that the molecule only exhibits in vitro activity. White means that the molecule exhibits neither in vitro nor in vivo activity. [Figure 12-2]Exemplary compounds from single-single IAJD libraries 1-4 are shown. Dark yellow means that the molecule is active both in vitro and in vivo, with the exception of IAJD19, 23, and 45, which only show in vivo activity and not in vitro activity. Light yellow means that the molecule only shows in vitro activity. White means that the molecule shows no activity in vitro or in vivo. [Figure 12-3] Exemplary compounds from single-single IAJD libraries 1-4 are shown. Dark yellow means that the molecule is active both in vitro and in vivo, with the exception of IAJD19, 23, and 45, which only show in vivo activity and not in vitro activity. Light yellow means that the molecule only shows in vitro activity. White means that the molecule shows no activity in vitro or in vivo.

[0078] [Figure 13] Shown are exemplary compounds from Twin-Twin IAJD Library 5. Dark yellow means the molecule is active both in vitro and in vivo. Light yellow means the molecule is only active in vitro. White means the molecule is neither active in vitro nor in vivo.

[0079] [Figure 14-1] Shown are exemplary compounds from Hybrid Twin-Mix IAJD Library 6. Dark yellow means the molecule is active both in vitro and in vivo. Light yellow means the molecule is active only in vitro. White means the molecule is not active in vitro or in vivo. [Figure 14-2] Shown are exemplary compounds from Hybrid Twin-Mix IAJD Library 6. Dark yellow means the molecule is active both in vitro and in vivo. Light yellow means the molecule is active only in vitro. White means the molecule is not active in vitro or in vivo. [Figure 14-3]Shown are exemplary compounds from Hybrid Twin-Mix IAJD Library 6. Dark yellow means the molecule is active both in vitro and in vivo. Light yellow means the molecule is active only in vitro. White means the molecule is not active in vitro or in vivo.

[0080] [Figure 15] A representative MALDI-TOF MS spectrum of compound 91c (4 / 2DMBA 1,3 Bn 2 PEG 4) is shown. The right side shows a magnified spectrum.

[0081] [Figure 16] 1 shows results demonstrating the effect of vortex time on aggregate size of the exemplary dendrimer IAJD9 (4.0 mg / mL in Tris buffer).

[0082] [Figure 17] Representative DLS data of the assembly of IAJD9 (8.00–1.00 mg / ml) in Tris buffer (pH = 7.4) are shown.

[0083] [Figure 18] Representative DLS data of the aggregates of IAJD22 (8.0–1.0 mg / ml) in Tris buffer (pH = 7.4) are shown.

[0084] [Figure 19] Representative DLS data of the aggregates of IAJD27 (8.0–1.0 mg / ml) in Tris buffer (pH = 7.4) are shown.

[0085] [Figure 20] Representative DLS data of DNPs assembled from IAJD1 and IAJD2 are shown.

[0086] [Figure 21] Representative DLS data of DNPs assembled from IAJD8 and IAJD9 are shown.

[0087] [Figure 22] Representative DLS data of DNPs assembled from IAJD10 and IAJD17 are shown.

[0088] [Figure 23] Representative DLS data of DNPs assembled from IAJD18 and IAJD19 are shown.

[0089] [Figure 24] Representative DLS data of DNPs assembled from IAJD20 and IAJD21 are shown.

[0090] [Figure 25] Representative DLS data of DNPs assembled from IAJD22 and IAJD23 are shown.

[0091] [Figure 26] Representative DLS data of DNPs assembled from IAJD24 and IAJD25 are shown.

[0092] [Figure 27] Representative DLS data of DNPs assembled from IAJD26 and IAJD27 are shown.

[0093] [Figure 28] Representative DLS data of DNPs assembled from IAJD28 and IAJD29 are shown.

[0094] [Figure 29] Representative DLS data of DNPs assembled from IAJD30 and IAJD31 are shown.

[0095] [Figure 30] Representative DLS data of DNPs assembled from IAJD33 and IAJD34 are shown.

[0096] [Figure 31]Representative DLS data of DNPs assembled from IAJD37 and IAJD40 are shown.

[0097] [Figure 32] Representative DLS data of DNPs assembled from IAJD43 and IAJD44 are shown.

[0098] [Figure 33] Representative DLS data of DNPs assembled from IAJD45 and IAJD46 are shown.

[0099] [Figure 34] Representative DLS data of DNPs assembled from IAJD47 are shown.

[0100] [Figure 35] Representative results are shown showing examples of good stability of DNP assembled from IAJD27 and poor stability of DNP assembled from IAJD30 and IAJD31.

[0101] [Figure 36] Representative results showing the dimensions of DNPs assembled from IAJD33 + IAJD32 (2%) in 1% fetal bovine serum are shown.

[0102] [Figure 37] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0103] [Figure 38] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0104] [Figure 39] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0105] [Figure 40]1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0106] [Figure 41] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0107] [Figure 42] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0108] [Figure 43] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0109] [Figure 44] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0110] [Figure 45] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0111] [Figure 46] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0112] [Figure 47-1] 1 shows a schematic diagram of additional compounds of the present invention as well as the pKa values ​​determined for these compounds. [Figure 47-2] 1 shows a schematic diagram of additional compounds of the present invention as well as the pKa values ​​determined for these compounds. [Figure 47-3] 1 shows a schematic diagram of additional compounds of the present invention as well as the pKa values ​​determined for these compounds.

[0113] [Figure 48]Representative results showing the distribution of nanoparticles in mice as a function of IAJD are shown.

[0114] [Figure 49] Representative results showing a comparison of in vivo and in vitro luminescence for different IAJDs are shown.

[0115] [Figure 50] Representative DLS data of DNPs assembled from IAJD64, IAJD65, IAJD66, IAJD70, IAJD71, IAJD74, IAJD75, IAJD76, and IAJD77 are shown.

[0116] [Figure 51] Representative DLS data of DNPs assembled from IAJD78, IAJD79, IAJD81, IAJD82, IAJD83, IAJD84, IAJD85, IAJD86, and IAJD87 are shown.

[0117] [Figure 52] Representative DLS data of DNPs assembled from IAJD88, IAJD89, IAJD91, IAJD95, IAJD96, IAJD97, IAJD98, IAJD99, and IAJD103 are shown.

[0118] [Figure 53] Representative DLS data of DNPs assembled from IAJD105, IAJD106, IAJD107, and IAJD108 are shown.

[0119] [Figure 54] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0120] [Figure 55] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0121] [Figure 56]1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0122] [Figure 57] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0123] [Figure 58] Representative results showing a comparison of in vivo and in vitro efficacy are shown.

[0124] [Figure 59] Schematic diagram of the synthesis of asymmetric IAJDs.

[0125] [Figure 60-1] A schematic diagram of IAJD81 to IAJD159 is shown. [Figure 60-2] A schematic diagram of IAJD81 to IAJD159 is shown. [Figure 60-3] A schematic diagram of IAJD81 to IAJD159 is shown. [Figure 61] Representative pKa and emission spectra of selected IAJDs are shown.

[0126] [Figure 62] Representative results demonstrating luminescence of selected IAJDs by location in mice are shown.

[0127] [Figure 63] Representative DLS data for assembled DNPs from IAJD113 to IAJD120 and IAJD122 are shown.

[0128] [Figure 64] Representative DLS data for assembled DNPs IAJD124 to IAJD130, IAJD133, IAJD135, and IAJD136 are shown.

[0129] [Figure 65]Representative DLS data for IAJD138 and assembled DNPs from IAJD141 to IAJD148 are shown.

[0130] [Figure 66] Representative DLS data for assembled DNPs from IAJD149 to IAJD154, IAJD161, IAJD162, and IAJD171 are shown.

[0131] [Figure 67] Representative DLS data for assembled DNPs IAJD172, IAJD173, IAJD177, IAJD178, IAJD110, IAJD111, and IAJD155 to IAJD159 are shown.

[0132] [Figure 68] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0133] [Figure 69] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0134] [Figure 70] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0135] [Figure 71] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0136] [Figure 72-1] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule. [Figure 72-2] 1 shows a representative titration curve showing the change in solution pH upon addition of strong acid to an IAJD molecule.

[0137] [Figure 73]Representative results regarding the structure and activity of IAJD are shown.

[0138] [Figure 74] Schematic diagram of IAJD with asymmetric alkyl chains and representative results demonstrating selective in vivo delivery of Luc-mRNA to the spleen and lymph nodes by DNP and less selective in vivo delivery of Luc-mRNA to organs by DNP are shown.

[0139] [Figure 75] Representative results are shown showing the ratio of in vivo activity of an asymmetric IAJD with one C18 alkyl chain to the corresponding symmetric IAJD with two identical C18 alkyl chains.

[0140] [Figure 76] Representative results showing the dimensions of DNPs assembled from IAJD125 in 1% fetal bovine serum are shown.

[0141] [Figure 77] Representative results showing the dimensions of DNPs assembled from IAJD155 in 1% fetal bovine serum are shown.

[0142] [Figure 78] Representative DLS data of DNP125 and 178 before and after dialysis in 1×PBS buffer for 3 hours are shown.

[0143] [Figure 79] A schematic diagram of a glycerol amphiphilic JD is shown.

[0144] [Figure 80] Schematics of R-, S-, rac, and achiral glycerols with two protected OH groups, as well as homochiral (1R, 1S, 2R, 2S), racemic (1rac, 2rac), and achiral (3, 4) glycerol-JD are shown.

[0145] [Figure 81]Schematic diagram of the synthesis of glycerol-JD from Library 1. Reagents and conditions: (i) DCC, DPTS, DCM, 23°C, 12 hours; (ii) H2, Pd / C, EtOAc, 23°C, 8 hours; (iii) 1M HCl, MeOH, 23°C, 1 hour.

[0146] [Figure 82] Schematic diagram of the synthesis of glycerol-JD from Library 2. Reagents and conditions: (i) DCC, DPTS, DCM, 23°C, 12 hours; (ii) H2, Pd / C, EtOAc, 23°C, 8 hours; (iii) 1 M HCl, 1,4-dioxane, 60°C, 8 hours.

[0147] [Figure 83] Schematic diagram of the synthesis of achiral glycerol-JD. Reagents and conditions: (i) DCC, DPTS, DCM, 23 °C, 12 h; (ii) H2, Pd / C, EtOAc, 23 °C, 8 h; (iii) 1 M HCl, 1,4-dioxane, 60 °C, 8 h.

[0148] [Figure 84] Representative results are shown showing the concentration dependence of the diameter (Dh, in nm) and square diameter (Dh2) of DS assembled by glycerol-JD in water. The top two left graphs correspond to glycerol-JD in library 1. The bottom two right graphs correspond to achiral glycerol-JDs 3 and 4. The remaining graphs correspond to glycerol-JD in library 2.

[0149] [Figure 85] Representative cryo-TEM images of DS assembled with glycerol-JD2R, 2S, and 2rac are shown.

[0150] [Figure 86] Representative cryo-TEM images of glycerol-based JD1R (a), 1S (b), and 1rac (c) self-assembled DSs are shown. Scale bar: 100 nm.

[0151] [Figure 87] Representative cryo-TEM images of DS self-assembled by glycerol-based achiral JD3 are shown.

[0152] [Figure 88] Representative cryo-TEM images of DS self-assembled by glycerol-based achiral JD4 are shown.

[0153] [Figure 89] Representative results are shown showing histograms of the normalized frequency of the number of vesicular layers of DS assembled from glycerol-JD1R, 1S, 1rac, 2R, 2S, and 2rac by statistical analysis.

[0154] [Figure 90] Representative H NMR spectrum of (3,5)-12G1-GC-(R-BMPA-(3,4,5)-3EO-G1(1R). CDCl, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm and 2.04 ppm are due to partially deuterated residues of CDCl and EtOAc, respectively.

[0155] [Figure 91] Representative C NMR spectrum of (3,5)-12G1-GC-(R)-BMPA-(3,4,5)-3EO-G1(1R). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0156] [Figure 92] Representative H NMR spectrum of (3,5)-12G1-GC-(S-BMPA-(3,4,5)-3EO-G1(1S). CDCl3, 500 MHz, 298 K. The signals marked with an asterisk at δ 7.26 ppm and 2.04 ppm are due to partially deuterated residues of CDCl3 and EA, respectively.

[0157] [Figure 93]Representative C NMR spectrum of (3,5)-12G1-GC-(S-BMPA-(3,4,5)-3EO-G1(1S). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0158] [Figure 94] Representative H NMR spectrum of (3,5)-12G1-GC-(rac)-BMPA-(3,4,5)-3EO-G1(1rac). CDCl, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm, 5.30 ppm, and 1.66 ppm are due to partially undeuterated residues of CDCl, DCM, and water, respectively.

[0159] [Figure 95] Representative C NMR spectrum of (3,5)-12G1-GC-(rac)-BMPA-(3,4,5)-3EO-G1(1rac). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0160] [Figure 96] Representative H NMR spectrum of (3,5)-12G1-BMPA-GC-(R-(3,4,5)-3EO-G1(2R). CDCl3, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm and 1.68 ppm are due to partially undeuterated residues of CDCl3 and water, respectively.

[0161] [Figure 97] Representative C NMR spectrum of (3,5)-12G1-BMPA-GC-(R-(3,4,5)-3EO-G1(2R). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0162] [Figure 98]Representative H NMR spectrum of (3,5)-12G1-BMPA-GC-(S-(3,4,5)-3EO-G1(2S). CDCl3, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm and 1.65 ppm are due to partially undeuterated residues of CDCl3 and water, respectively.

[0163] [Figure 99] Representative C NMR spectrum of (3,5)-12G1-BMPA-GC-(S-(3,4,5)-3EO-G1(2S). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0164] [Figure 100] Representative H NMR spectrum of (3,5)-12G1-BMPA-GC-(rac)-(3,4,5)-3EO-G1(2rac). CDCl3, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm and 1.60 ppm are due to partially deuterated residues of CDCl3 and water, respectively.

[0165] [Figure 101] Representative C NMR spectrum of (3,5)-12G1-BMPA-GC-(rac)-(3,4,5)-3EO-G1(2rac). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0166] [Figure 102] Representative H NMR spectrum of (3,5)-12G1-GC-(achiral)-BMPA-(3,4,5)-3EO-G2(3). CDCl3, 500 MHz, 298 K. The asterisk signals at δ 7.26 ppm, 2.04 ppm, and 1.66 ppm are due to partially undeuterated residues of CDCl3, EtOAc, and water, respectively.

[0167] [Figure 103] Representative C NMR spectrum of (3,5)-12G1-GC-(achiral)-BMPA-(3,4,5)-3EO-G2(3). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0168] [Figure 104] Representative H NMR spectrum of (3,5)-12G2-BMPA-GC-(achiral)-(3,4,5)-3EO-G1(4) is shown. CDCl, 500 MHz, 298 K. The signals marked with an asterisk at δ 7.26 ppm, 5.30 ppm, and 1.67 ppm are due to partially undeuterated residues of CDCl, DCM, and water, respectively.

[0169] [Figure 105] Representative C NMR spectrum of (3,5)-12G2-BMPA-GC-(achiral)-(3,4,5)-3EO-G1(4). CDCl, 500 MHz, 298 K. The signal marked with an asterisk at δ 77.16 ppm is due to CDCl.

[0170] [Figure 106] A representative MALDI-TOF-MS spectrum of a glycerol-based JD from Library 1 is shown.

[0171] [Figure 107] A representative MALDI-TOF-MS spectrum of a glycerol-based JD from Library 2 is shown.

[0172] [Figure 108] Representative MALDI-TOF-MS spectra of achiral glycerol-based JD3 (left) and 4 (right) are shown.

[0173] [Figure 109] A representative HPLC trace of glycerol-based JD from Library 1 is shown.

[0174] [Figure 110] A representative HPLC trace of a glycerol-based JD from Library 2 is shown.

[0175] [Figure 111] Representative HPLC traces of glycerol-based achiral JD3 (left) and 4 (right) are shown.

[0176] [Figure 112] Representative results are shown showing the standard QQ plot of sample 1rac. The distribution of 1rac is standard-like.

[0177] [Figure 113] Representative results are shown showing the standard QQ plot of sample 2rac. The distribution of 2rac is standard-like. DETAILED DESCRIPTION OF THE INVENTION

[0178] The present invention is based, in part, on the unexpected finding that nanoparticles comprising at least one ionic amphiphilic Janus dendrimer having the structure of Formula (I) effectively and efficiently deliver drugs to a desired target. Accordingly, in one aspect, the present invention relates to an ionic amphiphilic Janus dendrimer having the structure of Formula (I). In another aspect, the present invention relates to nanoparticles comprising at least one ionic amphiphilic Janus dendrimer of the present invention. In some embodiments, the nanoparticles further comprise at least one drug. In some embodiments, the nanoparticles further comprise at least one drug encapsulated by the ionic amphiphilic Janus dendrimer of the present invention. In another aspect, the present invention relates to compositions comprising at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles thereof. In some embodiments, the composition is a vaccine.

[0179] In one aspect, the present invention relates to a method for delivering a drug to a target of interest using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof. In another aspect, the present invention relates to a method for preventing or treating a disease or disorder in a subject using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof. In another aspect, the present invention relates to a method for inducing an adaptive immune response in a subject using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof. definition

[0180] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0181] As used herein, each of the following terms has the meaning associated with it in this section.

[0182] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0183] As used herein, when referring to a numerical value and / or range of a measurable value, e.g., amount, duration, etc., "about" is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, where such variations are appropriate for carrying out the disclosed methods. For example, "about 40 [units]" can mean within ±25% of 40 (e.g., 30-50), ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, less than ±1%, or any other value or range of values ​​therein or below. Furthermore, the phrases "less than about [value]" or "greater than about [value]" should be understood in light of the definition of the term "about" provided herein.

[0184] The term "compound," as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. In one embodiment, the term also refers to stereoisomers and / or optical isomers (including racemic mixtures) or enantiomerically enriched mixtures of the disclosed compounds.

[0185] As used herein, the terms "analog," "analogue," or "derivative" are meant to refer to a chemical compound or molecule created from a parent compound or molecule by one or more chemical reactions. As such, an analog may have a structure similar to that of a small molecule therapeutic described herein, or may be based on the scaffold of a small molecule therapeutic described herein, but may differ therefrom with respect to certain components or structural makeup that may have metabolically similar or opposite effects. An analog or derivative can also be a small molecule that differs in structure from a reference molecule but retains essential properties of the reference molecule. An analog or derivative may alter its interactions with certain other molecules compared to the reference molecule. An analog or derivative molecule may also include salts, adducts, tautomers, isomers, prodrugs, or other variants of the reference molecule.

[0186] As used herein, the term "prodrug" refers to an agent that is converted into the parent drug in vivo. For example, the term "prodrug" refers to a derivative of a known direct-acting drug, which has enhanced delivery properties and therapeutic value compared to the drug and is converted into an active drug by an enzymatic or chemical process. In some embodiments, a "prodrug" refers to an inactive or relatively less active form of an active drug that becomes active by undergoing chemical conversion via one or more metabolic processes. In one embodiment, a prodrug is chemically converted into a biologically, pharmaceutically, or therapeutically active form of the compound upon in vivo administration. In another embodiment, a prodrug is enzymatically metabolized into a biologically, pharmaceutically, or therapeutically active form of the compound by one or more steps or processes. For example, the compounds of the present invention can be administered to a subject as a prodrug containing an initiator attached to the active drug, which is degraded by metabolic processes to release the active drug in its active form.

[0187] The term "tautomers" refers to structural isomers of organic compounds that are readily interconvertible by chemical processes (tautomerization).

[0188] The term "isomers" or "stereoisomers" refers to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0189] As used herein, the term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, an alkyl group having the specified number of carbon atoms (i.e., C1- 50 means 1 to 50 carbon atoms), straight or branched chain hydrocarbons containing straight, branched, or cyclic substituents. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.

[0190] As used herein, the term "substituted alkyl" means an alkyl as defined above substituted with one, two, or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH, amino, azido, -N(CH), -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C-C)alkyl, -C(=O)NH, -SONH, -C(=NH)NH, and -NO. Examples of substituted alkyl include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0191] As used herein, the term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight- or branched-chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom may be positioned at any position in the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment to which it is attached, or may be attached to the most distal carbon atom in the heteroalkyl group. Examples include -O-CH-CH-CH, -CH-CH-CH-OH, -CH-CH-NH-CH, -CH-S-CH-CH, and -CHCH-S(=O)-CH. Up to two heteroatoms may be consecutive, such as, for example, -CH-NH-OCH or -CH-CH-SS-CH.

[0192] The term "amino" refers to a group of formula -NRaRa, -NHRa, or -NH2, where each Ra is independently an alkyl, alkenyl, or alkynyl group as defined above containing 1 to 20 carbon atoms. Unless stated otherwise specifically in the specification, an alkylamino group may be optionally substituted.

[0193] The term "hydroxy" or "hydroxyl" refers to a radical of the formula OH.

[0194] As used herein, unless otherwise stated, the term "alkoxy," used alone or in combination with other terms, means an alkyl group, as defined above, having the designated number of carbon atoms, attached to the remainder of the molecule through an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologs and isomers.

[0195] The terms “halo,” “halogen-substituted,” or “halogen,” as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0196] "Haloalkyl" or "halogenated alkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2 trifluoroethyl, 1,2 difluoroethyl, 3 bromo 2 fluoropropyl, 1,2 dibromoethyl, etc. Unless stated otherwise specifically in the specification, a haloalkyl group may be optionally substituted.

[0197] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In one embodiment, a cycloalkyl group is saturated or partially unsaturated. In another embodiment, a cycloalkyl group is fused to an aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties: [ka]

[0198] Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes "unsaturated non-aromatic carbocyclyl" or "non-aromatic unsaturated carbocyclyl" groups, both of which refer to a non-aromatic carbocycle, as defined herein, containing at least one carbon-carbon double bond or one carbon-carbon triple bond.

[0199] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a cyclic group containing 1 to 4 ring heteroatoms, each selected from O, S, and N. In one embodiment, each heterocycloalkyl group has 4 to 10 atoms in its ring system, provided that the ring of the group does not contain two adjacent O atoms. In another embodiment, the heterocycloalkyl group is fused to an aromatic ring. In one embodiment, the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic ring system may be attached at any heteroatom or carbon atom that results in a stable structure, unless otherwise specified. The heterocycle may be aromatic or non-aromatic in nature. In one embodiment, the heterocycle is heteroaryl.

[0200] Examples of 3-membered heterocycloalkyl groups include, but are not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidine and beta-lactam. Examples of 5-membered heterocycloalkyl groups include, but are not limited to, pyrrolidine, oxazolidine, and thiazolidinedione. Examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidine, morpholine, and piperazine. Other non-limiting examples of heterocycloalkyl groups are: [ka] is.

[0201] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepine, and hexamethylene oxide.

[0202] As used herein, the term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings and having aromatic character, i.e., having (4n+2) delocalized π (pi) electrons, where n is an integer.

[0203] As used herein, unless otherwise specified, the term "aryl," used alone or in combination with other terms, means a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), which rings may be joined together in a pendant fashion, such as biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl.

[0204] An "aralkyl" or "arylalkyl" is an alkyl group of the formula -R b -R c refers to the radical of R b is an alkylene group as defined above, and R cis one or more aryl radicals as defined above, e.g., benzyl, diphenylmethyl, etc. Unless specifically stated otherwise in the specification, an aralkyl group may be optionally substituted. For example, as used herein, the term "aryl-(C1-C3)alkyl" refers to a functional group in which an alkylene chain of one to three carbons is attached to an aryl group, e.g., -CH2CH2-phenyl, -CH2-phenyl(benzyl), aryl-CH2-, and aryl-CH(CH3)-. The term "substituted aryl-(C1-C3)alkyl" refers to an aryl-(C1-C3)alkyl functional group in which the aryl group is substituted. Similarly, the term "heteroaryl-(C1-C3)alkyl" refers to a functional group in which an alkylene chain of one to three carbons is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. The term "substituted heteroaryl-(C1-C3)alkyl" refers to a heteroaryl-(C1-C3)alkyl functional group in which the heteroaryl group is substituted.

[0205] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. Polycyclic heteroaryls can contain one or more rings that are partially saturated. Examples include the following moieties: [ka]

[0206] Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (especially 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (especially 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (especially 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0207] Examples of polycyclic heterocycles and heteroaryls include indolyl (especially 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (especially 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (especially 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (especially 3-, 4-, 5-, 6-, and 7-indolyl), and the like. benzothienyl (especially 3-, 4-, 5-, 6-, and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (especially 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (especially 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (especially 2-benzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolidinyl, and quinolizidinyl.

[0208] As used herein, the term "substituted" means that an atom or group of atoms replaces a hydrogen as a substituent attached to another group. The term "substituted" further refers to any level of substitution, i.e., mono-, di-, tri-, tetra-, or penta-substitution, and such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In one embodiment, the number of substituents varies from 1 to 4. In another embodiment, the number of substituents varies from 1 to 3. In yet another embodiment, the number of substituents varies from 1 to 2.

[0209] As used herein, the term "optionally substituted" means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group may be optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional groups individually and independently selected from the groups described herein.

[0210] In one embodiment, the substituents are independently selected from oxo, halogen, —CN, —NH, —OH, —NH(CH, —N(CH), alkyl, alkyl (including straight chain, branched chain and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, —S-alkyl, S(═O)alkyl, S(═O)N[H, alkyl, or aryl], —C(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —C(═O)N[H, or substituted or unsubstituted alkyl or aryl], —OC(═O)N[substituted or unsubstituted alkyl], —NHC(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O)[substituted or unsubstituted alkyl], -C(OH)[substituted or unsubstituted alkyl] and -C(NH)[substituted or unsubstituted alkyl]. In another embodiment, by way of example, the optional substituents are selected from the group consisting of oxo, fluorine, chlorine, bromine, iodine, -CN, -NH, -OH, -NH(CH, -N(CH) , -CH3, -CH2CH3, -CH(CH3)2, -CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, -OCH2CF3, -S(=O)2-CH3, -C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and -C(=O)OH. 1-6 Alkyl, -OH, C 1-6 It is selected from the group consisting of alkoxy, halo, amino, acetamido, oxo and nitro. As used herein, when a substituent is an alkyl or alkoxy group, the carbon chain can be branched, straight, or cyclic.

[0211] Integers and R, R 1 , R 2 , R 3 , R4 , R 5 , R 6 and the like are made in the chemical structures and moieties disclosed and described herein. 1 , R 2 , R 3 , R 4 , R 5 , R 6 Any notation such as R, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 The above is applicable to any structure or moiety listed above.

[0212] The term "nanoparticle" refers to a particle having at least one dimension on the nanometer scale (e.g., 1-1,000 nm) comprising one or more amphiphilic Janus dendrimers of Formula (I). In some embodiments, the nanoparticles are included in a formulation comprising a nucleoside-modified RNA described herein. In some embodiments, such nanoparticles comprise an ionic hydrophilic group and a lipophilic (hydrophobic) group. In one embodiment, the nanoparticles further comprise one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In one embodiment, the nanoparticles do not comprise additional excipients. In one embodiment, the nanoparticles do not comprise any additional lipids, additional cationic polymers, steroids, neutral lipids, charged lipids, or polymer-conjugated lipids other than at least one compound of Formula (I). In some embodiments, the nucleoside-modified RNA is encapsulated in the lipid portion of the nanoparticle or in an aqueous space enclosed by some or all of the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, such as a harmful immune response.

[0213] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; J. Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0214] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0215] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring configurations.

[0216] "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0217] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein or amino acid sequence specifying the antibody, and which DNA sequence or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art. The term should also be taken to mean an antibody produced by synthesis of an RNA molecule encoding the antibody, which RNA molecule expresses the antibody protein or amino acid sequence specifying the antibody, and which RNA has been obtained by transcribing DNA (synthetic or cloning) or by other techniques available and well known in the art.

[0218] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response can include either antibody production or the activation of specific immunogenically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Those skilled in the art will understand that any DNA or RNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an adaptive immune response will thus encode an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be synthesized, recombinantly produced, or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0219] The term "adjuvant" as used herein is defined as any molecule that enhances the antigen-specific adaptive immune response.

[0220] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to its mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA can be said to encode a protein or other product of that gene or cDNA.

[0221] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid inside a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0222] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0223] As used herein, "homology" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0224] "Immunogen" refers to any substance introduced into the body to generate an immune response. The substance can be a physical molecule such as a protein, or can be encoded by a vector such as DNA, mRNA, or a virus.

[0225] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment (e.g., a host cell).

[0226] In the context of the present invention, the following abbreviations are used for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond): "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0227] The term "modulate," as used herein, means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical, but untreated, subject. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0228] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs may contain introns. Furthermore, the nucleotide sequence may contain modified nucleosides that can be translated by the translational machinery in a cell. For example, an mRNA in which all uridines are replaced with pseudouridine, 1-methylpseudouridine, or another modified nucleoside.

[0229] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to link two protein-coding regions, in the same reading frame.

[0230] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means (i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques, PCR™, etc.), as well as by synthetic means.

[0231] In certain instances, the polynucleotide or nucleic acid of the present invention is a "nucleoside-modified nucleic acid," which refers to a nucleic acid containing at least one modified nucleoside. A "modified nucleoside" refers to a nucleoside having a modification. For example, over 100 different nucleoside modifications have been identified in RNA (Rozenski et al., 1999, The RNA Modification Database: 1999 update. Nucleic Acids Res 27:196-197).

[0232] In certain embodiments, "pseudouridine" refers to, in another embodiment, m 1acp 3 Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In another embodiment, the term refers to m 1 In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to m 3 " refers to Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that has not been further modified. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the pseudouridines above. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0233] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that a nucleotide sequence encoding a protein may, in some versions, contain introns.

[0234] As used herein, the terms "amino acid," "amino acid monomer," or "amino acid residue" refer to any of the 20 naturally occurring amino acids, including synthetic amino acids with unnatural side chains, and including both the D and L optical isomers.

[0235] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which many forms exist. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0236] The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence, e.g., the promoter recognized by bacteriophage RNA polymerase and used to generate mRNA by in vitro transcription.

[0237] The term "specifically binds" as used herein with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such cross-species reactivity does not, in itself, change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction involving labeled "A" and the antibody.

[0238] As used herein, the terms "transfect" or "transformation" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0239] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0240] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, reducing, ameliorating, or eradicating at least one sign or symptom of a disease or disorder state.

[0241] As used herein, the terms "therapeutic compound," "therapeutic agent," "drug," "active pharmaceutical agent," and "active pharmaceutical ingredient" are used interchangeably to refer to a chemical substance that exhibits a specific pharmacological effect in the body and is administered for such purpose. Non-limiting examples of therapeutic agents include, but are not limited to, hydrophilic therapeutic agents, hydrophobic therapeutic agents, antibiotics, antibodies, small molecules, anti-cancer agents, chemotherapeutic agents, immunomodulatory agents, RNA molecules, siRNA molecules, DNA molecules, gene editing agents, gene silencing agents, CRISPR-associated agents (e.g., guide RNA molecules, endonucleases, and variants thereof), painkillers, vaccines, anticonvulsants; antidiabetic agents, antifungal agents, antitumor agents, antiparkinsonian agents, antirheumatic agents, appetite suppressants, biological response modifiers, cardiovascular agents, central nervous system stimulants, contraceptives, dietary supplements, vitamins, vitamins, vitamin D, and the like. These include drugs for treating various conditions, including steroids, lipids, sugars, metals, amino acids (and precursors), nucleic acids and precursors, imaging agents, diagnostic agents, dopamine receptor agonists, erectile dysfunction drugs, contraceptives, gastrointestinal drugs, hormones, immunomodulators, antihypercalcemic agents, mast cell stabilizers, muscle relaxants, nutritional supplements, ophthalmic drugs, osteoporosis drugs, psychotherapeutic drugs, parasympathomimetics, parasympatholytics, respiratory drugs, sedative-hypnotics, skin and mucous membrane drugs, smoking cessation drugs, steroids, sympatholytics, urinary tract drugs, uterine relaxants, vaginal drugs, vasodilators, antihypertensive drugs, hyperthyroidism drugs, antihyperthyroidism drugs, antiasthmatic drugs, and vertigo drugs. In certain embodiments, one or more therapeutic agents are water-soluble, poorly water-soluble drugs, or drugs with low, medium, or high melting points. The therapeutic agents may be provided with or without stabilizing salt(s).

[0242] Some examples of active ingredients suitable for use in the pharmaceutical formulations and methods of the present invention include those that are hydrophilic, lipophilic, amphiphilic, or hydrophobic, and those that can be solubilized, dispersed, or partially solubilized and dispersed on or around the nanoclusters. The active agent-nanocluster combination can be further coated to encapsulate the drug-nanocluster combination, and can be targeted, for example, by functionalizing the nanoclusters with aptamers and / or antibodies. Alternatively, the active ingredient can be provided separately from the solid pharmaceutical composition, such as for co-administration. Such an active ingredient can be any compound or mixture of compounds that has therapeutic or other value when administered to an animal, particularly a mammal, such as a drug, nutrient, cosmeceutical, dietary supplement, diagnostic agent, nutritional agent, etc. While the active agents described herein can be found in their natural state, they are generally provided in the form of a salt. The active agents described herein include their isomers, analogs, and derivatives.

[0243] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0244] The term "therapeutically effective amount" refers to an amount of a compound of interest that elicits the biological or medical response of a tissue, system, or subject that is being sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the onset of, or alleviate to some extent, one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0245] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0246] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate if the disease is not ameliorated.

[0247] In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health state.

[0248] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0249] As used herein, "treating a disease or disorder" means reducing the frequency with which a patient experiences symptoms of the disease or disorder. Disease and disorder are used interchangeably herein.

[0250] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which a patient experiences such sign or symptom, or both, is reduced.

[0251] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical, but untreated, subject. The term encompasses perturbing and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0252] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0253] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the invention. Thus, the description of a range should be considered to have all the possible subranges specifically disclosed, as well as each individual numerical value within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed each individual number within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This is true regardless of the breadth of the range.

[0254] detail The present invention is based, in part, on the unexpected finding that nanoparticles comprising at least one ionic amphiphilic Janus dendrimer having the structure of Formula (I) effectively and efficiently deliver drugs to a target of interest. Accordingly, in one aspect, the present invention relates to an ionic amphiphilic Janus dendrimer having the structure of Formula (I). In another aspect, the present invention relates to nanoparticles comprising at least one ionic amphiphilic Janus dendrimer of the present invention. In some embodiments, the nanoparticles further comprise at least one drug. In some embodiments, the nanoparticles further comprise at least one drug encapsulated by the ionic amphiphilic Janus dendrimer of the present invention. In another aspect, the present invention relates to compositions comprising at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles thereof. In some embodiments, the composition is a vaccine.

[0255] In one aspect, the present invention relates to a method for delivering a drug to a target of interest using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof. In another aspect, the present invention relates to a method for preventing or treating a disease or disorder in a subject using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof. In another aspect, the present invention relates to a method for inducing an adaptive immune response in a subject using at least one ionic amphiphilic Janus dendrimer of the present invention or nanoparticles or compositions thereof.

[0256] Compounds of the Invention In one aspect, the present invention provides an amphiphilic Janus dendrimer of formula (I): [ka] or a racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, or derivative thereof. In various embodiments, the amphiphilic Janus dendrimer is an ionic amphiphilic Janus dendrimer.

[0257] In some embodiments, A has the following structure: [ka] or any combination thereof. In some embodiments, A is a polyvalent group comprising at least one selected from [ka] or any combination thereof. In some embodiments, A is optionally substituted.

[0258] In some embodiments, the dashed line represents a binding site for one of X, Y, or Z.

[0259] In some embodiments, X is a hydrophilic group comprising at least one amine. In some embodiments, the hydrophilic group is optionally substituted. In some embodiments, the amine is optionally substituted.

[0260] In some embodiments, Y is at least one C-C 50 In some embodiments, Y is a lipophilic group comprising an alkyl chain. 30 In some embodiments, the lipophilic group is optionally substituted. In some embodiments, the lipophilic group is a C-C alkyl group. 50- The alkyl chain is optionally substituted.

[0261] In some embodiments, Z is a group comprising at least one selected from ethylene glycol, diethylene glycol, triethylene glycol, or a polyethylene glycol chain. In some embodiments, Z is optionally substituted. In some embodiments, the ethylene glycol, diethylene glycol, triethylene glycol, and / or the polyethylene glycol chain are optionally substituted.

[0262] In some embodiments, R A is hydrogen, halogen-substituted, hydroxy, C1-C 50 -Alkyl, C1-C 50 -Alkyl halides, C1-C 50 -Alkoxy, C1-C 50 -halogenated alkoxy, or any combination thereof. In some embodiments, R A is hydrogen, halogen-substituted, hydroxy, C1-C 30 -Alkyl, C1-C 30 -Alkyl halides, C1-C 30 -Alkoxy, C1-C 30 -halogenated alkoxy, or any combination thereof. In some embodiments, hydroxy, C-C 50 -Alkyl, C1-C 50-Alkyl halides, C1-C 50 -alkoxy and / or C1-C 50 The halogenated alkoxy is optionally substituted.

[0263] In some embodiments, R B is hydrogen, halogen-substituted, hydroxy, C1-C 50 -Alkyl, C1-C 50 -Alkyl halides, C1-C 50 -Alkoxy, C1-C 50 -halogenated alkoxy, or any combination thereof. In some embodiments, R B is hydrogen, halogen-substituted, hydroxy, C1-C 30 -Alkyl, C1-C 30 -Alkyl halides, C1-C 30 -Alkoxy, C1-C 30 -halogenated alkoxy, or any combination thereof. In some embodiments, hydroxy, C-C 50 -Alkyl, C1-C 50 -Alkyl halides, C1-C 50 -alkoxy and / or C1-C 50 The halogenated alkoxy is optionally substituted.

[0264] In some embodiments, s is an integer from 0 to 5. In some embodiments, s is an integer from 1 to 5. For example, in one embodiment, s is the integer 5. In one embodiment, s is the integer 4. In one embodiment, s is the integer 3. In one embodiment, s is the integer 2. In one embodiment, s is the integer 1. In one embodiment, s is the integer 0.

[0265] In some embodiments, t is an integer from 0 to 5. In some embodiments, t is an integer from 1 to 5. For example, in one embodiment, t is the integer 5. In one embodiment, t is the integer 4. In one embodiment, t is the integer 3. In one embodiment, t is the integer 2. In one embodiment, t is the integer 1. In one embodiment, t is the integer 0.

[0266] In some embodiments, u is an integer from 0 to 4. In some embodiments, u is an integer from 1 to 4. For example, in one embodiment, u is the integer 4. In one embodiment, u is the integer 3. In one embodiment, u is the integer 2. In one embodiment, u is the integer 1. In one embodiment, u is the integer 0.

[0267] In some embodiments, the sum of s, t, and u is equal to the valence of A. For example, in one embodiment, the valence of A is 5, and the sum of s, t, and u is 5. In one embodiment, the valence of A is 4, and the sum of s, t, and u is 4. In one embodiment, s and t are each 2. In one embodiment, s is 1 and t is 3. In one embodiment, s is 3 and t is 1. In one embodiment, s is 1, t is 1, and u is 2. In one embodiment, s is 2, t is 1, and u is 1. In one embodiment, s is 1, t is 2, and u is 1. In one embodiment, the valence of A is 2, and the sum of s, t, and u is 2. In one embodiment, s and t are each 1.

[0268] For example, in some embodiments, A is [ka] where s and t are each 2.

[0269] In some embodiments, A is [ka] where t is 2, s is 1, and u is 0 or 1.

[0270] In some embodiments, A is [ka] where s and t are each 1.

[0271] In one embodiment, u is 0. Thus, in one embodiment, the amphiphilic Janus dendrimer having the structure of formula (I) is an amphiphilic Janus dendrimer having the structure of formula (II): [ka] or a racemate, enantiomer, diastereomer, pharmaceutically acceptable salt, or derivative thereof.

[0272] In one embodiment, X is a hydrophilic group comprising one amine. In one embodiment, X is a hydrophilic group comprising two amines. In one embodiment, X is a hydrophilic group comprising three amines. In one embodiment, X comprises at least one primary amine. In one embodiment, X comprises at least one secondary amine. In one embodiment, X comprises at least one tertiary amine. For example, in one embodiment, X comprises at least two tertiary amines.

[0273] In one embodiment, X comprises at least one amine that is protonated under biological conditions. In one embodiment, X comprises at least one amine that has a formal charge of +1 under biological conditions. In one embodiment, X comprises at least one carbohydrate.

[0274] In one embodiment, each occurrence of X is independently selected from the following structures: [ka] Or any combination of these.

[0275] In some embodiments, the dashed line indicates a connection to A.

[0276] In some embodiments, each occurrence of m, n, and o is independently an integer from 1 to 5.

[0277] In some embodiments, each occurrence of W is independently selected from C=O, C(R W )(R W ), NR W , O, or S. In some embodiments, R W Each occurrence of is independently selected from hydrogen, halogen-substituted, hydroxy, alkyl, halogenated alkyl, aryl, halogenated aryl, alkoxy, halogenated alkoxy, or any combination thereof. In one embodiment, each occurrence of W is NH. In one embodiment, each occurrence of W is O. In one embodiment, at least one occurrence of W is NH and at least one occurrence of W is O.

[0278] In some embodiments, L 1 , L 2 , L 3 , and L 4 Each occurrence of is independently a covalent bond or a divalent linking group selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. 1 , L 2 , L 3 , and L 4 At least one occurrence of L is a poly(alkyl ether) or oligo(alkyl ether). 1 , L 2 , L 3 , and L 4 Each occurrence of L is polyethylene glycol (PEG) or oligoethylene oxide. 1 , L 2 , L3 , and L 4 Each occurrence of independently represents the structure -[CHCHO] n -, and n is an integer of 0 to 10.

[0279] In some embodiments, R 11 , R 12 , R 13 , and R 14 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. 11 , R 12 , R 13 , or R 14 At least one of R comprises an amine. 11 , R 12 , and R 13 In some embodiments, one of R 11 , R 12 , and R 13 In some embodiments, two of R 11 , R 12 , and R 13 In one embodiment, three of R 11 , R 12 , and R 13 At least one of R contains two amines. 11 , R 12 , and R 13 In one embodiment, the amine-free R 11 , R 12 , and R 13 Any of the groups may include an alkyl group, an aryl group, or a combination thereof.

[0280] In one embodiment, R 11 , R 12 , R 13 , or R 14 At least one occurrence of has the structure: --C(O)(CH2) m -N(R 1 )(R 2 ).

[0281] In some embodiments, each occurrence of m is an integer from 1 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0282] In some embodiments, R 1 and R 2 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. 1 and R 2 can join together to form a ring.

[0283] In one embodiment, R 1 and R 2 are each alkyl. In one embodiment, R 1 and R 2 are each methyl. In one embodiment, R 1 and R 2 taken together form a 6-membered heterocycle. In one embodiment, R 1 and R 2 are taken together with the N to which they are attached to form a piperidine ring. In one embodiment, R 1 and R 2 are taken together with the N to which they are attached to form an N-alkylpiperazine ring. In another embodiment, R 1 and R 2 are each hydrogen.

[0284] In one embodiment, R 11 , R 12 , R 13 , or R 14 At least one occurrence of has the structure: -C(O)(CR 5 R 6 ) m -N(R 1 )(R2 ).

[0285] In some embodiments, each occurrence of m is an integer from 1 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0286] In some embodiments, R 1 and R 2 Each occurrence of is independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof.

[0287] In some embodiments, R 5 and R 6 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. 1 , R 2 , R 5 , and R 6 Any two or more of may be joined together to form a ring.

[0288] In one embodiment, the structure -C(O)(CR 5 R 6 ) m -N(R 1 )(R 2 ) is derived from an amino acid. In one embodiment, the amino acid is a standard amino acid. In one embodiment, the amino acid is a non-standard amino acid. In one embodiment, the amino acid is a β-amino acid (i.e., m is at least 2 and R 5 and R 6 (One geminal pair of R is H). 5 and R 6 One of them is H and the other is R 5 and R 6and the other is not H. In one embodiment, the group can be chiral. In one embodiment, the group can be enantiomerically enriched or enantiomerically pure. In one embodiment, the group can be racemic.

[0289] In one embodiment, R 11 , R 12 , R 13 , and R 14 Each occurrence of is independently selected from the following structures: [ka]

[0290] In some embodiments, each occurrence of m is an integer from 1 to 10. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0291] In one embodiment, each occurrence of X is independently selected from the following structures: [ka] Or any combination of these.

[0292] In some embodiments, each occurrence of n is independently an integer from 1 to 20. In some embodiments, each occurrence of n is independently an integer from 1 to 10.

[0293] In some embodiments, each occurrence of m is independently an integer from 1 to 20. In some embodiments, each occurrence of m is independently an integer from 1 to 10.

[0294] In some embodiments, R 1 and R 2 Each occurrence of R is independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. 1 and R 2 can join together to form a ring.

[0295] In some embodiments, R 3 Each occurrence of is independently hydrogen, (CH2) n , (CH2) n -OH, or any combination thereof.

[0296] In some embodiments, R 4 Each occurrence of is independently selected from hydrogen, deuterium, alkyl, aryl, or any combination thereof.

[0297] For example, in some embodiments, each occurrence of X is [ka] or any combination thereof.

[0298] In one embodiment, Y is at least one C-C 50 In one embodiment, Y is a lipophilic (hydrophobic) group comprising an alkyl chain. 30 In one embodiment, Y is a lipophilic (hydrophobic) group comprising an alkyl chain. 30 For example, in one embodiment, Y is a lipophilic (hydrophobic) group containing at least two C-C alkyl chains. 30 In one embodiment, Y is a lipophilic (hydrophobic) group comprising an alkyl chain. In one embodiment, Y is at least two C-C alkyl groups having different numbers of carbon atoms. 30 It is a lipophilic (hydrophobic) group containing an alkyl chain.

[0299] In one embodiment, Y is a lipophilic group further comprising at least one linking group selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, Y comprises alkylene, arylene, alkenylene, alkynylene, disulfide, ether, or any combination thereof. In one embodiment, Y does not comprise an amine. In one embodiment, Y comprises at least one C6 alkyl chain, at least one C7 alkyl chain, at least one C8 alkyl chain, at least one C9 alkyl chain, at least one C 10 Alkyl chain, at least one C 11 Alkyl chain, at least one C 12 Alkyl chain, at least one C 13 Alkyl chain, at least one C 14 Alkyl chain, at least one C 15 Alkyl chain, at least one C 20 Alkyl chain, at least one C 21 Alkyl chain, at least one C 23 Alkyl chain, at least one C 27 Alkyl chain, at least one C 30 Alkyl chain, at least one C 35 Alkyl chain, at least one C 40 Alkyl chain or at least one C 50 In one embodiment, Y is a lipophilic group comprising an alkyl chain. In one embodiment, Y comprises at least one carbohydrate.

[0300] In one embodiment, each occurrence of Y is independently selected from the following structures: [ka] Or any combination of these.

[0301] In some embodiments, the dashed line indicates a connection to A.

[0302] In some embodiments, each occurrence of W is independently selected from C=O, C(R W )(R W ), NR W , O, or S. In some embodiments, R W Each occurrence of is independently selected from hydrogen, halogen-substituted, hydroxy, alkyl, halogenated alkyl, aryl, halogenated aryl, alkoxy, halogenated alkoxy, or any combination thereof. In one embodiment, each occurrence of W represents O. In one embodiment, each occurrence of W represents NH. In one embodiment, at least one occurrence of W represents O and at least one occurrence of W represents NH.

[0303] In some embodiments, R 21 , R 22 , R 23 , and R 24 Each occurrence of is independently C1-C 50 In some embodiments, R 21 , R 22 , R 23 , and R 24 each occurrence is independently a straight or branched chain C-C 50 In some embodiments, R 21 , R 22 , R 23 , and R 24 Each occurrence of is independently C1-C 30 In some embodiments, R 21 , R 22 , R 23 , and R 24 each occurrence is independently a straight or branched chain C-C 30 In one embodiment, R 21 , R 22 , R 23 , and R 24 Each occurrence of is independently C8-C 12 Represents alkyl.

[0304] In one embodiment, each occurrence of Y is independently selected from the following structures: [ka]

[0305] In some embodiments, each occurrence of W is independently selected from C=O, C(R W )(R W ), NR W , O, or S. In some embodiments, R W Each occurrence of is independently selected from hydrogen, halogen-substituted, hydroxy, alkyl, halogenated alkyl, aryl, halogenated aryl, alkoxy, halogenated alkoxy, or any combination thereof. In one embodiment, each occurrence of W represents O. In one embodiment, each occurrence of W represents NH. In one embodiment, at least one occurrence of W represents O and at least one occurrence of W represents NH.

[0306] In some embodiments, n is an integer from 1 to 30. In some embodiments, n is an integer from 6 to 18.

[0307] In some embodiments, each occurrence of Y is independently selected from: [ka] Or any combination of these.

[0308] In some embodiments, each occurrence of Y is independently selected from: [ka] Or any combination of these.

[0309] In some embodiments, the dashed line indicates a connection to A.

[0310] In some embodiments, each occurrence of n is independently an integer from 1 to 20.

[0311] In some embodiments, -(CH) n CH3, -O(CH2) n - and -O(CH2) n Each occurrence of CH3 is independently straight or branched chain.

[0312] In some embodiments, t is an integer from 2 to 5. Thus, in some embodiments, the amphiphilic Janus dendrimer comprises a first Y and a second Y. In some embodiments, the first Y comprises an alkyl chain having a different number of carbon atoms than the second Y comprising an alkyl chain. In some embodiments, the first Y comprises an alkyl chain having an even number of carbon atoms, and the second Y comprises an alkyl chain having an odd number of carbon atoms. In some embodiments, the ratio between the carbon atoms in the first Y and the carbon atoms in the second Y is 3 or more and less than 7.

[0313] In some embodiments, u is 1 or 2. In one embodiment, u is 1. In one embodiment, u is 2.

[0314] In one embodiment, Z does not comprise an amine.

[0315] In one embodiment, Z is not a lipophilic group.

[0316] In one embodiment, Z comprises at least one carbohydrate.

[0317] In one embodiment, each occurrence of Z is independently selected from the following structures: [ka] Or any combination of these.

[0318] In some embodiments, the dashed line indicates a connection to A.

[0319] In some embodiments, L 1 , L 2 , L 3, and L 4 Each occurrence of is independently a covalent bond or a divalent linking group selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. 1 , L 2 , L 3 , and L 4 At least one occurrence of represents a single bond. 1 , L 2 , L 3 , and L 4 At least one of the groups has the formula -WC(O)(CH2) p represents a group of C(O)-W-, where p is an integer from 1 to 10, and W represents NH, O, or S. In one embodiment, L 1 , L 2 , L 3 , and L 4 At least one of the groups comprises polyethylene glycol.

[0320] In some embodiments, R 31 , R 32 , R 33 , and R 34 Each occurrence of is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof.

[0321] In some embodiments, each occurrence of m, n, and o is independently an integer from 1 to 5.

[0322] In one embodiment, each occurrence of Z is independently selected from the following structures: [ka]

[0323] In some embodiments, each occurrence of n is independently an integer from 1-100.

[0324] In some embodiments, each occurrence of p is independently an integer from 1 to 10.

[0325] In some embodiments, R 4 Each occurrence of is independently selected from hydrogen, deuterium, alkyl, aryl, or any combination thereof.

[0326] In some embodiments, the amphiphilic Janus dendrimer is an amphiphilic Janus dendrimer having a structure selected from at least one structure in Figure 12, at least one structure in Figure 13, at least one structure in Figure 14, at least one structure in Figure 47, at least one structure in Figure 60, at least one structure in Figure 80, or any combination thereof.

[0327] In some embodiments, the amphiphilic Janus dendrimer is an amphiphilic Janus dendrimer having a structure selected from the following: [ka] [ka] [ka]

[0328] In some embodiments, the amphiphilic Janus dendrimer having the structure of formula (II) contains sufficient amines (ionizable amines) in X to provide dual hydrophilic and binding functionality in the IAJD. For example, Figure 47 shows the IAJDs described herein, synthesized, and investigated in vitro and in vivo, their schematic diagrams, and pK aThe pKa values ​​are summarized below. The pKa values ​​were determined as described in the Examples. Co-assembly of all IAJDs from Figure 47 with Luc-mRNA was performed by injecting an ethanolic solution of IAJD into acetate buffer at pH=4. The acetate buffer contained mRNA added to the buffer from neutral water. Further details can be found in the Examples section below. Co-assembly forms nanoparticles (DNPs). Co-assembly is shown schematically in Figure 1. Figure 48 shows the in vivo transfection results of DNPs co-assembled from the IAJDs shown in Figure 1. The IAJD number is shown in the top left, and the IAJD pKa a The luminescence values, size in nm, are shown above each mouse image, along with the polydispersity (PDI) of the resulting DNPs. The scale of the luminescence values ​​is also shown. Representative images of mRNA delivery to different organs are shown at the bottom of the figure. Figure 49 shows a comparison of the activity of DNPs assembled from IAJD, shown schematically in vitro (blue) and in vivo (red) at the top of the figure. Thus, in some embodiments, in a dendrimer having the structure of formula (II), the valency of A can be 2, and the sum of s and t can be 2.

[0329] In some embodiments, the amphiphilic Janus dendrimer comprises homochiral, racemic, or achiral branding points. Thus, in some embodiments, the ionic amphiphilic Janus dendrimer is a homochiral ionic amphiphilic Janus dendrimer, a racemic ionic amphiphilic Janus dendrimer, or an achiral ionic amphiphilic Janus dendrimer.

[0330] In some embodiments, the amphiphilic Janus dendrimer is a symmetric amphiphilic Janus dendrimer.

[0331] In some embodiments, the amphiphilic Janus dendrimer is an asymmetric amphiphilic Janus dendrimer.

[0332] In some embodiments, the amphiphilic Janus dendrimer is an asymmetric amphiphilic Janus dendrimer.

[0333] For example, in some embodiments, the successful design of the hydrophobic region of amphiphilic Janus dendrimers is based, in part, on the use of different alkyl lengths and the discovery of an unexpectedly important function of the primary structure of the hydrophobic portion of IAJD, which increases the activity of targeted delivery of the desired cargo to the target site by up to 90.2-fold. In some embodiments, asymmetric one-component amphiphilic Janus dendrimers do not require the microfluidic or T-tube technology used by four-component LNPs to coassemble with mRNA. In some embodiments, one-component systems can coassemble with mRNA into DNPs with approximately 97% nucleic acid encapsulation efficiency by simply injecting their ethanolic solutions into an acidic buffer containing mRNA, rather than by the microfluidic or T-tube technology required by LNPs.

[0334] For example, Figure 2 shows an overview of LNP structure and co-assembly with mRNA. Figure 1 shows the structures of sSS, SS, and TM IAJDs with different and similar alkyl groups in the hydrophobic moiety and their co-assembly with mRNA. IAJDs can be described as single-single (SS, a single hydrophilic dendron connected to a single lipophilic dendron), twin-twin (TT, two hydrophilic dendrons connected to two lipophilic dendrons), and twin-mix (TM, two different hydrophilic dendrons connected to two lipophilic dendrons) IAJDs.

[0335] For example, Figure 59 shows the synthesis of asymmetric IAJD. In the first step, the 3-benzyl ether of methyl 3,5-dihydroxybenzoate was produced by etherification of 1 with BNCl in DMF at 80 °C in 5 h in 39% isolated yield. Subsequently, 2 was alkylated with 1-bromoundecane or 1-bromopentadecane in DMF using KCO base at 120 °C to produce 3 in 80–100% isolated yield. Hydrogenolysis of 3 (H / Pd, DCM / MeOH, 12 h) produced 4 in 100% isolated yield. Alkylation of 4 with various alkyl lengths, from 1-bromooctane to 1-bromooctadecane, in DMF using KCO base at 120 °C produced the asymmetric compound 5 in 74–92% isolated yield. Reduction of compound 5 with LiAlH4 in THF (0–23 °C, 1 h) produced benzyl alcohol 6 in 93–100% isolated yield. Compound 6 was reacted with 4-bromobutyric acid via its acid chloride, generated with SOCl2 at 23 °C catalyzed by DMF in CHCl2, followed by either esterification in the presence of NEt3 / DMAP (0–23 °C, 2 h) or direct esterification with DCC / DPTS for 12 h to produce compound 7 in 76–98% isolated yield. Reaction of compound 7 with methylpiperazine or hydroxyethylpiperazine (K2CO3, MeCN, 95 °C, 3 h) afforded IAJD8 (70–90% isolated yield) and 9 (76–98% isolated yield). Their purity, determined by a combination of HPLC, MALDI-TOF, and NMR, was greater than 99%. Their structures are shown in Figure 60 (IAJD113-178). IAJD133 has a similar structure to IAJD105, except that in 133 the interconnecting ester group of 105 is replaced with an amide. The benzylamine precursor of 133 was generated from the corresponding benzyl alcohol via its benzyl chloride obtained with SOCl2, followed by reaction with K-phthalimide followed by hydrazine, as reported.

[0336] Single-single (SS) IAJDs, previously reported to exhibit very high activity for pulmonary delivery, were synthesized with asymmetric alkyl groups in their lipophilic moieties. These are IAJDs 110–159 from Figure 60. Their sequence-defined hydrophilic dendrons were synthesized as reported in Zhang et al., Am. Chem. Soc. 2021, 143, 12315–12327. The hydrophilic dendrons were reacted with selected asymmetric lipophilic dendrons 6 or their amines. For convenience, IAJDs are referred to by their number followed by the ratio between their two alkyl groups forming the asymmetric lipophilic moiety. This nomenclature, along with their overall and schematic structures shown in Figure 60, facilitates discussion of their in vitro and in vivo activity versus molecular structure. For example, 116 (11 / 13) and 117 (11 / 13) both contain 11 and 13 carbon combinations in their lipophilic moieties, but 116 contains a methylpiperazine and 117 contains a hydroxyethylpiperazine ionic amine. The large red dot at the top of the sketch of 117 indicates the hydroxyethyl, and the thin blue line on 116 indicates the methyl group, both of which are attached to the piperazine (Figure 60). The combinations of 33 IAJDSSs and 7 IAJDSSs are shown in Figure 60. IAJDs 81, 86, 105, 106, and 107, marked in blue in the upper left corner of Figure 60, were previously reported (Zhang et al., J. Am. Chem. Soc. 2021, 143, 17975-17982). Transfection experiments using Luc-mRNA were carried out both in vitro and in vivo by following the methods reported in Zhang et al., J. Am. Chem. Soc. 2021, 143, 12315-12327 and Zhang et al., J. Am. Chem. Soc. 2021, 143, 17975-17982).

[0337] The overall transfection activity in vivo was analyzed according to its target selectivity and is summarized in Figure 62. The first important result of the transfection experiment was that the 11 IAJDs were approximately 10 8The symmetric IAJDs showed activity in the lungs, two in the liver, and six in the pancreas and lymph nodes (marked in pink in Figure 62 and Figure 74). Symmetric IAJDs 110 (12 / 12) and 111 (11 / 11), which contain an amide interconnecting group, were previously reported to show the highest activity in the lungs, as were IAJDs 33 (12 / 12) and 34 (11 / 11). The new IAJDs 110 (12 / 12) and 111 (11 / 11), which contain an interconnecting ester rather than an amide group, also showed very high activity in the lungs. The asymmetric IAJDs are stable in serum and PBS buffer and show very high activity in the lungs by a mechanism distinct from aggregation (Table below and Figures 76-78). The highest activity of all IAJDs was for 178 (13 / 18), which showed a 4.05 x 10 8 The total flux of MC3 p / s was 90.2-fold higher than that of the symmetric 99 (18 / 18) with the same head group, but only 4.2-fold lower than that of MC3 (Figure 61). It is also important to note that the transition from 158 (11 / 17) to 159 (11 / 17), the second of which is an IAJD containing an amide interconnecting group and the first an ester, resulted in an approximately six-fold increase in activity. This demonstrates the importance of the amide interconnecting group for pulmonary delivery, but also reveals that the presence of oligooxyethylene in the hydrophilic portion may be important for targeted delivery to the lung. These experiments demonstrate the important function of different alkyl groups derived from the hydrophobic portion of IAJD.

[0338] Finally, Figure 61 summarizes the activity of all IAJDs produced in the Examples and compares them with their symmetric (marked in light blue) and asymmetric (marked in blue) IAJDs, which can be used as control experiments. The results from Figure 61 show that up to a 90.2-fold increase in the activity of IAJDs was observed by changing their primary structure in the lipophilic moiety from symmetric to asymmetric. The ratio between the two alkyl lengths, preferably from odd-even combinations, may be greater than or equal to 3 and less than 7, and appears to result in the greatest increase in activity. Selected examples of IAJDs that support this conclusion are provided by IAJD119 (11 / 15), 125 (11 / 14), 127 (11 / 17), 128 (11 / 18), 130 (11 / EH), 153 (15 / 18), 155 (11 / EH), and 159 (11 / 17).

[0339] In some embodiments, the asymmetric amphiphilic Janus dendrimer is stable at about 5°C.

[0340] nanoparticles In one aspect, the present invention relates to nanoparticles comprising at least one amphiphilic Janus dendrimer of the present invention.

[0341] In various embodiments, the nanoparticles are single-component nanoparticles.

[0342] In various embodiments, the nanoparticles are quaternary nanoparticles.

[0343] In some embodiments, the nanoparticles comprise homochiral ionic amphiphilic Janus dendrimers, achiral ionic amphiphilic Janus dendrimers, or any combination thereof.

[0344] In some embodiments, the nanoparticles are single-layer nanoparticles.

[0345] In some embodiments, the nanoparticles are multi-layered nanoparticles.

[0346] In some embodiments, the nanoparticles are onion multilayer nanoparticles.

[0347] In some embodiments, the nanoparticles are racemic ionic amphiphilic Janus dendrimers.

[0348] In some embodiments, the nanoparticles are dendrimersome nanoparticles (DNPs).

[0349] In some embodiments, the nanoparticles comprise at least two amphiphilic Janus dendrimers. Thus, in some embodiments, the nanoparticles comprise a first ionic amphiphilic Janus dendrimer and a second ionic amphiphilic Janus dendrimer. In some embodiments, the first ionic amphiphilic Janus dendrimer has a different structure than the second ionic amphiphilic Janus dendrimer.

[0350] In some embodiments, the nanoparticles further comprise at least one amphiphilic Janus dendrimer as disclosed below (Wang et al., J. Am. Chem. Soc. 2020, 142, 9525-9536; Xiao et al., J. Am. Chem. Soc. 2016, 138, 12655-12663; Torre et al., Proc. Natl. Acad. Sci. USA 2019, 116, 15378-15385; Percec et al., J. Am. Chem. Soc. 2021, 143, 17724-17743; Wilson et al., J. Polym. Sci. Part A: Polymer Chemistry, 2010, 2498-2508; Xiao et al., Proc. Natl. Acad. Sci. USA 2017, E7045, E7053; and U.S. Patent Application Publication No. 2012277460; and U.S. Patent No. 8,614,347; (each of which is incorporated by reference in its entirety herein)).

[0351] In various embodiments, the nanoparticles are from about 10 nm to about 100,000 nm, from about 30 nm to about 1000 nm, from about 30 nm to about 500 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or from about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm , 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 200 nm, 250 nm, 300 nm, 310 nm, 375 nm, 400 nm, 500 nm, 800 nm, 1000 nm, 1250 nm, 1400 nm, or 1500 nm. For example, in some embodiments, the nanoparticles have an average diameter of about 10 nm to about 1,000 nm.

[0352] In various embodiments, the nanoparticles are substantially non-toxic.

[0353] In various embodiments, the nanoparticles are biodegradable.

[0354] In one aspect of the present invention, the nanoparticles comprise at least one cargo. In various aspects, the present invention is not limited to any particular cargo or other agent that the nanoparticles can carry or transport. Rather, the present invention includes any agent that can be carried by the nanoparticles. For example, agents that can be carried by the nanoparticles of the present invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents. Thus, in various embodiments, the nanoparticles comprise at least one agent. In other embodiments, the nanoparticles encapsulate at least one agent.

[0355] In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 1:1 to about 10,000:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 2:1 to about 1,000:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 3:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 4:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 5:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 6:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 7:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 8:1 to about 10:1. In some embodiments, the nanoparticles comprise or encapsulate at least one agent. In some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 9:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one agent, hi some embodiments, the weight ratio of amphiphilic Janus dendrimer to at least one agent is about 9.5:1 to about 10:1.

[0356] In various embodiments, the nanoparticles are suitable for delivering at least one cargo to cells of interest.

[0357] For example, in some embodiments, the cargo is at least one agent comprising a diagnostic agent, a detectable agent, a therapeutic agent, a nucleic acid molecule, a gene editing agent, a vaccine, a composition for protein replacement therapy, or any combination thereof. In some embodiments, the at least one agent is selected from an mRNA, an siRNA, a microRNA, a CRISPR-Cas9, an sgRNA, a small molecule, a protein, an antibody, a peptide, a protein, or any combination thereof. In some embodiments, the at least one agent comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes at least one selected from an antigen, an antibody, a gene editing molecule, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is selected from a cDNA, a cRNA, a cirRNA, an mRNA, a miRNA, an siRNA, an sgRNA, a modified RNA, a tRNA, an antagomir, an antisense molecule, a target nucleic acid, or any combination thereof. In some embodiments, the modified RNA is a nucleoside-modified RNA. In some embodiments, the nucleoside-modified RNA comprises a pseudouridine. In some embodiments, the nucleoside modified RNA comprises pseudouridine + 5-methyl-cytosine. In some embodiments, the nucleoside modified RNA comprises 5-methyl-uridine. In some embodiments, the nucleoside modified RNA comprises 1-methyl-pseudouridine.

[0358] Thus, in one embodiment, the nanoparticles can be used to deliver nucleoside-modified RNA to a subject in need thereof. In certain embodiments, delivering nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with at least one dendrimer of formula (I) prior to the contacting step. In another embodiment, the method of the present invention further comprises administering the nucleoside-modified RNA together with at least one dendrimer of formula (I).

[0359] In some embodiments, customizable targeting can be achieved based on the identity of linking group A. In addition, the identity of linking group A affects the delivery of nanoparticle cargo. For mRNA cargo, it has been found that ester linking groups lead to delivery to the liver and / or spleen, while amide groups favor delivery to the lung. This allows nanoparticles to be tailored to facilitate delivery to the desired target organ. An example is the delivery of anti-inflammatory drugs to the lung.

[0360] In another embodiment, the transfection reagent forms nanoparticles that are liposomes. In another embodiment, liposomes increase intracellular stability, increase uptake efficiency, and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of dendrimers arranged in a manner similar to the lipids that make up cell membranes. They have an internal aqueous space for entrapment of water-soluble compounds and range in size from 0.05 to several microns in diameter. In another embodiment, nanoparticle liposomes can deliver RNA to cells in a biologically active form.

[0361] In various embodiments, the nanoparticles are about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm to about 150 nm. In certain embodiments, the nucleoside-modified RNA has an average diameter of 0 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and is substantially non-toxic. In certain embodiments, the nucleoside-modified RNA, when present in the nanoparticles, is resistant to degradation by nucleases in aqueous solution.

[0362] Small molecule therapeutics In various embodiments, the agent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, the small molecule can be obtained using standard methods known to those of skill in the art. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis, and in vitro translation systems using methods well known in the art. In one embodiment, the small molecule therapeutic agent includes an organic molecule, an inorganic molecule, a biomolecule, a synthetic molecule, etc.

[0363] Combinatorial libraries of molecularly diverse compounds potentially useful in treating various diseases and conditions are well known in the art, as are methods for generating the libraries. The methods can use a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrays, deconvolution strategies, tagging techniques, and generation of unbiased molecular landscapes for lead discovery versus biased structures for lead development. In some embodiments of the present invention, therapeutic agents are synthesized and / or identified using combinatorial techniques.

[0364] In a general method for small library synthesis, an activated core molecule is condensed with many building blocks, resulting in a combinatorial library of covalently linked core-building block ensembles. The shape and rigidity of the core determine the orientation of the building blocks in shape space. These libraries can be biased by varying the core, linkages, or building blocks to target specified biological structures ("focused libraries"), or can be synthesized with less structural bias using flexible cores. In some embodiments of the invention, therapeutic agents are synthesized via small library synthesis.

[0365] The small molecules and small molecule compounds described herein may exist as salts even when no salt is indicated, and it is understood that the present invention encompasses all salts and solvates of the therapeutic agents described herein, as well as non-salt and non-solvated forms of the therapeutic agents, as will be appreciated by those of skill in the art. In some embodiments, the salts of the therapeutic agents of the present invention are pharmaceutically acceptable salts.

[0366] Where tautomeric forms may exist for any of the therapeutic agents described herein, it is intended that each and every tautomeric form be included in the invention, even if only one or some of the tautomeric forms may be explicitly depicted. For example, where a 2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridone tautomer is also intended.

[0367] The present invention also includes any or all stereochemical forms, including any enantiomeric or diastereomeric forms, of the described therapeutic agents. Recitation of a structure or name herein is intended to encompass all possible stereoisomers of the indicated therapeutic agent. All forms of a therapeutic agent, e.g., crystalline or amorphous forms of a therapeutic agent, are also encompassed by the present invention. Compositions comprising a therapeutic agent of the present invention, e.g., a composition of a substantially pure therapeutic agent containing its particular stereochemical form, or a composition comprising a mixture of a therapeutic agent of the present invention in any ratio containing two or more stereochemical forms, such as a racemic or non-racemic mixture, are also contemplated.

[0368] The present invention also includes any or all active analogs or derivatives, e.g., prodrugs, of any of the therapeutic agents described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. Thus, in certain instances, analogs of the small molecules described herein with tailored potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and development. Thus, in certain instances, new analogs are designed during optimization, taking into account issues of drug delivery, metabolism, novelty, and safety.

[0369] In some instances, the small molecule therapeutics described herein are derivatives or analogs of known therapeutic agents, as is well known in the art of combinatorial chemistry and medicinal chemistry. Analogs or derivatives can be prepared by adding and / or substituting functional groups at various positions. Thus, the small molecules described herein can be converted into derivatives / analogs using well-known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Additionally, linking atoms or groups can be modified to have carbon backbones or longer or shorter linkers with heteroatoms. Additionally, ring groups can be altered to have different numbers of atoms in the ring and / or to include heteroatoms. Furthermore, aromatic rings can be converted into cyclic rings, and vice versa. For example, rings can be 5-7 atoms and can be carbocyclic or heterocyclic.

[0370] As used herein, the terms "analog," "analogue," or "derivative" are meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog may have a structure similar to that of a small molecule therapeutic described herein, or may be based on the scaffold of a small molecule therapeutic described herein, but may differ therefrom with respect to specific components or structural makeup that may have metabolically similar or opposite effects. Analogs or derivatives of any of the small molecule inhibitors according to the present invention can be used to treat a disease or disorder.

[0371] In one embodiment, the small molecule therapeutics described herein can be independently derivatized, or analogs can be prepared therefrom by modifying hydrogen groups independently of each other to other substituents. That is, each atom on each molecule can be modified independently of other atoms on the same molecule. Any conventional modification for generating derivatives / analogs can be used. For example, the atoms and substituents can independently consist of hydrogen, alkyl, aliphatic, straight-chain aliphatic, aliphatic with chain heteroatoms, branched-chain aliphatic, substituted aliphatic, cycloaliphatic, heterocyclic aliphatic with one or more heteroatoms, aromatic, heteroaromatic, polyaromatic, polyamino acid, peptide, polypeptide, combinations thereof, halogen, halo-substituted aliphatic, etc. Furthermore, any ring group on the compound can be derivatized to increase and / or decrease ring size and change backbone atoms to carbon atoms or heteroatoms.

[0372] Nucleic acid therapeutics In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA, or miRNA molecule. In one embodiment, the isolated nucleic acid molecule encodes a therapeutic peptide such as an antithrombotic protein, including thrombomodulin, endothelial protein C receptor (EPCR), plasminogen activators and their mutants, or an antioxidant protein, including catalase, superoxide dismutase (SOD), and iron-sequestering proteins. In some embodiments, the therapeutic agent is an siRNA, miRNA, shRNA, or antisense molecule that inhibits a target nucleic acid, including those encoding proteins involved in the exacerbation of a pathological process.

[0373] In one embodiment, the nucleic acid comprises a promoter / regulatory sequence such that the nucleic acid can direct expression of the nucleic acid. Thus, the present invention encompasses expression vectors and methods for the introduction of an exogenous nucleic acid into a cell with co-expression of the exogenous nucleic acid in the cell, e.g., as described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as well as elsewhere herein.

[0374] In one embodiment, siRNA is used to reduce the level of a target protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types leads to the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs then assemble with protein components into the RNA-induced silencing complex (RISC), unwinding in the process. The activated RISC then binds to the complementary transcript through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, and sequence-specific degradation of the mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature, 395:854; Montgomery et al., 1998, TIG 14(7):255-258; David R. Engelke Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications to siRNA that facilitate intravenous systemic delivery. Optimization of siRNA involves consideration of overall G / C content, C / T content at the ends, Tm, and nucleotide content of the 3' overhang. See, e.g., Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Thus, the present invention also includes methods for reducing the level of PTPN22 using RNAi technology.

[0375] In one aspect, the present invention includes a vector comprising an siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide can inhibit the expression of a target polypeptide. The incorporation of a desired polynucleotide into a vector and the selection of the vector are well known in the art, for example, as described in Sambrook et al. (2012) and Ausubel et al. (1997), and are described elsewhere herein.

[0376] In certain embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) therapeutic agent.shRNA molecules are well known in the art and are directed against target mRNA, thereby reducing the expression of target.In certain embodiments, the coded shRNA is expressed by cells and then processed into siRNA.For example, in certain cases, cells have the natural enzyme (for example, Dicer) that cuts shRNA to form siRNA.

[0377] To assess the expression of siRNA, shRNA, or antisense polynucleotides, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification of expressing cells from a population of cells desired to be transfected or infected using the delivery vehicle of the present invention. In other embodiments, the selectable marker can be carried on a separate piece of DNA and can also be included in the delivery vehicle. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0378] Thus, in one aspect, the delivery vehicle may comprise a vector containing the nucleotide sequence or construct to be delivered. The choice of vector depends on the host cell into which the vector is subsequently introduced. In certain embodiments, the vector of the present invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Prokaryotic and / or eukaryotic vector-based systems can be used for use in the present invention to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0379] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which, when introduced into a cell, may or may not be integrated into the genome of the host cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the invention or the genetic constructs of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0380] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0381] In one embodiment, the recombinant expression vector may also include a nucleic acid molecule encoding the peptide or peptidomimetic.

[0382] A promoter can be one naturally associated with a gene or polynucleotide sequence, such as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer can be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter (which refers to a promoter not normally associated with a polynucleotide sequence in its natural environment). A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or expression-altering mutations. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques (including PCR™) in connection with the compositions disclosed herein (U.S. Pat. Nos. 4,683,202 and 5,928,906). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria and chloroplasts, can similarly be used.

[0383] Naturally, it is important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression; see, for example, Sambrook et al. (2012). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0384] The recombinant expression vector may also contain a selectable marker gene that facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, such as the Fc portion of immunoglobulins, preferably IgG. The selectable marker may be introduced on a vector separate from the nucleic acid of interest.

[0385] Following generation of an siRNA polynucleotide, one skilled in the art will appreciate that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Thus, siRNA polynucleotides can be further designed to resist degradation by modification to include phosphorothioate or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate ester, etc. (see, e.g., Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0386] Any polynucleotide can be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends, the use of phosphorothioate or 2'O-methyl rather than phosphodiester linkages in the backbone, and / or the inclusion of non-conventional bases such as inosine, queosine, and vabutosine, as well as acetyl-, methyl-, thio-, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0387] In one embodiment of the present invention, antisense nucleic acid sequences expressed by a plasmid vector are used as therapeutic agents to inhibit expression of a target protein. The antisense expression vector is used to transfect mammalian cells or the mammal itself, thereby causing a reduction in endogenous expression of the target protein.

[0388] Antisense molecules and their use to inhibit gene expression are well known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40) (as this term is defined elsewhere herein). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule, thereby inhibiting translation of the gene.

[0389] The use of antisense method to inhibit gene translation is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal.Biochem.172:289).Such antisense molecule can be provided to cell through gene expression using DNA encoding antisense molecule, as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0390] Alternatively, antisense molecules of the present invention can be synthetically produced and then delivered to cells. Antisense oligomers of about 10 to about 30 nucleotides, more preferably about 15 nucleotides, are preferred because they are easily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

[0391] In one embodiment of the present invention, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes useful for inhibiting the expression of target molecules can be designed, for example, by incorporating a target sequence complementary to the mRNA sequence encoding the target molecule into the basic ribozyme structure. Ribozymes that target target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or can be genetically expressed from DNA that encodes them.

[0392] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system in which a guide RNA (gRNA) that targets a gene encoding a target molecule and a CRISPR-associated (Cas) peptide form a complex to induce a mutation in the target gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding the gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding the Cas peptide.

[0393] In one embodiment, the agent comprises an miRNA or a miRNA mimic.In one embodiment, the agent comprises a nucleic acid molecule encoding an miRNA or a miRNA mimic.

[0394] miRNAs are small, non-coding RNA molecules that can cause post-transcriptional silencing of specific genes in cells by inhibiting translation or through degradation of target mRNAs. miRNAs can be perfectly complementary or have regions of non-complementarity with the target nucleic acid, resulting in "bulges" in the non-complementary regions. miRNAs can inhibit gene expression by suppressing translation, such as when the miRNA is not perfectly complementary to the target nucleic acid, or by triggering target RNA degradation, which is thought to occur only when the miRNA binds to its target with perfect complementarity. The present disclosure also includes double-stranded precursors of miRNAs. miRNAs or pri-miRNAs can be 18-100 nucleotides in length, or 18-80 nucleotides in length. Mature miRNAs can be 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides in length. miRNA precursors are typically about 70-100 nucleotides in length and have a hairpin conformation. miRNAs are generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha, which specifically process long pre-miRNAs into functional miRNAs. Hairpin or mature microRNAs, or pri-microRNA agents featured in this disclosure, can be synthesized in vivo by cell-based systems or in vitro by chemical synthesis.

[0395] In various embodiments, the agent comprises an oligonucleotide comprising the nucleotide sequence of a disease-associated miRNA. In certain embodiments, the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in its pre-miRNA, mature form, or hairpin form. In other embodiments, combinations of oligonucleotides comprising the sequence of one or more disease-associated miRNAs, any pre-miRNA, any fragment, or any combination thereof are contemplated.

[0396] miRNAs can be synthesized to contain modifications that confer desired characteristics, such as improved stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability (e.g., via endocytosis-dependent or endocytosis-independent mechanisms).

[0397] Modifications can also increase sequence specificity, thereby reducing off-site targeting. Methods of synthesis and chemical modification are described in more detail below. If desired, miRNA molecules may be modified to stabilize the miRNA against degradation, extend half-life, or otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Application Publication Nos. 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, each of which is incorporated by reference in its entirety. For increased nuclease resistance and / or binding affinity to the target, the single-stranded oligonucleotide agents featured in this disclosure may contain 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to targets. The inclusion of pyranose sugars in the oligonucleotide backbone can also reduce endonucleolytic cleavage. Oligonucleotides can be further modified by including a 3' cationic group or by inverting the 3'-terminal nucleoside with a 3-3' linkage. In another alternative, the 3' end can be blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. Without being bound by theory, the 3' may inhibit exonucleolytic cleavage by sterically blocking exonucleases from binding to the 3' end of the oligonucleotide. Small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can also block 3'-5' exonucleases.

[0398] In one embodiment, the miRNA comprises a 2'-modified oligonucleotide containing an oligodeoxynucleotide gap with some or all internucleotide linkages modified to phosphorothioate for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA, thus decreasing the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure can be used in conjunction with any technology that can be developed to enhance the stability or efficacy of inhibitory nucleic acid molecules.

[0399] miRNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in the internucleoside backbone are also considered to be nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts, and free acid forms are also included.

[0400] The miRNAs described herein, which may be in mature or hairpin form, can be provided as naked oligonucleotides. In some cases, it may be desirable to utilize formulations that aid in the delivery of miRNAs or other nucleotide oligomers to cells (see, e.g., U.S. Patent Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is incorporated herein by reference).

[0401] In some instances, the miRNA composition is at least partially crystalline, homogeneously crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the miRNA composition is in an aqueous phase, e.g., in a solution containing water. Aqueous phase or crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly in the case of an aqueous phase) or particle (e.g., a microparticle, as may be appropriate for a crystalline composition). Generally, the miRNA composition is formulated in a manner compatible with the intended method of administration. The miRNA composition can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes the oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., broad-spectrum specific RNAse inhibitors). In one embodiment, the miRNA composition comprises another miRNA, e.g., a second miRNA composition (e.g., a microRNA different from the first). Still other preparations can include at least 3, 5, 10, 20, 50, or 100 or more different oligonucleotide species.

[0402] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of an miRNA. In certain embodiments, the composition comprises an oligonucleotide having nucleobase identity to the nucleobase sequence of an miRNA, thereby designed to mimic the activity of the miRNA. In certain embodiments, the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin or a processed miRNA duplex.

[0403] In one embodiment, the oligonucleotide shares identity with an endogenous miRNA or miRNA precursor nucleobase sequence. Oligonucleotides selected for inclusion in the compositions of the invention can be one of several lengths. Such oligonucleotides can be 7 to 100 linked nucleosides in length. For example, oligonucleotides that share nucleobase identity with an miRNA can be 7 to 30 linked nucleosides in length. Oligonucleotides that share identity with an miRNA precursor can be up to 100 linked nucleosides in length. In certain embodiments, the oligonucleotide comprises 7 to 30 linked nucleosides. In certain embodiments, the oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, the oligonucleotide comprises 19 to 23 linked nucleosides. In certain embodiments, oligonucleotides are 40 to 50, 60, 70, 80, 90, or up to 100 linked nucleosides in length.

[0404] In certain embodiments, the oligonucleotides have sequences with specific identity to miRNAs or their precursors. The nucleobase sequences of mature miRNAs described herein and their corresponding stem-loop sequences are sequences found in miRBase, an online searchable database of miRNA sequences and annotations. Entries in the miRBase sequence database represent predicted hairpin portions (stem-loops) of miRNA transcripts, along with information about the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs) but may, in some cases, include some flanking sequence from the pre-miRNA and the predicted primary transcript. The miRNA nucleobase sequences described herein encompass any version of a miRNA, including those listed in Release 10.0 of the miRBase Sequence Database and any previous release of the miRBase Sequence Database. Sequence database releases may result in name changes for certain miRNAs. Sequence database releases may result in variations in the mature miRNA sequence. The compositions of the invention encompass oligomeric compounds comprising oligonucleotides with specific identity to any nucleobase sequence version of a miRNA described herein.

[0405] In certain embodiments, an oligonucleotide has a nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to an miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Thus, in certain embodiments, the nucleobase sequence of an oligonucleotide may have one or more non-identical nucleobases with respect to the miRNA.

[0406] In certain embodiments, the composition comprises a nucleic acid molecule encoding the miRNA, its precursor, mimic, or fragment. For example, the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, its precursor, mimic, or fragment in a desired mammalian cell or tissue. nucleic acid

[0407] In one embodiment, the invention comprises nanoparticles comprising or encapsulating one or more nucleic acid molecules. In one embodiment, the nucleic acid molecule is a nucleoside-modified mRNA molecule. In one embodiment, the nucleoside-modified mRNA molecule encodes an antigen. In one embodiment, the nucleoside-modified mRNA molecule encodes multiple antigens. In a particular embodiment, the nucleoside-modified mRNA molecule encodes an antigen that induces an adaptive immune response against the antigen. In one embodiment, the invention comprises a nucleoside-modified mRNA molecule encoding an adjuvant.

[0408] Alternatively, the nucleotide sequences encoding the antigens or adjuvants described herein may include sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting polynucleotide encodes a polypeptide according to the invention. Thus, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences recited herein and that encode an antigen or adjuvant of interest.

[0409] As used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein if the nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95%. Nucleotide sequences that are substantially homologous to a nucleotide sequence encoding an antigen can typically be isolated from the producing organism of the antigen based on the information contained in the nucleotide sequence, for example, by introducing conservative or non-conservative substitutions. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides at either end of the sequence, or the deletion of one or more nucleotides at any end or within the sequence. The degree of identity between two polynucleotides can be determined using computer algorithms and methods well known to those skilled in the art.

[0410] Additionally, the scope of the present invention includes nucleotide sequences that encode amino acid sequences that are substantially homologous to the amino acid sequences recited herein and that preserve the immunogenic function of the original amino acid sequences.

[0411] As used herein, an amino acid sequence is "substantially homologous" to any of the amino acid sequences described herein if the amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95%. Identity between two amino acid sequences is preferably determined using the BLASTN algorithm (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215:403-410 (1990)).

[0412] In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an antigen. In one embodiment, the construct comprises multiple nucleotide sequences encoding multiple antigens. For example, in certain embodiments, the construct encodes 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more antigens. In one embodiment, the present invention relates to a construct comprising a nucleotide sequence encoding an adjuvant. In one embodiment, the construct comprises a first nucleotide sequence encoding an antigen and a second nucleotide sequence encoding an adjuvant.

[0413] In one embodiment, the composition comprises a plurality of constructs, each construct encoding one or more antigens. In certain embodiments, the composition comprises one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more constructs. In one embodiment, the composition comprises a first construct comprising a nucleotide sequence encoding an antigen and a second construct comprising a nucleotide sequence encoding an adjuvant.

[0414] In another specific embodiment, the construct is operably linked to a translational control element. The construct may incorporate operably linked regulatory sequences for expression of the nucleotide sequence of the present invention, thereby forming an expression cassette.

[0415] vector The nucleic acid sequences encapsulated in the nanoparticles of the present invention can be obtained using recombinant methods known in the art, for example, by screening libraries from cells that express the gene, by deriving the gene from a vector known to contain the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the nucleic acid molecules of interest can be produced synthetically.

[0416] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0417] In one embodiment, a composition of the invention comprises an in vitro transcribed (IVT) RNA encoding an antigen. In one embodiment, a composition of the invention comprises an IVT RNA encoding multiple antigens. In one embodiment, a composition of the invention comprises an IVT RNA encoding an adjuvant. In one embodiment, a composition of the invention comprises an IVT RNA encoding one or more antigens and one or more adjuvants.

[0418] Nucleoside-modified RNA In one embodiment, the nucleic acid molecule comprises nucleoside-modified RNA.Nucleoside-modified mRNA has certain advantages over unmodified mRNA, including, for example, increased stability, low or no natural immunogenicity, and enhanced translation.The nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, the entire contents of which are incorporated herein by reference.

[0419] In certain embodiments, nucleoside-modified mRNAs do not activate any pathophysiological pathways, are highly efficiently translated, and serve as templates for continuous in vivo protein production for several days almost immediately after delivery (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small, making them applicable to human therapy. For example, as described herein, nucleoside-modified mRNAs encoding antigens have demonstrated the ability to induce CD4+ and CD8+ T cells and antigen-specific antibody production. For example, in certain instances, antigens encoded by nucleoside-modified mRNAs induce greater production of antigen-specific antibodies compared to antigens encoded by unmodified mRNAs.

[0420] In certain instances, expressing proteins by delivering encoding mRNA has many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence for the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with the plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, mRNA does not carry the risk of genomic integration, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15 to 30 minutes of in vivo injection of encoding mRNA. In certain embodiments, using mRNA rather than protein also has many advantages. Proteins often have a short half-life in the circulation, thus requiring frequent dosing, whereas mRNA provides a template for continuous protein production over several days. Purification of proteins can be problematic as they can contain aggregates and other contaminants that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0421] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified nucleoside pseudouridine. In certain embodiments, the inclusion of pseudouridine makes mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0422] The presence of modified nucleosides, including pseudouridine, in RNA has been demonstrated to suppress their natural immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, protein-coding in vitro transcribed RNA containing pseudouridine can be translated more efficiently than RNA that does not contain the modified nucleoside or RNA that contains other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it has been shown that the presence of pseudouridine improves RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and attenuates both PKR activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A key preparative HPLC purification procedure has been established to obtain pseudouridine-containing RNA with excellent translational potential and no natural immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified pseudouridine-containing RNA encoding erythropoietin to mice and macaques resulted in a significant increase in serum EPO levels (Kariko et al., 2012, Mol Ther 20:948-953), thereby confirming the suitability of pseudouridine-containing mRNA for in vivo protein therapy.

[0423] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules that contain pseudouridine or modified nucleosides. In certain embodiments, a composition comprises an isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises pseudouridine or modified nucleosides. In certain embodiments, a composition comprises a vector comprising an isolated nucleic acid encoding an antigen, an adjuvant, or a combination thereof, wherein the nucleic acid comprises pseudouridine or modified nucleosides.

[0424] In one embodiment, the nucleoside modified RNA of the present invention is IVT RNA.For example, in certain embodiments, the nucleoside modified RNA is synthesized by T7 phage RNA polymerase.In another embodiment, the nucleoside modified mRNA is synthesized by SP6 phage RNA polymerase.In another embodiment, the nucleoside modified RNA is synthesized by T3 phage RNA polymerase.

[0425] In one embodiment, the modified nucleoside is m 1 acp 3 In another embodiment, the modified nucleoside is m 1 In another embodiment, the modified nucleoside is Ψm(2'-O-methylpseudouridine). In another embodiment, the modified nucleoside is m 5 D(5-methyldihydrouridine). In another embodiment, the modified nucleoside is m 3 Ψ(3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the pseudouridines described above. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0426] In another embodiment, the modified nucleoside in the nucleoside-modified RNA of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0427] In another embodiment, the modified nucleoside of the present invention is m 5 In another embodiment, the modified nucleoside is m 5U (5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 In another embodiment, the modified nucleoside is s 2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0428] In other embodiments, the modified nucleoside is m 1 A (1-methyladenosine), m 2 A (2-methyladenosine), Am (2'-O-methyladenosine), ms 2 m 6 A(2-methylthio-N 6 -methyladenosine), i 6 A(N 6 -isopentenyl adenosine), ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine), io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine), ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine), g 6 A(N 6 -glycine carbamoyl adenosine), t 6 A(N 6 -threonylcarbamoyl adenosine), ms 2 t 6 A(2-methylthio-N 6 -threonylcarbamoyl adenosine), m 6 t 6 A(N 6 -methyl-N 6 -threonylcarbamoyl adenosine), hn 6 A(N 6 -hydroxynorvalylcarbamoyl adenosine), ms 2 hn 6 A(2-methylthio)N 6-hydroxynorvalylcarbamoyl adenosine), Ar(p) (2'-O-ribosyladenosine (phosphate)), I (inosine), m 1 I (1-methylinosine), m 1 Im (1,2'-O-dimethylinosine), m 3 C(3-methylcytidine), Cm(2'-O-methylcytidine), s 2 C(2-thiocytidine), ac 4 C(N 4 -acetylcytidine), f 5 C(5-formylcytidine), m 5 Cm (5,2'-O-dimethylcytidine), ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine), k 2 C (lycidin), m 1 G (1-methylguanosine), m 2 G(N 2 -methylguanosine), m 7 G (7-methylguanosine), Gm (2'-O-methylguanosine), m 2 2G(N 2 ,N 2 -dimethyladenosine), m 2 Gm(N 2 ,2'-O-dimethylguanosine), m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine), Gr(p)(2'-O-ribosylguanosine (phosphate)), yW (wybutosine), o2yW (peroxywybutosine), OHyW (hydroxywybutosine), OHyW * (undermodified hydroxywybutosine), IMg (wybutosine), mimG (methylwybutosine), Q (queuosine), oQ (epoxyqueuosine), galQ (galactosyl-queuosine), manQ (mannosyl-queuosine), preQ0 (7-cyano-7-deazaguanosine), preQ1 (7-aminomethyl-7-deazaguanosine), G + (Archaeosin), D (Dihydrouridine), m 5 Um (5,2'-O-dimethyluridine), s 4U(4-thiouridine), m 5 s 2 U(5-methyl-2-thiouridine), s 2 Um (2-thio-2'-O-methyluridine), acp 3 U(3-(3-amino-3-carboxypropyl)uridine), ho 5 U (5-hydroxyuridine), mo 5 U(5-methoxyuridine), cmo 5 U (uridine 5-oxyacetic acid), mcmo 5 U (uridine 5-hydroxyacetic acid methyl ester), chm 5 U(5-(carboxyhydroxymethyl)uridine)), mchm 5 U (5-(carboxyhydroxymethyl)uridine methyl ester), mcm 5 U (5-methoxycarbonylmethyluridine), mcm 5 Um (5-methoxycarbonylmethyl-2'-O-methyluridine), mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine), nm 5 s 2 U (5-aminomethyl-2-thiouridine), mnm 5 U (5-methylaminomethyluridine), mnm 5 s 2 U (5-methylaminomethyl-2-thiouridine), mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine), ncm 5 U (5-carbamoylmethyluridine), ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine), cmnm 5 U (5-carboxymethylaminomethyluridine), cmnm 5 Um (5-carboxymethylaminomethyl-2'-O-methyluridine), cmnm 5 s 2 U (5-carboxymethylaminomethyl-2-thiouridine), m 6 2A(N 6 ,N 6-dimethyladenosine), Im (2'-O-methylinosine), m 4 C(N 4 -methylcytidine), m 4 Cm(N 4 ,2'-O-dimethylcytidine), hm 5 C (5-hydroxymethylcytidine), m 3 U (3-methyluridine), cm 5 U (5-carboxymethyluridine), m 6 Am(N 6 ,2'-O-dimethyladenosine), m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine), m 2,7 G(N 2 ,7-methylguanosine), m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um (3,2'-O-dimethyluridine), m 5 D (5-methyldihydrouridine), f 5 Cm (5-formyl-2'-O-methylcytidine), m 1 Gm (1,2'-O-dimethylguanosine), m 1 Am(1,2'-O-dimethyladenosine), τm 5 U (5-taurinomethyluridine), τm 5 s 2 U (5-taurinomethyl-2-thiouridine)), imG-14 (4-demethylyosine), imG2 (isoyosine), or ac 6 A(N 6 -acetyladenosine).

[0429] In another embodiment, the nucleoside-modified RNA of the present invention comprises a combination of two or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of three or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of four or more of the above modifications.

[0430] In another embodiment, between 0.1% and 100% of the residues in a modified nucleoside of the invention are modified (e.g., by the presence of either pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the percentage of modified residues is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0431] In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0432] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the percentage of a given nucleotide that is modified is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0433] In another embodiment, the percentage of a given nucleotide that is modified is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0434] In another embodiment, the nucleoside-modified RNA of the present invention is translated intracellularly more efficiently than an unmodified RNA molecule having the same sequence. In another embodiment, the nucleoside-modified RNA exhibits an enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In another embodiment, translation is enhanced 3-fold. In another embodiment, translation is enhanced 5-fold. In another embodiment, translation is enhanced 7-fold. In another embodiment, translation is enhanced 10-fold. In another embodiment, translation is enhanced 15-fold. In another embodiment, translation is enhanced 20-fold. In another embodiment, translation is enhanced 50-fold. In another embodiment, translation is enhanced 100-fold. In another embodiment, translation is enhanced 200-fold. In another embodiment, translation is enhanced 500-fold. In another embodiment, translation is enhanced 1000-fold. In another embodiment, translation is enhanced 2000-fold. In another embodiment, the fold is 10-1000-fold. In another embodiment, the fold is 10-100-fold. In another embodiment, the fold is 10-200 fold. In another embodiment, the fold is 10-300 fold. In another embodiment, the fold is 10-500 fold. In another embodiment, the fold is 20-1000 fold. In another embodiment, the fold is 30-1000 fold. In another embodiment, the fold is 50-1000 fold. In another embodiment, the fold is 100-1000 fold. In another embodiment, the fold is 200-1000 fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0435] In another embodiment, the nucleoside-modified antigen-encoding RNA of the invention induces a significantly greater adaptive immune response than an unmodified in vitro-synthesized RNA molecule having the same sequence. In another embodiment, the modified RNA molecule exhibits a 2-fold greater adaptive immune response than its unmodified counterpart. In another embodiment, the adaptive immune response is increased 3-fold. In another embodiment, the adaptive immune response is increased 5-fold. In another embodiment, the adaptive immune response is increased 7-fold. In another embodiment, the adaptive immune response is increased 10-fold. In another embodiment, the adaptive immune response is increased 15-fold. In another embodiment, the adaptive immune response is increased 20-fold. In another embodiment, the adaptive immune response is increased 50-fold. In another embodiment, the adaptive immune response is increased 100-fold. In another embodiment, the adaptive immune response is increased 200-fold. In another embodiment, the adaptive immune response is increased 500-fold. In another embodiment, the adaptive immune response is increased 1000-fold. In another embodiment, the adaptive immune response is increased 2000-fold. In another embodiment, the adaptive immune response is increased by another fold difference.

[0436] In another embodiment, "inducing significantly more adaptive immune response" refers to a detectable increase in the adaptive immune response. In another embodiment, the term refers to a fold increase in the adaptive immune response (e.g., one of the fold increases listed above). In another embodiment, the term refers to an increase such that the nucleoside-modified RNA can be administered at a lower dose or frequency than an unmodified RNA molecule of the same species while still inducing an effective adaptive immune response. In another embodiment, the increase is such that the nucleoside-modified RNA can be administered using a single dose to induce an effective adaptive immune response.

[0437] In another embodiment, the nucleoside-modified RNA of the present invention exhibits significantly lower natural immunogenicity than an unmodified in vitro synthesized RNA molecule having the same sequence. In another embodiment, the modified RNA molecule exhibits a 2-fold lower natural immune response than its unmodified counterpart. In another embodiment, the natural immunogenicity is reduced 3-fold. In another embodiment, the natural immunogenicity is reduced 5-fold. In another embodiment, the natural immunogenicity is reduced 7-fold. In another embodiment, the natural immunogenicity is reduced 10-fold. In another embodiment, the natural immunogenicity is reduced 15-fold. In another embodiment, the natural immunogenicity is reduced 20-fold. In another embodiment, the natural immunogenicity is reduced 50-fold. In another embodiment, the natural immunogenicity is reduced 100-fold. In another embodiment, the natural immunogenicity is reduced 200-fold. In another embodiment, the natural immunogenicity is reduced 500-fold. In another embodiment, the natural immunogenicity is reduced 1000-fold. In another embodiment, the natural immunogenicity is reduced 2000-fold. In another embodiment, the natural immunogenicity is reduced by another fold difference.

[0438] In another embodiment, "exhibiting significantly lower natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In another embodiment, the term refers to a fold reduction in natural immunogenicity (e.g., one of the fold reductions listed above). In another embodiment, the term refers to a reduction such that an effective amount of nucleoside-modified RNA can be administered without eliciting a detectable natural immune response. In another embodiment, the term refers to a reduction such that the nucleoside-modified RNA can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the recombinant protein. In another embodiment, the reduction is such that the nucleoside-modified RNA can be repeatedly administered without eliciting a natural immune response sufficient to eliminate detectable production of the recombinant protein.

[0439] Polypeptide Therapeutics In another related aspect, the therapeutic agent comprises an isolated peptide that modulates a target. For example, in one embodiment, a peptide of the invention directly inhibits or activates a target by binding to the target and thereby modulating the target's normal functional activity. In one embodiment, a peptide of the invention modulates a target by competing with an endogenous protein. In one embodiment, a peptide of the invention modulates the activity of a target by acting as a transdominant-negative mutant.

[0440] A variant of a polypeptide therapeutic may be (i) one in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), where such substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one or more modified amino acid residues are present, e.g., residues modified by the attachment of a substituent group; (iii) the polypeptide is an alternative splice variant of a polypeptide of the invention; (iv) a fragment of the polypeptide; and / or (v) the polypeptide is fused to another polypeptide, e.g., a leader or secretory sequence or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are deemed to be within the scope of one of ordinary skill in the art in light of the teachings herein.

[0441] The nanoparticles may further comprise any lipid capable of forming particles to which one or more nucleic acid molecules are bound or particles in which one or more nucleic acid molecules are encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three classes: (1) "simple lipids," including fats, oils, and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. In one embodiment, the nanoparticles do not further comprise any lipid capable of forming particles to which one or more nucleic acid molecules are bound or particles in which one or more nucleic acid molecules are encapsulated. In one embodiment, the nanoparticles do not further comprise any or all of simple lipids, complex lipids, or derived lipids.

[0442] In some embodiments, the nanoparticles comprise cationic lipids. As used herein, the term "cationic lipid" refers to lipids that are cationic or become cationic (protonated) as the pH decreases below the pK of the ionic group of the lipid, but become progressively more neutral at higher pH values. At pH values ​​below the pK, the lipids can associate with negatively charged nucleic acids. In certain embodiments, the cationic lipids comprise zwitterionic lipids, which become positively charged when the pH decreases. In one embodiment, the nanoparticles do not comprise cationic lipids.

[0443] In certain embodiments, the cationic lipid, optionally present or absent in the nanoparticle, comprises any of several lipid species that have a net positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-( Cationic lipids include 2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).In addition, many commercially available preparations of cationic lipids can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) manufactured by GIBCO / BRL, Grand Island, NY), LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) manufactured by GIBCO / BRL), and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol manufactured by Promega Corp., Madison, Wis.).The following lipids are cationic and have a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0444] In one embodiment, the cationic lipid is an amino lipid. Such amino lipids include those described in International Publication No. 2012 / 016184, the entire contents of which are incorporated herein by reference. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA). .Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dioleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0445] In various embodiments, the nanoparticles further comprise a steroid or steroid analog. A "steroid" is a compound that includes the following carbon skeleton: [ka]

[0446] In certain embodiments, the steroid or steroid analog is cholesterol. In one embodiment, the nanoparticles do not comprise a steroid or steroid analog. In one embodiment, the nanoparticles do not comprise cholesterol.

[0447] In one embodiment, the nanoparticles further comprise a stabilizer. In one embodiment, the stabilizer comprises an oligooxyethylene. In one embodiment, the stabilizer comprises a water-soluble polymer. In one embodiment, the stabilizer comprises a water-soluble oligomer. In one embodiment, the stabilizer comprises a carbohydrate.

[0448] In certain embodiments, the nanoparticles comprise one or more targeting moieties that can target the nanoparticle to a cell or population of cells. For example, in one embodiment, the targeting moiety is a ligand that directs the nanoparticle to a receptor found on the surface of a cell.

[0449] In certain embodiments, the nanoparticles comprise one or more internalization domains. For example, in one embodiment, the nanoparticles comprise one or more domains that bind to cells and induce internalization of the nanoparticles. For example, in one embodiment, the one or more internalization domains bind to receptors found on the cell surface to induce receptor-mediated uptake of the nanoparticles. In certain embodiments, the nanoparticles can bind to biomolecules in vivo, which can then be recognized by cell surface receptors to induce internalization. For example, in one embodiment, the nanoparticles bind to systemic ApoE, which results in uptake of the nanoparticles and associated cargo.

[0450] composition In one aspect, the present invention relates to a composition comprising at least one amphiphilic Janus dendrimer of the present invention and / or nanoparticles thereof. In some embodiments, the composition further comprises at least one pharmaceutical agent described herein.

[0451] The present invention also relates to compositions comprising at least one compound of formula (I) and methods of use thereof for delivering an encapsulated agent to a site of interest. Exemplary agents that may be encapsulated in the compositions of the present invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents.

[0452] In one embodiment, the composition comprises nanoparticles comprising a compound of formula (I) and at least one agent encapsulated by the nanoparticles. In some embodiments, the encapsulated agent comprises an agent for inducing an immune response in a subject. In certain embodiments, the present invention provides a composition comprising nanoparticles encapsulating a nucleic acid molecule encoding an agent for inducing an immune response in a subject. For example, in certain embodiments, the composition comprises a vaccine comprising a nucleic acid molecule encoding an antigen.

[0453] In one embodiment, the composition is a vaccine.

[0454] In one embodiment, a composition comprises nanoparticles and one or more nucleic acid molecules described herein. For example, in one embodiment, a composition comprises nanoparticles and one or more nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or a combination thereof.

[0455] In one embodiment, the composition can be prepared by injecting a mixture containing a compound described herein into an appropriate solution, such as a solution containing the drug to be encapsulated. In one embodiment, microfluidic technology, such as that required for the formation of lipid nanoparticles (LNPs), is not required for the production of the nanoparticles of the present invention.

[0456] In one embodiment, a composition of the present invention comprises an in vitro transcribed (IVT) RNA molecule. For example, in certain embodiments, a composition of the present invention comprises an IVT RNA molecule encoding a drug. In certain embodiments, the IVT RNA molecule of the composition is a nucleoside-modified mRNA molecule. In certain embodiments, the drug is at least one of a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor-specific antigen, or a tumor-associated antigen. However, the present invention is not limited to any particular drug or drug combination. In certain embodiments, a composition comprises an adjuvant. In certain embodiments, a composition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, a composition comprises a nucleoside-modified RNA encoding an adjuvant.

[0457] In one embodiment, the composition comprises at least one nucleoside-modified RNA molecule encoding a combination of at least two agents. In one embodiment, the composition comprises a combination of two or more nucleoside-modified RNA molecules encoding a combination of two or more agents.

[0458] In one embodiment, the present invention provides a method for inducing an immune response in a subject. For example, the method can be used to provide immunity in a subject against viruses, bacteria, fungi, parasites, cancer, etc. In some embodiments, the method includes administering to a subject a composition comprising one or more nanoparticles comprising one or more nucleoside-modified RNAs encoding at least one antigen, adjuvant, or a combination thereof.

[0459] In one embodiment, the method comprises systemic administration of the composition to the subject, including, for example, intradermal administration. In certain embodiments, the method comprises administering multiple doses to the subject. In another embodiment, the method comprises administering a single dose of the composition, wherein the single dose is effective to induce a therapeutic response.

[0460] vaccine In one embodiment, the present invention provides an immunogenic composition for inducing an immune response in a subject. For example, in one embodiment, the immunogenic composition is a vaccine. As used herein, an "immunogenic composition" may include an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding the antigen, a cell expressing or presenting the antigen or a cellular component, or a combination thereof. In certain embodiments, the composition comprises or encodes all or a portion of any peptide antigen, or an immunogenically functional equivalent thereof. In other embodiments, the composition comprises a mixture of mRNA molecules encoding one or more additional immunostimulatory agents. Immune stimulatory agents include, but are not limited to, additional antigens, immunomodulators, or adjuvants. In the context of the present invention, the term "vaccine" refers to a substance that induces immunity upon inoculation into an animal.

[0461] Vaccines of the present invention may vary in the composition of their nucleic acid components. In a non-limiting example, the nucleic acid encoding the antigen may also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. Vaccines of the present invention and their various components may be prepared and / or administered by any of the methods disclosed herein or as would be known to one of skill in the art in light of the present disclosure.

[0462] The induction of immunity by expression of an antigen can be detected by observing the response of all or any part of the immune system in a host to the antigen in vivo or in vitro.

[0463] For example, methods for detecting the induction of cytotoxic T lymphocytes are well known. Foreign substances that enter the body are presented to T cells and B cells by the action of APCs. T cells that respond to antigens presented by APCs in an antigen-specific manner differentiate into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) upon stimulation with the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as "activation" of T cells. Therefore, CTL induction by epitopes of polypeptides or peptides, or combinations thereof, can be evaluated by presenting epitopes of polypeptides or peptides, or combinations thereof, to T cells by APCs and detecting the induction of CTLs. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0464] Methods for evaluating the CTL induction effect using dendritic cells (DCs) as APCs are well known in the art. DCs are a representative APC with robust CTL induction activity. In the method of the present invention, an epitope of a polypeptide or peptide, or a combination thereof, is first expressed by DCs, and then the DCs are contacted with T cells. Detection of T cells with cytotoxic effects against target cells after contact with DCs indicates that the epitope of the polypeptide or peptide, or a combination thereof, has the activity of inducing cytotoxic T cells. Furthermore, the induced immune response can also be tested by measuring IFN-γ produced and released by CTLs in the presence of antigen-presenting cells bearing immobilized peptides or peptide combinations by visualization using an anti-IFN-γ antibody (e.g., ELISPOT assay).

[0465] Besides DCs, peripheral blood mononuclear cells (PBMCs) can also be used as APCs. It has been reported that the induction of CTLs is enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, CTLs have been shown to be induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0466] Antigens confirmed to have CTL-inducing activity by these methods are antigens that have DC activation effects and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity through antigen presentation by APCs can also be used as vaccines against antigen-associated disorders.

[0467] The induction of immunity by expression of an antigen can be further confirmed by observing the induction of antibody production against the antigen. For example, if antibodies against the antigen are induced in an experimental animal immunized with a composition encoding the antigen, and if antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

[0468] The induction of immunity by antigen expression can be further confirmed by observing the induction of CD4+ T cells. CD4+ T cells can also lyse target cells, but primarily help induce other types of immune responses, including CTL and antibody production. The types of CD4+ T cell help can be Th1, Th2, Th9, Th17, T regulatory, or T follicular helper (T fh Each subtype of CD4+ T cell is instrumental in a particular type of immune response. Of particular interest to the present invention are T fh Subtyping is useful for generating high affinity antibodies.

[0469] In some embodiments, therapeutic compounds or compositions of the invention can be administered prophylactically (i.e., to prevent the disease or disorder) or therapeutically (i.e., to treat the disease or disorder) to a subject suffering from a disease or disorder or at risk of (or susceptible to) developing a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the onset of overt clinical symptoms of the disease, such that the disease or disorder is prevented or its progression is delayed. In the context of the medical field, the term "preventing" encompasses any activity that reduces the burden of mortality or morbidity due to a disease. Prevention can be carried out at primary, secondary, and tertiary levels. Primary prevention avoids the onset of the disease, while secondary and tertiary levels of prevention encompass activities aimed at preventing the progression and manifestations of the disease, as well as reducing the adverse effects of an already established disease by restoring function and reducing disease-related complications.

[0470] Targeting Domains In one embodiment, the composition includes a targeting domain that directs the delivery vehicle to a site. In one embodiment, the site is a site in need of the agent contained within the delivery vehicle. The targeting domain may include a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, etc., that specifically targets the particle to a site in need of a therapeutic agent. In certain embodiments, the particle includes multivalent targeting, i.e., the particle includes multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with the site in need of the agent contained within the delivery vehicle. For example, the targeting domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target may be a protein, protein fragment, antigen, or other biomolecule associated with the target site. In some embodiments, the targeting domain is an affinity ligand that specifically binds to the target. In certain embodiments, the target (e.g., an antigen) is associated with the site in need of treatment with the agent. In some embodiments, the targeting domain may be copolymerized with a composition comprising the delivery vehicle. In some embodiments, the targeting domain can be covalently attached to a composition comprising a delivery vehicle, for example, via a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. Targeting domains of the present invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0471] In various embodiments, the targeting domain binds to a cell surface molecule of a cell of interest, for example, in various embodiments, the targeting domain binds to a cell surface molecule of an endothelial cell, a stem cell, or an immune cell.

[0472] peptide In one embodiment, a targeting domain of the invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a target of interest.

[0473] The peptides of the present invention can be produced using chemical methods. For example, peptides can be synthesized by solid-phase techniques (Roberge JY et al. (1995) Science 269:202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A Peptide Synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer.

[0474] Alternatively, the peptide may be produced by recombinant means or by cleavage from a longer polypeptide. The composition of the peptide may be confirmed by amino acid analysis or sequencing.

[0475] Variants of peptide therapeutics according to the present invention may be (i) those in which one or more amino acid residues are replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residues may or may not be those encoded by the genetic code; (ii) those in which one or more modified amino acid residues are present, e.g., residues modified by the attachment of a substituent; (iii) those in which the peptide is an alternative splice variant of a peptide of the invention; (iv) a fragment of the peptide; and / or (v) those in which the peptide is fused to another peptide, e.g., a leader or secretory sequence, or a sequence used for purification (e.g., a His tag) or detection (e.g., an Sv5 epitope tag). Fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are deemed to be within the scope of those skilled in the art in light of the teachings herein.

[0476] As is known in the art, "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conserved amino acid substitutions with the sequence of a second peptide. A variant is defined to include a peptide sequence that differs from the original sequence, preferably by fewer than 40% of the residues per segment, more preferably by fewer than 25% of the residues per segment, more preferably by fewer than 10% of the residues per segment, and most preferably by only a few residues per segment, while remaining sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides can be determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215:403-410 (1990)].

[0477] The peptides of the present invention can be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation can be tested by adding dog microsomal membranes or Xenopus egg extract (U.S. Patent No. 6,103,489) to a standard translation reaction.

[0478] The peptides of the invention can include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

[0479] nucleic acid In one embodiment, the targeting domain of the present invention comprises an isolated nucleic acid, including, for example, DNA oligonucleotides and RNA oligonucleotides. In certain embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0480] Alternatively, the nucleotide sequence of the nucleic acid targeting domain can include sequence variations relative to the original nucleotide sequence, e.g., substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid functions as the original and specifically binds to the intended target.

[0481] As used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein if the nucleotide sequence has a degree of identity of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% to the nucleotide sequence described herein. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides at either end of the sequence, or the deletion of one or more nucleotides at any end or within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined using the BLASTN algorithm [BLAST Manual, Altschul, S. et al., NCBI NLM, NIH, Bethesda, Md. 20894, Altschul, S. et al., J. Mol. Biol. 215:403-410 (1990)].

[0482] antibody In one embodiment, the targeting domain of the present invention comprises an antibody or antibody fragment. In certain embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single-chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0483] The antibodies can be intact monoclonal or polyclonal antibodies, as well as immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single-chain Fv molecules (Ladner et al., U.S. Pat. No. 4,946,778), or chimeric antibodies, e.g., antibodies that contain the binding specificity of a murine antibody but whose remaining portions are human. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art.

[0484] Such antibodies can be produced in a variety of ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. The choice of production method depends on several factors, including the desired antibody structure, the importance of carbohydrate moieties on the antibody, ease of culture and purification, and cost. Many different antibody structures can be produced using standard expression techniques, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies containing components from different species. Small antibody fragments, such as Fab and Fv fragments, which lack effector functions and have limited pharmacokinetic activity, can be produced in bacterial expression systems. Single-chain Fv fragments exhibit low immunogenicity.

[0485] Adjuvants In one embodiment, the composition comprises an adjuvant. In one embodiment, the composition comprises a nucleic acid molecule encoding the adjuvant. In one embodiment, the nucleic acid molecule encoding the adjuvant is IVT RNA. In one embodiment, the nucleic acid molecule encoding the adjuvant is nucleoside-modified mRNA.

[0486] Exemplary adjuvants include, but are not limited to, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 lacking the signal sequence and optionally containing the signal peptide from IgE). Other genes that may be useful adjuvants include those encoding MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, and CD4 0, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase-ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and their functional fragments.

[0487] Antibody therapy The present invention also contemplates delivery vehicles that include antibodies or antibody fragments specific for a target, i.e., the antibody can inhibit the target to provide a beneficial effect.

[0488] The antibodies may be intact monoclonal or polyclonal antibodies, as well as immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single-chain Fv molecules (Ladner et al., U.S. Pat. No. 4,946,778), or chimeric antibodies (e.g., antibodies that contain the binding specificity of a mouse antibody, but whose remaining portions are human). Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, may be prepared using methods known to those skilled in the art.

[0489] Antibodies can be prepared using intact polypeptides or fragments containing the immunizing antigen of interest. The polypeptides or oligopeptides used to immunize animals can be obtained from the translation of RNA or can be chemically synthesized and, if desired, can be conjugated to a carrier protein. Suitable carriers that can be chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled polypeptide can then be used to immunize animals (e.g., mice, rats, or rabbits).

[0490] antigen The present invention provides a composition for inducing a therapeutic response in a subject. In one embodiment, the composition comprises an antigen. In one embodiment, the composition comprises a nucleic acid sequence encoding the antigen. For example, in certain embodiments, the composition comprises a nucleoside-modified RNA encoding the antigen. The antigen can be any molecule or compound, including, but not limited to, a polypeptide, peptide, or protein, that induces a therapeutic response, such as an adaptive immune response, in a subject.

[0491] In one embodiment, the antigen comprises a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the antigen and thus the pathogen. In one embodiment, the antigen comprises a fragment of a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the pathogen.

[0492] In certain embodiments, the antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an antigen described herein and that retains the immunogenic function of the original amino acid sequence, for example, in certain embodiments, the amino acid sequence of the antigen has a degree of identity to the original amino acid sequence of at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95%.

[0493] In one embodiment, the antigen is encoded by a nucleic acid sequence of a nucleic acid molecule. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. In one embodiment, the nucleic acid sequence comprises a modified nucleic acid sequence. For example, in one embodiment, the nucleic acid sequence encoding the antigen comprises nucleoside-modified RNA, as described in detail elsewhere herein. The nucleic acid sequence also includes additional sequences encoding linker, leader, or tag sequences that are linked to the antigen by a peptide bond.

[0494] In certain embodiments, the antigen encoded by the nucleoside-modified nucleic acid molecule comprises a protein, peptide, fragment, or variant thereof, or combination thereof, derived from any number of organisms, e.g., a virus, a parasite, a bacterium, a fungus, or a mammal. For example, in certain embodiments, the antigen is associated with an autoimmune disease, an allergy, or asthma. In other embodiments, the antigen is associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV), Ebola, pneumococcus, Haemophilus influenzae, meningococcus, dengue fever, tuberculosis, malaria, norovirus, or human immunodeficiency virus (HIV). In certain embodiments, the antigen comprises a consensus sequence based on the amino acid sequences of two or more different organisms. In certain embodiments, the nucleic acid sequence encoding the antigen is optimized for efficient translation in the organism to which the composition is delivered.

[0495] In one embodiment, the antigen comprises a tumor-specific antigen or a tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In one embodiment, the antigen comprises a fragment of a tumor-specific antigen or a tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In a particular embodiment, the tumor-specific antigen or tumor-associated antigen is a mutational variant of a host protein.

[0496] Viral antigens In one embodiment, the antigen comprises a viral antigen, or a fragment thereof, or a variant thereof. In certain embodiments, the viral antigen is derived from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In certain embodiments, the viral antigen is a papillomavirus, e.g., human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (variola major and variola minor), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, hantavirus (hemorrhagic fever), rabies virus, or the like. The virus may be derived from viruses such as Ebola, Marburg, measles, mumps, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella zoster, also known as chickenpox), cytomegalovirus (CMV), e.g., human CMV, Epstein-Barr virus (EBV), flavivirus, foot-and-mouth disease virus, chikungunya virus, Lassa virus, arenavirus, severe acute respiratory syndrome (SARS) virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or an oncogenic virus.

[0497] parasite antigen In certain embodiments, the antigen comprises a parasitic antigen or a fragment or variant thereof. In certain embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In certain embodiments, the helminth (i.e., worm) is a flatworm (e.g., flukes and tapeworms), an ancylohead, or a roundworm (e.g., pinworm). In certain embodiments, the ectoparasite is a lice, flea, tick, or mite.

[0498] In certain embodiments, the parasite is any parasite that causes the following diseases: acanthamoeba keratitis, amebic dysentery, ascariasis, babesiosis, balantidiosis, raccoon ascariasis, Chagas' disease, clonorchiasis, cochliomyiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, trophozoites, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, malaria, tumefacilitatoriasis, myiasis, onchocerciasis, pediculosis, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

[0499] In certain embodiments, the parasite is Acanthamoeba, Anisakis, Ascaris lumbricoides, bot flies, Balantidium coli, bed bugs, Cestoda (tapeworms), chigger mites, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, hookworms, Leishmania, Rhinoglossus nigricans, Clonorchis liver fluke, Bacillus loa, Paragonimus westermani, Pinworms, Plasmodium falciparum, Schistosoma haematobium, Strongyloides stercoralis, ticks, tapeworms, Toxoplasma gondii, Trypanosoma brucei, Trichuris trichiura, or Wuchereria bancrofti.

[0500] bacterial antigen In one embodiment, the antigen comprises a bacterial antigen or a fragment or variant thereof. In a specific embodiment, the bacterium is from any one of the following phyla: Akidobacterium, Actinobacteria, Aquifex, Caldicellicum, Chlamydia, Chlorobium, Chloroflexus, Chrysiogenes, Cyanobacteria, Deferibacter, Deinococcus-Thermus, Dictyoglomi, Yersinia, Fibrobacterium, Firmicutes, Fusobacterium, Gemmatimonas, Lentisphaera, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistes, Tenericutes, Thermodesulfobacteria, Thermotoga, and Verrucomicrobium.

[0501] In certain embodiments, the bacterium is a gram-positive bacterium or a gram-negative bacterium. In certain embodiments, the bacterium is an aerobic bacterium or an anaerobic bacterium. In certain embodiments, the bacterium is an autotrophic bacterium or a heterotrophic bacterium. In certain embodiments, the bacterium is a mesophilic bacterium, a neutrophilic bacterium, an extremophilic bacterium, an acidophilic bacterium, an alkaliphilic bacterium, a thermophilic bacterium, a psychrophilic bacterium, a halophilic bacterium, or an aeruginosa.

[0502] In certain embodiments, the bacterium is anthrax bacteria, antibiotic-resistant bacteria, disease-causing bacteria, food poisoning bacteria, infectious bacteria, salmonella, staphylococcus, streptococcus, or tetanus, hi certain embodiments, the bacterium is mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0503] fungal antigen In one embodiment, the antigen comprises a fungal antigen or a fragment or variant thereof. In certain embodiments, the fungus is an Aspergillus species, Blastomyces dermatitidis, a Candida yeast (e.g., Candida albicans), a Coccidioides species, a Cryptococcus neoformans, a Cryptococcus gattii, a dermatophyte, a Fusarium species, a Histoplasma capsulatum, a Mucoromycotina, a Pneumocystis jirovecii, a Sporothrix schenckii, an Exserohilum, or a Cladosporium.

[0504] tumor antigens In certain embodiments, the antigen comprises a tumor antigen, including, for example, a tumor-associated antigen or a tumor-specific antigen. In the context of the present invention, a "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigen of the present invention is derived from a cancer, including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma (e.g., breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.).

[0505] Tumor antigens are proteins produced by tumor cells that induce immune responses, particularly T cell-mediated immune responses. In one embodiment, the tumor antigens of the present invention comprise one or more antigenic cancer epitopes that are immunogenically recognized by tumor-infiltrating lymphocytes (TILs) derived from mammalian cancer tumors. The choice of antigen depends on the specific type of cancer to be treated or prevented by the compositions of the present invention.

[0506] Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0507] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute true tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, with limited success.

[0508] The type of tumor antigen referred to in the present invention can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not exist in other cells in the body. TAA-associated antigens are not unique to tumor cells, but are instead expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. The expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or it can be an antigen that is usually present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0509] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA19-9, CA72-4, CAM 17.1, NuMA, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3, CA27.29, BCAA, CA195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0510] In preferred embodiments, antigens include, but are not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

[0511] combination In one embodiment, the compositions of the present invention comprise a combination of drugs described herein. In certain embodiments, compositions comprising a combination of drugs described herein have an additive effect, where the overall effect of the combination is approximately equal to the sum of the effects of each individual drug. In other embodiments, compositions comprising a combination of drugs described herein have a synergistic effect, where the overall effect of the combination is greater than the sum of the effects of each individual drug.

[0512] A composition comprising a combination of drugs may contain the individual drugs in any suitable ratio. For example, in one embodiment, the composition may contain two individual drugs in a 1:1 ratio. However, the combination is not limited to a specific ratio. Rather, any ratio that is shown to be effective is encompassed.

[0513] Conjugation In various embodiments of the present invention, the delivery vehicle is conjugated to a targeting domain. Exemplary conjugation methods may include, but are not limited to, covalent bonding, electrostatic interactions, and hydrophobic ("van der Waals") interactions. In one embodiment, the conjugation is reversible, such that the delivery vehicle can dissociate from the targeting domain upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is irreversible, such that the delivery vehicle does not dissociate from the targeting domain under normal conditions.

[0514] In some embodiments, the conjugation comprises a covalent bond between the activated polymer-conjugated lipid and the targeting domain. The term "activated polymer-conjugated lipid" refers to a molecule comprising a lipid portion and a polymer portion that has been activated through functionalization of the polymer-conjugated lipid with a first coupling group. In one embodiment, the activated polymer-conjugated lipid comprises a first coupling group that can react with a second coupling group. In one embodiment, the activated polymer-conjugated lipid is an activated PEGylated lipid. In one embodiment, the first coupling group is attached to the lipid portion of the PEGylated lipid. In another embodiment, the first coupling group is attached to the polyethylene glycol portion of the PEGylated lipid. In one embodiment, the second functional group is covalently attached to the targeting domain.

[0515] The first and second coupling groups may be any functional groups known to those skilled in the art that form a covalent bond together, for example, under mild reaction conditions or physiological conditions. In some embodiments, the first or second coupling group is selected from the group consisting of maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazides, pentafluorophenyl (PFP) esters, phosphines, hydroxymethylphosphines, psoralens, imidoesters, pyridyl disulfides, isocyanates, vinyl sulfones, α-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenones, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctynes, aldehydes, and sulfhydryl groups. In some embodiments, the first or second coupling group is selected from the group consisting of free amines (-NH), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group reactive to sulfhydryl groups, such as maleimide, pyridyl disulfide, or haloacetyl. In one embodiment, the first coupling group is maleimide.

[0516] In one embodiment, the second coupling group is a sulfhydryl group. Sulfhydryl groups can be introduced onto the targeting domain using any method known to those skilled in the art. In one embodiment, the sulfhydryl group is present on a free cysteine ​​residue. In one embodiment, the sulfhydryl group appears via reduction of a disulfide on the targeting domain, for example, via reaction with 2-mercaptoethylamine. In one embodiment, the sulfhydryl group is introduced via a chemical reaction such as the reaction between a free amine and 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA).

[0517] In some embodiments, the polymer-conjugated lipid and targeting domain are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves the reaction between a functional group with a 1,3-dipole, such as an azide, nitrile oxide, nitrone, or isocyanide, and a linker with an alkene or alkyne dipolarophile. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those skilled in the art, including, but not limited to, cyclooctyne, dibenzocyclooctyne, monofluorinated cyclooctyne, difluorinated cyclooctyne, and biarylazacyclooctynone.

[0518] Pharmaceutical Composition The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparative methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into a desired single- or multi-dose unit.

[0519] Although the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for approved administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and perform such modifications with no more than routine experimentation, if any. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals (including non-human primates, commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs).

[0520] Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include extruded nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0521] The pharmaceutical composition of the present invention can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is approximately equal to the dosage of the active ingredient administered to a subject, or a convenient fraction of such a dosage, such as half or one-third of such a dosage.

[0522] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0523] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutically active agents.

[0524] Controlled- or sustained-release formulations of the pharmaceutical compositions of the invention can be prepared using conventional techniques.

[0525] As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physical disruption of the target tissue, and administration of the pharmaceutical composition through tissue disruption. Thus, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration by application of the composition through a surgical incision, administration by application of the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and kidney dialysis infusion techniques.

[0526] Pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0527] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using, for example, a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include formulations comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials (e.g., emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts).

[0528] Pharmaceutical compositions of the present invention can be prepared, packaged, or sold as formulations suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles containing the active ingredient and having diameters ranging from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device containing a dry powder reservoir into which a stream of propellant can be directed to disperse the powder, or using a self-propelling solvent / powder dispensing container, such as a device containing the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably contain a solid fine powder diluent, such as sugar, and are conveniently provided in unit dosage form.

[0529] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid nonionic surfactant or a solid anionic surfactant, or a solid diluent (preferably having a particle size similar to that of the particles comprising the active ingredient).

[0530] Pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0531] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using, for example, a non-toxic, parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials (e.g., emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts).

[0532] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffer salts; thickening agents; fillers; emulsifiers; antioxidants, antibiotics, antifungals, stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that can be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0533] Treatment method The present invention provides methods for delivering an agent to a cell, tissue, or organ of a subject. In some embodiments, the agent is a diagnostic agent for detecting at least one marker associated with a disease or disorder. In some embodiments, the agent is a therapeutic agent for treating or preventing a disease or disorder. Thus, in some embodiments, the present invention provides methods for diagnosing, treating, or preventing a disease or disorder, comprising administering an effective amount of a composition comprising one or more diagnostic or therapeutic agents, one or more adjuvants, or a combination thereof.

[0534] In some embodiments, the method provides immunity in a subject against an infection, disease, or disorder associated with the antigen. Thus, the present invention provides methods for treating or preventing an infection, disease, or disorder associated with an antigen. For example, the method can be used to treat or prevent a viral infection, bacterial infection, fungal infection, parasitic infection, or cancer, depending on the type of antigen in the administered composition. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0535] In one embodiment, the composition is administered to a subject having an infection, disease, or cancer associated with the antigen. In one embodiment, the composition is administered to a subject at risk of developing an infection, disease, or cancer associated with the antigen. For example, the composition may be administered to a subject at risk of contact with a virus, bacteria, fungus, parasite, etc. In one embodiment, the composition is administered to a subject who has a high likelihood of developing cancer through genetic factors, environmental factors, etc.

[0536] In one embodiment, the method comprises administering a composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens and one or more adjuvants. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding one or more antigens and a second nucleoside-modified nucleic acid molecule encoding one or more adjuvants. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens and a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0537] In certain embodiments, the methods comprise administering to the subject multiple nucleoside-modified nucleic acid molecules encoding multiple antigens, adjuvants, or combinations thereof.

[0538] In certain embodiments, the methods of the present invention allow for sustained expression of an antigen or adjuvant described herein for at least several days after administration. However, the methods also provide for transient expression in certain embodiments, such that the nucleic acid does not integrate into the subject's genome.

[0539] In certain embodiments, the methods comprise administering a nucleoside-modified RNA that provides stable expression of an antigen or adjuvant described herein. In some embodiments, administration of the nucleoside-modified RNA induces an effective adaptive immune response while resulting in little or no innate immune response.

[0540] Administration of the compositions of the present invention in therapeutic methods can be accomplished in many different ways using methods known in the art. In one embodiment, the methods of the present invention involve systemic administration to a subject, including, for example, enteral or parenteral administration. In certain embodiments, the methods involve intradermal delivery of the composition. In another embodiment, the methods involve intravenous delivery of the composition. In some embodiments, the methods involve intramuscular delivery of the composition. In one embodiment, the methods involve subcutaneous delivery of the composition. In one embodiment, the methods involve inhalation of the composition. In one embodiment, the methods involve intranasal delivery of the composition.

[0541] It will be understood that the compositions of the present invention can be administered to a subject either alone or in combination with another agent.

[0542] Thus, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions encoding the antigens, adjuvants, or combinations thereof described herein to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention can be administered to deliver doses ranging from ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates the administration of a dose resulting in a concentration of a compound of the present invention in a mammal of 10 nM to 10 μM.

[0543] Typically, dosages that can be administered to a mammal, preferably a human, in the methods of the present invention range from 0.01 μg to about 50 mg per kilogram of mammalian body weight, although the exact dosage administered will vary depending on any number of factors, including, but not limited to, the type of mammal and type of disease state being treated, the age of the mammal, and the route of administration. Preferably, the dosage of the compound varies from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. More preferably, the dosage varies from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0544] The composition may be administered to the mammal several times daily, or less frequently, such as once daily, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. The frequency of dosage will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the mammal.

[0545] In certain embodiments, administration of the immunogenic compositions or vaccines of the invention may be by single administration or may be boosted by multiple administrations.

[0546] In one embodiment, the invention includes a method comprising administering one or more compositions encoding one or more antigens or adjuvants described herein. In certain embodiments, the method has an additive effect, where the overall effect of administering the combination is approximately equal to the sum of the effects of administering each antigen or adjuvant. In other embodiments, the method has a synergistic effect, where the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant.

[0547] Treatment for SARS-CoV-2 In one embodiment, the present invention provides a method of inducing an adaptive immune response to SARS-CoV-2 in a subject, the method comprising administering an effective amount of a composition comprising an amphiphilic Janus dendrimer of formula (I) and one or more isolated nucleic acids encoding one or more SARS-CoV-2 antigens.

[0548] In one embodiment, the method provides immunity to SARS-CoV-2, SARS-CoV-2 infection, or a disease or disorder associated with SARS-CoV-2 in a subject. Accordingly, the present invention provides methods for treating or preventing an infection, disease, or disorder associated with SARS-CoV-2. In one embodiment, the disease or disorder associated with SARS-CoV-2 infection is COVID-19 or a comorbidity of COVID-19.

[0549] In some embodiments, the invention is a method of administering to a subject a composition comprising at least one nucleoside-modified RNA encoding at least one SARS-CoV-2 antigen.

[0550] In one embodiment, the composition is administered to a subject with an infection, disease, or disorder associated with SARS-CoV-2. In one embodiment, the composition is administered to a subject at risk of developing an infection, disease, or disorder associated with SARS-CoV-2. For example, the composition is administered to a subject at risk of contact with SARS-CoV-2. In one embodiment, the composition is administered to a subject who lives in, has traveled to, or is expected to travel to a geographic area where SARS-CoV-2 is prevalent. In one embodiment, the composition is administered to a subject who is in contact with, or is expected to come into contact with, another person who lives in, has traveled to, or is expected to travel to a geographic area where SARS-CoV-2 is prevalent. In one embodiment, the composition is administered to a subject who has been intentionally exposed to SARS-CoV-2 through occupation or contact.

[0551] In one embodiment, the method comprises administering a composition comprising at least one dendrimer of Formula (I), one or more nucleoside-modified nucleic acid molecules encoding one or more SARS-CoV-2 antigens, and one or more adjuvants. In one embodiment, the method comprises administering a composition comprising a first nucleoside-modified nucleic acid molecule encoding one or more SARS-CoV-2 antigens and a second nucleoside-modified nucleic acid molecule encoding one or more adjuvants. In one embodiment, the method comprises administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more SARS-CoV-2 antigens and a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0552] In certain embodiments, the method comprises administering to a subject multiple nucleoside-modified nucleic acid molecules encoding multiple SARS-CoV-2 antigens, adjuvants, or combinations thereof.

[0553] In certain embodiments, the methods of the invention allow for sustained expression of a SARS-CoV-2 antigen or adjuvant described herein for at least several days after administration. In certain embodiments, the methods of the invention allow for sustained expression of a SARS-CoV-2 antigen or adjuvant described herein for at least two weeks after administration. In certain embodiments, the methods of the invention allow for sustained expression of a SARS-CoV-2 antigen or adjuvant described herein for at least one month after administration. However, the methods also provide for transient expression, in certain embodiments, such that the nucleic acid is not integrated into the subject's genome.

[0554] In certain embodiments, the methods involve administering a nucleoside-modified RNA that provides stable expression of a SARS-CoV-2 antigen or adjuvant described herein. In some embodiments, administration of the nucleoside-modified RNA induces an effective adaptive immune response while resulting in little or no innate immune response.

[0555] In certain embodiments, the method provides durable protection against SARS-CoV-2. For example, in certain embodiments, the method provides durable protection against SARS-CoV-2. In certain embodiments, the method provides durable protection against SARS-CoV-2 for one month or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for two months or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for three months or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for four months or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for five months or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for six months or more. In certain embodiments, the method provides durable protection against SARS-CoV-2 for one year or more.

[0556] In one embodiment, a single immunization with the composition induces durable protection against SARS-CoV-2 for one month or more, two months or more, three months or more, four months or more, five months or more, six months or more, or one year or more.

[0557] Administration of the compositions of the present invention in therapeutic methods can be accomplished in many different ways using methods known in the art. In one embodiment, the methods of the present invention involve systemic administration to a subject, including, for example, enteral or parenteral administration. In certain embodiments, the methods involve intradermal delivery of the composition. In another embodiment, the methods involve intravenous delivery of the composition. In some embodiments, the methods involve intramuscular delivery of the composition. In one embodiment, the methods involve subcutaneous delivery of the composition. In one embodiment, the methods involve inhalation of the composition. In one embodiment, the methods involve intranasal delivery of the composition.

[0558] It will be understood that the compositions of the present invention can be administered to a subject either alone or in combination with another agent.

[0559] Accordingly, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions encoding the SARS-CoV-2 antigens, adjuvants, or combinations thereof described herein to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention can be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates the administration of a dose resulting in a concentration of a compound of the present invention in a mammal of 10 nM to 10 μM.

[0560] Typically, dosages that can be administered to a mammal (e.g., a human) in the methods of the present invention range from about 0.01 μg to about 50 mg per kilogram of mammalian body weight, although the exact dosage administered will vary depending on any number of factors, including, but not limited to, the type of mammal and type of disease state being treated, the age of the mammal, and the route of administration. In certain embodiments, the dosage of the compound varies from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. In certain embodiments, the dosage varies from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0561] The composition may be administered to the mammal several times daily, or less frequently, such as once daily, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or once a year or less. The frequency of dosing will be readily apparent to one of skill in the art and will depend on any number of factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0562] In certain embodiments, administration of the immunogenic compositions or vaccines of the invention may be by single administration or may be boosted by multiple administrations.

[0563] In one embodiment, the invention includes a method comprising administering one or more compositions encoding one or more SARS-CoV-2 antigens or adjuvants described herein. In certain embodiments, the method has an additive effect, where the overall effect of administering the combination is approximately equal to the sum of the effects of administering each SARS-CoV-2 antigen or adjuvant. In other embodiments, the method has a synergistic effect, where the overall effect of administering the combination is greater than the sum of the effects of administering each SARS-CoV-2 antigen or adjuvant.

[0564] Experimental example The present invention will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0565] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. Thus, the following examples are not to be construed as limiting in any way the remainder of the disclosure.

[0566] Example 1: One-component multifunctional sequence-defined ionic amphiphilic Janus dendrimer (IAJD) delivery system of mRNA for vaccines and drugs The present invention relates, in part, to a one-component, multifunctional, sequence-defined, ionic amphiphilic Janus dendrimers (IAJDs) delivery system that co-assembles with mRNA by simple injection into dendrimersome nanoparticles (DNPs) (Figure 1). Screening experiments using six libraries containing 52 IAJDs were performed both in vitro and in vivo. We have demonstrated proof of concept for DNPs, their potential applications, and their utility as a model for elucidating fundamental aspects of non-viral vectors.

[0567] One of the major advantages of synthetic vectors used for mRNA delivery is their unlimited synthetic capacity. The conversion of a four-component LNP (Figure 2) into a one-component DNP represents a demonstration of this synthetic capacity.

[0568] Figure 2 shows the process involved in the assembly of four-component LNPs. A four-component composition containing various ratios of ionic lipids, phospholipids, PEG-lipids, and cholesterol is prepared as a solution in ethanol. This ethanol solution is mixed with a pH 3-5 buffer and an aqueous solution of mRNA using a microfluidic device. The mRNA used in acidic buffer is produced and stored in neutral water. The resulting nanoparticles are analyzed by dynamic light scattering (DLS) to determine the diameter (D, nm) and polydispersity (PDI) of the LNPs. They are then dialyzed to pH 7.4, analyzed again by DLS, and stored at -70 °C before in vitro or in vivo experiments. Figure 1 shows the same process for one-component DNPs. IAJDs containing ionic amines incorporated in the correct order are dissolved in ethanol. The ethanol solution is injected into an acidic buffer (pH 3-5.2) containing mRNA. Depending on the original pH of the buffer, the resulting DNPs containing mRNA already have a pH between 4.5 and 7.3 and can be used for in vitro and in vivo experiments without dialysis after DLS analysis. Long-term storage of DNPs is possible at 5°C. Figure 1 also illustrates the transition from extracellular to intracellular processes for both LNPs and DNPs. Upon injection, both LNPs and DNPs approach the corresponding cells and are encapsulated via endocytosis. The extracellular pH is 7.4, and therefore, LNPs and DNPs enter the cell at a nearly neutral surface. Endocytosis of LNPs or DNPs then deposits them in endosomes, the pH of which drops from 6.8 to 4.5 during their morphogenesis into lysosomes due to ATP-dependent proton pumping on the endosomal membrane (Huotari and Helenius, EMBO J. 2011, 30, 3481-3500). Therefore, both LNPs and DNPs reprotonate and interact with naturally occurring anionic lipids, releasing mRNA that helps ribosomes generate new proteins. If the ionic amines from LNPs are dissociated at their centers, reprotonation of their periphery cannot occur, and therefore, mRNA release in cells has very low efficiency (1-2%).

[0569] Accelerated Modular Orthogonal Synthesis of Six Libraries of IAJDs: A modular orthogonal method was used for the synthesis of sequence-defined amphiphilic Janus glycodendrimers (JGDs). The accelerated modular orthogonal methods for the synthesis of single-single (a single hydrophobic dendron combined with a single hydrophilic dendron), twin-twin (two identical hydrophobic dendrons and two identical hydrophilic dendrons), and hybrid twin-mix (two identical hydrophobic dendrons and two different hydrophilic dendrons) (Figures 3 and 4) rely on a related, improved, and accelerated synthetic principle originally used for the synthesis of sequence-defined JGDs. Two different orthogonal protecting groups, 4-methoxybenzyl ether and benzyl ether, were used in this method (Horita et al., Tetrahedron 1986, 42, 3021-3028). The six libraries synthesized are shown schematically in Figure 4. Four of these libraries are based on single-single IAJDs. Nine IAJDs are available in Library 1, seven in Library 2, six in Library 3, and three in Library 4. Library 5 is based on twin-twin IAJDs generated from IAJD1-IAJD9 in Library 1 and JAJD33 in Library 3. Library 6 contains 17 hybrid twin-mix IAJDs selected from all libraries. The selection process was determined by the in vivo and in vitro activities of their single-single components. The hydrophilic portions of these IAJDs contain sequence-defined compositions based on ionizable amines: dimethylaminobutanoate (DMBA), dimethylaminopropanoate (DMPA), dimethylaminoacetate (DMA), piperidinebutanoate (PIP), and methylpiperazinebutanoate (MPRZ) (Figure 3). They are based on the pK values ​​of their corresponding ionizable lipids available in the literature. aThese sequences were selected based on their sequence-defined IAJD structure (Ramishetti et al., Adv. Mater. 2020:32-1906128; Kim et al., Sci. Adv. 2021, 7, eabf4398). The symbols used in the schematic diagrams of these sequence-defined IAJDs are shown in boxes at the bottom of Figure 4. The benzyl ether groups marked in red were used to isolate ionic amines and construct different sequences in the hydrophilic portion of the IAJD. Aromatic groups such as benzyl ethers have been suggested as hydrogen bond acceptors in molecular recognition, interacting with cations containing ammonium groups in both biology and synthetic supramolecular chemistry (Levitt et al., Philos. Trans. R. Soc., A. 1993, 345, 105-112; Perutz et al., J. Am. Chem. Soc. 1986, 108, 1064-1078; Loewenthal et al., J. Mol. Biol. 1992:224, 759-770; Zacharias et al., Trends Pharmacol. Sci. 2002, 23, 281-287; Gallivan et al., Proc. Natl. Acad. Sci. USA 1999, 96, 9459-9464; Dougherty, Science 1996, 271, 163-168; Dougherty, Acc. Chem. Res. 2013, 46, 885-893; Ma et al., Chem. Rev. 1997, 97, 1303-1324; Dougherty et al., Science 1990, 250, 1558-1560). This cation-π interaction is weaker than a conventional H-bond (approximately 3 kcal / mol) and therefore lowers the pK of ionic amines. a In the protonated state of the amine, it mediates the dynamic control of its pK aWhile the unprotonated state may promote the interaction of the benzyl ether with the nucleobases of mRNA, enhancing coassembly, or may segregate into the hydrophobic portion of DNP. This cation-π interaction has not previously been used in delivery vectors for mRNA. The hydrophobic portion of the IAJD contains both linear and branched alkyl groups of different lengths (Figures 3 and 4). The hydrophilic acid components of these IAJDs are shown in Figure 3 (Module A). Their synthesis is described elsewhere herein. The structures of the hydrophobic benzylamines are shown in Figure 3 (Module C). Their synthesis is also described elsewhere herein. Orthogonal combination of all these modules provides six libraries of IAJDs in an accelerated manner, as shown in Figure 4. The synthesis of the twin-twin portion of the hydrophobic IAJDs is described elsewhere herein. Detailed structures of all the libraries of IAJDs are shown in Figure 3 (Module A). Their pK a The values ​​and abbreviations are shown in Figure 12 (libraries 1, 2, 3, 4), Figure 13 (library 5), and Figure 14 (library 6).

[0570] In vitro transfection activity of DNP in human embryonic kidney (HEK) 293T cells: HEK 293T cells were seeded in 96-well plates (20,000 cells / well / 200 μL) and cultured in complete cell culture medium at 37°C for 24 hours in 5% CO2. Screening experiments were performed using unoptimized DNP containing naked nucleoside-modified mRNA with a molecular weight of 664,341 encoding firefly luciferase (mRNA-Luc) (Kariko et al., Immunity 2005, 23, 165-175; Pardi et al., Methods Mol. Biol. 2013, 969, 29-42). A fixed concentration of 125 ng / well of mRNA-Luc was used. TransIT (TransIT-mRNA transfection kit from Mirus Bio) and MC3-based LNPs (MC3:DLin-MC3-DMA, an FDA-approved LNP for mRNA delivery) were used as positive controls for cell transfection at an mRNA-Luc concentration of 125 ng / well, the same as the tested concentration of DNPs. Subsequently, cells were cultured for 24 hours, the medium was aspirated under vacuum using a glass pipette, and the cells were lysed with 30 μL / well of cell culture lysis reagent (Promega). The luminescence intensity corresponding to the expressed luciferase protein was determined and analyzed.

[0571] All 52 IAJDs from Figure 4 were co-assembled by injection with mRNA in DNPs and used in these experiments. Tables 1–5 (see below) summarize the conditions used to prepare the DNPs, their diameters (D, in nm) and polydispersities (PDI) obtained by DLS experiments, and luminescence results. Each experiment was performed at least three times (Figure 5). Of the 52 DNPs, 42 (81%) showed activity in vitro (Figure 5). Without any optimization, two of them, DNP9 and DNP22, showed higher activity than the positive controls MC3 and TransIT, while four of them, DNP8, DNP9, DNP21, and DNP22, showed higher activity than the most commonly used positive control, MC3.

[0572] For in vivo mRNA delivery in mice using DNP, 6- to 8-week-old female mice were used for these experiments. Four to 7 hours after injection of 100 μL of DNP solution containing 10 μg of mRNA-Luc, mice were imaged 10 minutes after intraperitoneal injection of D-luciferin (15 mg / mL, 10 μL / g body weight). The exposure time was 1 minute. For organ imaging, mice were sacrificed, organs were immediately collected, and bioluminescence imaging was performed. Table 7 (see below) summarizes all DNP injection data, including the D (nm) and PDI determined by DLS, along with the results obtained in vivo. Figure 6 shows the D (nm) and PDI of DNP (both in black above the mouse image) and the corresponding IAJD pK of all compounds used for delivery. a Figure 6 summarizes all mouse experiments including the 1000-kJ / mL (also shown in blue above) data. The results from Figures 6 and 7 provide proof of concept for a one-component multifunctional sequence-defined ionic amphiphilic Janus dendrimer delivery system for mRNA.

[0573] A summary of the results from Figure 7 shows that there appears to be no correlation between the activity of DNP in vivo and the same experiments in vitro (Figure 5), and that the most active IAJDs 33, 34, and 31 have the lowest pK a The results from Figure 6 also demonstrate that there is considerable tolerance of DNP activity to the diameter and polydispersity of the DNP. Diameters greater than 100 nm appear to be as active as DNPs with diameters less than 100 nm (Figure 6). However, the pK aA quantitative correlation between the dimensional stability and the dependence on DNP size needs to be studied. The dimensional stability of 40 DNPs was examined as a function of time at 5°C. Non-optimized experiments showed that 19 of the 40 DNPs were highly stable after storage at 5°C for up to 120 days. Almost all of these DNPs were assembled from IAJDs containing benzyl ethers in their hydrophilic moieties (Table 9, see below). Comparing the data from Figures 7 and 11, a series of very interesting activity trends are observed. As shown in Figure 11, the dimensional stability of DNPs is excellent for up to 120 days when stored at 5°C. The IAJD9, IAJD22, IAJD33, and IAJD34 single-single compounds form remarkably stable DNPSs that also exhibit high in vivo activity (Figure 7). IAJD46 is a twin-twin of IAJD33. However, the activity of DNP36 is approximately half that of DNP33 (Figures 6 and 7). Hybrid twin-mix IAJD47 is also based on the structure of IAJD33 or half of the structure of IAJD46. However, the activity of DNP47 is much lower than that of DNP33 and DNP46. Even more interesting is the comparison between single-single IAJD9 and hybrid twin-mix IAJD32 (Figures 6 and 7).

[0574] Figure 7 plots the results from Figure 6. Without any optimization, 28 of the 52 DNPs investigated (54%) show in vivo activity. Two of them, DNP33 and DNP34, show very high activity in the lung. The single-single IAJD9-derived DNP9 shows good activity (Figure 7) and stability (Figure 11). At the same time, the corresponding hybrid twin-mix IAJD32-based DNP32 (Figures 3 and 4) based on IAJD9 and a single PEG with a degree of polymerization of 45 also shows excellent stability (Figure 11), but is completely inactive in mice (Figure 7). To clarify this result, several co-assembled DNPs were prepared based on highly active IAJD and low concentrations of IAJD32. An example is a DNP assembled from IAJD33 and 2% IAJD32 (Figure 11, bottom row, second from the left) and Figure 7. The stability of this combined DNP was excellent (Figure 11), comparable to that of DNP32 assembled from IAJD32 alone. However, the in vivo activity of DNP coassembled with 2% IAJD32 from IAJD33 was only a fraction of that of DNP33 (Figure 7). This confirms the PEG Enigma mechanism. Thus, although insertion of a small fraction of the PEG attached to the IAJD can dramatically increase the stability of the resulting DNP, it also dramatically reduces its activity in vivo. Incorporation of short oligooxyethylene fragments into the structure of single-single, twin-twin, or hybrid twin-mix IAJDs may help address this issue.

[0575] The role of ionic amine concentration and sequence on the activity of the corresponding DNP is as follows: The binding activity of sugars located on the surface of glycodendrimersomes (GDSs) assembled from amphiphilic Janus glycodendrimers (JGDs) to sugar-binding proteins increases by decreasing the sugar concentration during the sequence definition process (Percec et al., J. Am. Chem. Soc. 2013, 135, 9055-9077). This unexpected trend was explained by the self-assembly of glycodendrimersomes at their periphery in a form that promotes higher binding activity between sugars and proteins at lower sugar concentrations.

[0576] Figure 8 plots representative activity data for sequence-defined IAJD-derived DNPs from both in vitro and in vivo experiments. Low or no activity was observed at high concentrations of ionizable amines in the IAJD structure, while extremely high activity was observed at lower ionizable amine concentrations in highly specific sequences. Without going into detail, the results from Figure 8 provide a mechanism for manipulating the activity of DNPs via the sequence and concentration of their ionizable amines. The change in activity observed in Figure 8 is much higher than that observed with sequence-defined dendrimersomes.

[0577] Some comments regarding the potential targeted delivery of mRNA: Figures 9 and 10 summarize selected representative organ delivery data from the experiments reported in Figure 6. They show the emission intensity reflecting delivery activity within the heart, lungs, liver, and spleen as a function of the structure of the IAJD used to design the structure of the DNP used in the delivery of mRNA. The highest emission was observed within the lungs for DNP (10 8 The next highest are again IAJD31, IAJD46, and IAJD27 (10 7 ) based on DNP, also intrapulmonary.

[0578] These lung activities are higher than the activity in the lungs of the control experiment for MC3 (Figure 9). It is important to understand that the organ activities from Figure 10 are much higher than the overall mouse activities from Figures 7 and 9. The next highest activity is in the liver (Figures 9 and 10). Without optimization, the luminescence in the liver was 10 for DNP in IAJD31 and IAJD34. 6 and 10 for IAJD30, IAJD33 and IAJD46 based DNP. 5 These are 10 8 The highest activity in the spleen was observed in IAJD29, IAJD30, and IAJD37-based DNP (10 5 ) and IAJD27-based DNP(10 4) in the control experiment using MC3. 7 These results indicate that single-component IAJD may provide a potential strategy for targeting different organs.

[0579] We report the design and accelerated modular orthogonal synthesis of six libraries containing 52 multifunctional, sequence-defined, ionic amphiphilic Janus dendrimers (IAJDs). All 52 IAJDs coassemble with mRNA by simple injection in acidic buffer to generate dendrimersome nanoparticles (DNPs) with predictable dimensions and narrow dispersity. The dimensions of these DNPs are stable for over 120 days at 5°C. Of the 52 DNPs encapsulating luciferase-mRNA, 42 (81%) were active in vitro, four of which showed greater activity than the four-component lipid nanoparticles obtained from the MC3 FDA-approved control experiment. 28 (54%) DNPs were active in vivo, two of which showed greater activity than the MC3 control experiment in the lung. These unoptimized preliminary experiments provide proof of concept for one-component multifunctional, sequence-defined, amphiphilic Janus dendrimers as an efficient delivery system for mRNA. This one-component delivery platform can be used to elucidate the mechanisms of mRNA encapsulation and release from supramolecular virus-like assemblies and for the production of vaccines and drugs.

[0580] mRNA vaccines rely on a delivery system for nucleic acids based on four-component ionic lipid nanoparticles (LNPs) containing phospholipids, cholesterol for mechanical properties, PEG-conjugated lipids for stability, and ionic amines.

[0581] In this report, the current four-component LNP delivery system is converted into a simpler and more precise one-component multifunctional JD system, with the highest activity mediated by sequence-defined low concentrations of ionic amines.

[0582] We designed and synthesized a new class of JDs, termed ionic amphiphilic Janus dendrimers (IAJDs). This concept provides the first attempt to elucidate the function of ionic amines (IAs) and their mRNA encapsulation and release capabilities as a function of concentration, structural isomerism, number of ionic amines, and their sequence by using IAJD-based DNPs as a delivery tool. The assembly of one-component DNPs relies on a simple injection method. This process is much simpler than the microfluidic device required to assemble four-component LNPs.

[0583] Six libraries containing 52 sequence-defined IAJDs were synthesized via an accelerated modular orthogonal method.

[0584] Of 52 DNPs assembled without any optimization from IAJD encapsulating luciferase-mRNA, 45 (87%) were active in vitro, with two of them (IAJD9 and 22) exhibiting higher activity than the positive controls, MC3 and transIT. Twenty-eight (54%) were active in vivo, with two of them (IAJD33 and 34) exhibiting very high activity in the lung.

[0585] The size stability of 40 DNPs was tested. Unoptimized screening experiments showed that 19 of the 40 DNPs were highly stable in size after storage at 5°C for up to 120 days. Almost all of these DNPs were assembled from IAJDs containing benzyl ethers in the hydrophilic moiety.

[0586] This study provides proof of concept for one-component IAJD as an efficient delivery system for mRNA, which can be used to produce vaccines and drugs by mRNA delivery.

[0587] The materials and methods used in these experiments are described here.

[0588] Triethylene glycol monoethyl ether (TCI, 98%), triethylene glycol (Alfa Aesar, 99%), benzyl chloride (Alfa Aesar, 99%), 4-methoxybenzyl chloride (TCI, 98%), p-toluenesulfonyl chloride (Alfa Aesar, 98%), gallic acid (Chem Impex, anhydrous, ACS grade), triethyl orthoformate (TCI, 98+%), Amberlyst-15(H) (Alfa Aesar), 1-bromooctane (Aldrich, 99%), 1-bromononane (Lancaster, 99%), 1-bromoundecane (Aldrich, 99%), 1-bromododecane (Alfa Aesar, 99%), (rac)-3-(bromomethyl)heptane (Aldrich, 95%), (rac)-1-bromo-3,7-dimethyloctane (TCI, 93%), palladium on activated carbon catalyst (Spectrum, 10 wt % loaded), pentaerythritol (Aldrich, 98%), 4-(dimethylamino)butyric acid hydrochloride (Alfa Aesar, 98%), 3-(dimethylamino)propionic acid hydrochloride (TCI, 98%), N,N-dimethylglycine hydrochloride (Acros, 99%), piperidine (Beantown Chemical, 99%), 1-methylpiperazine (Alfa Aesar, 98%), citrate buffer (100 mM, pH 3.0, TEKnova), Tris buffer (1 M, Thermo Fisher Scientific), and other reagents and solvents were obtained from commercial sources and used as received. 4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) was prepared according to literature procedures. 2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) was prepared according to literature procedures. Citrate buffer (100 mM, pH 3.0, TEKnova) CHCl (DCM) was dried over CaH and freshly distilled before use.

[0589] The purity and structural identity of the final products and intermediates were confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), 1 H and 13The cleavage was determined by a combination of techniques including 1C NMR and matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry.

[0590] 1 H and 13 C NMR spectra were recorded at 400 MHz and 101 MHz on a Bruker NEO (400 MHz) equipped with an autosampler and at 500 MHz and 126 MHz on a Bruker DRX (500 MHz) NMR spectrometer. All NMR spectra were measured in CDCl3 at 23 °C. Residual protic solvent CDCl3 ( 1 H, δ 7.26 ppm; 13 C, δ 77.16 ppm), and tetramethylsilane (TMS, δ 0 ppm), 1 H and 13 It was used as an internal standard in the C-NMR spectrum. Absorption is expressed as wavenumber (cm -1 NMR spectra were analyzed and exported by TopSpin 4.07 (Bruker) or MNova 14.

[0591] Molecular purity was determined by high-performance liquid chromatography (HPLC) using a Shimadzu LC-20AD high-performance liquid chromatography pump, a PE Nelson Analytical 900 Series Integral Data Station, a Shimadzu SPD-10A VP (UV-vis, λ = 254 nm), and three AM gel columns (a guard column, two 500 Å, 10 μm columns). 5% NEt in THF was used as the solvent at an oven temperature of 23 °C. Detection was by UV absorbance at 254 nm.

[0592] Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a PerSeptive Biosystems Voyager-DE (Framingham, MA) mass spectrometer equipped with a nitrogen laser (337 nm) operating in linear mode. Internal calibration was performed using angiotensin II and bombesin as standards. Analytical sample solutions were prepared by mixing a THF solution of the sample (5–10 mg / mL) with a THF solution of the matrix (2,5-dihydroxybenzoic acid, 10 mg / mL) at a 1:5 (v / v) ratio. The prepared sample solution (0.5 μL) was loaded onto a MALDI plate and dried at 23 °C. The plate was then inserted into the vacuum chamber of the instrument. The laser intensity and applied voltage were adjusted according to the molecular weight and nature of each analyte.

[0593] Dynamic light scattering (DLS) of DNP was performed in buffer solution on a Malvern Instruments particle sizer (Zetasizer Nano S, Malvern Instruments, UK) equipped with a 4 mW He-Ne laser at 633 nm and an avalanche photodiode positioned at 175 degrees to the beam and a temperature-controlled cuvette holder. Instrument parameters were determined automatically along with the measurement time.

[0594] pK of individual IAJD molecules a Measurement: The IAJD molecule was dissolved in ethanol (saturated with NaCl) at a concentration of 1.5 mg / mL in a volume of 3 mL. 0.1 M aqueous HCl was added in 7.5 μL increments. The resulting pH was measured using a Hach H170 pH meter. pK a was calculated using half-equivalent point titration.

[0595] Preparation of DNP containing mRNA-Luc: IAJD was dissolved in ethanol at various initial concentrations (5-160 mg / mL). Nucleoside-modified mRNA encoding firefly luciferase (mRNA-Luc) was dissolved in water at various initial concentrations (1-4 mg / mL). 12.5 μL of the mRNA solution was placed in a clean, RNA-free Eppendorf flask (1.5 mL) and mixed with 463 μL of citrate buffer (10 mM, pH 3.0) / acetate buffer (10 mM). For mRNA encapsulation in IAJD, 25 μL of the ethanolic stock solution of IAJD was quickly injected into the mRNA solution in citrate or acetate buffer, followed by vortexing for 5 seconds.

[0596] Dialysis of DNP: 0.5 mL of DNP solution was dialyzed against 10 mM Tris buffer (10 mM, pH 7.4) or 1X PBS buffer (pH 7.4) for 2 h in 3,500–14,000 molecular weight cutoff dialysis tubing (Spectrum Medical Instruments Inc. Spectra / Por molecular porous membrane tubing, flat width: 45 mm; diameter: 29 mm; volume / length: 6.4 mL / cm).

[0597] In vitro mRNA delivery: Human embryonic kidney (HEK) 293T cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) (complete medium) supplemented with 10% inactivated fetal bovine serum (FBS) (Gemini Bio-Products), 2 mM L-glutamine, and 100 U / mL penicillin / streptomycin (Life Technologies). For in vitro screening experiments, HEK293T cells were seeded into 96-well plates (20,000 cells / well / 200 μL) and cultured in complete medium at 37°C and 5% CO for 24 hours. Cells were transfected with IAJD containing encapsulated naked nucleoside-modified mRNA encoding firefly luciferase (mRNA-Luc) at a fixed concentration of 125 ng of mRNA-Luc per well. transIT (TransIT®-mRNA Transfection Kit, Mirus Bio) was used as a positive control for cell transfection according to the manufacturer's protocol. The same mRNA-Luc as the test particles was added to the cells at a concentration of 125 ng / well. The cells were cultured for an additional 24 hours, after which the medium was aspirated and the cells were lysed with 30 μL / well of cell culture lysis reagent (Promega). To determine luminescence intensity, 2.5 μL of lysed cells were mixed with 10 μL of luciferase assay substrate, and luminescence was analyzed using a MiniLumat LB 9506 luminometer (Berthold / EG&G; Wallac).

[0598] In vivo mRNA delivery: Six- to eight-week-old female BALB / c mice were purchased from Charles River Laboratories. Mice were injected retroorbitally with 100 μL of buffer solution containing 10 μg of IAJD-encapsulated mRNA-Luc under isoflurane anesthesia. Four to seven hours after injection, mice were imaged on a Perkin Elmer IVIS Spectrum CT system 10 minutes after intraperitoneal injection of 15 mg / mL D-luciferin (Regis Technologies) at 10 μL / g body weight. The exposure time was set to 1 minute with medium binning (binning = 8). For IVIS imaging of organs, mice were sacrificed, and then organs were immediately collected and bioluminescence imaging was performed. Synthesis of hydrophilic acids [ka]

[0599] Reagents and conditions: (i) TsCl, NaOH, THF-water, 0-5°C, 3 hours; (ii) benzyl chloride / NaOH, water, 100°C, 12 hours; (iii) 4-methoxybenzyl chloride, NaOH, water, 100°C, 12 hours; (iv) H2SO4 (catalyst), MeOH, reflux, 2 hours; (v) triethyl orthoformate, Amberlyst-15(H), toluene, Dean-Stark, reflux (vi) K2CO3, DMF, 70°C, 12 hours; (vii) 2M HCl, MeOH, 23°C, 2 hours; (viii) (a) KOH, EtOH, reflux, 2 hours; (b) HCl (1M); (ix) KHCO3, KI, 4-methoxybenzyl chloride, DMF, 60°C, 24 hours; (x) H2, Pd / C, DCM / MeOH, 23°C, 12 hours; (xi) TFA, DCM, 23°C, 2 hours; (xii) KHCO3, KI, DMF, 60°C, 24 hours.

[0600] The syntheses of 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (11), 2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethan-1-ol (14), and 2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (15) were adapted from previously reported literature procedures. The procedure was optimized to increase the yields of compounds 11, 14, and 15 to 78.02 g, 20.21 g, and 28.81 g, respectively. The synthesis of methyl 2-ethoxy-7-hydroxybenzo[d][1,3]dioxole-5-carboxylate (20) and methyl 4-(benzyloxy)-3,5-dihydroxybenzoate (24a) was adapted from literature procedures (Wang et al., J. Am. Chem. Soc. 2020, 142, 9525-9536). Methyl 3,4,5-tris(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)benzoate (30a), methyl 3,4,5-tris(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoate (30b), 3,4,5-tris(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)benzoic acid (5), and 3,4,5-tris(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoic acid (10) were prepared from literature procedures (Percec et al., Science 2010, 328, 1009-1014; Percec et al., J. Am. Chem. Soc. 2013, 135, 4129-4148). [ka]

[0601] 2-(2-(2-((4-Methoxybenzyl)oxy)ethoxy)ethoxy)ethan-1-ol (16). Triethylene glycol (13, 34.60 g, 229.75 mmol, 4 equivalents) was added to 120 mL of aqueous NaOH (50%). The mixture was stirred at 23 °C for 30 minutes. Then, 4-methoxybenzyl chloride (9.00 g, 57.47 mmol, 1 equivalent) was added, and the mixture was stirred at 100 °C for 24 hours. After cooling to 23 °C, the reaction mixture was diluted with water (80 mL) and extracted with diethyl ether (60 mL × 3). The organic phase was dried over anhydrous magnesium sulfate (MgSO) and filtered. The filtrate was concentrated and purified by column chromatography (SiO) using ethyl acetate (EtOAc) / hexane = 1 / 1 as the eluent to give the title compound as a reddish-brown oil (9.82 g, 60%). [ka]

[0602] 2-(2-(2-((4-Methoxybenzyl)oxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (17). Compound 16 (8.31 g, 30.74 mmol, 1 equiv.) and NaOH (1.84 g, 46.11 mmol, 1 equiv.) were dissolved in a mixture of THF and water (1 / 1, 40 mL), and the mixture was cooled to 0 °C. 4-Toluenesulfonyl chloride (TsCl, 5.86 g, 30.74 mmol, 1 equiv.) dissolved in 20 mL of THF was added to the mixture at 0 °C over 30 min. The reaction mixture was stirred at 0-5 °C for 2 h. The reaction mixture was poured into an ice-water bath (100 mL), and the mixture was extracted with DCM (50 mL × 3). The organic phase was dried over anhydrous MgSO and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using EtOAc / hexane=1 / 1 as the eluent to give the title compound as a reddish-brown oil (11.45 g, 88%). [ka]

[0603] Methyl 7-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-2-ethoxybenzo-[d][1,3]dioxole-5-carboxylate (21a). Methyl 2-ethoxy-7-hydroxybenzo[d][1,3]dioxole-5-carboxylate (20, 0.80 g, 3.33 mmol, 1 equiv.) and KCO (1.38 g, 10.00 mmol, 3 equiv.) were stirred in dry DMF (50 mL). 2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (15, 1.58 g, 4.00 mmol, 1.2 equiv.) was added, and the mixture was stirred at 70 °C under a N atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and the DMF was removed under reduced pressure. Water (30 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO and filtered. The filtrate was concentrated and purified by column chromatography (SiO) using hexane / EtOAc = 1 / 1 as an eluent to give the title compound (1.43 g, 93%) as a light yellow oil. [ka]

[0604] Methyl 2-ethoxy-7-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo-[d][1,3]dioxole-5-carboxylate (21b). Compound 20 (5.00 g, 20.82 mmol, 1 eq.) and K2CO3 (8.63 g, 62.46 mmol, 3 eq.) were stirred in dry DMF (70 mL). Compound 12 (7.95 g, 24.98 mol, 1.2 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (50 mL) was then added, and the mixture was extracted with DCM (50 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using hexane / EtOAc=1 / 2 as the eluent to give the title compound as a light yellow oil (6.23 g, 86%). [ka]

[0605] Methyl 2-ethoxy-7-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)-benzo[d][1,3]dioxole-5-carboxylate (21c). Compound 20 (0.70 g, 2.91 mmol, 1 eq.) and K2CO3 (1.21 g, 8.73 mmol, 3 eq.) were stirred in dry DMF (30 mL). Compound 17 (1.36 g, 3.20 mmol, 1.1 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (30 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using hexane / EtOAc=1 / 1 as the eluent to give the title compound as a light yellow oil (1.22 g, 85%). [ka]

[0606] Methyl 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-dihydroxybenzoate (22a). Compound 21a (0.97 g, 2.10 mmol) was dissolved in 20 mL of MeOH. Then, HCl (2 M, 10.0 mL, 20.00 mmol) was added. The mixture was stirred at 23 °C for 2 h. The reaction mixture was extracted with DCM (20 mL × 3), and the organic phase was dried over anhydrous MgSO. Filtration and evaporation of the solvent gave the title compound as a light yellow oil (0.85 g, 100%). [ka]

[0607] Methyl 3,4-dihydroxy-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoate (22b). Compound 21b (5.36 g, 13.88 mmol) was dissolved in 30 mL of MeOH. Then, HCl (2 M, 30.0 mL, 60.00 mmol) was added. The mixture was stirred at 23 °C for 2 h. The reaction mixture was extracted with DCM (30 mL × 3), and the organic phase was dried over anhydrous MgSO 4 MgSO 4. Filtration and evaporation of the solvent gave the title compound as a light yellow oil (4.58 g, 100%). [ka]

[0608] Methyl 3,4-dihydroxy-5-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)-benzoate (22c). Compound 21c (0.90 g, 1.83 mmol) was dissolved in 10 mL of MeOH. Then, HCl (2 M, 4.0 mL, 8.00 mmol) was added. The mixture was stirred at 23 °C for 2 h. The reaction mixture was extracted with DCM (20 mL × 3) and the organic phase was dried over anhydrous MgSO. Filtration and evaporation of the solvent gave the title compound as a light yellow oil (0.80 g, 100%). [ka]

[0609] Methyl 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoate (23a). Compound 22a (0.84 g, 2.07 mmol, 1 eq.) and K2CO3 (1.72 g, 12.42 mmol, 6 eq.) were stirred in dry DMF (30 mL). Compound 12 (1.45 g, 4.55 mol, 2.2 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (30 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using EtOAc / MeOH=30 / 1 as the eluent to give the title compound as a yellow oil (1.19 g, 82%). [ka]

[0610] Methyl 3,4-bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoate (23b). Compound 22b (4.00 g, 12.11 mmol, 1 eq.) and K2CO3 (10.04 g, 72.66 mmol, 6 eq.) were stirred in dry DMF (70 mL). Compound 15 (10.98 g, 27.83 mol, 2.3 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (50 mL) was then added, and the mixture was extracted with DCM (50 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using EtOAc / MeOH=50 / 1 as the eluent to give the title compound as a light yellow oil (8.42 g, 90%). [ka]

[0611] Methyl 3,4-bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)benzoate (23c). Compound 22c (0.79 g, 1.81 mmol, 1 eq) and K2CO3 (1.50 g, 10.86 mmol, 6 eq) were stirred in dry DMF (30 mL). Compound 15 (1.50 g, 3.80 mol, 2.1 eq) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (30 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using hexane / EtOAc=1 / 3 as the eluent to give the title compound as a yellow oil (1.31 g, 82%). [ka]

[0612] Methyl 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)benzoate (23d). Compound 22a (1.80 g, 4.43 mmol, 1 eq) and K2CO3 (3.67 g, 26.58 mmol, 6 eq) were stirred in dry DMF (50 mL). Compound 17 (3.95 g, 9.30 mol, 2.1 eq) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (30 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using EtOAc as the eluent to afford the title compound as a tan oil (3.65 g, 91%). [ka]

[0613] 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoic acid (1). Compound 23a (1.10 g, 1.57 mmol, 1 equiv.) was dissolved in ethanol (EtOH, 15 mL). KOH (0.62 g, 10.99 mmol, 7 equiv.) was added, and the mixture was stirred under reflux for 2 h. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to pH 1. The solution was extracted with DCM (20 mL × 3), and the organic phase was dried over anhydrous MgSO. Filtration and evaporation of the solvent afforded the title compound as a yellow oil (1.07 g, 100%). [ka]

[0614] 3,4-Bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoic acid (3). Compound 23b (7.88 g, 10.17 mmol, 1 equiv.) was dissolved in ethanol (50 mL). KOH (3.99 g, 71.19 mmol, 7 equiv.) was added and the mixture was stirred under reflux for 2 h. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to pH ∼1. The solution was extracted with DCM (40 mL × 3) and the organic phase was dried over anhydrous MgSO4. Filtration and evaporation of the solvent afforded the title compound as a yellow oil (7.73 g, 100%). [ka]

[0615] 3,4-Bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)benzoic acid (6). Compound 23c (0.98 g, 1.11 mmol, 1 equiv) was dissolved in ethanol (15 mL). KOH (0.44 g, 7.77 mmol, 7 equiv) was added and the mixture was stirred under reflux for 2 h. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to pH < 2. The solution was extracted with DCM (30 mL × 3) and the organic phase was dried over anhydrous MgSO. Filtration and evaporation of the solvent gave the title compound as a tan oil (0.96 g, 100%). [ka]

[0616] 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)benzoic acid (8). Compound 23d (2.80 g, 3.07 mmol, 1 equiv) was dissolved in ethanol (30 mL). KOH (1.20 g, 21.49 mmol, 7 equiv) was added and the mixture was stirred under reflux for 2 h. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to pH < 2. The solution was extracted with DCM (30 mL × 3) and the organic phase was dried over anhydrous MgSO. Filtration and evaporation of the solvent gave the title compound as a tan oil (2.73 g, 100%). [ka]

[0617] Methyl 3,5-dihydroxy-4-((4-methoxybenzyl)oxy)benzoate (24b). Methyl 3,4,5-trihydroxybenzoate (19, 3.25 g, 17.66 mmol, 1 equiv.), 4-methoxybenzyl chloride (2.77 g, 17.66 mmol, 1 equiv.), KHCO (2.53 g, 35.32 mmol, 3 equiv.), and KI (18 mg, 0.11 mmol, 0.006 equiv.) were stirred in dry DMF (80 mL) under a N atmosphere at 60 °C for 24 h. The reaction mixture was cooled to 23 °C, and the DMF was removed under reduced pressure. Water (50 mL) was then added, and the mixture was extracted with DCM (50 mL × 3). The organic phase was dried over anhydrous MgSO and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using hexane / EtOAc=2 / 1 as the eluent to give the title compound as a white solid (2.30 g, 76%). [ka]

[0618] Methyl 4-(benzyloxy)-3,5-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzoate (25a). Methyl 4-(benzyloxy)-3,5-dihydroxybenzoate (24a, 4.00 g, 14.59 mmol, 1 eq.) and K2CO3 (12.08 g, 87.41 mmol, 6 eq.) were stirred in dry DMF (70 mL). Compound 12 (10.64 g, 33.42 mol, 2.3 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and the DMF was removed under reduced pressure. Water (50 mL) was then added, and the mixture was extracted with DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO 2 ) using EtOAc as the eluent to afford the title compound as a light yellow oil (7.27 g, 88%). [ka]

[0619] Methyl 3,5-bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4-((4-methoxybenzyl)oxy)benzoate (25b). Compound 24b (4.00 g, 13.14 mmol, 1 eq.) and K2CO3 (10.90 g, 78.86 mmol, 6 eq.) were stirred in dry DMF (60 mL). Compound 15 (11.90 g, 30.17 mol, 2.3 eq.) was added, and the mixture was stirred at 70 °C under a N2 atmosphere for 12 h. The reaction mixture was cooled to 23 °C, and DMF was removed under reduced pressure. Water (50 mL) was then added, and the mixture was extracted with DCM (50 mL × 3). The organic phase was dried ...

Claims

**Claim 1** An ionic amphiphilic Janus dendrimer having the structure of the following formula (I): 【Chemical 1】 [wherein, A is [Chemical 2] a polyvalent group containing at least one selected from the group consisting of and any combination thereof, the dashed line represents a bonding site of one of X, Y, or Z, X is a hydrophilic group containing at least one amine, Y is a lipophilic group containing at least one C １ -C ３０ -alkyl chain, and Z contains at least one selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, and polyethylene glycol chains, R Ａ and R Ｂ each independently is selected from the group consisting of hydrogen, halogen substitution, hydroxy, C １ -C ３０ -alkyl, C １ -C ３０ -halogenated alkyl, C １ -C ３０ -alkoxy, C １ -C ３０ -halogenated alkoxy, and any combination thereof s is an integer from 0 to 5, t is an integer from 0 to 5, u is an integer from 0 to 4, the sum of s, t, and u is equal to the valence of A].

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