One component delivery system for nucleic acids

Abstract

The present invention relates to an amphiphilic Janus dendritic polymer that can form nanoparticles. The invention also relates to a method of 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, and to a method of delivering an agent to a subject in need thereof, the method comprises the step of delivering a composition comprising an agent encapsulated by nanoparticles to a subject.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and interest in 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 entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was completed with government support from National Science Foundation grants 1720530, 1807127, and 2104554, and National Institutes of Health grant TR002776. The government holds certain rights to this invention. Background Technology

[0005] Delivering exogenously generated nucleic acids into cells and / or their nuclei via viral and nonviral vectors to alter protein expression is one of the most fundamental concepts in nanomedicine. Both viral and nonviral delivery systems exhibit advantages and disadvantages. Viral vectors offer high transfection efficiency (95%) and greater specificity for cell targeting, including non-native cells. Some disadvantages of viral gene delivery include immunogenicity, cytotoxicity, difficulty in their assembly, inflammatory responses to repeated administration, and the possibility of insertional mutations. Nonviral delivery is biosafe, exhibiting lower toxicity and immunogenicity, but its transfection efficiency is lower (1-2%), and the vectors are less stable than viral vectors. Covalent and supramolecular dendrimers (dendritic macromolecules) complexed with nucleic acids on their cationic periphery have been used as nonviral vectors for DNA cell transfection.

[0006] Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, and cholesterol to improve mechanical properties, and poly(ethylene glycol) (PEG)-coupled lipids to provide stability, represent current non-viral leading vectors for mRNA delivery. The deficiencies in LNP production and stability are demonstrated by the microfluidic devices required for their assembly and the need for storage at extremely low temperatures (-70°C). Their design, synthesis, and assembly have been inspired by the development of stealth liposomes for delivering low-molar-mass drugs.

[0007] Because RNA is less stable than DNA, it must be protected by encapsulation before being released into the cell. At acidic pH (pH 3 to 5), when the pK of ionizable amines... aAt pH less than 7, LNPs can encapsulate large amounts of mRNA. At physiological pH (7.4), LNPs have a nearly neutral surface charge and a high positive charge at endosome pH. Electrostatic interactions between the cationicly charged LNPs and naturally occurring anionic lipids within the endosome membrane are thought to be the cause of RNA release. One of the main limitations of the four-component carrier is the unknown distribution of its four components within the LNP. The separation of neutral ionizable lipids as an oil phase within the LNP nucleus is thought to be the reason for its very low transfection efficiency (1–2%). A second drawback of LNPs is provided by PEG-conjugated lipids and is known as the “PEG dilemma.” PEG conjugated to LNPs increases circulation time in the blood after intravenous injection. However, the same PEG is known to reduce gene expression by up to four orders of magnitude by decreasing intracellular transport through cellular uptake and endosome escape.

[0008] Charge-change release transporters (CARTS) have also been demonstrated for mRNA delivery. This delivery concept is independent of the aforementioned viral and nonviral LNP-based methodologies. Artificial and synthetic vesicles, such as liposomes and polymersomes, have been elaborated in detail for drug delivery and as mimics of natural cells. Dendritic bodies (DS) assembled from amphiphilic Janus dendritic polymers (JD) have been shown to exhibit excellent mechanical properties and stability (including in serum). Amphiphilic JD, along with sugars conjugated to their hydrophilic portions, termed Janus sugar dendritic polymers (JGD), self-assemble into sugar dendritic bodies (GDS), which mimic the glycans of biological membranes and bind sugar-binding proteins. Both JD and JGD self-assemble into monodisperse DS and GDS with monolayer or multilayer structures via simple injection rather than microfluidic techniques, and their sizes are predictable. Sequence-defined JGDs self-assemble via injection of GDS. They have demonstrated higher biological activity for sugar-binding proteins at lower sugar densities in the defined sequences.

[0009] Therefore, there is a need in the art for compositions and methods for delivering mRNA. This invention addresses this unmet need. Summary of the Invention

[0010] In one aspect, the present invention relates in part to ionizable amphiphilic Janus dendritic polymers having the structure of formula (I):

[0011]

[0012]

[0013] In some embodiments, A is a multivalent group comprising a group selected from...

[0014] At least one, or any combination thereof. In some embodiments, A is a multivalent group comprising a group selected from... At least one of them, or any combination thereof.

[0015] In some implementations, dashed lines represent binding sites for one of the X, Y, or Z.

[0016] In some embodiments, X is a hydrophilic group comprising at least one amine.

[0017] In some implementations, Y comprises at least one C1-C 30 The lipophilic group of the alkyl chain. In some embodiments, Y is a C1-C group comprising at least two C1-C groups with different numbers of carbon atoms. 30 Lipophilic groups in alkyl chains.

[0018] In some embodiments, Z comprises at least one selected from ethylene glycol, diethylene glycol, triethylene glycol, or polyethylene glycol chains.

[0019] In some implementations, R A and R B Independently selected from hydrogen, halides, hydroxyl groups, C1-C 30 Alkyl, C1-C 30 Alkyl halides, C1-C 30 Alkoxy, C1-C 30 Alkyl halides, or any combination thereof.

[0020] In some implementations, s is an integer from 0 to 5. In some implementations, s is an integer from 1 to 5.

[0021] In some implementations, t is an integer from 0 to 5. In some implementations, t is an integer from 1 to 5.

[0022] In some implementations, u is an integer from 0 to 4.

[0023] In some implementations, the sum of s, t, and u equals the valence of A.

[0024] For example, in some implementations, A is... This indicates that s and t are each 2.

[0025] In some implementations, A is... This indicates that t is 2, s is 1, and u is 0 or 1.

[0026] In some implementations, A is... This indicates that s and t are each 1.

[0027] In some embodiments, the ionizable amphiphilic Janus dendritic polymer having the structure of formula (I) is an ionizable amphiphilic Janus dendritic polymer having the structure of formula (II):

[0028] (X) s -A-(Y) t

[0029] Equation (II).

[0030] In some implementations, each occurrence of X is independently selected from...

[0031]

[0032] Or any combination thereof.

[0033] In some implementations, the dashed line indicates a connection to A.

[0034] In some implementations, each occurrence of m, n, and o is an independent integer from 1 to 20. In some implementations, each occurrence of m, n, and o is an independent integer from 1 to 10. In some implementations, each occurrence of m, n, and o is an independent integer from 1 to 5.

[0035] In some implementations, each occurrence of W is independently selected from C=O, C(R) W (R) W ), NR W , O, or S. In some implementations, each occurrence of R... W It is independently selected from hydrogen, halide, hydroxyl, alkyl, alkyl halide, aryl, aryl halide, alkoxy, alkoxy halide, or any combination thereof.

[0036] In some implementations, each occurrence of L 1 L 2 L 3 and L 4 Independently a covalent or divalent linking group, selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, ynylene, arylene, heteroarylene, silylene, amine, amide, ester, ether, carbonyl, urethane, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate ester, poly(alkyl ether), heteroatom, or any combination thereof.

[0037] In some implementations, each occurrence of R 11 R 12 R 13 and R 14Independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R appears at least once. 11 R 12 R 13 、or R 14 For -C(O)(CH2) m -N(R 1 (R) 2 In some implementations, each occurrence of m is an independent integer from 1 to 10. In some implementations, each occurrence of R... 1 and R 2 Independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R 1 and R 2 They can form a ring together. In some implementations, each occurrence of R... 11 R 12 R 13 and R 14 Selected independently Or any combination thereof.

[0038] In some implementations, X comprises at least two tertiary amines.

[0039] In some implementations, each occurrence of X is independently selected from...

[0040]

[0041] , or any combination thereof.

[0042] In some implementations, each occurrence of R 3 Independently selected from hydrogen and (CH2) n (CH2) n -OH, or any combination thereof.

[0043] In some implementations, each occurrence of X is independently selected from... Or any combination thereof.

[0044] In some implementations, each occurrence of Y is independently selected from... Or any combination thereof.

[0045] In some implementations, each occurrence of R 21 R 22 R 23 and R 24 Independently contains either straight or branched C1-C chains 50Alkyl group. In some embodiments, each occurrence of R... 21 R 22 R 23 and R 24 Independently contains C1-C 30 alkyl.

[0046] In some implementations, each occurrence of Y is independently selected from...

[0047] Or any combination thereof. In some implementations, n is an integer from 1 to 30.

[0048] In some implementations, each occurrence of Y is independently selected from... Or any combination thereof.

[0049] In some implementations, each occurrence of -(CH2) n CH3, -O(CH2) n -、and -O(CH2) n CH3 can be either straight-chain or branched.

[0050] In some embodiments, the ionizable amphiphilic Janus dendritic polymer 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 of carbon atoms in the first Y to carbon atoms in the second Y is greater than or equal to 3 and less than 7.

[0051] In some implementations, u is 1 or 2.

[0052] In some implementations, each occurrence of Z is independently selected from... Or any combination thereof.

[0053] In some implementations, each occurrence of R 31 R 32 R 33 and R 34 The radical is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, each occurrence of Z is independently selected from... In some implementations, n is an independent integer from 1 to 100 for each occurrence. In some implementations, p is an independent integer from 1 to 10 for each occurrence. In some implementations, R is an independent integer from 1 to 10 for each occurrence. 4It is independently selected from hydrogen, deuterium, alkyl, aryl, or any combination thereof.

[0054] In some embodiments, the ionizable amphiphilic Janus dendritic polymer includes chiral, racemic, or achiral branding points.

[0055] In some embodiments, the ionizable amphiphilic Janus dendritic polymer is a chiral ionizable amphiphilic Janus dendritic polymer, a racemic ionizable amphiphilic Janus dendritic polymer, or a chiral ionizable amphiphilic Janus dendritic polymer.

[0056] In some embodiments, the ionizable amphiphilic Janus dendritic polymer is an ionizable amphiphilic Janus dendritic polymer having a structure selected from the group consisting of: Figure 12 at least one structure, Figure 13 at least one structure, Figure 14 at least one structure, Figure 47 at least one structure, Figure 60 at least one structure, Figure 80 At least one structure, and any combination thereof.

[0057] In one aspect, the present invention relates in part to nanoparticles comprising at least one of the ionizable amphiphilic Janus dendritic polymers of the present invention.

[0058] In some embodiments, the nanoparticles comprise a first ionizable amphiphilic Janus dendritic polymer and a second ionizable amphiphilic Janus dendritic polymer. In some embodiments, the first ionizable amphiphilic Janus dendritic polymer has a different structure than the second ionizable amphiphilic Janus dendritic polymer.

[0059] In some embodiments, the nanoparticles comprise a chiral, ionizable, amphiphilic Janus dendritic polymer, a chiral, ionizable, amphiphilic Janus dendritic polymer, or any combination thereof.

[0060] In some embodiments, the nanoparticles are monolayer nanoparticles or onion-like multilayer nanoparticles. In some embodiments, the nanoparticles comprise racemic, ionizable, amphiphilic Janus dendritic polymers. In some embodiments, the nanoparticles are multilayer nanoparticles.

[0061] In some embodiments, the nanoparticles further comprise at least one reagent. In some embodiments, the at least one reagent includes diagnostic reagents, detectable reagents, therapeutic agents, nucleic acid molecules, or any combination thereof. In some embodiments, the at least one reagent is selected from mRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, small molecules, proteins, antibodies, peptides, proteins, or any combination thereof.

[0062] In some embodiments, at least one reagent 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 molecules, targeting nucleic acids, or any combination thereof. In some embodiments, the nucleic acid molecule encodes at least one selected from antigens, antibodies, gene-editing molecules, chimeric antigen receptors (CARs), or any combination thereof. In one embodiment, the nucleoside-modified RNA comprises pseudouridine. In one embodiment, the nucleoside-modified RNA comprises pseudouridine plus 5-methylcytosine. In one embodiment, the nucleoside-modified RNA comprises 5-methyluridine. In one embodiment, the nucleoside-modified RNA comprises 1-methyl-pseudouridine.

[0063] In one aspect, the present invention relates to compositions comprising at least one dendritic polymer nanoparticle as described herein.

[0064] In one aspect, the present invention relates in part to a composition comprising at least one ionizable amphiphilic Janus dendritic polymer of the present invention and / or at least one nanoparticle of the present invention.

[0065] In one embodiment, the composition further comprises an adjuvant.

[0066] In one embodiment, the composition is a pharmaceutical composition.

[0067] In one embodiment, the composition is a vaccine.

[0068] In one aspect, the present invention relates in part to a method for delivering a reagent to a subject in need using at least one nanoparticle or a composition of the present invention containing the nanoparticle.

[0069] In some implementations, the method also includes delivering the reagent to the subject's liver.

[0070] In some implementations, the method also includes delivering the reagent to the subject's spleen.

[0071] In some implementations, the method also includes delivering the reagent to the lungs of the subject.

[0072] In some embodiments, the method treats or prevents at least one condition selected from viral infection, bacterial infection, fungal infection, parasitic infection, cancer, cancer-related disease or condition, autoimmune disease or condition, or any combination thereof.

[0073] In some embodiments, the reagent is encapsulated within nanoparticles. In one embodiment, the reagent is any of the reagents described herein. For example, in one embodiment, the reagent is a composition for protein replacement therapy. In one embodiment, the reagent is a composition for gene editing. In one embodiment, the reagent is a vaccine.

[0074] In one aspect, the invention relates in part to a method of preventing or treating a disease or condition in a subject with such need using at least one nanoparticle or a composition of the invention comprising the nanoparticle. In some embodiments, the disease or condition is selected from viral infections, bacterial infections, fungal infections, parasitic infections, cancer, cancer-related diseases or conditions, autoimmune diseases or conditions, or any combination thereof.

[0075] In one aspect, the present invention relates in part to a method for inducing an immune response in a subject in need of such an immune response using at least one nanoparticle or a composition of the present invention containing the nanoparticle.

[0076] In one aspect, the present invention relates to a method for delivering a reagent to a subject in need, the method comprising administering to the subject at least one dendritic nanoparticle described herein or a composition comprising the dendritic nanoparticle. Attached Figure Description

[0077] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0078] Figure 1 A schematic diagram of an exemplary single-component nanoparticle (DNP) for mRNA delivery is depicted.

[0079] Figure 2 A schematic diagram of a four-component lipid nanoparticle (LNP) system for mRNA delivery is depicted.

[0080] Figure 3 A schematic diagram depicts the hydrophilic and hydrophobic acids used in an exemplary DNP, as well as the connector.

[0081] Figure 4A schematic diagram of six libraries containing 52 ionizable amphiphilic Janus dendritic polymers (IAJD) is depicted.

[0082] Figure 5 Representative luciferase expression was depicted in HEK293T cells with DNPs that encapsulate luciferase mRNA.

[0083] Figure 6 Representative assays of in vivo transfection of single-component DNPs are depicted. The diameter and polydispersity of the DNPs (both shown in black) and the pKa value of the IAJDs (shown in blue) are shown below the IAJD molecule number. All these data are printed on top of each mouse image. Luminescence values ​​are also shown.

[0084] Figure 7 Representative results of quantifying luciferase signals in in vivo images were presented.

[0085] Figure 8 Representative results comparing the concentration and sequence of the IAJD with its in vitro and in vivo activities are presented. A schematic diagram of the IAJD used is shown on the right.

[0086] Figure 9 Representative in vivo images of organs are depicted.

[0087] Figure 10 This depicts representative images of single-component DNPs delivering mRNA to different organs.

[0088] Figure 11 Representative results are depicted for examples demonstrating the superior stability of DNPs assembled from IAJD9, IAJD22, IAJD33, IAJD34, IAJD32, IAJD33+IAJD32(2%), IAJD46, and IAJD47.

[0089] Figure 12 Exemplary compounds from single-to-single IAJD libraries 1-4 are depicted. Deep yellow indicates molecules that exhibit activity both in vitro and in vivo, except for IAJD19, 23, and 45, which are active only in vivo and in vitro. Light yellow indicates molecules that exhibit activity only in vitro. White indicates molecules that exhibit no activity either in vitro or in vivo.

[0090] Figure 13 Exemplary compounds from the bis-bisIAJD library 5 are depicted. Deep yellow indicates that the molecule exhibits activity both in vitro and in vivo. Light yellow indicates that the molecule exhibits activity only in vitro. White indicates that the molecule exhibits no activity both in vitro and in vivo.

[0091] Figure 14Exemplary compounds from the hybrid bi-mixed IAJD library 6 are depicted. Deep yellow indicates that the molecule exhibits activity both in vitro and in vivo. Light yellow indicates that the molecule exhibits activity only in vitro. White indicates that the molecule exhibits no activity either in vitro or in vivo.

[0092] Figure 15 The compound 91c (4 / The representative MALDI-TOF MS spectrum is shown. The spectrum on the right is magnified.

[0093] Figure 16 Results depicting the effect of vortexing time on the assembly size of an exemplary dendritic polymer IAJD9 (4.0 mg / mL in tris buffer).

[0094] Figure 17 Representative DLS data for the components of IAJD9 (8.00 to 1.00 mg / ml) in tris buffer (pH = 7.4) were depicted.

[0095] Figure 18 Representative DLS data for the components of IAJD22 (8.0 to 1.0 mg / ml) in tris buffer (pH = 7.4) were depicted.

[0096] Figure 19 Representative DLS data for components of IAJD27 (8.0 to 1.0 mg / ml) in tris buffer (pH = 7.4) were depicted.

[0097] Figure 20 Representative DLS data of DNPs assembled from IAJD1 and IAJD2 are depicted.

[0098] Figure 21 Representative DLS data of DNPs assembled from IAJD8 and IAJD9 are depicted.

[0099] Figure 22 Representative DLS data of DNPs assembled from IAJD10 and IAJD17 are depicted.

[0100] Figure 23 Representative DLS data of DNPs assembled from IAJD18 and IAJD19 are depicted.

[0101] Figure 24 Representative DLS data of DNPs assembled from IAJD20 and IAJD21 are depicted.

[0102] Figure 25 Representative DLS data of the DNP assembled from IAJD22 and IAJD23 are depicted.

[0103] Figure 26 Representative DLS data of DNPs assembled from IAJD24 and IAJD25 are depicted.

[0104] Figure 27 Representative DLS data of DNPs assembled from IAJD26 and IAJD27 are depicted.

[0105] Figure 28 Representative DLS data of DNPs assembled from IAJD28 and IAJD29 are depicted.

[0106] Figure 29 Representative DLS data of DNPs assembled from IAJD30 and IAJD31 are depicted.

[0107] Figure 30 Representative DLS data of the DNP assembled from IAJD33 and IAJD34 are depicted.

[0108] Figure 31 Representative DLS data of DNPs assembled from IAJD37 and IAJD40 are depicted.

[0109] Figure 32 Representative DLS data of the DNP assembled from IAJD43 and IAJD44 are depicted.

[0110] Figure 33 Representative DLS data of DNPs assembled from IAJD45 and IAJD46 are depicted.

[0111] Figure 34 Representative DLS data of DNP assembled by IAJD47 are depicted.

[0112] Figure 35 Representative results are depicted for examples showing the good stability of DNPs assembled from IAJD27 and the poor stability of DNPs assembled from IAJD30 and IAJD31.

[0113] Figure 36 Representative results depicting the size of DNPs assembled from IAJD33+IAJD32 (2%) in 1% fetal bovine serum were described.

[0114] Figure 37 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0115] Figure 38 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0116] Figure 39Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0117] Figure 40 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0118] Figure 41 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0119] Figure 42 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0120] Figure 43 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0121] Figure 44 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0122] Figure 45 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0123] Figure 46 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0124] Figure 47 A schematic diagram depicts other compounds of the present invention and the pKa values ​​measured for these compounds.

[0125] Figure 48 Representative results demonstrating that the distribution of nanoparticles in mice is a function of IAJD are presented.

[0126] Figure 49 Representative results comparing in vivo and in vitro luminescence of different IAJDs are described.

[0127] Figure 50 Representative DLS data of DNPs assembled from IAJD64, IAJD65, IAJD66, IAJD70, IAJD71, IAJD74, IAJD75, IAJD76, and IAJD77 are depicted.

[0128] Figure 51Representative DLS data of DNPs assembled from IAJD78, IAJD79, IAJD81, IAJD82, IAJD83, IAJD84, IAJD85, IAJD86, and IAJD87 are depicted.

[0129] Figure 52 Representative DLS data of DNPs assembled from IAJD88, IAJD89, IAJD91, IAJD95, IAJD96, IAJD97, IAJD98, IAJD99, and IAJD103 are depicted.

[0130] Figure 53 Representative DLS data of DNPs assembled from IAJD105, IAJD106, IAJD107, and IAJD108 are depicted.

[0131] Figure 54 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0132] Figure 55 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0133] Figure 56 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0134] Figure 57 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0135] Figure 58 Representative results demonstrating efficacy comparisons in vivo and in vitro are described.

[0136] Figure 59 A schematic diagram of asymmetric IAJD synthesis is depicted.

[0137] Figure 60 Schematic diagrams of IAJD 81 to IAJD 159 are depicted.

[0138] Figure 61 The representative pKa and luminescence of the selected IAJD are depicted.

[0139] Figure 62 Representative results demonstrating the luminescence of IAJDs by selective localization in mice are depicted.

[0140] Figure 63 Representative DLS data of DNPs assembled from IAJD113 to IAJD120 and IAJD122 are depicted.

[0141] Figure 64 Representative DLS data of DNPs assembled from IAJD124 to IAJD130, IAJD133, IAJD135 and IAJD136 are depicted.

[0142] Figure 65 Representative DLS data of DNPs assembled from IAJD138 and IAJD141 to IAJD148 are depicted.

[0143] Figure 66 Representative DLS data for DNPs assembled from IAJD149 to IAJD154, IAJD161, IAJD162, and IAJD171 are depicted.

[0144] Figure 67 Representative DLS data of DNPs assembled from IAJD172, IAJD173, IAJD177, IAJD178, IAJD110, IAJD111, and IAJD155 to IAJD159 are depicted.

[0145] Figure 68 Representative titration curves showing the response of the IAJD molecule to changes in the pH of the solution upon the addition of a strong acid were plotted.

[0146] Figure 69 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0147] Figure 70 Representative titration curves showing the response of the IAJD molecule to changes in the pH of the solution upon the addition of a strong acid were plotted.

[0148] Figure 71 Representative titration curves showing the response of the IAJD molecule to the pH change of the solution upon the addition of a strong acid were plotted.

[0149] Figure 72 Representative titration curves were plotted to show the pH change of the solution in response to the addition of a strong acid for the IAJD molecule.

[0150] Figure 73 Representative results related to the structure and activities of the IAJD are described.

[0151] Figure 74 A schematic diagram of IAJD with asymmetric alkyl chains is depicted, along with representative results demonstrating that DNP selectively delivers Luc-mRNA to the spleen and lymph nodes in vivo and that DNP delivers Luc-mRNA to organs with lower selectivity in vivo.

[0152] Figure 75 Representative results demonstrating the ratio between the in vivo activities of asymmetric IAJDs with one C18 alkyl chain and their corresponding symmetric IAJDs with two identical C18 alkyl chains are presented.

[0153] Figure 76 Representative results demonstrating the size of DNPs assembled from IAJD125 in 1% fetal bovine serum were depicted.

[0154] Figure 77 Representative results demonstrating the size of DNPs assembled from IAJD155 in 1% fetal bovine serum were depicted.

[0155] Figure 78 Representative DLS data for DNP 125 and 178 were depicted before and after dialysis in 1X PBS buffer for 3 hours.

[0156] Figure 79 A schematic diagram depicting the amphiphilic JD of glycerol is shown.

[0157] Figure 80 A schematic diagram depicts the R-, S-, rac- and achiral glycerols of OH with two protective properties and isochiral (1R, 1S, 2R, 2S), racemic (1rac, 2rac), and achiral (3, 4) glycerol-JD.

[0158] Figure 81 A schematic diagram of the synthesis of glycerol-JD from Library 1 is depicted. Reagents and conditions: (i) DCC, DPTS, DCM, 23°C, 12 h; (ii) H2, Pd / C, EtOAc, 23°C, 8 h; (iii) 1M HCl, MeOH, 23°C, 1 h.

[0159] Figure 82 A schematic diagram of the synthesis of glycerol-JD from Library 2 is depicted. Reagents and conditions: (i) DCC, DPTS, DCM, 23°C, 12 h; (ii) H2, Pd / C, EtOAc, 23°C, 8 h; (iii) 1M HCl, 1,4-dioxane, 60°C, 8 h.

[0160] Figure 83 A schematic diagram of the synthesis of glycerol-aachitic JD was drawn. Reagents and conditions: (i) DCC, DPTS, DCM, 23℃, 12 h; (ii) H2, Pd / C, EtOAc, 23℃, 8 h; (iii) 1M HCl, 1,4-dioxane, 60℃, 8 h.

[0161] Figure 84 The diameter (D) of the DS demonstrating the assembly of glycerol-JD in water was depicted. h(in nm) and square diameter (D) h 2 The following are representative results regarding the concentration dependence of glycerol-JD in library 1. The top two graphs correspond to glycerol-JD 3 and 4 in library 1. The remaining graphs correspond to glycerol-JD in library 2.

[0162] Figure 85 Representative cryo-TEM images of DS assembled from glycerol-JD 2R, 2S, and 2rac are depicted.

[0163] Figure 86 Representative cryo-TEM images of DS self-assembled from glycerol-JD 1R(a), 1S(b), and 1rac(c) are depicted. Scale bar is 100 nm.

[0164] Figure 87 Representative cryo-TEM images of DS self-assembled via glycerol-based achiral JD 3 are depicted.

[0165] Figure 88 Representative cryo-TEM images of DS self-assembled via glycerol-based achiral JD 4 are depicted.

[0166] Figure 89 The results are presented as representative of the normalized frequency histograms of the number of vesicle layers in DS assembled from glycerol-JD 1R, 1S, 1rac, 2R, 2S, and 2rac, as demonstrated by statistical analysis.

[0167] Figure 90 The representative of (3,5)-12G1-GC-(R)-BMPA-(3,4,5)-3EO-G1(1R) is described. 1 1H NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm and 2.04 ppm are due to partially undeuterated residues in CDCl3 and EtOAc, respectively.

[0168] Figure 91 The representative of (3,5)-12G1-GC-(R)-BMPA-(3,4,5)-3EO-G1(1R) is described. 13 C10 NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0169] Figure 92 The representative of (3,5)-12G1-GC-(S)-BMPA-(3,4,5)-3EO-G1(1S) is described. 11H NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm and 2.04 ppm are due to partially undeuterated residues in CDCl3 and EA, respectively.

[0170] Figure 93 The representative of (3,5)-12G1-GC-(S)-BMPA-(3,4,5)-3EO-G1(1S) is described. 13 C10 NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0171] Figure 94 The representative (3,5)-12G1-GC-(rac)-BMPA-(3,4,5)-3EO-G1(1rac) was described. 1 1H NMR spectrum. CDCl3, 500MHz, 298K. The asterisks at δ 7.26ppm, 5.30ppm, and 1.66ppm are due to partially undeuterated residues in CDCl3, DCM, and water, respectively.

[0172] Figure 95 The representative (3,5)-12G1-GC-(rac)-BMPA-(3,4,5)-3EO-G1(1rac) was described. 13 C10 NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0173] Figure 96 The representative of (3,5)-12G1-BMPA-GC-(R)-(3,4,5)-3EO-G1(2R) is described. 1 1H NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm and 1.68 ppm are due to partially undeuterated residues in CDCl3 and water, respectively.

[0174] Figure 97 The representative of (3,5)-12G1-BMPA-GC-(R)-(3,4,5)-3EO-G1(2R) is described. 13 C10 NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0175] Figure 98 The representative of (3,5)-12G1-BMPA-GC-(S)-(3,4,5)-3EO-G1(2S) is described. 11H NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm and 1.65 ppm are due to partially undeuterated residues in CDCl3 and water, respectively.

[0176] Figure 99 The representative of (3,5)-12G1-BMPA-GC-(S)-(3,4,5)-3EO-G1(2S) is described. 13 C10 NMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0177] Figure 100 The representative of (3,5)-12G1-BMPA-GC-(rac)-(3,4,5)-3EO-G1(2rac) is described. 1 HNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm and 1.60 ppm are due to partially undeuterated residues in CDCl3 and water, respectively.

[0178] Figure 101 The representative of (3,5)-12G1-BMPA-GC-(rac)-(3,4,5)-3EO-G1(2rac) is described. 13 CNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0179] Figure 102 The representative of (3,5)-12G1-GC-(chiral)-BMPA-(3,4,5)-3EO-G2(3) is described. 1 HNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm, 2.04 ppm, and 1.66 ppm are due to partially undeuterated residues in CDCl3, EtOAc, and water, respectively.

[0180] Figure 103 The representative of (3,5)-12G1-GC-(chiral)-BMPA-(3,4,5)-3EO-G2(3) is described. 13 CNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0181] Figure 104 The representative of (3,5)-12G2-BMPA-GC-(chiral)-(3,4,5)-3EO-G1(4) is described. 1HNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisks at δ 7.26 ppm, 5.30 ppm, and 1.67 ppm are due to partially undeuterated residues in CDCl3, DCM, and water, respectively.

[0182] Figure 105 The representative of (3,5)-12G2-BMPA-GC-(chiral)-(3,4,5)-3EO-G1(4) is described. 13 CNMR spectrum. CDCl3, 500 MHz, 298 K. The asterisk signal at δ 77.16 ppm is due to CDCl3.

[0183] Figure 106 Representative MALDI-TOF-MS spectra of glycerol-based JD from Library 1 were depicted.

[0184] Figure 107 Representative MALDI-TOF-MS spectra of glycerol-based JD from Library 2 were depicted.

[0185] Figure 108 Representative MALDI-TOF-MS spectra of glycerol-based achiral JD 3 (left) and 4 (right) were depicted.

[0186] Figure 109 A representative HPLC trace of glycerol-based JD from Library 1 was depicted.

[0187] Figure 110 Representative HPLC traces of glycerol-based JD from Library 2 were depicted.

[0188] Figure 111 Representative HPLC traces of glycerol-based achiral JD 3 (left) and 4 (right) were depicted.

[0189] Figure 112 Representative results are depicted to demonstrate the normality of the QQ plot for sample 1rac. The distribution of 1rac is similar to a normal distribution.

[0190] Figure 113 Representative results are depicted to demonstrate the normality of the QQ plot for sample 2rac. The distribution of 2rac is similar to a normal distribution. Detailed Implementation

[0191] This invention is partly based on the unexpected result that nanoparticles comprising at least one ionizable amphiphilic Janus dendritic polymer having the structure of formula (I) effectively and efficiently deliver reagents to targets of interest. Therefore, in one aspect, the invention relates to ionizable amphiphilic Janus dendritic polymers having the structure of formula (I). In another aspect, the invention relates to nanoparticles comprising at least one of the ionizable amphiphilic Janus dendritic polymers of the present invention. In some embodiments, the nanoparticles further comprise at least one reagent. In some embodiments, the nanoparticles further comprise at least one reagent encapsulated by the ionizable amphiphilic Janus dendritic polymer of the present invention. In another aspect, the invention relates to compositions comprising at least one of the ionizable amphiphilic Janus dendritic polymers of the present invention or nanoparticles thereof. In some embodiments, the composition is a vaccine.

[0192] In one aspect, the present invention relates to a method for delivering a reagent to a target of interest using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention. In another aspect, the present invention relates to a method for preventing or treating a disease or condition in a subject using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention. In yet another aspect, the present invention relates to a method for inducing an adaptive immune response in a subject using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention.

[0193] definition

[0194] 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 pertains.

[0195] As used herein, the following terms have the meanings relating to them in this section.

[0196] The articles “one” and “a” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” refers to one or more elements.

[0197] As used herein, when referring to measurable values ​​(e.g., numerical values ​​and / or ranges, such as quantities, durations, etc.), “about” is intended to include variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods. For example, “about 40 [units]” can refer to within ±25% of 40, within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, less than ±1% (e.g., 30 to 50), or any other value or range of values ​​thereunder or below. Furthermore, the phrases “less than about [one value]” or “greater than about [one value]” should be understood in accordance with the definition of the term “about” provided herein.

[0198] Unless otherwise stated, the term "compound" as used herein refers to any particular compound disclosed herein. In one embodiment, the term also refers to stereoisomers and / or optical isomers (including racemic mixtures) or mixtures enriched with enantiomers of the disclosed compound.

[0199] As used herein, the terms "analog," "analogue," or "derivative" refer to a compound or molecule formed from a parent compound or molecule through one or more chemical reactions. Therefore, an analog may have a structure similar to that of the small molecule therapeutic agent described herein, or it may be based on the skeleton of the small molecule therapeutic agent described herein but differ from it in certain components or structural compositions that may have similar or opposite metabolic effects. Analogs or derivatives may also be small molecules with structures different from the reference molecule but retaining the essential properties of the reference molecule. Analogs or derivatives may alter their interactions with certain other molecules relative to the reference molecule. Analog or derivative molecules may also include salts, adducts, tautomers, isomers, prodrugs, or other variants of the reference molecule.

[0200] As used herein, the term "prodrug" refers to an agent that is converted into a parent drug in vivo. For example, the term "prodrug" refers to a derivative of a known directly acting drug that has enhanced delivery properties and therapeutic value compared to the drug and is converted into an active drug through an enzymatic or chemical process. In some embodiments, "prodrug" refers to an inactive or relatively less active form of an active agent that becomes active through chemical conversion via one or more metabolic processes. In one embodiment, upon administration in vivo, the prodrug is chemically converted into a biological, pharmaceutical, or therapeutically active form of the compound. In another embodiment, the prodrug is enzymatically metabolized into a biological, pharmaceutical, or therapeutically active form of the compound through one or more steps or processes. For example, the compounds of the present invention can be administered to a subject as a prodrug comprising an initiator bound to an active agent, and the active agent is released in its active form through metabolic degradation.

[0201] The term "tautomer" refers to the constituent isomers of an organic compound that can easily interconvert through a chemical process (tautomerization).

[0202] The term "isomer" or "stereoisomer" refers to compounds that have the same chemical composition but differ in the spatial arrangement of atoms or groups.

[0203] As used herein, the term "alkyl" itself, or as part of another substituent, refers to having a specified number of carbon atoms (i.e., C12). 1-50 A straight-chain or branched hydrocarbon (representing one to fifty carbon atoms) and including straight-chain, branched, or cyclic substituents, unless otherwise specified. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.

[0204] As used herein, the term "substituted alkyl" refers to an alkyl group as defined above, which is substituted by one, two, or three substituents selected from the group consisting of: halogen, -OH, alkoxy, -NH2, amino, azide, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C=N, -C(=O)O(C1-C4)alkyl, -C(=O)NH2, -SO2NH2, -C(=NH)NH2, and -NO2. Examples of substituted alkyl groups include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0205] As used herein, the term "heteroalkyl" on its own or in combination with another term refers to a stable straight-chain or branched 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, unless otherwise stated. Heteratoms may be placed at any position on the heteroalkyl group, including between the remainder of the heteroalkyl group and the segment to which it is attached, and attached to the most terminal carbon atom of the heteroalkyl group. Examples include -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-SS-CH3.

[0206] The term "amino" refers to a group of the formula -NRaRa, -NHRa, or -NH2, wherein each R a Independently, it is an alkyl, alkenyl, or ynyl group containing 1 to 20 carbon atoms as defined above. Unless otherwise specified in the specification, alkylamino groups may optionally be substituted.

[0207] The term "hydroxyl" or "hydroxyl" refers to a group having the formula OH.

[0208] As used herein, the term "alkoxy" or otherwise used alone or in combination with other terms means an alkyl group having the specified number of carbon atoms as defined above, which is attached by an oxygen atom to the remainder of the molecule, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher homologues and isomers, unless otherwise stated.

[0209] As used herein, the terms “halo,” “halide,” or “halogen” refer alone or as part of another substituent to a fluorine, chlorine, bromine, or iodine atom, unless otherwise stated.

[0210] "Halogenated alkyl" or "alkyl halide" refers to an alkyl group as defined above, which is substituted with one or more halogenated radicals as defined above, such as trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Unless otherwise specified in the specification, the alkyl group may optionally be substituted.

[0211] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group in which each atom forming the ring (i.e., the skeleton atom) is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. Exemplary examples of cycloalkyl groups include, but are not limited to, the following:

[0212]

[0213] Monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyl groups include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyl groups include adamantane and norbornene. The term cycloalkyl includes "unsaturated non-aromatic carbocyclic" or "non-aromatic unsaturated carbocyclic" groups, both of which refer to non-aromatic carbocyclic groups as defined herein, containing at least one carbon double bond or one carbon triple bond.

[0214] As used herein, the term "heterocyclic alkyl" or "heterocyclic group" refers to a cyclic group containing one to four heteroatoms selected from O, S, and N. In one embodiment, each heterocyclic alkyl 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 heterocyclic alkyl group is fused with an aromatic ring. In one embodiment, the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. Unless otherwise stated, the heterocyclic system may be attached to any heteroatom or carbon atom that provides a stable structure. The heterocycle may be aromatic or non-aromatic in nature. In one embodiment, the heterocycle is a heteroaryl group.

[0215] Examples of 3-membered heterocyclic alkyl groups include, but are not limited to, aziridine. Examples of 4-membered heterocyclic alkyl groups include, but are not limited to, aziridine and β-lactam. Examples of 5-membered heterocyclic alkyl groups include, but are not limited to, pyrrolidine, oxazolidine, and thiazolidinediones. Examples of 6-membered heterocyclic alkyl groups include, but are not limited to, piperidine, morpholine, and piperazine. Other non-limiting examples of heterocyclic alkyl groups are:

[0216]

[0217] Examples of non-aromatic heterocycles include monocyclic groups such as azirropropane, ethylene oxide, cyclothioethane, azirrobutane, oxacyclobutane, thiohexacyclobutane, pyrrolidine, pyrrololine, pyrazolidine, imidazoline, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, tetrahydrothiophene, 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-dioxane-heptane, 4,7-dihydro-1,3-dioxane-heptene, and hexamethylene oxide.

[0218] As used herein, the term "aromatic" refers to a carbon ring or heterocycle having one or more polyunsaturated rings and exhibiting aromatic characteristics, i.e., having (4n+2) delocalized π electrons, where n is an integer.

[0219] As used herein, the term "aryl" alone or in combination with other terms refers to a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), wherein these rings may be attached together in a pendulous manner (e.g., biphenyl) or may be fused together (e.g., naphthalene), unless otherwise stated. Examples of aryl groups include phenyl, anthracene, and naphthyl.

[0220] "Arylalkyl" or "arylalkyl" refers to the formula -R b -R c The group, wherein R b It is an alkylene group as defined above, and R c It is one or more aryl groups as defined above, such as benzyl, diphenylmethyl, etc. Unless otherwise specified in the specification, the aryl group may optionally be substituted. For example, as used herein, the term "aryl-(C1-C3)alkyl" refers to a functional group in which one to three carbonylene chains are attached to an aryl group, such as -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 one to three carbonylene chains are attached to a heteroaryl group, such as -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.

[0221] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle with aromatic characteristics. Polycyclic heteroaryl groups may include one or more partially saturated rings. Examples include the following:

[0222]

[0223] Examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (especially 2- and 4-pyrimidinyl), pyridazinyl, thiophenyl, furanyl, pyrroleyl (especially 2-pyrroleyl), 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.

[0224] Examples of polycyclic heterocyclic and heteroaryl groups include indolyl (especially 3-, 4-, 5-, 6-, and 7-indolyl), indololinyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl (especially 1- and 5-isoquinolinyl), 1,2,3,4-tetrahydroisoquinolinyl, cinnolinyl, quinoxalinyl (especially 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthidyl, 1,4-benzodioxane, coumarin, dihydrocoumarin, 1,5-naphthidine, and benzofuran. (especially 3-, 4-, 5-, 6- and 7-benzofuranyl), 2,3-dihydrobenzofuranyl, 1,2-benzoisoxazolyl, benzothiophene (especially 3-, 4-, 5-, 6- and 7-benzothiophene), benzoxazolyl, benzothiazolyl (especially 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (especially 2-benzoimidazolyl), benzotriazolyl, thioxanthineyl, carbazolyl, carbaolinyl, acridineyl, pyrrolizidineyl and quinolizidineyl.

[0225] As used herein, the term "substituted" means that one atom or group of atoms replaces hydrogen as a substituent attached to another group. The term "substituted" further refers to any level of substitution, i.e., monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted, if such substitutions are permitted. The substituents are chosen independently, and substitution can occur at any chemically accessible position. In one embodiment, the number of substituents varies between one and four. In another embodiment, the number of substituents varies between one and three. In yet another embodiment, the number of substituents varies between one and two.

[0226] As used herein, the term "optionally substituted" means that the reference group may be substituted or unsubstituted. In one embodiment, the reference group is optionally substituted with zero substituents, i.e., the reference group is unsubstituted. In another embodiment, the reference group is optionally substituted with one or more additional groups, individually and independently selected from the groups described herein.

[0227] In one embodiment, the substituents are independently selected from the group consisting of: oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight-chain, branched 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)2alkyl, S(=O)2N [H, alkyl, or aryl], -C(=O)NH [substituted or unsubstituted alkyl, or substituted...]. -C(=O)N[H or substituted or unsubstituted alkyl or aryl]2, -OC(=O)N[substituted or unsubstituted alkyl]2, -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]2, and -C(NH2)[substituted or unsubstituted alkyl]2. In another embodiment, for example, the optional substituents are selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -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. In yet another embodiment, the substituents are independently selected from the group consisting of: C 1-6 Alkyl, -OH, C 1-6 Alkoxy, halogenated, amino, acetamido, oxo, and nitro groups. As used herein, the substituents are alkyl or alkoxy, and the carbon chain can be branched, linear, or cyclic.

[0228] In the chemical structures and portions disclosed and described herein, for integers and R, R 1 R 2 R 3 R 4 R 5 R 6 The references to integers and R, R are made multiple times. The specification mentions integers and R, R... 1 R 2 R 3 R 4 R 5 R 6 Any description of etc. applies to representing integers and R, R, respectively.1 R 2 R 3 R 4 R 5 R 6 Any structure or part thereof.

[0229] The term "nanoparticle" refers to a particle having at least one nanometer-scale size (e.g., 1-1,000 nm) comprising one or more amphiphilic Janus dendritic polymers of formula (I). In some embodiments, the nanoparticle is included in a formulation containing nucleoside-modified RNA as described herein. In some embodiments, such nanoparticles comprise ionizable hydrophilic groups and lipophilic (hydrophobic) groups. In one embodiment, the nanoparticle further comprises one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In one embodiment, the nanoparticle does not contain any additional excipients. In one embodiment, the nanoparticle does not contain 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 encapsulated by some or all of the lipids of the nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cells, such as adverse immune responses.

[0230] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this invention can exist in a variety of 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; 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).

[0231] The term "antibody fragment" refers to a portion of a complete antibody and specifically to the antigenic determination variable region of the complete 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.

[0232] As used in this article, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation.

[0233] As used in this article, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. κ and λ light chains refer to the two main isotypes of antibody light chains.

[0234] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as antibodies expressed by bacteriophages, for example. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding an antibody and the DNA molecule expressing an antibody protein or an amino acid sequence specifying an antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well-known in the art. The term should also be interpreted as referring to an antibody produced by synthesizing an RNA molecule encoding an antibody. The RNA molecule expresses an antibody protein or an amino acid sequence specifying an antibody, wherein the RNA is obtained by transcribing DNA (synthesis or cloning) or other techniques available and well-known in the art.

[0235] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an adaptive immune response. This immune response may involve antibody production, or activation of specific immunogenic competent cells, or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can be used 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 encoding a protein that elicits an adaptive immune response encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded by a "gene". It is apparent that antigens can be synthesized from or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0236] As used herein, the term "adjuvant" is defined as any molecule that enhances an antigen-specific adaptive immune response.

[0237] "Encoding" refers to the inherent properties of a specific nucleotide sequence (such as a gene, cDNA, or mRNA) within a polynucleotide sequence, used as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system, then the gene encodes a protein. The coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a template for gene or cDNA transcription) can be referred to as the protein or other product encoding that gene or cDNA.

[0238] A “vector” is a composition of material containing isolated nucleic acids and used to deliver those nucleic acids into cells. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0239] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as clomids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0240] As used herein, "homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. The molecules are homologous at that position when a position in two compared sequences is occupied by the same base or amino acid monomer subunit—for example, if each of two DNA molecules is occupied by adenine. 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 compared positions multiplied by 100. For example, if six out of ten positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology. Typically, comparisons are made when the two sequences are aligned to give the maximum homology.

[0241] An "immunogen" is any substance introduced into the body to produce an immune response. This substance can be a physical molecule (such as a protein), or it can be encoded by a vector (such as DNA or mRNA), or a virus.

[0242] "Separated" means altered or removed from its native state. For example, nucleic acids or peptides naturally present in living organisms are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their native coexisting substances are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-native environments (such as, for example, host cells).

[0243] In the context of this invention, the following abbreviations for common nucleosides (nucleobases bound to ribose or deoxyribose via N-glycosidic bonds) are used. “A” refers to adenosine, “C” to cytidine, “G” to guanosine, “T” to thymidine, and “U” to uridine.

[0244] As used herein, the term "modulation" refers to a detectable increase or decrease in a subject's response level compared to the response level of a subject without treatment or the compound, and / or compared to the response level of other subjects who are otherwise identical but untreated. This term includes interfering with and / or influencing native signals or responses, thereby mediating a beneficial therapeutic response in the subject (preferably human).

[0245] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns. Furthermore, nucleotide sequences may contain modified nucleosides capable of being translated through cellular translation mechanisms. For example, mRNA in which all uridines are replaced by pseudouridine, 1-methylpseudouridine, or another modified nucleoside.

[0246] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, the first and second nucleic acid sequences are operably linked when they are functionally related. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Typically, operably linked DNA or RNA sequences are contiguous, and two protein-coding regions are linked within the same reading frame when necessary.

[0247] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art will generally recognize that nucleic acids are polynucleotides, and that polynucleotides can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant methods (i.e., using common cloning techniques and PCR). TM (e.g., cloning nucleic acid sequences from recombinant libraries or cell genomes) and through synthetic methods.

[0248] In some cases, the polynucleotides or nucleic acids of the present invention are “nucleoside-modified nucleic acids,” which refer to nucleic acids containing at least one modified nucleoside. “Modified nucleoside” refers to a modified nucleoside. For example, more than one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0249] In some embodiments, "pseudouridine" refers to m 1 acp 3 Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In another embodiment, the term refers to m 1 Ψ(1-methylpseudouridine). 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, this term refers to m 3 Ψ(3-methylpseudouridine). In another embodiment, the term refers to the unmodified pseudouridine moiety. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above-described pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the invention.

[0250] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Phrasal nucleotide sequences encoding proteins or RNA may also include introns, to the extent that nucleotide sequences encoding proteins may contain introns in some versions.

[0251] As used herein, the terms “amino acid,” “amino acid monomer,” or “amino acid residue” refer to any one of the twenty naturally occurring amino acids, including synthetic amino acids with non-natural side chains and those including D and L optical isomers.

[0252] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to short chains and long chains; short chains are generally also referred to in the art, for example, as peptides, oligopeptides, and oligomers, while long chains are generally referred to in the art, for example, as proteins (there are many types of proteins). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0253] As used herein, the term "promoter" is defined as a DNA sequence recognized by a cellular or introduced synthetic mechanism required to initiate specific transcription of a polynucleotide sequence. For example, promoters recognized by bacteriophage RNA polymerases are used to produce mRNA via in vitro transcription.

[0254] As used herein, the term "specific binding" for antibodies refers to antibodies that recognize a specific antigen but substantially do not 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 antigens from one or more other species. However, this cross-species reactivity itself does not change the antibody's specific classification. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity itself does not change the antibody's specific classification. In some cases, the terms "specific binding" or "specifically binding" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance in a way that depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure rather than the protein it normally binds to. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0255] As used herein, the terms “transfection” or “transformation” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected” or “transformed” or “transduction” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0256] As used in this article, the phrase “under transcriptional control” or “operably linked” refers to the promoter being in the correct position and orientation relative to the polynucleotide to control the initiation of RNA polymerase transcription and the expression of the polynucleotide.

[0257] As used herein, the term "therapeutic" refers to treatment and / or prevention. A therapeutic effect is achieved by suppressing, reducing, alleviating, or eradicating at least one sign or symptom of a disease or condition.

[0258] As used herein, the terms “therapeutic compound,” “therapeutic agent,” “drug,” “active drug,” and “active drug ingredient” are used interchangeably to refer to a chemical entity that exhibits certain pharmacological effects in the body and is administered for this purpose. Non-limiting examples of therapeutic agents include, but are not limited to, hydrophilic therapeutic agents, hydrophobic therapeutic agents, antibiotics, antibodies, small molecules, anticancer agents, chemotherapeutic agents, immunomodulators, RNA molecules, siRNA molecules, DNA molecules, gene editing agents, gene silencing agents, CRISPR-related agents (e.g., guide RNA molecules, endonucleases, and their variants), analgesics, vaccines, anticonvulsants, antidiabetic agents, antifungal agents, antitumor agents, anti-Parkinson's agents, antirheumatic agents, appetite suppressants, biological response modifiers, cardiovascular agents, central nervous system stimulants, contraceptives, dietary supplements, vitamins, minerals, lipids, carbohydrates, metals, amino acids (and precursors), nucleic acids and precursors, contrast agents, diagnostic agents, dopamine receptor agonists, erectile dysfunction agents, and fertility agents. Agents, gastrointestinal agents, hormones, immunomodulators, anti-hypercalcemic agents, mast cell stabilizers, muscle relaxants, nutritional agents, ophthalmic agents, osteoporosis agents, psychotherapeutic agents, parasympathomimetic blockers, parasympathetic blockers, respiratory agents, sedative-hypnotic agents, skin and mucous membrane agents, smoking cessation agents, steroids, sympathomimetic blockers, urinary tract agents, uterine relaxants, vaginal agents, vasodilators, antihypertensive agents, hyperthyroidism agents, anti-hyperthyroidism agents, antiasthmatic agents, and vertigo agents. In some embodiments, one or more therapeutic agents are water-soluble, poorly water-soluble drugs, or drugs with low, medium, or high melting points. Therapeutic agents with or without one or more stable salts may be provided.

[0259] Examples of active ingredients suitable for pharmaceutical formulations and methods of the present invention include those that are hydrophilic, lipophilic, amphiphilic, or hydrophobic, and that can be dissolved, dispersed, or partially dissolved and dispersed on or near nanoclusters. The active agent-nanocluster combination can be further coated to encapsulate the pharmaceutical agent-nanocluster combination, and can be directed to a target by functionalizing the nanoclusters with, for example, aptamers and / or antibodies. Alternatively, the active ingredient can also be provided separately from the solid pharmaceutical composition, for example, for co-administration. Such active ingredients can be any compound or mixture of compounds that has therapeutic or other value when administered to animals (particularly mammals), such as pharmaceuticals, nutrients, cosmeceuticals, nutritional supplements, diagnostic agents, nutritional agents, etc. The active agents described herein can exist in their natural state; however, they are typically provided in the form of salts. The active agents described herein include their isomers, analogues, and derivatives.

[0260] As used in this article, "effective amount" refers to the amount that provides therapeutic or preventative benefits.

[0261] The term "therapeuticly effective amount" refers to the amount of a subject compound that will elicit a biological or medical response in the tissue, system, or subject being sought by an investigator, veterinarian, physician, or other clinician. The term "therapeuticly effective amount" includes, when administered, an amount sufficient to prevent the development of or to partially alleviate one or more signs or symptoms of the condition or disease being treated. Therapeuticly effective amounts will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0262] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, suitable for the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0263] "Disease" is a state of health in animals in which the animal is unable to maintain homeostasis, and in which the animal's health condition will continue to deteriorate if the disease is not improved.

[0264] In contrast, an animal's "illness" refers to a healthy state in which the animal can maintain homeostasis, but the animal's health is not as favorable as when it is free of illness. Without treatment, an illness does not necessarily lead to a further decline in the animal's health.

[0265] As used in this article, “treatment” refers to reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by the subject.

[0266] As used in this article, “treating a disease or condition” means reducing the frequency of a patient experiencing symptoms of a disease or condition. Disease and condition are used interchangeably in this article.

[0267] The disease or condition is "relieved" if the severity of the signs or symptoms of the disease or condition, the frequency with which the patient experiences such signs or symptoms, or both, decreases.

[0268] As used herein, the term "modulation" refers to a detectable increase or decrease in a subject's response level compared to the response level of a subject without treatment or the compound, and / or compared to the response level of other subjects who are otherwise identical but untreated. This term includes interfering with and / or influencing native signals or responses, thereby mediating a beneficial therapeutic response in the subject (preferably human).

[0269] "Parenteral" administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0270] Scope: Throughout this disclosure, various aspects of the invention can be presented in a scope format. It should be understood that the scope format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Therefore, it should be assumed that the description of the scope specifically discloses all possible sub-scopes and individual numerical values ​​within that scope. For example, a description of a scope such as 1 to 6 should be considered to have specifically disclosed sub-scopes, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0271] describe

[0272] This invention is partly based on the unexpected result that nanoparticles comprising at least one ionizable amphiphilic Janus dendritic polymer having the structure of formula (I) effectively and efficiently deliver reagents to targets of interest. Therefore, in one aspect, the invention relates to ionizable amphiphilic Janus dendritic polymers having the structure of formula (I). In another aspect, the invention relates to nanoparticles comprising at least one of the ionizable amphiphilic Janus dendritic polymers of the present invention. In some embodiments, the nanoparticles further comprise at least one reagent. In some embodiments, the nanoparticles further comprise at least one reagent encapsulated by the ionizable amphiphilic Janus dendritic polymer of the present invention. In another aspect, the invention relates to compositions comprising at least one of the ionizable amphiphilic Janus dendritic polymers of the present invention or nanoparticles thereof. In some embodiments, the composition is a vaccine.

[0273] In one aspect, the present invention relates to a method for delivering a reagent to a target of interest using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention. In another aspect, the present invention relates to a method for preventing or treating a disease or condition in a subject using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention. In yet another aspect, the present invention relates to a method for inducing an adaptive immune response in a subject using at least one ionizable amphiphilic Janus dendritic polymer or nanoparticles or compositions thereof of the present invention.

[0274] The compounds of the present invention

[0275] In one aspect, the present invention relates to amphiphilic Janus dendritic polymers of formula (I):

[0276]

[0277] Or its racemic mixture, enantiomer, diastereomer, pharmaceutically acceptable salt, or derivative. In various embodiments, the amphiphilic Janus dendritic polymer is an ionizable amphiphilic Janus dendritic polymer.

[0278] In some embodiments, A is a multivalent group that includes at least one selected from the following structures:

[0279] Or any combination thereof. In some embodiments, A is a multivalent group comprising a group selected from... At least one of, or any combination thereof. In some embodiments, A may optionally be replaced.

[0280] In some implementations, dashed lines represent binding sites for one of X, Y, or Z.

[0281] In some embodiments, X is a hydrophilic group comprising at least one amine. In some embodiments, the hydrophilic group may optionally be substituted. In some embodiments, the amine may optionally be substituted.

[0282] In some implementations, Y comprises at least one C1-C 50 The lipophilic group of the alkyl chain. In some embodiments, Y is a group containing at least one C1-C. 30 The lipophilic group of the alkyl chain. In some embodiments, the lipophilic group may optionally be substituted. In some embodiments, C1-C 50 The alkyl chain may be optionally substituted.

[0283] In some embodiments, Z is a group comprising at least one chain selected from ethylene glycol, diethylene glycol, triethylene glycol, or polyethylene glycol. In some embodiments, Z is optionally substituted. In some embodiments, the ethylene glycol, diethylene glycol, triethylene glycol, and / or polyethylene glycol chains are optionally substituted.

[0284] In some implementations, R A Selected from hydrogen, halides, hydroxyl groups, C1-C 50 Alkyl, C1-C 50 Alkyl halides, C1-C 50 Alkoxy, C1-C 50 Alkyl halides, or any combination thereof. In some embodiments, R A Selected from hydrogen, halides, hydroxyl groups, C1-C 30 Alkyl, C1-C 30 Alkyl halides, C1-C 30 Alkoxy, C1-C 30 Alkyl halides, or any combination thereof. In some embodiments, hydroxyl groups, C1-C...50 Alkyl, C1-C 50 Alkyl halides, C1-C 50 Alkoxy and / or C1-C 50 Alkoxy halides may be substituted.

[0285] In some implementations, R B Selected from hydrogen, halides, hydroxyl groups, C1-C 50 Alkyl, C1-C 50 Alkyl halides, C1-C 50 Alkoxy, C1-C 50 Alkyl halides, or any combination thereof. In some embodiments, R B Selected from hydrogen, halides, hydroxyl groups, C1-C 30 Alkyl, C1-C 30 Alkyl halides, C1-C 30 Alkoxy, C1-C 30 Alkyl halides, or any combination thereof. In some embodiments, hydroxyl groups, C1-C... 50 Alkyl, C1-C 50 Alkyl halides, C1-C 50 Alkoxy and / or C1-C 50 Alkoxy halides may be substituted.

[0286] In some implementations, s is an integer from 0 to 5. In some implementations, s is an integer from 1 to 5. For example, in one implementation, s is an integer 5. In one implementation, s is an integer 4. In one implementation, s is an integer 3. In one implementation, s is an integer 2. In one implementation, s is an integer 1. In one implementation, s is an integer 0.

[0287] In some implementations, t is an integer from 0 to 5. In some implementations, t is an integer from 1 to 5. For example, in one implementation, t is an integer 5. In one implementation, t is an integer 4. In one implementation, t is an integer 3. In one implementation, t is an integer 2. In one implementation, t is an integer 1. In one implementation, t is an integer 0.

[0288] In some implementations, u is an integer from 0 to 4. In some implementations, u is an integer from 1 to 4. For example, in one implementation, u is an integer 4. In one implementation, u is an integer 3. In one implementation, u is an integer 2. In one implementation, u is an integer 1. In one implementation, u is an integer 0.

[0289] In some embodiments, the sum of s, t, and u equals 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.

[0290] For example, in some implementations, A is... This indicates that s and t are each 2.

[0291] In some implementations, A is... This indicates that t is 2, s is 1, and u is 0 or 1.

[0292] In some implementations, A is... This indicates that s and t are each 1.

[0293] In one embodiment, u is 0. Therefore, in one embodiment, the amphiphilic Janus dendritic polymer having the structure of formula (I) is an amphiphilic Janus dendritic polymer having the structure of formula (II):

[0294] (X) s -A-(Y) t

[0295] Equation (II),

[0296] Or its racemic mixture, enantiomer, diastereomer, pharmaceutically acceptable salt, or derivative.

[0297] 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.

[0298] In one embodiment, X comprises at least one amine that is protonated under biological conditions. In one embodiment, X comprises at least one amine having a formal charge of +1 under biological conditions. In one embodiment, X comprises at least one carbohydrate.

[0299] In one implementation, each occurrence of X is independently selected from the following structures:

[0300] Or any combination thereof.

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

[0302] In some implementations, each occurrence of m, n, o is an independent integer from 1 to 5.

[0303] In some implementations, each occurrence of W is independently selected from C=O, C(R) W (R) W ), NR W , O, or S. In some implementations, each occurrence of R... W The component is independently selected from hydrogen, halides, hydroxyl groups, alkyl groups, alkyl halides, aryl groups, aryl halides, alkoxy groups, alkoxy halides, or any combination thereof. In one embodiment, W is NH each time it appears. In one embodiment, W is O each time it appears. In one embodiment, W is NH at least once, and W is O at least once.

[0304] In some implementations, each occurrence of L 1 L 2 L 3 and L 4 Independently a covalent or divalent linking group, selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenyl, ynylene, aryl, heteroaryl, silyl, amine, amide, ester, ether, carbonyl, urethane, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate ester, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, L appears at least once. 1 L 2 L 3 and L 4 It is a poly(alkyl ether) or an oligo(alkyl ether). In one embodiment, each occurrence of L... 1 L 2 L 3 and L 4 It is polyethylene glycol (PEG) or oligomeric ethylene oxide. In one embodiment, each occurrence of L... 1 L 2 L 3 and L 4 It independently possesses the structure - [CH2CH2O] n -; where n is an integer from 0 to 10.

[0305] In some implementations, each occurrence of R 11 R 12 R 13 and R 14 Independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R 11 R 12 R 13 、or R 14 At least one of them contains an amine. In one embodiment, R 11 R 12 and R 13 One of them contains an amine. In one embodiment, R 11 R 12 and R 13 Two of them contain amines. In one embodiment, R 11 R 12 and R 13 The three components contain amines. In one embodiment, R 11 R 12 and R 13 At least one of them contains two amines. In one embodiment, R 11 R 12 and R 13 One of the amines is protonated under biological conditions. In one embodiment, R does not contain an amine. 11 R 12 and R 13 Any one of them contains alkyl, aryl, or combinations thereof.

[0306] In one implementation, R appears at least once. 11 R 12 R 13 、or R 14 It has the structure -C(O)(CH2) m -N(R 1 (R) 2 ).

[0307] In some implementations, m is an integer from 1 to 10 each time it appears. In one implementation, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0308] In some implementations, each occurrence of R 1 and R 2 Independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R 1 and R 2Together they can form a ring.

[0309] In one implementation, R 1 and R 2 Each is an alkyl group. In one embodiment, R 1 and R 2 Each is a methyl group. In one embodiment, R 1 and R 2 Together they form a 6-membered heterocyclic ring. In one implementation, R 1 and R 2 Together with the N atoms they bind to, they form a piperidine ring. In one embodiment, R... 1 and R 2 Together with the N atoms they bind to, they form an N-alkylpiperazine ring. In one embodiment, R... 1 and R 2 Each is hydrogen.

[0310] In one implementation, R appears at least once. 11 R 12 R 13 、or R 14 It has the structure -C(O)(CR) 5 R 6 ) m -N(R 1 (R) 2 ).

[0311] In some implementations, m is an integer from 1 to 10 each time it appears. In one implementation, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0312] In some implementations, each occurrence of R 1 and R 2 It is independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof.

[0313] In some implementations, each occurrence of R 5 and R 6 Independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R 1 R 2 R 5 and R 6 Any two or more of them can form a ring together.

[0314] In one embodiment, it has the structure -C(O)(CR) 5 R 6 ) m-N(R 1 (R) 2 The group ) is derived from an amino acid. In one embodiment, the amino acid is a typical amino acid. In one embodiment, the amino acid is an atypical 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 A geminal pair is H). In one implementation, R 5 and R 6 One of them is H, and R 5 and R 6 The other one is not H. In one embodiment, the group may be chiral. In one embodiment, the group may be enantiomer-enriched or enantiomer-pure. In one embodiment, the group may be racemic.

[0315] In one implementation, each occurrence of R 11 R 12 R 13 and R 14 Independently selected from the following structures:

[0316]

[0317] In some implementations, m is an integer from 1 to 10 each time it appears. In one implementation, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0318] In one implementation, each occurrence of X is independently selected from the following structures:

[0319]

[0320] Or any combination thereof.

[0321] In some implementations, n is an integer from 1 to 20 each time it appears. In some implementations, n is an integer from 1 to 10 each time it appears.

[0322] In some implementations, each occurrence of m is an integer from 1 to 20. In some implementations, each occurrence of m is an integer from 1 to 10.

[0323] In some implementations, each occurrence of R 1 and R 2 Independently selected from hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, or any combination thereof. In some embodiments, R 1 and R 2 They can form a ring together.

[0324] In some implementations, each occurrence of R 3 Independently selected from hydrogen and (CH2) n (CH2) n -OH, or any combination thereof.

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

[0326] For example, in some implementations, each occurrence of X is independently selected from... Or any combination thereof.

[0327] In one implementation, Y is a subset of at least one C1-C 50 The lipophilic (hydrophobic) group of the alkyl chain. In one embodiment, Y is a group containing at least one C1-C group. 30 The lipophilic (hydrophobic) group of the alkyl chain. In one embodiment, Y is a group containing at least one C4-C group. 30 The lipophilic (hydrophobic) group of the alkyl chain. For example, in one embodiment, Y is a group containing at least two C1-C groups. 30 The lipophilic (hydrophobic) group of the alkyl chain. In one embodiment, Y is a C1-C group comprising at least two C1-C groups with different numbers of carbon atoms. 30 The lipophilic (hydrophobic) group of the alkyl chain.

[0328] 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, alkenyl, alkyneylene, aryl, heteroaryl, silylene, amine, amide, ester, ether, carbonyl, urethane, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, Y comprises alkylene, arylene, alkenylene, alkyneylene, disulfide, ether, or any combination thereof. In one embodiment, Y does not contain an amine. In one embodiment, Y is a lipophilic group, comprising 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 21Alkyl 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 Alkyl chain. In one embodiment, Y comprises at least one carbohydrate.

[0329] In one implementation, each occurrence of Y is independently selected from the following structures:

[0330] Or any combination thereof.

[0331] In some implementations, the dashed line indicates a connection to A.

[0332] In some implementations, each occurrence of W is independently selected from C=O, C(R) W (R) W ), NR W , O, or S. In some implementations, each occurrence of R... W The element is independently selected from hydrogen, halides, hydroxyl groups, alkyl groups, alkyl halides, aryl groups, aryl halides, alkoxy groups, alkoxy halides, 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, W represents O at least once, and W represents NH at least once.

[0333] In some implementations, each occurrence of R 21 R 22 R 23 and R 24 Independently contains C1-C 50 Alkyl group. In some embodiments, each occurrence of R... 21 R 22 R 23 and R 24 Independently contains either straight or branched C1-C chains 50 Alkyl group. In some embodiments, each occurrence of R... 21 R 22 R 23 and R 24 Independently contains C1-C 30 Alkyl group. In some embodiments, each occurrence of R... 21 R 22 R 23 and R 24 Independently contains either straight or branched C1-C chains 30Alkyl group. In one embodiment, each occurrence of R... 21 R 22 R 23 and R 24 Independently represent C8-C 12 alkyl.

[0334] In one implementation, each occurrence of Y is independently selected from the following structures:

[0335]

[0336] In some implementations, each occurrence of W is independently selected from C=O, C(R) W (R) W ), NR W , O, or S. In some implementations, each occurrence of R... W The element is independently selected from hydrogen, halides, hydroxyl groups, alkyl groups, alkyl halides, aryl groups, aryl halides, alkoxy groups, alkoxy halides, 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, W represents O at least once, and W represents NH at least once.

[0337] In some implementations, n is an integer from 1 to 30. In some implementations, n is an integer from 6 to 18.

[0338] In some implementations, each occurrence of Y is independently selected from...

[0339] Or any combination thereof.

[0340] In some implementations, each occurrence of Y is independently selected from...

[0341] , or any combination thereof.

[0342] In some implementations, the dashed line indicates a connection to A.

[0343] In some implementations, each occurrence of n is an integer from 1 to 20.

[0344] In some implementations, each occurrence of -(CH2) n CH3, -O(CH2) n -、and -O(CH2) n CH3 can be either straight-chain or branched.

[0345] In some embodiments, t is an integer from 2 to 5. Therefore, in some embodiments, the amphiphilic Janus dendritic polymer 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 alkyl chain comprised in the 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 of carbon atoms in the first Y to carbon atoms in the second Y is greater than or equal to 3 and less than 7.

[0346] In one implementation, u is 1 or 2. In one implementation, u is 1. In one implementation, u is 2.

[0347] In one implementation, Z does not contain an amine.

[0348] In one implementation, Z is not a lipophilic group.

[0349] In one embodiment, Z comprises at least one carbohydrate.

[0350] In one implementation, each occurrence of Z is independently selected from the following structures:

[0351] Or any combination thereof.

[0352] In some implementations, the dashed line indicates a connection to A.

[0353] In some implementations, each occurrence of L 1 L 2 L 3 and L 4 Independently a covalent or divalent linking group, selected from alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenyl, ynylene, aryl, heteroaryl, silyl, amine, amide, ester, ether, carbonyl, urethane, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate ester, poly(alkyl ether), heteroatom, or any combination thereof. In one embodiment, L appears at least once in group Z. 1 L 2 L 3 and L 4 Indicates a single bond. In one implementation, L 1 L 2 L 3 and L 4 At least one of the expressions in -WC(O)(CH2) pThe group is 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 them includes polyethylene glycol.

[0354] In some implementations, each occurrence of R 31 R 32 R 33 and R 34 It is independently selected from hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, or any combination thereof.

[0355] In some implementations, each occurrence of m, n, o is an independent integer from 1 to 5.

[0356] In one implementation, each occurrence of Z is independently selected from the following structures:

[0357]

[0358] In some implementations, each occurrence of n is an integer from 1 to 100.

[0359] In some implementations, each occurrence of p is an integer from 1 to 10.

[0360] In some embodiments, each occurrence of R4 is independently selected from hydrogen, deuterium, alkyl, aryl, or any combination thereof.

[0361] In some embodiments, the amphiphilic Janus dendritic polymer is an amphiphilic Janus dendritic polymer having a structure selected from the following: Figure 12 At least one structure in Figure 13 At least one of the structures, Figure 14 At least one of the structures, Figure 47 At least one of the structures, Figure 60 At least one structure in Figure 80 At least one of the structures, or any combination thereof.

[0362] In some embodiments, the amphiphilic Janus dendritic polymer is an amphiphilic Janus dendritic polymer having a structure selected from the following:

[0363]

[0364]

[0365]

[0366] In some embodiments, the amphiphilic Janus dendritic polymer having the structure of formula (II) contains an amine (ionizable amine) in X, which is sufficient to provide dual hydrophilicity and binding action in IAJD. For example, Figure 47 This paper summarizes the IAJDs synthesized and studied in vitro and in vivo, along with their schematic diagrams and pK. a Value. pK a The values ​​were determined as described in the examples. The ethanol solution of IAJD was injected into an acetate buffer solution at pH 4 to obtain the values ​​from... Figure 47 All IAJDs were co-assembled with Luc-mRNA. Acetate buffer containing mRNA was added to a buffer solution from neutral water. More details are available in the Examples section below. Co-assembly formed nanoparticles (DNPs). Co-assembly is schematically shown... Figure 1 middle. Figure 48 The in vivo transfection results of DNPs co-assembled by IAJD are shown, such as... Figure 1 As shown. The IAJD value is displayed in the upper left corner, along with IAJD pK. a The values, dimensions (in nm), and polydispersity (PDI) of the resulting DNPs are shown above each mouse image. The proportions of the luminescence values ​​are also shown. Representative images of mRNA delivery to different organs are shown at the bottom of the figure. Figure 49 The activity comparison of DNP assembled from IAJD in vitro (blue) and in vivo (red) is shown, schematically illustrated at the top of the figure. Therefore, in some embodiments, in the dendritic polymer having the structure of formula (II), the valence of A can be 2, and the sum of s and t can be 2.

[0367] In some embodiments, the amphiphilic Janus dendritic polymer includes isochiral, racemic, or achiral imprinting sites. Therefore, in some embodiments, the ionizable amphiphilic Janus dendritic polymer is an isochiral ionizable amphiphilic Janus dendritic polymer, a racemic ionizable amphiphilic Janus dendritic polymer, or an achiral ionizable amphiphilic Janus dendritic polymer.

[0368] In some embodiments, the amphiphilic Janus dendritic polymer is a symmetrical amphiphilic Janus dendritic polymer.

[0369] In some embodiments, the amphiphilic Janus dendritic polymer is an asymmetric amphiphilic Janus dendritic polymer.

[0370] In some embodiments, the amphiphilic Janus dendritic polymer is an asymmetric amphiphilic Janus dendritic polymer.

[0371] For example, in some embodiments, the successful design of the hydrophobic regions of the amphiphilic Janus dendritic polymer is partly based on the use of different alkyl lengths and the unexpectedly important role discovered in the primary structure of the IAJD hydrophobic moiety, which enhances the activity of targeted delivery of the desired cargo to the target site by up to 90.2-fold. In some embodiments, the asymmetric one-component amphiphilic Janus dendritic polymer does not require the microfluidic or T-tube techniques employed by the four-component LNP for co-assembly with mRNA. In some embodiments, the one-component system can be co-assembled with mRNA into DNPs with approximately 97% nucleic acid encapsulation efficiency by simply injecting its ethanol solution into an acidic buffer containing mRNA, instead of using the microfluidic or T-tube techniques required for LNPs.

[0372] For example, Figure 2 The structure of LNPs and their co-assembly with mRNA are summarized. Figure 1 The structures of sSS, SS, and TM IAJDs with different and similar alkyl groups in their hydrophobic regions and their co-assembly with mRNA are shown. IAJDs can be described as mono-mono (SS, a mono-hydrophilic dendrite attached to a mono-lipophilic dendrite), bis-bis (TT, two hydrophilic dendrites attached to two lipophilic dendrites), and bis-mixed (TM, two different hydrophilic dendrites attached to two lipophilic dendrites) IAJDs.

[0373] For example, Figure 59The synthesis of asymmetric IAJD is shown. In the first step, 3,5-dihydroxymethylbenzoic acid 3-benzyl ether was produced in 39% isolated yield by etherification of 1 with BnCl in DMF at 80 °C over 5 hours. Subsequently, 2 was alkylated in DMF with 1-bromoundecane or 1-bromopentadecane at 120 °C with a K2CO3 base to produce 3 in 80-100% isolated yield. Hydrogenolysis of 3 (H2 / Pd, DCM / MeOH, 12 hours) produced 4 in 100% isolated yield. Alkylation of 4 with different alkyl lengths from 1-bromooctane to 1-bromooctadecane with a K2CO3 base at 120 °C in DMF produced asymmetric compound 5 in 74-92% isolated yield. Compound 5 was reduced in THF with LiAlH4 (0-23 °C, 1 hour) to produce benzyl alcohol 6 in 93-100% isolated yield. Compound 6 was reacted with 4-bromobutyric acid (by generating its acyl chloride with SOCl2 in CH2Cl2 at 23 °C via DMF catalysis, followed by esterification in the presence of NEt3 / DMAP (0–23 °C, 2 h); or by direct esterification with DCC / DPTS over 12 h) to produce compound 7 in separation yields of 76%–98%. Compound 7 was reacted with methylpiperazine or hydroxyethylpiperazine (K2CO3, MeCN, 95 °C, 3 h) to produce IAJD 8 (70–90% separation yield) and 9 (76–98% separation yield). Their purity was determined by a combination of HPLC, MALDI-TOF, and NMR, and the purity was greater than 99%. Their structures are as follows: Figure 60 As shown (IAJD 113 to 178). IAJD133 has a similar structure to IAJD105, except that the interconnecting ester groups in 105 are replaced by amides in 133. The benzylamine precursor of 133 is produced by reacting the corresponding benzyl alcohol with its benzyl chloride obtained by SOCl2, followed by the reaction with K-phthalimide and subsequently hydrazine, as reported.

[0374] Previously reported, synthesized mono-single (SS)IAJDs with asymmetric alkyl groups in their lipophilic moiety exhibited very high lung delivery activity. They are Figure 60 IAJD 110 to 159. Their sequence-defined hydrophilic dendritic units were synthesized, as reported in Zhang et al., J. Am. Chem. Soc. 2021, 143, 12315-12327. The hydrophilic dendritic units reacted with selected asymmetric lipophilic dendritic units 6 or their amines. For convenience, IAJD refers to their number, followed by the ratio between their two alkyl groups forming their asymmetric lipophilic moieties. This nomenclature and... Figure 60The overall and schematic structures shown help to discuss the relationship between their in vitro and in vivo activities and molecular structure. For example, 116 (11 / 13) and 117 (11 / 13) both contain combinations of 11 and 13 carbons in their lipophilic moieties, but 116 contains methylpiperazine while 117 contains hydroxyethylpiperazine ionizable amines. The large red dot at the top of the animation for 117 indicates the hydroxyethyl group, while the thin blue line on 116 represents the methyl group; both are attached to the piperazine (…). Figure 60 The combination of 33 IAJD sSS and 7 IAJD SS is as follows: Figure 60 As shown. Figure 60 The IAJDs 81, 86, 105, 106, and 107, marked in blue in the upper left corner, have been previously reported (Zhang et al., J. Am. Chem. Soc. 2021, 143, 17975-17982). Transfection experiments using Luc-mRNA were performed in vitro and in vivo using 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.

[0375] Based on its target selectivity, the overall transfection activity in vivo was analyzed, and the tissue was... Figure 62 The first important result of the transfection experiment was that 11 IAJDs showed approximately 10 8 Active, 2 for the lungs, 3 for the liver, 6 for the spleen and lymph nodes ( Figure 62 (marked in pink) Figure 74 Previously reported, symmetrical IAJDs 110 (12 / 12) and 111 (11 / 11), containing amide interconnect groups, exhibited the highest lung activity, as did IAJDs 33 (12 / 12) and 34 (11 / 11). The novel IAJDs 110 (12 / 12) and 111 (11 / 11), containing interconnect esters instead of amide groups, also showed very high lung activity. Asymmetric IAJDs are stable in serum and PBS buffer and exhibit very high lung activity through a mechanism different from aggregation (as shown in the table below). Figures 76 to 78 The highest activity of all IAJDs was 178 (13 / 18), which showed 4.05 x 10⁻⁶. 8 The total flux emission per p / s is 90.2 times higher than that of symmetric 99 (18 / 18) with the same headgroup, and only 4.2 times lower than that of MC3. Figure 61Equally important is the transition from 158 (11 / 17) to 159 (11 / 17), where the first is an ester and the second is an IAJD containing an amide interconnecting group, resulting in an approximately 6-fold increase in activity. This demonstrates the importance of the amide interconnecting group for lung delivery, but also reveals that the presence of oligooxyethylene in the hydrophilic moiety may be crucial for targeted lung delivery. These experiments demonstrate the important role of the different alkyl groups in the hydrophobic moiety of IAJD.

[0376] at last, Figure 61 The activities of all IAJDs fabricated in the examples were summarized and compared with their symmetrical (light blue marked) and asymmetrical (blue marked) IAJDs, which could be used as control experiments. Figure 61 The results showed that by changing the primary structure of their lipophilic moieties from symmetric to asymmetric, the activity of IAJDs increased by up to 90.2-fold. Preferably, the ratio between the lengths of the two alkyl groups from the odd-even combinations could be equal to or greater than 3 and less than 7, and appeared to result in the maximum increase in activity. Selected examples of IAJDs supporting this conclusion are provided by IAJDs 119 (11 / 15), 125 (11 / 14), 127 (11 / 17), 128 (11 / 18), 130 (11 / EH), 153 (15 / 18), 155 (11 / EH), and 159 (11 / 17).

[0377] In some embodiments, the asymmetric amphiphilic Janus dendritic polymer is stable at about 5°C.

[0378] Nanoparticles

[0379] In one aspect, the present invention relates to nanoparticles comprising at least one of the amphiphilic Janus dendritic polymers of the present invention.

[0380] In various implementations, the nanoparticles are single-component nanoparticles.

[0381] In various implementations, the nanoparticles are four-component nanoparticles.

[0382] In some embodiments, the nanoparticles comprise a chiral, ionizable, amphiphilic Janus dendritic polymer, a chiral, ionizable, amphiphilic Janus dendritic polymer, or any combination thereof.

[0383] In some implementations, the nanoparticles are monolayer nanoparticles.

[0384] In some implementations, the nanoparticles are multilayer nanoparticles.

[0385] In some implementations, the nanoparticles are onion-shaped multilayer nanoparticles.

[0386] In some embodiments, the nanoparticles are racemic, ionizable, amphiphilic Janus dendritic polymers.

[0387] In some embodiments, the nanoparticles are dendritic polymer nanoparticles (DNP).

[0388] In some embodiments, the nanoparticles comprise at least two amphiphilic Janus dendritic polymers. Therefore, in some embodiments, the nanoparticles comprise a first ionizable amphiphilic Janus dendritic polymer and a second ionizable amphiphilic Janus dendritic polymer. In some embodiments, the first ionizable amphiphilic Janus dendritic polymer has a different structure than the second ionizable amphiphilic Janus dendritic polymer.

[0389] In some embodiments, the nanoparticles further comprise at least one amphiphilic Janus dendritic polymer, disclosed in 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. 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 herein by reference in its entirety.

[0390] In various embodiments, the nanoparticles have a size of about 10 nm to about 100,000 nm, about 30 nm to about 1,000 nm, about 30 nm to about 500 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 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm. The average diameter can be 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.

[0391] In all implementations, the nanoparticles are essentially non-toxic.

[0392] In various implementation methods, the nanoparticles are biodegradable.

[0393] In one aspect of the invention, the nanoparticles comprise at least one cargo. In various aspects, the invention is not limited to any particular cargo or other reagent that the nanoparticles can carry or transport. Rather, the invention includes any reagent that can be carried by the nanoparticles. For example, reagents that can be carried by the nanoparticles of the invention include, but are not limited to, diagnostic reagents, detectable reagents, and therapeutic agents. Thus, in various embodiments, the nanoparticles comprise at least one reagent. In other embodiments, the nanoparticles encapsulate at least one reagent.

[0394] In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 1:1 to about 10,000:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 2:1 to about 1,000:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 3:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 4:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 5:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 6:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 7:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 8:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 9:1 to about 10:1. In some embodiments, the nanoparticles contain or encapsulate at least one reagent. In some embodiments, the weight ratio of amphiphilic Janus dendritic polymer to at least one reagent is from about 9.5:1 to about 10:1.

[0395] In various embodiments, nanoparticles are suitable for delivering at least one cargo to cells of interest.

[0396] For example, in some embodiments, the cargo is at least one reagent, including diagnostic reagents, detectable reagents, therapeutic reagents, nucleic acid molecules, gene editing agents, vaccines, compositions for protein replacement therapy, or any combination thereof. In some embodiments, at least one reagent is selected from mRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, small molecules, proteins, antibodies, peptides, proteins, or any combination thereof. In some embodiments, at least one reagent includes a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes at least one selected from antigens, antibodies, gene editing molecules, chimeric antigen receptors (CARs), 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 cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecules, targeting nucleic acids, or any combination thereof. In some embodiments, the modified RNA is nucleoside-modified RNA. In some embodiments, the nucleoside-modified RNA comprises pseudouridine. In some embodiments, the nucleoside-modified RNA comprises pseudouridine plus 5-methylcytosine. In some embodiments, the nucleoside-modified RNA comprises 5-methyluridine. In some embodiments, the nucleoside-modified RNA comprises 1-methylpseuuridine.

[0397] Therefore, in one embodiment, the nanoparticles can be used to deliver nucleoside-modified RNA to a subject in need. In some embodiments, delivering the nucleoside-modified RNA to a subject comprises mixing the nucleoside-modified RNA with at least one dendritic polymer of formula (I) prior to the contact step. In another embodiment, the method of the present invention further comprises applying the nucleoside-modified RNA together with at least one dendritic polymer of formula (I).

[0398] In some implementations, customizable targeting can be achieved based on the identity of the linker group A. Furthermore, the identity of linker group A influences the delivery of nanoparticle cargo. For mRNA cargo, ester linkers have been found to lead to delivery to the liver and / or spleen, while amide groups favor delivery to the lungs. This allows for the customization of nanoparticles to facilitate delivery to desired target organs. One example is the delivery of anti-inflammatory drugs to the lungs.

[0399] In another embodiment, the transfection reagent forms nanoparticles, which are liposomes. In another embodiment, liposomes increase intracellular stability, increase uptake efficiency, and enhance biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of dendritic polymers arranged in a manner similar to that constituting the cell membrane. In another embodiment, they have internal aqueous spaces for encapsulating water-soluble compounds and their size ranges from 0.05 micrometers to several micrometers in diameter. In another embodiment, nanoparticle liposomes can deliver RNA into cells in a biologically active form.

[0400] In various embodiments, the nanoparticles have an average diameter of 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 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or 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, or 150 nm, and are substantially non-toxic. In some embodiments, the nucleoside-modified RNA, when present in nanoparticles, is resistant to nuclease degradation in aqueous solution.

[0401] Small molecule therapeutic agents

[0402] In various embodiments, the reagent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, it can be obtained using standard methods known to those skilled in the art. These methods include chemical organic synthesis or biological methods. Biological methods 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 comprises organic molecules, inorganic molecules, biomolecules, synthetic molecules, etc.

[0403] Combinatorial libraries of molecularly diverse compounds with the potential to treat a variety of diseases and conditions, and methods for preparing these libraries, are well known in the art. These methods can utilize 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 sequences, deconvolution strategies, labeling techniques, and the generation of unbiased molecular landscapes for lead compound discovery and biased structures for lead compound development. In some embodiments of the invention, combinatorial techniques are used to synthesize and / or identify therapeutic agents.

[0404] In general methods of small-library synthesis, an activated core molecule condenses with a number of building blocks to produce a covalently linked core-building block composite library. The shape and stiffness of the core determine the orientation of the building blocks in shape space. These libraries can be biased to target characteristic biological structures (“focused libraries”) by altering the core, connections, or building blocks, or synthesized with smaller structural biases using flexible cores. In some embodiments of the invention, therapeutic agents are synthesized via small-library synthesis.

[0405] Even if salts are not described, the small molecules and small molecule compounds described herein can also exist as salts, and it should be understood that this invention covers all salts and solvates of the therapeutic agents described herein, as well as non-salt and non-solvent forms of the therapeutic agents, as well as those well known to those skilled in the art. In some embodiments, the salts of the therapeutic agents of this invention are pharmaceutically acceptable salts.

[0406] When any therapeutic agent described herein can exist in tautomer forms, each tautomer is intended to be included in the invention, even if only one or some tautomer forms may be explicitly described. For example, when the 2-hydroxypyridinyl moiety is described, the corresponding 2-pyridone tautomer is also referred to.

[0407] This invention also includes any or all stereochemical forms, including any enantiomers or diastereomers of the described therapeutic agents. References to structures or names herein are intended to cover all possible stereoisomers of the described therapeutic agents. This invention also covers all forms of the therapeutic agents, such as crystalline or amorphous forms. Compositions comprising the therapeutic agents of this invention are also intended, such as compositions of substantially pure therapeutic agents (including their specific stereochemical forms), or compositions comprising mixtures of the therapeutic agents of this invention in any proportion (including two or more stereochemical forms), such as racemic or non-racemic mixtures.

[0408] This invention also includes any or all active analogs or derivatives, such as prodrugs, of any therapeutic agent described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecule described herein is a candidate for derivatization. Thus, in certain cases, analogs of the small molecules described herein, having modulated potency, selectivity, and solubility, are included herein and provide useful lead compounds for drug discovery and drug development. Therefore, in certain cases, new analogs are designed with consideration of drug delivery, metabolism, novelty, and safety issues during optimization.

[0409] In some cases, the small molecule therapeutics described herein are derivatives or analogs of known therapeutics, as are well known in the fields of combinatorial and medicinal chemistry. Analogs or derivatives can be prepared by adding and / or substituting functional groups at different positions. Therefore, the small molecules described herein can be transformed into derivatives / analogs using well-known chemical synthesis procedures. For example, all hydrogen atoms or substituents can be selectively modified to produce novel analogs. Furthermore, linking atoms or groups can be modified to have longer or shorter linkers with a carbon backbone or heteroatoms. Additionally, cyclic groups can be modified to have different numbers of atoms and / or include heteroatoms in the ring. Furthermore, aromatic compounds can be converted into rings and vice versa. For example, the ring can be 5-7 atoms and can be a carbon ring or a heterocycle.

[0410] As used herein, the terms "analog," "analogue," or "derivative" refer to a chemical compound or molecule prepared from a parent compound or molecule through one or more chemical reactions. Therefore, an analog may have a structure similar to that of the small molecule therapeutic agents described herein, or may be based on the backbone of the small molecule therapeutic agents described herein but differ from them in some component or structural composition, and may have similar or opposite metabolic effects. Analogs or derivatives of any small molecule inhibitor according to the present invention can be used to treat diseases or conditions.

[0411] In one embodiment, the small molecule therapeutic agents described herein can be independently derivatized, or analogs can be prepared, by modifying hydrogen groups, which are independent of each other, with other substituents. That is, each atom on each molecule can be modified independently relative to other atoms on the same molecule. Any conventional modifications used to generate derivatives / analytes can be used. For example, atoms and substituents can be independently composed of: hydrogen, alkyl, aliphatic compounds, straight-chain aliphatic compounds, aliphatic compounds having chain heteroatoms, branched aliphatic compounds, substituted aliphatic compounds, cyclic aliphatic compounds, heterocyclic aliphatic compounds having one or more heteroatoms, aromatic compounds, heteroaromatic compounds, polyaromatic compounds, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halogenated aliphatic compounds, etc. In addition, any cyclic group on the compound can be derivatized to increase and / or decrease the ring size and change the main chain atom to a carbon atom or a heteroatom.

[0412] Nucleic acid therapy

[0413] In other related aspects, the therapeutic agent is an isolated nucleic acid. In some embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In some 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 a thrombotic regulatory protein, an endothelial protein C receptor (EPCR), an antithrombotic protein including plasminogen activator and its mutants, an antioxidant protein including catalase, superoxide dismutase (SOD), and an iron chelating protein. In some embodiments, the therapeutic agent is siRNA, miRNA, shRNA, or an antisense molecule that inhibits targeted nucleic acids, including those encoding proteins involved in the exacerbation of the pathological process.

[0414] In one embodiment, the nucleic acid contains a promoter / regulatory sequence that enables the nucleic acid to direct its expression. Therefore, this invention covers expression vectors and methods for introducing exogenous nucleic acids into cells and simultaneously expressing them within the cells, such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, ColdSpringHarbor Laboratory, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as well as those described elsewhere herein.

[0415] In one implementation, siRNA is used to reduce the level of a targeted protein. RNA interference (RNAi) is the phenomenon in which double-stranded RNA (dsRNA) is introduced into a wide variety of organisms and cell types, leading to the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short interfering RNAs, or siRNAs, by a ribonuclease called Dicer. The siRNA then assembles with the protein components to form an RNA-induced silencing complex (RISC), which unwinds in the process. The activated RISC then binds to the complementary transcript via base-pairing interactions between the antisense strand of the siRNA and the mRNA. The bound mRNA is cleaved, and the 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., RNAiA Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) described the chemical modification of siRNA, which facilitates intravenous systemic delivery. Optimizing siRNA requires consideration of overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' extrusion. See, for example, Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, this invention also includes methods for reducing PTPN22 levels using RNAi technology.

[0416] On one hand, the present invention includes a vector comprising siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide. The incorporation of the desired polynucleotide into the vector and the choice of the vector are well known in the art, as described, for example, in Sambrook et al. (2012) and Ausubel et al. (1997) and elsewhere herein.

[0417] In some embodiments, the expression vectors described herein encode short hairpin RNA (shRNA) therapeutics. shRNA molecules are well-known in the art and target mRNAs that reduce the expression of the target. In some embodiments, the encoded shRNA is expressed by a cell and then processed into siRNA. For example, in some cases, the cell possesses a natural enzyme (e.g., dicer) that cleaves shRNA to form siRNA.

[0418] To assess the expression of siRNA, shRNA, or antisense polynucleotides, the expression vector to be introduced into cells may also contain a selective marker gene or a reporter gene, or both, to facilitate the identification of expressing cells from a cell population attempting to transfect or infect using the delivery vector of the present invention. In other embodiments, the selective marker may be carried on a separate DNA fragment or may be contained within the delivery vector. Both the selective marker and the reporter gene may have suitable regulatory sequences flanking them to enable expression in host cells. Useful selective markers are known in the art and include, for example, antibiotic resistance genes, such as neomycin resistance.

[0419] Therefore, on the one hand, the delivery vector may contain a vector that contains a nucleotide sequence or construct to be delivered. The choice of vector will depend on the host cell into which it will be subsequently introduced. In a particular embodiment, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a specific embodiment, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic vectors can be used in the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are already widely available commercially.

[0420] For example, the vector for introducing the nucleic acid sequence can be a plasmid, which, when introduced into a cell, may or may not integrate into the host cell's genome. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the present invention or gene constructs of the present invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0421] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector that can be used to transform animal cells.

[0422] In one embodiment, the recombinant expression vector may also contain a nucleic acid molecule encoding a peptide or a peptide mimic.

[0423] Promoters can be promoters naturally associated with a gene or polynucleotide sequence, such as those obtained by isolating a 5' non-coding sequence located upstream of a coding segment and / or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a polynucleotide sequence, located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter (i.e., a promoter not typically associated with a polynucleotide sequence in the natural environment). Recombinant or heterologous enhancers also refer to enhancers not typically associated with a polynucleotide sequence in the natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cells, and promoters or enhancers that are not "naturally present" (i.e., containing different elements of different transcriptional regulatory regions and / or mutations altering expression). In addition to synthetically generating the nucleic acid sequences of promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can also be used. TM The sequences are generated by combining the components disclosed herein (US Patent 4,683,202, US Patent 5,928,906). Furthermore, it is considered that control sequences guiding the transcription and / or expression of sequences within non-nuclear organelles (such as mitochondria, chloroplasts, etc.) may also be employed.

[0424] Naturally, it is important to employ promoters and / or enhancers that effectively guide the expression of DNA segments in the selected cell types, organelles, and organisms 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 promoters employed can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to guide high-level expression of the introduced DNA segment, such as facilitating the large-scale production of recombinant proteins and / or peptides. Promoters can be heterologous or endogenous.

[0425] Recombinant expression vectors can also contain selective marker genes that facilitate host cell selection. Suitable selective marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof (such as the Fc portion of immunoglobulin, preferably IgG). Selective markers can be introduced into vectors separate from the target nucleic acid.

[0426] After the siRNA polynucleotide is produced, the technicians will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve siRNA as a therapeutic compound. Therefore, siRNA polynucleotides can be further engineered to resist degradation by modifying them to include thiophosphates or other bonds, methylphosphonates, sulfones, sulfates, carbonyl radicals (ketyl), dithiophosphates, aminophosphates, phosphates, etc. (see, for example, 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: Oligodoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0427] Any polynucleotide can be further modified to increase its in vivo stability. Possible modifications include, but are not limited to, adding flanking sequences at the 5′ and / or 3′ ends; using thiophosphates or 2′O-methyl groups in the main chain instead of phosphodiester bonds; and / or including non-traditional bases such as inosine, queuosine, and wybutosine, as well as acetyl-, methyl-, thio-, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0428] In one embodiment of the invention, the antisense nucleic acid sequence expressed by the plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein. The antisense expression vector is used to transfect mammalian cells or mammals themselves, thereby resulting in a reduction in the endogenous expression of the target protein.

[0429] Antisense molecules and their use in suppressing gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). An antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a specific mRNA molecule, as the term is defined elsewhere herein (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule, thereby suppressing gene translation.

[0430] The use of antisense methods to suppress gene translation is known in the art and described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172: 289). Such antisense molecules can be provided to cells through the genetic expression of DNA encoding antisense molecules, as taught in Inoue, 1993, U.S. Patent No. 5,190,931.

[0431] Alternatively, the antisense molecules of the present invention can be synthesized and then provided to cells. Antisense oligomers of about 10 to about 30 nucleotides, and more preferably about 15 nucleotides, are preferred because they are readily synthesized and introduced into target cells. Synthetic antisense molecules contemplated in this invention include oligonucleotide derivatives known in the art that exhibit improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0432] In one embodiment of the invention, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes that can be used to inhibit the expression of target molecules can be designed by incorporating a target sequence into a basic ribozyme structure, such as a structure complementary to the mRNA sequence encoding the target molecule. Ribozymes targeting target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA), or they can be genetically expressed from the DNA encoding them.

[0433] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, wherein a guide RNA (gRNA) of a gene encoding a target molecule and a CRISPR-associated (Cas) peptide form a complex to induce a mutation within the targeted gene. In one embodiment, the therapeutic agent comprises gRNA or a nucleic acid molecule encoding gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0434] In one embodiment, the reagent comprises miRNA or a miRNA mimic. In another embodiment, the reagent comprises a nucleic acid molecule encoding miRNA or a miRNA mimic.

[0435] miRNAs are small non-coding RNA molecules that can induce post-transcriptional silencing of specific genes in cells by inhibiting the translation of the targeted mRNA or by degradation. miRNAs can be perfectly complementary to the target nucleic acid or can have non-complementary regions, resulting in a "bulge" at the non-complementary region. miRNAs can suppress gene expression by inhibiting translation, such as when the miRNA is not perfectly complementary to the target nucleic acid, or by inducing degradation of the target RNA (which is thought to occur only when the miRNA binds to its target with perfect complementarity). This disclosure may also include double-stranded precursors of miRNAs. miRNAs or precursor miRNAs can be 18-100 nucleotides or 18-80 nucleotides in length. Mature miRNAs can have 19-30 nucleotides or 21-25 nucleotides in length, particularly 21, 22, 23, 24, or 25 nucleotides. Precursor miRNAs are typically about 70-100 nucleotides in length and have a hairpin conformation. miRNAs are generated in vivo from precursor miRNAs via the enzymes Dicer and Drosha (which specifically process long precursor miRNAs into functional miRNAs). The hairpin or mature microRNA or pre-microRNA reagents characterized in this disclosure can be synthesized in vivo via cell-based systems or in vitro via chemical synthesis.

[0436] In various embodiments, the reagent comprises an oligonucleotide containing the nucleotide sequence of a disease-associated miRNA. In some embodiments, the oligonucleotide contains the nucleotide sequence of a precursor microRNA, a mature or hairpin-form disease-associated miRNA. In other embodiments, combinations of oligonucleotides comprising sequences of one or more disease-associated miRNAs, any precursor miRNA, any fragment thereof, or any combination thereof are contemplated.

[0437] miRNAs can be synthesized to include modifications that confer desired properties. For example, modifications can improve stability, hybridization thermodynamics with target nucleic acids, targeting of specific tissues or cell types, or cell permeability (e.g., through endocytosis-dependent or endocytosis-independent mechanisms).

[0438] Modifications can also increase sequence specificity, thereby reducing off-site targeting. Methods for synthesis and chemical modification are described in more detail below. If desired, miRNA molecules can be modified to stabilize the miRNA against degradation, extend its half-life, or otherwise enhance its efficacy. The desired modifications are described, for example, in U.S. Patent Publications Nos. 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, each of which is incorporated herein by reference in its entirety. To increase nuclease resistance and / or binding affinity to the target, the single-stranded oligonucleotide reagents characterized in this disclosure may include 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, 2′-O-aminopropyl, 2′-amino, and / or phosphate thioester bonds. Including locked nucleic acids (LNAs), ethylene nucleic acids (ENAs) (e.g., 2′-4′-ethylene-bridged nucleic acids), and certain nucleotide modifications can increase binding affinity to targets. Including pyranose in the oligonucleotide backbone can also reduce endonucleolytic cleavage. Oligonucleotides can be further modified by including 3′ cationic groups or by reversing the 3′-terminal nucleoside using a 3-3′ bond. Alternatively, the 3′ terminus can be blocked with an aminoalkyl group. Other 3′ conjugates can inhibit 3′-5′ exonucleolytic cleavage. Although not bound by theory, the 3′ can inhibit exonuclease cleavage by sterically blocking the binding of exonucleases to the 3′ end of the oligonucleotide. Even small alkyl chains, aryl or heterocyclic conjugates, or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3′-5′ exonucleases.

[0439] In one embodiment, the miRNA comprises a 2′-modified oligonucleotide containing an oligodeoxynucleotide internucleotide space, wherein some or all of the internucleotide bonds are modified with phosphate thioesters to acquire nuclease resistance. The presence of methylphosphonate modification increases the affinity of the oligonucleotide for its target RNA and thus reduces IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It should be understood that the methods and reagents of this disclosure can be used in conjunction with any techniques that can be developed to enhance the stability or efficacy of repressive nucleic acid molecules.

[0440] miRNA molecules include nucleotide oligomers containing modified backbones or non-natural nucleoside interchains. Oligomers with modified backbones include those that retain a phosphorus atom in the backbone and those that do not. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their nucleoside interchain are also considered nucleotide oligomers. Nucleonucleotide oligomers with modified oligonucleotide backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, methyl and other alkylphosphonates (including 3′-alkylenephosphonates and chiral phosphonates), hypophosphonates, aminophosphates, thioaminophosphates, thioalkylphosphates, thioalkyl phosphates, and borophosphates. Various salts, mixed salts, and free acid forms are also included.

[0441] The miRNAs described herein may be in mature or hairpin form and may be provided as naked oligonucleotides. In some cases, it may be necessary to utilize formulations that facilitate the delivery of miRNAs or other nucleotide oligomers to cells (see, for example, 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).

[0442] 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 instance, the miRNA composition is in an aqueous phase, such as a solution comprising water. The aqueous phase or crystalline composition may be incorporated into a delivery carrier, such as liposomes (particularly for the aqueous phase) or particles (e.g., microparticles suitable for the crystalline composition). Typically, the miRNA composition is formulated in a manner compatible with the intended method of administration. The miRNA composition may be formulated in combination with another reagent (e.g., another therapeutic agent or a reagent stabilizing an oligonucleotide reagent (e.g., a protein complexed with an oligonucleotide reagent)). Other reagents include chelating agents, such as EDTA (e.g., to remove divalent cations, such as Mg), salts, and RNase inhibitors (e.g., broadly specific RNase inhibitors). In one embodiment, the miRNA composition includes another miRNA, such as a second miRNA composition (e.g., a microRNA different from the first miRNA composition). Other formulations may also include at least three, five, ten, twenty, fifty, or one hundred or more different oligonucleotide species.

[0443] In some embodiments, the composition comprises an oligonucleotide composition that mimics miRNA activity. In some embodiments, the composition comprises an oligonucleotide having nucleobase identity with the nucleobase sequence of the miRNA and is therefore designed to mimic miRNA activity. In some embodiments, the oligonucleotide composition mimicking miRNA activity comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin or a processed miRNA double-strand.

[0444] In one embodiment, the oligonucleotide shares identity with the nucleobase sequence of an endogenous miRNA or miRNA precursor. The oligonucleotide selected for inclusion in the compositions of the present invention can be of a variety of lengths. Such an oligonucleotide can be 7 to 100 linked nucleosides in length. For example, an oligonucleotide sharing nucleobase identity with a miRNA can be 7 to 30 linked nucleosides in length. An oligonucleotide sharing identity with a miRNA precursor can be up to 100 linked nucleosides in length. In some embodiments, the oligonucleotide comprises 7 to 30 linked nucleosides. In some 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 some embodiments, the oligonucleotide comprises 19 to 23 linked nucleosides. In some embodiments, the oligonucleotide is 40 to a maximum of 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0445] In some embodiments, the oligonucleotide has a sequence that is identical to the miRNA or its precursor. The nucleobase sequence of the mature miRNA described herein and its corresponding stem-loop sequence are sequences found in miRBase (an online searchable database of miRNA sequences and annotations). Entries in the miRBase sequence database represent the predicted hairpin portion of the miRNA transcript (stem-loop), as well as information about the location and sequence of the mature miRNA. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs) and in some cases may include the precursor miRNA and some flanking sequences from a presumed primary transcript. The miRNA nucleobase sequences described herein cover any version of the miRNA, including sequences described in miRBase sequence database version 10.0 and sequences described in any earlier versions of the miRBase sequence database. The release of the sequence database may lead to the renaming of certain miRNAs. The release of the sequence database may lead to changes in the mature miRNA sequence. The compositions of the present invention cover oligomeric compounds comprising oligonucleotides that are identical to any version of the miRNA described herein.

[0446] In some embodiments, the oligonucleotide has a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the miRNA in a region of 7, 8, 9, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Therefore, in some embodiments, the oligonucleotide's nucleotide sequence may have one or more nucleotides that are different from the miRNA.

[0447] In some embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, its precursor, its mimic, or a fragment thereof. For example, the composition may comprise a viral vector, plasmid, granule, or other expression vector suitable for expressing the miRNA, its precursor, its mimic, or a fragment thereof in desired mammalian cells or tissues.

[0448] Nucleic acid

[0449] In one embodiment, the present invention includes 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 some embodiments, the nucleoside-modified mRNA molecule encodes an antigen that induces an adaptive immune response against the antigen. In one embodiment, the present invention includes a nucleoside-modified mRNA molecule encoding an adjuvant.

[0450] As described herein, the nucleotide sequence encoding an antigen or adjuvant may alternatively include sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the obtained multiple nucleotides encode a polypeptide according to the invention. Therefore, the scope of the invention includes nucleotide sequences substantially homologous to the nucleotide sequences described herein and encoding antigens or adjuvants of interest.

[0451] As used herein, a nucleotide sequence is considered “substantially homologous” to any nucleotide sequence described herein when its nucleotide sequence has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with respect to a nucleotide sequence. A nucleotide sequence substantially homologous to a nucleotide sequence encoding an antigen can generally be isolated from the antigen-producing organism based on information contained in the nucleotide sequence, for example, by introducing conserved or non-conserved substitutions. Other examples of possible modifications include inserting one or more nucleotides into the sequence, adding one or more nucleotides at any end of the sequence, or deleting one or more nucleotides at any end of the sequence or within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods widely known to those skilled in the art.

[0452] Furthermore, the scope of this invention includes nucleotide sequences that encode amino acid sequences substantially homologous to the amino acid sequences described herein and retain the immunogenic function of the original amino acid sequences.

[0453] As used herein, an amino acid sequence is considered “substantially homologous” to any amino acid sequence described herein when its amino acid sequence has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with respect to an amino acid sequence. 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)).

[0454] 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 some embodiments, the construct encodes one or more, two or more, five or more, ten or more, fifteen or more, or twenty 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.

[0455] In one embodiment, the composition comprises a plurality of constructs, each construct encoding one or more antigens. For example, in some 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 containing a nucleotide sequence encoding an antigen; and a second construct containing a nucleotide sequence encoding an adjuvant.

[0456] In another specific embodiment, the construct is operatively bound to a translation control element. The construct may be combined with an operatively binding regulatory sequence for expressing the nucleotide sequence of the present invention, thereby forming an expression cassette.

[0457] carrier

[0458] The nucleic acid sequences encapsulated in the nanoparticles of this invention can be obtained using recombination methods known in the art, such as, for example, by using standard techniques, screening libraries from cells expressing genes, deriving genes from vectors known to contain genes, or directly isolating genes from cells and tissues containing genes. Alternatively, the nucleic acid molecules of interest can be synthesized.

[0459] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0460] In one embodiment, the composition of the present invention comprises in vitro transcribed (IVT) RNA encoding an antigen. In one embodiment, the composition of the present invention comprises IVT RNA encoding multiple antigens. In one embodiment, the composition of the present invention comprises IVT RNA encoding an adjuvant. In one embodiment, the composition of the present invention comprises IVT RNA encoding one or more antigens and one or more adjuvants.

[0461] Nucleoside-modified RNA

[0462] In one embodiment, the nucleic acid molecule comprises nucleoside-modified RNA. Nucleoside-modified mRNA has particular advantages over unmodified mRNA, including, for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNAs that can be used in this invention are further described in U.S. Patent No. 8,278,036, the entire contents of which are incorporated herein by reference.

[0463] In some implementations, the nucleoside-modified mRNA does not activate any pathophysiological pathways, is highly effective and translates almost immediately after delivery, and serves as a template for continuous protein production in vivo over several days (Karikó et al., 2008, Mol Ther 16: 1833-1840; Karikó et al., 2012, Mol Ther 20: 948-953). The small amount of mRNA required to exert its physiological effects makes it suitable for human therapy. For example, as described herein, nucleoside-modified mRNA encoding antigens has been shown to induce the production of CD4+ and CD8+ T cells and antigen-specific antibodies. For example, in some cases, antigens encoded by nucleoside-modified mRNA induce greater production of antigen-specific antibodies compared to antigens encoded by unmodified mRNA.

[0464] In some cases, expressing proteins by delivering mRNA offers numerous advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to the cell, thus avoiding all the side effects associated with the plasmid backbone, viral genes, and viral proteins. More importantly, unlike DNA- and virus-based vectors, mRNA does not carry the risk of integration into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating proteins can be measured within 15 to 30 minutes after in vivo injection of mRNA. In some implementations, using mRNA instead of protein also has many advantages. Circulating proteins typically have short half-lives, thus protein therapy requires frequent administration, while mRNA provides a template for continuous protein production over several days. Protein purification is problematic, and they may contain aggregates and other impurities that can cause side effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050: 257-265).

[0465] In some embodiments, the nucleoside-modified RNA comprises a naturally occurring modified nucleoside pseudouridine. In some embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable. 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; Karikó et al., 2011, Nucleic Acids Research 39:e142; Karikó et al., 2012, Mol Ther 20:948-953; Karikó et al., 2005, Immunity 23:165-175).

[0466] It has been demonstrated that the presence of modified nucleosides (including pseudouridine) in RNA inhibits their innate immunogenicity (Karikó et al., 2005, Immunity 23: 165-175). Furthermore, protein-coding, in vitro transcribed RNA containing pseudouridine can be translated more efficiently than RNA without modified nucleosides or containing other modified nucleosides (Karikó et al., 2008, Mol Ther 16: 1833-1840). Subsequently, it has been shown that the presence of pseudouridine increases RNA stability (Anderson et al., 2011, Nucleic Acids Research 39: 9329-9338) and attenuates PKR activation and translational repression (Anderson et al., 2010, Nucleic Acids Res 38: 5884-5892). A preparative HPLC purification procedure has been established, which is crucial for obtaining pseudouridine-containing RNA with excellent translational potential and no innate immunogenicity (Karik6 et al., 2011, Nucleic Acids Research 39: e142). Administration of HPLC-purified pseudouridine-containing erythropoietin-encoding RNA to mice and macaques resulted in a significant increase in serum EPO levels (Karik6 et al., 2012, Mol Ther 20: 948-953), thus demonstrating that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0467] This invention includes RNA, oligonucleotides, and polynucleotide molecules comprising pseudouridine or modified nucleosides. In some embodiments, the composition comprises isolated nucleic acid encoding an antigen, wherein the nucleic acid comprises pseudouridine or modified nucleosides. In some embodiments, the composition comprises a vector comprising isolated nucleic acid encoding an antigen, an adjuvant, or a combination thereof, wherein the nucleic acid comprises pseudouridine or modified nucleosides.

[0468] In one embodiment, the nucleoside-modified RNA of the present invention is IVT RNA. For example, in some 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 yet another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0469] In one embodiment, the modified nucleoside is m 1 acp 3 Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In another embodiment, the modified nucleoside is m 1 Ψ(1-methylpseudouridine). 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-methylpseuuridine). In another embodiment, the modified nucleoside is the unmodified pseudouridine moiety. In another embodiment, the modified nucleoside is any of the above-described pseudouridine monophosphate, diphosphate, or triphosphate. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0470] 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).

[0471] In another embodiment, the modified nucleoside of the present invention is m 5 C(5-methylcytidine). In another embodiment, the modified nucleoside is m 5 U(5-methyluridine). In another embodiment, the modified nucleoside is m 6 A(N 6 -methyladenosine). In another embodiment, the modified nucleoside is S. 2U(2-thiouridine). In another embodiment, the modified nucleoside is Ψ(pseudouridine). In another embodiment, the modified nucleoside is Um(2′-O-methyluridine).

[0472] 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 (Isopentenyl adenosine); 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 -glycylcarbamoyladenosine); t 6 A(N 6 (-Threonylcarbamoyladenosine); ms 2 t 6 A(2-methylthio-N) 6 -Threonylcarbamoyladenosine); m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyladenosine); hn 6 A(N 6 -hydroxyn-valinecarbamoyladenosine); ms 2 hn 6 A(2-methylthio-N) 6 -hydroxyvaline carbamoyl adenosine); Ar(p)(2′-O-ribosyl adenosine (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 5Cm(5,2′-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2′-O-methylcytidine); k 2 C (lysicytoside); 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 -dimethylguanosine); m 2 Gm(N 2 ,2′-O-dimethylguanosine); m 2 2Gm(N 2 N 2 , 2′-O-trimethylguanosine); Gr(p)(2′-O-ribosylguanosine (phosphate ester)); yW (huaitin); o2yW (peroxyhuaitin); OHyW (hydroxyhuaitin); OHyW* (undermodified hydroxyhuaitin); imG (huaoside); mimG (methylhuaoside); Q (pigmentoside); oQ (epoxypigmentoside); galQ (galactosylpigmentoside); manQ (mannosylpigmentoside); preQ0 (7-cyano-7-deazoguanosine); preQ1 (7-aminomethyl-7-deazoguanosine); G + (archaeosine); D (dihydrouridine); m 5 Um(5,2′-O-dimethyluridine); s 4 U(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-oxyacetic 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 s2 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-dimethylguanosine); 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 5U(5-Taurine methyluridine); τm 5 s 2 U (5-Tauratemethyl-2-thiouridine); imG-14 (4-Demethylwyoside); imG2 (Isowyosine); or ac 6 A(N 6 -acetyl adenosine.

[0473] In another embodiment, the nucleoside-modified RNA of the present invention comprises a combination of two or more of the above-described modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of three or more of the above-described modifications. In yet another embodiment, the nucleoside-modified RNA comprises a combination of more than three of the above-described modifications.

[0474] In another embodiment, 0.1% to 100% of the residues in the nucleoside modification of the present invention are modified (e.g., by the presence of pseudouridine or modified nucleoside bases). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the score is 18%. In another embodiment, the score is 20%. In another embodiment, the score is 25%. In another embodiment, the score is 30%. In another embodiment, the score is 35%. In another embodiment, the score is 40%. In another embodiment, the score is 45%. In another embodiment, the score is 50%. In another embodiment, the score is 60%. In another embodiment, the score is 70%. In another embodiment, the score is 80%. In another embodiment, the score is 90%. In another embodiment, the score is 100%.

[0475] In another embodiment, the score is less than 5%. In another embodiment, the score is less than 3%. In another embodiment, the score is less than 1%. In another embodiment, the score is less than 2%. In another embodiment, the score is less than 4%. In another embodiment, the score is less than 6%. In another embodiment, the score is less than 8%. In another embodiment, the score is less than 10%. In another embodiment, the score is less than 12%. In another embodiment, the score is less than 15%. In another embodiment, the score is less than 20%. In another embodiment, the score is less than 30%. In another embodiment, the score is less than 40%. In another embodiment, the score is less than 50%. In another embodiment, the score is less than 60%. In another embodiment, the score is less than 70%.

[0476] 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 modified nucleotides 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 score is 25%. In another embodiment, the score is 30%. In another embodiment, the score is 35%. In another embodiment, the score is 40%. In another embodiment, the score is 45%. In another embodiment, the score is 50%. In another embodiment, the score is 60%. In another embodiment, the score is 70%. In another embodiment, the score is 80%. In another embodiment, the score is 90%. In another embodiment, the score is 100%.

[0477] In another embodiment, the percentage of a given nucleotide 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%.

[0478] In another embodiment, the nucleoside-modified RNA of the present invention is translated more efficiently in cells than unmodified RNA molecules having the same sequence. In another embodiment, the nucleoside-modified RNA exhibits enhanced translation ability by target cells. In another embodiment, translation is enhanced by a 2-fold factor relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the coefficient is 10-100 times. In another embodiment, the coefficient is 10-200 times. In another embodiment, the coefficient is 10-300 times. In another embodiment, the coefficient is 10-500 times. In another embodiment, the coefficient is 20-1000 times. In another embodiment, the coefficient is 30-1000 times. In another embodiment, the coefficient is 50-1000 times. In another embodiment, the coefficient is 100-1000 times. In another embodiment, the coefficient is 200-1000 times. In another embodiment, the translation is enhanced by any other significant amount or range.

[0479] In another embodiment, the RNA encoding the nucleoside-modified antigen of the present invention induces a significantly stronger adaptive immune response compared to an unmodified in vitro synthesized RNA molecule having the same sequence. In another embodiment, the modified RNA molecule exhibits an adaptive immune response that is twice as strong as its unmodified counterpart. In another embodiment, the adaptive immune response is increased by a factor of 3. In another embodiment, the adaptive immune response is increased by a factor of 5. In another embodiment, the adaptive immune response is increased by a factor of 7. In another embodiment, the adaptive immune response is increased by a factor of 10. In another embodiment, the adaptive immune response is increased by a factor of 15. In another embodiment, the adaptive immune response is increased by a factor of 20. In another embodiment, the adaptive immune response is increased by a factor of 50. In another embodiment, the adaptive immune response is increased by a factor of 100. In another embodiment, the adaptive immune response is increased by a factor of 2000. In another embodiment, the adaptive immune response is increased by a factor of 500. In another embodiment, the adaptive immune response is increased by a factor of 1000. In another embodiment, the adaptive immune response is increased by a factor of 2000. In another implementation, the adaptive immune response adds another fold difference.

[0480] In another embodiment, “inducing a significantly stronger 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., 1 in the fold increase mentioned above). In another embodiment, the term refers to an increase such that nucleoside-modified RNA can be administered at a lower dose or frequency than unmodified RNA molecules of the same kind, while still inducing an effective adaptive immune response. In another embodiment, this increase allows nucleoside-modified RNA to be used with a single dose to induce an effective adaptive immune response.

[0481] In another embodiment, the nucleoside-modified RNA of the present invention exhibits significantly lower innate immunogenicity than unmodified in vitro synthesized RNA molecules having the same sequence. In another embodiment, the modified RNA molecule exhibits a 2-fold lower innate immune response than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

[0482] In another embodiment, “exhibiting significantly lower innate immunogenicity” refers to a detectable reduction in innate immunogenicity. In another embodiment, the term refers to a fold reduction in innate immunogenicity (e.g., 1 in the fold reduction mentioned above). In another embodiment, the term refers to a reduction such that an effective amount of nucleoside-modified RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a reduction such that nucleoside-modified RNA can be repeatedly administered without triggering an innate immune response sufficient to detectably reduce recombinant protein production. In another embodiment, this reduction allows nucleoside-modified RNA to be repeatedly administered without triggering an innate immune response sufficient to eliminate detectable recombinant protein production.

[0483] peptide therapy

[0484] In other related aspects, therapeutic agents include peptides that modulate the separation of a target. For example, in one embodiment, the peptides of the present invention directly inhibit or activate a target by binding to it, thereby modulating the normal functional activity of the target. In one embodiment, the peptides of the present invention modulate a target by competing with endogenous proteins. In one embodiment, the peptides of the present invention modulate target activity by acting as transdominant-negative mutants.

[0485] Variants of the peptide therapeutic agent may be (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be amino acid residues encoded by the genetic code; (ii) variants in which one or more modified amino acid residues are present (e.g., residues modified by attachment of substituents); (iii) variants in which the peptide is an alternative splice variant of the peptide of the present invention; (iv) fragments of the peptide; and / or (v) variants in which the peptide is fused with another peptide (such as a leader sequence or secretion sequence or a sequence for purification (e.g., a His tag) or a sequence for detection (e.g., an Sv5 epitope tag)). Fragments include peptides produced by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of those skilled in the art based on the teachings herein.

[0486] Nanoparticles may further include any lipids capable of forming particles, one or more nucleic acid molecules attached to the particle, or one or more nucleic acid molecules encapsulated within the particle. The term "lipid" refers to a group of organic compounds that are derivatives of fatty acids (such as esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) "simple lipids," including fats and oils as well as waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. In one embodiment, the nanoparticles do not further include any lipids capable of forming particles, one or more nucleic acid molecules attached to the particle, or one or more nucleic acid molecules encapsulated within the particle. In one embodiment, the nanoparticles do not further comprise any or all of simple lipids, complex lipids, or derived lipids.

[0487] In one embodiment, the nanoparticles comprise cationic lipids. As used herein, the term "cationic lipid" refers to a lipid that is cationic or becomes cationic (protonated) when the pH decreases below the pK of its ionizable groups, but gradually becomes more neutral at higher pH values. Below the pK pH, the lipid is then able to bind to negatively charged nucleic acids. In some embodiments, cationic lipids comprise zwitterionic lipids that exhibit a positive charge as the pH decreases. In one embodiment, the nanoparticles do not contain cationic lipids.

[0488] In some embodiments, the cationic lipids (optionally present or absent in the nanoparticles) comprise any of a variety of lipid species that carry a net positive charge at a selective pH (e.g., physiological pH). Such lipids include, but are not limited to, N,N-diolenoyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-diolenoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearate-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-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA), bis(octadecylamido)glycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylpropane ammonium (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Furthermore, many commercial formulations of the cationic lipids used in this invention are available. These include, for example... (Commercially available cationic liposomes containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), sourced from GIBCO / BRL, Grand Island, NY); (Commercially available cationic liposomes containing N-(1-(2,3-diolenoyloxy)propyl)-N-(2-(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA) and (DOPE), from GIBCO / BRL); and (Commercially available cationic lipids containing bis(octadecylamidoglycylcarboxyspermine) (DOGS) (in ethanol), from Promega Corp., Madison, Wis.). The following lipids are cationic and carry a positive charge below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0489] In one embodiment, the cationic lipid is an amino lipid. Such amino lipids include those described in WO 2012 / 016184, which is incorporated herein by reference in its entirety. 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-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), and 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl). 1,2-Dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazine)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dilinoleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0490] In various embodiments, the nanoparticles further comprise a steroid or a steroid analogue. A "steroid" is a compound comprising the following carbon skeleton:

[0491]

[0492] In some embodiments, the steroid or steroid analogue is cholesterol. In one embodiment, the nanoparticles do not contain steroids or steroid analogues. In one embodiment, the nanoparticles do not contain cholesterol.

[0493] In one embodiment, the nanoparticles further comprise a stabilizer. In one embodiment, the stabilizer comprises polyethylene oligomer. In one embodiment, the stabilizer comprises a water-soluble macromolecule. In one embodiment, the stabilizer comprises a water-soluble oligomer. In one embodiment, the stabilizer comprises a carbohydrate.

[0494] In some embodiments, the nanoparticles include one or more targeting portions that enable the nanoparticles to be targeted to cells or cell populations. For example, in one embodiment, the targeting portion is a ligand that directs the nanoparticles toward a receptor present on the cell surface.

[0495] In some embodiments, the nanoparticles include one or more internalization domains. For example, in one embodiment, the nanoparticles include one or more domains that bind to cells to induce nanoparticle internalization. For example, in one embodiment, one or more internalization domains bind to receptors present on the cell surface to induce receptor-mediated nanoparticle uptake. In some embodiments, the nanoparticles are capable of binding biomolecules in vivo, wherein the biomolecules bound to the nanoparticles can then be recognized by cell surface receptors to induce internalization. For example, in one embodiment, the nanoparticles bind to the ApoE system, which leads to the uptake of the nanoparticles and associated cargo.

[0496] Composition

[0497] In one aspect, the present invention relates to compositions comprising at least one of the amphiphilic Janus dendritic polymers and / or nanoparticles of the present invention. In some embodiments, the compositions further comprise at least one reagent described herein.

[0498] The present invention also relates to compositions comprising at least one compound of formula (I) and methods of using the same for delivering an encapsulating agent to a site of interest. Exemplary reagents that can be encapsulated in the compositions of the present invention include, but are not limited to, diagnostic reagents, detectable reagents, and therapeutic agents.

[0499] In one embodiment, the composition comprises nanoparticles containing a compound of formula (I) and at least one reagent encapsulated by the nanoparticles. In some embodiments, the encapsulated reagent includes an agent for inducing an immune response in a subject. In some 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 some embodiments, the composition comprises a vaccine containing a nucleic acid molecule encoding an antigen.

[0500] In one embodiment, the composition is a vaccine.

[0501] In one embodiment, the composition comprises nanoparticles and one or more nucleic acid molecules (described herein). For example, in one embodiment, the composition comprises nanoparticles and one or more nucleoside-modified RNA molecules encoding one or more antigens, adjuvants, or combinations thereof.

[0502] In one embodiment, the composition can be prepared by injecting a mixture comprising the compounds described herein into a suitable solution (e.g., a solution containing the reagent to be encapsulated). In one embodiment, the production of the nanoparticles of the present invention does not require microfluidic techniques (e.g., those required for the formation of lipid nanoparticles (LNPs)).

[0503] In one embodiment, the composition of the present invention comprises an in vitro transcribed (IVT) RNA molecule. For example, in some embodiments, the composition of the present invention comprises an IVT RNA molecule encoding a reagent. In some embodiments, the IVT RNA molecule of the composition is a nucleoside-modified mRNA molecule. In some embodiments, the reagent is at least one of a viral antigen, bacterial antigen, fungal antigen, parasitic antigen, tumor-specific antigen, or tumor-associated antigen. However, the present invention is not limited to any particular reagent or combination of reagents. In some embodiments, the composition comprises an adjuvant. In some embodiments, the composition comprises a nucleic acid molecule encoding an adjuvant. In one embodiment, the composition comprises nucleoside-modified RNA encoding an adjuvant.

[0504] In one embodiment, the composition comprises at least one nucleoside-modified RNA molecule encoding a combination of at least two reagents. In another embodiment, the composition comprises a combination of two or more nucleoside-modified RNA molecules encoding a combination of two or more reagents.

[0505] 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 against viruses, bacteria, fungi, parasites, cancer, etc., in a subject. In some embodiments, the method includes administering to the subject a composition comprising one or more nanoparticles, the nanoparticles comprising one or more nucleoside-modified RNA encoding at least one antigen, adjuvant, or a combination thereof.

[0506] In one embodiment, the method includes systemic administration of the composition to a subject, including, for example, intradermal administration. In some embodiments, the method includes administering multiple doses to the subject. In another embodiment, the method includes administering a single dose of the composition, wherein the single dose is effective in inducing a therapeutic response.

[0507] vaccine

[0508] 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 comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, or a combination thereof. In certain embodiments, the composition comprises or encodes all or part of any peptide antigen, or an immunogenic equivalent thereof. In other embodiments, the composition comprises a mixture of mRNA molecules encoding one or more additional immunostimulants. Immunostimulants include, but are not limited to, additional antigens, immunomodulators, or adjuvants. In the context of this invention, the term “vaccine” refers to a substance that induces immunity when administered to an animal.

[0509] The 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 together with an adjuvant. It should be understood, of course, that the various compositions described herein may further include additional components. According to this disclosure, the vaccines of the present invention and their various components may be prepared and / or administered by any of the methods disclosed herein or any methods known to those skilled in the art.

[0510] Induced immunity through antigen expression can be detected by observing the response of all or any part of the host's immune system to the antigen, either in vivo or in vitro.

[0511] For example, methods for detecting the induction of cytotoxic T lymphocytes are well known. Foreign substances entering the living body are presented to T cells and B cells via the action of antigen-presenting cells (APCs). T cells that respond to antigens presented by APCs in an antigen-specific manner differentiate into cytotoxic T cells (also known as cytotoxic T lymphocytes or CTLs) due to antigen stimulation. These antigen-stimulated cells then proliferate. This process is referred to herein as the “activation” of T cells. Therefore, CTL induction by epitopes of polypeptides, peptides, or combinations thereof can be assessed by presenting epitopes of polypeptides, peptides, or combinations thereof to T cells via APCs and detecting CTL induction. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0512] Methods for assessing CTL induction using dendritic cells (DCs) as APCs are well known in the art. DCs are representative APCs with potent CTL induction. In the method of this invention, an epitope of a polypeptide, or peptide, or a combination thereof, is initially expressed by a DC, which is then contacted with T cells. Detection of cytotoxic T cells on cells of interest after contact with DCs reveals that the epitope of the polypeptide, or peptide, or a combination thereof has cytotoxic T cell induction activity. Furthermore, the induced immune response (such as an ELISPOT assay) can be detected by measuring IFN-γ produced and released by CTLs in the presence of antigen-presenting cells carrying fixed peptides or peptide combinations, and visualized using anti-IFN-γ antibodies.

[0513] Besides dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs) can also be used as apoptotic cells (APCs). CTL induction has been reported to be 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.

[0514] These methods confirm that antigens with CTL-inducing activity are antigens that possess both DC-activating and subsequent CTL-inducing activity. Furthermore, CTLs that acquire cytotoxicity through APC antigen presentation can also be used as vaccines against antigen-related diseases.

[0515] Induced immunity via antigen expression can be further confirmed by observing the induction of antibody production against the antigen. For example, the composition is identified as induced when antibodies against the antigen are induced in laboratory animals immunized with a composition encoding the antigen, and when antigen-associated pathology is suppressed by these antibodies.

[0516] Observing the induction of CD4+ T cells can further confirm the induction of immunity by antigen expression. CD4+ T cells can also lyse target cells, but their primary role is to help induce other types of immune responses, including CTLs and antibody production. The types of CD4+ T cell helper cells can be characterized as Th1, Th2, Th9, Th17, regulatory T cells, or follicular helper T cells (T cells). fh CD4+ T cells. Each subtype of CD4+ T cells contributes to certain types of immune responses. This invention is of particular interest to T cells. fh Subtypes help produce high-affinity antibodies.

[0517] In some embodiments, the therapeutic compounds or compositions of the present invention may be administered prophylactically (i.e., to prevent disease or condition) or therapeutically (e.g., to treat disease or condition) to subjects who have or are at risk of developing (or being susceptible to) disease or condition. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs before the onset of overt clinical symptoms of the disease, thereby preventing or alternatively delaying the progression of the disease or condition. In the context of the medical field, the term "prevention" includes any activity that reduces the mortality or morbidity burden of a disease. Prevention may occur at primary, secondary, and tertiary levels of prevention. While primary prevention avoids the development of disease, secondary and tertiary levels of prevention include activities aimed at preventing disease progression and the onset of symptoms, as well as reducing the negative effects of an established disease by restoring function and reducing disease-related complications.

[0518] Targeted domain

[0519] In one embodiment, the composition includes a targeting domain that directs the delivery vector to a site. In one embodiment, the site is a site for a reagent that needs to be contained within the delivery vector. The targeting domain may include nucleic acids, peptides, antibodies, small molecules, organic molecules, inorganic molecules, glycans, sugars, hormones, etc., which target the particles to a site that specifically requires a therapeutic agent. In some embodiments, the particles include multivalent targets, wherein the particles incorporate multiple targeting mechanisms described herein. In some embodiments, the targeting domain of the delivery vector specifically binds to a target associated with a site for which a reagent contained within the delivery vector is required. 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 specific disease state. Such a target may be a protein, protein fragment, antigen, or other biomolecule associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand that specifically binds to a target. In some embodiments, the target (e.g., an antigen) is associated with a site that requires treatment with a reagent. In some embodiments, the targeting domain may copolymerize with the composition containing the delivery vector. In some embodiments, the targeting domain may be covalently attached to a composition comprising a delivery vector, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vector. In some embodiments, the targeting domain is an additive in the delivery vector. The targeting domains of the present invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0520] In various embodiments, the targeting domain binds to cell surface molecules of the cell of interest. For example, in various embodiments, the targeting domain binds to cell surface molecules of endothelial cells, stem cells, or immune cells.

[0521] Cong

[0522] In one embodiment, the targeting domain of the present invention comprises a peptide. In some embodiments, the peptide targeting domain specifically binds to a target of interest.

[0523] The peptides of this invention can be prepared using chemical methods. For example, the peptides can be synthesized using solid-phase techniques (Roberge JY et al. (1995) Science 269:202-204), cleaved from resin, and purified by preparative high-performance liquid chromatography. For example, automated synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions.

[0524] Alternatively, peptides can be prepared using recombinant methods or by cleaving longer peptides. The composition of the peptide can be confirmed by amino acid analysis or sequencing.

[0525] Variants of the peptide according to the invention may be (i) variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code; (ii) variants in which one or more modified amino acid residues are present, such as residues modified by attachment of substituents; (iii) variants in which the peptide is an alternative splice variant of the peptide of the invention; (iv) fragments of the peptide; and / or (v) variants in which the peptide is fused with another peptide (e.g., a leader sequence or secretion sequence or a sequence for purification (e.g., a His tag) or a sequence for detection (e.g., an Sv5 epitope tag)). These fragments include peptides produced by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of those skilled in the art based on the teachings herein.

[0526] As is known in the art, the “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 as a peptide sequence that differs from the original sequence, preferably by less than 40% residues / target segment, more preferably by less than 25% residues / target segment, even more preferably by less than 10% residues / target segment, and even more preferably by only a few residues / target segment, while simultaneously being sufficiently homologous to the original sequence to preserve its functionality. This invention includes amino acid sequences having at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similarity or identity to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. Preferably, the identity between two amino acid sequences is determined by 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)].

[0527] The peptides of this invention can undergo post-translational modifications. For example, post-translational modifications falling within the scope of this invention include signal peptide cleavage, glycosylation, acetylation, isoprenelation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modification or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation can be examined by adding canine microsomal membranes or Xenopus laevis egg extract (US Patent No. 6,103,489) to a standard translation reaction.

[0528] The peptides of the present invention may include non-natural amino acids formed by post-translational modification or by introducing non-natural amino acids during translation.

[0529] Nucleic acid

[0530] In one embodiment, the targeting domain of the present invention comprises isolated nucleic acids, including, for example, DNA oligonucleotides and RNA oligonucleotides. In some 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.

[0531] Alternatively, the nucleotide sequence of the nucleic acid targeting domain may contain sequence variations relative to the original nucleotide sequence, such as substitutions, insertions, and / or deletions of one or more nucleotides, provided that the resulting nucleic acid functions as the original nucleic acid and specifically binds to the target of interest.

[0532] In the sense used herein, a nucleotide sequence is considered "substantially homologous" to any nucleotide sequence described herein when it has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with respect to such a sequence. Other examples of possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides to either end of the sequence, or the deletion of one or more nucleotides at either 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. Preferably, the identity between two amino acid sequences is 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)].

[0533] Antibody

[0534] In one embodiment, the targeting domain of the present invention comprises an antibody or antibody fragment. In some embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and its single-chain Fv (scFv) fragments, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0535] Antibodies can be complete 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. Patent No. 4,946,778), or chimeric antibodies, such as antibodies containing binding specificity of mouse antibodies, but the remainder of which is of human origin. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art.

[0536] These antibodies can be produced in various ways, including hybridoma cultures, recombinant expression in bacterial or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing method depends on a number of factors, including the desired antibody structure, the importance of the carbohydrate moiety to the antibody, the 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-sized antibody fragments, such as Fab and Fv fragments, that lack effector function and have limited pharmacokinetic activity can be produced in bacterial expression systems. Single-chain Fv fragments exhibit low immunogenicity.

[0537] adjuvant

[0538] 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.

[0539] Exemplary adjuvants include, but are not limited to, interferon-alpha, interferon-gamma, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell attraction chemokine (CTACK), thymic chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with a missing signal sequence and optionally including a signal peptide from IgE). Other genes that may be useful adjuvants include those encoding the following: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-I, VLA-I, Mac-1, p150.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, and ICE (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 gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig, and their functional fragments.

[0540] Antibody therapy

[0541] The present invention also envisions a delivery vector comprising a target-specific antibody or antibody fragment. That is, the antibody can inhibit the target to provide a beneficial effect.

[0542] Antibodies can be complete 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. Patent No. 4,946,778), or chimeric antibodies (e.g., antibodies containing binding specificity of mouse antibodies but with the remainder derived from human sources). Antibodies (including monoclonal and polyclonal antibodies, fragments, and chimeras) can be prepared using methods known to those skilled in the art.

[0543] Antibodies can be prepared using complete polypeptides or fragments containing the immunogenic antigen of interest. Polypeptides or oligopeptides used for immunizing animals can be obtained from RNA translation or chemically synthesized, and (if desired) can be conjugated to a carrier protein. Suitable carriers that can be chemically conjugated to peptides include bovine serum albumin and thyroglobulin, and keyhole hemocyanin. The conjugated polypeptide can then be used to immunize animals (e.g., mice, rats, or rabbits).

[0544] antigen

[0545] This invention provides a composition for inducing a therapeutic response in a subject. In one embodiment, the composition comprises an antigen. In another embodiment, the composition comprises a nucleic acid sequence encoding the antigen. For example, in some embodiments, the composition comprises nucleoside-modified RNA encoding the antigen. The antigen can be any molecule or compound, including but not limited to polypeptides, peptides, or proteins that induce a therapeutic response (e.g., an adaptive immune response) in a subject.

[0546] In one embodiment, the antigen comprises a pathogen-associated polypeptide or peptide, such that the antigen induces an adaptive immune response against the antigen and thus against the pathogen. In another embodiment, the antigen comprises a fragment of a pathogen-associated polypeptide or peptide, such that the antigen induces an adaptive immune response against the pathogen.

[0547] In some embodiments, the antigen comprises an amino acid sequence substantially homologous to the amino acid sequence of the antigen described herein and retaining the immunogenic function of the original amino acid sequence. For example, in some embodiments, the amino acid sequence of the antigen has at least 60%, advantageously at least 70%, preferably at least 85%, and more preferably at least 95% identity with respect to the original amino acid sequence.

[0548] In one embodiment, the antigen is encoded by a nucleic acid sequence of a nucleic acid molecule. In some embodiments, the nucleic acid sequence comprises DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. In one embodiment, the nucleic acid sequence includes 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. In some cases, the nucleic acid sequence includes an additional sequence encoding a adapter or tag sequence that is linked to the antigen via a peptide bond.

[0549] In some embodiments, the antigen encoded by the nucleoside-modified nucleic acid molecule comprises proteins, peptides, fragments thereof, variants thereof, or combinations thereof from any number of organisms (e.g., viruses, parasites, bacteria, fungi, or mammals). For example, in some embodiments, the antigen is associated with autoimmune diseases, allergic reactions, 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 some embodiments, the antigen comprises a common sequence based on the amino acid sequences of two or more different organisms. In some embodiments, the nucleic acid sequence encoding the antigen is optimized for efficient translation in the organism in which the composition is delivered.

[0550] In one embodiment, the antigen includes a tumor-specific antigen or a tumor-associated antigen, such that the antigen induces an adaptive immune response against the tumor. In another embodiment, the antigen includes 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 some embodiments, the tumor-specific antigen or tumor-associated antigen is a mutant variant of a host protein.

[0551] Viral antigens

[0552] In one embodiment, the antigen includes a viral antigen, a fragment thereof, or a variant thereof. In some embodiments, the viral antigen is derived from viruses belonging to one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepatoviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Clonorviridae. In some embodiments, the viral antigen is derived from papillomaviruses, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), poliovirus, hepatitis B virus, hepatitis C virus, smallpox virus (major and minor), vaccinia virus, influenza virus, rhinovirus, dengue virus, equine encephalitis virus, rubella virus, yellow fever virus, and 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, Ebola virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex virus type 1 (oral herpes), herpes simplex virus type 2 (genital herpes), herpes zoster (varicella-zoster virus), 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 oncogenic virus.

[0553] parasite antigen

[0554] In some embodiments, the antigen includes a parasitic antigen or a fragment or variant thereof. In some embodiments, the parasite is a protozoan, worm, or ectoparasite. In some embodiments, the worm (i.e., the worm) is a flatworm (e.g., a fluke and a tapeworm), a thorny-headed worm, or a roundworm (e.g., a pinworm). In some embodiments, the ectoparasite is a lice, a flea, a tick, or a mite.

[0555] In some implementations, the parasite is any parasite that causes the following diseases: Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, balantidiasis, Bayliss scariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Sparganosis, Gynematode infection, Echinococcus infection, elephantiasis, pinworm infection, Fascioliasis, Fascioliasis, filariasis, Giardiasis, Gnathostomiasis, Hymenolepis, Isosporidiosis, Katayama fever, leishmaniasis, Lyme disease, malaria, Metaclonorchiasis, myiasis, Onchocerciasis, lice infestation, scabies, schistosomiasis, and sleeping sickness. (Sickness), Strongyloides disease, Taeniasis, Toxocara disease, Toxoplasmosis, Trichinosis, and Whipworm disease.

[0556] In some embodiments, the parasites are Acanthamoeba, Anisakis, Ascaris, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chigger mite, Spiraling test fly, Entamoeba Histolytica, Fasciolopsis buski, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loaloa, Paragonimus-lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosome, Whipworm, or Wuchereria bancrofti. bancrofti).

[0557] bacterial antigens

[0558] In one embodiment, the antigen includes a bacterial antigen or a fragment or variant thereof. In some embodiments, the bacteria are from any of the following phyla: Acidobacteria, Actinobacteria, Aquagenic Bacteria, Bacteroidetes, Thermophilic Bacteria, Chlamydia, Chlorobacteria, Chlorophytes ...

[0559] In some embodiments, the bacteria are Gram-positive or Gram-negative. In some embodiments, the bacteria are aerobic or anaerobic. In some embodiments, the bacteria are autotrophic or heterotrophic. In some embodiments, the bacteria are thermophiles, neutrophils, extremophiles, acidophiles, alkaliphiles, thermophiles, psychrophiles, halophiles, or hyperosmophiles.

[0560] In some embodiments, the bacteria are Bacillus anthracis, antibiotic-resistant bacteria, pathogenic bacteria, food poisoning bacteria, infectious bacteria, Salmonella, Staphylococcus, Streptococcus, or Clostridium tetani. In some embodiments, the bacteria are mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0561] Fungal antigens

[0562] In one embodiment, the antigen includes a fungal antigen or a fragment or variant thereof. In some embodiments, the fungus is an Aspergillus species, a Blastomyces dermatitidis genus, Candida yeast (e.g., Candida albicans), a Coccidioides genus, Cryptococcus neoformans, Cryptococcus gattii, dermatophytes, Fusarium genus, Histoplasma capsulatum, Mucormycetes, Pneumocystis jirovecii, Sporothrix schenckii, Helicobacter genus, or Cladosporium genus.

[0563] Tumor antigens

[0564] In some embodiments, the antigen includes tumor antigens, including, for example, tumor-associated antigens or tumor-specific antigens. In the context of this invention, "tumor antigen," "hyperplastic disease antigen," or "antigen associated with a hyperplastic disease" refers to an antigen commonly found in a particular hyperplastic disease. In some aspects, the hyperplastic disease antigens of this invention are derived from cancer, including but not limited to primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma (such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer), etc.

[0565] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T-cell-mediated immune response. In one embodiment, the tumor antigen of the present invention comprises one or more antigenic oncotopes 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 being treated or prevented by the compositions of the present invention.

[0566] Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mutHsp70-2, 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, liver glycoside B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0567] In one implementation, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignancy. Malignant tumors express a number of 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 GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to a single tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for monoclonal antibody passive immunotherapy with limited success.

[0568] The tumor antigen types mentioned in this invention can also be tumor-specific antigens (TSA) or tumor-associated antigens (TAAs). TSAs are unique to tumor cells and do not occur on other cells in the body. TAA-associated antigens are not unique to tumor cells but are also expressed on normal cells under conditions that do not induce an immune tolerance state to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they can be antigens that are normally present in normal cells at very low levels but expressed at much higher levels in tumor cells.

[0569] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multipotent antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, 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, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, 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 / cyclic protein C-related protein, TAAL6, TAG72, TLP, and TPS.

[0570] In a preferred embodiment, the antigens include, but are not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1TCR, MAGE A3 TCR, etc.

[0571] combination

[0572] In one embodiment, the composition of the present invention comprises a combination of the reagents described herein. In some embodiments, the composition comprising the combination of the reagents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual reagent. In other embodiments, the composition comprising the combination of the reagents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual reagent.

[0573] Compositions containing a combination of reagents may contain individual reagents in any suitable proportions. For example, in one embodiment, the composition contains two individual reagents in a 1:1 ratio. However, the combination is not limited to any particular ratio. Rather, any ratio that has proven effective is included.

[0574] Adhesion

[0575] In various embodiments of the invention, the delivery carrier is conjugated to the target domain. Exemplary methods of conjugation may include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic (“van der Waals”) interactions. In one embodiment, the conjugation is reversible, such that the delivery carrier can dissociate from the target domain upon exposure to certain conditions or chemical reagents. In another embodiment, the conjugation is irreversible, such that the delivery carrier does not dissociate from the target domain under normal conditions.

[0576] In some embodiments, the conjugation includes 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 moiety and a polymer moiety activated by functionalizing the polymer-conjugated lipid with a first coupling group. In one embodiment, the activated polymer-conjugated lipid includes a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer-conjugated lipid is an activated polyethylene glycol-modified lipid. In one embodiment, the first coupling group is bound to the lipid moiety of the polyethylene glycol-modified lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol moiety of the polyethylene glycol-modified lipid. In one embodiment, a second functional group is covalently attached to the targeting domain.

[0577] The first and second coupling groups can be any functional groups known to those skilled in the art to 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: maleimide, N-hydroxysuccinimide (NHS) ester, carbodiimide, acylhydrazine, pentafluorophenyl (PFP) ester, phosphine, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, vinyl sulfone, α-haloacetyl, aryl azide, acyl azide, alkyl azide, diaziridine, benzophenone, epoxide, carbonate, acid anhydride, sulfonyl chloride, cyclooctyne, aldehyde, and mercapto. In some embodiments, the first or second coupling group is selected from the group consisting of: free amine (-NH2), free mercapto (-SH), free hydroxide group (-OH), carboxylate, acylhydrazine, and alkoxyamine. In some embodiments, the first coupling group is a functional group reactive to a thiol group, such as maleimide, pyridyl disulfide, or haloacetyl. In one embodiment, the first coupling group is maleimide.

[0578] In one embodiment, the second coupling group is a thiol group. The thiol group can be mounted on the target domain using any method known to those skilled in the art. In one embodiment, the thiol group is present on a free cysteine ​​residue. In one embodiment, the thiol group is revealed by the reduction of a disulfide on the target domain (e.g., by reaction with 2-mercaptoethylamine). In one embodiment, the thiol group is mounted by a chemical reaction, such as a reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0579] In some embodiments, the polymer-conjugated lipids and targeting domains are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves reactions between functional groups having 1,3-dipolar polarity (such as azides, nitrile oxides, nitrones, isocyanates, etc.) and linkers (having olefin or alkyne dipolarophilic molecules). Exemplary dipolarophilic molecules include any strain of cyclic alkenes and cyclic alkynes known to those skilled in the art, including but not limited to cyclooctyne, dibenzocyclooctyne, monofluorinated cyclooctyne, difluorinated cyclooctyne, and diarylazirazine octanone.

[0580] Pharmaceutical Composition

[0581] The pharmaceutical compositions described herein can be formulated by any method known or subsequently developed in the field of pharmacology. Typically, such formulations involve the step of associating the active ingredient with a carrier or one or more other excipient components, and then, if necessary or desired, shaping or packaging the product into the desired single- or multi-dose units.

[0582] Although the description of the pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all species of animals. Modifications to pharmaceutical compositions suitable for human administration are well known to be made to make them suitable for administration to a variety of animals, and such modifications can be designed and performed by ordinary technical veterinary pharmacologists through routine (if any) experiments. Subjects considering administration of the pharmaceutical compositions of the present invention include, but are not limited to, humans and other primates and mammals, including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0583] Pharmaceutical compositions that can be used in the methods of this invention can be prepared, included, or marketed in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, oral, intravenous, intraventricular, intradermal, intramuscular, or other routes of administration. Other formulations considered include anticipated nanoparticles, liposome formulations, resealed red blood cells containing the active ingredient, and immunogenicity-based formulations.

[0584] The pharmaceutical compositions of the present invention can be prepared, packaged, or marketed as a single unit dose or in batches of multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a suitable fraction of that dose, such as one-half or one-third of the dose.

[0585] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, body type, and condition of the subject being treated and, more importantly, on the route of administration of the composition. For example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0586] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharmaceutical active agents.

[0587] The controlled-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques.

[0588] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physically destroying the subject’s tissue and administering the pharmaceutical composition through a tear in the tissue. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, application through a surgical incision, or application through a non-surgical wound that penetrates the tissue. In particular, parenteral administration considers, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and renal dialysis infusion techniques.

[0589] Pharmaceutical compositions suitable for parenteral administration are formulated to contain an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or marketed in a form suitable for bolus or continuous administration. Injectable formulations can be prepared, packaged, or marketed in unit doses, such as in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations in oily or aqueous carriers. Such formulations may further contain one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0590] Pharmaceutical compositions can be prepared, packaged, or marketed as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated using known techniques and may contain additional components besides the active ingredient, such as dispersants, wetting agents, or suspending agents described herein. For example, such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents (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 fixative oils, such as synthetic monoglycerides or diglycerides. Other useful parenterally applicable formulations include those containing the active ingredient in microcrystalline form, in liposome formulations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, microsoluble polymers, or microsoluble salts.

[0591] The pharmaceutical compositions of the present invention can be prepared, packaged, or marketed in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles containing the active ingredient and having a diameter ranging from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. This composition is conveniently available in dry powder form for administration using a device comprising a dry powder reservoir (to which a propellant flow can be directed to disperse the powder), or using a self-propelled solvent / powder dispensing container (such as a device containing the active ingredient dissolved or suspended in a low-boiling-point propellant in a sealed container). Preferably, the powder comprises particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nanometers and at least 95% by number of the particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter less than 6 nanometers. The dry powder composition preferably comprises a solid fine powder diluent (such as sugar) and is conveniently provided in unit dose form.

[0592] Low-boiling-point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant can constitute 50 to 99.9% (w / w) of the composition, and the active ingredient can constitute 0.1 to 20% (w / w) of the composition. The propellant may further include additional components such as liquid nonionic or solid anionic surfactants or solid diluents (preferably having a particle size on the same order of magnitude as the particles containing the active ingredient).

[0593] Pharmaceutical compositions suitable for parenteral administration are formulated to contain an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or marketed in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or marketed in unit doses, such as in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations in oily or aqueous carriers. Such formulations may further contain one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0594] Pharmaceutical compositions can be prepared, packaged, or marketed as sterile, injectable aqueous or oily suspensions or solutions. Suspensions or solutions can be formulated using known techniques and may contain additional components besides the active ingredient, such as dispersants, wetting agents, or suspending agents described herein. For example, such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents (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 fixative oils, such as synthetic monoglycerides or diglycerides. Other useful parenterally applicable formulations include those containing the active ingredient in microcrystalline form, in liposome formulations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, microsoluble polymers, or microsoluble salts.

[0595] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; biodegradable compositions such as gelatin; aqueous carriers and solvents; oily carriers and solvents; suspending agents; dispersants or wetting agents; emulsifiers, demulcents; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the present invention are known in the art and described, for example, in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which are incorporated herein by reference.

[0596] Treatment

[0597] This invention provides a method for delivering a reagent to the cells, tissues, or organs of a subject. In some embodiments, the reagent is a diagnostic reagent for detecting at least one biomarker associated with a disease or condition. In some embodiments, the reagent is a therapeutic agent for treating or preventing a disease or condition. Therefore, in some embodiments, this invention provides a method for diagnosing, treating, or preventing a disease or condition, comprising administering an effective amount of a composition comprising one or more diagnostic or therapeutic agents, one or more adjuvants, or a combination thereof.

[0598] In some embodiments, the method provides a subject with immunity against an antigen-associated infection, disease, or condition. Therefore, the present invention provides methods for treating or preventing antigen-associated infections, diseases, or conditions. For example, the method can be used to treat or prevent viral infections, bacterial infections, fungal infections, parasitic infections, or cancer, depending on the type of antigen in the applied composition. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0599] In one embodiment, the composition is administered to a subject who has an antigen-related infection, disease, or cancer. In another embodiment, the composition is administered to a subject who is at risk of developing an antigen-related infection, disease, or cancer. For example, the composition may be administered to a subject who is at risk of exposure to viruses, bacteria, fungi, parasites, etc. In yet another embodiment, the composition is administered to a subject who has an increased likelihood of developing cancer (despite genetic factors, environmental factors, etc.).

[0600] In one embodiment, the method includes administering a composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens and one or more adjuvants. In another embodiment, the method includes 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 yet another embodiment, the method includes administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more antigens, and administering a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0601] In some implementations, the method includes administering to a subject a nucleic acid molecule modified with multiple nucleosides that encodes multiple antigens, adjuvants, or combinations thereof.

[0602] In some embodiments, the method of the present invention allows the antigen or adjuvant described herein to be expressed continuously for at least several days after administration. However, in some embodiments, the method also provides transient expression because, in some embodiments, the nucleic acid is not integrated into the subject's genome.

[0603] In some embodiments, the method includes administering nucleoside-modified RNA that provides stable expression of the antigen or adjuvant described herein. In some embodiments, the administration of nucleoside-modified RNA results in little to no innate immune response while inducing an effective adaptive immune response.

[0604] The administration of the compositions of the present invention in therapeutic methods can be achieved in a variety of different ways using methods known in the art. In one embodiment, the method of the present invention includes systemic administration to a subject, including, for example, enteric or parenteral administration. In some embodiments, the method includes intradermal delivery of the composition. In another embodiment, the method includes intravenous delivery of the composition. In some embodiments, the method includes intramuscular delivery of the composition. In one embodiment, the method includes subcutaneous delivery of the composition. In one embodiment, the method includes inhalation of the composition. In one embodiment, the method includes intranasal delivery of the composition.

[0605] It should be understood that the compositions of the present invention can be administered to the subject alone or in combination with another reagent.

[0606] Therefore, the treatment and prevention methods of the present invention include the use of pharmaceutical compositions encoding the antigens, adjuvants, or combinations thereof described herein for carrying out the methods of the present invention. Pharmaceutical compositions that can be used to carry out the present invention may be administered to deliver doses from ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates that the administered dose results in a concentration of the compound of the present invention in mammals of 10 nM to 10 μM.

[0607] Typically, in the method of the present invention, the dosage that can be applied to mammals (preferably humans) ranges from 0.01 μg to about 50 mg per kilogram of mammalian body weight, and the precise dosage will depend on many factors, including but not limited to the type of mammal and the type of disease state being treated, the age of the mammal, and the route of administration. Preferably, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. More preferably, the dosage will vary from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0608] The composition may be administered to mammals several times a day, or less frequently (e.g., once a day, once a week, once every two weeks, once a month), or even less frequently (e.g., once every few months, or even once a year or less). The frequency of dosing will be apparent to those skilled in the art and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0609] In some embodiments, the immunogenic composition or vaccine of the present invention may be administered by a single administration or by multiple administrations.

[0610] In one embodiment, the invention includes a method of administering one or more compositions encoding one or more antigens or adjuvants described herein. In some embodiments, the method has an additive effect, wherein 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, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant.

[0611] Treatment methods for SARS-CoV-2

[0612] In one embodiment, the present invention provides a method for inducing an adaptive immune response against SARS-CoV-2 in a subject, comprising administering an effective amount of a composition comprising an amphiphilic Janus dendritic polymer of formula (I) and one or more isolated nucleic acids encoding one or more SARS-CoV-2 antigens.

[0613] In one embodiment, the method provides a subject with immunity against SARS-CoV-2, SARS-CoV-2 infection, or a disease or condition associated with SARS-CoV-2. Therefore, the present invention provides a method for treating or preventing SARS-CoV-2-related infections, diseases, or conditions. In one embodiment, the SARS-CoV-2-related disease or condition is COVID-19 or a comorbidity of COVID-19.

[0614] In some embodiments, the present invention is a method of administering a composition to a subject, the composition comprising at least one nucleoside-modified RNA encoding at least one SARS-CoV-2 antigen.

[0615] In one embodiment, the composition is administered to a subject who has a SARS-CoV-2-related infection, disease, or condition. In another embodiment, the composition is administered to a subject at risk of developing a SARS-CoV-2-related infection, disease, or condition. For example, the composition may be administered to a subject at risk of exposure to SARS-CoV-2. In one embodiment, the composition is administered to a subject who lives in, travels 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 in contact with, or is expected to be in contact with, another person in a geographic area where SARS-CoV-2 is prevalent. In one embodiment, the composition is administered to a subject who has been knowingly exposed to SARS-CoV-2 through their occupation or contact.

[0616] In one embodiment, the method includes administering a composition comprising at least one dendritic polymer of formula (I) and 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 includes 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 includes administering a first composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more SARS-CoV-2 antigens, and administering a second composition comprising one or more nucleoside-modified nucleic acid molecules encoding one or more adjuvants.

[0617] In some implementations, the method includes administering to a subject a nucleic acid molecule modified with multiple nucleosides that encodes multiple SARS-CoV-2 antigens, adjuvants, or combinations thereof.

[0618] In some embodiments, the method of the present invention allows the SARS-CoV-2 antigen or adjuvant described herein to be persistently expressed for at least several days after administration. In some embodiments, the method of the present invention allows the SARS-CoV-2 antigen or adjuvant described herein to be persistently expressed for at least 2 weeks after administration. In some embodiments, the method of the present invention allows the SARS-CoV-2 antigen or adjuvant described herein to be persistently expressed for at least 1 month after administration. However, in some embodiments, the method also provides transient expression because, in some embodiments, the nucleic acid is not integrated into the subject's genome.

[0619] In some embodiments, the method includes administering nucleoside-modified RNA that provides stable expression of the SARS-CoV-2 antigen or adjuvant described herein. In some embodiments, the administration of nucleoside-modified RNA results in little to no innate immune response while inducing an effective adaptive immune response.

[0620] In some implementations, the method provides sustained protection against SARS-CoV-2. For example, in some implementations, the method provides sustained protection against SARS-CoV-2 for more than 2 weeks. In some implementations, the method provides sustained protection against SARS-CoV-2 for 1 month or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 2 months or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 3 months or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 4 months or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 5 months or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 6 months or longer. In some implementations, the method provides sustained protection against SARS-CoV-2 for 1 year or longer.

[0621] In one embodiment, a single immunization of the composition provides sustained protection against SARS-CoV-2 for 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, or 1 year or longer.

[0622] The administration of the compositions of the present invention in therapeutic methods can be achieved in a variety of different ways using methods known in the art. In one embodiment, the method of the present invention includes systemic administration to a subject, including, for example, enteric or parenteral administration. In some embodiments, the method includes intradermal delivery of the composition. In another embodiment, the method includes intravenous delivery of the composition. In some embodiments, the method includes intramuscular delivery of the composition. In one embodiment, the method includes subcutaneous delivery of the composition. In one embodiment, the method includes inhalation of the composition. In one embodiment, the method includes intranasal delivery of the composition.

[0623] It should be understood that the compositions of the present invention can be administered to the subject alone or in combination with another reagent.

[0624] Therefore, the treatment and prevention methods of the present invention include the use of pharmaceutical compositions encoding the SARS-CoV-2 antigen, adjuvant, or combinations thereof described herein for carrying out the methods of the present invention. Pharmaceutical compositions that can be used to carry out the present invention may be administered to deliver doses from 1 ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates that the administered dose results in a concentration of the compound of the present invention of 10 nM to 10 μM in mammals.

[0625] Typically, in the methods of this invention, the dosage that can be administered to mammals (e.g., humans) ranges from 0.01 μg to about 50 mg per kilogram of mammalian body weight, and the precise dosage will depend on many factors, including but not limited to the type of mammal and the type of disease state being treated, the age of the mammal, and the route of administration. In some embodiments, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of mammalian body weight. In some embodiments, the dosage will vary from about 1 μg to about 1 mg per kilogram of mammalian body weight.

[0626] The composition may be administered to mammals several times a day, or less frequently (e.g., once a day, once a week, once every two weeks, once a month) or even less frequently (e.g., once every few months or even once a year or less). The dosage frequency will be apparent to those skilled in the art and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0627] In some embodiments, the immunogenic composition or vaccine of the present invention may be administered by a single administration or by multiple administrations.

[0628] In one embodiment, the invention includes a method of administering one or more compositions encoding one or more SARS-CoV-2 antigens or adjuvants described herein. In some embodiments, the method has an additive effect, wherein 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, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each SARS-CoV-2 antigen or adjuvant.

[0629] Experimental Examples

[0630] The invention is described in further detail with reference to the following experimental embodiments. Unless otherwise specified, these embodiments are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be construed as limited to the following embodiments, but should be interpreted as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0631] Without further description, it is believed that those skilled in the art can make and utilize the invention and implement the claimed methods using the foregoing description and the following illustrative embodiments. Therefore, the following working examples should not be construed as limiting the remainder of this disclosure in any way.

[0632] Example 1: Ionizable amphiphilicity defined by a single-component multifunctional sequence for mRNA used in vaccines and drugs Janus Dendritic Polymer (IAJD) Delivery System

[0633] This invention relates in part to a single-component, multifunctional, sequence-defined, ionizable amphiphilic Janus dendritic polymer (IAJD) delivery system, which is achieved by simple injection into dendritic polymer nanoparticles (DNPs) co-assembled with mRNA. Figure 1 In vitro and in vivo screening experiments were conducted using six libraries containing 52 IAJDs. These studies demonstrated proof of concept for DNPs, their potential applications, and their role as a model for elucidating fundamental aspects of nonviral vectors.

[0634] One of the main advantages of synthetic vectors used for mRNA delivery is their unlimited synthetic capacity. (The text then abruptly shifts to a seemingly unrelated topic: "A four-component LNP (...") Figure 2 The conversion to a single-component DNP represents proof of this synthetic capability.

[0635] Figure 2 The assembly process involving a four-component LNP is illustrated. A four-component composition containing varying proportions of ionizable lipids, phospholipids, PEG lipids, and cholesterol was prepared as a solution in ethanol. This ethanol solution was mixed with a microfluidic device containing a buffer solution with pH 3 to 5 and an aqueous solution containing mRNA. The mRNA was generated for use in the acidic buffer solution and stored in neutral water. The resulting nanoparticles were analyzed by dynamic light scattering (DLS) to determine the diameter (D, nm) and polydispersity (PDI) of the LNPs. The nanoparticles were dialyzed to pH 7.4, analyzed again by DLS, and stored at -70°C before in vitro or in vivo experiments. Figure 1 The same procedure for single-component DNPs is shown. IAJDs containing ionizable amines, incorporated in precise order, are dissolved in ethanol. The ethanol solution is then infused into an acidic buffer solution (pH 3 to 5.2) containing mRNA. Depending on the initial pH of the buffer, the resulting mRNA-containing DNPs have a pH of 4.5 to 7.3 and can be used for in vitro and in vivo experiments after DLS analysis without dialysis. DNPs can be stored long-term at 5°C. Figure 1The process of LNP and DNP transitioning from the extracellular to the intracellular environment is also illustrated. Upon injection, both LNP and DNP approach the corresponding cell and are encapsulated via endocytosis. The extracellular pH is 7.4, and thus LNP and DNP enter the cell through a nearly neutral surface. Endocytosis of either LNP or DNP then deposits them into the endosome, where the pH decreases from 6.8 to 4.5 during their transformation into lysosomes due to ATP-dependent proton pumps on the endosome membrane (Huotari and Helenius, EMBO J. 2011, 30, 3481-3500). Consequently, LNP and DNP undergo reprotonation, interacting with naturally occurring anionic lipids and releasing mRNA that helps the ribosome produce new proteins. While ionizable amines from LNPs dissociate at their centers, reprotonation does not occur at their peripheries, and therefore the release of mRNA into the cell is very inefficient (1-2%).

[0636] Accelerated Modular-Orthogonal Synthesis of Six Libraries from IAJD: This method utilizes a modular orthogonal approach to synthesize sequence-defined amphiphilic Janus sugar dendritic polymers (JGD). An accelerated modular orthogonal method is employed for synthesizing single-to-single (a single hydrophobic dendritic unit combined with a single hydrophilic dendritic unit), double-double (two identical hydrophobic dendritic units and two identical hydrophilic dendritic units), and heterodouble-mixture (two identical hydrophobic dendritic units and two distinct hydrophilic dendritic units). Figure 3 and Figure 4 This method relies on the relevant but improved and accelerated synthetic principles of JGD, which were originally used to define the synthetic sequences. 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 as follows: Figure 4 Schematic illustration. Four of these libraries are based on single-single IAJDs. Library 1 has 9 IAJDs, Library 2 has 7 IAJDs, Library 3 has 6 IAJDs, and Library 4 has 3 IAJDs available. Library 5 is based on a bis-bis IAJD generated from IAJD1 to IAJD9 of Library 1 and JAJD33 of Library 3. Library 6 contains 17 hetero-bis-mixed IAJDs selected from all libraries. The selection process was determined by the in vivo and in vitro activity of their single-single components. The hydrophilic moieties of these IAJDs contain compositions defined by the sequence of ionizable amines based on dimethylaminobutyrate (DMBA), dimethylaminopropionate (DMPA), dimethylaminoacetic acid ester (DMA), piperidine butyrate (PIP), and methylpiperazine butyrate (MPRZ). Figure 3 These are based on the pK values ​​of their corresponding ionizable lipids, which are available in the literature.a The selected (Ramishetti, et al., Adv. Mater. 2020, 32, 1906128; Kim, et al., Sci. Adv. 2021, 7, eabf4398). The symbols used in the schematic diagram of these sequence-defined IAJDs are as follows: Figure 4 The square at the bottom is shown. The benzyl ether group marked in red is used to separate ionizable amines and to construct different sequences in the hydrophilic part of the IAJD. In biology and synthetic supramolecular chemistry, aromatic groups (such as benzyl ethers) have been proposed as hydrogen bond acceptors in molecular recognition, interacting with cations including ammonium groups (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 conventional hydrogen bonding (approximately 3 kcal / mol) and therefore can mediate the pK of ionizable amines. a Dynamic control. In the protonated state of the amine, it can increase its pK. a In its deprotonated state, it can promote the interaction between benzyl ether and the nucleic acid bases of mRNA to enhance co-assembly, or to separate within the hydrophobic moiety of DNP. This cation-π interaction has not been previously used in delivery vectors for mRNA. The hydrophobic moiety of IAJD contains straight-chain and branched alkyl groups of varying lengths. Figure 3 and Figure 4 These IAJD hydrophilic acid components, such as Figure 3 (Module A) is shown; their synthesis is described elsewhere in this document. The structure of hydrophobic benzylamine is shown below. Figure 3 (Module C) is shown. Their composition is also described elsewhere in this paper. All these modules are combined in an orthogonal manner (such as...). Figure 4 (As shown) Six libraries of IAJD are provided in an accelerated manner. The synthesis of bi-bipartite hydrophobic IAJDs is described elsewhere in this paper. Detailed structures of all IAJD libraries and their pK values ​​are also provided. a Values ​​and abbreviations such as Figure 12 (Document 1, 2, 3, 4) Figure 13 (Document 5) and Figure 14 As shown in (Document 6).

[0637] In vitro transfection activity of DNP in human embryonic kidney (HEK) 293T cells: HEK293T cells were seeded into 96-well plates (20,000 cells / well / 200 μL) and cultured in complete cell culture medium at 37°C and 5% CO2 for 24 h. Screening was performed using unoptimized DNP containing naked nucleotide-modified mRNA with a molar mass of 664,341 encoding firefly luciferase (mRNA-Luc) (Karikó, et al., Immunity 2005, 23, 165-175; Pardi, et al., Methods Mol. Biol. 2013, 969, 29-42). A constant concentration of 125 ng / well of mRNA-Luc was used. transIT (from MirusBio's TransIT-mRNA Transfection Kit) and MC3-based LNP (MC3: DLin-MC3-DMA, an FDA-approved LNP for mRNA delivery) were used as positive controls for cell transfection. The concentration of mRNA-Luc was 125 ng / well, the same as the concentration of DNP tested. Cells were then cultured for 24 hours, and the culture medium was aspirated under reduced pressure using glass pipettes, followed by lysis with 30 μL / well of cell culture lysis reagent (Promega). The luminescence intensity corresponding to the expressed luciferase protein was characterized and analyzed.

[0638] Figure 4 All 52 IAJDs were co-assembled by injecting mRNA into DNPs and used in these experiments. Tables 1 through 5 (see below) summarize the conditions used to prepare the DNPs, their diameter (D, in nm) and polydispersity (PDI) obtained by DLS experiments, and the 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.

[0639] In vivo mRNA delivery of DNP in mice: Six- to eight-week-old female mice were used in these experiments. Four to seven hours after injection of 100 μL of a DNP solution containing 10 μg mRNA-Luc, mice were imaged 10 minutes after intraperitoneal injection of 10 μL / g body weight of 15 mg / mL D-luciferin. Exposure time was 1 minute. For organ imaging, mice were euthanized, organs were immediately collected, and bioluminescent imaging was performed. Table 7 (see below) summarizes all DNP injection assembly data, including D and PDI in nm as determined by DLS, and results obtained in vivo. Figure 6 All mouse experiments were summarized, including D and PDI in nm for DNP (both indicated in black at the top of mouse images), and pK for the corresponding IAJD of all compounds used for delivery. a Values ​​(also indicated in blue at the top). Figure 6 and Figure 7 The results provide proof of concept for an ionizable amphiphilic Janus dendritic polymer delivery system for mRNA with a single-component, multifunctional sequence definition.

[0640] Figure 7 The results concluded that there appeared to be no correlation between in vivo DNP activity and the same in vitro experiments. Figure 5 Furthermore, the most active IAJDs 33, 34, and 31 have the lowest pK values. a value. Figure 6 The results also confirmed that the activity of DNPs is quite tolerant of their diameter and polydispersity. DNPs with a diameter greater than 100 nm appear to have the same activity as DNPs with a diameter less than 100 nm. Figure 6 However, the activity and pK must be studied. a Quantitative correlation of the size dependence of DNPs was investigated. The stability of the size of 40 DNPs as a function of time at 5 °C was studied. Unoptimized experiments showed that 19 of the 40 DNPs were very size 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). In comparison... Figure 7 and Figure 11 When analyzing the data, a series of very interesting activity trends were observed. For example... Figure 11 As shown, when DNPs are stored at 5°C, their size stability is excellent for up to 120 days. The single-mono compounds IAJD9, IAJD22, IAJD33, and IAJD34 form very stable DNPS and also exhibit high in vivo activity. Figure 7 IAJD46 is a bis-bis-IAJD33. However, the activity of DNP36 is about half that of DNP33. Figure 6 and Figure 7 The hetero-bi-hybrid IAJD47 is also based on the structure of IAJD33 or half 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 mono-mono IAJD9 and hetero-bi-hybrid IAJD32. Figure 6 and Figure 7 ).

[0641] Figure 7 Drawing from Figure 6 The results showed that, without any optimization, 28 (54%) of the 52 DNPs studied exhibited activity in vivo. Two of these, DNP33 and DNP34, showed very high activity in the lungs. Single-to-single IAJD9-derived DNP9s showed good activity. Figure 7 ) and stability ( Figure 11 Simultaneously, DNP32 based on IAJD32, which is a corresponding hetero-bi-mixed IAJD32-based DNP32 with a degree of polymerization of IAJD9 and a single PEG of 45, is also produced. Figure 3 and Figure 4 It also exhibits excellent stability. Figure 11 However, it had no activity in mice. Figure 7 To elucidate these results, several co-assembled DNPs were prepared based on highly active IAJD and low concentrations of IAJD32. One example is a DNP assembled from IAJD33 and 2% IAJD32. Figure 11 (The second one from the left in the bottom row) and Figure 7 This combination of DNPs exhibits excellent stability. Figure 11 The stability of DNP32 assembled from IAJD33 alone is comparable to that of DNP32 assembled from IAJD32 alone. However, the in vivo activity of DNP assembled from IAJD33 and 2% IAJD32 is only a fraction of that of DNP33 alone. Figure 7 This confirms the mechanism of the PEG puzzle. Therefore, while inserting a small fraction of PEG conjugated to IAJD can significantly improve the stability of the resulting DNP, it also reduces its in vivo activity even more significantly. Incorporating short oligooxyethylene fragments into the structure of mono-mono, bis-bis, or even hetero-bis-hybrid IAJDs can help address this problem.

[0642] The effects of ionizable amine concentration and sequence on the activity of corresponding DNPs: The binding activity of sugars on the surface of sugar dendritic bodies (GDS) assembled from amphiphilic Janus sugar dendritic polymers (JGDs) to sugar-binding proteins increases by decreasing the sugar concentration during sequence definition (Percec, et al., J. Am. Chem. Soc. 2013, 135, 9055-9077). This unexpected trend is explained by the self-organization of the periphery of the sugar dendritic bodies, whose morphology contributes to higher binding activity between sugars and proteins at lower sugar concentrations.

[0643] Figure 8 Representative activity data for sequence-defined IAJD-derived DNPs were plotted in in vitro and in vivo experiments. Low or no activity was observed at high concentrations of ionizable amines in the IAJD structure, and extremely high activity was observed at lower ionizable amine concentrations in very specific sequences. Without further discussion, Figure 8 The results provide a mechanism for designing NDP activity based on the sequence and concentration of its ionizable amines. Figure 8 The changes in activity observed were far greater than those observed in the case of sequence-defined dendritic polymer bodies.

[0644] Some comments on potential targeted delivery of mRNA: Figure 9 and Figure 10 A summary of selections Figure 6 Representative organ delivery data from the experiments reported in the study. They show luminescence intensities reflecting delivery activity in the heart, lung, liver, and spleen as a function of the IAJD structure used to design DNP structures for mRNA delivery. DNP(10) formation in the lung... 8 The single-component IAJD33 and IAJD34 exhibited the highest luminescence. The next highest luminescence was again based on IAJD31, IAJD46, and IAJD27 (10). 7 DNP is present in the lungs.

[0645] These lung activities were higher than those in the lungs of the MC3 control group. Figure 9 It is important to recognize that Figure 10 The organ activity in it is much higher than Figure 7 and Figure 9 Overall mouse activity. The next highest activity is in the liver ( Figure 9 and Figure 10 Without optimization, the luminescence of DNPs based on IAJD31 and IAJD34 in the liver was 10. 6 The luminescence of DNPs based on IAJD30, IAJD33, and IAJD46 is 10.5 They are smaller than MC3 (i.e., 10). 8 The highest activity in the spleen is DNP (10) based on IAJD29, IAJD30, and IAJD37. 5 ) and DNP (10) based on IAJD27 4 The control experiment using MC3 in the spleen was 10. 7 These results suggest that single-component IAJD could provide a potential strategy for targeting different organs.

[0646] This report describes the design and accelerated modular orthogonal synthesis of six libraries containing ionizable amphiphilic Janus dendritic polymers (IAJDs) with 52 multifunctional sequences. All 52 IAJDs were co-assembled with mRNA via simple injection in acidic buffer to generate dendritic polymer body nanoparticles (DNPs) with predictable size and narrow polydispersity. The size of these DNPs was stable for over 120 days at 5 °C. Of the 52 DNPs encapsulating luciferase mRNA, 42 (81%) showed activity in vitro, four of which showed higher activity than the four-component lipid nanoparticles obtained from MC3 in an FDA-approved control experiment. 28 (54%) DNPs showed activity in vivo, two of which showed higher activity in the lungs than the MC3 control experiment. These unoptimized preliminary experiments provide proof of concept for single-component amphiphilic Janus dendritic polymers with multifunctional sequences as an efficient mRNA delivery system. This single-component delivery platform could be used to elucidate the mechanisms of mRNA encapsulation and release from supramolecular virus-like assemblies and for the production of vaccines and drugs.

[0647] mRNA vaccines rely on a nucleic acid delivery system based on four-component ionizable lipid nanoparticles (LNPs), which contain phospholipids, cholesterol for mechanical properties, PEG-conjugated lipids for stability, and ionizable amines.

[0648] In this report, the current four-component LNP delivery system is transformed into a simpler and more precise single-component multifunctional JD system with the highest activity mediated by low concentrations of ionizable amines defined by the sequence.

[0649] A novel class of JDs, named ionizable amphiphilic Janus dendritic polymers (IAJDs), was designed and synthesized. This concept is the first attempt to elucidate the role of ionizable amines (IAs) and their ability to encapsulate and release mRNA (as a function of the concentration, structural isomericness, quantity, and sequence of ionizable amines) by using IAJD-based DNPs as delivery tools. Assembly of single-component DNPs relies on a simple injection method. This process is significantly simpler than the microfluidic devices required for assembling four-component LNPs.

[0650] Six libraries containing IAJDs with 52 sequence definitions were synthesized using an accelerated modular orthogonal method.

[0651] Of the 52 DNPs assembled from IAJD encapsulated luciferase-mRNA without any optimization, 45 (87%) showed activity in vitro, two of which (IAJD9 and 22) showed higher activity than the positive controls MC3 and transIT. 28 (54%) showed activity in vivo, two of which (IAJD33 and 34) showed very high activity in the lungs.

[0652] The size stability of 40 DNPs was tested. Unoptimized screening experiments showed that 19 of the 40 DNPs were very size stable after storage at 5°C for up to 120 days. Almost all of these DNPs were assembled from IAJDs containing benzyl ether in their hydrophilic moieties.

[0653] This work provides proof of concept for single-component IAJDs as an efficient mRNA delivery system. They will be used to produce vaccines and drugs via mRNA delivery.

[0654] The materials and methods used in these experiments are now described.

[0655] Triethylene glycol monomethyl 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 (ChemImpex, anhydrous, ACS grade), triethyl orthoformate (TCI, 98+%), Albert-15(H) (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 (spectral, 10 wt% loading), pentaerythritol (Aldrich (98%)), 4-(dimethylamino)butyrate (Alfa Aesar (98%)), 3-(dimethylamino)propionate (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 is. 4-(dimethylamino)pyridinium 4-toluenesulfonate (DPTS) was prepared according to a literature procedure. 2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) was prepared according to a literature procedure. Citrate buffer (100 mM, pH 3.0, TEKnova) CH2Cl2 (DCM) was dried on CaH2 and freshly distilled before use.

[0656] Methods including thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used. 1 H and 13 A combination of techniques, including C NMR and matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, was used to determine the purity and structural identity of the final products and intermediates.

[0657] 1 H and 13C10 NMR spectra were recorded at 400 MHz and 101 MHz on a Bruker NEO (400 MHz) NMR spectrometer 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. 1 H, δ 7.26 ppm; 13 The residual proton solvent (C, δ 77.16 ppm) and tetramethylsilane (TMS, δ 0 ppm) were used as... 1 H- and 13 Internal reference in C-NMR spectrum. Absorption in wavenumber (cm²) -1 () indicates. NMR spectra were analyzed and derived using TopSpin 4.07 (Bruker) or MNova 14.

[0658] Molecular purity was determined by high-performance liquid chromatography (HPLC) using a Shimadzu LC-20AD HPLC pump, a PE Nelson Analytical 900 series integrated data station, a Shimadzu SPD-10A VP (UV-vis, λ = 254 nm) and three AM gel columns (one guard column, two...). The experiment was performed on a 10 μm column. THF containing 5% NEt3 was used as the solvent in an oven at 23 °C. Measurements were taken by UV absorbance at 254 nm.

[0659] Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a PerSeptive Biosystem-Voyager-DE (Framingham, MA) mass spectrometer equipped with a nitrogen laser (337 nm) and operated 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) and a THF solution of the matrix (2,5-dihydroxybenzoic acid, 10 mg / mL) at a 1 / 5 (v / v) ratio. The prepared sample solutions (0.5 μL) were loaded onto MALDI plates and dried at 23 °C before being inserted into the instrument's vacuum chamber. The applied laser intensity and voltage were adjusted according to the molecular weight and properties of each compound analyzed.

[0660] Dynamic light scattering (DLS) of DNP was performed in buffer solution using a Malvern Instruments particle size analyzer (Zetasizer Nano S, Malvern Instruments, UK). This analyzer is equipped with a 4mW He-Ne laser at 633nm and an avalanche photodiode positioned at 175° to the beam, along with a temperature-controlled cuvette holder. Instrument parameters were automatically determined over time.

[0661] pK of a single IAJD molecule a Measurement: Dissolve IAJD molecules at a concentration of 1.5 mg / mL in 3 mL of ethanol (saturated solution containing NaCl). Add 0.1 M HCl aqueous solution in increments of 7.5 μL, and measure the pH using a HachH170 pH meter. Calculate pK using half-equivalence point titration. a .

[0662] Formulations of DNP containing mRNA-Luc: IAJD is dissolved in ethanol at varying initial concentrations (5-160 mg / mL). Nucleoside-modified mRNA encoding firefly luciferase (mRNA-Luc) is dissolved in water at varying initial concentrations (1-4 mg / mL). 12.5 μL of the mRNA solution is placed in a clean, RNA-free eppendorf tube (1.5 mL) and mixed with 463 μL of citrate buffer (10 mM, pH 3.0) / acetate buffer (10 mM). To encapsulate the mRNA into IAJD, 25 μL of the ethanol stock solution of IAJD is rapidly injected into the mRNA solution in citrate or acetate buffer, followed by vortexing for 5 seconds.

[0663] DNP dialysis: Dialyze 0.5 mL of DNP solution in a dialysis tube with a molecular weight cutoff of 3,500-14,000 (Spectrum Medical Instruments Inc.) for 2 hours with 10 mM Tris buffer (pH 7.4) or 1X PBS buffer (pH 7.4). Spectra / Por molecular porous membrane tube folded width: 45 mm; diameter: 29 mm & volume / length: 6.4 mL / cm.

[0664] In vitro mRNA delivery: Human embryonic kidney (HEK) 293T cells (US Type Culture Collection) were cultured in Duchenne Modified Eagle 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 at 37°C and 5% CO2 in complete medium for 24 hours. Cells were transfected with IAJD containing naked nucleoside-modified mRNA (mRNA-Luc) encapsulated with firefly luciferase at a fixed concentration of 125 ng / well. transIT was administered according to the manufacturer's protocol. The mRNA transfection kit (Mirus Bio) was used as a positive control for cell transfection, and mRNA-Luc was added to cells at a concentration of 125 ng / well, the same as the test particles. Cells were cultured for another 24 hours, then the culture medium was aspirated and cells were lysed with 30 μL / well of cell culture lysis reagent (Promega). To characterize 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 spectrophotometer (Berthold / EG&G; Wallac).

[0665] In vivo mRNA delivery: 6-8 week old female BALB / c mice were purchased from the Charles River Laboratory. Under isoflurane anesthesia, 100 μL of a buffer solution containing IAJD encapsulating 10 μg of mRNA-Luc was retro-orbitally injected into the mice. 4-7 hours post-injection, mice were imaged on a Perkin Elmer IVIS spectral CT system 10 minutes after intraperitoneal injection of 10 μL / g body weight of D-luciferin (15 mg / mL) (Regis Technologies). Exposure time was set to 1 minute using a binning medium (binning = 8). For IVIS imaging of organs, mice were euthanized, and organs were immediately collected for bioluminescence imaging.

[0666] Synthesis of hydrophilic acids

[0667]

[0668] Reagents and conditions: (i) TsCl, NaOH, THF-water, 0-5℃, 3 hours; (ii) Benzyl chloride, NaOH, water, 100℃, 12 hours; (iii) 4-Methoxybenzyl chloride, NaOH, water, 100℃, 12 hours; (iv) H2SO4 (catalyst), MeOH, reflux, 2 hours; (v) Triethyl orthoformate, Amberlyst-15(H) toluene, Dean-Stark, reflux, 18 hours; vi) K₂CO₃, DMF, 70℃, 12 hours; (vii) 2M HCl, MeOH, 23℃, 2 hours; (viii) (a) KOH, EtOH, reflux, 2 hours; (b) HCl (1M); (ix) KHCO₃, KI, 4-methoxybenzyl chloride, DMF, 60℃, 24 hours; (x) H₂, Pd / C, DCM / MeOH, 23℃, 12 hours; (xi) TFA, DCM, 23℃, 2 hours. (xii) KHCO₃, KI, DMF, 60℃, 24 hours.

[0669] The synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (11), 2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethyl-1-ol (14) and 2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (15) was adapted from a previously reported literature procedure. 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]dioxane-5-carboxylate (20) and methyl 4-(benzyloxy)-3,5-dihydroxybenzoate (24a) was adapted from a literature procedure (Wang, et al., J. Am. Chem. Soc. 2020, 142, 9525-9536). 3,4,5-tris(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)benzoate (30a), 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 according to literature procedures (Percec, et al., Science 2010, 328, 1009-1014; Percec, et al., J.Am.Chem.Soc. 2013, 135, 4129-4148).

[0670]

[0671] 2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethyl-1-ol (16). Triethylene glycol (13, 34.60 g, 229.75 mmol, 4 equivalents) was added to 120 mL of 50% aqueous NaOH solution. The mixture was stirred at 23 °C for 30 min. Then 4-methoxybenzyl chloride (9.00 g, 57.47 mmol, 1 equivalent) was added, and the mixture was stirred at 100 °C for 24 h. The reaction mixture was cooled to 23 °C, diluted with water (80 mL), and extracted with diethyl ether (60 mL × 3). The organic phase was dried over anhydrous magnesium sulfate (MgSO4) and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with ethyl acetate (EtOAc) / hexane = 1 / 1 as the eluent to give the title compound (9.82 g, 60%) as a brownish-red oil.

[0672]

[0673] 2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (17). Compound 16 (8.31 g, 30.74 mmol, 1 equivalent) and NaOH (1.84 g, 46.11 mmol, 1 equivalent) 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 equivalent) dissolved in 20 mL of THF was added to the mixture over 30 minutes at 0 °C. The reaction mixture was stirred at 0–5 °C for 2 hours. 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 MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) using EtOAc / hexane = 1 / 1 as the eluent to give the title compound (11.45 g, 88%) as a brownish-red oil.

[0674]

[0675] 7-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)-2-ethoxybenzo-[d][1,3]dioxacyclopentene-5-carboxylate (21a). Methyl 2-ethoxy-7-hydroxybenzo[d][1,3]dioxacyclopentene-5-carboxylate (20, 0.80 g, 3.33 mmol, 1 equivalent) and K₂CO₃ (1.38 g, 10.00 mmol, 3 equivalents) 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 equivalents) was added, and the mixture was stirred at 70 °C under a N₂ atmosphere for 12 hours. 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 by DCM (30 mL × 3). The organic phase was dried over anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with hexane / EtOAc = 1 / 1 as the eluent to give the title compound (1.43 g, 93%) as a pale yellow oil.

[0676]

[0677] 2-Ethoxy-7-(2-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzo-[d][1,3]dioxane-5-carboxylate (21b). Compound 20 (5.00 g, 20.82 mmol, 1 equivalent) and K₂CO₃ (8.63 g, 62.46 mmol, 3 equivalent) were stirred in dry DMF (70 mL). Compound 12 (7.95 g, 24.98 mol, 1.2 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO₄ and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with hexane / EtOAc = 1 / 2 as the eluent to give the title compound (6.23 g, 86%) as a pale yellow oil.

[0678]

[0679] 2-Ethoxy-7-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)ethoxy)-benzo[d][1,3]dioxane-5-carboxylic acid methyl ester (21c). Compound 20 (0.70 g, 2.91 mmol, 1 equivalent) and K2CO3 (1.21 g, 8.73 mmol, 3 equivalent) were stirred in dry DMF (30 mL). Compound 17 (1.36 g, 3.20 mmol, 1.1 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with hexane / EtOAc = 1 / 1 as the eluent to give the title compound (1.22 g, 85%) as a pale yellow oil.

[0680]

[0681] 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 hours. The reaction mixture was extracted with DCM (20 mL × 3) and the organic phase was dried over anhydrous MgSO4. The solvent was evaporated after filtration to give the title compound (0.85 g, 100%) as a pale yellow oil.

[0682]

[0683] 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 hours. The reaction mixture was extracted with DCM (30 mL × 3) and the organic phase was dried over anhydrous MgSO4. The solvent was evaporated after filtration to give the title compound (4.58 g, 100%) as a pale yellow oil.

[0684]

[0685] 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 hours. The reaction mixture was extracted with DCM (20 mL × 3) and the organic phase was dried over anhydrous MgSO4. The solvent was evaporated after filtration to give the title compound (0.80 g, 100%) as a pale yellow oil.

[0686]

[0687] 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)methyl benzoate (23a). Compound 22a (0.84 g, 2.07 mmol, 1 equivalent) and K2CO3 (1.72 g, 12.42 mmol, 6 equivalent) were stirred in dry DMF (30 mL). Compound 12 (1.45 g, 4.55 mol, 2.2 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with EtOAc / MeOH = 30 / 1 as the eluent to give the title compound (1.19 g, 82%) as a yellow oil.

[0688]

[0689] 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 equivalent) and K₂CO₃ (10.04 g, 72.66 mmol, 6 equivalent) were stirred in dry DMF (70 mL). Compound 15 (10.98 g, 27.83 mol, 2.3 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO₄ and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) using EtOAc / MeOH = 50 / 1 as the eluent to give the title compound (8.42 g, 90%) as a pale yellow oil.

[0690]

[0691] Methyl 3,4-bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)benzoate (23c). Compound 22c (0.79 g, 1.81 mmol, 1 equivalent) and K2CO3 (1.50 g, 10.86 mmol, 6 equivalent) were stirred in dry DMF (30 mL). Compound 15 (1.50 g, 3.80 mol, 2.1 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with hexane / EtOAc = 1 / 3 as the eluent to give the title compound (1.31 g, 82%) as a yellow oil.

[0692]

[0693] Methyl 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)benzoate (23d). Compound 22a (1.80 g, 4.43 mmol, 1 equivalent) and K2CO3 (3.67 g, 26.58 mmol, 6 equivalent) were stirred in dry DMF (50 mL). Compound 17 (3.95 g, 9.30 mol, 2.1 equivalent) was added and the mixture was stirred at 70 °C under 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 on anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) using EtOAc as the eluent to give the title compound (3.65 g, 91%) as a brownish-yellow oil.

[0694]

[0695] 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 equivalent) was dissolved in ethanol (EtOH, 15 mL). KOH (0.62 g, 10.99 mmol, 7 equivalent) was added, and the mixture was refluxed and stirred for 2 hours. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to a pH of about 1. The solution was extracted with DCM (20 mL × 3), and the organic phase was dried over anhydrous MgSO4. The solvent was filtered and evaporated to give the title compound (1.07 g, 100%) as a yellow oil.

[0696]

[0697] 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 equivalent) was dissolved in ethanol (50 mL). KOH (3.99 g, 71.19 mmol, 7 equivalent) was added, and the mixture was refluxed and stirred for 2 hours. The mixture was cooled to 23 °C and acidified with dilute HCl (1 M) to a pH of about 1. The solution was extracted with DCM (40 mL × 3), and the organic phase was dried over anhydrous MgSO4. The solvent was filtered and evaporated to give the title compound (7.73 g, 100%) as a yellow oil.

[0698]

[0699] 3,4-Bis(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-5-(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)benzoic acid (6). Compound 23c (0.98 g, 1.11 mmol, 1 equivalent) was dissolved in ethanol (15 mL). KOH (0.44 g, 7.77 mmol, 7 equivalent) was added, and the mixture was refluxed and stirred for 2 hours. 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 MgSO4. The solvent was filtered and evaporated to give the title compound (0.96 g, 100%) as a brownish-yellow oil.

[0700]

[0701] 3-(2-(2-(2-(benzyloxy)ethoxy)ethoxy)ethoxy)-4,5-bis(2-(2-(2-((4-methoxybenzyl)oxy)ethoxy)ethoxy)benzoic acid (8). Compound 23d (2.80 g, 3.07 mmol, 1 equivalent) was dissolved in ethanol (30 mL). KOH (1.20 g, 21.49 mmol, 7 equivalent) was added, and the mixture was refluxed and stirred for 2 hours. 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 MgSO4. The solvent was filtered and evaporated to give the title compound (2.73 g, 100%) as a brownish-yellow oil.

[0702]

[0703] Methyl 3,5-dihydroxy-4-((4-methoxybenzyl)oxy)benzoate (24b). Methyl 3,4,5-trihydroxybenzoate (19 g, 3.25 g, 17.66 mmol, 1 equivalent), 4-methoxybenzyl chloride (2.77 g, 17.66 mmol, 1 equivalent), KHCO3 (2.53 g, 35.32 mmol, 3 equivalent), and KI (18 mg, 0.11 mmol, 0.006 equivalent) were stirred in dry DMF (80 mL) at 60 °C under a N2 atmosphere 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 on anhydrous MgSO4 and filtered. The filtrate was concentrated and purified by column chromatography (SiO2) with hexane / EtOAc = 2 / 1 as the eluent to give the title compound (2.30 g, 76%) as a white solid.

[0704]

[0705] 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 equivalent) and K₂CO₃ (12.08 g, 87.41 mmol, 6 equivalents) were stirred in dry DMF (70 mL). Compound 12 (10.64 g, 33.42 mol, 2.3 equivalents) 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 (SiO2) using Et0Ac as the eluent to give the title compound (7.27 g, 88%) as a pale yellow oil.

[0706]

[0707] 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 equivalent) and K₂CO₃ (10.90 g, 78.86 mmol, 6 equivalent) were stirred in dry DMF (60 mL). Compound 15 (11.90 g...

Claims

1. An ionizable amphiphilic Janus dendrimer having the structure of formula (I): in: A is a multivalent group including at least one selected from the group consisting of: and any combination thereof; The dashed line indicates the binding site for one of X, Y, or Z; X is a hydrophilic group comprising at least one amine; Y is a compound containing at least one C1-C 30 The lipophilic group of the alkyl chain; Z comprises at least one selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, and a polyethylene glycol chain; R A and R B independently selected from the group consisting of hydrogen, halide, hydroxyl, C1-C 30 Alkyl, C1-C 30 Alkyl halides, C1-C 30 Alkoxy, C1-C 30 Alkoxy halides 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; and The sum of s, t and u is equal to the valence of A.

2. The ionizable amphiphilic Janus dendrimer according to claim 1, wherein the ionizable amphiphilic Janus dendrimer having the structure of formula (I) is an ionizable amphiphilic Janus dendrimer having the structure of formula (II): (X) s -OH) t Formula (II) in: A is a multivalent group including at least one selected from the group consisting of: and any combination thereof; The dashed line indicates the binding site of one of X and Y; R A and R B independently selected from the group consisting of hydrogen, halide, hydroxyl, C1-C 30 Alkyl, C1-C 30 Alkyl halides, C1-C 30 Alkoxy, C1-C 30 Alkoxy halides and any combination thereof; s is an integer from 1 to 5; t is an integer from 1 to 5; and The sum of s and t is equal to the valence of A.

3. The ionizable amphiphilic Janus dendrimer according to claim 1 or 2, wherein Y is a C1-C1-C2-d-i-n-butyl group having at least two different carbon number groups. 30 The lipophilic group of the alkyl chain.

4. The ionizable amphiphilic Janus dendrimer according to any one of claims 1 to 3, wherein A is composed of indicates; and s and t are each 2.

5. The ionizable amphiphilic Janus dendrimer according to any one of claims 1 to 3, wherein A is composed of express; t is 2; s is 1; and u is 0 or 1.

6. The ionizable amphiphilic Janus dendrimer according to any one of claims 1 to 3, wherein A is composed of indicates; and s and t are each 1.

7. The ionizable amphiphilic Janus dendrimer according to any one of claims 1 to 6, wherein each occurrence of X is independently selected from the group consisting of: and any combination thereof; The dotted line indicates the connection with A; Each occurrence of W is independently selected from the group consisting of: C=O, C(R W )(R W ), NR W , O and S; in, Each occurrence of R W independently selected from the group consisting of hydrogen, halide, hydroxy, alkyl, alkyl halide, aryl, aryl halide, alkoxy, alkoxy halide, and any combination thereof; and Each time L appears 1 , L 2 , L 3 and L 4 is independently a covalent bond or a divalent linking group selected from the group consisting of an alkylene group, a cycloalkylene group, a heteroalkylene group, a heterocycloalkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, a silyl group, an amine, an amide, an ester, an ether, a carbonyl group, a carbamate, a thioether, a thioester, a disulfide, a hydrazine, a urea, a thiourea, a phosphate ester, a poly(alkyl ether), a heteroatom, and any combination thereof; Each occurrence of R 11 , R 12 , R 13 and R 14 independently selected from the group consisting of hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, and any combination thereof; and Each occurrence of m, n, o is independently an integer from 1 to 5.

8. The ionizable amphiphilic Janus dendrimer according to claim 7, wherein at least one occurrence of R 11 , R 12 , R 13 or R 14 -C(O)(CH2) m -N(R 1 )(R 2 ); Each occurrence of m is independently an integer from 1 to 10; and Each occurrence of R 1 and R 2 are independently selected from the group consisting of hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, and any combination thereof; wherein R 1 and R 2 Can form a ring together.

9. The ionizable amphiphilic Janus dendrimer of claim 7, wherein each occurrence of R 11 , R 12 , R 13 and R 14 Independently selected from the group consisting of: and any combination thereof; wherein each occurrence of m is independently an integer from 1 to 10.

10. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein X comprises at least two tertiary amines.

11. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein each occurrence of X is independently selected from the group consisting of: and any combination thereof; The dotted line indicates the connection with A; Each occurrence of R 1 and R 2 are independently selected from the group consisting of hydrogen, deuterium, alkyl, aryl, cycloalkyl, amine, heterocycloalkyl, carbonyl, and any combination thereof; wherein R 1 and R 2 can form a ring together; Each occurrence of R 3 are independently selected from the group consisting of: hydrogen, (CH2) n 、(CH2) n -OH and any combination thereof; and Each occurrence of n is independently an integer from 1 to 20.

12. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein each occurrence of X is independently selected from the group consisting of: and any combination thereof.

13. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein each occurrence of Y is independently selected from the group consisting of: and any combination thereof; The dotted line indicates the connection with A; Each occurrence of W is independently selected from the group consisting of: C=O, C(R W )(R W ), NR W , O and S; Each occurrence of R W independently selected from the group consisting of hydrogen, halide, hydroxy, alkyl, alkyl halide, aryl, aryl halide, alkoxy, alkoxy halide, and any combination thereof; and Each occurrence of R 21 , R 22 , R 23 and R 24 Independently contain straight or branched C1-C 50 alkyl.

14. The ionizable amphiphilic Janus dendrimer of claim 13, wherein each occurrence of R 21 , R 22 , R 23 and R 24 Independently contains C1-C 30 -alkyl.

15. The ionizable amphiphilic Janus dendrimer of claim 13, wherein each occurrence of Y is independently selected from the group consisting of: and any combination thereof; wherein each occurrence of W is independently selected from the group consisting of: C=O, C(R W )(R W ), NR W , O and S; Each occurrence of R W independently selected from the group consisting of hydrogen, halide, hydroxy, alkyl, alkyl halide, aryl, aryl halide, alkoxy, alkoxy halide, and any combination thereof; and n is an integer of 1 to 30.

16. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein each occurrence of Y is independently selected from the group consisting of: and any combination thereof; The dotted line indicates the connection with A; Each occurrence of n is independently an integer from 1 to 20; and Each occurrence of -(CH2) n CH3, -O(CH2) n - and -O(CH2) n CH3 is independently linear or branched.

17. The ionizable amphiphilic Janus dendrimer of any preceding claim, wherein the ionizable amphiphilic Janus dendrimer comprises a first Y and a second Y, wherein 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.

18. The ionizable amphiphilic Janus dendrimer of claim 17, wherein 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.

19. The ionizable amphiphilic Janus dendrimer according to claim 1, wherein u is 1 or 2; Each occurrence of Z is independently selected from the group consisting of: and any combination thereof; The dotted line indicates the connection with A; Each time L appears 1 , L 2 , L 3 and L 4 is independently a covalent bond or a divalent linking group selected from the group consisting of an alkylene group, a cycloalkylene group, a heteroalkylene group, a heterocycloalkylene group, an alkenylene group, an alkynylene group, an arylene group, a heteroarylene group, a silyl group, an amine, an amide, an ester, an ether, a carbonyl group, a carbamate, a thioether, a thioester, a disulfide, a hydrazine, a urea, a thiourea, a phosphate ester, a poly(alkyl ether), a heteroatom, and any combination thereof; Each occurrence of R 31 , R 32 , R 33 and R 34 independently selected from the group consisting of hydrogen, deuterium, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, phenoxy, amine, heterocycloalkyl, carbonyl, and any combination thereof; and Each occurrence of m, n, o is independently an integer from 1 to 5.

20. The ionizable amphiphilic Janus dendrimer of claim 19, wherein each occurrence of Z is independently selected from the group consisting of: wherein each occurrence of n is independently an integer from 1 to 100; Each occurrence of p is independently an integer from 1 to 10; and Each occurrence of R 4 Independently selected from the group consisting of hydrogen, deuterium, alkyl, aryl, and any combination thereof.

21. The ionizable amphiphilic Janus dendrimer of any preceding claim, wherein the ionizable amphiphilic Janus dendrimer comprises homochiral, racemic, or achiral imprinting sites.

22. The ionizable amphiphilic Janus dendrimer of any preceding claim, wherein the ionizable amphiphilic Janus dendrimer is a homochiral ionizable amphiphilic Janus dendrimer, a racemic ionizable amphiphilic Janus dendrimer, or an achiral ionizable amphiphilic Janus dendrimer.

23. The ionizable amphiphilic Janus dendrimer of any of the preceding claims, wherein the ionizable amphiphilic Janus dendrimer is an ionizable amphiphilic Janus dendrimer having a structure selected from the group consisting of at least one structure of FIG. 12 , at least one structure of FIG. 13 , at least one structure of FIG. 14 , at least one structure of FIG. 47 , at least one structure of FIG. 60 , at least one structure of FIG. 80 , and any combination thereof.

24. The ionizable amphiphilic Janus dendrimer according to any one of the preceding claims, wherein the ionizable amphiphilic Janus dendrimer is an ionizable amphiphilic Janus dendrimer having a structure selected from the group consisting of:

25. A nanoparticle comprising at least one ionizable amphiphilic Janus dendrimer according to any one of the preceding claims.

26. The nanoparticle of claim 25, wherein the nanoparticle comprises a first ionizable amphiphilic Janus dendrimer and a second ionizable amphiphilic Janus dendrimer, The first ionizable amphiphilic Janus dendrimer has a different structure from the second ionizable amphiphilic Janus dendrimer.

27. The nanoparticle of claim 25, wherein the nanoparticle comprises a homochiral ionizable amphiphilic Janus dendrimer, an achiral ionizable amphiphilic Janus dendrimer, or any combination thereof.

28. The nanoparticle according to claim 27, wherein the nanoparticle is a single-layer nanoparticle or an onion-like multi-layer nanoparticle.

29. The nanoparticle of claim 25, wherein the nanoparticle comprises a racemic ionizable amphiphilic Janus dendrimer.

30. The nanoparticle of claim 29, wherein the nanoparticle is a multilayer nanoparticle.

31. The nanoparticle of any one of claims 25-30, wherein the nanoparticle further comprises at least one agent.

32. The nanoparticle of claim 31, wherein the at least one agent comprises a diagnostic agent, a detectable agent, a therapeutic agent, a nucleic acid molecule, or any combination thereof.

33. The nanoparticle of claim 31, wherein the at least one agent is selected from the group consisting of mRNA, siRNA, microRNA, CRISPR-Cas9, sgRNA, small molecules, proteins, antibodies, peptides, proteins, and any combination thereof.

34. The nanoparticle of claim 31, wherein the at least one agent comprises a nucleic acid molecule.

35. The nanoparticle of claim 34, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule.

36. The nanoparticle of claim 34, wherein the nucleic acid molecule is selected from the group consisting of cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecules, targeting nucleic acids, and any combination thereof.

37. The nanoparticle of claim 34, wherein the nucleic acid molecule encodes at least one selected from the group consisting of an antigen, an antibody, a gene editing molecule, a chimeric antigen receptor (CAR), and any combination thereof.

38. A composition comprising at least one selected from the group consisting of: at least one ionizable amphiphilic Janus dendrimer of any one of claims 1-24, at least one nanoparticle of any one of claims 25-37, and any combination thereof.

39. The composition of claim 38, wherein the composition further comprises an adjuvant.

40. The composition of claim 38, wherein the composition is a pharmaceutical composition.

41. The composition of claim 38, wherein the composition is a vaccine.

42. A method of delivering an agent to a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle of any one of claims 31-37 or a composition comprising the nanoparticle.

43. The method of claim 42, wherein the composition further comprises an adjuvant.

44. The method of claim 42, wherein the agent is encapsulated within the nanoparticle.

45. The method of claim 42, wherein the method treats or prevents at least one condition selected from the group consisting of viral infection, bacterial infection, fungal infection, parasitic infection, cancer, a disease or condition associated with cancer, an autoimmune disease or condition, and any combination thereof.

46. ​​The method of claim 42, wherein the agent is a composition for protein replacement therapy.

47. The method of claim 42, wherein the agent is a composition for gene editing.

48. The method of claim 42, wherein the agent is a vaccine.

49. The method of claim 42, wherein the method further comprises delivering the agent to the subject's liver.

50. The method of claim 42, wherein the method further comprises delivering the agent to the spleen of the subject.

51. The method of claim 42, wherein the method further comprises delivering the agent to the lungs of the subject.

52. The method of claim 42, wherein the agent comprises at least one selected from the group consisting of: cDNA, cRNA, CirRNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, tRNA, antagomir, antisense molecule, targeting nucleic acid, and any combination thereof.

53. The method of claim 52, wherein the modified RNA is a nucleoside-modified RNA.

54. The method of claim 53, wherein the nucleoside-modified RNA comprises pseudouridine.

55. The method of claim 53, wherein the nucleoside-modified RNA comprises pseudouridine plus 5-methyl-cytosine.

56. The method of claim 53, wherein the nucleoside-modified RNA comprises 5-methyl-uridine.

57. The method of claim 53, wherein the nucleoside-modified RNA comprises 1-methyl-pseudouridine.

58. A method of preventing or treating a disease or condition in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle of any one of claims 25-37 or a composition comprising the nanoparticle.

59. The method of claim 58, wherein the disease or condition is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, a parasitic infection, cancer, a disease or condition associated with cancer, an autoimmune disease or condition, and any combination thereof.

60. A method of inducing an immune response in a subject in need thereof, wherein the method comprises administering to the subject at least one nanoparticle of any one of claims 25-37 or a composition comprising the nanoparticle.

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