Methods and compositions for targeted delivery by polymerosomes

Abstract

The present disclosure relates to copolymers and polymersomes for targeted delivery of biomolecules to living organisms. Exemplary copolymers include an initiator block, a propagator block, and a linker connecting the initiator block and the propagator block. The initiator block includes a glycan head configured to provide targeted delivery, and the propagator block includes a functional moiety configured to provide desired properties to the polymersome.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 458,102 filed on 8 April 2023, U.S. Provisional Patent Application No. 63 / 587,231 filed on 2 October 2023, and U.S. Provisional Patent Application No. 63 / 575,056 filed on 5 April 2024, the entirety of the aforementioned applications is incorporated herein by reference.

[0002] This disclosure generally pertains to compositions and methods relating to polymeric nanocarriers, and in particular to pharmaceutical formulations comprising polymersomes capable of selective / targeted delivery of payloads to desired target regions or cells within tissues. [Background technology]

[0003] Nanotechnology-based delivery is widely used in scientific, industrial, and clinical applications. It has become a promising method for drug delivery, offering advantages including improved solubility and permeability of drug molecules. In a recent example, mRNA vaccines developed against the COVID-19 virus utilized special lipid nanoparticles (LNPs) adapted to encapsulate and stabilize mRNA molecules, given the general instability of mRNA molecules and the need for low-temperature storage (e.g., -70°C). Typical lipid nanoparticles are usually composed of several types of lipids. The ratios of these lipids require fine-tuning, the production of lipid nanoparticles can be costly, and lipid nanoparticles generally cannot selectively deliver mRNA molecules. Therefore, there is a need in this field for nanoparticles with simpler construction and the ability to selectively deliver mRNA. [Overview of the project] [Means for solving the problem]

[0004] (Summary of the invention) In one embodiment, the disclosure provides a copolymer for forming polymersomes. The copolymer comprises an initiation block comprising a glycan head, a propagator block further comprising a functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof, and a linkage comprising a disulfide bond covalently connecting the initiation block and the propagator block.

[0005] In one embodiment, the present disclosure provides a polymerosome, which comprises a membrane defining an internal space, the membrane comprising an exemplary copolymer of the present disclosure.

[0006] In one embodiment, the Disclosure provides a pharmaceutical formulation comprising the polymersomes of the Disclosure.

[0007] In one embodiment, the present disclosure provides a kit for preparing polymersomes. The kit comprises an initiator, which is a first reagent comprising a glycan head and an initiator linker, and a propagator, which comprises a functional moiety and a propagator linker, which comprises a second reagent comprising a guanidine group, a zwitterionic group, diethylenetriamine or a combination thereof, wherein the initiator linker is configured to be coupled to the propagator linker via a linker comprising a disulfide bond.

[0008] In one embodiment, the Disclosure provides a method for targeted delivery of a payload to a subject. The method comprises administering an effective amount of a pharmaceutical formulation comprising polymerosomes to a subject, wherein the polymerosomes comprise a membrane encapsulating the payload, and the membrane comprises the copolymer of the Disclosure.

[0009] In one embodiment, the present disclosure provides a method for preventing or treating a disease in a subject, comprising administering an effective amount of a pharmaceutical formulation comprising polymersomes to the subject, wherein the polymersomes comprise a membrane, the membrane comprises a polymer component according to any one of claims 1 to 34 and a payload encapsulated within the membrane, and the payload is a therapeutic agent or induces a therapeutic agent.

[0010] In one embodiment, the present disclosure provides a method for boosting an adaptive immune response, comprising administering an effective amount of a pharmaceutical formulation comprising polymerosomes to a target, wherein the polymerosomes comprise a membrane encapsulating a payload, the membrane comprises the copolymer of the present disclosure, and the payload is immunogenic or induces immunogenic biomolecules. [Brief explanation of the drawing]

[0011] [Figure 1A] This diagram illustrates the exemplary synthesis of the exemplary copolymers of this disclosure. Both X and Y are integers, independently ranging from 9 to 14. [Figure 1B] The structures of several exemplary polymerosomes, including copolymers according to some embodiments of this disclosure, are illustrated. [Figure 2A] The results of an agarose gel electrophoresis assay are shown, demonstrating the encapsulation efficiency of several exemplary copolymers according to certain embodiments of this disclosure. The assay payload demonstrating this efficacy was approximately 920 bp of GFP mRNA. Copolymers, with or without GFP mRNA, had a size greater than 10 kDa. [Figure 2B] This disclosure provides a graphical representation showing the average fluorescence intensity of HEK293 cells after transfection with polymerosomes containing GFP-mRNA, according to some operational embodiments of this disclosure. [Figure 3A] The results of an agarose gel electrophoresis assay demonstrating the encapsulation efficiency of several copolymers by certain working embodiments of this disclosure are shown. The payload in this demonstration assay was spike mRNA of approximately 2550 bp in size. Exemplary copolymers, with or without spike mRNA, were larger than 10 kDa. The N / P ratio (ratio of positively chargeable polymer amine groups (N=nitrogen) to negatively charged nucleic acid phosphate groups (P)) for each experimental group is indicated by the number shown in each lane. The N / P ratios tested in this experiment were 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20. [Figure 3B] Along with CryoEM images and insert images, a graph display showing the particle size and zeta (ζ) potential of mRNA-PNP(P1 / P5) is provided. [Figure 3C] This image shows chemiluminescent imaging demonstrating spike protein expression mediated by spike mRNA-PNP (I1-P1 / P5) in HEK293T cells. The first lane shows protein expression from cells transfected with spike mRNA, and the second lane shows protein expression from cells transfected with spike mRNA-PNP (I1-P1 / P5). β-actin was used as the baseline expression in this experiment. [Figure 4] This document provides a graphical representation of the co-localization of intracellular lysosomes and mRNA-PNPs, investigated using confocal fluorescence imaging over a 4-hour period. Images were collected at time points 1, 1.5, 2, 2.5, 3, 3.5, and 4, respectively. mRNA-PNPs were labeled with FITC for detection. [Figure 5] This report provides a graphical representation of the cellular uptake fluorescence signals of BMDCs, B cells, and T cells one hour after treatment with spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I2-P1 / P4-FITC / P5), targeting Siglec-2. Data were collected using flow cytometry analysis. [Figure 6] This report provides a graphical representation of the cellular uptake fluorescence signals of BMDCs, B cells, and T cells one hour after treatment with spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I5-P1 / P4-FITC / P5), targeting DC-SIGN. Data were collected using flow cytometry analysis. [Figure 7] This disclosure provides a graphical representation of the binding analysis of DC-SIGN, MMR, MINCLE, Dectin-2, and Langerin (0.625 μg / mL) to polymerosomes at pH 7.4 according to some embodiments of this disclosure. [Figure 8A] This diagram shows a schematic representation of the design for an animal immunization experiment. [Figure 8B] A graph is provided showing the serum spike-specific IgG titers induced by immunization of I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control), measured 28 days after immunization. [Figure 8C] A graph is provided showing the neutralization titers (ID50) of I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control) measured 28 days after immunization. The neutralization titer was calculated as the reciprocal of the serum dilution factor that resulted in a 50% reduction in RLU compared to the viral control well, after subtracting the background RLU. The ID50 values ​​are labeled on the plot along with the standard error of the mean. [Figure 9] This graph shows FACS data of cellular uptake into C2C12 muscle of polymerosomes (0 and 1 hour after treatment) and polymerosomes without the target glycan (1 hour after treatment) according to this disclosure. [Modes for carrying out the invention]

[0012] Nanoparticle delivery is widely used in a variety of applications. In addition to lipid nanoparticles (LNPs), which are almost certainly the most common type of nanoparticle, another type of nanoparticle, polymerosomes, has also gained increasing attention in industrial and clinical applications. Polymerosomes (i.e., polymer-based nanoparticles, polymer vesicles, or polymer nanoparticles (PNPs)) are self-assembling enclosures from amphiphilic block copolymers. These amphiphilic block copolymers are macromolecules containing at least one hydrophobic polymer block and at least one hydrophilic polymer block. When hydrated, these amphiphilic block copolymers self-assemble to form an enclosure, so that the hydrophobic blocks tend to associate with each other to minimize direct exposure to water and form the inner surface of the enclosure, while the hydrophilic blocks tend to face outward and form the outer surface of the enclosure. The hydrophobic core of these water-soluble polymerosomes can provide an environment for solubilizing additional hydrophobic molecules. Thus, these water-soluble polymerosomes can act as carrier polymers for hydrophobic molecules encapsulated within the polymerosome. Furthermore, the self-assembly of amphiphilic block polymers occurs without the presence of stabilizers, which in other respects provides colloidal stability and prevents aggregation.

[0013] Polymerosomes offer many advantages, such as high stability in storage, high availability in manufacturing stages, ease of surface modification, high biocompatibility, and controlled release mechanisms. However, the industry still lacks polymerosomes that efficiently deliver drug molecules to specific areas of a living organism. This is likely due to the fact that conventional polymerosomes do not effectively encapsulate the payload (e.g., the biomolecule administered for their therapeutic effect) and are generally unstable in serum. Furthermore, conventional polymerosomes do not allow for selective delivery.

[0014] polymerosomes Accordingly, one aspect of the present disclosure provides a polymerosome. The polymerosomes of the present disclosure are designed to provide selective delivery (or targeted delivery) and good encapsulation efficiency, particularly for nucleic acid-type payloads. An exemplary polymerosome comprises a membrane, the membrane defining an internal space configured to encapsulate or hold a payload. The membrane of the polymerosome of the present disclosure comprises a copolymer comprising an initiation block, a propagator block, and a ligator, the ligator covalently connecting the initiation block and the propagator block and comprising a disulfide bond. The initiation block comprises a glycan head, and the propagator block comprises a functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof.

[0015] Without being constrained by theory, the disulfide bond of the ligation site (i.e., the disulfide ligation site) is selected for its biodegradability and / or thiol-mediated uptake in the intracellular environment. Thus, the disulfide bond can facilitate the uptake of the polymerosomes of this disclosure and their post-uptake degradation for release of the encapsulated payload, for example, via glutathione-mediated cleavage in the cell.

[0016] In some embodiments, the copolymer may include one or more start blocks and one or more propagator blocks. For example, the copolymer may include one start block, a first propagator block and a second propagator block, and both the start block and the first propagator block, and the first propagator block and the second propagator block are connected via couplings.

[0017] Start block In certain embodiments, the initiation block includes a glycan head configured to result in selective delivery. For this purpose, the glycan head may have a targeting portion, which is the ligand of the target (e.g., a receptor on the target cell). In some embodiments, the targeting portion may be the terminal portion of the glycan head to increase the probability of interacting with and binding to the target. However, this disclosure is not limited to this configuration. In some embodiments, the target cell for selective delivery is an antigen-presenting cell (APC, e.g., a dendritic cell). In some embodiments, the target cell may be another type of immune cell. And in some other embodiments, the target may be any living cell designed to interact with the payload.

[0018] In certain embodiments, the initiation block is configured to bind to lectin receptors, e.g., Siglec-1 (sialoadhesin), Siglec-2, Siglec-5 / E, and DC-SIGN, with a certain degree of affinity, thus exhibiting better uptake by certain types of APCs. In some embodiments, the glycan head of the initiation block may contain mannosidase, which may be a terminal mannose configured to bind to DC-SIGN on dendritic cells. In some embodiments, the glycan head may contain sialoside. In some embodiments, to target Siglec-1, the glycan head may contain 9-N-(4H-thieno[3,2-c]chromen-2-carbamoyl)-Neu5Ac-α2,3-Gal-GlcNAc. In some embodiments, to target Siglec-2, the glycan head may contain 9-biphenylNeu5Ac-α2,6-Gal. In some embodiments, the glycan head may contain Neu5Ac-α2,3-Gal-GlcNAc to target Siglec-5 / E.

[0019] Bonding under acidic conditions. In some embodiments, the bond between the start block and the target is Ca 2+The relationship is such that calcium coordination is reduced in low pH environments, potentially resulting in lower binding affinity. Therefore, to obtain better binding affinity under acidic conditions, the glycan head of the starting block may contain an aryl group. Without wishing to be constrained by any theory, the aryl group may be involved in CH-π and hydrophobic interactions that enhance the bond under acidic conditions. The aryl group can be an unsubstituted benzene or a benzene substituted with a halide or alkyl halide (e.g., CF3). In some embodiments, the aryl group is coupled to the targeting moiety. For example, the glycan head of the starting block may contain an O-arylmannoside.

[0020] Structural configuration of the glycan head. In certain embodiments, the glycan head can be a linear or branched structure. In some embodiments, the glycan head may have multiple targeting moieties, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting moieties. Multiple targeting moieties can be arranged in a linear, branched, or star-shaped configuration. For example, the glycan head of the initiation block may contain a monomannoside, dimannoside, or trimannoside, and if the glycan head contains a trimannoside, the trimannoside can be in a linear form or a branched structure, e.g., α-1,3-α-1,6-trimannoside. In certain embodiments, in some situations, a branched configuration (e.g., a trimannoside glycan head) has been shown to exhibit superior binding affinity to its target receptor.

[0021] Initiation spacer. In some embodiments, the initiation block further includes an initiation spacer. The initiation spacer is configured to provide structural flexibility to the glycan heads and / or hydrophobicity to the copolymer to facilitate the assembly of polymerosomes. Without wishing to be constrained by theory, the flexibility allows the glycan heads to move between the initiation block and the target during the interaction, thereby facilitating binding between them.

[0022] The preferred spacer is biocompatible. In some embodiments, the starting spacer includes a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof. For example, the spacer may be a polyethylene glycol (PEG) portion formed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65, or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, for example, 2-72, 2-60, 2-48, 2-36, 2-24, 2-18, 2-15, 2-10, 4-72, 4-60, 4-48, 4-36, 4-24, 4-18, 4-15, 4-10, 8-72, 8-60, 8-48, 8-36, 8-24, 8-18, 8-15, or 8-10 (OCH2CH2) subunits. In some embodiments, the PEG portion can have a linear, branched, or star-shaped structure.

[0023] In some embodiments, the spacer is a saturated carbon portion, which may be a lipid tail connected to the glycan head. In some embodiments, the saturated carbohydrate contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or any range defined by the aforementioned endpoints, e.g., 2-15, 2-12, 2-10, 2-8, 2-6, 2-4, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, or 6-8 carbons.

[0024] Binding affinity. In some embodiments, the binding affinity between the glycan head of the initiation block and the target is the dissociation constant (K). D ) can be defined by. In some embodiments, K at pH 7.4 DThe numbers are 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3250, 3500, 3750, 4000, 4250, 4500, and 47 50, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750 or 8000 nM, or any range defined by the aforementioned endpoints, e.g., 5-8000, 5-7000, 5-6000, 5-5000, 5-4000, 5-3000, 5-25 00, 5~2000, 5~1500, 5~1250, 5~1000, 5~900, 5~800, 5~700, 5~600, 5~500, 5~400, 5~300, 5~200, 5~150, 5~100, 5~75, 5~50, 5~30, 5~20, 10~8000, 10~7000, 10~6000, 10~5000, 10~4000, 10 It can be ~3000, 10~2500, 10~2000, 10~1500, 10~1250, 10~1000, 10~900, 10~800, 10~700, 10~600, 10~500, 10~400, 10~300, 10~200, 10~150, 10~100, 10~75, 10~50, 10~30, or 10~20 nM.

[0025] In some other embodiments, K at pH 5 DThis can be 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 1750 or 2000 nM, or any range defined by the aforementioned endpoints, e.g., 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-750, 1-700, 1-650, 1-600, 1-550, 1-50 It can be 0, 1~450, 1~400, 1~350, 1~300, 1~250, 1~200, 1~150, 1~100, 1~75, 1~50, 1~40, 1~30, 1~20, 1~10 or ~5, 5~2000, 5~1500, 5~1000, 5~900, 5~800, 5~750, 5~700, 5~650, 5~600, 5~550, 5~500, 5~450, 5~400, 5~350, 5~300, 5~250, 5~200, 5~150, 5~100, 5~75, 5~50, 5~40, 5~30, 5~20, 5~10 nM.

[0026] Example: In some embodiments, the start block's glycan head is 9 BPC Neu5Ac conjugate N-glycan head (e.g., IB2), Neu5Ac conjugate N-glycan (e.g., IB3), 9 TCC N This includes eu5Ac conjugate N-glycans (e.g., IB4) or combinations thereof. In certain embodiments, the glycan head includes at least one of the following structures (Note: "IB" represents an initiate block, which describes the initiate conjugate in the copolymer of this disclosure. However, "IB" can be used interchangeably with "I" to mean a concisely described initiator):

[0027] [ka] (In the formula, black circles represent mannoside, white circles represent galactose, black squares represent GlcNAc, and diamonds represent Neu5Ac).

[0028] Propagator Block In certain embodiments, the exemplary polymerosomes of the present disclosure exhibit desired properties, such as efficient payload encapsulation, reduced serum protein adsorption, enhanced membrane fusion, and efficient payload release after uptake. At least one of the desired properties is provided by the propagator block of the present disclosure. For this purpose, the propagator block of the present disclosure comprises a functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof.

[0029] In some embodiments, the copolymer of the Disclosure comprises a single propagator block that provides at least one of the desired properties. In some embodiments, the copolymer of the Disclosure comprises a plurality of propagator blocks, each providing at least one of the desired properties.

[0030] Efficient payload encapsulation. In some embodiments, the polymerosomes of this disclosure are designed to carry nucleic acid-type payloads, such as mRNA or DNA molecules. In such embodiments, the propagator block of the copolymer of the polymerosome may contain guanidine groups. In some embodiments, the propagator block contains one, two, three, four, five, or more guanidine groups. Without wishing to be constrained by theory, multiple guanidine groups in the propagator block provide stronger salt bridges between the guanidinium groups of the copolymer and the phosphate groups of the nucleic acid molecule (e.g., mRNA). In certain embodiments, the propagator block contains three guanidine groups.

[0031] Reduced serum protein adsorption and enhanced membrane fusion. As mentioned above, one common drawback of polymerosomes is that they are said to be unstable in serum due to serum protein adsorption. To reduce serum protein adsorption and enhance serum stability, the copolymers of the present disclosure may contain zwitterions, which also enhance the membrane fusion of the polymerosomes with target cells. The zwitterions of the present disclosure may be molecules / parts with an overall zero charge by having an equal number of positively charged and negatively charged functional groups at pH 4.5 to 7.5. In some embodiments, the zwitterions of the present disclosure have an isoelectric point between pH 4.5 and 7.5. The zwitterions may be, but are not limited to, choline phosphate (CP), sulfothetins, phosphonium sulfonates, or psilocybin. In some embodiments, the zwitterions of the present disclosure contain alkylphosphobetaine groups containing phosphate and amine groups, which provide negative and positive charges, respectively.

[0032] Efficient release of the payload after uptake. Another desired property of polymerosomes is their ability to efficiently release the contained / encapsulated payload after uptake. The release of the contained / encapsulated payload occurs in the lysosome of the target cell, and this release results from escape from the endosomal / lysosomal pathway or degradation of the polymerosome. In some embodiments, the zwitterion of the propagator block can facilitate endosomal escape. In even more embodiments, the propagator block may further contain alkyl chains, which also contribute to endosomal escape.

[0033] On the other hand, in some embodiments, the propagator block of the present disclosure may include a diethylene-triamine moiety to facilitate lysosomal degradation. The terminal amine residues of the diethylene-triamine moiety may also be used for additional functionalization. Alternatively, the propagator block of the present disclosure may include ethylenediamine, 1-(2-aminoethyl)piperazine, and / or tris(2-aminoethyl)amine.

[0034] Propagator spacer. In some embodiments, the propagator block of the present disclosure includes a propagator spacer primarily configured to impart the hydrophobicity to the copolymer required for assembly into polymersomes. In some embodiments, the propagator spacer includes a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0035] In some embodiments, the spacer may be a polyethylene glycol (PEG) portion formed of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65, or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, for example, 2-72, 2-60, 2-48, 2-36, 2-24, 2-18, 2-15, 2-10, 4-72, 4-60, 4-48, 4-36, 4-24, 4-18, 4-15, 4-10, 8-72, 8-60, 8-48, 8-36, 8-24, 8-18, 8-15, or 8-10 (OCH2CH2) subunits. In some embodiments, the PEG portion can have a linear, branched, or star-shaped structure.

[0036] In some embodiments, the saturated carbon portion may be a lipid tail extending from the propagator block. In some embodiments, the saturated carbohydrate contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or any range defined by the aforementioned endpoints, e.g., 2-15, 2-12, 2-10, 2-8, 2-6, 2-4, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, or 6-8 carbons. In certain embodiments, the propagator spacer comprises a monocarboxylic acid amide moiety (e.g., a lipoamide moiety) or other biocompatible structures, which provide saturated carbohydrates and functional groups for conjugation.

[0037] Example. In some embodiments, the propagator block of the present disclosure comprises at least one of the following structures (Note: “PB” represents a propagator block, which describes a propagator conjugate in the copolymer of the present disclosure. However, for the sake of brevity, “PB” may be used interchangeably with “P” to mean propagator):

[0038] [ka]

[0039] Copolymer Propagator Blocks. In some embodiments, the copolymer comprises a plurality of propagator blocks and a plurality of linkers, where each of the plurality of propagator blocks is connected to at least one other propagator block or initiation block of the plurality of propagator blocks via one of the plurality of linkers. Without wishing to be bound by theory, this disclosure assumes that it is beneficial to have two or more propagators within the structure of the copolymer. Each of the two or more propagators can provide at least one of the desired properties: efficient payload encapsulation, reduced serum protein adsorption, enhanced membrane fusion, and efficient payload release after uptake. In some embodiments, each of the two or more propagators has a different structure and / or desired property that it provides, thus forming the copolymer as a hetero-copolymer.

[0040] In some embodiments, the hetero-copolymer comprises any two or more propagator blocks PB1, PB2, PB3, PB4, and PB5. In some embodiments, the copolymer comprises at least two propagator blocks, which are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) PB1, PB4, and PB5, and two or more propagators in the copolymer can be represented by the formula numbers of the two or more propagators separated by the symbol " / ". For example, the two propagator blocks PB1 and PB5 of the copolymer can be represented as PB1 / PB5. However, the order and quantity of the propagator blocks in the referenced copolymer are not limited by how they are named. Without being constrained by theory, this disclosure finds that PB1 / PB4, PB2 / PB4, PB1 / PB5, and PB2 / PB5 copolymers exhibit superior intracellular delivery through efficient membrane fusion and payload release.

[0041] In certain embodiments, hetero-copolymer copolymers having two or more different types of propagator blocks may consist of any of the initiators I2, I3, I4, I5, I6, I7, I8, I9, and I10. Initiator-propagator conjugations can be represented by the formula numbers of the initiator and propagator, separated by a hyphen "-". For example, a copolymer containing initiator I5 and propagator P5 can be represented as I5-P5. However, the order and quantity of the initiator blocks and / or propagator blocks of the referenced copolymer are not limited by how they are named. In some embodiments, the copolymer of hetero-copolymers having two or more different types of propagators conjugated with one initiator of the present disclosure is I5-P1 / P5, I5-P1 / P4, I5-P2 / P5, I5-P2 / P4, I5-P1 / P4 / P5, I6-P1 / P5, I6-P1 / P4, I6-P2 / P5, I6-P2 / P4, I6-P1 / P4 / P5, I7-P1 / P5, I7-P1 / P4, I7- You can choose from the group consisting of P2 / P5, I7-P2 / P4, I7-P1 / P4 / P5, I8-P1 / P5, I8-P1 / P4, I8-P2 / P5, I8-P2 / P4, I8-P1 / P4 / P5, I9-P1 / P5, I9-P1 / P4, I9-P2 / P5, I9-P2 / P4, I9-P1 / P4 / P5, I10-P1 / P5, I10-P1 / P4, I10-P2 / P5, I10-P2 / P4, and I10-P1 / P4 / P5.

[0042] In some embodiments, the hetero-copolymer copolymer comprises several first propagator blocks and several second propagator blocks. In a particular embodiment, the number of first propagator blocks is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or any range defined by the aforementioned endpoints, for example, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 9-20, 9-18, 9-16, 9-14, 9-12, 12-20, 12-18, 12-16. In a particular embodiment, the number of second propagator blocks is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, or any range defined by the aforementioned endpoints, for example, 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, 3-20, 3-18, 3-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3-4, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 6-8, 9-20, 9-18, 9-16, 9-14, 9-12, 12-20, 12-18, 12-16. In some embodiments, the number of first propagator blocks and / or second propagator blocks can be determined using mass spectrometry, but are not limited to this.

[0043] Polymerosomes containing copolymers of the present disclosure The polymerosomes of the present disclosure comprise a membrane, and the membrane comprises copolymers of the present disclosure. In some embodiments, the membrane comprises multiple copolymers of the present disclosure, which aggregate to form a membrane having propagator blocks that couple to one another via hydrophobic interactions. The starting block of each copolymer of the polymerosome extends from the membrane and is exposed to the surrounding environment.

[0044] In some embodiments, the copolymer of the Disclosure constitutes at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the polymerosome membrane, or any range defined by the aforementioned endpoints, for example, 50%-99%, 50%-95%, 50%-90%, 50%-85%, 50%-80%, 50%-75%, 50%-70%, 50%-65% of the polymerosome membrane. It comprises percentages such as %, 50%~60%, 60%~99%, 60%~95%, 60%~90%, 60%~85%, 60%~80%, 60%~75%, 60%~70%, 60%~65%, 70%~99%, 70%~95%, 70%~90%, 70%~85%, 70%~80%, 50%~75%, 80%~99%, 80%~95%, 80%~90%, 80%~85%, 90%~99%, or 90%~95%.

[0045] In some embodiments, the film comprises at least two different types of copolymers of the Disclosure. In certain embodiments, the film comprises a copolymer comprising at least two different types of propagator blocks of the Disclosure. For example, the membrane comprises a copolymer containing a first propagator block and a second propagator block, wherein the molecular ratio of the first propagator block to the second propagator block in the membrane is 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 15, 20, or 25, or any range defined by the aforementioned endpoints, e.g., 1-25, 1-20, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 1-2.5, 1-2, 1-1.5, 2-25, 2-20, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 2-3, or 2-2.5.

[0046] In some embodiments, the polymersomes of the present disclosure are dendritic cell-targeted vaccines (DCTVs) for the specific targeted delivery of a payload to dendritic cells, thereby achieving and / or improving the immunogenic response of the vaccine.

[0047] Size of polymerosomes. In some embodiments, the polymerosomes of the present disclosure have diameters of 0.001, 0.005, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 microns, or any range defined by the aforementioned endpoints, e.g., 0.001-5, 0.001-4, 0.001-3, 0.001-2, 0.001-1, 0.001-0.8, 0.001-0.6, 0.001-0.4, 0.001-0.2, 0.001-0. They have a diameter of 1, 0.001~0.05, 0.001~0.01, 0.001~0.005, 0.05~5, 0.05~4, 0.05~3, 0.05~2, 0.05~1, 0.05~0.8, 0.05~0.6, 0.05~0.4, 0.05~0.2, 0.05~0.1, 0.1~5, 0.1~4, 0.1~3, 0.1~2, 0.1~1, 0.1~0.8, 0.1~0.6, 0.1~0.4, 0.1~0.2, 0.5~5, 0.5~4, 0.5~3, 0.5~2, 0.5~1, 0.5~0.8, 1~5, 1~4, 1~3, or 1~2 microns. The size of the polymerosomes can be determined by using dynamic light scattering (DLS), although these are not limited to these values. In some embodiments, the polymerosomes of this disclosure have a polydispersity index (PDI) of approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1, or any range defined by the aforementioned endpoints, e.g., 0.01 to 1, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0. 7. It has a polyvariance index (PDI) of 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, 0.01-0.05, 0.1-1, 0.1-0.9, 0.1-0.8, 0.1-0.7, 0.1-0.6, 0.1-0.5, 0.1-0.4, 0.1-0.3, or 0.1-0.2.

[0048] Zeta potential and molecular weight. Without wishing to be constrained by theory, the zeta potential and molecular weight of polymerosomes may influence cellular uptake of polymerosomes. In some embodiments, the polymerosomes of this disclosure include zeta potentials of approximately -50, -40, -30, -20, -15, -10, -5, 0, +5, +10, +15, +20, +30, +40, or +50, or any range defined by the aforementioned endpoints, e.g., -50 to +50, -50 to +40, -50 to +30, -50 to +20, -50 to +15, -50 to +10, -50 to +5, -50 to -5, -50 to -10, -50 to -15, -50 to Includes zeta potentials of -20, -20 to +50, -20 to +40, -20 to +30, -20 to +20, -20 to +15, -20 to +10, -20 to +5, -20 to -5, -20 to -10, -20 to -15, -15 to +50, -15 to +40, -15 to +30, -15 to +20, -15 to +15, -15 to +10, -15 to +5, -15 to -5, -15 to -10, +5 to +50, +5 to +40, +5 to +30, +5 to +20, +5 to +15, or +5 to +10. In some embodiments, the polymerosomes of this disclosure include molecular weights of approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, and 50 kDa, or any range defined by the aforementioned endpoints, for example, 1-50 kDa, 1-40 kDa, 1-30 kDa, 1-20 kDa, 1-15 kDa, 1-10 kDa, 1-5 kDa, 2-50 kDa, 2-40 kDa, 2-30 kDa, 2-20 kDa, 2-15 kDa, and 2-10 kDa. This includes molecular weights of kDa, 2-5kDa, 5-50kDa, 5-40kDa, 5-30kDa, 5-20kDa, 5-15kDa, 5-10kDa, 8-50kDa, 8-45kDa, 8-40kDa, 8-35kDa, 8-30kDa, 8-25kDa, 8-20kDa, 8-15kDa, 8-10kDa, 12-50kDa, 12-45kDa, 12-35kDa, 12-25kDa, 12-15kDa, 25-50kDa, 25-40kDa, or 25-30kDa.

[0049] Payload. In some embodiments, the polymerosome membrane defines an internal space configured to encapsulate or carry a payload. Where used herein, “encapsulate a payload,” “encapsulated in the internal space,” or similar descriptions refer to a state in which the payload is held, wrapped, or surrounded by the polymerosome membrane. The payload may move freely within the internal space or may be covalently or noncovalently bound to the membrane. Encapsulation may be substantial, complete, or partial and does not preclude the possibility that a portion of the payload may be exposed to the environment outside the polymerosome. In embodiments of partial encapsulation, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the payload is held, wrapped, or surrounded by the polymerosome membrane. In some embodiments, the payload may be a biomolecule, e.g., nucleic acids, compounds, polypeptides, proteins, glycan heads, or combinations thereof.

[0050] In some embodiments, the payload is ribonucleic acid (RNA, e.g., mRNA) or deoxyribonucleic acid (DNA, e.g., double-stranded or single-stranded DNA), which can be delivered to target cells using the polymerosomes of this disclosure and then encode polypeptides or proteins in vivo. The nucleic acids, e.g., mRNA molecules used in this disclosure, can be prepared by in vitro transcription from a reference nucleic acid. In vitro transcription can be carried out as described in PCT patent publication WO2014 / 152027, filed March 13, 2014, which incorporates its entirety by reference.

[0051] In some embodiments, the polypeptide or protein is immunogenic (e.g., antigenic) to the organism to which the polymerosome is administered. In such embodiments, the polymerosomes of the present disclosure are used to encapsulate and carry immunogenic proteins or nucleic acids configured to encode the immunogenic protein in vivo, for example, to encapsulate and retain mRNA molecules in RNA vaccines. The immunogenic protein can be a protein of a pathogen of viral (e.g., SARS-CoV-2, influenza (flu), respiratory syncytial virus (RSV), EBV, dengue fever, VZV, HIV, ZIKA, or NIPAH) origin, bacterial, or fungal origin. In some embodiments, the immunogenic protein can be a viral spike protein. In certain embodiments, the spike protein can be of coronavirus (CoV) origin, for example, SARS-CoV, MERS-CoV, and SARS-CoV-2. In some embodiments, examples of coronaviruses (CoVs) described herein include, but are not limited to, alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, omicron-SARS-CoV2, and their variants.

[0052] In some embodiments, the payload is a nucleic acid, which can be a polynucleotide having a reading frame configured to encode a polypeptide or protein in vivo. Such a polynucleotide may be modified with a 5' end cap, which is generated during the in vitro transcription reaction using the following chemical RNA cap analogs: 3”-O-Me-m7G(5)ppp(5')G[ARCA cap], G(5)ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, or m7G(5')ppp(5')G (New England Bio Labs, Ipswich, Mass.). 5'-capping of the modified polynucleotide can be completed post-transcriptionally using a cowpox virus capping enzyme to generate a “CAP 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The CAP 1 structure can be generated by producing m7G(5')ppp(5')G-2'-O-methyl using both cowpox virus capping enzyme and 2'-O-methyltransferase. The CAP 2 structure can be generated from the CAP 1 structure, followed by 2'-O-methylation of the 5'-third-to-last nucleotide using 2'-O-methyltransferase. The CAP 3 structure can be generated from the CAP 2 structure, followed by 2'-O-methylation of the 5'-fourth-to-last nucleotide using 2'-O-methyltransferase. The enzymes may be derived from recombinant sources. After transfection in mammalian cells, the modified polynucleotides have stability for 12–18 hours or longer, e.g., 24, 36, 48, 60, 72, or longer than 72 hours.

[0053] In some embodiments, nucleic acids may be modified. In some embodiments, nucleic acids may have several (more than one) modifications, which may be identical or different from one another. In some embodiments, nucleic acids may contain one, two, or more (optionally different) nucleoside or nucleotide modifications in a particular region. In some embodiments, modified nucleic acids (e.g., modified mRNA polynucleotides) exhibit reduced degradation in cells or organisms compared to unmodified nucleic acids. In some embodiments, modified nucleic acids may exhibit reduced immunogenicity in organisms (e.g., reduced innate response).

[0054] In some embodiments, the modifications may include chemical modifications. In some embodiments, the modifications may be naturally occurring modifications, unnaturally occurring modifications, or both. Some exemplary modifications useful in this disclosure, but not limited to, include modifications of sugar, nucleic acid base, nucleoside linkages (e.g., phosphate linkages, phosphodiester linkages, or linkages to phosphodiester backbones) or combinations thereof. In some embodiments, the nucleic acids (e.g., RNA) used as payloads in this disclosure are codon-optimized. For example, nucleic acids can be modified to increase their G / C content. The G / C content of nucleic acids can affect their stability. Nucleic acids with a higher amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids with a higher amount of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO2002 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modifications within a transformed region. Due to the degenerate nature of the gene code, modifications work by substituting existing codons with codons that promote higher RNA stability, without altering the amino acids produced.

[0055] In some embodiments, the nucleic acid may further include a sequence encoding a signal peptide. The signal peptide may comprise three regions: (1) an N-terminal region of different lengths, which typically contains positively charged amino acids; (2) a hydrophobic region; and (3) a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the trans-transport of the growing peptide chain. While the signal peptide is not typically involved in the final destination of the mature protein, this is not limited to this disclosure. The signal peptide is usually cleaved from the precursor protein by ER-resident signal peptidases. They can also remain uncleaved and function as membrane anchors. In some embodiments, the signal peptide may also be condensed with a polypeptide or protein and designed so that the payload is encoded at its C-terminus or N-terminus.

[0056] In some embodiments, the payload may be a therapeutic or prophylactic reagent for treating or preventing a disease (e.g., cancer or infection). For example, the payload may include, but is not limited to, antiviral agents such as ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (GS-5734), and favipiravir (T-705), interferon, adefovir, tenofovir, acyclovir, brivudine, cidofovir, fomivirsen, foscarnet, ganciclovir, amantadine, rimantadine, zanamivir, remdesivir, mornupiravir, and pachyrovid. In other embodiments, the payload may be an anticancer agent. In certain embodiments, the payload is a nucleic acid configured to encode a therapeutic or prophylactic reagent.

[0057] In some embodiments, the N / P ratio (ratio of positively chargeable polymeramine (N=nitrogen) groups to negatively charged nucleic acid phosphate groups (P)) of the polymerosome encapsulating the nucleic acid is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or The range is 50, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, 8-40, 8-20, 8-12, 9-50, 9-30, or 9-15. In another embodiment, the polymerosome encapsulating the mRNA has a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

[0058] In some embodiments, where the polymerosome payload is a nucleic acid configured to encode a polypeptide or protein within the target cell after being taken up by the target cell, the polymerosome encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 copies of the polypeptide or protein in vivo, or any range defined by the aforementioned endpoints, including or excluding the endpoints. For example, the polymerosomes are configured to encode a range of copies, such as 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 5-8, 4-50, 4-45, 4-35, 4-25, 4-15, 4-9, 4-6, 7-50, 7-45, 7-35, 7-25, 7-15, or 7-9 copies. In some embodiments, after uptake by target cells, the polymerosomes are configured to continuously and immediately encode polypeptides or proteins in vivo until the nucleic acid (i.e., payload) is inactivated in vivo.

[0059] Composition / Formulation One aspect of this disclosure relates to a composition (i.e., a formulation) comprising the polymersomes of this disclosure. The polymersomes of the composition can encapsulate a payload and are configured to deliver the payload to a target region of a biological organism. The payload may include nucleic acids, compounds, peptides, proteins, glycan heads, or combinations thereof, as described herein. In some embodiments, the payload may be an immunogenic protein or nucleic acid configured to encode an immunogenic protein in vivo. In some embodiments, the formulation further includes pharmaceutically acceptable excipients, adjuvants, or combinations thereof. In certain embodiments, the composition is a pharmaceutical composition or a pharmaceutical formulation.

[0060] In some embodiments, the composition includes 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95% (w / w) of the polymersomes of this disclosure, with or without a payload, or any range defined by the aforementioned endpoints, with or without the endpoints, for example, 0.01% to 95% (w / w), 0.01% to 90% (w / w). , 0.01%~80%(w / w), 0.01%~70%(w / w), 0.01%~60%(w / w), 0.01%~50%(w / w), 0.01%~40%(w / w), 0.01%~30%(w / w), 0.01%~20% (w / w), 0.01%~10%(w / w), 0.01%~5%(w / w), 0.01%~1%(w / w), 0.01%~0.1%(w / w), 0.1%~95%(w / w), 0.1%~90%(w / w), 0.1%~80% (w / w), 0.1%~70%(w / w), 0.1%~60%(w / w), 0.1%~50%(w / w), 0.1%~40%(w / w), 0.1%~30%(w / w), 0.1%~20%(w / w), 0.1%~10%(w) / w), 0.1%~5%(w / w), 0.1%~1%(w / w), 1%~95%(w / w), 1%~90%(w / w), 1%~80%(w / w), 1%~70%(w / w), 1%~60%(w / w), 1%~50%(w / w) The composition comprises polymersomes of the Disclosure in the range of 1%-40% (w / w), 1%-30% (w / w), 1%-20% (w / w), 1%-10% (w / w), 1%-5% (w / w), 5%-95% (w / w), 5%-90% (w / w), 5%-80% (w / w), 5%-70% (w / w), 5%-60% (w / w), 5%-50% (w / w), 5%-40% (w / w), 5%-30% (w / w), 5%-20% (w / w), or 5%-10% (w / w). The remainder of the composition may be excipients as described herein.

[0061] In some embodiments, the composition is an mRNA vaccine, and the polymerosome encapsulates mRNA configured to encode an immunogenic protein in vivo. The immunogenic protein may be the viral spike protein of a pathogen or other antigenic molecule. In certain embodiments, the composition of the present invention may be a COVID-19 mRNA vaccine.

[0062] The exemplary COVID-19 mRNA vaccines described herein can be designed based on mRNA technology to remove the glycan shield of the coronavirus (e.g., SARS-CoV-2) spike protein to better expose the conserved region of the spike protein. By having a deletion of the glycosylation site in the receptor-binding domain (RBD) or subunit 2 (S2) domain, coronavirus spike protein mRNA vaccines expose highly conserved epitopes, induce antibody and CD8 T-cell responses, and provide broader protection against alpha, beta, gamma, delta, omicron, and various variants compared to unmodified mRNA. The vaccine may be a low-sugar universal vaccine (LSUV) as described in WO2022 / 221835 (mRNA containing a sequence selected from SEQ ID NOs. 1-52), WO2022 / 221837A2 (mRNA containing a sequence selected from SEQ ID NOs. 1-21), and US20200046826A1 (mRNA containing a sequence selected from SEQ ID NOs. 1-20), which are incorporated herein by reference in their entirety.

[0063] In some embodiments, the compositions of the present invention are configured to treat or prevent a disease (e.g., cancer). In such embodiments, the payload carried by the polymerosome may be a therapeutic reagent, a prophylactic reagent, or a nucleic acid configured to encode a therapeutic or prophylactic reagent in vivo. For example, the composition may be a personalized cancer vaccine (e.g., melanoma), a KRAS vaccine (KRAS-inducing), or a checkpoint vaccine (e.g., PD-1, PDL-1 related).

[0064] In some embodiments, the compositions of the present invention can be administered together with other compositions (e.g., vaccines or drugs). Examples of other compositions, but not limited to, include influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines.

[0065] Combination composition In some embodiments, the composition may be a combo composition (e.g., a combo vaccine) comprising a first polymerosome encapsulating a first payload and a second polymerosome encapsulating a second payload. The first and second polymerosomes may be polymerosomes described herein, but they may differ from each other in terms of the structure or properties of their copolymers. For example, the first and second polymerosomes may differ in size, the copolymer forming their membrane, the payload encapsulated within the polymerosome, or a combination thereof.

[0066] For example, the first polymerosome contains a glycan head configured to bind to DC-SIGN, while the second polymerosome contains a glycan head configured to bind to Siglec-1. In another example, the first polymerosome contains a glycan configured to target antigen-presenting cells, while the second polymerosome contains a glycan configured to target cancer cells.

[0067] In some embodiments, the first and second payloads are different from each other. For example, the first payload may be a protein or peptide, while the second payload may be a nucleic acid. In certain embodiments, both the first and second payloads may be mRNA molecules but may encode different proteins. For example, the first payload may be an mRNA configured to encode the delta-SARS-CoV-2 spike protein, while the second payload may be an mRNA configured to encode the omicron-SARS-CoV-2 spike protein.

[0068] Additional components of the composition In some embodiments, the compositions disclosed herein may further include adjuvants and / or inactive substances, such as pharmaceutically acceptable excipients. In certain embodiments, the adjuvant may be, but is not limited to, C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide (α-GalCer), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts), squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), CpG1018 (Dynavax), or a combination thereof.

[0069] In certain embodiments, pharmaceutically acceptable excipients may include solvents, dispersion media, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidases, or mixtures thereof. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (Remington: The Science and Practice of Pharmacy, 22 ndSee Edition, Edited by Allen, Loyd V., Jr., Pharmaceutical Press. The use of conventional excipients may be envisioned within the scope of this disclosure, except where any conventional excipient is incompatible with the substance or its derivatives by interacting adversely with any other component(s) of the pharmaceutical composition, for example, by generating any undesirable biological effects or otherwise. The formulation of standard pharmaceutically acceptable excipients can be carried out using prescribed methods in the field of pharmacy (see Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA).

[0070] In some embodiments, the composition further comprises a phosphate conjugate. Without being bound by theory, phosphate conjugates can increase the in vivo circulation time and / or increase the targeted delivery of the polymerosomes of this disclosure. Phosphate conjugates for use in this disclosure can be prepared using the methods described in PCT publication WO2013 / 033438 filed August 30, 2012, or U.S. publication US2013 / 0196948 filed June 23, 2011, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, phosphate conjugates may include compounds of any of the formulas described in PCT publication WO2013 / 033438 filed August 30, 2012, the contents of which are incorporated herein by reference in their entirety.

[0071] In some embodiments, the composition further comprises a conjugate for enhancing the delivery of the polymerosomes of this disclosure. Without wishing to be constrained by theory, the conjugate selected for use can inhibit the phagocytic clearance of polymerosomes in the subject. In some examples, the conjugate may be the human membrane protein CD47 or a “self-peptide” derived therefrom (e.g., the “self” particle described by Rodriguez et al. (Science 2013, 339, 971-975) which is incorporated herein by reference in its entirety).

[0072] In some embodiments, where the payload is an immunogenic agent or a nucleic acid configured to encode an immunogenic agent, the composition further comprises an immunostimulant that enhances the immune response induced by the immunogenic agent. In non-limiting examples, the composition may comprise a Th1 immunostimulant, which can enhance the Th1-based response of the immune system (see PCT publication WO2010 / 123569 and U.S. publication 2011 / 0223201, each incorporated herein by reference in whole).

[0073] In some embodiments, the composition does not contain viral components (e.g., viral capsids, viral enzymes, or other viral proteins, e.g., those required for virus-based replication) or have them packaged, encapsulated, linked, or otherwise attached within the virus or viral particles.

[0074] Kit for preparing polymersomes One aspect of the present disclosure relates to a kit for preparing polymersomes of the present disclosure. The kit comprises a first reagent and a second reagent, the first reagent comprising an initiator comprising a glycan head and an initiator linker, and the second reagent comprising a propagator comprising a functional moiety and a propagator linker, the functional moiety comprising a guanidine group, a zwitterionic group, diethylenetriamine or a combination thereof, and the initiator linker is configured to be coupled to the propagator linker via a linker comprising a disulfide bond.

[0075] In some embodiments, the initiator linkage is configured to couple with the propagator linkage, thereby forming the linkage of the copolymer of the Disclosure. In some embodiments, the initiator linkage and the propagator linkage are independently thiol groups or dithioane groups.

[0076] In some embodiments, the kit further comprises a reagent containing a payload encapsulated by polymerosomes prepared using the kit of this disclosure. In certain embodiments, the payload may be as described herein.

[0077] Initiator The initiator includes a glycan head and an initiator linking portion. In some embodiments, the glycan head is described above in the initiator block of the copolymer of the present disclosure.

[0078] In some embodiments, the initiator molecule further comprises an initiator spacer described herein in the copolymer of the Disclosure. In certain embodiments, the initiator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0079] In some embodiments, the initiator is

[0080] [ka] The data is selected from the group consisting of TIFF0007851666000004.tif145165.

[0081] PropaGe The propagator comprises a functional moiety and a propagator linking moiety, the functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof. In some embodiments, the functional moiety of the propagator molecule is as described above in the propagator block of the copolymer of the present disclosure.

[0082] In some embodiments, the propagator further comprises a propagator spacer described herein in the copolymer of the Disclosure. In certain embodiments, the propagator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0083] In some embodiments, the second reagent comprises two types of propagators, each differing from the others in the structure or desired properties it provides. For example, the second reagent may comprise a first propagator and a second propagator, each independently comprising a functional moiety containing a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In certain embodiments, the first propagator comprises a guanidine group and the second propagator comprises a zwitterionic group. In other embodiments, the first propagator comprises a guanidine group and the second propagator comprises diethylenetriamine.

[0084] In some other embodiments, the kit further comprises a third reagent, which comprises a propagator different from the second reagent's propagator in terms of the structure or desired properties it provides. For example, the second reagent's propagator is the first propagator molecule, and the third reagent comprises the second propagator. The first and second propagators may independently contain functional moieties comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In certain embodiments, the first propagator comprises a guanidine group and the second propagator comprises a zwitterionic group. In other embodiments, the first propagator comprises a guanidine group and the second propagator comprises diethylenetriamine.

[0085] In some embodiments, the propagator is

[0086] [ka] It is selected from the group consisting of the following.

[0087] packaging All components of the kit of this disclosure can be packaged individually in physical containers. In some embodiments, the first reagent and the second reagent are contained in the same container; in other words, they are in ready-to-use packaging. In some other embodiments, the first reagent and the second reagent are contained in separate containers, so that the user can decide whether and when to mix them.

[0088] How to use One aspect of the present disclosure relates to a method using the polymersomes of the present disclosure. In particular, the method is carried out to obtain a desired effect in a subject, such as targeting and delivering a payload, preventing or treating a disease, or boosting an adaptive immune response. In some embodiments, the subject may be an animal or a human, but is not limited thereto, and the payload is designed to demonstrate its efficacy against these subjects, and there is a need to treat or prevent a disease in these subjects, or to boost an adaptive immune response in these subjects.

[0089] A method for targeting and delivering a payload to a target. In some embodiments, a method is provided for targeted delivery of a payload to a subject, comprising administering an effective amount of the polymersomes of this disclosure to the subject. In some embodiments, a method is provided for targeted delivery of a payload to a subject, comprising administering an effective amount of the pharmaceutical formulation of this disclosure to the subject. The polymersomes and payloads are as described herein, and the polymersomes encapsulate the payload within an internal space defined by the polymersome membrane.

[0090] Without being constrained by any theory, targeted delivery is achieved by the initiation block of copolymers that form polymerosomes. In particular, the initiation block provides the desired binding affinity / specificity target to the desired region of the target via its glycan head.

[0091] Methods for preventing or treating diseases in the target population. In some embodiments, a method is provided for preventing or treating a disease in a subject, comprising administering an effective amount of the polymersomes of the present disclosure to the subject. The polymersomes of the method encapsulate a payload within an internal space defined by the polymersome membrane, the payload being a therapeutic agent or inducer configured to prevent and / or treat a disease.

[0092] Without wishing to be bound by any theory, the polymersomes of this disclosure provide targeted delivery via the glycan head of their copolymer. Therefore, by using the polymersomes of this disclosure to deliver a payload, the efficacy of the payload can be more effectively implemented. For example, in embodiments comprising a structure in which the glycan head specifically binds to DC-SIGN on dendritic cells, an antigenic or immunogenic payload can be effectively delivered to dendritic cells to induce an immune response for the prevention of a disease of concern. This strategy is beneficial for delivering nucleic acids encoding antigens of antigens or vaccines. Several other examples involve having a glycan head designed to target cancer cells so that an antitumor reagent can be effectively delivered to the tumor microenvironment. This strategy can increase the efficacy of the antitumor reagent and reduce therapeutic side effects.

[0093] In some embodiments, the disease is characterized by dysfunction or abnormal protein or polypeptide activity. For example, the disease is selected from the group consisting of rare diseases, infections, cancer and proliferative disorders, genetic disorders (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal diseases, and metabolic diseases.

[0094] In some embodiments, the disease may be cancer or an infectious disease. In certain embodiments, the disease may be a viral infection, but may include, without limitation, infections associated with human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19).

[0095] How to boost the adaptive immune response In some embodiments, a method is provided for boosting an adaptive immune response, comprising administering an effective amount of the polymerosomes of the present disclosure to a subject. The polymerosomes of the present method encapsulate a payload within an internal space defined by the polymerosome membrane, the payload being a therapeutic agent configured to induce or trigger an adaptive immune response in a subject.

[0096] Without wishing to be bound by any theory, the polymerosomes of this disclosure provide targeted delivery to immune cells via glycan heads of their copolymers. In some embodiments, the glycan heads include structures that bind to antigen-presenting cells with desired specificity or affinity. For example, the glycan heads may include structures that specifically bind to DC-SIGNs on dendritic cells so that the polymerosomes can specifically deliver an immunogenic payload to dendritic cells to facilitate the initiation of an adaptive immune response.

[0097] In some embodiments, the boosted adaptive immune response is against diseases, including but not limited to cancer or infectious diseases. For example, infectious diseases can be viral infections, including, but not limited to, infections associated with human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19).

[0098] Administration In some embodiments of the methods of the present disclosure, a subject is administered a single-dose polymersome or formulation of the present disclosure, with or without a payload encapsulated. In some embodiments, a subject is administered an initial dose by polymersome, followed by at least one booster dose, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional doses, with intervals between doses of approximately 1, 2, 3, 4, 5, 6, 7 days, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or approximately The administrations are performed at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or within any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, between 1 and 7 days, 1 and 5 days, 1 and 3 days, 1 and 10 weeks, 1 and 8 weeks, 1 and 6 weeks, 1 and 4 weeks, 1 and 2 weeks, 1 and 12 months, 1 and 8 months, 1 and 6 months, 1 and 4 months, 1 and 2 months, or between 6 and 12 months. In a particular embodiment, the polymerosomes of the Disclosure encapsulating the payload are administered twice, either identically or in different doses, at intervals of 1 day, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, between 1 and 5 days, 1 and 2 weeks, 1 and 3 months, 1 and 6 months, 1 and 1 year, 3 and 1 year, or 6 and 1 year.

[0099] Route of administration. The polymerosomes or compositions described herein may be administered by any route. Suitable routes include, but are not limited to, oral, nasal, mucosal, submucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, percutaneous, and oral buccal routes. Some practical topical applications include, but are not limited to, the application of drops, sprays, aerosols, gels, or ointments to the mucosal epithelium of the eyes, nose, mouth, anus, or vagina. Other possible routes of administration are the application of sprays, aerosols, or powders by inhalation through the respiratory tract.

[0100] Effective dose. As used herein, an effective dose means an amount sufficient to produce the desired effect. In embodiments where the purpose of administering the polymerosomes of this disclosure is to treat a disease, the effective dose means a therapeutic effective dose, while in some other embodiments where the purpose is to prevent a disease, the effective dose means a prophylactic effective dose.

[0101] In some other embodiments, where the purpose of administering polymerosomes with a payload is to boost an adaptive immune response, the effective dose can be determined as an amount sufficient to induce an antigen-specific immune response in the subject to which the polymerosomes and payload are administered. The antigen-specific immune response can be characterized by measuring the antibody titer of the anti-antigenic polypeptide (i.e., the payload or its products) produced in the subject to which the polymerosomes and payload are administered. In some embodiments, the measurement can be performed using enzyme-linked immunosorbent assay (ELISA).

[0102] In some embodiments, antibody titers are used to assess whether a subject has had an infection or to determine whether immunization is required. In some embodiments, antibody titers are used to determine the intensity of an autoimmune response, to determine whether booster immunization is required or whether immunization has been boosted, to determine whether a past vaccine was effective, and / or to identify any recent or previous infection.

[0103] The effective amount of the method of this disclosure can be determined based on several factors, including, but not limited to, the condition of the subject (age, sex, species, weight, health status, etc.), the progression of the disease to be treated, the route of administration, the dose and interval of administration, and the nature of the payload. With respect to the nature of the payload, for example, in embodiments in which the polymerosome of this disclosure is used to carry mRNA, such as in the case of an mRNA vaccine, the effective amount can be determined based on the effective amount of mRNA required to evoke a sufficient immune response in the subject. Therefore, in some embodiments where the payload is mRNA, the effective amount of the method of this disclosure is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 micrograms (μg or ug) or any range defined by the aforementioned endpoint, including or excluding the endpoint, for example, 5 micrograms to 1000 micrograms, 5 micrograms to 900 micrograms, 5 micrograms to 800 micrograms, 5 micrograms to 700 micrograms, 5 micrograms to 600 micrograms, 5 micrograms to 500 micrograms, 5 micrograms to 400 micrograms Chlorograms, 5 micrograms to 300 micrograms, 5 micrograms to 200 micrograms, 5 micrograms to 175 micrograms, 5 micrograms to 150 micrograms, 5 micrograms to 125 micrograms, 5 micrograms to 100 micrograms, 5 micrograms to 90 micrograms, 5 micrograms to 80 micrograms, 5 micrograms to 70 micrograms, 5 micrograms to 60 micrograms, 5 micrograms to 50 micrograms, 5 micrograms to 40 micrograms, 5 micrograms to 30 micrograms, 5 micrograms to 20 micrograms, 5 micrograms to 10 micrograms, 10 micrograms to 1000 micrograms, 10 micrograms to 900 micrograms, 10 micrograms to 800 micrograms, 10 micrograms to 700 micrograms,10 micrograms to 600 micrograms, 10 micrograms to 500 micrograms, 10 micrograms to 400 micrograms, 10 micrograms to 300 micrograms, 10 micrograms to 200 micrograms, 10 micrograms to 175 micrograms, 10 micrograms to 150 micrograms, 10 micrograms to 125 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 90 micrograms, 10 micrograms to 80 micrograms, 10 micrograms to 70 micrograms 10 micrograms to 60 micrograms, 10 micrograms to 50 micrograms, 10 micrograms to 40 micrograms, 10 micrograms to 30 micrograms, 10 micrograms to 20 micrograms, 50 micrograms to 1000 micrograms, 50 micrograms to 900 micrograms, 50 micrograms to 800 micrograms, 50 micrograms to 700 micrograms, 50 micrograms to 600 micrograms, 50 micrograms to 500 micrograms, 50 micrograms to 400 micrograms 50 micrograms to 300 micrograms, 50 micrograms to 200 micrograms, 50 micrograms to 175 micrograms, 50 micrograms to 150 micrograms, 50 micrograms to 125 micrograms, 50 micrograms to 100 micrograms, 50 micrograms to 90 micrograms, 50 micrograms to 80 micrograms, 50 micrograms to 70 micrograms, or 50 micrograms to 60 micrograms, 100 micrograms to 1000 micrograms, 100 micrograms to 900 micrograms Micrograms, 100 micrograms to 800 micrograms, 100 micrograms to 700 micrograms, 100 micrograms to 600 micrograms, 100 micrograms to 500 micrograms, 100 micrograms to 400 micrograms, 100 micrograms to 300 micrograms, 100 micrograms to 200 micrograms, 100 micrograms to 175 micrograms, 100 micrograms to 150 micrograms, 300 micrograms to 1000 micrograms, 300 micrograms to 900 micrograms,The ranges are 300 micrograms to 800 micrograms, 300 micrograms to 700 micrograms, 300 micrograms to 600 micrograms, 300 micrograms to 500 micrograms, 300 micrograms to 400 micrograms, 500 micrograms to 1000 micrograms, 500 micrograms to 900 micrograms, 500 micrograms to 800 micrograms, 500 micrograms to 700 micrograms, 500 micrograms to 600 micrograms, 600 micrograms to 800 micrograms, or 700 micrograms to 900 micrograms.

[0104] However, given the targeted delivery provided by the polymersomes of this disclosure, it can be expected that the effective amount required for the method of this disclosure may be lower than the effective amount required for other untargeted delivery methods. For example, the effective amount required for the method of this disclosure may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, or 99% lower than the effective amount required for other untargeted delivery methods, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1-99%, 1-95%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-99%, 5-95%, 5-90%, 5-80%, 5- 70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 30-99%, 30-95%, 30-90%, 30-80%, 30-70%, 30-60 The percentage may be low in the range of %, 30-50%, 30-40%, 50-99%, 50-95%, 50-90%, 50-80%, 50-70%, 50-60%, 70-99%, 70-95%, 70-90%, 70-80%, 80-99%, 80-95%, 80-90%, 90-99%, or 95-99%.

[0105] Furthermore, in some embodiments in which the polymersomes of the Disclosure are used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an antibody against the antigenic agent, the titer of the antibody induced by the Disclosure increases by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log compared to the titer of the antibody induced by a non-targeted delivery method, or increases by any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1 to 10 log, 1 to 8 log, 1 to 6 log, 1 to 4 log, 2 to 9 log, 2 to 7 log, 2 to 5 log, 3 to 10 log, 3 to 8 log, 3 to 5 log, or 4 to 6 log.

[0106] In some other embodiments in which polymersomes of the Disclosure are used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an immune response to the antigenic agent, the antibody titer against the antigenic agent induced by the Disclosure is 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the antibody titer of the untargeted delivery method, or within any range defined by the aforementioned endpoints, for example, 0.1 to 10, 0.1 to 9, 0.1, with or without the endpoints. Higher in the range of ~8, 0.1~7, 0.1~6, 0.1~5, 0.1~4, 0.1~3, 0.1~2, 0.1~1, 0.1~0.5, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 0.5~2, 0.5~1, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 5~10, 5~9, 5~8, 5~7, 5~6, 7~10, 7~9, 7~8, or 8~10 times.

[0107] In some embodiments, the polymersomes of the Disclosure are used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an immune response to the antigenic agent. In some embodiments, the immune response is induced by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 days earlier than an immune response induced by a non-targeted delivery method, or by any range defined by the aforementioned endpoints, for example, 1-20, 1-18, 1-14, 1-10, 1-6, 2-20, 2-18, 2-14, 2-10, 2-6, 5-20, 5-18, 5-14, or 5-10 days earlier.

[0108] In some embodiments, the polymerosomes described herein are administered in a method of this disclosure to deliver a payload (body weight of one subject, per day, once or more times per day) of about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg (body weight of one subject, per day, once or more times per day) at a dose level sufficient to obtain the desired effect in vivo.

[0109] definition Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which this disclosure pertains. Unless otherwise stated, the technologies used or assumed herein are standard methodologies well known to those skilled in the art. Unless otherwise pointed out, the implementation of this disclosure utilizes prior art in microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the scope of the skills of those skilled in the art. Materials, methods and examples are for illustrative purposes only and are not intended to limit the scope of this disclosure. The following are presented as examples and are not intended to limit the scope of this disclosure.

[0110] It should be understood that in some cases the numbers used to describe and claim certain embodiments of this disclosure, such as the amount of raw material components, properties such as molecular weight, reaction conditions, and results, are modified with the term "approximately." Those skilled in the art will understand the meaning of the value modified by the term "approximately" in context. The numerical values ​​presented in some embodiments of this disclosure may include certain errors derived from the standard deviation in each of these test measurements. For example, the term "approximately," as used herein, refers to a measurable value such as a quantity or temporary period and is intended to include variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from a specified value, thus the variation is appropriate.

[0111] As used herein, “substantially” means sufficiently effective for the intended purpose. The term “substantially” thus allows for minor, slight variations from an absolute or perfect state, dimension, measurement, result, etc., such as variations that would be expected by a person skilled in the art but do not have a noticeable effect on the overall performance. When used in reference to numerical values, parameters, or characteristics, “substantially” means within 10 percent.

[0112] As used herein, "treat", "treatment" and "treating" refer to methods for obtaining beneficial or desired results, such as clinical results. In the present disclosure, beneficial or desired results can include inhibiting or suppressing the onset or progression of an infectious disease or disorder, preventing the onset of an infectious disease or disorder, restoring or reducing the symptoms of an infectious disease or disorder; or combinations thereof.

[0113] As used herein, "preventing" and "prevention" are used interchangeably with "prevention method" and can mean complete prevention of an infectious disease or prevention of the onset of symptoms of that infectious disease, delaying the onset of a disease or its symptoms or reducing the severity of an subsequently developed infectious disease or its symptoms.

[0114] As used herein, "glycan" refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or branched. Glycans can include residues of natural sugars (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.).

Examples

[0115] [Example 1] Synthesis of Exemplary Polymersomes Chemicals and Methods For chemical synthesis, all starting materials and commercially available reagents were purchased from Sigma-Aldrich and used as received unless otherwise specified. All reactions were carried out using dry solvents in oven-dried glassware under a nitrogen atmosphere. 1 H and 13 C NMR spectra were recorded on a Brucker AV-600 spectrometer using the solvent used as a reference (1 H and 13 For C, the values ​​were as follows: CDCl3: δ 7.24 and 77.23, CD3OD: δ 3.31 and 49.2, D2O: δ 4.80, DMSO-d6: δ 2.5 and 39.51). Chemical shifts (δ) are reported in ppm using the following rules: chemical shift, multiplicity (s=singular, d=double, t=tripular, q=quadruplicate, m=multiplex), integral, and binding constant (J), where J is reported in Hz. High-resolution mass spectra were recorded under ESI-TOF mass spectrometry conditions. Silica gel (E, Merck) was used for flash chromatography. The IMPACT® system (Intein Mediated Purification with Affinity Chitinbinding Tag) was purchased from New England Biolabs. His-tag purification resin was purchased from Roche. HiTrap IMAC columns (5 mL) were purchased from GE Healthcare Life Sciences. Gel permeation chromatography (GPC) with an Ultimate 3000 liquid chromatography system equipped with a 101 refractive index detector and Shodex column was performed using THF as the eluent, at 30°C, 1 mL per minute. -1 Polymer products were analyzed by flow rate. Calibration was based on a narrow linear poly(styrene) Shodex standard (SM-105). Mw and dispersion of polymer products were calculated using DIONEX Chromeleon software. Transmission electron microscope (TEM) images were obtained using an FEI Tecnai G2 F20 S-Twin.

[0116] The chemicals and methods described herein apply to all examples described herein.

[0117] Preparation of Exemplary Propagators of the Present Disclosure Five exemplary propagators, propagators P1, P2, P3, P4, and P5, are described herein. The preparation processes for propagators P1 (compound 2), P2 (compound 3), and P4 (compound 8) are illustrated in Scheme 1 below. Propagator P3 (compound 11) was prepared according to Scheme 2 below. Propagator P5 (compound 14) was prepared according to Scheme 3 below. The compounds obtained at each step of the scheme were verified by NMR. Additional details and data are provided below.

[0118] [ka]

[0119] [ka]

[0120] [ka]

[0121] compound 1

[0122] [ka]

[0123] Based on the published procedure, compound 1 was synthesized and characterized. ¹H NMR (600 MHz, CDCl₃): δ 5.92 (br, ¹H), 3.60–3.57 (m, ¹H), 3.29 (dt, J = 11.2 Hz, ²H), 3.21–3.10 (m, ²H), 2.86–2.79 (m, ²H), 2.55–2.31 (m, ¹H), 2.21 (t, ²H, J = 7.4 Hz), 1.90–1.85 (m, ¹H), 1.77–1.41 (m, ⁸H). The synthesis was carried out according to Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153–12160, which is incorporated herein by reference in its entirety.

[0124] compound 2

[0125] [ka]

[0126] Based on the published procedure, compound 2 was synthesized and characterized. 1 ¹H NMR (600 MHz, MeOD): δ 3.98 (s, 1H), 3.61-3.39 (m, 1H), 3.30-3.22 (m, 4H), 3.22-2.88 (m, 2H), 2.56-2.30 (m, 1H), 2.21 (t, J = 7.4 Hz, 2H), 1.98-1.72 (m, 1H), 1.79-1.30 (m, 6H). The synthesis was carried out according to Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153-12160, which is incorporated herein by reference in its entirety.

[0127] compound 3

[0128] [ka]

[0129] Based on the published procedure, compound 3 was synthesized and characterized. 1 ¹H NMR (600 MHz, CDCl3): δ 3.61 - 3.56 (m, 1H), 3.41 - 3.34 (m, 1H), 3.31 - 3.16 (m, 8H), 3.14 - 3.06 (m, 2H), 2.54 - 2.40 (m, 1H), 2.08 (t, J = 7.4 Hz, 2H), 1.95 - 1.86 (m, 1H), 1.69 (s, 1H), 1.55 (m, 3H), 1.47 - 1.31 (m, 2H). The synthesis is incorporated herein by reference in its entirety, from Guo, J. et al., Rational Design of Poly(disulfide)s as a Universal Platform for Delivery of CRISPR-Cas9 Machineries toward Therapeutic Genome The study was conducted in accordance with Editing.ACS Central Science 2021, 7, 990-1000.

[0130] compound 4

[0131] [ka]

[0132] Compound 4 was synthesized and characterized according to the published protocol. 1 ¹H NMR (600 MHz, DMSO-d6): δ 7.44–7.12 (m, 5H), 5.02 (s, 2H), 3.62–3.41 (m, 4H), 3.35 (t, 1H, J = 7.2 Hz), 2.98 (d, 2H, J = 5.9 Hz), 1.79–1.03 (m, 6H). The synthesis was carried out according to Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153–12160, which is incorporated herein by reference in its entirety.

[0133] compound 5

[0134] [ka]

[0135] Compound 5 was synthesized and characterized according to the published protocol. 1 ¹H NMR (600 MHz, CDCl3): δ 11.52 (br, 1H), 8.62 (s, 1H), 3.45 (q, J = 4.00 Hz, 2H), 2.86 (t, J = 4.00 Hz, 2H), 1.49 (s, 9H), 1.48 (s, 9H). The synthesis was carried out according to Kuppusamy, R. et al., Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents. Eur. J. Med. Chem. 2018, 143, 1702-1722, which is incorporated herein by reference in its entirety.

[0136] compound 6

[0137] [ka]

[0138] A solution of 4 (0.126 mmol) in DMF (1 mL) was pre-activated under nitrogen for 30 minutes using EDC (0.506 mmol), HOBt (0.506 mmol), and trimethylamine (0.57 mmol). Then, 5 (0.506 mmol) in DMF (1 mL) was added to the above solution, and the resulting solution was stirred at room temperature for 12 hours. The mixture was concentrated and dried under vacuum, and then diluted with ethyl acetate. The organic layer was washed three times with H2O, dried over MgSO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel (MeOH / DCM1:20) to produce 6 (134 mg, 74%). 1 H NMR (600 MHz, CDCl3): δ 11.45-11.33 (m, 3H), 8.53-8.45(m, 3H), 8.35 (t, J = 4.9 Hz, 2H), 7.33-7.25 (m, 5H), 5.04 (s, 2H), HRMS (ESI) C 57 H 97 N 14 O 17 [M+H] + Calculated value in m / z: 1249.7156; Measured value: 1249.7166.

[0139] compound 7

[0140] [ka]

[0141] A solution of 6 (0.12 mmol) in MeOH (2 mL) was added to charcoal-supported palladium (Pd / C, 10% Pd content, 13 mg). The mixture was stirred at room temperature for 6 hours under a hydrogen gas atmosphere. The solution was filtered through a Celite pad. The residues were concentrated and dried under vacuum, and the resulting residues were dissolved in DCM (2 mL). Lipoic acid (0.152 mmol), EDCI (0.304 mmol), HOBt (0.304 mmol), and TEA (0.304 mmol) were added to the mixture, and the mixture was stirred at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and then diluted with ethyl acetate. The organic layer was washed three times with H2O, dried over MgSO4, filtered, and concentrated. The crude product was purified by column chromatography on silica gel (MeOH / DCM 1:50) to produce 7 (94 mg, 82%). 1 H NMR (600 MHz, CDCl3): δ 11.48-11.30 (m, 3H), 8.57-8.46 (m, 3H), 8.40 (t, J = 5.2 Hz, 2H), 8.15-8.12 (m, 1H), 6.22-6.18 (m, 1H), 3.60-3.34 (m, 16H), 3.28-3.04 (m, 7H), 2.48-2.38 (m, 1H), 2.17-2.14 (t, J = 7.4 Hz, 2H), 1.92-1.71 (m, 11H), 1.71-1.59 (m, 4H), 1.57-1.34 (m, 64H). HRMS (ESI) C 57 H 103 N 14 O 16 S2[M+H] + Calculated value in m / z: 1303.7118; Measured value: 1303.7129.

[0142] compound 8

[0143] [ka]

[0144] Compound 7 (0.08 mmol) was added to a solution of 1,4-dioxane (0.5 mL) in 4 M HCl (0.5 mL), and the mixture was stirred at room temperature for 12 hours. The solution was then removed and dried under vacuum to produce compound 8 (24 mg, 89%). 1 H NMR (600 MHz, D2O): δ 3.60-3.40 (m, 3H), 3.4-3.16 (m, 16H), 3.02 (s, 2H), 2.76 (s, 2H), 2.20-2.03 (m, 2H), 1.98-1.76 (m, 2H), 1.62-1.16 (m, 11H). 13 C NMR (150 MHz, D2O): δ 176.61, 175.62, 173.68, 172.92, 156.88 (x3), 66.01, 65.73, 56.57, 55.36, 55.24, 40.40 (x2), 40.27, 38.69, 37.99 (x3), 37.82, 35.44, 33.57, 28.66, 28.18, 27.73, 24.99, 22.71. HRMS (ESI) C 27 H 55 N 14 O4S2[M+H] + Calculated value in m / z: 703.3967; Measured value: 703.3995.

[0145] compound 9

[0146] [ka]

[0147] A solution of lipoic acid (2.5 mmol) in DMF (3 mL) was pre-activated under nitrogen for 30 minutes using EDCI (3 mmol), HOBt (3 mmol), and trimethylamine (3 mmol). Then, lysine (1 mmol) in DMF (3 mL) was added to the above solution, and the resulting solution was stirred at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and then diluted with ethyl acetate. The organic layer was washed three times with H2O, dried over MgSO4, filtered, and concentrated. The crude product was purified by column chromatography (MeOH / DCM1:20) on silica gel to produce 9 (230 mg, 90%). 1 H NMR (600 MHz, MeOD): δ 4.37-4.32 (dd, J = 4.6, 9.2 Hz, 1H), 3.63-3.57 (qui, J =6.8 Hz, 2H), 3.22-3.17 (m, 4H), 3.14-3.09 (m, 2H), 2.51-2.45 (m, 2H), 2.29 (t, J = 7.0 Hz, 2H), 2.21 (t, J = 7.0 Hz, 2H), 1.95-1.86 (m, 3H), 1.79-1.62 (m, 9H), 1.60-1.40 (m, 8H). HRMS (ESI)C 57 H 97 N 14 O 17 [M+H] + Calculated value in m / z: 1249.7156; Measured value: 1249.7166.

[0148] compound 10

[0149] [ka]

[0150] A solution of 9 (0.141 mmol) in DMF (1 mL) was pre-activated under nitrogen for 30 minutes using EDC (0.211 mmol), HOBt (0.211 mmol), and trimethylamine (0.282 mmol). Then, 5 (0.183 mmol) in DMF (1 mL) was added to the above solution, and the resulting solution was stirred at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and then diluted with ethyl acetate. The organic layer was washed three times with H2O, dried over MgSO4, filtered, and concentrated. The crude product was purified by column chromatography (MeOH / DCM1:20) on silica gel to produce 10 (92 mg, 84%). 1 H NMR (600 MHz, CDCl3): δ 11.39 (s, 1H), 8.58 (t, J = 6.0 Hz, 1H), 7.98 (t, J = 4.6 Hz, 1H), 6.41 (d, J = 7.4 Hz, 1H), 5.79-5.74, (m, 1H), 4.37 (q, J = 7.5 Hz, 1H), 3.57-3.48 (m, 4H), 3.44-3.35 (m, 2H), 3.26-3.12 (m, 4H), 3.11-3.05 (m, 2H), 2.46-2.39 (m, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.13 (t, J = 7.4 Hz, 2H), 1.91-1.84 (m, 2H), 1.82-1.75 (m, 1H), 1.71-1.57 (m, 9H), 1.54-1.35 (m, 20H), 1.33-1.25 (m, 2H). 13 C NMR (150 MHz, CDCl3): δ 172.91, 172.55, 171.72, 162.87, 157.42, 153.03, 83.72, 79.97, 56.46, 56.44, 52.78, 41.19, 40.27, 40.26, 40.20, 38.96, 38.48 (x2), 36.48, 36.37, 36.30, 34.64, 34.61, 32.60, 29.18, 28.95, 28.88, 28.29 (x2), 28.05 (x2), 25.46, 25.36, 25.32, 22.29. HRMS (ESI) C35 H 63 N6O7S4[M+H] + Calculated value in m / z: 807.3641; Measured value: 807.3655.

[0151] compound 11

[0152] [ka]

[0153] Compound 10 (0.1 mmol) was added to a solution of 1,4-dioxane (0.5 mL) in 4 M HCl (0.5 mL), and the mixture was stirred at room temperature for 12 hours. The solution was then removed, and the mixture was dried under vacuum to produce compound 8 (59 mg, quantitative). The compound was of high purity and was used without further purification.

[0154] compound 12

[0155] [ka]

[0156] A solution of lipoic acid (3 mmol) and CDI (3.9 mmol) was dissolved in 25 ml of anhydrous DCM. This solution was added dropwise at 0°C to 5 ml of anhydrous DCM containing 8 mmol of N-methyl-1,3-propanediamine. The reaction mixture was stirred at 0°C for 40 minutes and at room temperature for 30 minutes, then washed three times with H2O, dried over MgSO4, filtered, and concentrated to obtain compound 12 (589 mg, 66%). 1H NMR (600 MHz, CDCl3): δ 3.57-3.50 (m, 1H), 3.31 (q, J = 5.8 Hz, 2H), 3.17-3.12 (m, 1H), 3.11-3.05 (m, 1H), 2.65 (t, J = 6.0 Hz, 2H), 2.46-2.40 (m, 1H), 2.39 (s, 3H), 2.15-2.11 (m, 2H), 1.91-1.84 (m, 1H), 1.72-1.54 (m, 6H), 1.49-1.37 (m, 2H). HRMS (ESI) C 12 H 26 N2OS2[M+H] + Calculated value in m / z: 277.1408; Measured value: 277.1399.

[0157] compound 13

[0158] [ka]

[0159] Compound 13 was synthesized and characterized according to the published protocol. 1 ¹H NMR (600 MHz, CDCl3): δ 4.50-4.38 (m, 4H), 4.19-4.15 (m, 2H), 1.74-1.70 (m, 2H), 1.38-1.20 (m, 12H), 0.89 (t, J = 7.2 Hz, 3H). The synthesis was carried out according to Liu, S. et al., Zwitterionic Phospholipidation of Cationic Polymers Facilitates Systemic mRNA Delivery to Spleen and Lymph Nodes. J. Am. Chem. Soc. 2021, 143, 21321-21330, which is incorporated in its entirety herein by reference.

[0160] compound 14

[0161] [ka]

[0162] A 1 ml solution of 12 (0.25 mmol) in anhydrous DMF was added to 13 (0.25 mmol), and the reaction mixture was stirred at 70°C for 24 hours. The mixture was concentrated and dried under vacuum to obtain 14 (116 mg, 90%). 1 H NMR (600 MHz, MeOD): δ 7.90 (s, 1H), 4.14-4.01 (m, 1H), 3.91-3.83 (m, 1H), 3.73-3.67 (m, 1H), 3.62-3.55 (m, 1H), 3.28 (t, J = 6.6 Hz, 2H), 3.21-3.15 (m, 1H), 3.13-3.06 (m, 1H), 2.98 (t, J = 7.2 Hz, 2H), 2.69 (s, 3H), 2.50-2.41 (m, 1H), 2.24 (t, J = 7.2 Hz, 2H), 1.93-1.84 (m, 1H), 1.76-1.59 (m, 6H), 1.51-1.43 (m, 2H), 1.43-1.37 (m,1H), 1.36-1.25 (m, 5H), 0.92-0.88 (m, 3H). 13 C NMR (150 MHz, MeOD): δ 177.08, 79.68, 57.79, 48.91, 48.77, 48.04, 41.54, 39.55, 37.06, 36.91, 35.91, 33.85, 33.20, 32.09, 32.05, 30.44, 30.12, 27.73, 27.16, 26.81, 23.92, 14.65. HRMS (ESI) C 22 H 44 N2O5PS2[MH] - Calculated value in m / z: 511.2429; Measured value: 511.2423.

[0163] Preparation of the exemplary initiators of this disclosure Nine exemplary initiators, initiators I2, I3, I4, I5, I6, I7, I8, I9, and I10, are described herein. Initiators I2 (compound 34), I3 (compound 39), and I4 (compound 37) were prepared according to Schemes 4, 5, and 6. Initiators I5 (compound 16), I6 (compound 43), I7 (compound 44), I8 (compound 40), I9 (compound 49), and I10 (compound 54) were prepared according to Schemes 7, 8, 9, and 10. The compounds obtained at each step of the scheme were verified by NMR. Additional details and data are provided below.

[0164] Furthermore, a control initiator I1 was prepared in this example. I1, which does not retain a glycan head, is an initiator without a specific targeting priority and was prepared to characterize the propagator by forming a copolymer of the exemplary propagator of this disclosure. Initiator I1 has the following structure:

[0165] [ka]

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] [ka]

[0171]

Chem.

[0172]

Chem.

[0173]

Chem.

[0174] Compound 15 8

[0175]

Chem.

[0176] Compound 15 was synthesized and characterized according to the published protocol. 1H NMR (600 MHz, MeOD): δ 6.93 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 5.3 (s, 1H), 4.00 - 3.99 (m, 1H), 3.89 - 3.88 (m, 1H), 3.81 - 3.79 (m, 1H), 3.79 - 3.70 (m,  2H), 3.69 - 3.67 (m, 1H).

[0177] Compound 16

[0178]

Chem.

[0179] A solution of 15 (0.24 mmol) in DMF (2 mL) was added to EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol). The resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified by column chromatography (MeOH / DCM1:2) on silica gel to produce 16 (72%). 1 H NMR (600 MHz, MeOD) δ 6.94 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 5.30 (s, 1H), 4.00 (dd, J = 3.4, 1.8 Hz, 1H), 3.89 (dd, J = 9.7, 3.4 Hz, 1H), 3.81-3.67 (m, 4H), 2.72 (t, J = 6.8 Hz, 2H), 2.60 (t, J = 6.8 Hz, 2H). 13 C NMR (150 MHz, MeOD) δ 176.55, 151.13, 143.23, 119.25(x2), 117.93 (x2), 101.49, 101.32, 75.10, 72.43, 72.18, 68.42, 62.66, 40.34, 20.70. HRMS (ESI) C 15 H 22 NO7S [M+H] + Calculated value: 360.1117, Measured value: 360.1101.

[0180] Compounds 17-20. Compounds 17-20 were synthesized according to Peng, W.; Paulson, JC, CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells. J.Am.Chem.Soc. 2017, 139, 12450-12458, which is incorporated herein by reference in its entirety.

[0181] Compounds 21-25 were synthesized according to Chien, W.-T., et al., Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions. Chem.Commun. 2014, 50, 5786-5789.

[0182] Compound 26. Compound 25 (0.43 mmol) in DCM (5 mL) was added to EDC (0.52 mmol), HOBt (0.52 mmol), trimethylamine (0.86 mmol), and 3-mercaptopropionic acid (0.47 mmol). The resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified by column chromatography (MeOH / DCM 1:10) on silica gel to produce compound 26 (184 mg, 80%). 1 H NMR (600 MHz, CDCl3) δ 5.96 (d, J = 8.6 Hz, 1H), 5.28 (t, J = 9.6 Hz, 1H), 5.05 (t, J = 9.4 Hz, 1H), 4.64 (d, J = 8.4 Hz, 1H), 4.24 (dd, J = 12.3, 4.7 Hz, 1H), 4.11 (dd, J = 12.3, 2.5 Hz, 1H), 3.86-3.80 (m, 2H), 3.68-3.66 (m, 1H), 3.47-3.43 (m, 1H), 3.34-3.29 (m, 1H), 3.22-3.16 (m, 1H), 2.81 (t, J = 8.2 Hz, 2H), 2.51-2.45 (m, 2H), 2.06 (s, 3 H), 2.01 (s, 3 H), 2.00 (s, 3 H), 1.93 (s, 3 H), 1.57-1.51 (m, 4H), 1.48 (t, J = 7.0 Hz, 2H) 1.37-1.29 (m, 4H). HRMS (ESI) C 23 H 38 N2O 10 S [M+H] +The calculated value was 535.2325, and the measured value was 535.2314. The entire sample is incorporated herein by reference. The synthesis was carried out according to Maklakova, S. et al., Cellular uptake of N-acetyl-d-galactosamine-, N-acetyl-d-glucosamine- and d-mannose-containing fluorescent glycoconjugates investigated by liver intravital microscopy. Carbohydr. Res. 2020, 489, 107928.

[0183] Compound 27. Compound 26 was added to NaOMe in MeOH, and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, concentrated and dried under vacuum to produce compound 27 (92 mg, quantitative). 1 H NMR (600 MHz, MeOD) δ 4.38 (d, J = 9.2 Hz, 1H), 3.90-3.86 (m, 2H), 3.68 (dd, J = 12.9, 5.9 Hz, 1H), 3.65-3.62 (m, 1H), 3.48-3.43 (m, 2H), 3.28-3.24 (m, 1H), 3.20-3.14 (m, 2H), 2.94 (t, J = 7.3 Hz, 1H), 2.73 (t, J = 7.3 Hz, 1H), 2.59 (t, J = 7.3 Hz, 1H), 2.47 (t, J = 7.3 Hz, 1H), 1.97 (s, 3H), 1.56-1.48 (m, 4H), 1.39-1.35 (m, 4H). 13 C NMR (150 MHz, MeOD): δ173.70, 169.75, 102.74, 77.92, 76.07, 70.50, 62.76, 57.35, 41.02, 40.32, 36.45, 35.19, 30.49, 27.66, 26.73, 23.03. HRMS (ESI) C 17 H 32N2O 17 S [M+H] + Calculated value: 409.2008, Measured value: 409.2017.

[0184] 9 Am Neu5Ac-α2,3-SCT30,9 Am Preparation of Neu5Ac-α2,6-SCT31 and Neu5Ac-α2,3-SCT38. 30 mg of sialyl glycopeptide (SGP) was decomposed in Tris-HCl buffer with Endo-S WT (300 μg) at 37°C for 48 hours, purified by Sephadex G-25 gel filtration chromatography, and the product was analyzed by ESI-MS. The entire product is incorporated herein by reference: Lin, C.-W., et al., Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer. Proceedings of the National Academy of Sciences 2021, 118, e2114774118, to obtain SCT compound 28. Neuraminidase (5 U / ml, 12 μL) in Tris-HCl buffer was added to the mixture and incubated at 37°C for 12 hours. The mixture was purified by Sephadex G-25 gel filtration chromatography to obtain desialylated N-glycan 29. Subsequently, 100 mM 9 Am The reaction was carried out in 0.5 mL of HEPES buffer (50 mM, pH 8.5) containing Neu5Ac (14.5 mg, 47 μmol), 110 mM CTP (27.2 mg, 52 μmol), and 20 mM MgCl2. The pH of the reaction mixture was adjusted to 8.5 by adding 2N NaOH. Then, 0.5 mg / mL of NmCSS was added to the above solution. The resulting mixture was incubated at 37°C for 8 hours and then CMP-9 was applied. Am The formation of Neu5Ac was monitored by TLC analysis.

[0185] 9 Am Regarding Neu5Ac-α2,6-SCT, hST6Gal-I (0.5 mg / mL) is combined with N-glycan for 9 AmIn addition to the reaction mixture of Neu5Ac, the whole was incubated at 37 °C according to Peng, W., et al., Recent H3N2 Viruses Have Evolved Specificity for Extended, Branched Human-type Receptors, Conferring Potential for Increased Avidity. Cell Host Microbe 2017, 21, 23 - 34, which is incorporated herein by reference. 9 Am For Neu5Ac-α2,3-SCT, PmST3 (0.3 mg / mL) was added to the reaction mixture of Neu5Ac with N-glycan and incubated at 37 °C. 9 Am In addition to the reaction mixture of Neu5Ac, it was incubated at 37 °C. For Neu5Ac-α2,3-SCT, PmST3 (0.3 mg / mL) was added to the reaction mixture of Neu5Ac with N-glycan and incubated at 37 °C. The above reaction was monitored by mass spectrometry analysis and TLC. After the acceptor was consumed, the reaction was centrifuged, and the supernatant was filtered through a centrifugal filter with a molecular weight cut-off of 10 kDa (Amicon Ultra, Millipore) to remove proteins. The filtrate was purified by P-2 gel filtration chromatography to obtain 9 Am Neu5Ac-α2,3-SCT 30, 9 Am Neu5Ac-α2,6-SCT 31 and Neu5Ac-α2,3-SCT 38.

[0186] 9 BPC Preparation of Neu5Ac-α2,6-SCT-SH 34, 9 TCC Neu5Ac-α2,3-SCT-SH 37 and Neu5Ac-α2,3-SCT-SH 39. 9 AmNeu5Ac-α2,6-SCT and DIEA (5.0 equivalents) were dissolved in H2O, followed by addition of biphenylcarboxylic acid-N-hydroxysuccinimide (hydrozysuccinimide) ester (BPC-NHS) (3 equivalents) in THF. The reaction mixture was stirred at 0 °C until the starting material was consumed. The reaction mixture was then purified on a Sep-Pak C18 column (2 g, Waters Corp.) and eluted with H2O-MeOH to give compound 32 in 91% yield. 9 Am Neu5Ac-α2,3-SCT was prepared in the same manner as described above by stirring 4H-thieno[3,2-c]chromene-2-carbamoyl-NHS (TCC-NHS) in THF and H2O to give compound 35. After purification, the product was obtained in 91% yield.

[0187] 9 BPC An aqueous mixture of Neu5Ac-α2,6-SCT 32 or 9 TCC Neu5Ac-α2,3-SCT 35 was added to CDMBI and TEA and incubated at 4 °C for 1 h to give the corresponding oxazoline N-glycans 33 and 36, respectively. These were then purified by Sephadex G-25 gel filtration chromatography and characterized by ESI-MS. GlcNAc-SH 27 (0.25 mg) and Endo-M (N175Q) (1.6 U / mL) were added to a solution of 50 mM phosphate buffer (pH 7) together with 9 BPC Neu5Ac-α2,6-SCT-oxazoline and incubated at 30 °C for 30 min. The product of transglycosylation was isolated by P-2 gel filtration chromatography to give compound 34, which was characterized by ESI-MS. 9 TCC Neu5Ac-α2,3-SCT-SH 37 and Neu5Ac-α2,3-SCT-SH 39 were prepared in the same manner as described above.

[0188] Compound 34. 9 BPC Neu5Ac-α2,6-SCT-SH. 1H NMR (600 MHz, DMSO-d6): δ = 8.33 (s, 3H, NH), 8.00 (d, J = 8.6 Hz, 4H), 7.72 (dd, J = 8.0, 14.2 Hz, 9H), 7.49 (t, J = 8.3 Hz, 4H), 7.40 (t, J = 8.3 Hz, 2H), 4.98-4.95 (m, 2H), 4.76 (s, 1H), 4.54 (s, 1H), 4.44-4.39 (m, 2H), 4.25-4.22 (m, 3H), 3.99 (s, 1H), 3.87 (s, 1H), 3.83-3.73 (m, 5H), 3.41-3.25 (m, 58H), 3.21 - 3.16 (m, 3H), 3.11 - 2.99 (m, 4H), 2.87 (t, J = 6.9 Hz, 2H), 2.61 (d, J = 8.4 Hz, 2H), 2.44 (t, J = 7.4 HRMS (ESI) C 119 H 171 N9O 63 S 2- [M-2H] 2- Calculated value: 1383.0093, measured value 1383.0077.

[0189] Compound 37.9 TCC Neu5Ac-α2,3-SCT-SH. 1H NMR (600 MHz, DMSO-d6): δ = 8.22 (s, 3H, NH), 8.10-8.09 (m, 1H, NH), 7.96-7.93 (m, 1H, NH), 7.81-7.79 (m, 1H, NH), 7.74-7.72 (m, 1H, NH), 7.35 (d, J = 7.8 Hz, 2H), 7.29 (s, 2H), 7.20 (t, J = 7.8 Hz, 2H), 6.97 (t, J = 7.8 Hz, 2H), 6.92 (d, J = 7.9 Hz, 2H), 5.29 (s, 4H), 5.01-4.95 (m, 2H), 4.77 (s, 1H), 4.54 (s, 1H), 4.44-4.36 (m, 2H), 4.27-4.19 (m, 3H), 3.99 (s, 1H), 3.88 (s, 1H), 3.85-3.71 (m, 3H), 3.50-3.22 (m, 52H), 3.19 (d, J = 9.5 Hz, 2H), 3.10-3.00 (m, 4H), 2.87 (t, J = 6.9 Hz, 2H), 2.64 - 2.58 (m, 2H), 2.44 (t, J = 7.4 Hz, 2H), 1.93-1.78 (m, 18H), 1.45-1.39 (m, 2H), 1.39-1.29 (m, 4H), 1.28-1.20 (m, 4H), 1.16 (t, J = 6.9 Hz, 1H). HRMS (ESI) C 117 H 169 N9O 65 S3 2- [M-2H] 2- Calculated value: 1416.9606, measured value 1416.9623.

[0190] Compound 39. Neu5Ac-α2,3-SCT-SH 1H NMR (600 MHz, DMSO-d6): δ = 8.33 (s, 3H, NH), 8.10 (s, 1H, NH), 7.96-7.94 (m, 1H, NH), 7.81-7.80 (m, 1H, NH), 7.74-7.73 (m, 1H, NH), 4.98-4.95 (m, 2H), 4.76-4.75 (m, 1H), 4.54 (s, 1H), 4.46-4.38 (m, 2H), 4.25-4.22 (m, 3H), 3.99 (s, 1H), 3.87 (s, 1H), 3.80-3.71 (m, 4H), 3.41-3.25 (m, 59H), 3.23 - 3.18 (m, 3H), 3.08 - 3.01 (m, 3H), 2.87 (t, J = 6.9 Hz, 2H), 2.63-2.60 (m, 2H), 2.44 (t, J = 7.4 Hz, 2H), 1.87-1.79 HRMS (ESI) C 93 H 153 N7O 63 S 2- [M-2H] 2- Calculated value: 1203.9357, measured value 1203.9377.

[0191] Compound 40

[0192]

change

[0193] Compound 15 (0.12mmol), EDC (0.12mmol), HOBt (0.12mmol), DMAP (0.12mmol), トリメチルアミン (0.2mmol) and びCT (PEG) in DMF (1mL) 12(0.1 mmol) was added, and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified by column chromatography on silica gel to produce 40 (59%). ¹H NMR (600 MHz, MeOD): δ6.93 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 5.3 (s, 1H), 4.00-3.99 (m, 1H), 3.89-3.88 (m, 1H), 3.81-3.70 (m, 5H), 3.70-3.59 (m, 48H), 2.68 (t, J = 6.8 Hz, 2H), 2.50-2.47 (m, 2H). 13 C NMR (150 MHz, MeOD): δ170.17, 151.03, 143.50, 119.26(x2), 117.82(x2), 101.36, 75.10, 74.08, 72.45, 72.19, 71.45, 71.41, 71.37, 71.30, 71.24, 71.11, 70.93(x18), 68.43, 62.68, 24.67. HRMS (ESI) C 39 H 70 NO 19 S [M+H] + Calculated value: 888.4263, Measured value: 888.4241.

[0194] Compounds 41-42. Compounds 41 and 42 were synthesized according to Lee, H.-K. et al., Reactivity-Based One-Pot Synthesis of Oligomannoses: Defining Antigens Recognized by 2G12, a Broadly Neutralizing Anti-HIV-1 Antibody. Angew. Chem. Int. Ed. 2004, 43, 1000-1003, which is incorporated herein by reference in its entirety.

[0195] compound 43

[0196] [ka]

[0197] A solution of 42 (0.24 mmol) in DMF (2 mL) was added to EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol). The resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified by column chromatography (MeOH / DCM1:3) on silica gel to produce 16 (81%). 1 H NMR (600 MHz, MeOD) δ 4.91 (s, 1H), 3.84 (dd, J = 3.4, 1.8 Hz, 1H), 3.82-3.76 (m, 2H), 3.75-3.67 (m, 2H), 3.62 (m, 1H), 3.56-3.41 (m, 2H), 2.94 (t, J = 7.0 Hz, 2H), 2.70 (t, J = 7.2 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H). HRMS (ESI) C 14 H 28 NO7S [M+H] + Calculated value: 354.1586, Measured value: 354.1602.

[0198] compound 44

[0199] [ka]

[0200] Compound 42 (0.12 mmol) in DMF (1 mL) was mixed with EDC (0.12 mmol), HOBt (0.12 mmol), trimethylamine (0.2 mmol), and CT (PEG). 12 (0.1 mmol) was added, and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified by column chromatography on silica gel to produce 44 (62%). 1H NMR (600 MHz, D2O): δ4.77 (d, J = 8.6 Hz, 2H), 3.85-3.82 (m, 1H), 3.81-3.77 (m, 1H), 3.69-3.67 (m, 1H), 3.66-3.64 (m, 2H), 3.63-3.57 (m, 41H), 3.55-3.45 (m, 4H), 2.88 (t, J = 6.8 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 1.66-1.51 (m, 4H), 1.40-1.31 (m, 2H). 13 C NMR (150 MHz, D2O): δ171.01, 99.62, 72.72, 70.57, 69.99, 69.24 (x22), 69.04, 67.90, 67.39, 66.73, 60.92, 39.36, 37.57, 27.96, 26.70, 22.91, 22.43. HRMS (ESI) C 38 H 76 NO 19 S [M+H] + Calculated value: 882.4732, measured value 882.4750.

[0201] Compound 45

[0202]

change

[0203] Compound 15b (5 mmol) in MeOH was added to NaOMe (0.2 equivalents), and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, filtered, and concentrated and dried under vacuum. This was then dissolved in anhydrous DCM (40 mL) and treated with imidazole (7.5 mmol) at 0°C, followed by the addition of TBDPSCl (5.5 mmol). The mixture was stirred under a nitrogen atmosphere at room temperature for 2.5 hours. The reaction was quenched by the addition of MeOH. After stirring at room temperature for 10 minutes, the solvent was removed under reduced pressure to obtain a dry residue. This was purified by column chromatography using MeOH / DCM (1 / 10) to obtain compound 45 (75%). 1 H NMR (600 MHz, CDCl3) δ 7.61 (m, 4H), 7.41-7.29 (m, 11H), 7.23-7.18 (m, 2H), 6.92 (d, J = 9.3 Hz, 2H), 5.41 (s, 1H), 5.16 (s, 2H), 4.09 (s, 1H), 4.02-4.00 (dd, J = 9.3, 3.4 Hz, 1H), 3.91 (t, J = 9.3 Hz, 1H), 3.85 (d, J = 5.1 Hz, 2H), 3.72-3.69 (m, 1H), 1.01, (s, 9H). 13 C NMR (150 MHz, CDCl3): 162.61, 135.64(x4), 135.54(x4), 132.73, 129.95(x4), 128.64(x4), 128.38, 128.33, 127.84(x2), 127.81(x2), 98.09, 71.41, 71.22, 70.22, 70.13, 64.89, 36.53, 31.48, 26.83(x3), 19.19. 36 H 42 NO8Si [M+H] + Calculated value: 644.2680, Measured value: 644.2699.

[0204] compound 46

[0205] [ka]

[0206] To a solution of compound 45 (3 mmol) and a catalytic amount of CSA (0.3 mmol) in CH3CN (60 mL), trimethyl orthobenzoate (9 mmol) was added at room temperature under nitrogen atmospheric pressure. After stirring for 30 minutes, Et3N was added to quench the reaction, and the resulting mixture was dried under reduced pressure. The residues were purified by column chromatography using EA / Hex(1 / 2) to obtain compound 46 (81%). 1 H NMR (600 MHz, CDCl3) δ 7.65-7.59 (m, 2H), 7.57-7.52 (m, 4H), 7.41-7.27 (m, 14H), 7.26-7.20 (m, 2H), 6.93 (d, J = 9.2 Hz, 2H), 5.77 (s, 1H), 5.17 (s, 2H), 4.70 (d, J = 6.1 Hz,1H), 4.58 (dd, J = 9.3, 3.4 Hz, 1H), 3.79-3.76 (m, 2H), 3.74-3.70 (m, 1H), 3.69-3.66 (m, 1H), 3.22 (s, 3H), 2.53 (d, J = 3.9 Hz, 1H), 0.93, (s, 9H). 13 C NMR (150 MHz, CDCl3): 171.23, 153.49, 152.24, 137.09, 136.09, 135.68(x4), 135.48(x4), 132.98, 132.72, 129.86, 129.84(x2), 129.18, 128.65(x2), 128.39, 128.37, 128.34, 127.78(x2), 127.71(x2), 126.23, 121.11, 117.18, 95.69, 79.52, 69.57, 69.45, 67.03, 63.75, 60.44, 51.16, 26.76(x3), 19.15, 14.22. HRMS (ESI) C 44 H 48 NO9Si [M+H] + Calculated value: 762.3098, Measured value: 762.3072.

[0207] compound 47

[0208] [ka]

[0209] Compound 46 (2 mmol) was dissolved in DCM (20 mL) and sequentially mixed with DIPEA (6 mmol), benzoic anhydride (4 mmol), and DMAP (0.2 mmol). After stirring for 30 minutes, the solvent was evaporated under reduced pressure to obtain a dry residue, which was then poured into EA (20 mL) and 2N HCl (20 mL) with vigorous stirring for 30 minutes. The solvent was removed by evaporation, followed by extraction with EA. The collected organic layer was washed with ice-cold saturated NaHCO3 (aqueous solution), water, and brine, and dried over MgSO4. The filtrate was evaporated under reduced pressure and redissolved in THF (20 mL). AcOH (4 mmol) and 1 M TBAF (2.4 mmol in THF) were added at 0°C. The resulting mixture was gradually warmed to room temperature, stirred for another 2 hours, and then diluted with EA. The organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The dried residue was purified by column chromatography using EA / Hex(1 / 2) to obtain compound 47 (65%). 1 H NMR (600 MHz, CDCl3) δ 8.10-8.05 (m, 4H), 7.61-7.56 (m, 2H), 7.48-7.41 (m, 4H), 7.39-7.27 (m, 7H), 7.03 (d, J = 9.2 Hz, 2H), 5.69 (s, 1H), 5.62-5.55 (m, 2H), 5.16, (s, 2H), 4.62 (dd, J = 9.3, 3.4 Hz, 1H), 4.06-4.01 (m, 1H), 3.76-3.67 (m, 2H). 13C NMR (150 MHz, CDCl3): 167.42, 166.05, 153.55, 152.01, 136.03, 133.84, 133.76, 130.00 (x4), 129.09, 128.96, 128.69(x4), 128.65(x4), 128.62(x4), 128.34, 117.11 (x2), 96.18, 72.61, 71.30, 70.01, 68.53, 61.16. HRMS (ESI) 34 H 32 NO 10 [M+H] + Calculated value: 614.2026, Measured value: 614.2038.

[0210] compound 48

[0211] [ka]

[0212] A stirred solution of 47 (0.2 mmol) and 4A molecular sieve (0.2 g) in anhydrous DCM (2 mL) was cooled to -40°C, and then BF3(OEt)2 (0.02 mmol) was added dropwise to the solution. The solution of 15a in anhydrous DCM was added dropwise to the above mixture and stirred at -40°C for 1 hour. Then the reaction mixture was gradually warmed to room temperature and stirred for another hour. The solution was quenched by the addition of triethylamine, then filtered, saturated aqueous solution NaHCO3 was added, and extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography using silica gel to obtain the trisaccharide product. The product was then dissolved in MeOH, NaOMe (0.2 equivalents) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized with IR-120, filtered, and concentrated and dried under vacuum. The deacetylated mixture was purified using Bio-Gel P-2 Gel (Biorad) with H2O as the eluent to obtain a pure trisaccharide. The compound was freeze-dried, then dissolved in MeOH (2 mL), 10% Pd-C (30 mg) was added, and the mixture was vigorously stirred overnight under H2 atmosphere. The solution was filtered through Celite and concentrated and dried to obtain compound 48 (42%).1 H NMR (600 MHz, D2O) δ 7.03 (d, J = 9.2 Hz, 2H), 6.88 (d, J = 9.2 Hz, 2H), 5.48 (s, 1H), 5.19 (s, 1H), 4.76 (s, 1H), 4.32 (s, 1H), 4.14 (dd, J = 9.3, 3.0 Hz, 1H), 4.11 (s, 1H), 3.95-3.63 (m, 15H). 13 C NMR (150 MHz, D2O): 151.38, 143.39, 121.23, 120.70, 105.19, 101.60, 101.39, 80.93, 76.13, 75.38, 74.04, 73.27, 73.12, 72.79, 72.66, 72.22, 69.50, 69.41, 68.70, 67.90, 63.71, 63.65. HRMS (ESI) C 24 H 37 NO 16 Na[M+Na] + Calculated value: 618.2010, Measured value: 618.2029.

[0213] compound 49

[0214] [ka]

[0215] Compound 48 (0.12 mmol) in DMF (1 mL) is mixed with EDC (0.12 mmol), HOBt (0.12 mmol), DMAP (0.12 mmol), trimethylamine (0.2 mmol), and CT (PEG). 12 (0.1 mmol) was added, and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified with Bio-Gel P-2Gel using H2O as the eluent to produce 49 (54%). 1H NMR (600 MHz, D2O): 7.20 (d, J = 9.2 Hz, 2H), 7.13 (d, J = 9.2 Hz, 2H), 5.53 (s, 1H), 5.09 (s, 1H), 4.65 (s, 1H), 4.25 (s, 1H), 4.06 (dd, J = 9.3, 3.0 Hz, 1H), 4.01 (m, 1H), 3.83-3.67 (m, 11H), 3.61-3.56 (m, 52H), 2.64 (t, J = 6.4 Hz, 2H), 2.48 (t, J = 6.4 Hz, 2H). 13 C NMR (150 MHz, D2O): 178.06, 153.84, 149.84, 123.02, 118.11, 120.42, 98.8, 97.78, 78.05, 73.39, 72.61, 72.17, 71.47, 70.52, 70.35, 70.01, 69.85, 69.55, 69.38, 69.31, 69.17, 66.97, 66.74, 66.60, 65.82, 65.10, 60.96, 60.86, 35.82, 23.03. HRMS (ESI) C 51 H 89 NO 29 SNa[M+Na] + Calculated value: 1234.5139, measured value 1234.5114.

[0216] Compound 51

[0217]

change

[0218] Trichloroacetonitrile and DBU were added to a 50 (1 mmol) stirred solution in 10 mL of anhydrous DCM, and the solution was stirred at room temperature for 2 hours. The solvent was removed, and the residues were purified by flash column chromatography using silica gel to obtain the imidate product. The stirred solution of benzyl(4-hydroxyphenyl) carbamate (1.2 mmol) and 4A molecular sieve (1 g) in 10 mL of anhydrous DCM was cooled to -40°C, and then BF3(OEt)2 (0.1 mmol) was added dropwise to the solution. The imidate donor (1 mmol) in anhydrous DCM was added dropwise to the above mixture, and the mixture was stirred at -40°C for 1 hour. The reaction mixture was then gradually warmed to room temperature and stirred for another hour. The solution was quenched by the addition of triethylamine, then filtered, saturated aqueous solution NaHCO3 was added, and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residues were purified by flash column chromatography using silica gel to obtain compound 51 (72%). 1 H NMR (600 MHz, CDCl3) δ 7.40-7.28 (m, 18H), 7.19 (d, J = 7.9 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 7.00 (d, J = 8.1 Hz, 2H), 5.57-5.55 (m, 2H), 5.12 (s, 2H), 4.91 (d, J = 10.5 Hz, 1H), 4.80 (d, J = 10.5 Hz, 1H), 4.69 (d, J = 10.5 Hz, 1H), 4.65 (d, J = 10.5 Hz, 1H), 4.53 (d, J = 10.5 Hz, 1H), 4.46 (d, J = 10.5 Hz, 1H), 4.22 (dd, J = 9.4, 3.6 Hz, 1H), 4.07-4.04 (t, J = 9.7 Hz, 1H), 3.93 (d, J = 9.5 Hz, 1H), 3.83 (dd, J = 10.9, 3.9 Hz, 1H), 3.68 (d, J = 10.8 Hz, 1H), 2.21 (s, 3H). 13C NMR (150 MHz, CDCl3):170.44, 156.25, 154.62, 138.27, 138.04, 137.82, 136.51, 132.71, 129.79, 128.50, 128.43, 128.31, 128.27, 128.10, 128.08, 127.83, 127.80, 127.64, 127.60, 116.63, 96.13, 77.95, 76.66, 75.22, 74.00, 73.34, 71.99, 71.92, 68.55, 68.5, 66.63, 21.09. HRMS (ESI) C 43 H 43 NO 10 [M+H] + Calculated value: 718.3016, Measured value: 718.3041.

[0219] compound 52

[0220] [ka]

[0221] 0.6 mmol of 51 in MeOH was stirred, to which 0.1 equivalent of NaOMe was added, and the resulting solution was stirred at room temperature for 1 hour. The mixture was neutralized with IR-120, filtered, and concentrated and dried under vacuum. The deacetylated product was then dissolved in anhydrous DCM (5 mL) and 0.5 g of 4A molecular sieve was added. The solution was cooled to -40°C, and then 0.05 mmol of BF3(OEt)2 was added dropwise. 0.5 mmol of imidate donor in anhydrous DCM was added dropwise to the mixture, and the mixture was stirred at -40°C for 1 hour. The reaction mixture was then gradually warmed to room temperature and stirred for another hour. The solution was quenched by the addition of triethylamine, filtered, saturated aqueous solution NaHCO3 was added, and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. Next, the product was dissolved in MeOH, NaOMe (0.1 equivalent) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized with IR-120, filtered, and concentrated and dried under vacuum. The residues were purified by flash column chromatography using silica gel to obtain compound 52 (69%). 1 H NMR (600 MHz, CDCl3) δ 7.36-7.25 (m, 25H), 7.22-7.13 (m, 10H), 6.99-6.93 (m, 4H), 5.66 (s, 1H), 5.17 (s, 1H), 5.06 (s, 2H), 4.86 (d, J = 10.5 Hz, 1H), 4.79 (d, J = 10.5 Hz, 1H), 4.73, (s, 2H), 4.65 (d, J = 10.5 Hz, 1H), 4.58-4.57 (m, 2H), 4.55-4.52 (m, 2H), 4.47-4.43 (m, 3H), 4.19-4.14 (m, 2H), 3.99-3.96 (m, 2H), 3.88 (dd, J = 9.1, 3.1 Hz, 1H), 3.84-3.76 (m, 3H), 3.67-3.63 (m, 3H). 13C NMR (150 MHz, CDCl3): 156.22, 154.70, 138.47, 138.35, 138.18, 138.14, 138.05, 137.90, 136.51, 132.30, 129.74, 129.63, 128.46, 128.44, 128.31, 128.28, 128.26, 128.19, 128.06, 127.93, 127.85, 127.83, 127.74, 127.68, 127.60, 127.54, 127.46, 127.38, 127.32, 116.63, 101.12, 96.95, 79.97, 79.42, 77.21, 77.00, 76.78, 75.12, 75.02, 74.66, 74.45, 74.33, 73.23, 73.16, 72.44, 72.41, 72.16, 71.69, 68.99, 68.45, 66.57. HRMS (ESI) C 68 H 70 NO 13 [M+H] + Calculated value: 1108.4847, Measured value: 1108.4819

[0222] compound 53

[0223] [ka]

[0224] A stirred solution of 52 (0.3 mmol) in anhydrous DCM (2.5 mL) was added together with 4A molecular sieve (0.25 g). The solution was cooled to -40°C, and then BF3(OEt)2 (0.03 mmol) was added dropwise. A solution of imidate donor (0.3 mmol) in anhydrous DCM was added dropwise to the above mixture, and the mixture was stirred at -40°C for 1 hour. The reaction mixture was then gradually warmed to room temperature and stirred for another hour. The solution was quenched by adding triethylamine, then filtered, saturated aqueous solution NaHCO3 was added, and it was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residues were purified by flash column chromatography using silica gel to obtain the trisaccharide product. The product was then dissolved in MeOH, NaOMe (0.1 equivalent) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized with IR-120, filtered, and concentrated and dried under vacuum. Next, the compound was dissolved in MeOH (2 mL), 10% Pd-C (30 mg) was added, and the mixture was vigorously stirred overnight under H2 air. The solution was filtered through Celite and concentrated and dried to obtain compound 53 (60%). 1 H NMR (600 MHz, D2O) δ 7.03 (d, J = 9.0 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 3.94-3.55 (m, 16H), 3.48 (t, J = 9.6 Hz, 1H), 3.29-3.27 (m, 1H). 13 C NMR (150 MHz, D2O): 151.36, 143.37, 121.20, 120.67, 101.59, 101.23, 96.33, 78.46, 75.34, 74.69, 73.51, 73.11, 72.97, 72.52, 71.53, 69.28, 69.14, 68.90, 65.51, 63.26, 62.91. HRMS (ESI) C 24 H 38 NO 16 [M+H] + Calculated value: 596.2191, Measured value: 596.2044.

[0225] compound 54

[0226] [ka]

[0227] Compound 53 (0.02 mmol) in DMF (0.2 mL) is mixed with EDC (0.02 mmol), HOBt (0.02 mmol), DMAP (0.02 mmol), trimethylamine (0.04 mmol), and CT (PEG). 12 (0.02 mmol) was added, and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The mixture was concentrated and dried under vacuum, and the crude product was purified with Bio-Gel P-2 Gel using H2O as the eluent to produce 54 (58%). 1 H NMR (600 MHz, D2O): 7.03 (d, J = 9.2 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 3.84-3.46 (m, 65H), 3.30-3.26 (m, 1H), 2.65 (t, J = 6.5 Hz, 2H), 2.52 (s, 2H). 13 C NMR (150 MHz, D2O):173.22, 151.36, 143.37, 121.37, 120.59, 101.20, 101.67, 97.04, 79.17, 76.06, 75.40, 75.22, 74.23, 73.69, 73.23, 72.60, 72.45, 72.22, 71.68, 71.21, 69.85, 69.61, 63.97, 38.52, 26.08. HRMS (ESI) C 51 H 90 NO 29 S[M+H] + Calculated value: 1212.5319, Measured value: 1212.5146.

[0228] Preparation of polymerosomes In this example, polymerosomes of the present disclosure were prepared by encapsulating mRNA to form mRNA-polymer nanoparticles (PNPs) for delivery. The term "PNP" is used interchangeably with "polymerosome" to describe the polymerosomes of the present disclosure. Exemplary PNPs include I2-P1 / P5 mRNA-PNP, I3-P1 / P5 mRNA-PNP, I4-P1 / P5 mRNA-PNP, I5-P1 / P5 mRNA-PNP, I6-P1 / P5 mRNA-PNP, I7-P1 / P5 mRNA-PNP, I8-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA PNP, and I10-P1 / P5 mRNA PNP, which are illustrated in Figure 1B.

[0229] Construction of WT spike DNA. pMRNA XP mRNA synthesis vectors were obtained from System Biosciences. The spike DNA sequences of WT (Wuhan / WH01 / 2019 strain) containing K986P and K987P mutations (2P) were codon-optimized for human (Homo sapiens) pMRNA. XPThe vector was digested with EcoRI and BamHI at 37°C for 1 hour. The spike protein of the DNA sequence was amplified using KOD One® PCR Master Mix (TOYOBO Bio-Technology). The linearized pMRNAXP vector and PCR fragments of spike protein DNA were cleaned with Wizard SV Gel and PCR Clean-Up System (Promega). The PCR fragments of spike protein DNA were cloned into the linearized pMRNAXP vector using In-Fusion HD Cloning Kit (Clontech Laboratories, Inc.). The cloning mixture was converted to One Shot® TOP10 Chemically Competent E. coli (Invitrogen®) and incubated overnight at 37°C. Successful builds were screened using Quick Taq HS DyeMix (TOYOBO Bio-Technology). Insert-specific and backbone-specific primers were designed for colony PCR. Single clones were selected at the pipette tip and PCR was performed. PCR products were analyzed using agarose gel electrophoresis. Possible candidates were selected and analyzed by DNA sequencing.

[0230] Polymer synthesis. The polymerization process was carried out according to the published procedure described in Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153-12160, which is incorporated herein by reference. Modifications were made as necessary. Briefly, stock solutions of monomers (i.e., propagator, 2 M in DMF), initiator (50 mM in DMF, freshly prepared), polymerization inhibitor (iodoacetamide, 0.5 M in H2O, freshly prepared), and triethanolamine (TEOA) buffer (1 M, pH=7.0) were prepared. The initiator was added to 80 μL of mixed buffer (DMF / TEOA=1 / 1) and 10 μL of monomer stock solution (a 1:1 v / v ratio was used for heteropolymers P1 / P3, P2 / P3, P1 / P4, P2 / P4, P1 / P5, and P2 / P5). See Figure 1A. After stirring at room temperature for 30 minutes, the polymerization reaction was quenched by adding 1.9 mL of polymerization inhibitor stock solution. On the same day, the resulting polymer was dialyzed against H2O. The solution was freeze-dried, and the polymer was maintained at -20°C. For in vitro and in vivo experiments, the initiator was mixed with 5% IP, followed by the same protocol as described above.

[0231] For the formulation of mRNA-PNPs, mRNA was encapsulated within the corresponding polymer using a self-assembly process. Specifically, the polymer in the ethanol phase (10 mg / mL) was mixed with an aqueous solution of mRNA (1 mg / mL) at pH 4.0 with an N / P ratio of 3–1. See Figure 1B. The mRNA-PNPs were dialyzed overnight at 4°C in PBS buffer (pH 7.4) using a Micro Float-A-Lyzer (10 kDa MWCO, spectrum lab) and stored at -40°C until further use.

[0232] Characterization of polymerosomes. To investigate the geometry of mRNA-polymer complexes, we first mixed P1 polymers with mRNA and found that this formed nearly spherical nanoparticles. The complex of P1 / P5 copolymer with mRNA resembled liposomes. The polymers likely form a positively charged layer that can encapsulate mRNA and facilitate subsequent cellular uptake through the involvement of zwitterions.

[0233] Molecular weight and polymerization index were characterized by gel permeation chromatography (GPC). While the polymers exhibited a single mode in the GPC chromatogram, they showed a broad molecular weight distribution and eluted with relatively short elution times, indicating their polymeric state. The peak molecular weight for mRNA-PNP (P1 / P5) was 10.2 kDa (PDI=1.33).

[0234] [Example 2] Polymerosome encapsulation and transfection efficiency To identify the optimal polymer as a model for efficient intracellular delivery of GFP-mRNA, homopolymers and hetero-copolymers were synthesized by copolymerization of different propagators, and their encapsulation ability and transfection efficiency were evaluated in HEK293T cells.

[0235] Quantification of encapsulated mRNA The encapsulation efficiency was determined using Quant-iT® RiboGreen® RNA reagent and kit (Thermo Scientific®). Prepared mRNA polymerosomes (mRNA-PNPs) were treated overnight with 10 mM GSH. The solution was then diluted 250-fold with 1X TE buffer and further diluted 2-fold with TE buffer or TE buffer containing 2% Triton X-100. mRNA was prepared at concentrations of 100, 50, 25, 12.5, and 0 ng / ml in TE or TE buffer containing 1% Triton X-100, and a calibration curve was established. After incubation at 37°C for 10 minutes, Quant-iT® RiboGreen® RNA Reagent was added to the wells. Fluorescence intensity was measured using CLARIOstar® Plus (BMG Labtech).

[0236] result All copolymers containing a guanidine group were able to encapsulate GFP-mRNA. In particular, the I1-P2 / P3, I1-P2 / P4, and I1-P2 / P5 copolymers, which have a trivalent guanidine moiety, showed a higher ability to encapsulate mRNA, achieving results comparable to those obtained with the conventional transfection agent polyethyleneimine (PEI). See Figure 2A.

[0237] Formulation of WT spike mRNA for mRNA-PNP formation. To obtain WT spike mRNA, linear DNA containing a T7 promoter, 50 untranslated regions, 30 untranslated regions, S-2P, and poly(A) tail signal sequence was amplified for 1 hour at 37°C according to the manufacturer's protocol using TOOLS Ultra High Fidelity DN polymerase (BIOTOOLS Co., Ltd.) with 1 μL of DNA template in an mMESSAGEmMACHINE® kit (Thermo Scientific). The mRNA was purified using an RNA cleanup kit (BioLabs) according to the manufacturer's protocol and stored at -80°C until further use. For mRNA-PNP formulation, mRNA was encapsulated in the corresponding polymer using a self-assembly process. Specifically, the polymer in the ethanol phase (10 mg / mL) was mixed with an aqueous solution of mRNA (1 mg / mL) at pH 4.0 in different N / P ratios. mRNA-PNPs were dialyzed overnight at 4°C in PBS buffer (pH 7.4) using a Micro Float-A-Lyzer (10kDa MWCO, spectrum lab) and stored at -40°C until further use.

[0238] result The results showed that the P1 / P5 copolymer had excellent ability to capture spike mRNA at N / P ratios of 1, 5, 10, or 20 (Figure 3A), and the average particle size of the resulting mRNA-P1 / P5 copolymer complex was approximately 127 nm, as revealed by CryoEM, TEM, and Dynamic Light Scattering (DLS) analysis (Figure 3B). The measured zeta potential was approximately -4.2 mV. For the next experiment, an N / P ratio of 3 was selected to ensure complete inclusion. mRNA release and translation occurred in a time-dependent manner in the presence of 10 mM GSH, and could be the result of GSH-mediated polymer degradation (data not shown).

[0239] HEK293T cell transfection. Transfection of polymerosomes encapsulating GFP-mRNA. Next, the transfection efficiency of GFP-mRNA in HEK293T cells was evaluated using different copolymers. HEK293T cells were 5 × 10⁶ cells per well in a 6-well plate in 2.5 mL DMEM medium. 5 Cells were plated. 1 μg of GFP mRNA or 3 μg of WT spike mRNA was formulated using the corresponding polymer according to the procedure described above and then added to the cells. 18 hours after transfection, GFP and spike expression were monitored by fluorescence microscopy and Western blotting, respectively. For Western blotting, cells containing spike protein were dissolved in 200 μL of RIPA lysis buffer containing a protease inhibitor and incubated for 10 minutes. The cells were then vortexed, centrifuged, and analyzed by Western blotting using polyclonal anti-SARS-CoV-2 S protein antibody (1:5000 in 1% BSA), followed by HRP conjugate anti-rabbit antibody (1:10000). Spike protein was detected with a chemiluminescent HRP substrate and visualized with a trans-illuminator (FUJIFILM LAS3000).

[0240] result The results showed that copolymer P1 demonstrated comparable transfection ability to HEK293T cells to the conventional transfection agent polyethyleneimine (PEI), while heteropolymers P1 / P4, P2 / P4, P1 / P5, and P2 / P5 exhibited superior transfection efficiency and were able to release mRNA for translation to GFP. In particular, polymers containing zwitterionic groups, P1 / P5, and P2 / P5 (Figure 2B) were extremely effective, likely due to membrane fusion as described above. All polymerosomes tested showed less impact on cell viability than the conventional transfection agent PEI, and none of the polymerosomes showed apparent cytotoxicity (data not shown).

[0241] Transfection of polymerosomes encapsulating WT spike mRNA was performed. Subsequently, HEK293T cells were transfected with a spike mRNA-P1 / P5 complex (3 μg), and Western blotting was performed. 48 hours after transfection, cells were analyzed for spike-protein translation by Western blotting using a spike-specific antibody. A significant band was observed at approximately 250 kDa, corresponding to the SARS-CoV-2 spike protein (second lane), compared to spike mRNA as a negative control (first lane) (Figure 3C). This experiment confirmed that the P1 / P5 copolymer is an effective nanocarrier for in vitro mRNA transfection.

[0242] Location of mRNA-PNPs in cells To visualize the intracellular location of mRNA-PNPs, FITC-labeled polymers were synthesized from P1 / P4 / P5, where FITC was conjugated to the polymer via an amine group on P4. As shown in Figure 4, co-localization of lysosomes with mRNA-PNPs (I1-P1 / P4 / P5) was lower than that of mRNA-PNPs (I1-P1 / P4), indicating that alkylated zwitterionic residues can significantly improve membrane fusion and lysosomal escape.

[0243] [Example 3] Targeted (selective) delivery To selectively deliver mRNA vaccines to antigen-presenting cells (APCs), particularly dendritic cells, we designed initiators with different glycan heads recognized by lectin receptors, such as Siglec-1, Siglec-2, Siglec-5 / E, and DC-SIGN. These are primarily expressed in DCs and macrophages. To evaluate Siglecs-mediated mRNA-PNP uptake, we compared the binding and internalization of mRNA-PNP to T cells, B cells, and bone marrow-derived dendritic cells (BMDCs).

[0244] method Splenocyte preparations and BMDC cultures. To prepare spleen cells, mouse spleens were homogenized on ground-glass ends of glass slides, treated with RBC lysis buffer (Sigma) to deplete red blood cells (RBCs), and then passed through a cell strainer (BD Biosciences). Bone marrow-derived dendritic cells (BMDCs) were prepared as described. Briefly, bone marrow was isolated from mouse femur and tibia and treated with RBC lysis buffer (Sigma-Aldrich) to deplete RBCs. The cells were then cultured at a density of 2 × 10⁵ cells / ml in RPMI-1640 containing 10% heat-inactivated FBS (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 20 ng / ml recombinant mouse GM-CSF (eBioscience). The cells were replenished with an equal volume of the complete medium described above on day 3, and half the volume of the medium was replaced on day 6. On day 8, the suspended cells were collected.

[0245] PNP treatment of spleen cells and BMDCs. Spleen cells or BMDCs were incubated with 1:2000 mRNA-PNP (diluted at 10 mg / mL) in RPMI-1640 at 37°C for 24 hours. Cells were blocked with an Fc receptor binding inhibitor (clone: ​​93, eBioscience) for 20 minutes. Spleen cells were stained with antibodies against CD3 (clone: ​​17A2, BV421-conjugate, Biolegend) and CD19 (clone: ​​1D3, PECy7-conjugate, BD Biosciences). BMDCs were stained with antibody against CD11c (clone N418 APC-conjugate, Biolegend). Labeled cells were analyzed using FACSC and a Flow Cytometer (BD Biosciences).

[0246] C2C12 cell culture. The mouse myomyogonia cell line C2C12 was purchased from Taiwan's Bioresource Collection and Research Center. C2C12 cells were cultured in DMEM with high glucose (ATCC) supplemented with 10% FBS and 1× antibiotic-antifungal agent. The cells were incubated at 37°C under controlled conditions of 5% CO2 and humidified air. The culture medium was changed every 2-3 days.

[0247] Treatment of polymerosomes with C2C12. Cultured C2C12 myobiocytes were isolated from petri dishes using 0.25% Trypsin-EDTA (Gibco) and neutralized with growth medium containing 10% FBS. mRNA-PNP (I1-P1 / P4-FITC-P5) or mRNA-PNP (I9-P1 / P4-FITC / P5) were added to 200 μL of C2C12 cells (2 × 10⁶) in growth medium. 5 In addition to the cells, the final dilutions relative to the original stock (10 mg / mL) were increased to 1:1000, 1:2000, 1:4000, or 1:8000. Measurements were taken at three time points: 5 minutes, 1 hour, and 24 hours.

[0248] Flow cytometry. After incubation with mRNA-PNP, BMDC cells were washed with ice-cold FACS buffer (1% FBS in 1× DPBS containing 0.1% sodium azide), incubated with purified anti-mouse CD16 / 32 antibody (BioLegend) in FACS buffer on ice for 20 minutes, and then washed with FACS buffer. BMDCs were stained with APC anti-mouse CD11c antibody (BioLegend) at 4°C for 30 minutes and washed with FACS buffer. Finally, BMDCs were stained with propidium iodide (Sigma-Aldrich). C2C12 cells were centrifuged and washed with FACS buffer. Cells were stained with propidium iodide. Flow cytometry was performed using a FACSCanto flow cytometer (BD Bioscience).

[0249] ELISA-mediated Glycan-PNP and DC-SIGN binding assay. To evaluate the binding of DC-SIGN to mannoside-modified PNPs, ELISA plates were coated overnight at 4°C with mRNA-PNP(I5-P1 / P5), mRNA-PNP(I6-P1 / P5), mRNA-PNP(I7-P1 / P5), mRNA-PNP(I8-P1 / P5), mRNA-PNP(I9-P1 / P5), or mRNA-PNP(I10-P1 / P5) (10 mg / mL) in PBS. The plates were incubated with diluted DC-SIGN ECD (15-0.075 nM in HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, and 0.1% BSA) at pH 7.4, 6.0, and 5.0 for 1 hour at room temperature. HRP-conjugated anti-DC-SIGN(B2)IgG antibody (Santa Cruz Biotechnology) was used to detect conjugated DC-SIGN ECD. After incubation at room temperature for 1 hour, the plates were treated with tetramethylbenzidine (TMB) for 10 minutes. After adding 0.5 M sulfuric acid to the plates, the optical density was measured at 450 nm using a microplate reader. The apparent Kd was calculated using GraphPad Prism with a nonlinear regression curve fit to the total binding amount.

[0250] Glycan-PNP binding assay to DC-SIGN-Fc, MMR-Fc, MINCLE-Fc, Dectin-2-Fc, and Langerin-Fc via ELISA. To evaluate the binding of receptor proteins to mannoside-modified PNPs, ELISA plates were coated overnight in PBS at 4°C with mRNA-PNP(I1-P1 / P5), mRNA-PNP(I8-P1 / P5), mRNA-PNP(I9-P1 / P5), or mRNA-PNP(I10-P1 / P5) (10 mg / mL). The plates were incubated with diluted DC-SIGN-Fc, MMR-Fc, MINCLE-Fc, Dectin-2-Fc, and Langerin-Fc (0.625 μg / mL in buffer) at pH 7.4 and room temperature for 1 hour. Binding proteins were detected using an HRP-conjugate anti-Fc IgG antibody. After incubation at room temperature for 1 hour, the plates were treated with tetramethylbenzidine (TMB) for 10 minutes. After adding 0.5 M sulfuric acid to the plates, the average optical density was measured at 450 nm using a microplate reader.

[0251] result Since evaluation is performed by flow cytometry, FITC-conjugated mRNA-PNPs were incubated with each cell line for 1 hour. I2 mRNA-PNPs with 9BPCNeu5Ac conjugated N-glycan, intended to target Siglec-2, showed higher cellular uptake by all APCs compared to I1 mRNA-PNPs without glycan modification (Figure 5). Specifically, in this experiment, I2 polymerosomes showed increases of 33.1%, 32.6%, and 27.8% in BMDC, B cell, and T cell uptake, respectively, compared to I1 polymerosomes. Similar results were obtained by using I3 and I4 mRNA-PNPs to target Siglec-5 / E and Siglec-1, respectively. Here, all glycan-modified polymerosomes showed better uptake by all APCs than glycan-free mRNA-PNPs. Similarly, I9-P1 / P4-FITC / P5 mRNA-PNP showed higher uptake by C2C12 muscle cells (approximately a 1x increase, Figure 9) compared to I1-P1 / P4-FITC-P5 mRNA-PNP.

[0252] As shown in Figure 6, I5 polymerosomes with arylmannose heads (I5-P1 / P4-FITC / P5) showed approximately 34% higher (33.8% increase) cell uptake by BMDCs compared to I1 polymerosomes without glycan heads (I1-P1 / P4-FITC / P5). On the other hand, B cells and T cells with low DC-SIGN expression showed only a slight increase in fluorescence signaling (16.2% and 12.8% increases, respectively) when treated with polymerosomes. This data demonstrates that efficient internalization and selective uptake of mRNA-PNP (I5-P1 / P4-FITC / P5) by dendritic cells can be achieved through targeting of the DC-SIGN receptor.

[0253] Evaluation of DC-SIGN binding to mRNA polymerosomes generated from I5-P1 / P5, I6-P1 / P5, I7-P1 / P5, I8-P1 / P5, I9-P1 / P5 and I10-P1 / P5 under different pH values ​​was performed on those polymerosomes at lower K D We demonstrated that these mRNAs can bind to DC-SIGN (Table 1). mRNA polymerosomes from I5-P1 / P5, I8-P1 / P5, I9-P1 / P5, and I10-P1 / P5 bound to the DC-SIGN extracellular domain (ECD) with nearly the same affinity at pH 7.4 and 5.0. In comparison, binding of mRNA polymerosomes from I6-P1 / P5 and I7-P1 / P5 was not detected at lower pH values. This suggests that coordination to calcium ions is reduced at low pH values.

[0254] [Table 1]

[0255] Without being constrained by theory, these binding results indicate that aryl trimannosides interact with DC-SIGN in the acidic endosomal compartment. Such binding stability enhances DC-SIGN-mediated signaling and its synergy with endosomal-resident Toll-like receptors, such as TLR7. The strong binding of aryl-mannoside-containing PNPs to DC-SIGN may be due to the high density display of the ligand, where the aryl group may be involved in CH-π and hydrophobic interactions. In addition, for receptor-targeted delivery, ligands are generally ligated to a carrier, and the distance between the carrier and ligand is regulated by the presence of a spacer. mRNA-PNPs with longer ligands (I8-P1 / P5 with Man-Ar-PEG12 and I7-P1 / P5 with Man-PEG12) showed slightly higher affinity for DC-SIGN. Overall, mRNA-PNPs (I9-P1 / P5) containing aryl-trimannosides showed the highest affinity and lowest KD for DC-SIGN. The increased affinity may be due to ligand clustering and spatial arrangement in the polymer, and the aryl portion may enhance its hydrophobic interactions.

[0256] To investigate whether the uptake of mannosylated mRNA-PNPs is dependent on DC-SIGN, further binding affinity tests were performed on DC-SIGN, macrophage mannose receptor (MMR), MINCLE, Dectin-2, and langerin. As shown in Figure 7, I8, I9, and I10 polymerosomes all showed selective binding compared to I1. Among these, the branched aryl-trimannoside I9 showed a better preference for DC-SIGN compared to the linear aryl-trimannoside I10. The fact that I9 exhibited more selective binding to DC-SIGN supports the idea that efficient uptake of mRNA-I9-P1 / P5 by DCs is likely mediated by DC-SIGN.

[0257] [Example 4] Immunization using polymerosomes of the present disclosure Next, we evaluated the effects of WT spike mRNA-PNPs with and without arylmannoside heads on vaccination and immune response.

[0258] Methods and immunotherapy design animal Balb / c mice (8 weeks old) were purchased from the National Laboratory Animal Center, Taiwan. All mice were maintained in an environment free of specific pathogens. 8-week-old Balb / c mice were immunized twice with im at a 2-week interval. Each vaccine dose contained PBS (100 μl). Ten days after the last immunization, serum collected from the immunized mice was subjected to ELISA analysis. The experimental protocol was approved by Academia Sinica's Institutional Animal Care and Utilization Committee (Approval No. 22-08-1901).

[0259] Animal immunization. BALB / c mice (n=5) aged 6-8 weeks were immunized intramuscularly with 15 μg of mRNA-PNP in phosphate-buffered saline (PBS). The animals were immunized at week 0 and boosted with a second vaccination at week 2 (Figure 8A). Serum samples were collected from each mouse one week after the second immunization. A group treated with spike mRNA-LNP (including ALC-0315, DSPC, ALC-0159, and cholesterol, which are commonly used in current mRNA vaccine formulations) was included as a positive control.

[0260] Measurement of serum IgG titer. Mouse serum IgG titers were determined using ELISA. Wells of a 96-well ELISA plate (Greiner Bio-One) were coated overnight with 100 ng of SARS-CoV-2 spike protein (ACROBiosystems) in 100 mM sodium bicarbonate at pH 8.8 and 4°C. The wells were blocked at 37°C for 1 hour with 200 μl of 5% skim milk in 1× PBS and washed three times with 200 μl of PBST (1× PBS, 0.05% Tween 20, pH 7.4). Serially diluted mouse serum samples were added to the wells and incubated at 37°C for 2 hours, then washed six times with 200 μl of PBST. The wells were incubated at 37°C for 1 hour with 100 μl of HRP-conjugated anti-mouse secondary antibody (1:10000, in PBS) and washed six times with 200 μl of PBST. 100 μl of horseradish peroxidase substrate (1-Step® Ultra TMB-ELISA Substrate Solution) (Thermo Scientific®) was added to the wells, followed by 100 μl of 1 M H2SO4. After incubation for 30 minutes, absorbance (OD450 nm) was measured using SpectraMax M5.

[0261] Pseudoviral Neutralization Assay. Pseudoviruses were constructed at the RNAi Core Facility of Academia Sinica using a procedure similar to that previously described. Briefly, a pseudo-lentivirus possessing the SARS-CoV-2 spike protein was generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91, pLAS2w.Fluc.Ppuro. HEK-293T cells were seeded one day prior to transfection, and the indicated plasmid was delivered to the cells using the TransITR-LT1 transfection reagent (Mirus). The medium was refreshed at 16 hours and collected at 48 and 72 hours after transfection. Cell debris was removed by centrifugation at 4,000xg for 10 minutes, and the supernatant was passed through a 0.45 μm syringe filter (Pall Corporation). The pseudo-lentiviruses were divided into equal volumes and then stored at -80°C. To predict lentiviral titer using the AlamaBlue assay (Thermo Scientific), the transduction units (TUs) of SARS-CoV-2 pseudolentivirals were predicted by using a cell viability assay responsive to limited dilutions of lentiviruses. Briefly, HEK-293T cells stably expressing the human ACE2 gene were plated into 96-well plates one day prior to lentiviral transduction. To determine the titer of the pseudolentivirals, different amounts of lentivirus were added to a medium containing polybrene (final concentration 8 μg / ml). Spin infection was performed in the 96-well plates at 1,100xg for 30 minutes at 37°C. After incubating the cells at 37°C for 16 hours, the medium containing the virus and polybrene was removed and replaced with fresh, complete DMEM containing 2.5 μg / ml puromycin. After 48 hours of treatment with puromycin, the medium was removed and cell viability was detected using 10% AlamaBlue reagent according to the manufacturer's instructions. The survival rate of uninfected cells (without puromycin treatment) was set to 100%. Viral titer (transduction units) was determined by plotting the ratio of surviving cells to diluted viral loads.For the neutralization assay, heat-inactivated serum or antibody was sequentially diluted and incubated with 1,000 TU of SARS-CoV-2 pseudolentivirus in DMEM at 37°C for 1 hour. The mixture was then inoculated into 10,000 HEK-293T cells stably expressing the human ACE2 gene in a 96-well plate. At 16 hours post-infection, the medium was replaced with fresh, complete DMEM (supplemented with 10% FBS and 100 U / mL penicillin / streptomycin), and the cells were cultured continuously for another 48 hours. Luciferase gene expression levels were determined using the Bright-Glo Luciferase Assay System (Promega). Relative light units (RLU) were detected using Tecan i-control (Infinite 500). The inhibition percentage was expressed as the ratio of the RLU reduction in the presence of diluted serum to the RLU value of the serum-free control, using the formula (RLU). control -RLU Serum Calculated using the RLU control.

[0262] result The results showed that both I1-P1 / P5 polymerosome treatment and mRNA-LNP treatment could generate levels of 10,000 anti-spike antibodies in serum on day 28. In contrast, I9-P1 / P5 polymerosome treatment could generate levels of approximately 30,000 anti-spike antibodies, which was at least three times more (Figure 8B). Next, serum was tested for its ability to neutralize pseudovirus-mediated entry into ACE2-expressing cells. Antiserum levels from I1-P1 / P5 polymerosomes were similar to those of the mRNA-LNP group, both below 2000. In comparison, significantly higher levels of neutralizing antibodies (over 6000) were observed in the I9-P1 / P5 polymerosome group (Figure 8C), and spike-specific antibody titers and pseudovirus neutralizing activity were well correlated.

[0263] Exemplary Embodiments Embodiment 1. A copolymer for forming polymerosomes, comprising an initiation block containing a glycan head, a propagator block containing a functional moiety containing a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, and a linking portion containing a disulfide bond that covalently connects the initiation block and the propagator block.

[0264] Embodiment 2. The copolymer according to Embodiment 1, wherein the glycan head contains a terminal mannoside.

[0265] Embodiment 3. The copolymer according to Embodiment 1 or Embodiment 2, wherein the glycan head comprises an O-arylmannoside containing an optionally substituted benzene ring.

[0266] Embodiment 4. The copolymer according to any one of Embodiments 1 to 3, wherein the glycan head comprises a monomannoside, a dimannoside, or a trimannoside.

[0267] Embodiment 5. The copolymer according to Embodiment 4, wherein the trimannoside is a linear or branched trimannoside.

[0268] Embodiment 6. The copolymer according to Embodiment 5, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0269] Embodiment 7. The copolymer according to any one of Embodiments 1 to 6, wherein the start block further includes a start spacer.

[0270] Embodiment 8. The copolymer according to Embodiment 7, wherein the starting spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0271] Embodiment 9. The copolymer according to Embodiment 8, wherein the saturated carbon moiety comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms (optionally 2 to 6 carbon atoms).

[0272] Embodiment 10. The copolymer according to Embodiment 8 or Embodiment 9, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0273] Embodiment 11. The copolymer according to Embodiment 10, wherein the PEG portion has a linear, branched, or star-shaped structure.

[0274] Embodiment 12. The copolymer according to any one of Embodiments 1 to 11, wherein the glycan head is configured to bind to dendritic cells.

[0275] Embodiment 13. The copolymer according to Embodiment 12, wherein the glycan head is configured to selectively bind to DC-SIGN.

[0276] Embodiment 14. The glycan head has a K content in the range of 5 to 8000 nM at pH 7.4. D The copolymer according to Embodiment 13, configured to bond to DC-SIGN.

[0277] Embodiment 15.K D The copolymer according to Embodiment 14, wherein the concentration is in the range of 5 to 500 nM at pH 7.4.

[0278] Embodiment 16. The glycan head has a K content in the range of 1 to 2000 nM at pH 5. D The copolymer according to any one of embodiments 13 to 15, configured to bond to DC-SIGN.

[0279] Embodiment 17.K D However, the copolymer according to Embodiment 16 has a concentration in the range of 1 to 600 nM at pH 5.

[0280] Embodiment 18. The glycan head is 9 BPC Neu5Ac conjugate N-glycan, Neu5Ac conjugate N-glycan, 9 TCC The copolymer according to Embodiment 1, comprising Neu5Ac conjugate N-glycan or a combination thereof.

[0281] Embodiment 19. The copolymer according to Embodiment 18, wherein the starting block is configured to bond to Siglec-2, Siglec-5 / E, Siglec-1, or a combination thereof.

[0282] Embodiment 20. The start block is

[0283] [ka] A copolymer according to any one of Embodiments 1 to 19, selected from the group consisting of (wherein black circles represent mannoside, white circles represent galactose, black squares represent GlcNAc, and diamonds represent Neu5Ac).

[0284] Embodiment 21. The copolymer according to any one of Embodiments 1 to 20, wherein the propagator block contains one or more guanidine groups.

[0285] Embodiment 22. The copolymer according to Embodiment 21, wherein the propagator block comprises three guanidine groups.

[0286] Embodiment 23. The copolymer according to any one of Embodiments 1 to 22, comprising a propagator spacer in which the propagator block comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0287] Embodiment 24. The copolymer according to Embodiment 23, wherein the saturated carbon moiety contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms (optionally 2 to 6 carbon atoms).

[0288] Embodiment 25. The copolymer according to Embodiment 23 or Embodiment 24, wherein the PEG portion comprises 2 to 72 (OCH2CH2) subunits.

[0289] Embodiment 26. The copolymer according to Embodiment 25, wherein the PEG portion has a linear, branched, or star-shaped structure.

[0290] Embodiment 27. The copolymer according to any one of Embodiments 1 to 26, comprising a plurality of propagator blocks and a plurality of connecting parts, wherein each of the plurality of propagator blocks is connected to at least one other propagator block or start block of the plurality of propagator blocks via one of the plurality of connecting parts.

[0291] Embodiment 28. The copolymer according to any one of Embodiments 1 to 27, wherein the propagator block is a first propagator block, the linking portion is a first linking portion, and the copolymer further comprises a second propagator block connected to the first propagator block via a second linking portion, wherein the first propagator block and the second propagator block independently comprise a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, and the second linking portion comprises a disulfide bond.

[0292] Embodiment 29. The copolymer according to Embodiment 28, wherein the first propagator block contains a guanidine group and the second propagator block contains a zwitterionic group.

[0293] Embodiment 30. The copolymer according to Embodiment 28, wherein the first propagator block contains a guanidine group and the second propagator block contains diethylenetriamine.

[0294] Embodiment 31. The copolymer according to any one of Embodiments 28 to 30, wherein the copolymer further comprises a third propagator block, the third propagator block being linked to a first propagator block or a second propagator block via a third linkage comprising a disulfide bond.

[0295] Embodiment 32. The propagator block is

[0296] [ka] A copolymer according to any one of embodiments 1 to 31, selected from the group consisting of the following.

[0297] Embodiment 33. The copolymer according to Embodiment 32, comprising at least two propagator blocks, wherein at least two propagator blocks are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) P1, P4 and P5.

[0298] Embodiment 34. The start block and at least two propagator blocks are IB5-PB1 / PB5, IB5-PB1 / PB4, IB5-PB2 / PB5, IB5-PB2 / PB4, IB5-PB1 / PB4 / PB5, IB6-PB1 / PB5, IB6-PB1 / PB4, IB6-PB2 / PB5, IB6-PB2 / PB4, IB6-PB1 / PB4 / PB5, IB7-PB1 / PB5, IB7-PB1 / PB4, IB7-PB2 / PB5, IB7-PB2 / PB4, IB7-PB1 / PB4 / PB5 The copolymer according to Embodiment 33, selected from the group consisting of IB8-PB1 / PB5, IB8-PB1 / PB4, IB8-PB2 / PB5, IB8-PB2 / PB4, IB8-PB1 / PB4 / PB5, IB9-PB1 / PB5, IB9-PB1 / PB4, IB9-PB2 / PB5, IB9-PB2 / PB4, IB9-PB1 / PB4 / PB5, IB10-PB1 / PB5, IB10-PB1 / PB4, IB10-PB2 / PB5, IB10-PB2 / PB4, and IB10-PB1 / PB4 / PB5.

[0299] Embodiment 35. A polymerosome comprising a membrane defining an internal space, wherein the membrane comprises a copolymer described in any one of Embodiments 1 to 34.

[0300] Embodiment 36. The polymersome according to Embodiment 35, wherein the copolymer constitutes at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0301] Embodiment 37. A polymerosome according to Embodiment 35 or Embodiment 36, wherein the membrane encapsulates a payload therein.

[0302] Embodiment 38. The polymerosome according to Embodiment 37, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0303] Embodiment 39. The polymerosome according to Embodiment 38, wherein the nucleic acid is RNA or DNA.

[0304] Embodiment 40. A polymerosome according to Embodiment 39, wherein the payload encodes a polypeptide.

[0305] Embodiment 41. A polymerosome according to any one of Embodiments 37 to 40, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0306] Embodiment 42. A polymerosome according to any one of Embodiments 35 to 41, wherein the copolymer is a first copolymer and the membrane further comprises a second copolymer, and the first copolymer and the second copolymer are independent of any one of Embodiments 1 to 34.

[0307] Embodiment 43.0. A polymersome according to any one of Embodiments 35 to 42, having a diameter of 0.001 to 5 microns or 0.01 to 5 microns.

[0308] Embodiment 44. A polymerosome according to any one of Embodiments 35 to 43, wherein the payload is a first payload and the membrane further encapsulates a second payload.

[0309] Embodiment 45. The polymerosome according to Embodiment 44, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof. Embodiment 46. The polymersome according to Embodiment 45, wherein the first payload and the second payload are different.

[0310] Embodiment 47. A formulation comprising a polymersome as described in any one of Embodiments 35 to 46.

[0311] Embodiment 48.0.01: The formulation according to Embodiment 47, comprising 95% (w / w) polymerosomes.

[0312] Embodiment 49. The formulation according to Embodiment 47 or Embodiment 48, wherein the polymerosome is a first polymerosome, and the composition further comprises a second polymerosome.

[0313] Embodiment 50. The formulation according to Embodiment 49, wherein the first polymerosome and the second polymerosome differ in size, copolymer forming their membrane, payload encapsulated within the polymerosome, or a combination thereof.

[0314] Embodiment 51. A formulation according to any one of Embodiments 47 to 50, further comprising a pharmaceutically acceptable excipient, adjuvant, or combination thereof.

[0315] Embodiment 52. The formulation according to Embodiment 51, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersant, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

[0316] Embodiment 53. The formulation according to Embodiment 51 or Embodiment 52, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in some vaccines that can be used in the compositions of the present disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salt, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03, MF59, and CpG 1018, or combinations thereof.

[0317] Embodiment 54. A kit for preparing polymerosomes, comprising an initiator, wherein the initiator is a first reagent comprising a glycan head and an initiator linker, and a propagator, wherein the propagator comprises a functional moiety and a propagator linker, wherein the functional moiety comprises a second reagent comprising a guanidine group, a zwitterionic group, diethylenetriamine or a combination thereof, and the initiator linker is configured to be coupled to the propagator linker via a linker comprising a disulfide bond.

[0318] Embodiment 55. The kit according to Embodiment 54, wherein the glycan head contains a terminal mannoside.

[0319] Embodiment 56. The kit according to Embodiment 54 or Embodiment 55, wherein the glycan head contains O-arylmannoside.

[0320] Embodiment 57. The kit according to any one of Embodiments 54 to 56, wherein the glycan head comprises a monomannoside, a dimannoside, or a trimannoside.

[0321] Embodiment 58. The kit according to Embodiment 57, wherein the trimannoside is a linear or branched trimannoside.

[0322] Embodiment 59. The kit according to Embodiment 58, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0323] Embodiment 60. The kit according to any one of Embodiments 54 to 59, wherein the initiator further comprises an initiation spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0324] Embodiment 61. The kit according to Embodiment 60, wherein the saturated carbon portion comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms (optionally 2 to 6 carbon atoms).

[0325] Embodiment 62. The kit according to Embodiment 61, wherein the PEG portion includes 2 to 72 (OCH2CH2) subunits.

[0326] Embodiment 63. The kit according to Embodiment 62, wherein the PEG portion has a linear, branched, or star-shaped configuration.

[0327] Embodiment 64. The kit according to any one of Embodiments 54 to 63, wherein the glycan head is configured to bind to dendritic cells.

[0328] Embodiment 65. The kit according to Embodiment 64, wherein the glycan head is configured to selectively bind to DC-SIGN.

[0329] Embodiment 66. The glycan head has a K content in the range of 5 to 8000 nM at pH 7.4. D The kit according to embodiment 65, configured to be coupled to DC-SIGN.

[0330] Embodiment 67.K D However, the kit according to Embodiment 66 has a concentration in the range of 5 to 500 nM at pH 7.4.

[0331] Embodiment 68. The glycan head has a K content in the range of 1 to 800 nM at pH 5. D The kit according to any one of embodiments 65 to 67, configured to couple to DC-SIGN.

[0332] Embodiment 69.K D However, the kit according to Embodiment 68 has a pH of 1 to 600 nM at pH 5.

[0333] Embodiment 70. The glycan head is 9 BPC Neu5Ac conjugate N-glycan (I2), Neu5Ac conjugate N-glycan (I3), 9 TCC A kit according to any one of embodiments 54 to 69, comprising Neu5Ac conjugate N-glycan (I4) or a combination thereof.

[0334] Embodiment 71. The kit according to Embodiment 70, wherein the glycan head is configured to be coupled to Siglec-2, Siglec-5 / E, Siglec-1, or a combination thereof.

[0335] Embodiment 72. The kit according to any one of Embodiments 54 to 71, wherein the initiator linking portion is a thiol group or a dithiolane group.

[0336] Embodiment 73. The initiator is

[0337] [ka] A kit according to any one of embodiments 54 to 72, selected from the group consisting of TIFF0007851666000050.tif198161.

[0338] Embodiment 74. The kit according to any one of Embodiments 54 to 73, wherein the propagator contains one or more guanidine groups.

[0339] Embodiment 75. The kit according to Embodiment 74, wherein the propagator comprises three guanidine groups.

[0340] Embodiment 76. The kit according to any one of Embodiments 54 to 75, further comprising a propagator spacer in which the propagator comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0341] Embodiment 77. The kit according to Embodiment 76, wherein the saturated carbon portion comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms (optionally 2 to 6 carbon atoms).

[0342] Embodiment 78. The kit according to Embodiment 76 or Embodiment 77, wherein the PEG portion includes 2 to 72 (OCH2CH2) subunits.

[0343] Embodiment 79. The kit according to Embodiment 78, wherein the PEG portion is linear PEG.

[0344] Embodiment 80. A kit according to any one of Embodiments 54 to 79, wherein the propagator of the second reagent is the first propagator, the second reagent further comprises the second propagator, or the kit further comprises a third reagent comprising the second propagator, and the first propagator and the second propagator independently comprise a functional moiety comprising a guanidine group, a zwitterionic group, a diethylenetriamine, or a combination thereof.

[0345] Embodiment 81. The kit according to Embodiment 80, wherein the first propagator comprises a guanidine group and the second propagator comprises a zwitterionic group.

[0346] Embodiment 82. The kit according to Embodiment 80, wherein the first propagator comprises a guanidine group and the second propagator comprises diethylenetriamine.

[0347] Embodiment 83. The kit according to any one of Embodiments 54 to 82, wherein the propagator linking portion is a thiol group or a dithiolane group.

[0348] Embodiment 84. The propagator is

[0349] [ka] A kit according to any one of embodiments 54 to 83, selected from the group consisting of the following.

[0350] Embodiment 85. The kit according to any one of Embodiments 54 to 84, wherein the first reagent and the second reagent are contained in the same container.

[0351] Embodiment 86. The kit according to any one of Embodiments 54 to 84, wherein the first reagent and the second reagent are contained in separate containers.

[0352] Embodiment 87. The kit according to any one of Embodiments 54 to 86, further comprising a payload, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0353] Embodiment 88. The kit according to Embodiment 87, wherein the nucleic acid is RNA or DNA.

[0354] Embodiment 89. The kit according to Embodiment 88, wherein the payload encodes a polypeptide.

[0355] Embodiment 90. The kit according to any one of Embodiments 87 to 89, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0356] Embodiment 91. A method for targeting and delivering a payload to a subject, comprising administering an effective amount of a pharmaceutical formulation containing polymersomes to the subject, wherein the polymersomes include a membrane encapsulating the payload, and the membrane contains the copolymer described in any one of Embodiments 1 to 34.

[0357] Embodiment 92. The method according to Embodiment 91, wherein the copolymer constitutes at least 50%, 70%, 80%, 90%, 95%, or 99% of the film.

[0358] Embodiment 93. The method according to Embodiment 92, wherein the copolymer is a first copolymer and the film further comprises a second copolymer, and the first copolymer and the second copolymer are independent of any one of Embodiments 1 to 34.

[0359] Embodiment 94. The method according to any one of Embodiments 91 to 93, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a glycan, or a combination thereof.

[0360] Embodiment 95. The method according to Embodiment 94, wherein the nucleic acid is RNA or DNA.

[0361] Embodiment 96. The method according to Embodiment 95, wherein the payload encodes a polypeptide.

[0362] Embodiment 97. The method according to any one of Embodiments 91 to 96, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0363] Embodiment 98. The method according to any one of Embodiments 91 to 97, wherein the payload is a first payload and the membrane further encapsulates a second payload.

[0364] Embodiment 99. The method according to Embodiment 98, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0365] Embodiment 100. The method according to Embodiment 99, wherein the first payload and the second payload are different.

[0366] Embodiment 101. A method for preventing or treating a disease in a subject, comprising administering an effective amount of a pharmaceutical formulation containing polymersomes to the subject, wherein the polymersomes include a membrane, the membrane includes a polymer component described in any one of Embodiments 1 to 34 and a payload encapsulated within the membrane, and the payload is a therapeutic agent or induces a therapeutic agent.

[0367] Embodiment 102. The method according to Embodiment 101, wherein the polymer component constitutes at least 50%, 70%, 80%, 90%, 95%, or 99% of the film.

[0368] Embodiment 103. The method according to Embodiment 101 or Embodiment 102, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a glycan, or a combination thereof.

[0369] Embodiment 104. The method according to Embodiment 103, wherein the nucleic acid is RNA or DNA.

[0370] Embodiment 105. The method according to Embodiment 104, wherein the payload encodes a polypeptide.

[0371] Embodiment 106. The method according to any one of Embodiments 101 to 105, wherein the payload is a first payload and the membrane further encapsulates a second payload.

[0372] Embodiment 107. The method according to Embodiment 106, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0373] Embodiment 108. The method according to Embodiment 107, wherein the first payload and the second payload are different.

[0374] Embodiment 109. The method according to any one of Embodiments 101 to 108, wherein polymerosomes are administered in an initial dose, followed by one, two, three, four, five or more booster doses.

[0375] Embodiment 110. The method according to Embodiment 109, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, six months, or thereafter, after the initial dose.

[0376] Embodiment 111. The method according to any one of Embodiments 101 to 110, wherein the effective amount is in the range of approximately 5 μg to 1000 μg.

[0377] Embodiment 112. A method for boosting an adaptive immune response, comprising administering an effective amount of a pharmaceutical formulation comprising polymerosomes to a target, wherein the polymerosomes comprise a membrane encapsulating a payload, the membrane comprises a copolymer according to any one of Embodiments 1 to 34, and the payload is immunogenic or induces an immunogenic biomolecule.

[0378] Embodiment 113. The method according to Embodiment 112, wherein the polymer component constitutes at least 50%, 70%, 80%, 90%, 95%, or 99% of the film.

[0379] Embodiment 114. The method according to Embodiment 83 or Embodiment 84, wherein the payload is a nucleic acid, compound, peptide, protein, glycan, or a combination thereof.

[0380] Embodiment 115. The method according to Embodiment 85, wherein the nucleic acid is RNA or DNA.

[0381] Embodiment 116. The method according to any one of Embodiments 112 to 115, wherein the biomolecule is a polypeptide or a protein.

[0382] Embodiment 117. The method according to any one of Embodiments 112 to 116, wherein the payload is a first payload and the membrane further encapsulates a second payload.

[0383] Embodiment 118. The method according to Embodiment 117, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0384] Embodiment 119. The method according to Embodiment 118, wherein the first payload and the second payload are different.

[0385] Embodiment 120. The method according to any one of Embodiments 112 to 119, wherein polymerosomes are administered in an initial dose, followed by one, two, three, four, five or more booster doses.

[0386] Embodiment 121. The method according to Embodiment 120, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, six months, or thereafter, following the initial dose.

[0387] Embodiment 122. The method according to any one of Embodiments 112 to 121, wherein the effective amount is in the range of approximately 5 μg to 1000 μg.

Claims

1. A copolymer for forming polymersomes, Starting block including Glycanhead, Propagator blocks comprising functional moieties including guanidine groups, zwitterionic groups, diethylenetriamine, or combinations thereof, and The start block and the propagator block are connected by a covalent bond, and the connecting part includes a disulfide bond. A copolymer containing [a specific component].

2. The copolymer according to claim 1, wherein the glycan head contains a terminal mannoside.

3. The copolymer according to claim 1, wherein the glycan head comprises an O-arylmannoside containing an optionally substituted benzene ring.

4. The copolymer according to claim 1, wherein the glycan head comprises a monomannoside, a dimannoside, or a trimannoside.

5. The copolymer according to claim 4, wherein the trimannoside is α-1,3-α-1,6-trimannoside.

6. The copolymer according to claim 1, wherein the starting block further comprises a starting spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

7. The copolymer according to claim 1, wherein the glycan heads are configured to bind to dendritic cells.

8. The copolymer according to claim 7, wherein the glycan head is configured to selectively bind to DC-SIGN.

9. The glycan head has a K content in the range of 5 to 8000 nM at pH 7.

4. Ｄ The copolymer according to claim 8, configured to bond to DC-SIGN.

10. Glycanhead, 9 ＢＰＣ Neu5Ac conjugate N-glycan, Neu5Ac conjugate N-glycan, 9 ＴＣＣ The copolymer according to claim 1, comprising Neu5Ac conjugate N-glycan or a combination thereof, wherein BPC represents biphenylcarboxyl, TCC represents 4H-thieno[3,2-c]chromen-2-carbamoyl, and Neu5Ac represents N-acetylneuraminic acid.

11. The starting block is, 【Chemistry 1】 The copolymer according to claim 1, selected from the group consisting of (wherein the formula, black circles represent mannoside, white circles represent galactose, black squares represent GlcNAc, and diamond represents Neu5Ac).

12. The copolymer according to claim 1, wherein the propagator block contains one or more guanidine groups.

13. The copolymer according to claim 12, wherein the propagator block comprises three guanidine groups.

14. The copolymer according to claim 1, wherein the propagator block comprises a propagator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

15. The system further includes a second propagator block, where the propagator block is a first propagator block, the connecting portion is a first connecting portion, and the copolymer is connected to the first propagator block via a second connecting portion. The first propagator block and the second propagator block independently comprise a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, and The copolymer according to claim 1, wherein the second linkage portion includes a disulfide bond.

16. The copolymer according to claim 15, wherein the first propagator block contains a guanidine group and the second propagator block contains a zwitterionic group, or the first propagator block contains a guanidine group and the second propagator block contains diethylenetriamine.

17. The propagator block 【Chemistry 2】 A copolymer according to claim 1, selected from the group consisting of the following.

18. The copolymer according to claim 17, comprising at least two propagator blocks, wherein at least two propagator blocks are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) PB1, PB4 and PB5.

19. The starting block is, 【Transformation 3】 Selected from the group consisting of (in the formula, black circles represent mannoside, white circles represent galactose, black squares represent GlcNAc, and diamonds represent Neu5Ac), The start block and at least two propagator blocks are IB5-PB1 / PB5, IB5-PB1 / PB4, IB5-PB2 / PB5, IB5-PB2 / PB4, IB5-PB1 / PB4 / PB5, IB6-PB1 / PB5, IB6-PB1 / PB4, IB6-PB2 / PB5, IB6-PB2 / PB4, IB6-PB1 / PB4 / PB5, IB7-PB1 / PB5, IB7-PB1 / PB4, IB7-PB2 / PB5, IB7-PB2 / PB4, IB7-PB1 / PB4 / PB5, IB The copolymer according to claim 18, selected from the group consisting of 8-PB1 / PB5, IB8-PB1 / PB4, IB8-PB2 / PB5, IB8-PB2 / PB4, IB8-PB1 / PB4 / PB5, IB9-PB1 / PB5, IB9-PB1 / PB4, IB9-PB2 / PB5, IB9-PB2 / PB4, IB9-PB1 / PB4 / PB5, IB10-PB1 / PB5, IB10-PB1 / PB4, IB10-PB2 / PB5, IB10-PB2 / PB4, and IB10-PB1 / PB4 / PB5.

20. A polymerosome comprising a membrane defining an internal space, wherein the membrane comprises the copolymer described in any one of claims 1 to 19.

21. The polymerosome according to claim 20, wherein the copolymer comprises at least 50% of the membrane.

22. The polymersome according to claim 20, wherein the membrane encapsulates a payload therein.

23. The polymerosome according to claim 22, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

24. The polymerosome according to claim 23, wherein the nucleic acid is RNA or DNA.

25. The polymersome according to claim 22, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

26. The polymerosome according to claim 20, wherein the copolymer is a first copolymer, and the membrane further comprises a second copolymer, the second copolymer being as described in any one of claims 1 to 19.

27. The polymersome according to claim 22, wherein the payload is a first payload, and the membrane further encapsulates a second payload, and the first payload and the second payload are different.

28. A formulation comprising polymerosomes according to claim 20.

29. The formulation according to claim 28, comprising 0.01 to 95% (w / w) polymerosomes.

30. The formulation according to claim 28, wherein the polymerosome is a first polymerosome, and the composition further comprises a second polymerosome, wherein the first polymerosome and the second polymerosome differ in size, copolymer forming its membrane, payload encapsulated within the polymerosome, or a combination thereof.

31. The formulation according to claim 28, further comprising pharmaceutically acceptable excipients, adjuvants, or combinations thereof.

32. The formulation according to claim 31, wherein the pharmaceutically acceptable excipients include a solvent, dispersion medium, diluent, dispersant, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

33. The formulation according to claim 31, wherein the adjuvant is C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03, MF59, CpG 1018, or a combination thereof.

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