Methods and compositions for targeted delivery using polymersomes

Abstract

FIELD: chemistry; pharmaceuticals.SUBSTANCE: copolymer intended for forming a polymersome; polymersome containing the proposed copolymer and a payload, such as a nucleic acid, polypeptide or protein; pharmaceutical composition for selective delivery of a payload containing the proposed polymersome; method for targeted delivery of a payload to a subject using the said polymersome; method for enhancing an adaptive immune response using the said polymersome; kit for producing the said polymersome. The proposed copolymer has a structurewhere x and y are integers and independently of each other are from 9 to 14, wherein the initiator block comprises a glycan head consisting of monomannoside, dimannoside or trimannoside, and the first block and the second block comprise a functional moiety comprising a guanidine group, diethylenetriamine or a zwitterionic group that is an alkylphosphobetaine group, choline phosphate or sulfotetin, or a combination thereof.EFFECT: selective delivery of payload and high encapsulation efficiency.47 cl, 15 dwg, 1 tbl, 4 ex

Description

[0001] Cross-references to related applications

[0002] This application claims priority from U.S. Provisional Patent Application No. 63 / 458,102, filed April 8, 2023, U.S. Provisional Patent Application No. 63 / 587,231, filed October 2, 2023, and U.S. Provisional Patent Application No. 63 / 575,056, filed April 5, 2024, all of which are hereby incorporated by reference in their entirety.

[0003] Technical field

[0004] The present invention generally relates to compositions and methods relating to polymeric nanocarriers, in particular pharmaceutical preparations containing polymersomes, capable of selective / targeted delivery of a payload to a target tissue site or cell.

[0005] Prior Art

[0006] Nanotechnology-based drug delivery is widely used in science, industry, and clinical applications. It has emerged as a promising approach to drug delivery, offering several advantages, including improved solubility and penetration of drug molecules. In a recent example, mRNA vaccines developed against COVID-19 viruses utilized a specialized lipid nanoparticle (LNP) designed to encapsulate and stabilize mRNA molecules, which are generally unstable and must be stored at low temperatures (e.g., -70°C). Typical lipid nanoparticles typically consist of several types of lipids. The ratio of these lipids requires careful adjustment, and the production of such lipid nanoparticles can be expensive. Furthermore, lipid particles typically cannot deliver mRNA molecules selectively. Therefore, there is a need in this area for a nanoparticle with a simpler design and the ability to selectively deliver mRNA molecules.

[0007] Brief description of the invention

[0008] In one aspect, the present invention describes a copolymer for forming a polymersome. The copolymer comprises an initiator block containing a glycan head and a propagator block further comprising a functional moiety that contains a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and a linker covalently linking the initiator block and the propagator block, wherein the linker comprises a disulfide bond.

[0009] In one aspect, the present invention describes a polymersome. The polymersome has a membrane defining an internal space, wherein the membrane contains a copolymer of the present invention.

[0010] In one aspect, the present invention provides a pharmaceutical preparation comprising a polymersome of the present invention.

[0011] In one aspect, the present invention describes a kit for producing a polymersome. The kit comprises a first reagent, which is an initiator, wherein the initiator comprises a glycan head and an initiator-binding moiety, and a second reagent, which is a propagator, wherein the propagator comprises a functional moiety and a propagator-binding moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator-binding moiety is capable of binding to the propagator-binding moiety via a linker containing a disulfide bond.

[0012] In one aspect, the present invention describes a method for targeted delivery of a payload to a subject. This method comprises administering to the subject an effective amount of a pharmaceutical preparation comprising a polymersome, wherein the polymersome has a membrane encapsulating the payload, and wherein the membrane comprises a copolymer of the present invention.

[0013] In one aspect, the present invention describes a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical preparation comprising a polymersome, wherein the polymersome has a membrane, wherein the membrane comprises a polymer component according to any one of claims 1 to 34; and a payload encapsulated within the membrane; and wherein the payload is a therapeutic agent or is convertible into a therapeutic agent.

[0014] In one aspect, the present invention describes a method for enhancing an adaptive immune response, comprising administering to a subject an effective amount of a pharmaceutical preparation comprising a polymersome, wherein the polymersome has a membrane encapsulating a payload, and wherein the membrane comprises a copolymer of the present invention, wherein the payload is immunogenic or is converted into an immunogenic biomolecule.

[0015] Brief description of drawings

[0016] Fig. 1A is a schematic illustration of a synthesis example of an example of a copolymer of the present invention. X and Y are both independently integers from 9 to 14.

[0017] Figure 1B illustrates the structures of some examples of polymersomes containing copolymers according to some embodiments of the present invention.

[0018] Figure 2A shows agarose gel electrophoresis results demonstrating the encapsulation efficiency of several exemplary copolymers according to certain embodiments of the present invention. The payload in this efficiency test was GFP mRNA, approximately 920 base pairs in size. Copolymers, with or without GFP mRNA, were larger than 10 kDa.

[0019] Fig. 2B is a graphical representation showing the average fluorescence intensity of HEK293 cells after transfection with polymersomes encapsulating GFP mRNA, according to some embodiments of the present invention.

[0020] Figure 3A shows the results of an agarose gel electrophoresis analysis demonstrating the encapsulation efficiency of several copolymers according to some embodiments of the present invention. The payload in these tests was an mRNA spike of approximately 2550 base pairs. Examples of copolymers, with or without the mRNA spike, had a size greater than 10 kDa. The N / P ratio (positively charged amino groups (N = nitrogen) to negatively charged phosphate (P) groups of nucleic acids) in each experimental group is indicated by the numbers listed in each row. The N / P ratios tested in this experiment were 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20.

[0021] Fig. 3B is a graphical representation showing the particle size and zeta (ζ) potential of mRNA-PNP (P1 / P5) with CryoEM image and insets.

[0022] Figure 3C shows a chemiluminescence image demonstrating the spike protein expression mediated by spike mRNA-PNP (I1-P1 / P5) in HEK293T cells. The first row shows the protein expression from cells transfected with spike mRNA, and the second row shows the protein expression from cells transfected with spike mRNA-PNP (I1-P1 / P5). Beta-actin was used as a benchmark expression in this experiment.

[0023] Figure 4 shows a graphical representation of the colocalization of lysosome and mRNA-PNP in cells, which was examined using confocal fluorescence imaging for 4 hours. Images were captured at 1, 1.5, 2, 2.5, 3, 3.5, and 4 hours, respectively. mRNA-PNP was labeled with FITC for detection.

[0024] Figure 5 is a graph showing the cellular uptake of fluorescence signal of BMDCs, B cells, and T cells treated with spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I2-P1 / P4-FITC / P5) targeting Siglec-2 after one hour. The data were obtained by flow cytometry.

[0025] Figure 6 is a graph showing the cellular uptake of fluorescent signal of BMDCs, B cells, and T cells treated with spike mRNA-PNP (I1-P1 / P4-FITC / P5) or spike mRNA-PNP (I5-P1 / P4-FITC / P5) targeting DC-SIGN after one hour. The data were obtained by flow cytometry.

[0026] Fig. 7 is a graphical representation showing the binding assay of DC-SIGN, MMR, MINCLE, Dectin-2, and Langerin (0.625 μg / mL) at pH 7.4 to polymersomes according to some embodiments of the present invention.

[0027] Fig. 8A schematically shows the design of animal immunization experiments.

[0028] Fig. 8B is a graphical representation showing the spike protein-specific IgG titer in blood plasma induced by immunization with I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control) measured on Day 28 after immunization.

[0029] Figure 8C is a graphical representation of the neutralization titer (ID50) measured on day 28 post-immunization with I1-P1 / P5 mRNA-PNP, I9-P1 / P5 mRNA-PNP, and LNP-mRNA (control). The neutralization titer was calculated as the reciprocal of the plasma dilution that resulted in a 50% reduction in RLU compared to the viral control wells, after subtracting the background RLU. ID50 values ​​are plotted with the standard error of the mean.

[0030] Fig. 9 is a graphical representation showing the FACS data of C2C12 muscle cell uptake by the polymersomes of the present invention (0 hours and 1 hour after administration) and the polymersomes without the targeting glycan (1 hour after administration).

[0031] Detailed description of the invention

[0032] Nanoparticle delivery is widely used in various applications. In addition to liquid nanoparticles (LNPs), which are probably the most common type of nanoparticle, polymersomes, another type of nanoparticle, are also attracting growing interest in industrial and clinical applications. Polymersomes (i.e., polymeric nanoparticles, polymeric vesicles, or polymeric nanoparticles (PNPs)) are self-assembled enclosures made of amphiphilic block copolymers. These amphiphilic copolymers are macromolecules containing at least one hydrophobic polymer block and at least one hydrophilic polymer block.Upon contact with water, these amphiphilic block copolymers self-assemble into closed particles. The hydrophobic blocks associate with each other to minimize direct contact with water and form the inner surface of the closed particle, while the hydrophilic blocks face outward, forming the outer surface of the particle. The hydrophobic core of these water-soluble polymersomes provides space for the solubilization of additional hydrophobic molecules. Therefore, these water-soluble polymersomes can act as carrier polymers for hydrophobic molecules encapsulated within the polymersomes. Furthermore, self-assembly of amphiphilic block copolymers occurs in the absence of stabilizers, which otherwise provide colloidal stability and prevent aggregation.

[0033] Polymersomes offer many advantages, such as high storage stability, ease of production, easy surface modification, high biocompatibility, and controlled release mechanisms. However, the industry still lacks polymersomes that effectively deliver drug molecules to specific sites in the body. This is likely due to the fact that known polymersomes do not effectively encapsulate the payload (e.g., the biomolecule administered to achieve a therapeutic effect) and are generally unstable in the blood. Furthermore, they are also unable to achieve selective delivery.

[0034] Polymersome

[0035] Accordingly, in one aspect of the present invention, a polymersome is described. The polymersome of the present invention serves to implement selective delivery (or targeted delivery) and achieve high encapsulation efficiency, in particular for a nucleic acid as a payload. Such a polymersome has a membrane that defines an internal space intended for encapsulating or transporting the payload. The membrane of the polymersome of the present invention comprises a copolymer including an initiator block, a propagator block, and a linker, wherein the linker covalently connects the initiator block and the propagator block and contains a disulfide bond. The initiator block contains a glycan head, and the propagator block contains a functional moiety containing a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0036] Without being bound by any theoretical justification, the disulfide bond (i.e., the disulfide linker) in the linker was selected due to its ability to be biodegraded in the intracellular environment and / or due to thiol uptake. Thus, the disulfide bond can facilitate the uptake of the polymersome of the present invention and its degradation after uptake to release the payload, for example, via the intracellular degradation mechanism induced by glutathione.

[0037] In some embodiments, the copolymer may comprise more than one initiator block and more than one propagator block. For example, the copolymer may comprise one initiator block, a first propagator block, and a second propagator block, wherein the initiator block and the first propagator block, as well as the first propagator block and the second propagator block, are connected by a linker.

[0038] Initiator Block

[0039] In certain embodiments, the initiating unit comprises a glycan head for selective delivery. For this purpose, the glycan head may have a targeting moiety, which is a ligand for the target (e.g., a receptor on the target cell). In some embodiments, the targeting moiety may be the terminal portion of the glycan head to have a greater chance of interacting with and binding to the target. However, the present invention 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 other types of immune cells. In some other embodiments, the target may be any biological cell with which the payload is intended to interact.

[0040] In particular embodiments, the initiator unit is designed to bind to a lectin receptor, such as Siglec-1 (sialoadhesin), Siglec-2, Siglec-5 / E, and DC-SIGN, with a certain degree of affinity, thereby achieving improved uptake by a certain type of APC. In some embodiments, the glycan head of the initiator unit comprises a mannosidase, which can be a terminal mannose designed to bind to DC-SIGN on a dendritic cell. In some embodiments, the glycan head comprises a sialoside. In some embodiments, to target Siglec-1, the glycan head can comprise 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Ac-α2,3-Gal-GlcNAc. In some embodiments, to target Siglec-2, the glycan head may comprise 9-biphenyl Neu5Ac-α2,6-Gal. In some embodiments, to target Siglec-5 / E, the glycan head may comprise Neu5Ac-α2,3-Gal-GlcNAc.

[0041] Binding under acidic conditions. In some embodiments, the binding between the initiator block and the target is Ca 2+ -correlated, and calcium coordination may decrease in low pH environments, resulting in reduced binding affinity. Therefore, to achieve greater binding affinity under acidic conditions, the glycan head of the initiator moiety may contain an aryl group. Without being bound by theory, the aryl group may participate in CH-π and hydrophobic interactions that enhance binding under acidic conditions. The aryl group may be unsubstituted benzene or benzene substituted with a halogen or haloalkyl (e.g., CF3). In some embodiments, the aryl group is linked to a targeting moiety. For example, the glycan head of the initiator moiety may contain O-aryl mannoside.

[0042] The structural configuration of the glycan head. In particular embodiments, the glycan head may have a linear structure or a branched structure. In some embodiments, the glycan head may have multiple targeting moieties, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting moieties. The multiple targeting moieties may have a linear, branched, or star-shaped configuration. For example, the glycan head of the initiator unit may comprise mono-mannoside, di-mannoside, or tri-mannoside, and when the glycan head comprises tri-mannoside, this tri-mannoside may be linear or have a branched structure, such as α-1,3-α-1,6-trimannoside. In particular embodiments, it should be noted that in some circumstances, a branched configuration (e.g., a tri-mannosidic glycan head) exhibits higher binding affinity to a target receptor.

[0043] Initiation Spacer. In some embodiments, the initiator block further comprises an initiator spacer. The initiator spacer is intended to impart structural flexibility to the glycan head and / or to impart hydrophobicity to the entire copolymer to facilitate polymersome assembly. Without being bound by theory, this flexibility allows the glycan head to move during interaction between the initiator block and the target, thereby facilitating the formation of a binding site between them.

[0044] A preferred spacer is biocompatible. In some embodiments, the initiating spacer is a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof. For example, the spacer may be a polyethyleneglycol (PEG) moiety 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 ranges between the above endpoints, such as 2 to 72, 2 to 60, 2 to 48, 2 to 36, 2 to 24, 2 to 18, 2 to 15, 2 to 10, 4 to 72, 4 to 60, 4 to 48, 4 to 36, 4 to 24, 4 up to 18, 4 to 15, 4 to 10, 8 to 72, 8 to 60, 8 to 48, 8 to 36, 8 to 24, 8 to 18, 8 to 15, or 8 to 10 (OCH2CH2) subunits. In some embodiments, the PEG moiety may have a linear, branched, or star-shaped structure.

[0045] In some embodiments, the spacer is a saturated carbon moiety, which may be a lipid tail, attached to the glycan head. In some embodiments, the saturated hydrocarbon comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms, or any range of carbon atom numbers between the above endpoints, such as 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, or 6 to 8 carbon atoms.

[0046] Binding affinity. In some embodiments, the binding affinity between the glycan head of the initiator block and the target may be determined by the dissociation constant (K D ). In some embodiments, K Dat pH 7.4 can be 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, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750 or 8000 nM, or any range between the above endpoints, such as 5 to 8000, 5 to 7000, 5 to 6000, 5 to 5000, 5 to 4000, 5 to 3000, 5 to 2500, 5 to 2000, 5 to 1500, 5 to 1250, 5 to 1000, 5 to 900, 5 to 800, 5 to 700, 5 to 600, 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 150, 5 to 100, from 5 to 75, from 5 to 50, from 5 to 30, from 5 to 20, from 10 to 8000, from 10 to 7000, from 10 to 6000, from 10 to 5000, from 10 to 4000, from 10 to 3000, from 10 to 2500, from 10 to 2000, from 10 to 1500, from 10 to 1250, from 10 to 1000, from 10 to 900, from 10 to 800, from 10 to 700, from 10 to 600, from 10 to 500, from 10 to 400, from 10 to 300, from 10 up to 200, from 10 to 150,from 10 to 100, from 10 to 75, from 10 to 50, from 10 to 30, or from 10 to 20 nM.,

[0047] In some other embodiments, K Dat pH 5 may 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 between the above endpoints, such as 1 to 2000, 1 to 1500, 1 to 1000, 1 to 900, 1 to 800, 1 to 750, from 1 to 700, from 1 to 650, from 1 to 600, from 1 to 550, from 1 to 500, from 1 to 450, from 1 to 400, from 1 to 350, from 1 to 300, from 1 to 250, from 1 to 200, from 1 to 150, from 1 to 100, from 1 to 75, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, or from 1 to 5, from 5 to 2000, from 5 to 1500, from 5 to 1000, from 5 to 900, from 5 to 800, from 5 to 750, from 5 to 700, from 5 to 650, from 5 to 600, from 5 to 550, from 5 to 500, from 5 to 450, from 5 to 400, from 5 to 350, from 5 to 300, from 5 to 250, from 5 to 200, from 5 to 150, from 5 to 100, from 5 to 75, from 5 to 50, from 5 to 40, from 5 to 30, from 5 to 20, from 5 to 10 nM.

[0048] Examples. In some embodiments, the glycan head of the initiator unit comprises 9 BPC Neu5Ac-conjugated N-glycan head (e.g., IB2), Neu5Ac-conjugated N-glycan (e.g., IB3), 9 TCC Neu5Ac-conjugated N-glycan (e.g., IB4) or a combination thereof. In particular embodiments, the glycan head comprises at least one of the following structures (it should be noted that “IB” means an initiator block, which is an initiator conjugated to the copolymer of the present invention. However, “IB” can be used interchangeably with “I,” meaning initiator, for brevity):

[0049] ;

[0050] where the filled circle represents mannoside, the open circle represents galactose, the filled square represents GlcNAc, and the diamond represents Neu5Ac.

[0051] Propagator block

[0052] In particular embodiments, the polymersome of the present invention exhibits targeted properties such as efficient payload encapsulation, reduced plasma protein adsorption, improved membrane fusion, and efficient payload release after uptake. At least one of the targeted properties is provided by the propagator unit of the present invention. For this purpose, the propagator unit of the present invention comprises a functional moiety comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0053] In some embodiments, the copolymer of the present invention comprises a single propagator block providing at least one of the target properties. In some embodiments, the copolymer of the present invention comprises multiple propagator blocks, each of which provides at least one of the target properties.

[0054] Efficient payload encapsulation. In some embodiments, the polymersome of the present invention serves to carry a nucleic acid as a payload, such as an mRNA molecule or a DNA molecule. In such embodiments, the propagator block of the polymersome copolymer may comprise a guanidine group. In some embodiments, the propagator block comprises 1, 2, 3, 4, 5, or more guanidine groups. Without being bound by theory, it should be noted that multiple guanidine groups in the propagator block provide a stronger salt bridge between the guanidine groups of the copolymer and the phosphate groups of the nucleic acid molecule (e.g., mRNA). In particular embodiments, the propagator block comprises three guanidine groups.

[0055] Reduced plasma protein adsorption and improved membrane fusion. As mentioned above, one of the common disadvantages of polymersomes is their suspected instability in plasma due to the adsorption of plasma proteins. In order to reduce the adsorption of plasma proteins in order to improve stability in blood plasma, the copolymer of the present invention may contain a zwitterion, which also improves the fusion of the polymersome membrane with the target cell. The zwitterion of the present invention may be a molecule / moiety with a net zero charge, bearing an equal number of positively charged and negatively charged functional groups at a pH of from 4.5 to 7.5. In some embodiments, the zwitterion of the present invention has an isoelectric point at a pH between 4.5 and 7.5. The zwitterion may be, but is not limited to, choline phosphates (CP), sulfotetins or phosphonium sulfonates.In some embodiments, the zwitterion of the present invention is an alkylphosphobetaine group comprising a phosphate group and an amino group having a negative charge and a positive charge, respectively.

[0056] Efficient payload release after uptake. Another target property of the polymersome is the ability to efficiently release the entrapped / encapsulated payload after uptake. Release of the entrapped / encapsulated payload occurs in the lysosome of the target cell and results from endosomal / lysosomal release or polymersome degradation. In some embodiments, the zwitterion of the propagator block can accelerate endosomal release. Furthermore, in some embodiments, the propagator block may further comprise an alkyl chain, which also participates in endosomal release.

[0057] On the other hand, in some embodiments, to facilitate lysosomal degradation, the propagator block of the present invention may comprise a diethylenetriamine moiety. The terminal amine residue of the diethylenetriamine moiety can also be used for additional functionalization. Alternatively, the propagator block of the present invention may comprise ethylenediamine, 1-(2-aminoethyl)piperazine, and / or tris(2-aminoethyl)amine.

[0058] Propagator spacer. In some embodiments, the propagator block of the present invention comprises a propagator spacer, primarily designed to impart the necessary hydrophobicity to the copolymer for assembly into a polymersome. In some embodiments, the propagator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0059] In some embodiments, the spacer may be a polyethylene glycol (PEG) moiety 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 the number thereof may be in any ranges between the above endpoints, such as from 2 to 72, from 2 to 60, from 2 to 48, from 2 to 36, from 2 to 24, from 2 to 18, from 2 to 15, from 2 to 10, from 4 to 72, from 4 to 60, from 4 to 48, from 4 to 36, from 4 to 24, from 4 to 18, from 4 to 15, from 4 to 10, from 8 to 72, from 8 to 60, from 8 to 48, from 8 to 36, from 8 to 24, from 8 to 18, from 8 to 15, or from 8 to 10 (OCH2CH2) subunits. In some embodiments, the PEG moiety can have a linear, branched, or star-shaped structure.

[0060] In some embodiments, the saturated carbon moiety may be a lipid tail extending from the propagator block. In some embodiments, the saturated hydrocarbon comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms, or any range of carbon atoms between the above endpoints, such as 2 to 15, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, or 6 to 8 carbon atoms.In particular embodiments, the propagator spacer comprises a moiety that is a monocarboxylic acid amide (e.g., a lipoamide moiety) or other biocompatible structure that provides a saturated hydrocarbon and a functional group for conjugation.

[0061] Examples. In some embodiments, the propagator block of the present invention comprises at least one of the following structures (note that “PB” means a propagator block that is a propagator conjugated to the copolymer of the present invention. However, “PB” can be used interchangeably with “P”, which means a propagator, for brevity):

[0062] PB1,

[0063] PB2,

[0064] PB3,

[0065] PB4, and

[0066] PB5.

[0067] Propagator block of the copolymer. In some embodiments, the copolymer comprises multiple propagator blocks and multiple linkers, wherein each propagator block of the multiple propagator blocks is connected to at least another propagator block of the multiple propagator blocks or to an initiator block via one of multiple linkers. Without being bound by any theory, it is believed in the present invention that the presence of two or more propagators in the copolymer structure provides an advantage. Each of the two or more propagators can provide at least one of the following desired properties: efficient encapsulation of the payload, reduced adsorption of plasma proteins, improved membrane fusion, and efficient release of the payload after absorption.In some embodiments, each of the two or more propagators has a different structure and / or target property that it provides, thereby forming the copolymer as a hetero-copolymer.

[0068] 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 that are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, (4) PB2 and PB5, or (5) PB1, PB4 and PB5, wherein the two or more propagators in the copolymer can be designated as formula numbers of the two or more propagators separated by the symbol “ / .” For example, two propagator blocks PB1 and PB5 in the copolymer can be designated as PB1 / PB5. However, the order and number of propagator blocks in the corresponding copolymer are not limited to those named. Without being bound by any theoretical basis, it was found in the present invention that the copolymer of PB1 / PB4, PB2 / PB4, PB1 / PB5 and PB2 / PB5 exhibits improved intracellular delivery due to efficient membrane fusion and payload release.

[0069] In particular embodiments, a hetero-copolymer copolymer containing two or more different types of propagator blocks can be configured with any initiator from I2, I3, I4, I5, I6, I7, I8, I9 and I10. The configuration of the initiator and propagator can be designated by the formula numbers of the initiator and propagator, separated by the symbol “-.” For example, a copolymer containing an initiator I5 and a propagator P5 can be designated as I5-P5. However, the order and number of initiator blocks and / or propagator blocks in the corresponding copolymer are not limited to those named.In some embodiments, a hetero-copolymer copolymer comprising two or more different types of propagators conjugated to a single initiator of the present invention may be selected from the group consisting of 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-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.

[0070] In some embodiments, the hetero-copolymer copolymer comprises a number of first propagator blocks and a number of second propagator blocks. In particular embodiments, the number of first propagator units 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 between the above endpoints, such as from 1 to 20, from 1 to 18, from 1 to 16, from 1 to 14, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 4, from 3 to 20, from 3 to 18, from 3 to 16, from 3 to 14, from 3 to 12, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 4, from 6 to 20, from 6 to 18, from 6 to 16, from 6 to 14, from 6 to 12, from 6 to 10, from 6 to 8, from 9 to 20, from 9 to 18, from 9 to 16, from 9 to 14, from 9 to 12, from 12 to 20, from 12 to 18, from 12 to 16.In particular embodiments, the number of second propagator units 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 between the above endpoints, such as 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 3 to 4, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 9 to 20, 9 to 18, 9 to 16, 9 to 14, 9 to 12, 12 to 20, 12 to 18, 12 to 16. In some embodiments, the number of first propagator blocks and / or the number of second propagator blocks can be determined using mass spectrometry, but is not limited to this method.

[0071] Polymersome containing the copolymer of the present invention

[0072] The polymersome of the present invention has a membrane that comprises a copolymer of the present invention. In some embodiments, the membrane comprises multiple copolymers of the present invention that assemble into a membrane via propagator blocks that connect to each other through hydrophobic interactions. The initiator block of each copolymer of the polymersome protrudes from the membrane and contacts the surrounding space.

[0073] In some embodiments, the copolymer of the present invention makes up at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the polymersome membrane, or any range between the above endpoints, such as from 50% to 99%, from 50% to 95%, from 50% to 90%, from 50% to 85%, from 50% to 80%, from 50% to 75%, from 50% to 70%, from 50% to 65%, from 50% to 60%, from 60% to 99%, from 60% to 95%, from 60% to 90%, from 60% to 85%, from 60% to 80%, from 60% to 75%, from 60% to 70%, from 60% to 65%, from 70% to 99%, from 70% to 95%, from 70% to 90%, from 70% to 85%, from 70% to 80%, from 50% to 75%, from 80% to 99%, from 80% to 95%, from 80% to 90%, 80% to 85%, 90% to 99%, or 90% to 95%.

[0074] In some embodiments, the membrane comprises at least two different types of copolymers of the present invention. In particular embodiments, the membrane comprises copolymers containing at least two different types of propagator blocks of the present invention. For example, the membrane comprises copolymers comprising 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 between the above end points, such as from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2.5, from 1 to 2, from 1 to 1.5, from 2 to 25, from 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2 to 2.5.

[0075] In some embodiments, the polymersome of the present invention may be a dendritic cell-targeted vaccine (DCTV) for specifically targeting the payload to dendritic cells, thereby achieving or improving the immunogenic response to the vaccine.

[0076] Polymersome size. In some embodiments, a polymersome of the present invention has a diameter 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 between the above endpoints, such as 0.001 to 5, 0.001 to 4, 0.001 to 3, 0.001 to 2, 0.001 to 1, 0.001 to 0.8, 0.001 to 0.6, 0.001 to 0.4, 0.001 to 0.2, from 0.001 to 0.1, from 0.001 to 0.05, from 0.001 to 0.01, from 0.001 to 0.005, from 0.05 to 5, from 0.05 to 4, from 0.05 to 3, from 0.05 to 2, from 0.05 to 1, from 0.05 to 0.8, from 0.05 to 0.6, from 0.05 to 0.4, from 0.05 to 0.2, from 0.05 to 0.1, from 0.1 to 5, from 0.1 to 4, from 0.1 to 3, from 0.1 to 2, from 0.1 to 1, from 0.1 to 0.8, from 0.1 to 0.6, from 0.1 to 0.4, 0.1 to 0.2, 0.5 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1, 0.5 to 0.8, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 microns.Polymersome size can be determined by (but is not limited to) dynamic light scattering (DLS). In some embodiments, a polymersome of the present invention has a polydispersity index (PI) of about 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 between the above endpoints, such as from 0.01 to 1, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, 0.01 to 0.1, 0.01 to 0.05, 0.1 to 1, 0.1 to 0.9, 0.1 to 0.8, 0.1 to 0.7, 0.1 to 0.6, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, or 0.1 to 0.2.

[0077] Zeta potential and molecular weight. Without any theoretical basis, the zeta potential and molecular weight of a polymersome may influence its cellular uptake. In some embodiments, a polymersome of the present invention has a zeta potential of about -50, -40, -30, -20, -15, -10, -5, 0, +5, +10, +15, +20, +30, +40, or +50, or any range between the above endpoints, such as -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 -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, a polymersome of the present invention has a molecular weight of about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50 kDa, or any range between the above endpoints, such as 1 to 50 kDa, 1 to 40 kDa, 1 to 30 kDa, 1 to 20 kDa, 1 to 15 kDa, 1 to 10 kDa, 1 to 5 kDa, 2 to 50 kDa, 2 to 40 kDa, 2 to 30 kDa, 2 to 20 kDa, 2 to 15 kDa, 2 to 10 kDa, 2 to 5 kDa, 5 to 50 kDa, 5 to 40 kDa, 5 to 30 kDa, 5 to 20 kDa, 5 to 15 kDa, 5 to 10 kDa, 8 to 50 kDa, 8 to 45 kDa, 8 to 40 kDa, 8 to 35 kDa, 8 to 30 kDa, 8 to 25 kDa, 8 to 20 kDa, 8 to 15 kDa, 8 to 10 kDa, 12 to 50 kDa, 12 to 45 kDa, 12 to 35 kDa, 12 to 25 kDa, 12 to 15 kDa, 25 to 50 kDa, 25 to 40 kDa, or 25 to 30 kDa.

[0078] Payload. In some embodiments, the polymersome membrane defines an internal space designed to encapsulate or contain the payload. As used herein, the term "encapsulated payload," "encapsulated within an internal space," or similar terms means that the payload is retained, enclosed, or surrounded by the polymersome membrane. The payload may be freely displaced within the internal space or may be covalently or non-covalently associated with the membrane. Encapsulation may be substantial, complete, or partial and does not preclude a portion of the payload from being in contact with the surrounding space outside the polymersome. In partial encapsulation embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the payload is retained, enclosed, or surrounded by the polymersome membrane.In some embodiments, the payload may be a biomolecule such as a nucleic acid, a compound, a polypeptide, a protein, a glycan head, or a combination thereof.

[0079] In some embodiments, the payload is ribonucleic acid (RNA, e.g., mRNA) or deoxyribonucleic acid (DNA, e.g., double-stranded DNA or single-stranded DNA), which, after delivery by the polymersome of the present invention to a target cell, can encode a polypeptide or protein in vivo. A nucleic acid, such as an mRNA molecule, used in the present invention can be obtained by in vitro transcription from an appropriate nucleic acid. In vitro transcription can be performed as described in PCT patent application WO2014 / 152027, filed March 13, 2014, which is incorporated herein by reference in its entirety.

[0080] In some embodiments, the polypeptide or protein is immunogenic (e.g., antigenic) for the organism to which the polymersome is administered. In such embodiments, the polymersome of the present invention is used to encapsulate and deliver an immunogenic protein or a nucleic acid designed to encode an immunogenic protein in vivo, such as an mRNA molecule in an RNA vaccine. The immunogenic protein may be a protein of a pathogen of viral (e.g., SARS-CoV-2, influenza, respiratory syncytial virus (RSV), EBV, dengue, VZV, HIV, Zika, or NIPAH), bacterial, or fungal origin. In some embodiments, the immunogenic protein may be the spike protein of a virus. In particular embodiments, the spike protein may be of coronavirus (CoV) origin, such as SARS-CoV, MERS-CoV, and SARS-CoV-2.In some embodiments, examples of the coronavirus (CoV) described in the present invention include, but are not limited to, alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, omicron-SARS-CoV2, and variants thereof.

[0081] In some embodiments, the payload is a nucleic acid, which may be a polynucleotide having an open reading frame designed to encode a polypeptide or protein in vivo. Such a polynucleotide may be modified with a 5'-terminal cap that is generated during an in vitro transcription reaction using the following chemical analogs of the RNA cap: 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 BioLabs, Ipswich, Mass.). 5'-capping of the modified polynucleotide can be completed after transcription using Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The Cap 1 structure can be generated using Vaccinia Virus Capping Enzyme and 2'-O-methyltransferase, generating m7G(5')ppp(5')G-2'-O-methyl.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 can be obtained from a recombinant source. After transfection into mammalian cells, the modified polynucleotides have a stability of 12-18 hours, or longer than 18 hours, for example, 24, 36, 48, 60, 72, or longer than 72 hours.

[0082] In some embodiments, a nucleic acid may be modified. In some embodiments, a nucleic acid may have multiple (more than one) modifications, the same or different from each other. In some embodiments, a nucleic acid comprises, in a specific region, one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified nucleic acid (e.g., a modified mRNA polynucleotide) exhibits reduced degradation in a cell or organism compared to an unmodified one. In some embodiments, a modified nucleic acid may exhibit reduced immunogenicity in an organism, respectively (e.g., a reduced innate response).

[0083] In some embodiments, the modification may include chemical modifications. In some embodiments, the modification may be natural, non-natural, or both. Some exemplary modifications that can be used in the present invention include, but are not limited to, modifications of the sugar, nucleobase, internucleotide linkage (e.g., with a phosphate linker, with a phosphodiester linkage, or with a phosphodiester backbone), or a combination thereof. In some embodiments, the nucleic acid (e.g., RNA) used as a payload in the present invention may be codon-optimized. For example, the nucleic acid can be modified to increase the G / C content. The G / C content of a nucleic acid can affect its stability.Nucleic acids with increased numbers of guanine (G) and / or cytosine (C) residues may be functionally more stable than those containing large numbers of adenine (A), thymine (T), or uracil (U) nucleotides. For example, WO2002 / 098443 describes a pharmaceutical composition comprising mRNA stabilized by a sequence modification in the translated region. Due to the degeneration of the genetic code, modifications work by replacing existing codons with those that impart greater RNA stability without changing the final amino acid.

[0084] In some embodiments, the nucleic acid may further comprise a sequence encoding a signal peptide. The signal peptide may comprise three regions: (1) an N-terminal region of variable length, 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 the nascent precursor protein (pre-protein) directs the ribosome to the membrane of the rough endoplasmic reticulum (ER) and initiates the transfer of the nascent peptide chain through it. The signal peptide is typically not responsible for the final destination of the mature protein, but this is not limited in the present invention. Signal peptides are cleaved from precursor proteins by a signal peptidase located in the endoplasmic reticulum (ER). They may remain unsplit and act as a membrane anchor.In some embodiments, the signal protein may be designed to bind to the encoded polypeptide or protein that is encoded by the payload at its C-terminus or N-terminus.

[0085] In some embodiments, the payload may be a therapeutic or prophylactic agent for treating or preventing a disease (e.g., cancer or an infectious disease). For example, the payload may be an antiviral agent, including (but not limited to) 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, molnupiravir and pasclovid. In other examples, the payload may be an anticancer agent. In particular embodiments, the payload is a nucleic acid designed to encode a therapeutic or prophylactic agent.

[0086] In some embodiments, the N / P ratio (positively charged amino groups of the polymer (N = nitrogen) to negatively charged phosphate (P) groups of nucleic acids) in the nucleic acid-encapsulating polymersome is about 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 50, or any range between the above endpoints, inclusive or inclusive, such as from 1 to 50, from 1 to 40, from 1 up to 30, from 1 to 20, from 1 to 10, from 1 to 5, from 5 to 50, from 5 to 40, from 5 to 30, from 5 to 20, from 5 to 10, from 10 to 50, from 10 to 40, from 10 to 30, from 10 to 20, from 8 to 40, from 8 to 20, from 8 to 12, from 9 to 50, from 9 to 30, or from 9 to 15. In another embodiment, the mRNA-encapsulating polymersome has a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

[0087] In some embodiments, wherein the payload in the polymersome is a nucleic acid encoding a polypeptide or protein in a target cell, upon uptake by the target cell, the polymersome is designed to encode in vivo 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, or any range between the above endpoints, inclusive or inclusive, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 5 to 8, 4 to 50, 4 to 45, 4 to 35, 4 to 25, 4 to 15, 4 to 9, 4 to 6, 7 to 50, 7 to 45, 7 to 35, 7 to 25, 7 to 15, or 7 to 9 copies.In some embodiments, after uptake by a target cell, the polymersome is designed to encode a polypeptide or protein in vivo continuously and continuously until the nucleic acid (i.e., the payload) is deactivated in vivo.

[0088] Compositions / drug

[0089] One aspect of the present invention relates to a composition (i.e., a preparation) comprising a polymersome of the present invention. The polymersome in the composition can encapsulate a payload and is intended to deliver the payload to a target site in the body. The payload of the present invention can include a nucleic acid, a compound, a peptide, a protein, a glycan head, or a combination thereof. In some embodiments, the payload can be an immunogenic protein or a nucleic acid encoding an immunogenic protein in vivo. In some embodiments, the preparation further comprises a pharmaceutically acceptable excipient, adjuvant, or a combination thereof. In particular embodiments, the composition is a pharmaceutical composition or a pharmaceutical preparation.

[0090] In some embodiments, the composition comprises 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 polymersome of the present invention, which does or does not encapsulate the payload, or any range between the above endpoints, such as (including or excluding) 0.01% to 95% (w / w), 0.01% to 90% (w / w), 0.01% to 80% (w / w), 0.01% to 70% (w / w), 0.01% to 60% (w / w), 0.01% up to 50% (w / w), from 0.01% to 40% (w / w), from 0.01% to 30% (w / w), from 0.01% to 20% (w / w), from 0.01% to 10% (w / w), from 0.01% to 5% (w / w), from 0.01% to 1% (w / w), from 0.01% to 0.1% (w / w), from 0.1% to 95% (w / w), from 0.1% to 90% (w / w), from 0.1% to 80% (w / w), from 0.1% to 70% (w / w), from 0.1% to 60% (w / w), from 0.1% to 50% (w / w), from 0.1% to 40% (w / w), from 0.1% up to 30% (weight / weight), from 0.1% to 20% (weight / weight), from 0.1% to 10% (weight / weight), from 0.1% to 5% (weight / weight), from 0.1% to 1% (w / w), 1% to 95% (w / w), 1% to 90% (w / w), 1% to 80% (w / w), 1% to 70% (w / w), 1% to 60% (w / w), 1% to 50% (w / w), 1% to 40% (w / w), 1% to 30% (w / w), 1% to 20% (w / w), 1% to 10% (w / w), 1% to 5% (w / w), 5% to 95% (w / w), 5% to 90% (w / w), 5% to 80% (w / w), 5% to 70% (w / w), 5% to 60% (w / w), 5% to 50% (weight / weight), 5% to 40% (weight / weight), 5% to 30% (weight / weight), 5% to 20% (weight / weight), or 5% to 10% (weight / weight). The remaining percentage of the composition may be an excipient as defined herein.

[0091] In some embodiments, the composition is an mRNA vaccine, wherein the polymersome encapsulates mRNA encoding an immunogenic protein in vivo. The immunogenic protein may be the viral spike protein or another pathogen antigenic molecule. In particular embodiments, the composition of the present invention may be a COVID-19 mRNA vaccine.

[0092] An example of a COVID-19 mRNA vaccine for use in the present invention can be obtained by using mRNA technology to remove glycan shields from the spike protein of a coronavirus (e.g., SARS-CoV-2) to better expose conserved regions of the spike protein. The mRNA vaccine of the coronavirus spike protein removes glycosylation sites in the receptor-binding domain or in the subunit 2 (S2) domain to expose highly conserved epitopes and generate antibodies and CD8 T cell responses with improved 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 (wherein the mRNA comprises a sequence selected from SEQ ID NOs 1-52), WO2022 / 221837A2 (wherein the mRNA comprises a sequence selected from SEQ ID Nos 1-21) and US20200046826A1 (wherein the mRNA comprises a sequence selected from SEQ ID Nos 1-20), which are incorporated herein in their entirety by reference.

[0093] In some embodiments, the composition of the present invention is for the treatment or prevention of a disease (e.g., cancer). In such embodiments, the payload carried by the polymersome may be a therapeutic agent, a prophylactic agent, or a nucleic acid designed to encode a therapeutic agent or prophylactic agent in vivo. For example, the composition may be a personalized cancer vaccine (e.g., melanoma), a KRAS vaccine (KRAS-induced), or a checkpoint vaccine (e.g., PD-1, PDL-1-related).

[0094] In some embodiments, the composition of the present invention can be administered together with another composition (e.g., a vaccine or a drug). Examples of the other composition include, but are not limited to, an influenza vaccine, an adenovirus vaccine, an anthrax vaccine, a cholera vaccine, a diphtheria vaccine, a hepatitis B vaccine, an HPV vaccine, a measles vaccine, a mumps vaccine, a smallpox vaccine, a rotavirus vaccine, a tuberculosis vaccine, a pneumococcal vaccine, and a Haemophilus influenzae type b vaccine.

[0095] Combined compositions

[0096] In some embodiments, the composition may be a combination composition (e.g., a combination vaccine) comprising a first polymersome encapsulating a first payload and a second polymersome encapsulating a second payload. The first polymersome and the second polymersome may be polymersomes according to the present invention, but differ from each other in the structure or properties of the copolymers therein. For example, the first polymersome and the second polymersome may differ in size, the copolymers forming their membranes, the payload encapsulated in the polymersomes, or a combination of these parameters.

[0097] For example, the first polymersome contains a glycan head designed to bind to DC-SIGN, and the second polymersome contains a glycan head designed to bind to Siglec-1. In another example, the first polymersome contains a glycan designed to bind to an antigen-presenting cell, and the second polymersome contains a glycan designed to bind to a cancer cell.

[0098] In some embodiments, the first payload and second payload are different from each other. For example, the first payload may be a protein or peptide, and the second payload may be a nucleic acid. In particular embodiments, the first payload and second payload may both be mRNA molecules, but encoding different proteins. For example, the first payload may be mRNA encoding the SARS-CoV2 delta spike protein, and the second payload may be mRNA encoding the SARS-CoV2 omicron spike protein.

[0099] Additional components of the composition

[0100] In some embodiments, the composition of the present invention may further comprise an adjuvant and / or an inactive substance, such as a pharmaceutically acceptable excipient. In particular embodiments, the adjuvant may be, but is not limited to, C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosidase (α-GalCer), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixtures of aluminum salts), squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), CpG 1018 (Dynavax), or a combination thereof.

[0101] In particular embodiments, the pharmaceutically acceptable excipient may be a solvent, dispersants, diluent, dispersing additive, suspending additive, surfactant, isotonic agent, thickening or emulsifying agent, preservative, polymer, peptide, protein, cell, hyaluronidase, or mixtures thereof. Various excipients for the preparation of pharmaceutical compositions and methods for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 22 ndEdition, Edited by Allen, Loyd V., Jr., Pharmaceutical Press). The use of commonly known excipients is within the scope of the present invention, except in cases where such commonly known excipients are incompatible with the substance or its derivatives, for example, by exerting any undesirable biological effect or otherwise interacting adversely with any other components of the pharmaceutical composition. The introduction of standard pharmaceutically acceptable excipients into the preparation can be accomplished by methods known in the pharmaceutical art (see Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA).

[0102] In some embodiments, the composition further comprises a phosphate conjugate. Without being bound by theory, the phosphate conjugate may increase the in vivo circulation time and / or enhance the targeted delivery of the polymersome of the present invention. Phosphate conjugates for use in the present invention can be prepared according to the techniques described in PCT Publication No. WO 2013 / 033438, filed April 30, 2012, or in US Patent Application No. US 2013 / 0196948, filed June 23, 2011, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, phosphate conjugates may include a compound of any of the formulas described in PCT Publication No. WO 2013 / 033438, filed August 30, 2012, which is incorporated herein by reference in its entirety.

[0103] In some embodiments, the composition further comprises a conjugate for enhancing delivery of the polymersome of the present invention. Without being bound by any theory, the conjugate selected for use can inhibit phagocytic clearance of the polymersome in a subject. In some examples, the conjugate can be the human membrane protein CD47 or a “self” peptide formed therefrom (e.g., the “self” particles described in Rodriguez et al. (Science 2013, 339, 971-975), which is incorporated herein by reference in its entirety).

[0104] In some embodiments, when the payload is an immunogenic agent or a nucleic acid encoding an immunogenic agent, the composition further comprises an immunostimulatory agent to enhance the immune response induced by the immunogenic agent. As a non-limiting example, the composition may comprise a Th1 immunostimulatory agent that can enhance a Th1-mediated response of the immune system (see PCT Publication WO 2010 / 123569 and US Patent Application No. 2011 / 0223201, each of which is incorporated herein in its entirety by reference).

[0105] In some embodiments, the composition does not include viral components (e.g., viral capsids, viral enzymes, or other viral proteins, such as those necessary for viral replication), and is not packaged, encapsulated, bound, or otherwise associated with a virus or viral particle.

[0106] Polymersome Preparation Kit

[0107] One aspect of the present invention relates to a kit for producing a polymersome according to the present invention. The kit comprises a first reagent and a second reagent, wherein the first reagent is an initiator comprising a glycan head and an initiator-binding moiety, and the second reagent is a propagator comprising a functional moiety and a propagator-binding moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator-binding moiety is capable of binding to the propagator-binding moiety via a linker comprising a disulfide bond.

[0108] In some embodiments, the initiator-binding moiety is capable of binding to the propagator-binding moiety, thereby forming a linker in the copolymer of the present invention. In some embodiments, the initiator-binding moiety and the propagator-binding moiety independently represent a thiol group or a dithiolane group.

[0109] In some embodiments, the kit further comprises a reagent that is a payload for encapsulation with a polymersome produced using the kit of the present invention. In particular embodiments, the payload may be as described herein.

[0110] Initiator

[0111] The initiator comprises a glycan head and an initiator-binding moiety. In some embodiments, the glycan head is as described above for the initiator block of the copolymer of the present invention.

[0112] In some embodiments, the initiator molecule further comprises an initiator spacer, as described herein for the copolymer of the present invention. In particular embodiments, the initiator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0113] In some embodiments, the initiator is selected from the group consisting of:

[0114] ,

[0115] ,

[0116] ,

[0117] ,

[0118] ,

[0119] ,

[0120] ,

[0121] , And

[0122] .

[0123] Propagator

[0124] The propagator comprises a functional moiety and a propagator-binding moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In some embodiments, the functional moiety of the propagator molecule is as described above for the propagator block in the copolymer of the present invention.

[0125] In some embodiments, the propagator further comprises a propagator spacer, as described herein for the copolymer of the present invention. In particular embodiments, the propagator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0126] In some embodiments, the second reagent comprises two types of propagators, each of which differs from the other in the structure or target properties it provides. For example, the second reagent may comprise a first propagator and a second propagator, each of which independently comprises a functional moiety comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In particular 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.

[0127] In some other embodiments, the kit further comprises a third reagent comprising a propagator that differs from the propagator in the second reagent in structure or the target properties it provides. For example, the propagator in the second reagent is a first propagator molecule, and the third reagent comprises a second propagator. The first propagator and the second propagator can independently comprise a functional moiety comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof. In particular 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.

[0128] In some embodiments, the propagator is selected from the group consisting of:

[0129]

[0130] Packaging

[0131] All components of the kit of the present invention can be packaged in a physical container, respectively. In some embodiments, the first reagent and the second reagent are contained in the same container; in other words, they are contained in a ready-to-use package. 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.

[0132] Methods of application

[0133] One aspect of the present invention concerns methods of using the polymersome of the present invention. In particular, these methods are used to achieve a desired effect, such as targeted delivery of a payload, prevention or treatment of a disease, or enhancement of the adaptive immune response in a subject. In some embodiments, the subject may be, but is not limited to, an animal or a human in which the payload serves to demonstrate its effectiveness, who requires treatment or prevention of a disease, or who requires enhancement of the adaptive immune response.

[0134] Methods of targeted delivery of payload in the subject

[0135] In some embodiments, a method for targeted delivery of a payload in a subject is described, which comprises administering to the subject an effective amount of a polymersome of the present invention. In some embodiments, a method for targeted delivery of a payload in a subject is described, which comprises administering to the subject an effective amount of a pharmaceutical preparation of the present invention. The polymersome and payload are as described herein, and the polymersome encapsulates the payload within an interior space defined by the polymersome membrane.

[0136] Without any theoretical justification, targeted delivery is achieved by the initiator blocks of the copolymers that form the polymersome. Specifically, the initiator blocks provide the desired binding affinity / specificity, targeting the desired region of the subject thanks to their glycan head.

[0137] Methods for preventing or treating a disease in a subject

[0138] In some embodiments, a method for preventing or treating a disease in a subject is described, which comprises administering to the subject an effective amount of a polymersome of the present invention. The polymersome in this method encapsulates a payload within the interior space defined by the polymersome membrane, and the payload is a therapeutic agent or is converted into a therapeutic agent intended for the prevention and / or treatment of the disease.

[0139] Without being bound by any theory, the polymersome of the present invention enables targeted delivery via the glycan heads of its copolymers. Therefore, when using the polymersome of the present invention to deliver a payload, the payload efficiency can be improved. For example, in embodiments in which the glycan head has a structure that specifically binds to DC-SIGN on dendritic cells, an antigenic or immunogenic payload can be efficiently delivered to dendritic cells to elicit an immune response that prevents the occurrence of a particular disease. This strategy provides an advantage in delivering an antigen or a nucleic acid encoding a vaccine antigen. Some other examples include the presence of a glycan head that targets cancer cells, so that an antitumor agent is efficiently delivered to the tumor microenvironment.This strategy has the potential to increase the effectiveness of anticancer drugs and reduce the side effects of treatment.

[0140] In some embodiments, the disease is characterized by dysfunctional or impaired activity of a protein or polypeptide. For example, the disease is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases.

[0141] In some embodiments, the disease may be cancer or infectious diseases. In particular embodiments, the disease may be a viral infection, including (but not limited to) human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or infections associated with SARS-CoV-2 (COVID-19).

[0142] Methods to enhance the adaptive immune response

[0143] In some embodiments, a method for enhancing an adaptive immune response is described, comprising administering to a subject an effective amount of a polymersome according to the present invention. The polymersome in this method encapsulates a payload within an internal space defined by the polymersome membrane, and the payload is a therapeutic agent or is converted into a therapeutic agent intended to elicit an adaptive immune response in the subject.

[0144] Without being bound by theory, the polymersome of the present invention enables targeted delivery to immune cells via glycan heads in its copolymers. In some embodiments, the glycan head comprises a structure that binds to the antigen-presenting cell with the desired specificity or affinity. For example, the glycan head may comprise a structure that binds specifically to DC-SIGN on dendritic cells, such that the polymersome is capable of delivering an immunogenic payload specifically to dendritic cells, facilitating an adaptive immune response.

[0145] In some embodiments, the enhanced adaptive immune response is directed against a disease, including but not limited to cancer or an infectious disease. The infectious disease, for example, can be a viral infection, including but not limited to human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or infections associated with SARS-CoV-2 (COVID-19).

[0146] Introduction

[0147] With respect to the methods of the present invention, in some embodiments, a single dose of a polymersome or preparation of the present invention is administered to a subject, which may or may not encapsulate a payload.In some embodiments, the subject is administered a polymersome or an initial dose followed by at least one booster dose, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more subsequent doses, with an interval between each dose of about 1, 2, 3, 4, 5, 6, 7 days, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or any range between the above endpoints, e.g., inclusive or inclusive, 1 to 7 days, 1 to 5 days, 1 to 3 days, 1 to 10 weeks, 1 to 8 weeks, 1 to 6 weeks, 1 to 4 weeks, 1 to 2 weeks, 1 to 12 months, 1 to 8 months, 1 to 6 months, 1 to 4 months, 1 to 2 months, or 6 to 12 months.In particular embodiments, the payload-encapsulating polymersome of the present invention is administered twice at the same or different dosage, and the two administrations are carried out at an interval of 1 day, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 1 to 5 days, 1 to 2 weeks, 1 to 3 months, 1 to 6 months, 1 month to 1 year, 3 months to 1 year, or 6 months to 1 year.

[0148] Route of Administration. The polymersome or composition of the present invention can be administered by any route. Suitable routes of administration include, but are not limited to, oral, nasal, mucosal, submucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, transdermal, and buccal administration. Some practical methods of topical application include, but are not limited to, drops, spray, aerosol, gel, or ointment applied to the mucous epithelium of the eye, nose, mouth, anus, or vagina. Other possible routes of administration include administration by spray, aerosol, or powder by inhalation into the respiratory tract.

[0149] Effective amount for administration. As used herein, an effective amount means an amount sufficient to achieve the desired effect. In embodiments where the purpose of administering the polymersome of the present invention is to treat a disease, an effective amount means a therapeutically effective amount, and in some other embodiments where the purpose is to prevent a disease, an effective amount means a prophylactically effective amount.

[0150] In some other embodiments, where the purpose of administering a polymersome with a payload is to enhance an adaptive immune response, an effective amount can be defined as an amount sufficient to elicit an antigen-specific immune response in a subject to whom the polymersome and payload are administered. An antigen-specific immune response can be characterized by measuring the titer of an anti-antigen antibody to the polypeptide (i.e., to the payload or a product formed from the payload) produced in a subject to whom the polymersome and payload have been administered. In some embodiments, such a measurement can be performed using an enzyme-linked immunosorbent assay (ELISA).

[0151] In some embodiments, the antibody titer is used to assess whether a subject has had an infection or to determine whether immunization is necessary. In some embodiments, the antibody titer is used to assess the strength of the autoimmune response, to determine whether a booster immunization is necessary or has occurred, to determine whether a previous vaccine was effective, and / or to identify recent or past infections.

[0152] The effective amount in the methods of the present invention can be determined based on several factors, including (but not limited to) the patient's condition (age, gender, species, body weight, health status, etc.), the progress of the disease in question, the route of administration, the dosage and interval between administrations, and the nature of the payload. With regard to the nature of the payload, for example, in embodiments where the polymersome of the present invention is used to transfer mRNA, as in the case of an mRNA vaccine, the effective amount can be determined by the effective amount of mRNA required to elicit a sufficient immune response in the subject. Accordingly, in some embodiments wherein the payload is mRNA, the effective amount in the methods of the present invention 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 1,000 micrograms (mcg), or any range between the above endpoints, such as, inclusive or non-inclusive, 5 micrograms to 1,000 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, 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, from 5 micrograms to 90 micrograms, from 5 micrograms to 80 micrograms, from 5 micrograms to 70 micrograms, from 5 micrograms to 60 micrograms, from 5 micrograms to 50 micrograms, from 5 micrograms to 40 micrograms, from 5 micrograms to 30 micrograms, from 5 micrograms to 20 micrograms, from 5 micrograms to 10 micrograms, from 10 micrograms to 1000 micrograms,from 10 micrograms to 900 micrograms, from 10 micrograms to 800 micrograms, from 10 micrograms to 700 micrograms, from 10 micrograms to 600 micrograms, from 10 micrograms to 500 micrograms, from 10 micrograms to 400 micrograms, from 10 micrograms to 300 micrograms,10 micrograms to 200 micrograms, from 10 micrograms to 175 micrograms, from 10 micrograms to 150 micrograms, from 10 micrograms to 125 micrograms, from 10 micrograms to 100 micrograms, from 10 micrograms to 90 micrograms, from 10 micrograms to 80 micrograms, from 10 micrograms to 70 micrograms, from 10 micrograms to 60 micrograms, from 10 micrograms to 50 micrograms, from 10 micrograms to 40 micrograms, from 10 micrograms to 30 micrograms, from 10 micrograms to 20 micrograms, from 50 micrograms to 1000 micrograms, from 50 micrograms to 900 micrograms, from 50 micrograms to 800 micrograms, from 50 micrograms to 700 micrograms, from 50 micrograms to 600 micrograms, from 50 micrograms to 500 micrograms, from 50 micrograms to 400 micrograms, from 50 micrograms to 300 micrograms,from 50 micrograms to 200 micrograms, from 50 micrograms to 175 micrograms, from 50 micrograms to 150 micrograms, from 50 micrograms to 125 micrograms, from 50 micrograms to 100 micrograms, from 50 micrograms to 90 micrograms, from 50 micrograms to 80 micrograms, from 50 micrograms to 70 micrograms, from or 50 micrograms to 60 micrograms. 100 micrograms to 1000 micrograms, 100 micrograms to 900 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, 300 micrograms to 800 micrograms, 300 micrograms to 700 micrograms, from 300 micrograms to 600 micrograms, from 300 micrograms to 500 micrograms, from 300 micrograms to 400 micrograms,from 500 micrograms to 1000 micrograms, from 500 micrograms to 900 micrograms, from 500 micrograms to 800 micrograms, from 500 micrograms to 700 micrograms, from 500 micrograms to 600 micrograms, from 600 micrograms to 800 micrograms, or from 700 micrograms to 900 micrograms.,

[0153] However, given that the targeted delivery is carried out by the polymersome of the present invention, it can be expected that the effective amount required in the methods of the present invention may be lower than the effective amount required in other methods without targeted delivery.For example, the effective amount required in the methods of the present invention may be lower than the effective amount required in other non-targeted methods by 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%, or any range between the above endpoints, such as, inclusive or inclusive, by 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%, 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%.

[0154] Furthermore, in some embodiments in which the polymersome of the present invention is used to deliver an antigenic agent or a nucleic acid encoding an antigenic agent to induce the production of antibodies against the antigen, the antibody titer generated by the present invention, compared to the antibody titer generated in non-targeted delivery methods, is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or in any range between the above endpoints, such as, inclusive or inclusive, 1 to 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, 2 to 9 times, 2 to 7 times, 2 to 5 times, 3 to 10 times, 3 to 8 times, 3 to 5 times, or 4 to 6 times.

[0155] In some other embodiments, in which the polymersome of the present invention is used to deliver an antigenic agent or a nucleic acid encoding an antigenic agent to induce an immune response against the antigenic agent, the antibody titer against the antigenic agent generated by the present invention, compared to the antibody titer generated in non-targeted delivery methods, is 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher, or in any range between the above endpoints, such as, inclusive or inclusive, 0.1 to 10, 0.1 to 9, 0.1 to 8, 0.1 to 7, 0.1 to 6, 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, from 0.1 to 1, from 0.1 to 0.5, from 0.5 to 10, from 0.5 to 9, from 0.5 to 8, from 0.5 to 7, from 0.5 to 6, from 0.5 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 7 to 10, 7 to 9, 7 to 8, or 8 to 10 times.

[0156] In some embodiments in which the polymersome of the present invention is used to deliver an antigenic agent or a nucleic acid encoding an antigenic agent to induce an immune response against the antigenic agent, the immune response occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 days earlier than the immune response induced by non-targeted delivery methods, or in any range between the above endpoints, such as, inclusive or inclusive, 1 to 20, 1 to 18, 1 to 14, 1 to 10, 1 to 6, 2 to 20, 2 to 18, 2 to 14, 2 to 10, 2 to 6, 5 to 20, 5 to 18, 5 to 14, or 5 to 10 days earlier.

[0157] In some embodiments, the polymersome in the methods of the present invention is administered at a dose sufficient to deliver a payload of from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, per subject's body weight per day, one or more times daily, to achieve the desired effect in vivo.

[0158] Definitions

[0159] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains. Unless otherwise indicated, the techniques used herein are standard techniques well known to those skilled in the art. The practice of the present invention, unless otherwise indicated, employs well-known techniques in microbiology, culture cultivation, molecular biology, chemistry, biochemistry, and recombinant DNA technology, which are known to those skilled in the art. The materials, methods, and examples described are illustrative only and not limiting. The following description is provided for illustrative purposes and in no way limits the scope of the present invention.

[0160] Numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions and results, etc., used to describe and claim certain embodiments of the present invention, should be understood as modified in some instances by the term "about" or "approximately." One skilled in the art will understand the meaning of the term "about" in the context of the value to which it refers. Numerical values ​​​​given in some embodiments of the present invention may contain certain errors resulting from standard deviations in the corresponding measurements. For example, the term "about" as used herein means a measured value, such as an amount, a time interval, etc., covering deviations of ±20%, ±10%, ±5%, ±1% or ±0.1% from the stated value, if such variations are acceptable.

[0161] As used in this text, the term "substantially" means sufficient to achieve the intended purpose. The term "substantially" allows for minor, insignificant deviations from the absolute or ideal state, size, value, result, etc., expected by a person skilled in the art and not materially affecting the overall result. When used in relation to numerical values ​​of parameters or characteristics expressed by numerical values, "substantially" means a deviation within ten percent.

[0162] As used in this text, the terms "treat" and "treatment" mean an approach to achieving beneficial or desired results, such as clinical results. As used herein, beneficial or desired results include inhibiting or suppressing the onset or progression of an infection or disease; alleviating or slowing the progression of symptoms of an infection or disease; or a combination thereof.

[0163] When used in this text, the terms "prevent" and "prevention" are used interchangeably with the term "prophylaxis" and may mean preventing an infection altogether or preventing the development of symptoms of that infection, delaying the onset of the disease or its symptoms; or reducing the severity of a subsequent infection or its symptoms.

[0164] As used herein, the term “glycan” means a polysaccharide, oligosaccharide, or monosaccharide. Glycans may be monomers or polymers of sugar residues and may be linear or branched. Glycan may include residues of natural sugars (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic 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.).

[0165] Example

[0166] Example 1: Synthesis of polymersome examples

[0167] Chemical Materials and Methods

[0168] For chemical synthesis, all starting compounds and commercially available reagents were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. All reactions were performed in oven-dried glassware under a nitrogen atmosphere using dry solvents. 1 H and 13 C NMR spectra were recorded on a Brucker AV-600 spectrometer, and solvent peaks (CDCl3 at δ 7.24 and 77.23, CD3OD at δ 3.31 and 49.2, and D2O at δ 4.80, and DMSO-d6 at δ 2.5 and 39.51 for 1 H and 13C, respectively). Chemical shift values ​​(δ) are given in ppm as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, qv = quartet, m = multiplet), integrated intensity, and spin–spin coupling constants (J), where J is in hertz (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. Histidine-tagged purification resin was purchased from Roche. A HiTrap IMAC column (5 mL) was purchased from GE Healthcare Life Sciences. Gel permeation chromatography (GPC) on an Ultimate 3000 liquid chromatograph with a 101 RI detector and Shodex columns was used to analyze polymer products with THF as eluent at 30°C and a flow rate of 1 mL / min.Calibration was performed using Shodex polystyrene standards with a narrow molecular weight distribution (SM-105). Mw values ​​and dispersions of the polymer products were calculated using DIONEX chromeleon software. Transmission electron microscopy (TEM) images were obtained on an FEI Tecnai G2 F20 S-Twin instrument.

[0169] The chemical reagents and methods described in this section were used in all examples described in the present invention.

[0170] Obtaining an example of a propagator according to the present invention

[0171] Five examples of propagators, propagators P1, P2, P3, P4, and P5, are described in this text. The method for preparing propagator P1 (Compound 2), P2 (Compound 3), and P4 (Compound 8) is 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 structures of the compounds obtained in each step of these schemes were confirmed by NMR. Additional details and data are provided below.

[0172]

[0173] Scheme 2

[0174]

[0175] Scheme 3

[0176]

[0177] Connection 1

[0178]

[0179] Compound 1 was synthesized and characterized according to a published procedure. 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 the work of Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153-12160, which is incorporated herein in its entirety by reference.

[0180] Connection 2

[0181]

[0182] Compound 2 was synthesized and characterized according to the published procedure. 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). Synthesis was performed 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.

[0183] Connection 3

[0184]

[0185] Compound 3 was synthesized and characterized according to the published procedure. 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). Synthesis was performed according to Guo, J. et al., Rational Design of Poly(disulfide)s as a Universal Platform for Delivery of CRISPR-Cas9 Machineries toward Therapeutic Genome Editing. ACS Central Science 2021, 7, 990-1000, which is incorporated herein by reference in its entirety.

[0186] Connection 4

[0187]

[0188] Compound 4 was synthesized and characterized according to the published procedure. 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). Synthesis was performed 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.

[0189] Connection 5

[0190]

[0191] Compound 5 was synthesized and characterized according to the published procedure. 1 H 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 performed 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.

[0192] Connection 6

[0193]

[0194] A solution of 4 (0.126 mmol) in DMF (1 mL) was preactivated by adding EDC (0.506 mmol), HOBt (0.506 mmol), and trimethylamine (0.57 mmol) under nitrogen atmosphere for 30 min. Then, a solution of 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 h. The reaction mixture was evaporated to dryness in vacuo and then diluted with ethyl acetate. The organic layer was washed with water three times, dried over MgSO4, filtered, and evaporated. The resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 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), 3.59-3.19 (m, 16H), 3.15-3.12 (kv, J = 6.0 Hz, 2H), 3.06-3.04 (t, J = 6.9 Hz, 1H), 1.77-1.71 (m, 1H), 1.55-1.32 (m, 59H). HRMS (ESI) calculated for C 57 H 97 N 14 O17 [M+H] + m / z 1249.7156; found: 1249.7166.

[0195] Connection 7

[0196]

[0197] Pd / C, 10% Pd content, 13 mg was added to a solution of 6 (0.12 mmol) in MeOH (2 mL). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 6 h. The solution was filtered through a pad of celite. The residue was evaporated to dryness in vacuo, and the resulting residue was 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 then stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo and then diluted with ethyl acetate. The organic layer was washed with water three times, dried over MgSO4, filtered, and evaporated. The resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:50) to afford 7 (94 mg, 82%). 1H 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), 6.15-8.12 (m, 1H), 6.2-18 (m, H). 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), . 1.57-1.34 (m, 64H). HRMS (ESI) calculated for C 57 H 103 N 14 O 16 S2[M+H] + m / z 1303.7118; found: 1303.7129.

[0198] Connection 8

[0199]

[0200] Compound 7 (0.08 mmol) was added to a solution of 4M HCl (0.5 mL) in 1,4-dioxane (0.5 mL), and the resulting mixture was stirred at room temperature for 12 hours. After that, the solution was evaporated and dried in vacuo to give 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), 1.8-2.03 (m, 2H), 1.8-3.6 (m, 2H). 1.62-1.16 (m, 11H). 13C 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) calculated for C 27 H 55 N 14 O4S2[M+H] + m / z 703.3967; found: 703.3995.

[0201] Connection 9

[0202]

[0203] A solution of lipoic acid (2.5 mmol) in DMF (3 mL) was preactivated by adding EDCI (3 mmol), HOBt (3 mmol), and trimethylamine (3 mmol) under a nitrogen atmosphere for 30 min. Then, lysine (1 mmol) in DMF (3 mL) was added to the above solution, and the reaction solution was stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo and then diluted with ethyl acetate. The organic layer was washed with water three times, dried over MgSO4, filtered, and evaporated. The resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 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 (quint, 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) calculated for C 57 H 97 N 14 O 17 [M+H] +m / z 1249.7156; found: 1249.7166.

[0204] Connection 10

[0205]

[0206] A solution of 9 (0.141 mmol) in DMF (1 mL) was preactivated by adding EDC (0.211 mmol), HOBt (0.211 mmol), and trimethylamine (0.282 mmol) under nitrogen atmosphere for 30 min. Then, 5 (0.183 mmol) in DMF (1 mL) was added to the above solution, and the reaction solution was stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo and then diluted with ethyl acetate. The organic layer was washed with water three times, dried over MgSO4, filtered, and evaporated. The resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:20) to give 10 (92 mg, 84%). 1H 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), 5.5, 7.4 H), 4.37 (sq, 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), 2.46-2.3.9 (m, 2H), 2.46-2.9 (m, J, 2H), (t = 2.2H). 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, 2H), 1.2-3 (H). 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, 5.2.2, 4.19, 4.19. 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, (28.28), (x2). 25.46, 25.36, 25.32, 22.29. HRMS (ESI) calculated for C 35 H 63 N6O7S4[M+H] + m / z 807.3641; found: 807.3655.

[0207] Connection 11

[0208]

[0209] Compound 10 (0.1 mmol) was added to a solution of 4 M HCl (0.5 mL) in 1,4-dioxane (0.5 mL), and the resulting mixture was stirred at room temperature for 12 h. Afterwards, the solution was evaporated and dried in vacuo, yielding 8 (59 mg, quant.). The resulting compound was sufficiently pure and was used further without further purification.

[0210] Connection 12

[0211]

[0212] A solution of lipoic acid (3 mmol) and CDI (3.9 mmol) was dissolved in 25 mL of anhydrous DCM. The resulting 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 for 40 min at 0°C and 30 min at room temperature, then washed with water three times and dried over MgSO4, filtered, and evaporated to yield 12 (589 mg, 66%). 1H NMR (600 MHz, CDCl3): δ 3.57-3.50 (m, 1H), 3.31 (kv, 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) calculated for C 12 H 26 N2OS2[M+H] + m / z 277.1408; found: 277.1399.

[0213] Connection 13

[0214]

[0215] Compound 13 was synthesized and characterized according to the published procedure. 1 H 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). Synthesis was performed 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 herein by reference in its entirety.

[0216] Connection 14

[0217]

[0218] A solution of 12 (0.25 mmol) in 1 mL of anhydrous DMF was added to 13 (0.25 mmol), and the reaction mixture was stirred at 70 o C for 24 h. The reaction mixture was evaporated to dryness in vacuo, yielding 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) calculated for C 22 H 44 N2O5PS2[MH] - m / z 511.2429; found: 511.2423.

[0219] Obtaining Examples of Initiators of the Present Invention

[0220] Nine examples of initiators, initiators I2, I3, I4, I5, I6, I7, I8, I9 and I10, are described in this application. Initiators I2 (Compound 34), I3 (Compound 39) and I4 (Compound 37) were prepared according to Scheme 4, Scheme 5 and Scheme 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 Scheme 7, Scheme 8, Scheme 9 and Scheme 10. The structure of the compounds obtained in each step in these schemes was confirmed by NMR. Additional details and data are described below.

[0221] In addition, a control initiator I1 was prepared in this example. I1, which lacks a glycan head group, is an initiator without targeting preferences and was prepared to form a copolymer for the propagators of the present invention to characterize the propagators. Initiator I1 has the following structure:

[0222] I1

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] Compound 15 8

[0232]

[0233] Compound 15 was synthesized and characterized according to the published methodology. 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, 3.91-Hz), 3.89 (m, 2H). (m, 1H), 3.79-3.70 (m, 2H), 3.69-3.67 (m, 1H).

[0234] Connection 16

[0235]

[0236] EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol) were added to a solution of 15 (0.24 mmol) in DMF (2 mL), and the resulting solution was stirred under nitrogen atmosphere at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:2) to give 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) calculated for C 15 H 22 NO7S [M+H] + : 360.1117, found: 360.1101.

[0237] Compound 17-20. Compounds 17-20 were synthesized according to the work of Peng, W.; Paulson, J.C., CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells. J.

[0238] Am. Chem. Soc. 2017, 139, 12450–12458, which is incorporated herein by reference in its entirety.

[0239] Compound 21-25. Compounds 21-25 were synthesized according to the work of 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.

[0240] Compound 26. To compound 25 (0.43 mmol) in DCM (5 mL) were added EDC (0.52 mmol), HOBt (0.52 mmol), trimethylamine (0.86 mmol), and 3-mercaptopropionic acid (0.47 mmol), and the resulting solution was stirred under nitrogen atmosphere at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:10) to give 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, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.93 (s, 3H), 1.57-1.51 (m, 4H), 1.48 (t, J = 7.0 Hz, 2H), 1.37-1.29 (m, 4H). HRMS (ESI) calculated for C 23 H 38 N2O 10S [M+H] + : 535.2325, found: 535.2314. Synthesis was performed according to the work of Maklakova, SY 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, which is incorporated herein by reference in its entirety.

[0241] Compound 27. NaOMe was added to compound 26 in MeOH, and the resulting solution was stirred under nitrogen at room temperature for 2 h. The reaction mixture was neutralized by adding IR-120, then filtered and evaporated to dryness in vacuo, yielding 27 (92 mg, quant.) 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): δ 173.70, 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) calculated for C 17 H 32 N2O 17 S [M+H] + : 409.2008, found: 409.2017.

[0242] Getting 9 Am Neu5Ac-α2,3-SCT 30, 9 AmNeu5Ac-α2,6-SCT 31 and Neu5Ac-α2,3-SCT 38. 30 mg of sialyl glycopeptide (SGP) was treated with Endo-S WT (300 μg) in Tris-HCl buffer at 37°C for 48 hours and then purified by gel permeation chromatography on Sephadex G-25, and the resulting product was analyzed by ESI-MS to yield SCT compound 28, according to 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, which is incorporated herein by reference in its entirety. Neuraminidase (5 U / ml, 12 μl) in Tris-HCl buffer was added to the mixture at 37°C for 12 h, and then purified by gel permeation chromatography on Sephadex G-25 to obtain desialylated N-glycan 29. The reaction was then carried out in 0.5 ml HEPES buffer (50 mM, pH 8.5) containing 100 mM 9 AmNeu5Ac (14.5 mg, 47 μmol), 110 mM CTP (27.2 mg, 52 μmol), and 20 mM MgCl2 were added. The pH of the reaction mixture was adjusted to 8.5 by adding 2 N NaOH. Then 0.5 mg / ml NmCSS was added to the above solution. The resulting mixture was incubated at 37°C for 8 hours, and CMP-9 formation was monitored. Am Neu5Ac by TLC analysis.

[0243] To get 9 Am Neu5Ac-α2,6-SCT was added with hST6Gal-I (0.5 mg / ml) to the reaction mixture 9 Am Neu5Ac with N-glycan and 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 in its entirety. To obtain 9 Am Neu5Ac-α2,3-SCT was added to the reaction mixture with PmST3 (0.3 mg / ml) 9 AmNeu5Ac with N-glycan and incubated at 37°C. To obtain 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 reaction mixtures were monitored by mass spectrometry and TLC. After complete disappearance of the acceptor, the reaction mixture was centrifuged, and the supernatant was transferred to a centrifugal filter with a molecular weight cutoff of 10 kDa (Amicon Ultra, Millipore) to separate the proteins. The filtrate was purified by gel permeation chromatography on P-2, yielding 9 Am Neu5Ac-α2,3-SCT 30, 9 Am Neu5Ac-α2,6-SCT 31 and Neu5Ac-α2,3-SCT 38.

[0244] Getting 9 BPC 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 equiv) were dissolved in H2O, then biphenylcarboxylic acid N-hydroxysuccinimide ester (BPC-NHS) (3 equiv) in THF was added. The reaction mixture was stirred at 0°C until the starting material completely disappeared. The reaction mixture was then purified on a Sep-Pak C18 column (2 g, Waters Corp.), eluting with water-MeOH, to afford compound 32 in 91% yield. 9 Am Neu5Ac-α2,3-SCT was prepared similarly to the above procedure by stirring with 4H-thieno[3,2-c]chromene-2-carbamoyl-NHS (TCC-NHS) in a mixture of THF and H2O to give compound 35. After purification, the desired product was obtained in 91% yield.

[0245] Into the mixture 9 BPC Neu5Ac-α2,6-SCT 32 or 9 TCCNeu5Ac-α2,3-SCT 35 in water was added with CDMBI and TEA and incubated at 4°C for 1 hour, generating the corresponding oxazoline N-glycan 33 and 36, respectively. The products were then purified by gel permeation chromatography on Sephadex G-25 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) with 9 BPC Neu5Ac-α2,6-SCT-oxazoline and incubated at 30 °C for 30 min. The transglycosylation products were isolated by gel permeation chromatography on P-2, yielding compound 34, and characterized by ESI-MS. 9 TCC Neu5Ac-α2,3-SCT-SH 37 and Neu5Ac-α2,3-SCT-SH 39 were prepared using the same method described above.

[0246] Connection 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 Hz, 2H), 1.91-1.77 (m, 18H), 1.44 (t, J = 6.9 Hz, 2H), 1.39-1.28 (m, 4H), 1.28-1.19 (m, 4H), 1.12 (t, J = 6.9 Hz, 1H). HRMS (ESI) calculated for C 119 H 171 N9O 63 S 2- [M-2H] 2- : 1383.0093, found: 1383.0077.

[0247] Connection 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) calculated for C 117 H 169 N9O 65 S3 2- [M-2H] 2- : 1416.9606, found: 1416.9623.

[0248] 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 (m, 18H), 1.44 (t, J = 6.9 Hz, 2H), 1.39-1.28 (m, 4H), 1.28-1.19 (m, 4H), 1.07 (t, J = 6.9 Hz, 1H). HRMS (ESI) calculated for C 93 H 153 N7O 63 S 2- [M-2H] 2- : 1203.9357, found: 1203.9377.

[0249] Connection 40

[0250]

[0251] EDC (0.12 mmol), HOBt (0.12 mmol), DMAP (0.12 mmol), trimethylamine (0.2 mmol) and CT(PEG) were added to compound 15 (0.12 mmol) in DMF (1 mL). 12(0.1 mmol), and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified by column chromatography on silica gel, yielding 40 (59%). 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), 3.81-3.70 (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): δ 170.17, 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) calculated for C 39 H 70 NO 19 S [M+H] + : 888.4263, found: 888.4241.

[0252] Compound 41-42. Compounds 41 and 42 were synthesized according to the work of 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.

[0253] Connection 43

[0254]

[0255] EDC (0.24 mmol), HOBt (0.24 mmol), trimethylamine (0.4 mmol), and 3-mercaptopropionic acid (0.2 mmol) were added to a solution of 42 (0.24 mmol) in DMF (2 mL), and the resulting solution was stirred under nitrogen atmosphere at room temperature for 2 h. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified by silica gel column chromatography (MeOH / DCM 1:3) to give 16 (81%). 1H 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) calculated for C 14 H 28 NO7S [M+H] + : 354.1586, found: 354.1602.

[0256] Connection 44

[0257]

[0258] EDC (0.12 mmol), HOBt (0.12 mmol), trimethylamine (0.2 mmol) and CT(PEG) were added to compound 42 (0.12 mmol) in DMF (1 ml). 12 (0.1 mmol), and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified by column chromatography on silica gel, yielding 44 (62%). 1H NMR (600 MHz, D2O): δ 4.77 (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): δ 171.01, 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) calculated for C 38 H 76 NO 19 S [M+H] + : 882.4732, found: 882.4750.

[0259] Connection 45

[0260]

[0261] NaOMe (0.2 equiv) was added to compound 15b (5 mmol) in MeOH, and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The reaction mixture was neutralized by adding IR-120, filtered, and evaporated to dryness in vacuo. The residue was then dissolved in anhydrous DCM (40 mL), imidazole (7.5 mmol) was added at 0o C, then TBDPSCl (5.5 mmol) was added. The reaction mixture was stirred at room temperature for 2.5 h under nitrogen. The reaction was quenched by adding MeOH. After stirring at room temperature for 10 min, the solvent was removed under reduced pressure, yielding a dry residue, which was purified by column chromatography with MeOH / DCM (1 / 10) to yield 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. HRMS (ESI) calculated for C 36 H 42 NO8Si [M+H] +: 644.2680, found: 644.2699.

[0262] Connection 46.

[0263]

[0264] Trimethyl orthobenzoate (9 mmol) was added to a solution of compound 45 (3 mmol) and a catalytic amount of CSA (0.3 mmol) in CH3CN (60 mL) at room temperature under a nitrogen atmosphere. After stirring for 30 min, Et3N was added to quench the reaction, and the resulting mixture was dried under reduced pressure. The residue was purified by column chromatography, eluting with ethyl acetate / hexane (1 / 2), to afford 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). 13C 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) calculated for C 44 H 48 NO9Si [M+H] + :762.3098, found: 762.3072.

[0265] Connection 47

[0266]

[0267] Compound 46 (2 mmol) was dissolved in DCM (20 mL) and sequentially added with stirring DIPEA (6 mmol), benzoic anhydride (4 mmol) and DMAP (0.2 mmol). After stirring for 30 min, the solvent was evaporated under reduced pressure to obtain a dry residue, then the residue was poured into ethyl acetate (20 mL) and 2N HCl solution (20 mL) and stirred vigorously for 30 min. The solvent was evaporated, the residue was extracted with ethyl acetate. The combined organic layer was washed with ice-cold saturated NaHCO3(aq) solution, water and saturated sodium chloride solution, then dried over MgSO4. The filtrate was evaporated under reduced pressure and redissolved in THF (20 mL). AcOH (4 mmol) and 1M TBAF (2.4 mmol in THF) were added at 0 oC. The resulting mixture was gradually warmed to room temperature, stirred for another 2 hours, and then diluted with ethyl acetate. The organic layer was washed with saturated NaHCO3 (aq.) solution, water, and saturated sodium chloride solution, dried over anhydrous MgSO4, and evaporated under reduced pressure. The dried residue was purified by column chromatography, eluting with ethyl acetate / hexane (1 / 2), to afford 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). 13 C 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) calculated for C 34 H 32 NO 10 [M+H] +:614.2026, found: 614.2038.

[0268] Connection 48

[0269]

[0270] A stirred solution of 47 (0.2 mmol) and 4A molecular sieves (0.2 g) in anhydrous DCM (2 mL) was cooled to -40°C and then added dropwise to the resulting solution of BF3(OEt)2 (0.02 mmol). A solution of 15a in anhydrous DCM was added dropwise and stirred for 1 h at -40°C. The reaction mixture was then gradually warmed to room temperature and stirred for another 1 h. The reaction was quenched by the addition of triethylamine, then filtered, saturated aqueous NaHCO3 solution was added, and the mixture was extracted with dichloromethane. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to afford the trisaccharide product. The resulting product was then dissolved in MeOH, NaOMe (0.2 equiv) was added, and the resulting solution was stirred at room temperature for 2 hours. The reaction mixture was neutralized by adding IR-120, filtered, and evaporated to dryness in vacuo.The deacetylated mixture was purified on Bio-Gel P-2 Gel (Biorad) with water as eluent, yielding pure trisaccharide. The resulting compound was lyophilized and then dissolved in MeOH (2 mL), 10% Pd-C (30 mg) was added, and the mixture was vigorously stirred under H2 overnight. The solution was filtered through Celite and evaporated to dryness, yielding 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) calculated for C 24 H 37 NO 16 Na[M+Na] + :618.2010, found: 618.2029.

[0271] Connection 49

[0272]

[0273] EDC (0.12 mmol), HOBt (0.12 mmol), DMAP (0.12 mmol), trimethylamine (0.2 mmol) and CT(PEG) were added to compound 48 (0.12 mmol) in DMF (1 mL). 12 (0.1 mmol), and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified on Bio-Gel P-2 Gel with water as eluent, yielding 49 (54%). 1 H 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) calculated for C 51 H 89 NO29 SNa[M+Na] + :1234.5139, found: 1234.5114.

[0274] Connection 51

[0275]

[0276] Trichloroacetonitrile and DBU were added to a stirred solution of 50 (1 mmol) in anhydrous DCM (10 mL), and the solution was stirred for 2 h at room temperature. The solvent was removed, and the residue was purified by flash column chromatography on silica gel to give the imidate product. A stirred solution of benzyl (4-hydroxyphenyl)carbamate (1.2 mmol) and 4A molecular sieves (1 g) in anhydrous DCM (10 mL) was cooled to -40 °C, then BF3(OEt)2 (0.1 mmol) was added dropwise. A solution of imidate donor (1 mmol) in anhydrous DCM was added dropwise to the above reaction mixture and stirred for 1 h at -40 °C. After this, the reaction mixture was gradually warmed to room temperature and stirred for another 1 h. The reaction was quenched by the addition of triethylamine, then filtered, saturated aqueous NaHCO3 was added, and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography on silica gel, yielding compound 51 (72%). 1H 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). 13 C 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) calculated for C 43 H 43 NO 10 [M+H] + :718.3016, found: 718.3041.

[0277] Connection 52

[0278]

[0279] NaOMe (0.1 equiv) was added to a stirred solution of 51 (0.6 mmol) in MeOH, and the resulting solution was stirred at room temperature for 1 h. The reaction mixture was neutralized by adding IR-120, filtered, and evaporated to dryness in vacuo. The deacetylated product was then dissolved in anhydrous DCM (5 mL) supplemented with 4A molecular sieves (0.5 g). The solution was cooled to -40°C, and BF3(OEt)2 (0.05 mmol) was added dropwise. A solution of imidate donor (0.5 mmol) in anhydrous DCM was added dropwise to the above reaction mixture and stirred for 1 h at -40°C. After this, the reaction mixture was gradually warmed to room temperature and stirred for another 1 h. The reaction was quenched by adding triethylamine, then filtered, saturated aqueous NaHCO3 was added, and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The resulting product was then dissolved in MeOH, NaOMe (0.1 equiv) was added, and the resulting solution was stirred at room temperature for 2 hours.The reaction mixture was neutralized by the addition of IR-120, filtered, and evaporated to dryness under vacuum. The residue was purified by column flash chromatography on silica gel to afford 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), 5.17 (s, 1H), , (d, 25H), (d, 25H). 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, H, 4.47), 4.47. 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) calculated for C 68 H 70 NO 13 [M+H] + :1108.4847, found: 1108.4819

[0280] Connection 53

[0281]

[0282] 4 A molecular sieves (0.25 g) were added to a stirred solution of 52 (0.3 mmol) in anhydrous DCM (2.5 mL). The solution was cooled to -40°C, and 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 reaction mixture and stirred for 1 h at -40°C. After this, the reaction mixture was gradually warmed to room temperature and stirred for another 1 h. The reaction was quenched by the addition of triethylamine, then filtered, saturated aqueous NaHCO3 was added, and the mixture was extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography on silica gel to yield the trisaccharide product. The resulting product was then dissolved in MeOH, NaOMe (0.1 equiv) was added, and the resulting solution was stirred at room temperature for 2 hours. The reaction mixture was neutralized with IR-120, filtered, and evaporated to dryness in vacuo.The resulting compound was then dissolved in MeOH (2 mL), 10% Pd-C (30 mg) was added, and the mixture was vigorously stirred under H2 overnight. The solution was filtered through Celite and evaporated to dryness, yielding 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) calculated for C 24 H 38 NO 16 [M+H] + :596.2191, found: 596.2044.

[0283] Connection 54

[0284]

[0285] EDC (0.02 mmol), HOBt (0.02 mmol), DMAP (0.02 mmol), trimethylamine (0.04 mmol) and CT(PEG) were added to compound 53 (0.02 mmol) in DMF (0.2 mL). 12(0.02 mmol), and the resulting solution was stirred under nitrogen at room temperature for 12 hours. The reaction mixture was evaporated to dryness in vacuo, and the resulting crude product was purified on Bio-Gel P-2 Gel with water as eluent, yielding 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) calculated for C 51 H 90 NO 29 S[M+H] + :1212.5319, found: 1212.5146.

[0286] Obtaining polymers

[0287] In this example, polymersomes of the present invention were prepared that encapsulate mRNA to form mRNA-polymer nanoparticles (PNPs) for delivery. The term “PNPs” is used interchangeably with the term “polymersome” to describe the polymersomes of the present invention. Examples of PNPs included 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 Fig. 1B.

[0288] WT spike DNA design. pmRNA mRNA synthesis vector XP were obtained from System Biosciences. The WT (Wuhan / WH01 / 2019 line) spike DNA sequence with K986P and K987P mutations (2P) was codon-optimized for Homo sapiens. 1 pmRNA XPThe vector was digested with EcoRI and BamHI at 37°C for 1 hour. The spike protein DNA sequence was amplified using KOD One™ PCR master mix (TOYOBO Bio-Technology). The linearized pmRNAXP vector and the spike protein DNA PCR fragment were purified using Wizard SV Gel and the PCR Clean-Up System (Promega). The spike protein DNA PCR fragment was cloned into the linearized pmRNAXP vector using the In-Fusion HD Cloning Kit (Clontech Laboratories, Inc.). The cloning mixture was transformed into One Shot™ TOP10 chemically competent E. coli cells (Invitrogen™) and incubated at 37°C overnight. Quick Taq HS DyeMix (TOYOBO Bio-Technology) was used to screen for a successful construct. An insert-specific primer and a base-specific primer were selected for colony PCR. A single clone was selected using a PCR pipette tip. PCR products were analyzed by agarose gel electrophoresis. Potential candidates were selected and analyzed by DNA sequencing.

[0289] Polymer Synthesis. The polymerization process was carried out according to published procedures as described in Jiaqi Fu et al., Journal of the American Chemical Society 2015 137 (37), 12153–12160, which is incorporated herein by reference in its entirety. Modifications were made where necessary. Briefly, stock solutions of monomers (i.e., 2 M propagator in DMF), initiators (50 mM in DMF, freshly prepared), terminator (iodoacetamide, 0.5 M in H2O, freshly prepared), and triethanolamine (TEOA) were prepared in buffer (1 M, pH 7.0). The initiator was added to 80 mL 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 Fig. 1A. After 30 min of vigorous stirring at room temperature, the polymerization reaction was quenched by adding 1.9 mL of terminator stock solution. The resulting polymer was dialyzed against H2O on the same day.The solution was lyophilized and the polymers were stored at -20. o C. For in vitro and in vivo experiments, the initiator was mixed with 5% IP and then the protocol described above was followed.

[0290] To prepare mRNA-PNP, mRNA was encapsulated in the corresponding polymer by self-assembly process; that is, the polymer in ethanol (10 mg / mL) was mixed with mRNA aqueous solution (1 mg / mL) at pH 4.0 in the ratio of N / P = 3 / 1. See Fig. 1B. mRNA-PNP was dialyzed against phosphate-buffered saline (pH 7.4) using Micro Float-A-Lyzer (10 kDa MWCO, spectrum lab) overnight at 4 o C and stored at -40°C until further use.

[0291] Characterization of polymersomes. To determine the morphology of the mRNA-polymer complex, P1 polymer was first mixed with mRNA, and a nearly spherical nanoparticle was observed to form. The P1 / P5 copolymer complex with mRNA was liposomal. Polymers tend to form a positively charged layer, which can encapsulate mRNA and facilitate subsequent cellular uptake due to the participation of a zwitterion.

[0292] Molecular weight and degree of polymerization were characterized by gel permeation chromatography (GPC). The polymers showed a unimodal but fairly broad molecular weight distribution according to the GPC chromatogram and eluted with a relatively short elution time, indicating a polymeric state. The peak molecular weight was 10.2 kDa (polydispersity coefficient KP = 1.33) for mRNA-PNP (P1 / P5).

[0293] Example 2: Encapsulation and transfection efficiency of polymersomes

[0294] To find the optimal polymer for efficient intracellular delivery of GFP-mRNA as a selected model, homopolymers and heteropolymers were synthesized by copolymerizing different propagators, and their encapsulation ability and transfection efficiency in HEK293T cells were evaluated.

[0295] Quantification of encapsulated mRNA

[0296] The encapsulation efficiency was determined using the Quant-iT™ RiboGreen™ RNA Reagent and Kit (Thermo Scientific™). The resulting mRNA polymersome (mRNA-PNP) was treated with 10 mM GSH overnight. The solution was then diluted 250-fold with 1X TE buffer, and then 2-fold dilutions were performed with TE buffer or TE buffer containing 2% Triton X-100. mRNAs were prepared at concentrations of 100, 50, 25, 12.5, and 0 ng / mL in TE or TE buffer containing 1% Triton X-100 to construct a standard curve. After incubation at 37ºC for 10 minutes, Quant-iT™ RiboGreen™ RNA reagent was added to the well. Fluorescence intensity was measured using CLARIOstar® Plus (BMG Labtech).

[0297] Result. All copolymers containing a guanidine group were able to encapsulate GFP-mRNA. In particular, I1-P2 / P3, I1-P2 / P4, and I1-P2 / P5 copolymers with trivalent guanidine moieties demonstrated stronger mRNA encapsulation ability and comparable results to the traditional transfection agent polyethyleneimine (PEI). See Fig. 2A.

[0298] Formulation of WT spike mRNA to generate mRNA-PNP. To obtain WT spike mRNA, linear DNA containing the T7 promoter, 50 untranslated regions, 30 untranslated regions, S-2P, and poly(A) signal terminal sequence was amplified using TOOLS Ultra High Fidelity DNA Polymerase (BIOTOOLS Co., Ltd.) with one μl of template DNA in the mMESSAGE mMACHINE™ Kit (Thermo Scientific) at 37°C for 1 hour according to the manufacturer's protocol. mRNA was purified using an RNA purification kit (BioLabs) according to the manufacturer's protocol and stored at -80°C until further use. To prepare mRNA-PNP, mRNA was encapsulated in an appropriate polymer by self-assembly; That is, the polymer in the ethanol phase (10 mg / ml) was mixed with an aqueous solution of mRNA (1 mg / ml) at pH 4.0 and different N / P ratios. mRNA-PNP was dialyzed against phosphate-buffered saline (pH 7.4) using a Micro Float-A-Lyzer (10 kDa MWCO, spectrum lab) overnight at 4o C and stored at -40°C until further use.

[0299] Result. The result showed that the P1 / P5 copolymer exhibited excellent mRNA spike trapping activity at the N / P ratio of 1, 5, 10, or 20 (Fig. 3A), and the average particle size of the corresponding mRNA-P1 / P5 copolymer complex was approximately 127 nm as analyzed by CryoEM, TEM, and dynamic light scattering (DLS) (Fig. 3B). The measured zeta potential was approximately -4.2 mV. An N / P ratio of 3 was chosen for further experiments to ensure complete encapsulation. The release and translation of mRNA could be attributed to GSH-mediated polymer degradation, since it occurred in a dose-dependent manner in the presence of 10 mM GSH (data not shown).

[0300] Transfection into HEK293T cells

[0301] Transfection of polymersomes encapsulating GFP-mRNA. Next, the efficiency of GFP-mRNA transfection into HEK293T cells was assessed using different copolymers. HEK293T cells were plated at a density of 5x10 5Cells per well in a 6-well plate were transfected in 2.5 ml of DMEM medium. A preparation of 1 μg of GFP mRNA or 3 μg of WT spike mRNA with the appropriate polymer was prepared using the method described above and then added to the cells. Eighteen hours after transfection, GFP expression and spike protein were monitored by fluorescence microscopy and Western blotting, respectively. For Western blotting, cells containing the spike protein were lysed by adding 200 μl of RIPA lysis buffer containing protease inhibitor and incubated for 10 minutes. The cells were then vortexed, centrifuged, and analyzed by Western blotting with polyclonal antibodies to SARS-CoV-2 S protein (1:5000 with 1% BSA), followed by HRP-conjugated rabbit antibodies (1:10,000). The spike protein was detected using a chemiluminescent HRP substrate and visualized with a transilluminator (FUJIFILM LAS3000).

[0302] Result. The obtained result showed that the copolymer P1 had the transfection ability of HEK293T cells comparable to the traditional transfection agent polyethyleneimine (PEI), while the heteropolymer P1 / P4, P2 / P4, P1 / P5, and P2 / P5 showed higher transfection efficiency and were able to release mRNA for translation into GFP. In particular, the polymer containing the zwitterionic group, P1 / P5, and P2 / P5 (Fig. 2B), showed high efficiency, probably due to the membrane fusion described above. All the tested polymersomes had less impact on cell viability than the traditional transfection agent PEI, and none of them showed significant cytotoxicity (data not shown).

[0303] Transfection of polymersomes encapsulating WT spike mRNA. Then, the spike mRNA-P1 / P5 complex (3 μg) was transfected into HEK293T cells and Western blotting was performed. 48 hours after transfection, the cells were analyzed for spike protein translation by Western blotting with spike-specific antibodies. A prominent band at ~250 kDa corresponding to the SARS-CoV-2 spike protein (second line) was observed compared with spike mRNA as a negative control (first line) (Fig. 3C). This study confirmed that the P1 / P5 copolymer is an effective nanocarrier for mRNA transfection in vitro.

[0304] Localization of mRNA-PNP in cells

[0305] To visualize the localization of mRNA-PNP in cells, a FITC-labeled polymer of P1 / P4 / P5 was synthesized in which FITC was conjugated to the polymer via the amino group on P4. As shown in Fig. 4, the colocalization of lysosome with mRNA-PNP (I1-P1 / P4 / P5) was lower than that with mRNA-PNP (I1-P1 / P4), indicating that the alkylated zwitterion residue can significantly enhance membrane fusion and lysosomal release.

[0306] Example 3: Address (selective) delivery

[0307] To selectively deliver vaccine mRNA to antigen-presenting cells (APCs), particularly dendritic cells, the authors prepared initiators with different glycan heads recognized by lectin receptors, such as Siglec-1, Siglec-2, Siglec-5 / E, and DC-SIGN, which are expressed primarily on dendritic cells and macrophages. To assess the uptake of mRNA-PNP through Siglec receptors, the authors compared the binding and internalization of mRNA-PNP in T cells, B cells, and bone marrow-derived dendritic cells (BMDCs).

[0308] Methods

[0309] Preparation of spleen cells and BMDC culture. To prepare spleen cells, mouse spleens were homogenized and RBC lysis buffer (Sigma) was added to dissolve red blood cells (RBCs), followed by sieving through a cell strainer (BD Biosciences). Bone marrow-derived dendritic cells (BMDCs) were prepared according to the literature. 3Briefly, bone marrow was isolated from mouse femurs and tibias and supplemented with RBC lysis buffer (Sigma) to degrade red blood cells (RBCs). Cells were then grown in RPMI-1640 medium 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) at a density of 2 × 10 5 cells / ml. The cells were supplemented with an equal volume of the complete culture medium described above on Day 3 and then refreshed with half the volume of medium on Day 6. On Day 8, the suspended cells were harvested.

[0310] Treatment of spleen cells and BMDCs with PNP. Spleen cells or BMDCs were incubated with 1:2000 mRNA-PNP (diluted to 10 mg / mL) in RPMI-1640 medium at 37°C for 24 hours. Cells were blocked with an Fc receptor-binding inhibitor (clone: ​​93, eBioscience) for 20 minutes. Splenocytes were stained with antibodies to CD3 (clone: ​​17A2, BV421-conjugated, Biolegend), CD19 (clone: ​​1D3, PECy7-conjugated, BD Biosciences). BMDCs were stained with antibodies to CD11c (clone N418, APC-conjugated, Biolegend). Labeled cells were analyzed using FACSC and a flow cytometer (BD Biosciences).

[0311] C2C12 cell culture. The mouse muscle myoblast cell line C2C12 was purchased from Taiwan's Bioresource Collection and Research Center. C2C12 cells were grown in high-glucose DMEM (ATCC) supplemented with 10% FBS and 1× antibiotic-antifungal agent. Cells were incubated at 37°C with 5% CO2 and a humidified atmosphere. The culture medium was changed every 2-3 days.

[0312] Treatment of C2C12 with polymersomes. The grown C2C12 myoblasts were detached from the culture dish 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) was added to 200 μl of C2C12 cells (2 × 10 5 cells) in the growth medium to a final dilution of 1:1000, 1:2000, 1:4000, or 1:8000 with the original stock solutions (10 mg / ml). Measurements were taken at three time points: 5 min, 1 h, and 24 h.

[0313] Flow cytometry. After incubation with mRNA-PNP, BMDCs were washed with ice-cold FACS buffer (1% FBS in 1×DPBS with 0.1% sodium azide) and incubated with purified mouse CD16 / 32 antibodies (BioLegend) in FACS buffer on ice for 20 minutes, then washed with FACS buffer. BMDCs were stained with APC mouse CD11c antibodies (BioLengend) 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. The cells were stained with propidium iodide. Flow cytometry was performed on a FACSCanto flow cytometer (BD Bioscience).

[0314] Assay of Glycan-PNP Binding to DC-SIGN by ELISA. To evaluate the binding of DC-SIGN to mannoside-modified PNP, ELISA plates were coated 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 at 4°C overnight, respectively. Plates were incubated with diluted DC-SIGN ECD (15 to 0.075 nM in HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, 0.1% BSA) at pH 7.4, 6.0, and 5.0 for 1 hour at room temperature. Bound DC-SIGN ECD was detected using HRP-conjugated anti-DC-SIGN (B2) IgG (Santa Cruz Biotechnology). After 1 hour of incubation at room temperature, plates were treated with tetramethylbenzidine (TMB) for 10 minutes. Optical density was measured at 450 nm after adding 0.5 M sulfuric acid to the plates using a microplate reader.The Kd value was calculated by fitting a nonlinear regression curve to the total binding using GraphPad Prism software.

[0315] Assay of glycan-PNP binding to DC-SIGN-Fc, MMR-Fc, MINCLE-Fc, Dectin-2-Fc, and Langerin-Fc by ELISA. To evaluate the binding of receptor proteins to mannoside-modified PNP, ELISA plates were coated 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) in PBS at 4°C overnight, respectively. 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 for 1 hour at room temperature. Bound proteins were detected using HRP-conjugated anti-Fc IgG antibodies. After 1 hour of incubation at room temperature, plates were treated with tetramethylbenzidine (TMB) for 10 minutes. The average optical density was measured at 450 nm after adding 0.5 M sulfuric acid to the plates using a microplate reader.

[0316] Results

[0317] As determined by flow cytometry, FITC-conjugated mRNA-PNP was incubated with each cell line for 1 hour. I2 mRNA-PNP with 9BPCNeu5Ac-conjugated N-glycan, designed to target Siglec-2, showed higher cellular uptake for all APCs compared to I1 mRNA-PNP without glycan modification (Fig. 5). Specifically, in this experiment, compared with I1 polymersomes, I2 polymersomes showed a 33.1% increase, a 32.6% increase, and a 27.8% increase in uptake by BMDC, B cells, and T cells, respectively. Similar results were obtained using I3 and I4 mRNA-PNP to target Siglec-5 / E and Siglec-1, respectively, where all glycan-containing polymersomes showed improved uptake by all APCs than glycan-free mRNA-PNP. Similarly, I9-P1 / P4-FITC / P5 mRNA-PNP showed higher uptake (approximately 1-fold higher, Fig. 9) by C2C12 muscle cells compared to I1-P1 / P4-FITC-P5 mRNA-PNP.

[0318] As shown in Fig. 6, the aryl mannose headgroup-tagged polymersome I5 (I5-P1 / P4-FITC / P5) exhibited ~34% higher (33.8% increase) cellular uptake by BMDCs compared with the glycan headgroup-tagged polymersome I1 (I1-P1 / P4-FITC / P5). On the other hand, B cells and T cells with insignificant DC-SIGN expression showed only a slight increase (16.2% increase and 12.8% increase, respectively) in fluorescent signal when treated with the polymersomes. These data indicate that efficient internalization and selective uptake of mRNA-PNP (I5-P1 / P4-FITC / P5) by dendritic cells can be achieved by targeting the DC-SIGN receptor.

[0319] A study of the binding of DC-SIGN to mRNA polymersomes generated from I5-P1 / P5, I6-P1 / P5, I7-P1 / P5, I8-P1 / P5, I9-P1 / P5 and I10-P1 / P5 at different pH values ​​showed that these polymersomes are able to bind to DC-SIGN with low K D(Table 1). mRNA polymersomes with I5-P1 / P5, I8-P1 / P5, I9-P1 / P5, and I10-P1 / P5 binding affinities bind to the DC-SIGN extracellular domain (ECD) at pH 7.4 and 5.0 with virtually identical affinity. In comparison, binding of mRNA polymersomes from I6-P1 / P5 and I7-P1 / P5 at lower pH was not detected, indicating that coordination with calcium ions is weakened at low pH.

[0320] Table 1

[0321] Glycan Valence Aril Linker Spacer Kd,app (nM) (pH 7.4) Kd,app (nM) (pH 5) I5 1 + - 448.1 ± 11.3 570.1 ± 21.2 I6 1 - - 6416 ± 282 But. I7 1 - + 2395 ± 49 But. I8 1 + + 278.8 ± 17.3 300.1 ± 25.9 I9 3 + a3, a6 + 3.6 ± 1.1 3.8 ± 1.4 I10 3 + a2, a2 + ± 4.6 24.2 ± 4.8

[0322] Without any theoretical justification, the obtained binding results indicate that aryl trimannoside interacts with DC-SIGN in acidic endosomal compartments. This binding stability enhances the DC-SIGN signal and its synergism with the signal of endosomal Toll-like receptors such as TLR7. The strong binding of PNP to aryl mannoside and DC-SIGN may be due to the density distribution in the ligand, and the aryl group may be involved in CH-π and hydrophobic interactions. In addition, for receptor-targeted delivery, ligands are often linked to a carrier, and the distance between the carrier and the ligand is regulated by the presence of a spacer. mRNA-PNP with a longer ligand (I8-P1 / P5 with Man-Ar-PEG12 and I7-P1 / P5 with Man-PEG12) showed slightly higher affinity for DC-SIGN. In conclusion, mRNA-PNP with aryl-trimannoside (I9-P1 / P5) showed the highest affinity and the lowest KD value for DC-SIGN.The increased affinity may be due to the clustering effect and spatial arrangement of the ligand in the polymer, and the aryl moiety may facilitate this hydrophobic interaction.

[0323] In addition to DC-SIGN, the binding affinity to the macrophage mannose receptor (MMR), MINCLE, Dectin-2, and langerin was examined to determine whether the uptake of mannosylated PNP mRNA was dependent on DC-SIGN. As shown in Fig. 7, compared with I1, polymersomes I8, I9, and I10 all showed selective binding. Of these, the branched aryl-trimannoside type I9 showed greater preference for DC-SIGN than the linear aryl-trimannoside type I10. The fact that I9 exhibited more selective binding to DC-SIGN confirms that the efficient uptake of I9-P1 / P5 mRNA by dendritic cells most likely occurs through DC-SIGN.

[0324] Example 4: Immunization using the polymersomes of the present invention

[0325] Next, the effect of WT spike mRNA-PNP with or without aryl mannoside head on vaccination and immune response was investigated.

[0326] Immunization Methodology

[0327] Animals. Balb / c mice (8 weeks old) were purchased from the National Laboratory Animal Center (Taiwan). All mice were maintained under pathogen-free conditions. Eight-week-old Balb / c mice were immunized intramuscularly twice, at 2-week intervals. Each vaccine contained PBS (100 μl). Plasma from vaccinated mice was subjected to ELISA analysis 10 days after the last immunization. The experimental procedure was approved by Academia Sinica's Institutional Animal Care and Utilization Committee (approval no. 22-08-1901).

[0328] Animal immunization. 6- to 8-week-old BALB / c mice (n = 5) were intramuscularly immunized with 15 μg of mRNA-PNP in phosphate-buffered saline (PBS). Animals were immunized at week 0 and additionally vaccinated for the second time at week 2 (Fig. 8A), and blood samples were collected from each mouse 1 week after the second immunization. A group injected with mRNA-LNP spike (including ALC-0315, DSPC, ALC-0159, and cholesterol, which is widely used in existing mRNA vaccine formulations) was included as a positive control.

[0329] Measurement of IgG titer in blood plasma. An ELISA method was used to determine the IgG titer in mouse plasma. Wells in a 96-well ELISA plate (Greiner Bio-One) were coated with 100 ng of SARS-CoV-2 spike protein (ACROBiosystems) in 100 mM sodium bicarbonate solution, pH 8.8, at 4°C overnight. The wells were blocked with 200 μl of 5% skim milk in 1X PBS at 37°C for 1 hour and washed with 200 μl of PBST (1X PBS, 0.05% Tween 20, pH 7.4) three times. Mouse plasma samples with 2-fold serial dilutions were added to wells for incubation at 37°C for 2 hours and washed six times with 200 µl PBST. Wells were incubated with 100 µl HRP-conjugated secondary mouse antibody (1:10,000, in PBS) at 37°C for 1 hour and washed six times with 200 µl PBST. 100 µl horseradish peroxidase substrate (1-Step™ Ultra TMB-ELISA Substrate Solution) (Thermo Scientific™) was added to the wells, followed by the addition of 100 µl 1M H2SO4.After 30 min of incubation, absorbance (OD 450 nm) was measured using a SpectraMax M5.

[0330] Pseudovirus neutralization assay. Pseudovirus was constructed at the RNAi Core Facility of the Chinese Academy of Sciences using a method similar to that described previously. Briefly, pseudotyped lentivirus carrying 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 before transfection, and the plasmids were delivered using TransITR-LT1 transfection reagent (Mirus). The culture medium was refreshed after 16 hours, and the cells were harvested at 48 and 72 hours posttransfection. Cell debris was removed by centrifugation at 4000 g for 10 minutes, and the supernatant was passed through a 0.45-μm syringe filter (Pall Corporation). Aliquots of pseudotyped lentivirus were collected and stored at -80°C.To determine lentivirus titers using the AlarmaBlue assay (Thermo Scientific), the transduction unit (TU) of SARS-CoV-2 pseudotyped lentivirus was determined using a cell viability assay in response to a limited dilution of lentivirus. Briefly, HEK-293T cells stably expressing the human ACE2 gene were seeded in a 96-well plate one day before lentivirus transduction. To determine the pseudotyped lentivirus titer, different amounts of lentivirus were added to culture medium containing polybrene (final concentration 8 μg / mL). The resulting cells were centrifuged at 1100 g in a 96-well plate for 30 minutes at 37°C. After incubating the cells at 37°C for 16 h, the culture medium containing virus and polybrene was removed and replaced with fresh complete DMEM medium containing 2.5 μg / ml puromycin.After 48 hours of puromycin treatment, the culture medium was removed, and cell viability was determined using 10% AlamarBlue reagents according to the manufacturer's instructions. The percentage of uninfected cell survival (without puromycin treatment) was considered 100%. Viral titer (transduction units) was determined by plotting cell survival versus the dose of diluted virus. For the neutralization assay, heat-inactivated plasma or antibodies were serially diluted and incubated with 1,000 TU of SARS-CoV-2 pseudotyped lentivirus in DMEM for 1 hour at 37°C. The reaction mixture was then inoculated with 10,000 HEK-293T cells stably expressing the human ACE2 gene in a 96-well plate. The culture medium was replaced with fresh complete DMEM medium (supplemented with 10% FBS and 100 U / ml penicillin / streptomycin) 16 h after addition and continuously grown for another 48 h.Luciferase gene expression levels were determined using the Bright-Glo Luciferase Assay System (Promega). Relative light units (RLU) were detected using the Tecan i-control (Infinite 500). Percent inhibition was calculated as the ratio of the RLU reduction in the presence of diluted plasma to the RLU value of the control sample without plasma using the formula (RLU. контроль - RLU Плазма ) / RLU контроль .

[0331] Results

[0332] The results showed that both I1-P1 / P5 polymersome treatment and mRNA-LNP treatment generated anti-spike protein antibody levels of approximately ten thousand in plasma on Day 28. In contrast, I9-P1 / P5 polymersome treatment was able to generate anti-spike protein antibody levels of approximately thirty thousand, which was at least threefold higher (Fig. 8B). The plasma was then tested for its ability to neutralize pseudovirus-mediated entry into ACE2-expressing cells. The antiserum level from I1-P1 / P5 polymersomes was similar to that of the mRNA-LNP group; both were below 2000. In comparison, a significantly higher level (above 6000) of neutralizing antibodies was observed in the I9-P1 / P5 polymersome group (Fig. 8C), and the spike-specific antibody titer correlated well with the pseudovirus neutralizing activity.

[0333] Illustrative Embodiments

[0334] Embodiment 1. A copolymer for forming a polymersome, wherein the copolymer comprises: an initiator block comprising a glycan head; a propagator block comprising a functional moiety that comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and a linker covalently linking the initiator block and the propagator block, wherein the linker comprises a disulfide bond.

[0335] Embodiment 2. The copolymer of embodiment 1, wherein the glycan head comprises a terminal mannoside.

[0336] Embodiment 3. The copolymer of embodiment 1 or embodiment 2, wherein the glycan head comprises an O-aryl mannoside containing an optionally substituted benzene ring.

[0337] Embodiment 4. The copolymer of any one of embodiments 1 to 3, wherein the glycan head comprises mono-mannoside, di-mannoside, or tri-mannoside.

[0338] Embodiment 5. The copolymer of embodiment 4, wherein the tri-mannoside is a linear or branched tri-mannoside.

[0339] Embodiment 6. The copolymer of embodiment 5, wherein the branched tri-mannoside is α-1,3-α-1,6-trimannoside.

[0340] Embodiment 7. The copolymer of any one of embodiments 1 to 6, wherein the initiator block further comprises an initiator spacer.

[0341] Embodiment 8. The copolymer of embodiment 7, wherein the initiator spacer comprises a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0342] Embodiment 9. The copolymer of 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, 2-6 carbon atoms).

[0343] Embodiment 10. The copolymer of embodiment 8 or embodiment 9, wherein the PEG moiety comprises 2-72 (OCH2CH2) subunits.

[0344] Embodiment 11. The copolymer of embodiment 10, wherein the PEG moiety has a linear, branched, or star-shaped configuration.

[0345] Embodiment 12. The copolymer of any one of embodiments 1 to 11, wherein the glycan head is designed to bind to a dendritic cell.

[0346] Embodiment 13. The copolymer of embodiment 12, wherein the glycan head is designed to selectively bind to DC-SIGN.

[0347] Embodiment 14. The copolymer of embodiment 13, wherein the glycan head is designed to bind to DC-SIGN at K D in the range from 5 to 8000 nM at pH 7.4.

[0348] Embodiment 15. The copolymer of Embodiment 14, where K Dis in the range from 5 to 500 nM at pH 7.4.

[0349] Embodiment 16. The copolymer of any one of embodiments 13-15, wherein the glycan head is designed to bind to DC-SIGN at K D in the range from 1 to 2000 nM at pH 5.

[0350] Embodiment 17. The copolymer of Embodiment 16, where K D ranges from 1 to 600 nM at pH 5.

[0351] Embodiment 18. The copolymer of embodiment 1, wherein the glycan head comprises 9 BPC Neu5Ac conjugated N-glycan, Neu5Ac conjugated N-glycan, 9 TCC Neu5Ac conjugated N-glycan or a combination of both.

[0352] Embodiment 19. The copolymer of embodiment 18, wherein the initiator block is configured to bind to Siglec-2, Siglec-5 / E, Siglec-1, or a combination thereof.

[0353] Embodiment 20. The copolymer of any one of embodiments 1 to 19, wherein the initiator block is selected from the group consisting of:

[0354] ;

[0355] where the filled circle represents mannoside, the open circle represents galactose, the filled square represents GlcNAc, and the diamond represents Neu5Ac.

[0356] Embodiment 21. The copolymer of any one of embodiments 1 to 20, wherein the propagator block comprises more than one guanidine group.

[0357] Embodiment 22. The copolymer of embodiment 21, wherein the propagator block comprises three guanidine groups.

[0358] Embodiment 23. The copolymer of any one of embodiments 1-22, wherein the propagator block comprises a propagator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0359] Embodiment 24. The copolymer of embodiment 23, 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, 2-6 carbon atoms).

[0360] Embodiment 25. The copolymer of embodiment 23 or embodiment 24, wherein the PEG moiety comprises 2-72 (OCH2CH2) subunits.

[0361] Embodiment 26. The copolymer of embodiment 25, wherein the PEG moiety has a linear, branched, or star-shaped configuration.

[0362] Embodiment 27. The copolymer of any one of embodiments 1-26, comprising multiple propagator blocks and multiple linkers, and each propagator block of the multiple propagator blocks is connected to at least another propagator block of the multiple propagator blocks or to an initiator block via one of the multiple linkers.

[0363] Embodiment 28. The copolymer of any one of embodiments 1-27, wherein the propagator block is a first propagator block, the linker is a first linker, and the copolymer further comprises a second propagator block connected to the first propagator block via a second linker; wherein the first propagator block and the second propagator block independently comprise a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the second linker comprises a disulfide bond.

[0364] Embodiment 29. The copolymer of embodiment 28, wherein the first propagator block comprises a guanidine group and the second propagator block comprises a zwitterionic group.

[0365] Embodiment 30. The copolymer of embodiment 28, wherein the first propagator block comprises a guanidine group and the second propagator block comprises diethylenetriamine.

[0366] Embodiment 31. The copolymer of any one of embodiments 28-30, wherein the copolymer further comprises a third propagator block, wherein the third propagator block is connected to the first propagator block or the second propagator block via a third linker comprising a disulfide bond.

[0367] Embodiment 32. The copolymer of any one of embodiments 1 to 31, wherein the propagator block is selected from the group consisting of:

[0368] PB1,

[0369] PB2,

[0370] PB3,

[0371] PB4, and

[0372] PB5.

[0373] Embodiment 33. The copolymer of embodiment 32 comprising at least two propagator blocks, wherein the 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.

[0374] Embodiment 34. The copolymer of embodiment 33, wherein the initiator block and the at least two propagator blocks are selected from the group consisting of 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, 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.

[0375] Embodiment 35. A polymersome comprising a membrane defining an internal space, wherein the membrane comprises a copolymer according to any one of embodiments 1 to 34.

[0376] Embodiment 36. The polymersome of embodiment 35, wherein the copolymer makes up at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0377] Embodiment 37. The polymersome of embodiment 35 or embodiment 36, wherein the membrane encapsulates the payload.

[0378] Embodiment 38 The polymersome of embodiment 37, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0379] Embodiment 39. The polymersome of embodiment 38, wherein the nucleic acid is RNA or DNA.

[0380] Embodiment 40. The polymersome of embodiment 39, wherein the payload encodes a polypeptide.

[0381] Embodiment 41. The polymersome of any one of embodiments 37-40, wherein the payload is immunogenic, or the payload is a nucleic acid encoding an immunogenic polypeptide or protein.

[0382] Embodiment 42. The polymersome of any one of embodiments 35-41, wherein the copolymer is a first copolymer and the membrane further comprises a second copolymer, wherein the first copolymer and the second copolymer are independent of any one of embodiments 1-34.

[0383] Embodiment 43. The polymersome of any one of embodiments 35-42, having a diameter of 0.001 to 5 microns or 0.01 to 5 microns.

[0384] Embodiment 44. The polymersome of any one of embodiments 35-43, wherein the payload is a first payload, and the membrane further encapsulates a second payload.

[0385] Embodiment 45. The polymersome of embodiment 44, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0386] Embodiment 46. The polymersome of embodiment 45, wherein the first payload and the second payload are different.

[0387] Embodiment 47. A preparation comprising a polymersome according to any one of embodiments 35-46.

[0388] Embodiment 48. The preparation of embodiment 47, comprising from 0.01 to 95% (w / w) of polymersome.

[0389] Embodiment 49. The preparation of embodiment 47 or embodiment 48, wherein the polymersome is a first polymersome and the composition further comprises a second polymersome.

[0390] Embodiment 50. The preparation of embodiment 49, wherein the first polymersome and the second polymersome differ in size, copolymers forming their membranes, payload encapsulated in these polymersomes, or a combination thereof.

[0391] Embodiment 51. The preparation according to any one of embodiments 47-50, further comprising a pharmaceutically acceptable excipient, adjuvant, or combination thereof.

[0392] Embodiment 52. The preparation according to claim 51, wherein the auxiliary substance is a solvent, dispersants, a diluent, a dispersing additive, a suspending additive, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase or mixtures thereof.

[0393] Embodiment 53. The preparation of embodiment 51 or embodiment 52, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosidase, aluminum salt, squalene, MF59 or QS-21. Other examples of adjuvants in some vaccines that can be used in the composition of the present invention are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixtures of aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03, MF59 and CpG 1018 or a combination thereof.

[0394] Embodiment 54. A kit for producing a polymersome, comprising: a first reagent that is an initiator, wherein the initiator comprises a glycan head and an initiator-binding moiety, and a second reagent that comprises a propagator, wherein the propagator comprises a functional moiety and a propagator-binding moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof; and wherein the initiator-binding moiety is capable of binding to the propagator-binding moiety through a linker that comprises a disulfide bond.

[0395] Embodiment 55. The kit of embodiment 54, wherein the glycan head comprises a terminal mannoside.

[0396] Embodiment 56. The kit of embodiment 54 or embodiment 55, wherein the glycan head comprises O-aryl mannoside.

[0397] Embodiment 57. The kit according to any one of embodiments 54-56, wherein the glycan head comprises mono-mannoside, di-mannoside, or tri-mannoside.

[0398] Embodiment 58. The kit of embodiment 57, wherein the tri-mannoside is a linear or branched tri-mannoside.

[0399] Embodiment 59. The kit of embodiment 58, wherein the branched tri-mannoside is α-1,3-α-1,6-trimannoside.

[0400] Embodiment 60. The kit according to any one of embodiments 54-59, wherein the initiator further comprises an initiator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0401] Embodiment 61. The kit of embodiment 60, 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, 2-6 carbon atoms).

[0402] Embodiment 62. The kit of embodiment 61, wherein the PEG moiety comprises 2 to 72 (OCH2CH2) subunits.

[0403] Embodiment 63. The kit of embodiment 62, wherein the PEG moiety has a linear, branched, or star-shaped configuration.

[0404] Embodiment 64. The kit according to any one of embodiments 54-63, wherein the glycan head is designed to bind to a dendritic cell.

[0405] Embodiment 65. The kit of embodiment 64, wherein the glycan head is designed to selectively bind to DC-SIGN.

[0406] Embodiment 66. The kit of embodiment 65, wherein the glycan head is designed to bind to DC-SIGN at K D in the range from 5 to 8000 nM at pH 7.4.

[0407] Embodiment 67. The kit according to embodiment 66, where K D is in the range from 5 to 500 nM at pH 7.4.

[0408] Embodiment 68. The kit according to any one of embodiments 65-67, wherein the glycan head is designed to bind to DC-SIGN at K D in the range from 1 to 800 nM at pH 5.

[0409] Embodiment 69. The kit according to embodiment 68, where K D ranges from 1 to 600 nM at pH 5.

[0410] Embodiment 70. The kit according to any one of embodiments 54 to 69, wherein the glycan head comprises 9 BPC Neu5Ac conjugated N-glycan (I2), Neu5Ac conjugated N-glycan (I3), 9 TCC Neu5Ac conjugated N-glycan (I4) or a combination of both.

[0411] Embodiment 71. The kit of embodiment 70, wherein the glycan head is designed to bind to Siglec-2, Siglec-5 / E, Siglec-1, or a combination thereof.

[0412] Embodiment 72. The kit according to any one of embodiments 54 to 71, wherein the initiator-binding moiety is a thiol group or a dithiolane group.

[0413] Embodiment 73. The kit according to any one of embodiments 54 - 72, wherein the initiator is selected from the group consisting of:

[0414] ,

[0415] ,

[0416] ,

[0417] ,

[0418] ,

[0419] ,

[0420] ,

[0421] , And

[0422] .

[0423] Embodiment 74. The kit according to any one of embodiments 54 to 73, wherein the propagator comprises more than one guanidine group.

[0424] Embodiment 75. The kit of embodiment 74, wherein the propagator comprises three guanidine groups.

[0425] Embodiment 76. The kit according to any one of embodiments 54-75, wherein the propagator further comprises a propagator spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

[0426] Embodiment 77. The kit of embodiment 76, 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, 2-6 carbon atoms).

[0427] Embodiment 78. The kit of embodiment 76 or embodiment 77, wherein the PEG moiety comprises 2 to 72 (OCH2CH2) subunits.

[0428] Embodiment 79. The kit of embodiment 78, wherein the PEG moiety is linear PEG.

[0429] Embodiment 80. The kit of any one of embodiments 54 to 79, wherein the propagator in the second reagent is a first propagator, and wherein the second reagent further comprises a second propagator, or the kit further comprises a third reagent comprising a second propagator; and wherein the first propagator and the second propagator independently comprise a functional moiety comprising a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof.

[0430] Embodiment 81. The kit of embodiment 80, wherein the first propagator comprises a guanidine group and the second propagator comprises a zwitterionic group.

[0431] Embodiment 82. The kit of embodiment 80, wherein the first propagator comprises a guanidine group and the second propagator comprises diethylenetriamine.

[0432] Embodiment 83. The kit according to any one of embodiments 54 to 82, wherein the propagator-binding moiety is a thiol group or a dithiolane group.

[0433] Embodiment 84. The kit according to any one of embodiments 54 - 83, wherein the propagator is selected from the group consisting of:

[0434]

[0435] Embodiment 85. The kit according to any one of embodiments 54-84, wherein the first reagent and the second reagent are contained in a single container.

[0436] Embodiment 86. The kit according to any one of embodiments 54-84, wherein the first reagent and the second reagent are contained in different containers.

[0437] Embodiment 87. The kit of any one of embodiments 54-86, further comprising a payload, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0438] Embodiment 88. The kit of embodiment 87, wherein the nucleic acid is RNA or DNA.

[0439] Embodiment 89. The kit of embodiment 88, wherein the payload encodes a polypeptide.

[0440] Embodiment 90. The kit according to any one of embodiments 87-89, wherein the payload is immunogenic, or the payload is a nucleic acid encoding an immunogenic polypeptide or protein.

[0441] Embodiment 91. A method for targeted delivery of a payload to a subject, comprising administering to the subject an effective amount of a pharmaceutical preparation comprising a polymersome, wherein the polymersome has a membrane encapsulating the payload, and wherein the membrane comprises a copolymer according to any one of embodiments 1-34.

[0442] Embodiment 92. The method of embodiment 91, wherein the copolymer makes up at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0443] Embodiment 93. The method of embodiment 92, wherein the copolymer is a first copolymer and the membrane further comprises a second copolymer, wherein the first copolymer and the second copolymer are independent of any of embodiments 1-34.

[0444] Embodiment 94. The method according to any one of embodiments 91-93, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a glycan, or a combination thereof.

[0445] Embodiment 95. The method of embodiment 94, wherein the nucleic acid is RNA or DNA.

[0446] Embodiment 96. The method of embodiment 95, wherein the payload encodes a polypeptide.

[0447] Embodiment 97. The method according to any one of embodiments 91-96, wherein the payload is immunogenic, or the payload is a nucleic acid encoding an immunogenic polypeptide or protein.

[0448] Embodiment 98. The method of any one of embodiments 91-97, wherein the payload is a first payload, and the membrane further encapsulates a second payload.

[0449] Embodiment 99. The method of embodiment 98, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0450] Embodiment 100. The method of embodiment 99, wherein the first payload and the second payload are different.

[0451] Embodiment 101. A method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical preparation comprising a polymersome, wherein the polymersome has a membrane, wherein the membrane comprises a polymer component according to any one of embodiments 1-34; and a payload encapsulated within the membrane; and wherein the payload is or is convertible to a therapeutic agent.

[0452] Embodiment 102. The method of embodiment 101, wherein the polymer component makes up at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0453] Embodiment 103. The method of embodiment 101 or embodiment 102, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a glycan, or a combination thereof.

[0454] Embodiment 104. The method of embodiment 103, wherein the nucleic acid is RNA or DNA.

[0455] Embodiment 105. The method of embodiment 104, wherein the payload encodes a polypeptide.

[0456] Embodiment 106. The method according to any one of embodiments 101-105, wherein the payload is a first payload, and the membrane further encapsulates a second payload.

[0457] Embodiment 107. The method of embodiment 106, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0458] Embodiment 108. The method of embodiment 107, wherein the first payload and the second payload are different.

[0459] Embodiment 109. The method according to any one of embodiments 101-108, wherein the polymersome is administered in an initial dose and followed by one, two, three, four, five or more booster doses.

[0460] Embodiment 110. The method of embodiment 109, wherein the booster doses are administered at about one month, about two months, about three months, about four months, about five months, or about six months or more after the initial dose is administered.

[0461] Embodiment 111. The method according to any one of embodiments 101-110, wherein the effective amount is in the range of from about 5 mcg to 1000 mcg.

[0462] Embodiment 112. A method for enhancing an adaptive immune response, comprising administering to a subject an effective amount of a pharmaceutical preparation comprising a polymersome; wherein the polymersome has a membrane encapsulating a payload, and wherein the membrane comprises a copolymer according to any one of embodiments 1-34; wherein the payload is immunogenic or is converted into an immunogenic biomolecule.

[0463] Embodiment 113. The method of embodiment 112, wherein the polymer component makes up at least 50%, 70%, 80%, 90%, 95%, or 99% of the membrane.

[0464] Embodiment 114. The method of embodiment 83 or embodiment 84, wherein the payload is a nucleic acid, a compound, a peptide, a protein, a glycan, or a combination thereof.

[0465] Embodiment 115. The method of embodiment 85, wherein the nucleic acid is RNA or DNA.

[0466] Embodiment 116. The method according to any one of embodiments 112-115, wherein the biomolecule is a polypeptide or a protein.

[0467] Embodiment 117. The method of any one of embodiments 112 - 116, wherein the payload is a first payload, and the membrane further encapsulates a second payload.

[0468] Embodiment 118. The method of embodiment 117, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0469] Embodiment 119. The method of embodiment 118, wherein the first payload and the second payload are different.

[0470] Embodiment 120. The method according to any one of embodiments 112-119, wherein the polymersome is administered in an initial dose and followed by one, two, three, four, five or more booster doses.

[0471] Embodiment 121. The method of embodiment 120, wherein the booster doses are administered at about one month, about two months, about three months, about four months, about five months, or about six months or more after the initial dose is administered.

[0472] Embodiment 122. The method according to any one of embodiments 112-121, wherein the effective amount is in the range of from about 5 mcg to 1000 mcg.

Claims

1. A copolymer for forming a polymersome, wherein the copolymer has the structure where both x and y are integers and are independently chosen from 9 to 14, wherein said initiating unit in the structure comprises a glycan head and wherein the glycan head comprises monomannoside, dimannoside or trimannoside; wherein the first block comprises a functional moiety that comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, and wherein the zwitterionic group is an alkylphosphobetaine group, choline phosphate (CP), or sulfotetin; wherein the second block comprises a functional moiety that comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, and wherein the zwitterionic group is an alkylphosphobetaine group, choline phosphate (CP), or sulfotetin.

2. The copolymer of claim 1, wherein the glycan head comprises a terminal mannoside.

3. The copolymer of claim 1, wherein the glycan head comprises an O-aryl mannoside containing an optionally substituted benzene ring.

4. The copolymer of claim 1, wherein the tri-mannoside is α-1,3-α-1,6-trimannoside.

5. The copolymer of claim 1, wherein the initiating block further comprises an initiating spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

6. The copolymer of claim 1, wherein the glycan head is designed to bind to a dendritic cell.

7. The copolymer of claim 6, wherein the glycan head is designed to selectively bind to DC-SIGN.

8. The copolymer of claim 7, wherein the glycan head is designed to bind to DC-SIGN at K D in the range from 5 to 8000 nM at pH 7.

4.

9. The copolymer according to claim 1, wherein the glycan head contains 9 BPCNeu5Ac conjugated N-glycan, Neu5Ac conjugated N-glycan, 9 TCC Neu5Ac conjugated N-glycan or a combination thereof; where BPC means biphenylcarboxyl, TCC means 4H-thieno[3,2-c]chromene-2-carbamoyl) and Neu5Ac means N-acetylneuraminic acid.

10. The copolymer of claim 1, wherein the initiator block is selected from the group consisting of: ; where the filled circle represents mannoside, the open circle represents galactose, the filled square represents GlcNAc, and the diamond represents Neu5Ac.

11. The copolymer of claim 1, wherein the first block or the second block contains more than one guanidine group.

12. The copolymer of claim 11, wherein the first block or the second block contains three guanidine groups.

13. The copolymer of claim 1, wherein the first block or the second block comprises a spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

14. The copolymer of claim 1, wherein the first block comprises a guanidine group and the second block comprises a zwitterionic group; or wherein the first block comprises a guanidine group and the second block comprises diethylenetriamine.

15. The copolymer of claim 1, wherein the first block or the second block is independently selected from the group consisting of: PB1, PB2, PB3, PB4 and PB5.

16. The copolymer of claim 15, wherein the first block and / or the second block are (1) PB1 and PB5, (2) PB1 and PB4, (3) PB2 and PB5, and (4) PB2 and PB5.

17. The copolymer of claim 16, wherein the initiator block is selected from the group consisting of: ; where the filled circle represents mannoside, the open circle represents galactose, the filled square represents GlcNAc, and the diamond represents Neu5Ac; and wherein the initiating block, the first block and the second block are selected from the group consisting of IB5, PB1 and PB5; IB5, PB1 and PB4; IB5, PB2 and PB5; IB5, PB2 and PB4; IB6, PB1 and PB5; IB6, PB1 and PB4; IB6, PB2 and PB5; IB6, PB2 and PB4; IB7, PB1 and PB5; IB7, PB1 and PB4; IB7, PB2 and PB5; IB7, PB2 and PB4; IB8, PB1 and PB5; IB8, PB1 and PB4; IB8, PB2 and PB5; IB8, PB2 and PB4; IB9, PB1 and PB5; IB9, PB1 and PB4; IB9, PB2 and PB5; IB9, PB2 and PB4; IB10, PB1 and PB5; IB10, PB1 and PB4; IB10, PB2 and PB5; and IB10, PB2 and PB4.

18. A polymersome for selective delivery of a payload, comprising a membrane defining an internal space, wherein the membrane comprises a copolymer according to any one of claims 1-17, wherein the payload is encapsulated in the membrane and wherein the payload is a nucleic acid, a polypeptide or a protein.

19. The polymersome of claim 18, wherein the nucleic acid is RNA or DNA.

20. The polymersome of claim 18, wherein the nucleic acid encodes an immunogenic polypeptide or protein.

21. The polymersome of claim 18, wherein said copolymer is a first copolymer, and wherein the membrane also comprises a second copolymer, and wherein the first copolymer and the second copolymer are independently selected from copolymers according to any one of claims 1-17.

22. A pharmaceutical composition for selective delivery of a payload, comprising a polymersome according to any one of claims 18-21 and a pharmaceutically acceptable excipient, adjuvant or a combination thereof.

23. The pharmaceutical composition according to claim 22, containing from 0.01 to 95% w / w polymersomes.

24. The pharmaceutical composition of claim 22, wherein the polymersome is a first polymersome, and the composition further comprises a second polymersome, wherein the first polymersome and the second polymersome are independently selected from a polymersome according to any one of claims 18-21 and differ in size, copolymers forming their membranes, payload encapsulated in these polymersomes, or a combination thereof.

25. The pharmaceutical composition according to claim 22, wherein the pharmaceutically acceptable excipient is a solvent, dispersant, diluent, dispersing additive, suspending additive, surfactant, isotonic agent, thickener or emulsifier, preservative, or mixtures thereof.

26. The pharmaceutical composition of claim 22, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosidase, aluminum salt, squalene, MF59, QS-21, complete Freund's adjuvant, incomplete Freund's adjuvant, AS03, CpG 1018, or a combination thereof.

27. A method for targeted delivery of a payload to a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a polymersome and a pharmaceutically acceptable excipient, adjuvant or combination thereof, wherein the polymersome is a polymersome according to any one of paragraphs 18-21.

28. A method for enhancing an adaptive immune response, comprising administering to a subject an effective amount of a pharmaceutical composition comprising a polymersome and a pharmaceutically acceptable excipient, adjuvant or combination thereof, wherein the polymersome is a polymersome according to any one of paragraphs 18-21 and the payload is a nucleic acid, polypeptide or protein that is immunogenic, or a nucleic acid encoding an immunogenic polypeptide or protein.

29. The method of claim 28, wherein the pharmaceutical composition is intended to be administered in an initial dose followed by administration of at least one, two, three, four, five or more booster doses.

30. The method of claim 29, wherein the booster doses are administered at about one month, about two months, about three months, about four months, about five months, or about six months or more after the initial dose.

31. The method of claim 28, wherein the pharmaceutical composition is intended for administration in an effective amount of from about 5 mcg to 1000 mcg.

32. A kit for producing a polymersome, wherein the polymersome is a polymersome according to any one of paragraphs 18-21, and said kit comprises: a first reagent that is an initiator, wherein the initiator comprises (1) a glycan head comprising a monomannoside, dimannoside, or trimannoside, and (2) an initiator-binding moiety, and a second reagent comprising a functional moiety and a linking moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, wherein the zwitterionic group is an alkylphosphobetaine group, choline phosphate (CP), or sulfotetin; and wherein the initiator-binding moiety is configured to be capable of binding to the binding moiety of the second reagent via a linker that is a disulfide bond, a third reagent comprising a functional moiety and a linking moiety, wherein the functional moiety comprises a guanidine group, a zwitterionic group, diethylenetriamine, or a combination thereof, wherein the zwitterionic group is an alkylphosphobetaine group, choline phosphate (CP), or sulfotetin; and payload, which is a nucleic acid, polypeptide, or protein.

33. The kit according to claim 32, wherein the glycan head comprises a terminal mannoside.

34. The kit of claim 32, wherein the glycan head comprises an O-aryl mannoside containing an optionally substituted benzene ring.

35. The kit of claim 32, wherein the tri-mannoside is α-1,3-α-1,6-trimannoside.

36. The kit of claim 32, wherein the initiator further comprises an initiating spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

37. The kit of claim 32, wherein the glycan head is configured to bind to a dendritic cell.

38. The kit of claim 37, wherein the glycan head is configured to selectively bind to DC-SIGN.

39. The kit of claim 38, wherein the glycan head is configured to bind to DC-SIGN at K D in the range from 5 to 8000 nM at pH 7.

4.

40. The kit according to claim 32, wherein the glycan head contains 9 BPC Neu5Ac conjugated N-glycan (I2), Neu5Ac conjugated N-glycan (I3), 9 TCC Neu5Ac conjugated N-glycan (I4) or a combination thereof, where BPC stands for biphenylcarboxyl, TCC stands for 4H-thieno[3,2-c]chromene-2-carbamoyl) and Neu5Ac stands for N-acetylneuraminic acid.

41. The set according to claim 32, wherein the initiator is selected from the group consisting of: , , , , , , , , And .

42. The kit according to claim 32, wherein the second reagent and / or the third reagent contains more than one guanidine group.

43. The kit of claim 32, wherein the second reagent and / or the third reagent further comprises a spacer comprising a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof.

44. The kit of claim 32, wherein the second reagent comprises a guanidine group and the third reagent comprises a zwitterionic group; or the second reagent comprises a guanidine group and the third reagent comprises diethylenetriamine.

45. The kit according to claim 32, wherein the second reagent and / or the third reagent is selected from the group consisting of:

46. ​​The kit according to claim 32, wherein the nucleic acid is RNA or DNA.

47. The kit of claim 46, wherein the payload is immunogenic or the payload is a nucleic acid encoding an immunogenic polypeptide or protein.

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