Polymer-lipid hybrid nanoparticles containing lipids and block copolymers and methods of making and using same - Patents.com

Abstract

The present invention relates to a polymer-lipid hybrid nanoparticle comprising a lipid and a block copolymer, wherein the amount of the lipid present in the polymer-lipid hybrid nanoparticle, expressed as a mole percent (mol%), is greater than the amount of the block copolymer present in the polymer-lipid hybrid nanoparticle, expressed as a mole percent, said mole percent being based on the total amount of all components forming the polymer-lipid nanoparticle. The present invention also relates to such a polymer-lipid hybrid nanoparticle, further comprising a soluble encapsulated antigen, which is a protein and / or a polynucleotide. Furthermore, the present invention relates to a method for encapsulating such an antigen in such a polymer-lipid hybrid nanoparticle, as well as a composition comprising such a polymer-lipid hybrid nanoparticle, and the use of such a polymer-lipid hybrid nanoparticle and / or composition as a vaccine, as a drug, as a means for targeting cells, tissues and / or organs, and / or as a non-viral delivery system capable of delivering nucleotides to the inside of cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to European patent application 22158324, filed with the European Patent Office on February 23, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0003] Technical Field The present invention relates to polymer-lipid hybrid nanoparticles comprising a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percent (mol%), present in the polymer-lipid hybrid nanoparticle is greater than the amount of said block copolymer, expressed in mole percent, present in the polymer-lipid hybrid nanoparticle. The present invention also relates to such polymer-lipid hybrid nanoparticles further comprising a soluble encapsulated antigen, which is a protein and / or a polynucleotide. Furthermore, the present invention relates to a method for encapsulating such antigens in such polymer-lipid hybrid nanoparticles, as well as compositions comprising such polymer-lipid hybrid nanoparticles, and the use of such polymer-lipid hybrid nanoparticles and / or compositions as vaccines, as drugs, as a means for targeting cells, tissues, and / or organs, and / or as a non-viral delivery system capable of delivering nucleotides, for example, to the interior of cells. In particular, in the course of the present invention, synthetic polymer-lipid hybrid nanoparticles comprising the block copolymers PBD-PEO (non-degradable) or poly(ε-caprolactone)-poly(ethylene glycol) (PCL-PEO) (biodegradable) were explored as novel platforms for polynucleotide (e.g., mRNA) delivery. [Background technology]

[0004] 2. Background of the Invention Immunization is a well-established process, but there are differences in the level of response induced between different immunogens or antigens. For example, membrane proteins make up a class of antigens that produce low response levels. As a result, this means that a large amount of membrane protein is required to generate or induce a desired level of immune response. Membrane proteins are notoriously difficult to synthesize and are insoluble in water without detergent. This makes it costly and difficult to obtain sufficient amounts of membrane protein for immunization. Furthermore, membrane proteins require proper folding to function properly. The immunogenicity of a properly folded native membrane protein is typically much greater than that of its solubilized form, which may not be folded in a physiologically relevant manner. Thus, adjuvants are sometimes used to enhance the immunogenicity of such solubilized antigens, but it is an inefficient method that does not provide much benefit (e.g., WO2014 / 077781A1).

[0005] Although transfected cells and lipid-based systems have been used to present membrane protein antigens with the aim of increasing the likelihood of isolating antibodies that may be effective in vivo, these systems are often unstable (e.g., oxidation sensitive), tedious, and expensive. Moreover, the current state of the art for such membrane protein antigens is the use of inactive virus-like particles for immunization.

[0006] On the other hand, vaccines are the most efficient approach to prevent diseases, mainly infectious diseases [e.g., Liu et al., 2016]. As of today, most licensed vaccines are made with either live or killed viruses. Despite their effectiveness in generating humoral responses (antibody-mediated responses) that block viral growth and cell entry, the safety of such vaccines remains a consideration. Over the past decades, scientific advances have helped to overcome such issues by engineering vaccine vectors that are non-replicating recombinant viruses. In parallel, protein-based or subunit antigens have been explored as safer alternatives. However, such protein-based vaccines typically induce insufficient immunity (both humoral and cellular responses). Several approaches have been used to improve the immunogenic properties of antigens. For example, microencapsulation of antigens in polymers has been extensively investigated, but enhances antigen immunogenicity, aggregation, and denaturation, remaining unsolved [e.g., Hilbert et al., 1999]. Furthermore, adjuvants (e.g., oil-in-water emulsions or polymer emulsions) [e.g., US9636397B2, US2015 / 0044242A1] are used together with antigens to induce more pronounced humoral and cellular responses. Despite these advances, they are inefficient in terms of uptake and cross-presentation. Based on the available information of the immune system during infection with viruses, virus-like particles that mimic such properties have been exploited to promote cross-presentation. Synthetic structures such as liposomes with encapsulated antigens are particularly attractive. Liposomes are unilamellar self-assembled structures made of lipids. Cationic liposomes are more attractive and promising than liposomes as delivery vehicles because they are efficiently taken up by antigen-presenting cells (APCs) [e.g., Maji et al., 2016]. Furthermore, this allows the incorporation of immunomodulatory agents such as monophosphoryl lipid A (MPL), CpG oligodeoxynucleotides, which are toll-like receptor (TLR) agonists that stimulate immune cells via their receptors.Despite these opportunities for such delivery vehicles, one of the limiting factors is the stability of liposomes in the presence of serum components. Loading high melting temperature lipids by PEGylation reduces some of the stability issues of liposomes. One such well-characterized example is the inter bilayered-crosslinked multilamellar vesicle (ICMV), formed by stabilizing multilamellar vesicles with short covalent bridges linking lipids [e.g., Moon et al., 2011]. Other nanoparticle structures have led to successful immunization with nanodiscs [e.g., Kuai et al., 2017] or pH-sensitive particles [e.g., Luo et al., 2017]. However, such strategies still require adjuvants or are not efficient outside of the prototypic ovalbumin (OVA) model.

[0007] Furthermore, polymersomes provide a stable alternative to liposomes and have been used to incorporate membrane proteins to induce immune responses [e.g., Quer et al., 2011, WO2014 / 077781A1]. Protein antigens have also been encapsulated within the chemically altered (but oxidation-sensitive) membrane of polymersomes to release antigens and adjuvants to dendritic cells [e.g., Stano et al., 2013].

[0008] On the other hand, messenger RNA (mRNA) has emerged as a promising strategy to prevent and treat various diseases, including infectious diseases, cancer, and genetic disorders. However, clinical translation of mRNA therapeutics is hindered by its instability and inefficient in vivo delivery. Recently, advanced lipid nanoparticle (LNP) systems have proven efficacy in preclinical trials and successfully entered the clinic. For example, LNP-Onpattro (LNP-ON), containing DMG-PEG, DSPC, MC3 and Chol (in a ratio of 1.5:10.0:50:38.5) encapsulated with therapeutic siRNA (pastisiran) to treat hereditary mediated amyloidosis, was approved by the FDA in 2018. To date, many other LNPs have been developed for mRNA delivery. Notably, mRNA-1273 and BNT162b have been used in clinics around the world to prevent coronavirus disease 2019 (COVID-19). Despite some positive results, maintaining the long-term stability and efficacy of mRNA-loaded LNPs remains a challenge. For example, mRNA-1273 and BNT162b are recommended to be stored at -80°C and -20°C, respectively. As cold chain transportation and storage are unavailable in many regions, there is an urgent need to develop therapeutic agents with improved long-term stability.

[0009] Thus, despite the progress made in using polymers, there remains a need for a method to overcome or at least alleviate the above problems and provide improved functionality, particularly in the treatment and / or prevention of infectious diseases, cancer, and autoimmune diseases, for efficient and stable uptake, delivery, and / or stable cross-presentation, particularly in the treatment and / or prevention of CD8 (+) There remains a need to provide delivery vehicles / systems and methods based thereon that have improved functionality in that they are also able to induce a T cell mediated immune response. Summary of the Invention

[0010] The present invention relates to polymer-lipid hybrid nanoparticles comprising a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percent (i.e., mol %), present in the polymer-lipid hybrid nanoparticle is greater than the amount of said block copolymer, expressed in mole percent, present in the polymer-lipid hybrid nanoparticle, said mole percent being based on the total amount of all components forming the polymer-lipid nanoparticle.

[0011] Further, the present invention relates to such polymer-lipid hybrid nanoparticles, wherein the lipid (e.g., ionizable lipid) is selected from the group consisting of the ionizable lipid DLin-MC3-DMA (also referred to as MC3) and the ionizable lipid C12-200. Further, the present invention relates to such polymer-lipid hybrid nanoparticles, wherein the block copolymer is selected from the group consisting of poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO) block copolymers, polycaprolactone (PCL)-PEO block copolymers, poly(lactide-co-glycolide) (PLGA)-PEO (e.g., with various LA:GA ratios), and DMG-PEG block copolymers. Further, the present invention relates to such polymer-lipid hybrid nanoparticles, wherein the lipid to block copolymer molar % ratio is 31.8-12 and about 35-2.5. Further, the present invention relates to such polymer-lipid hybrid nanoparticles, further comprising a stabilizer, e.g., comprising or consisting of cholesterol (also referred to as CHOL). Furthermore, the present invention relates to such polymer-lipid hybrid nanoparticles further comprising another lipid selected from the group consisting of DMPC, DSPC, DOPE, DOTAP, DODAP, DOTMA, DODMA, DDA, 18:1 PA (1,2-dioleoyl-sn-glycero-3-phosphate), 14:0 PA (1,2-dimyristoyl-sn-glycero-3-phosphate), 18:1 BMP (bis(monooleoylglycero)phosphate).The present invention further relates to such polymer-lipid hybrid nanoparticles consisting of: (i) PBD-PEO, MC3, CHOL; (ii) PBD-PEO, C12-200, CHOL; (iii) PBD-PEO, DOPE, C12-200, CHOL; (iv) PBD-PEO, DOPE, C12-200, CHOL; (v) PBD-PEO, DOPE, C12-200, CHOL; (vi) PBD-PEO, DOPE, C12-200, CHOL; (vii) DMG-PEG, DSPC, MC3, CHOL; (viii) PCL-PEO, DMPC, MC3, CHOL; (ix) PCL-PEO, DMPC, MC3, CHOL; (x) PCL-PEO, DMPC, MC3, CHOL; or (xi) PCL-PEO, DMPC, MC3, CHOL. Furthermore, the present invention relates to such polymer-lipid hybrid nanoparticles further comprising soluble encapsulated antigens which are proteins and / or polynucleotides.

[0012] Furthermore, the present invention relates to compositions comprising such polymer-lipid hybrid nanoparticles.

[0013] Furthermore, the present invention relates to a method for delivering nucleotides to the interior of a cell without the use of a viral vector as a delivery vehicle, comprising the steps of: (i) providing the polymer-lipid hybrid nanoparticles and / or compositions of the present invention; and (ii) contacting said polymer-lipid hybrid nanoparticles and / or compositions with a cell.

[0014] The exemplary polymer-lipid hybrid nanoparticles of the present invention exhibit favorable physicochemical properties and / or excellent encapsulation efficiency (about 100%). Compared to benchmark LNP-ON (i.e., LNP-Onpattro or LNP-ONP, which may be used interchangeably herein), the performance of the polymer-lipid hybrid nanoparticles of the present invention is superior, enhancing the in vitro transfection efficacy and / or long-term thermal stability of polynucleotides (e.g., mRNA). Furthermore, the polymer-lipid hybrid nanoparticle formulations of the present invention exhibit lower cytotoxicity compared to benchmark LNP-ON. Furthermore, the exemplary polymer-lipid hybrid nanoparticle formulations of the present invention can potently activate cDC1 and cDC2 in lymph nodes to promote antigen surface presentation. In summary, the present invention provides a novel class of polymer-lipid hybrid nanoparticles with efficient protein and antigen expression and enhanced thermal stability, which makes them suitable for delivering therapeutic mRNA across a wide range of diseases.

[0015] The present invention therefore meets this need by providing stable polymer-lipid hybrid nanoparticles comprising lipids and block copolymers as described herein, methods based thereon and methods for producing the same, as well as compositions comprising such polymer-lipid hybrid nanoparticles as described herein, as characterized in the claims and illustrated by the accompanying examples and figures.

[0016] Overview of sequence listing SEQ ID NO:1 is an exemplary firefly luciferase (Luc) mRNA sequence from Photinus pyralis.

[0017] SEQ ID NO:2 is an exemplary ovalbumin (OVA) mRNA (https: / / www.trilinkbiotech.com / media / folio3 / productattachments / product_insert / ova_orf_catno_l-7210_l-7610_.txt).

[0018] SEQ ID NO:3 is an exemplary Mus musculus CD19 mRNA sequence.

[0019] SEQ ID NO:4 is an exemplary OVA peptide. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1 shows cryo-TEM images (A, B) and particle size (C) of exemplary polymer-lipid hybrid nanoparticles (BNPs) of the present invention prepared from ionized lipid DLin-MC3-DMA and PBD-PEO block copolymers by a solvent dispersion method and encapsulating luciferase mRNA. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] FIG. 2 shows cryo-TEM images (A, B, C) and particle size (D) of exemplary polymer-lipid hybrid nanoparticles (BNPs) of the present invention prepared from ionized lipid DLin-MC3-DMA and PBD-PEO block copolymers by a solvent dispersion method and encapsulating ovalbumin mRNA. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Diagram 3]3A shows a cryo-TEM image of an exemplary polymer-lipid hybrid nanoparticle of the present invention, prepared from ionized lipid DLin-MC3-DMA and PBD-PEO block copolymer by mixing method (alternative methods can be, for example, T-Mixer method, homogenization and / or microfluidic chip-based mixing method) and encapsulated with luciferase mRNA. FIG. 3B shows a cryo-TEM image of an exemplary BNP-008 polymer-lipid hybrid nanoparticle of the present invention, prepared from ionized lipid C12-200 and PBD-PEO block copolymer by mixing method and encapsulated with luciferase mRNA. FIG. 3C shows a cryo-TEM image of an exemplary PCL-008 polymer-lipid hybrid nanoparticle of the present invention, prepared from ionized lipid DLin-MC3-DMA and PCL-PEO block copolymer by mixing method and encapsulated with luciferase mRNA. FIG. 3D shows a cryo-TEM image of an exemplary PCL-012 polymer-lipid hybrid nanoparticle of the invention prepared by a mixing method from the ionized lipid DLin-MC3-DMA and PCL-PEO block copolymer and encapsulating luciferase mRNA. [Figure 4A] Figure 4 shows electrophoretic analysis of exemplary polymer-lipid hybrid nanoparticles of the present invention. Figure 4A shows an agarose gel image of luciferase mRNA encapsulated by exemplary polymer-lipid hybrid nanoparticles BNP prepared from ionized lipids DLin-MC3-DMA and PBD-PEO block copolymers by solvent dispersion method compared to a control formulation. Luciferase mRNA remains intact after encapsulation in BNP polymer-lipid hybrid nanoparticles. Figure 4B shows RNAse protection assay using gel electrophoretic analysis. Here, exemplary polymer-lipid hybrid nanoparticles BNP samples were prepared by solvent dispersion method and stored at 4°C for 2 weeks before analysis. [Figure 4B] See legend to Figure 4A. [Diagram 5]Figure 1 shows in vitro transfection efficiency profiles in HEK293T cells after storage for more than 3 weeks (A, B) at 4°C of luciferase mRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the invention prepared by solvent dispersion method from ionized lipid DLin-MC3-DMA and PBD-PEO block copolymers compared to control formulation nanoparticles. Nanoparticles were prepared by solvent dispersion method. N / P=27. N / P ratio: N (nitrogen) in ionized cationic lipid and P (phosphorus) in mRNA. [Figure 6] Figure 1 shows ovalbumin protein expression in HEK293T cells 24 hours after transfection from OVA mRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the present invention prepared by solvent dispersion method from ionized lipid DLin-MC3-DMA and PBD-PEO block copolymers compared to control formulation nanoparticles (A, B). Nanoparticles were prepared by solvent dispersion method. N / P=27. N / P ratio: N (nitrogen) in ionized cationic lipid and P (phosphorus) in mRNA. [Figure 7] 1 shows an agarose gel image of OVA mRNA encapsulated by exemplary BNP and PCL polymer-lipid hybrid nanoparticles of the present invention prepared by the mixing method compared to control formulation nanoparticles. All samples contain intact mRNA, as no degradation from the gel was observed. [Figure 8] Figure 1 shows the stability assay of luciferase mRNA encapsulated by exemplary BNP and PCL polymer-lipid hybrid nanoparticles of the present invention after 1 month storage at 4°C. Transfection efficiency of in vitro luciferase mRNA nanoparticles (prepared by the mixing method) in HEK293T cells 24 hours after transfection. *25ng Luc mRNA was used as reference. After 1 month at 4°C, LNP ON and BNP008 appeared to degrade, whereas BNP002, PCL008, PCL012 remained unchanged. [Figure 9]FIG. 1 shows the in vitro cytotoxicity of Luc mRNA encapsulated by exemplary BNP and PCL polymer-lipid hybrid nanoparticles of the present invention (prepared by the mixing method) against HEK293T cells after 24 hours of incubation. [Figure 10] FIG. 1 shows ovalbumin protein expression (A, B) from OVA mRNA encapsulated by exemplary BNP and PCL polymer-lipid hybrid nanoparticles of the present invention (prepared by the mixing method) in HEK293T cells 24 hours after transfection. [Figure 11A] Figure 11 shows the expression analysis of Luc mRNA. Figure 11A shows the in vivo expression kinetics analysis of Luc mRNA encapsulated by exemplary BNP and PCL polymer-lipid hybrid nanoparticles of the present invention administered to mice by intramuscular (IM) injection. Figure 11B shows ex vivo IVIS bioimaging of Luc mRNA delivered in ACM nanoparticles administered to mice by IM. Figure 11C shows ex vivo IVIS bioimaging of Luc mRNA delivered in ACM nanoparticles administered to mice by IV. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12A] Figure 12 shows the tissue expression profile of the protein encoded by Luc mRNA in mice. Figure 12A shows the ex vivo imaging analysis of the tissue expression profile of the protein encoded by Luc mRNA in mice after IM injection (6 hours later at this point). Figure 12B shows the tissue expression profile of the protein encoded by Luc mRNA in mice 6 hours after IV injection. Figure 12C shows the tissue expression profile of the protein encoded by Luc mRNA in mice 6 hours after IV injection as percentage expression in individual tissues. [Figure 12B] See legend to Figure 12A. [Figure 12C]See legend to Figure 12A. [Figure 13] FIG. 1 shows dendritic cell (DC) activation in draining lymph nodes (ovalbumin mRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the invention). [Figure 14-1] FIG. 14 shows OVA peptide surface display, ovalbumin mRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the invention. [Figure 14-2] FIG. 14 shows OVA peptide surface display, ovalbumin mRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the invention. [Figure 15] We show Cas12a / gRNA encapsulation by an exemplary BNP 002 polymer-lipid hybrid nanoparticle of the present invention. Cas12a and gRNA were mixed with ASF p52 at 250 nM concentration. The solution was incubated at RT for 10-15 min so that Cas12a binds to gRNA. This was further encapsulated in BNP-002 using PNI system at TFF 12 ml / min and FRR 3:1 at 1 ml scale using the mixing method. After formulation was completed, DLS of the sample was performed and the rest of the sample was dialyzed using PBS buffer. After dialysis, the sample was collected and DLS was collected before and after sterile filtration. [Figure 16] FIG. 1 shows dynamic light scattering (DLS) analysis of Cas12a / gRNA encapsulated by exemplary BNP polymer-lipid hybrid nanoparticles of the invention prepared by the mixing method. [Figure 17] Figure 1 shows an ACM-OVA mRNA vaccine adaptive immunity study. Mice were immunized with ACM-OVA mRNA formulations. a. Immunization and bleeding schedule. b, c. Circulating SIINFEKL-specific CD8+ T cells. d, e. Serum OVA IgG titers. Two-way or one-way ANOVA with Tukey's multiple comparisons were performed as appropriate. [Figure 18A]FIG. 18 shows cryo-TEM images of exemplary BNP polymer-lipid hybrid nanoparticles of the invention prepared by microfluidizer. (A) Cryo-TEM of BNP-002.2 (carrying luciferase mRNA), where BNP-002.2 shows spherical nanoparticles (50-150 nm) with amorphous structure. (B) Cryo-TEM of BNP-012 (carrying luciferase mRNA), where BNP-012 shows predominant distribution: multicompartmental structure, vesicles consist of heterogeneous structure (i.e., vesicle fusion; vesicles with buddies, vesicles buddy surrounding by bilayer). (C) Cryo-TEM of BNP-025 (carrying luciferase mRNA), where BNP-025 shows vesicular structure (30-150 nm) with relatively high polydispersity. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19] Figure 1 shows an agarose gel image of Luc mRNA-loaded nanoparticles prepared by microfluidizer, indicating that all samples contained intact mRNA, as no degradation was observed in the gel. [Figure 20] Figure 1 shows in vitro luciferase mRNA nanoparticle transfection efficiency profile in HEK293T cells, showing that all formulations had high luciferase protein expression comparable to LNP-ON, with BNP-002.2 demonstrating significantly higher in vitro transfection efficacy compared to LNP-ON (p<0.05). [Figure 21A]Figure 21 shows the luciferase protein expression biodistribution percentage profile via IV (intravenous) administration. Here, BNP-002.2 accumulated luciferase protein in the liver (54%), spleen (44.5%), and 2.1% in the lung. BNP-012 expressed luciferase protein in the liver (0.9%), whereas 1.4% was expressed in the spleen and 92% in the lung. BNP-025 produced luciferase protein in the liver (2.7%), spleen (13%), and 76% in the lung. BNP-012 and BNP-025 containing cationic lipids (DOTAP and DOTMA) produced luciferase protein mainly in the lung. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 22A] Figure 22 shows the tissue expression profile (raw flux values) of protein encoded by Luc mRNA in mice 6 hours after IV administration. Raw flux values ​​(photons s-1). In the liver, LNP ONP produced significantly more Luc protein than other groups. In the spleen, BNP-008 produced significantly more Luc protein than other groups. The amount of Luc protein expression in the lungs between different groups is almost the same. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 23]Luciferase protein expression percent biodistribution profile (flux) via IV administration is shown, demonstrating that mRNA was delivered in an organ-specific manner to the liver, spleen, and lungs via engineered block copolymer-lipid hybrid nanoparticles. Other organs include the heart, kidney, and pancreas. LNP-ON produced luciferase protein in the liver (98%) versus 1.2% in the spleen and 0.5% in the lungs, BNP-002 produced luciferase protein in the liver (98%) versus 0.5% in the spleen and 0.3% in the lungs, BNP-008 facilitated even higher levels of luciferase protein expression in the spleen (67%), liver (26%) and 5% in the lungs, BNP-012 produced luciferase protein in the spleen (1.4%), liver (0.9%) and 92% in the lungs, and BNP-025 produced luciferase protein in the spleen (13%), liver (2.7%) and 76% in the lungs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention The following detailed description refers, by way of example, to the accompanying examples and figures which show specific details and embodiments in which the present invention can be implemented. These embodiments are described in sufficient detail to enable those skilled in the art to implement the present invention. Other embodiments may be utilized, such that structural, logical, and eclectic changes may be made without departing from the scope of the present invention. The various aspects of the present invention described herein are not necessarily mutually exclusive, as an aspect of the present invention may be combined with one or more other aspects to form a new embodiment of the present invention.

[0022] Messenger RNA (mRNA) has emerged as a promising strategy for preventing and treating a variety of diseases, including infectious diseases, cancer, and genetic disorders.

[0023] However, clinical translation of mRNA therapeutics is hindered by their instability and inefficient in vivo delivery. Recently, advanced lipid nanoparticle (LNP) systems have proven efficacy in preclinical trials and successfully entered the clinic. For example, LNP-Onpattro (LNP-ON or LNP-ONP), containing DMG-PEG, DSPC, MC3, and Chol (1.5:10.0:50:38.5), encapsulated with therapeutic siRNA (patisiran) to treat hereditary mediated amyloidosis, was approved by the FDA in 2018. To date, many other LNPs have been developed for mRNA delivery. Notably, mRNA-1273 and BNT162b have been used in clinics worldwide to prevent coronavirus disease 2019 (COVID-19).

[0024] However, despite the positive results, maintaining the long-term stability and efficacy of mRNA-loaded LNPs remains a challenge. For example, mRNA-1273 and BNT162b are recommended to be stored at -80°C and -20°C, respectively. As cold chain transportation and storage are unavailable in many regions, there is an urgent need to develop therapeutic agents with improved long-term stability.

[0025] In the course of this invention, synthetic polymer-lipid hybrid nanoparticles, including PBD-PEO (non-degradable) or poly(ε-caprolactone)-poly(ethylene glycol) (PCL-PEO) (biodegradable) block copolymers, PLGA-PEO, were explored as novel platforms for polynucleotide (e.g., mRNA) delivery.

[0026] PBD-PEO, PCL-PEO, PLGA-PEO polymer-lipid hybrid nanoparticles with well-defined molecular weights and narrow polydispersities were synthesized. For example, such synthetic polymers can be combined with helper lipids and ionized lipids and formulated to create a new class of polymer-lipid hybrid nanoparticles for, for example, mRNA delivery (e.g., PBD-PEO polymer-lipid hybrid nanoparticles may be interchangeably referred to herein as "BNPs" and PCL-PEO polymer-lipid hybrid nanoparticles may be interchangeably referred to herein as "PCLs"). The effects of composition and N / P ratio (N in ionized cationic lipids and P in mRNA) on the performance of BNPs (prepared by solvent dispersion method) were systematically evaluated in terms of particle size, polydispersity, surface charge, morphology, encapsulation efficiency, loading level, and in vitro transfection. Furthermore, the optimal formulations were generated by Precision Nanosystem Incorporation NanoasemblrPatform (PNI). Furthermore, the in vivo delivery efficacy of BNP and PCL was evaluated using a luciferase protein expression model in mice. Importantly, the optimal formulation demonstrated robust mRNA delivery both in vitro and in vivo, while exhibiting improved storage stability compared to the benchmark LNP-ON. Overall, BNP showed great potential to deliver therapeutic mRNA.

[0027] In the context of the present invention, the term "polynucleotide" (also referred to as "nucleic acid"; "nucleic acid" can be used interchangeably with the term "polynucleotide") refers to a polymer composed of nucleotide units that can be hydrolyzed to, for example, certain pyrimidine or purine bases (usually adenine, cytosine, guanine, thymine, uracil), d-ribose or 2-deoxy-d-ribose, and phosphate. Non-limiting examples of "polynucleotides" include DNA molecules (e.g., cDNA or genomic DNA), RNA (e.g., siRNA, mRNA, guide RNA, or self-amplifying mRNA (saRNA)), oligonucleotides (e.g., antisense oligonucleotides), combinations thereof, or hybrid molecules composed of DNA and RNA. Nucleic acids can be double-stranded or single-stranded, and can contain double-stranded and single-stranded fragments simultaneously. Double-stranded DNA and mRNA molecules are most preferred.

[0028] In the context of the present invention, the term "antisense oligonucleotide" refers to a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid present in a normal cell or present in an affected cell. Exemplary "antisense oligonucleotides" include antisense RNA, siRNA, and RNAi.

[0029] In the context of the present invention, polymersomes are vesicles with a polymer membrane that are typically, but not necessarily, formed from the self-assembly of a dilute solution of one or more amphiphilic block copolymers. The one or more amphiphilic block copolymers may be of different types, for example, diblock and triblock (ABA or ABC) amphiphilic block copolymers. Polymersomes may also be formed of tetrablock or pentablock copolymers. In the case of triblock copolymers, the central block is often protected from the environment by its neighboring blocks, whereas diblock copolymers self-assemble into a bilayer with two hydrophobic blocks arranged tail-to-tail, to a much greater effect. In most cases, the membrane of the vesicle has an insoluble middle layer and a soluble outer layer. The driving force for the formation of polymersomes by self-assembly is believed to be the microphase separation of the insoluble blocks, which tend to associate to protect themselves from contact with water. Polymersomes have such properties due to the large molecular weight of the constituent copolymers. Vesicle formation is facilitated by increasing the total molecular weight of the block copolymer. As a result, the diffusion of the (polymeric) amphiphile in these vesicles is much less compared to vesicles formed by lipids and surfactants. This small mobility of the polymer chains aggregated in the vesicle structure allows stable polymersome morphology to be obtained. Unless otherwise clearly defined, the terms "polymersome" and "vesicle" used herein are considered similar and may be used interchangeably. Importantly, polymersomes may be formed from one type of block copolymer or from two or more types of block copolymers. This also means that polymersomes may be formed from a mixture of polymersomes and therefore may contain two or more types of block copolymers.

[0030] In the context of the present invention, the polymer-lipid hybrid nanoparticles of the present invention comprise a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percent (mol%), present in the polymer-lipid hybrid nanoparticle is greater than the amount of said block copolymer, expressed in mole percent, present in the polymer-lipid hybrid nanoparticle, said mole percent being based on the total amount of all components forming the polymer-lipid nanoparticle. Such polymer-lipid hybrid nanoparticles are not polymersomes. They may have an electro-lucent amorphous internal structure surrounded by a peripheral bilayer. Exemplary polymer-lipid hybrid nanoparticles of the invention have one or more of the following characteristics: (i) a diameter of greater than 75 nm, e.g., the diameter is in the range of about 80 nm to about 450 nm, or the diameter is in the range of about 80 nm to about 140 nm, or the diameter is in the range of about 100 nm to about 140 nm (the diameter can be ascertained, e.g., by a dynamic light scattering (DLS) instrument using the preferred DLS parameter Z-average (d, nm). Z-average size is the intensity weighted harmonic mean particle diameter (see Figures 1 and 2)); (ii) a polydispersity index of greater than about 0.15. (iii) a zeta potential, preferably between -40 mV and +40 mV; (iv) physicochemical properties as set forth in one or more of Tables 2, 3, 6A, 6B, and Figures 1-16; and (v) an electrolucent amorphous interior structure surrounded by a peripheral bilayer membrane.

[0031] The polymer-lipid hybrid nanoparticles of the present invention may comprise soluble encapsulated antigens that are proteins and / or polynucleotides, preferably, said proteins are nucleases involved in gene editing or RNA editing, and the polynucleotides are selected from RNA (e.g., siRNA, mRNA, guide RNA, or self-amplifying mRNA (saRNA)) or DNA molecules.

[0032] In the context of the present invention, the term "encapsulated" means surrounded by a membrane (e.g., the membrane of the polymer-lipid hybrid nanoparticle of the present invention, e.g., embodied in the lumen of said polymer-lipid hybrid nanoparticle). The term "encapsulated" with respect to an antigen further means that said antigen is not incorporated into, covalently bound or conjugated to said membrane (e.g., the membrane of the polymer-lipid hybrid nanoparticle of the present invention).

[0033] In the context of the present invention, the term "antigen" refers to any substance that can specifically bind to a component of the immune system. Only antigens that can elicit (or raise or induce) an immune response are considered immunogenic and are called "immunogens". Exemplary non-limiting antigens are proteins and polynucleotides. Exemplary non-limiting protein antigens are nucleases involved in gene editing or RNA editing. Exemplary non-limiting polynucleotides are selected from RNA (e.g., siRNA, mRNA (e.g., as set forth in SEQ ID NO:1, 2, or 3), guide RNA, or self-amplifying mRNA (saRNA)) molecules, or DNA molecules. Antigens may originate from within the body ("self-antigens") or from the external environment ("non-self").

[0034] The term "polypeptide" is used herein equivalently to the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually peptides having less than 30 amino acids) comprise one or more amino acids linked together (resulting in an amino acid chain) via covalent peptide bonds. The term "polypeptide" as used herein describes a collection of molecules, e.g., molecules consisting of more than 30 amino acids. Polypeptides may further form multimers, e.g., dimers, trimers, and higher oligomers, i.e., may consist of multiple polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. As a result, the corresponding higher order structures of such multimers are called homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is an antibody molecule. An antibody molecule in its natural form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins. Here, the modifications are brought about, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well known in the art.

[0035] In the context of the present invention, the term "CD8(+) T cell-mediated immune response" refers to an immune response mediated by cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8(+) T cells, or killer T cells). Examples of cytotoxic T cells include, but are not limited to, antigen-specific effector CD8(+) T cells. In order for the T cell receptor (TCR) to bind to a class I MHC molecule, the former must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. These T cells are therefore called CD8(+) T cells. Once activated, TC cells undergo "clonal expansion" with the help of the cytokine interleukin-2 (IL-2), a growth and differentiation factor for T cells. This allows the number of cells specific for the target antigen to increase, which can then migrate throughout the body in search of antigen-positive somatic cells.

[0036] In the context of the present invention, the term "cellular immune response" refers to an immune response that does not involve antibodies, but rather the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.

[0037] In the context of the present invention, the term "humoral immune response" refers to immune responses mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Aspects involving antibodies are often referred to as antibody-mediated immunity.

[0038] In the context of the present invention, the term "stabilizer" may refer to a substance that renders or maintains a solution, mixture (e.g., polymer-lipid hybrid nanoparticles), suspension, or state resistant to chemical change. Exemplary, non-limiting stabilizers of the present invention include or consist of cholesterol, substituted or unsubstituted cholesterol moieties, or cholesterol derivatives, preferably, said cholesterol derivatives are hydroxylated cholesterol derivatives (e.g., hydroxycholesterol).

[0039] In the context of the present invention, the term "B cells", also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immune component of the adaptive immune system by secreting antibodies.

[0040] An "antibody," as used herein, is a protein comprising one or more polypeptides (including one or more binding domains, preferably antigen-binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes and the myriad immunoglobulin variable region genes. In particular, an "antibody," as used herein, is typically a tetrameric glycosylated protein composed of two light (L) chains, each of about 25 kDa, and two heavy (H) chains, each of about 50 kDa. Two types of light chains, called lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG being preferred in the context of the present invention. Antibodies according to the present invention are also envisaged that have an IgE constant domain or a part thereof bound by the Fcε receptor I. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide, called the J chain, and contain ten antigen-binding sites. In contrast, IgA antibodies contain two to five basic four-chain units that can polymerize and combine with the J chain to form multivalent assemblies. For IgG, the four-chain unit is generally about 150,000 daltons. Each light chain comprises an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain contains one N-terminal V domain (VH), three or four C domains (CH) and one hinge region. The constant domains are not directly involved in binding the antibody to the antigen, but can exhibit various effector functions, such as participating in antibody-dependent cellular cytotoxicity (ADCC). If the antibody must exert ADCC, it is preferably an IgG1 subtype antibody, whereas the IgG4 subtype does not have the ability to exert ADCC.

[0041] The term "antibody" also includes, but is not limited to, monoclonal, monospecific, polyspecific or multispecific antibodies, such as bispecific antibodies, humanized antibodies, camelized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutated antibodies, grafted antibodies, and antibodies generated in vitro, with chimeric or humanized antibodies being preferred. The term "humanized antibody" is generally defined for antibodies in which the specificities encoding the CDRs of the HC and LC have been transferred to a suitable human variable framework ("CDR grafting"). The term "antibody" also includes scFvs, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs) and nanobodies. In the context of the present invention, the term "antibody" also includes dimeric, trimeric or multimeric antibodies, or bifunctional, trifunctional or multifunctional antibodies having several antigen binding sites.

[0042] Furthermore, the term "antibody" as used in the present invention also relates to derivatives (including fragments) of the antibodies described herein. A "derivative" of an antibody includes an amino acid sequence that has been altered by the introduction of substitutions, deletions, or additions of amino acid residues. Furthermore, a derivative encompasses an antibody that has been modified by the covalent attachment of any type of molecule to the antibody or protein. Examples of such molecules include, but are not limited to, sugar, PEG, hydroxyl-, ethoxy-, carboxy-, or amine groups. Indeed, covalent modifications of antibodies can lead to, but are not limited to, glycosylation, pegylation, acetylation, phosphorylation, amidation.

[0043] The antibody in the context of the present invention is preferably an "isolated" antibody. When "isolated" is used to describe an antibody disclosed herein, it means an antibody that has been identified, separated, and / or recovered from components of its production environment. Preferably, an isolated antibody is free of association with all other components from its production environment. Contaminating components of its production environment, such as those resulting from recombinant transfected cells, are typically materials that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. However, an isolated antibody is usually prepared by at least one purification step.

[0044] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, e.g., an amino acid selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (GIn or Q); glutamic acid (GIu or E); glycine (GIy or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (VaI or V), although modified, synthetic, or rare amino acids may be used if desired. In general, amino acids can be grouped as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, VaI); negatively charged side chains (e.g., Asp, GIu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, GIn, GIy, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0045] A "polyclonal antibody" or "polyclonal antiserum" refers to an immune serum containing a mixture of antibodies specific for one antigen (monovalent, or specific, antiserum) or a mixture of antibodies specific for multiple antigens (polyvalent antiserum), which may be prepared from the blood of an animal immunized with one or more antigens.

[0046] The term "immunizing" refers to one or more steps of administering one or more antigens to a non-human animal so that antibodies can be produced in the animal.

[0047] Specifically, non-human animals are preferably immunized at least twice, and more preferably three times, with said polypeptide (antigen), optionally mixed with an adjuvant. An "adjuvant" is a non-specific stimulator of the immune response. An adjuvant may take the form of a composition containing either or both of the following components: (a) a substance designed to form a deposit that protects the antigen from rapid catabolism (e.g., mineral oil, alum, aluminum hydroxide, liposomes, or surfactants (e.g., pluronic polyols) and (b) a substance that non-specifically stimulates the immune response of the immunized host animal (e.g., by increasing lymphokine levels in the immunized host animal).

[0048] Exemplary molecules for increasing lymphokine levels include lipopolysaccharide (LPS) or its lipid A portion; Bordetalla pertussis; pertussis toxin; Mycobacterium tuberculosis; and muramyl dipeptide (MDP). Examples of adjuvants include Freund's adjuvant (optionally containing killed M. tuberculosis; complete Freund's adjuvant); aluminum hydroxide adjuvant; and monophosphoryl lipid A-synthetic trehalose dicorynomylcholeate (MPL-TDM).

[0049] The "non-human animal" immunized herein is preferably a rodent. A "rodent" is an animal belonging to the order Rodentia, a placental mammal. Exemplary rodents include mice, rats, guinea pigs, squirrels, hamsters, ferrets, etc., and mice are the preferred rodents for immunization according to the methods herein. Other non-human animals that can be immunized herein include non-human primates, such as Old World monkeys (e.g., baboons or macaques, including rhesus and cynomolgus monkeys; see U.S. Patent 5,658,570); birds (e.g., chickens); rabbits; goats; sheep; cows; horses; pigs; donkeys; dogs, etc.

[0050] "Screening" refers to subjecting one or more monoclonal antibodies (e.g., purified antibodies and / or hybridoma culture supernatants containing the antibodies) to one or more assays that qualitatively and / or quantitatively determine the ability of the antibodies to bind to an antigen of interest.

[0051] By "immunoassay" is meant an assay for determining the binding of an antibody to an antigen, in which either the antibody or the antigen, or both, are optionally adsorbed to a solid phase at some stage of the assay (i.e., an "immunosorbent" assay). Exemplary such assays include ELISA, radioimmunoassay (RIA), and FACS assay. Thus, in view of the above, the present invention provides monoclonal or polyclonal antibodies that can be obtained by the aforementioned methods for producing antibodies, i.e., by immunizing a non-human animal as described above.

[0052] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can form malignant tumors or cells that can invade adjacent tissues and metastasize through the lymphatic system or bloodstream to distant parts of the body.

[0053] Non-limiting examples of cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), stomach cancer, germ cell tumors, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic melanoma, e.g., cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers including asbestos-induced cancers, virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (which generate granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (which generate B, T, NK, and plasma cells), e.g., all types of leukemias, lymphomas, and myelomas, For example, acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (Ml), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant, lymphoproliferative disorder, true histiocytic lymphoma lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytoid lymphoma with Waldenström macroglobulinemia. lymphoma) (LPL); myelomas, e.g., IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called indolent myeloma), solitary, plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nerves, including seminoma, teratocarcinoma, astrocytoma, schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma, including small cell and cerebrospinal cell types, hematopoietic tumors of lymphoid lineage, including, but not limited to, T-cell tumors, e.g., T-cell prolymphocytic leukemia (T-PLL);Large granular lymphocyte leukemia (LGL), preferably of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of the above cancers. The methods described herein may also be used to treat metastatic cancer, refractory cancer (e.g., cancer refractory to previous immunotherapy, e.g., with blocking CTLA-4 or PD-1 or PD-L1 antibodies), and recurrent cancer.;

[0054] The term "subject" is intended to include living organisms. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. However, the subject (animal) may also be a non-mammalian animal, such as a bird or fish. In some preferred embodiments of the present invention, the subject is a human, while in some other preferred embodiments, the subject may be a livestock animal, which may be a mammal or a non-mammal. Examples of such non-mammals are birds (e.g., poultry, such as chickens, ducks, geese, or turkeys), fish (e.g., fish farmed in aquaculture, such as salmon, trout, or tilapia), or crustaceans (e.g., shrimp or prawns). Examples of mammalian (livestock) animals include goats; sheep; cows; horses; pigs; or donkeys. Other mammals include, for example, cats, dogs, mice, and rabbits. In an exemplary embodiment, the polymer-lipid hybrid nanoparticles of the present invention are used for vaccination or immunization of both mammalian and non-mammalian domestic animals (birds, fish, crustaceans) against viral infections (see the Examples section in this regard).Accordingly, in such cases, the polymer-lipid hybrid nanoparticles of the present invention may encapsulate soluble full-length viral proteins or soluble fragments of full-length viral proteins.

[0055] When used for vaccination of both humans and non-human animals, the polymer-lipid hybrid nanoparticles or compositions comprising the polymer-lipid hybrid nanoparticles of the present invention may be dissolved in an appropriate (pharmaceutical acceptable) buffer, such as phosphate buffered saline (PBS) or 0.9% saline (an isotonic solution of 0.90% w / v NaCl with an osmolality of 308 mOsm / L) and administered orally to the respective subject (see also the Examples section).

[0056] As used herein, the term "LNP-Onpattro" may be used synonymously with the terms "LNP-ON" or "LNP-ONP" and may refer to lipid nanoparticles containing DMG-PEG, DSPC, MC3, and Chol, for example, in a molar ratio of 1.5:10.0:50:38.5.

[0057] In exemplary embodiments of these polymer-lipid hybrid nanoparticles and oral formulations, the polymer-lipid hybrid nanoparticles used for vaccination have encapsulated therein a viral antigen, including a soluble portion of a viral antigen, including influenza hemagglutinin, swine influenza hemagglutinin, foot and mouth disease (FMD) viral proteins, such as VP1, VP2, or VP3 coat proteins (VP1 coat protein contains the major antigenic determinants of the FMD virion and therefore variations in its sequence should be responsible for the high viral antigenic variability), ovalbumin (OVA), or porcine epidemic diarrhea (PED) virus spike (SPIKE) protein.

[0058] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure, or at least partially halt, the disease and its complications in a patient already suffering from the disease. Amounts effective for this use depend on the severity of the infection and the general state of the subject's own immune system. The term "patient" includes humans and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0059] The appropriate dosage or therapeutically effective amount of the antibody or its antigen-binding portion depends on the condition to be treated, the severity of the condition, previous therapy, and the patient's medical history and response to the therapeutic agent.The appropriate dosage can be adjusted according to the judgment of the attending physician so that it can be administered to the patient at once or over a series of administrations.The pharmaceutical composition can be administered as the sole therapeutic agent or in combination with additional therapy as necessary.

[0060] If the pharmaceutical composition is lyophilized, the lyophilized material is first reconstituted with a suitable liquid prior to administration. The lyophilized material may be reconstituted, for example, with sterile water for injection (BWFI), saline, phosphate buffered saline (PBS), or the same formulation in which the protein was dissolved prior to lyophilization.

[0061] Pharmaceutical compositions for injection may be provided in unit dosage form, for example, in ampoules or multi-dose containers, together with added preservatives.In addition, many recent drug delivery approaches have been developed, and the pharmaceutical compositions of the present invention are suitable for administration using these new methods, for example, Inject-ease, Genject, injector pens, for example, Genen, and needleless devices, for example, MediJector and BioJector.The pharmaceutical compositions can also be adapted to administration methods that have not yet been discovered.See also Langer, 1990, Science, 249: 1527-1533.

[0062] Pharmaceutical compositions may also be formulated as depot preparations. Such long-acting formulations may be administered by implantation (e.g., subcutaneous, ligament or tendon, subsynovial or intramuscular), by subsynovial injection, or by intramuscular injection. Thus, for example, formulations may be modified with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or may be modified as sparingly soluble derivatives, e.g., sparingly soluble salts.

[0063] The pharmaceutical compositions may also take the form of a variety of conventional depots used for administration to provide the reactive compositions, including, for example, solid, semi-solid, and liquid dosage forms, such as liquid solutions or suspensions, slurries, gels, creams, balms, emulsions, lotions, powders, sprays, foams, pastes, ointments, salves, balms, and drops.

[0064] The pharmaceutical composition may be provided in a vial, pack, or dispenser device, which may contain one or more unit dosage forms containing the active ingredient, if desired.In one embodiment, the dispenser device may be equipped with a syringe with a single dose of the liquid formulation ready for injection.The syringe may be accompanied by instructions for administration.

[0065] The formulations described herein are useful as pharmaceutical compositions in the treatment and / or prevention of pathological medical conditions described herein in patients in need of such treatment and / or prevention. The term "treatment" refers to therapeutic treatment and prophylactic or preventative measures. Treatment includes application or administration of the formulation to the body, isolated tissue or cells from a patient with a disease / disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder for the purpose of curing, healing, alleviating, alleviating, altering, curing, ameliorating, improving, or affecting the disease, the symptom of a disease, or the predisposition to a disease.

[0066] As used herein, the terms "treat" and "treatment" refer to administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to a subject. A "therapeutically effective amount" refers to an amount of a pharmaceutical composition or antibody sufficient to treat or ameliorate a disease or disorder, delay the onset of a disease, or provide any therapeutic benefit in the treatment or management of a disease.

[0067] As used herein, the term "prevention" refers to the use of an agent to prevent the onset of a disease or disorder. A "prophylactically effective amount" defines the amount of an active ingredient or pharmaceutical agent sufficient to prevent the onset or recurrence of a disease.

[0068] As used herein, the terms "disorder" and "disease" are used interchangeably to refer to a condition in a subject. In particular, the term "cancer" is used interchangeably with the term "tumor."

[0069] Kits of the present invention typically include the container described above, as well as one or more other containers containing materials desirable from a commercial or user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts and instructions for use.

[0070] In the context of the present invention, the term "soluble antigen" as used herein refers to an antigen that can be dissolved or liquefied. The term "soluble antigen" includes antigens that have been "solubilized", i.e., made particularly soluble or more soluble in water by the action of detergents or other agents. Exemplary non-limiting soluble antigens of the present invention include polypeptides derived from insoluble portions of proteins, hydrophobic polypeptides that are made soluble for encapsulation, as well as aggregated polypeptides that dissolve as aggregates. In some aspects, antigens of the present invention (e.g., membrane proteins) are solubilized with the aid of detergents, detergents, temperature changes, or pH changes.

[0071] In some aspects, the present invention provides polymer-lipid hybrid nanoparticles comprising a lipid and a block copolymer, wherein the amount of said lipid present in the polymer-lipid hybrid nanoparticle, expressed as a mole percent (i.e., mol %), is greater than the amount of said block copolymer present in the polymer-lipid hybrid nanoparticle, expressed as a mole percent, said mole percent being based on the total amount of all components forming the polymer-lipid nanoparticle.

[0072] In some aspects, there is provided a polymer-lipid hybrid nanoparticle as described herein, wherein the lipid (e.g., ionizable lipid) is selected from the group consisting of the ionizable lipid DLin-MC3-DMA (also referred to as MC3) and the ionizable lipid C12-200.

[0073] In some aspects, the present invention provides a polymer-lipid hybrid nanoparticle as described herein, wherein the block copolymer is selected from the group consisting of PBD-PEO block copolymers, PCL-PEO block copolymers, and DMG-PEG block copolymers (e.g., Table 1).

[0074] Table 1. Exemplary polymers and lipids used in the polymer-lipid hybrid nanoparticles of the present invention. TIFF2025508798000001.tif32164TIFF2025508798000002.tif246164

[0075] In some aspects, the present invention provides polymer-lipid hybrid nanoparticles as described herein, having a lipid to block copolymer molar % ratio of about 31.8 to 12 and about 35 to 2.5.

[0076] In some aspects, the present invention provides polymer-lipid hybrid nanoparticles as described herein, further comprising a stabilizer, e.g., comprising or consisting of cholesterol (also known as CHOL).

[0077] In some aspects, the present invention provides polymer-lipid hybrid nanoparticles as described herein further comprising another lipid, wherein the other lipid is selected from the group consisting of DMPC, DSPC, DOPE, DOTAP, DODAP, DOTMA, DODMA, DDA, 18:1 PA (1,2-dioleoyl-sn-glycero-3-phosphate), 14:0 PA (1,2-dimyristoyl-sn-glycero-3-phosphate), 18:1 BMP (bis(monooleoylglycero)phosphate) (e.g., Table 1).

[0078] In some aspects, the present invention provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: (i) PBD-PEO, MC3, CHOL; (ii) PBD-PEO, C12-200, CHOL; (iii) PBD-PEO, DOPE, C12-200, CHOL; (iv) PBD-PEO, DOPE, C12-200, CHOL; (v) PBD-PEO, DOPE, C12-200, CHOL; (vi) PBD-PEO, DOPE, C12-200, CHOL; (vii) DMG-P The present invention provides a polymer-lipid hybrid nanoparticle as described herein, which consists of EG, DSPC, MC3, CHOL; (viii) PCL-PEO, DMPC, MC3, CHOL; (ix) PCL-PEO, DMPC, MC3, CHOL; (x) PCL-PEO, DMPC, MC3, CHOL; or (xi) PCL-PEO, DMPC, MC3, CHOL; (xii) PLGA-PEO, DMPC, MC3, CHOL.

[0079] In some aspects, the present invention provides a polymer-lipid hybrid nanoparticle as described herein, further comprising a soluble encapsulated antigen, which is a protein and / or a polynucleotide.

[0080] In some aspects, the present invention provides polymer-lipid hybrid nanoparticles as described herein that are capable of maintaining long-term stability and / or efficacy of said polynucleotides (e.g., mRNA, e.g., those set forth in SEQ ID NOs:1, 2, or 3).

[0081] In some aspects, the present invention provides compositions comprising the polymer-lipid hybrid nanoparticles described herein.

[0082] In some aspects, the present invention provides a method for delivering a nucleotide to the interior of a cell without the use of a viral vector as a delivery vehicle, the method comprising the steps of: (i) providing a polymer-lipid hybrid nanoparticle and / or composition of the present invention; and (ii) contacting said polymer-lipid hybrid nanoparticle and / or composition with a cell.

[0083] In some aspects of the invention, the polymer-lipid hybrid nanoparticles of the invention are selected from the group consisting of: (a) BNP-012 having 10 mM (mol%) DOTAP:cholesterol:DSPC:PBD-b-PEO (40:48:10:2) and / or BNP-025 having 10 mM (mol%) DOTMA:cholesterol:DSPC:PBD-b-PEO (40:48:10:2); (b) BNP-002 having 5 mM (mol%) DLin-MC3-DMA:cholesterol:PBD-b-PEO (49:39:12); or (c) BNP-002.2 having 5 mM (mol%) DLin-MC3-DMA:cholesterol:DSPC:PBD-b-PEO (49.3:39.0:10.1:1.6).

[0084] In some aspects of the invention, the polymer-lipid hybrid nanoparticles of the invention are capable of targeting (e.g., predominantly targeting) tissues and / or cells of an organ selected from the group consisting of liver, spleen, and lung, and preferably, said targeting is performed without the use of a functional ligand, and more preferably, (a) the following polymer-lipid hybrid nanoparticles are suitable for targeting (e.g., used for targeting) to the lung: BNP-012 with 10 mM (mol %) DOTAP:Cholesterol:DSPC:PBD-b-PEO (40:48:10:2) and / or 10 mM (mol %) DOTMA:Cholesterol:DSPC:PB (b) the following polymer-lipid hybrid nanoparticles are suitable for targeting to the liver (e.g., for use in targeting the liver): BNP-002 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:PBD-b-PEO (49:39:12); (c) the following polymer-lipid hybrid nanoparticles are suitable for targeting to the spleen (e.g., for use in targeting the spleen): BNP-002.2 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:DSPC:PBD-b-PEO (49.3:39.0:10.1:1.6).

[0085] Based on the above, a new class of lipid hybrid nanoparticles has been developed in the course of the present invention, which is particularly suitable for mRNA delivery. Exemplary optimal polymer-lipid hybrid nanoparticles of the present invention exhibit favorable physicochemical properties and / or excellent encapsulation efficiency (about 100%). As evidenced by high levels of Luc and OVA protein expression (see, e.g., Experimental Section below), optimal formulations of the polymer-lipid hybrid nanoparticles of the present invention outperform with improved in vitro transfection potency and / or long-term thermal stability compared to benchmark LNP-ONs. Furthermore, ACM polymer-lipid hybrid nanoparticle formulations exhibit lower cytotoxicity compared to benchmark LNP-ONs (see, e.g., Experimental Section below). Importantly, optimal formulations of the polymer-lipid hybrid nanoparticles of the present invention exhibit potent in vivo mRNA delivery potency comparable to benchmark LNP-ONs. Furthermore, OVA mRNA formulations can potently activate cDC1 and cDC2 in lymph nodes to promote antigen surface presentation. In summary, the present invention provides a novel class of polymer-lipid hybrid nanoparticles with efficient protein and antigen expression and improved thermal stability, thereby holding great potential for therapeutic mRNA delivery across a wide range of diseases.

[0086] The present invention is also characterized by the following items.

[0087] 1. A polymer-lipid hybrid nanoparticle comprising a lipid and a block copolymer, the amount of the lipid, expressed in mole percent (mol%), present in the polymer-lipid hybrid nanoparticle is greater than the amount of the block copolymer, expressed in mole percent, present in the polymer-lipid hybrid nanoparticle; the mole percentage is based on the total amount of all components forming the polymer-lipid nanoparticle; Preferably, more is at least about 2 times more, more preferably, more is at least about 3 times more, most preferably, more is at least about 4 times more, and even most preferably, more is at least about 5 times more. The polymer-lipid hybrid nanoparticles.

[0088] 2. The mole percent ratio of the lipid to the block copolymer is about 31.8:12 to about 35:2.5, and preferably, the mole percent ratio is as follows: a) 49:12, b) 35:2.5; c) 31.8:12, d) 35:12, e) 23.8:4.8, f) 45:10, g)49:8 The polymer-lipid hybrid nanoparticle according to any one of the preceding items, selected from the group consisting of:

[0089] 3. Features include: a) the polymer-lipid hybrid nanoparticles are synthetic; b) a diameter of more than 75 nm, preferably the diameter is in the range of about 80 nm to about 450 nm, more preferably the diameter is in the range of about 80 nm to about 140 nm, and most preferably the diameter is in the range of about 100 nm to about 140 nm; c) a polydispersity index (PDI) greater than about 0.15, preferably greater than about 0.17, more preferably a PDI of about 0.175 to about 0.245, and / or d) a zeta potential of about -40 mV to about +40 mV, preferably a zeta potential of more than 12 mV; e) spherical particles having an amorphous structure; f) having a multi-compartment structure; g) They are vesicles with a heterogeneous structure surrounded by a bilayer (e.g., capable of fusion and budding). The polymer-lipid hybrid nanoparticle of any one of the preceding claims, comprising one or more of:

[0090] 4. Further comprising a stabilizer; Preferably, the stabilizing agent is cholesterol (or a stabilizing agent represented by, for example, formula I: TIFF2025508798000003.tif21128, CHOL) and / or a substituted or unsubstituted cholesterol moiety, The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0091] 5. The polymer-lipid hybrid nanoparticle of any one of the preceding items, wherein the stabilizer is selected from the group consisting of cholesterol, substituted or unsubstituted cholesterol moieties, and cholesterol derivatives, preferably, the cholesterol derivative is a hydroxylated cholesterol derivative (e.g., hydroxycholesterol).

[0092] 6. Further comprising another lipid; Preferably, the lipid is: a) For example, formula II: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 14:0 PC), with TIFF2025508798000004.tif23128; b) For example, formula III: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, 18:0 PC), with TIFF2025508798000005.tif22128; c) For example, formula IV: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), with TIFF2025508798000006.tif22128; d) For example, formula V: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), with TIFF2025508798000007.tif25128; e) For example, formula VI: 1,2-dioleoyl-3-trimethylammonium propane (DODAP), with TIFF2025508798000008.tif22128; f) For example, formula VII: 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), with TIFF2025508798000009.tif14128; g) For example, formula VIII: 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), with TIFF2025508798000010.tif13128; h) For example, formula IX: Dimethyldioctadecylammonium (DDA) with TIFF2025508798000011.tif16128, i) For example, formula X: 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA), with TIFF2025508798000012.tif23128; j) For example, formula XI: 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), with TIFF2025508798000013.tif25128; k) For example, formula XII: Bis(monooleoylglycero)phosphate (e.g., S and / or R isomers) (18:1 BMP) with TIFF2025508798000014.tif36128 A cationic lipid, an ionized cationic lipid, and / or an anionic lipid selected from the group consisting of: The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0093] 7. The lipid (e.g., an ionizable lipid) is: a) Formula XIII: ionized lipid DLin-MC3-DMA (or MC3, i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), with TIFF2025508798000015.tif16128; b) For example, formula XIV: Ionized lipid C12-200, with TIFF2025508798000016.tif30128, c) For example, formula XVIII (e.g., CAS number 2322290-93-5): Ionized lipid 306O with TIFF2025508798000017.tif29128 i10 The polymer-lipid hybrid nanoparticle according to any one of the preceding items, selected from the group consisting of:

[0094] 8. A block copolymer comprising: a) poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymers (e.g., a PB-PEO diblock copolymer contains 5-50 blocks of PB and 5-50 blocks of PEO); b) poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymers (e.g., those of Formula XVII: TIFF2025508798000018.tif25170, or a linear chain having formula XVIII: Comb-type with TIFF2025508798000019.tif45128, c) poly(dimethylsiloxane)-poly(acrylic acid) (PDMS-PAA); d) a PBD-PEO diblock copolymer, the PBD-PEO diblock copolymer being For example, Formula XV: TIFF2025508798000020.tif18128, where n=22, b indicates block, m=12, *=OCH3, 5-50 blocks of PBD and 5-50 blocks of PEO, for example, PBD where k=1000 Da. 1.2k -b-PEO 0.6k The PBD-PEO block copolymer, e) a PCL-PEO block copolymer, the PCL-PEO diblock copolymer being For example, Formula XVI: I have TIFF2025508798000021.tif19128, In the formula, n=22, m=29, * = H, PCL having 5 to 50 blocks and PEO having 5 to 50 blocks, for example, k = 1000 Da, 3.3k -b-PEO 1k The PCL-PEO block copolymer, f) A PLGA-PEO block copolymer, the PLGA-PEO diblock copolymer being For example, Formula XVI: TIFF2025508798000022.tif30128, where x=23, y=4, m=26, n=29, 5-50 blocks of PLGA and 5-50 blocks of PEO, e.g., k=1000 Da, 1.9k -b-PEO 1k The PLGA-PEO block copolymer, g) PLA-PEO block copolymers, the PLA-PEO diblock copolymers comprising 5-50 blocks of PLA and 5-50 blocks of PEO; h) PDMS-PEO (poly(dimethylsiloxane)-poly(ethylene oxide) block copolymers or PMOXA-PDMS-PMOXA (poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) triblock copolymers, where the PDMS copolymers contain 5-60 blocks of PDMS, 5-50 blocks of PEO, and 5-50 blocks of PMOXA; i) PVP-PLA (polyvinylpyrrolidone-polylactic acid) block copolymer; j) Poly(N-vinylpyrrolidone-b-polylactic acid; k) PIB-PAA (polyisobutylene-polyacrylic acid); l) PIP-PEO (polyisoprene-polyethylene oxide); m) For example, formula XVII: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (i.e., DMG-PEG block copolymer, e.g., DMG-PEG having the formula: 2k ), n) Poly(ε-caprolactone-co-δ-valerolactone) P(CL-VL)-PEG diblock copolymer The polymer-lipid hybrid nanoparticle according to any one of the preceding items, selected from the group consisting of:

[0095] 9. The polymer-lipid hybrid nanoparticle according to item 8, wherein the block copolymer is PBD-b-PEO or PCL-PEO.

[0096] 10. The following: a) PBD-PEO, MC3, CHOL, preferably in a molar ratio of about 12:49:39; b) PBD-PEO, C12-200, CHOL, preferably in a molar ratio of about 12:49:39; c) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 2.5:16:35:46.5; d) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 12:12.8:31.8:43.4; e) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 12:6.5:35:46.5; f) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 4.8:23.8:23.8:47.6; g) DMG-PEG, DSPC, MC3, CHOL, preferably in a molar ratio of about 1.6:10.1:49.3:39.0; h) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 10:10:45.0:35.0; i) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 10:10:45.0:35.0; j) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 8:4:49.0:39.0; k) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 8:4:49.0:39.0; l) DLin-MC3-DMA, cholesterol, PBD-b-PEO, preferably in a molar ratio of about 49:39:12; m) DLin-MC3-DMA, cholesterol, DSPC:PBD-b-PEO, preferably in a molar ratio of about 49.3:39.0:10.1:1.6; n) DOTAP, cholesterol, DSPC:PBD-b-PEO, preferably in a molar ratio of about 40:48:10:2; o) DOTMA, cholesterol:DSPC, PBD-b-PEO, preferably in a molar ratio of about 40:48:10:2 2. The polymer-lipid hybrid nanoparticle of any one of the preceding claims, comprising or consisting of:

[0097] 11. Further comprising a soluble encapsulated antigen which is a protein and / or a polynucleotide; Preferably, the protein is a nuclease involved in gene editing or RNA editing, and the polynucleotide is selected from an RNA (e.g., an siRNA, an mRNA (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3), a guide RNA, or a self-amplifying mRNA (saRNA), or an antisense oligonucleotide) molecule, or a DNA molecule; The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0098] 12. The polymer-lipid hybrid nanoparticle of any one of the preceding items, preferably capable of maintaining long-term stability and / or efficacy of the polynucleotide (e.g., siRNA, mRNA, guide RNA, or self-amplifying mRNA (saRNA)) compared to LNP-ON.

[0099] 13. The nanoparticles comprising: a) characterized as set forth in one or more of Tables 1, 2, 3, 4, 5, 6A, and / or 6B, Tables 7, 8, 9, 10, and / or 11 herein; b) an electrolucent amorphous inner structure surrounded by a peripheral bilayer; c) expressing the polynucleotide (e.g., 6 hours or 24 hours after transfection), Preferably, the polynucleotide is selected from an RNA (e.g., an mRNA or a self-amplifying mRNA (saRNA) or an antisense oligonucleotide) molecule or a DNA molecule. said expressing d) eliciting a cellular and / or humoral immune response; e) (i) activation of dendritic cells (DCs) (e.g., in lymph nodes); and / or (ii) surface display of the polypeptide encoded by the polynucleotide eliciting an immune response comprising f) CD8 (+) Inducing a T cell mediated immune response, Preferably, the induction is in vivo, ex vivo or in vitro induction. The inducing, g) CD4 (+) Inducing a T cell mediated immune response, Preferably, the induction is in vivo, ex vivo or in vitro induction. The inducing, h) eliciting a humoral immune response, including the production of specific antibodies (e.g., against a polypeptide encoded by the polynucleotide), More preferably, the humoral immune response is an in vivo, ex vivo, or in vitro immune response. The inducing, i) is capable of targeting (e.g., 6 hours after IV injection) predominantly (e.g., at least 51%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, at least 95%, at least 98%, or at least 99%) to the spleen (e.g., BNP-008, e.g., a polymer-lipid hybrid nanoparticle having a molar ratio of PBD-PEO:DOPE:C12-200:CHOL of 12:6.5:35:46.5); j) being capable of targeting predominantly (e.g., at least 51%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, at least 95%, at least 98%, or at least 99%) to the liver (e.g., 6 hours after IV injection); Preferably, the polymer-lipid hybrid nanoparticles are BNP-002 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:PBD-b-PEO (49:39:12), more preferably, the polymer-lipid hybrid nanoparticles are PCL-008 (e.g., as described herein, e.g., composed of PCL-PEO:DMPC:MC3:CHOL with a molar ratio of 10:10:45.0:35.0) and / or PCL-012 (e.g., as described herein, e.g., composed of PCL-PEO:DMPC:MC3:CHOL with a molar ratio of 8:4:49.0:39.0). said targeting being possible, k) being able to target predominantly (e.g., at least 51%, e.g., at least 76%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, at least 95%, at least 98%, or at least 99%) the lungs (e.g., 6 hours after IV injection); Preferably, the polymer-lipid hybrid nanoparticles are BNP-012 with 10 mM (mol%) DOTAP:cholesterol:DSPC:PBD-b-PEO (40:48:10:2) and / or BNP-025 with 10 mM (mol%) DOTMA:cholesterol:DSPC:PBD-b-PEO (40:48:10:2). The ability to target The polymer-lipid hybrid nanoparticle according to any one of the preceding items, which is capable of performing one or more of and / or is characterized by one or more of the following:

[0100] 14. The nanoparticles can target antigen-presenting cells, the nanoparticles comprising: A ligand (e.g., an antibody) capable of targeting and / or binding to the antigen-presenting cell. 4. The polymer-lipid hybrid nanoparticle of any one of the preceding claims, wherein the polymer-lipid hybrid nanoparticle is not attached to a

[0101] 15. The nanoparticles are capable of targeting cells, the nanoparticles comprising: A ligand (e.g., an antibody) capable of targeting and / or binding to said target cell. The polymer-lipid hybrid nanoparticle of any one of the preceding claims, wherein the polymer-lipid hybrid nanoparticle is attached to

[0102] 16. The nanoparticles can selectively target tissues and / or organs (e.g., liver, spleen, lungs, and / or kidneys), and the nanoparticles are functionalized with Any ligand capable of targeting and / or binding to said target tissues and organs (e.g., N-acetylgalactosamine (GalNac), antibodies) The polymer-lipid hybrid nanoparticle of any one of the preceding claims, which is not attached to a

[0103] 17. The nanoparticles are capable of selectively targeting tissues and / or organs (e.g., the liver, spleen, lungs, and / or kidneys), and the nanoparticles are functionalized or Any ligand capable of targeting and / or binding to said target tissues and organs (e.g., N-acetylgalactosamine (GalNac), antibodies) The polymer-lipid hybrid nanoparticle of any one of the preceding claims, wherein the polymer-lipid hybrid nanoparticle is attached to

[0104] 18. It can be obtained by a solvent dispersion method or a micromixing method (for example, as described in the experimental section herein); For example, the N / P ratio (N in the ionized cationic lipid and P in the mRNA) is in the range of about 4 to about 40. The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0105] 19. The polymer-lipid hybrid nanoparticle of any one of the preceding items, which is not a polymersome.

[0106] 20. The polymer-lipid hybrid nanoparticles are a) Liver, b) spleen, c) Lungs and can target (e.g., predominantly, e.g., at least 51%) tissues and / or cells of an organ selected from the group consisting of: Preferably, the targeting is performed without the use of a functional ligand, and more preferably a) The following polymer-lipid hybrid nanoparticles are suitable for lung targeting: BNP-012 with 10 mM (mol%) DOTAP:Cholesterol:DSPC:PBD-b-PEO (40:48:10:2) and / or BNP-025 with 10 mM (mol%) DOTMA:Cholesterol:DSPC:PBD-b-PEO (40:48:10:2); b) The following polymer-lipid hybrid nanoparticles are suitable for targeting to the liver: BNP-002 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:PBD-b-PEO (49:39:12); PCL-008 (e.g., PCL-PEO:DMPC:MC3:CHOL (10:10:45.0:35.0) and / or PCL-012 (e.g., PCL-PEO:DMPC:MC3:CHOL (8:4:49.0:39.0), c) The following polymer-lipid hybrid nanoparticles are suitable for targeting the spleen: BNP-002.2 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:DSPC:PBD-b-PEO (49.3:39.0:10.1:1.6); The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0107] 21. The following: a) BNP-012 with 10 mM (mol%) DOTAP:Cholesterol:DSPC:PBD-b-PEO (40:48:10:2) and / or BNP-025 with 10 mM (mol%) DOTAP:Cholesterol:DSPC:PBD-b-PEO (40:48:10:2); b) BNP-002 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:PBD-b-PEO (49:39:12); c) BNP-002.2 with 5 mM (mol%) DLin-MC3-DMA:cholesterol:DSPC:PBD-b-PEO (49.3:39.0:10.1:1.6); d) PCL-008 with PCL-PEO:DMPC:MC3:CHOL (10:10:45.0:35.0); e) PCL-012 with PCL-PEO:DMPC:MC3:CHOL (8:4:49.0:39.0) The polymer-lipid hybrid nanoparticle according to any one of the preceding items, selected from the group consisting of:

[0108] 22. Has organ tropism (e.g., a preference for a particular organ, e.g., a preference for delivery to and / or interaction with a particular organ); Preferably, the organ tropism is selected from the group consisting of hepatotropism, spleentropism, and pneumotropism. The polymer-lipid hybrid nanoparticle according to any one of the preceding items.

[0109] 23. The polymer-lipid hybrid nanoparticle according to any one of the preceding paragraphs, which consists of less than five components, preferably three or four components, and more preferably comprises a block copolymer (e.g., PBD-b-PEO or PCL-PEO) according to any one of the preceding paragraphs.

[0110] 24. The polymer-lipid hybrid nanoparticle of any one of the preceding paragraphs, which consists of five or more components and includes a block copolymer (e.g., PBD-b-PEO or PCL-PEO) of any one of the preceding paragraphs.

[0111] 25. A composition comprising the polymer-lipid hybrid nanoparticles described in any one of the preceding items, preferably comprising one or more polymer-lipid hybrid nanoparticles described in any one of the preceding items.

[0112] 26. A composition according to any one of the preceding items, which is a pharmaceutical composition or a diagnostic composition.

[0113] 27. A composition according to any one of the preceding items, which is a therapeutic composition, an immunogenic composition, an antigenic composition, or an immunotherapeutic composition.

[0114] 28. A composition or polymer-lipid hybrid nanoparticle according to any one of the preceding items, comprising one or more oligonucleotides, nucleases, and guide RNAs for modifying and / or manipulating and / or disrupting genetic material (e.g., a genome and / or transcriptome) or a template (e.g., a nucleotide sequence, e.g., RNA or DNA) inside a cell.

[0115] 29. The composition of any one of the preceding items, which is a non-viral delivery system capable of delivering nucleotides to the interior of a cell.

[0116] 30. The composition of any one of the preceding items, which is a vaccine.

[0117] 31. An isolated antigen-presenting cell or hybridoma cell exposed to a polymer-lipid hybrid nanoparticle or composition according to any one of the preceding items.

[0118] 32. An antigen-presenting cell according to any one of the preceding items, comprising a dendritic cell.

[0119] 33. The antigen-presenting cell of any one of the preceding items, comprising a macrophage.

[0120] 34. An antigen-presenting cell according to any one of the preceding paragraphs, comprising a B cell.

[0121] 35. A composition according to any one of the preceding paragraphs, comprising the polymer-lipid hybrid nanoparticle, composition, antigen-presenting cell, and / or hybridoma according to any one of the preceding paragraphs, and further comprising a pharma- ceutically acceptable excipient or carrier.

[0122] 36. A kit comprising the polymer-lipid hybrid nanoparticle, composition, antigen-presenting cell, hybridoma, and / or vaccine described in any one of the preceding items.

[0123] 37. A method for inducing an immune response in a subject (e.g., a human), comprising the steps of: i) providing to said subject the polymer-lipid hybrid nanoparticle, composition, antigen presenting cell, hybridoma, and / or vaccine according to any one of the preceding paragraphs; ii) administering the polymer-lipid hybrid nanoparticles, compositions, antigen presenting cells, hybridomas, and / or vaccines to the subject, preferably by intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous injection, or non-invasive administration to a mucosal surface.

[0124] 38. A method for delivering a nucleotide to the interior of a cell without using a viral vector as a delivery means, comprising the steps of: i) providing a polymer-lipid hybrid nanoparticle or composition according to any one of the preceding paragraphs; ii) contacting the polymer-lipid hybrid nanoparticle or composition with a cell.

[0125] 39. A method of modifying and / or manipulating and / or disrupting genetic material (e.g., genome or transcriptome) or a template (e.g., a nucleotide sequence, e.g., RNA or DNA) inside a cell, comprising the steps of: i) providing a polymer-lipid hybrid nanoparticle or composition according to any one of the preceding paragraphs (e.g., comprising one or more oligonucleotides, a nuclease, and a guide RNA); ii) contacting the polymer-lipid hybrid nanoparticle or composition with a cell.

[0126] 40. A polymer-lipid hybrid nanoparticle, composition, antigen presenting cell, hybridoma, kit, and / or vaccine according to any one of the preceding items for use as a medicine and / or in therapy (e.g., veterinary use).

[0127] 41. The following method: i) methods of treating and / or preventing a disease or disorder; ii) methods for discovering and / or screening and / or preparing antibodies; iii) methods of producing or preparing immunogenic or immunostimulatory compositions; iv) A method for targeted delivery of one or more polypeptides encoded by a polynucleotide, and most preferably, the targeted delivery is performed within a subject; v) methods of stimulating an immune response to one or more polypeptides encoded by the polynucleotides; vi) CD8 (+) Methods for triggering cross-protection induced by T cell-mediated immune responses vii) CD8 (+) T cell-mediated immune response and / or CD4 (+) Methods for triggering an immune response, including a T cell mediated immune response; viii) A method for treating, ameliorating, preventing and / or diagnosing an infectious disease, preferably wherein the infectious disease is a viral or bacterial infectious disease, more preferably wherein the viral infectious disease is selected from the group consisting of influenza infection, PED virus infection, food and mouth virus infection, respiratory syncytial virus infection, herpes virus infection, ix) methods for treating, ameliorating, preventing, or diagnosing cancer or autoimmune diseases; x) methods for sensitizing cancer cells to chemotherapy; xi) methods for inducing apoptosis in cancer cells; xii) a method for stimulating an immune response in a subject; xiii) methods for immunizing non-human animals; xiv) methods for preparing hybridomas; xv) methods for modifying and / or manipulating and / or disrupting genetic material (e.g., genome and / or transcriptome) or templates (e.g., nucleotide sequences, e.g., RNA or DNA) inside a cell; xvi) methods for delivering nucleotides to the interior of a cell without using a viral vector as a delivery means; xvii) a method for targeting antigen presenting cells (e.g., the method does not include a step of attaching a ligand to the polymer-lipid hybrid nanoparticle); xviii) a method for targeting a cell, preferably wherein the cell is not an antigen-presenting cell (e.g., the method comprises attaching a ligand to the polymer-lipid hybrid nanoparticle); xix) methods for maintaining the long-term stability and / or efficacy of the polynucleotide (e.g., siRNA, mRNA (e.g., SEQ ID NO:1, 2, or 3), guide RNA, or self-amplifying mRNA (saRNA)); xx) A method according to any one of i) to xvix), which is an in vivo and / or ex vivo and / or in vitro method. The polymer-lipid hybrid nanoparticle, composition, antigen presenting cell, hybridoma, kit, and / or vaccine according to any one of the preceding items for use in one or more of the following:

[0128] 42. The following: i) treating and / or preventing a disease or disorder; ii) discovering and / or screening and / or preparing antibodies; iii) generating or preparing an immunogenic or immunostimulatory composition; iv) targeted delivery of one or more polypeptides encoded by the polynucleotides, most preferably within the subject; v) stimulating an immune response against one or more polypeptides encoded by the polynucleotides; vi) triggering cross-protection induced by CD8(+) T cell-mediated immune responses; vii) triggering an immune response, including a CD8(+) T cell-mediated immune response and / or a CD4(+) T cell-mediated immune response; viii) treating, ameliorating, preventing and / or diagnosing an infectious disease, preferably wherein the infectious disease is a viral or bacterial infectious disease, more preferably wherein the viral infectious disease is selected from the group consisting of influenza infection, PED virus infection, food and mouse virus infection, respiratory syncytial virus infection, herpes virus infection; ix) treating, ameliorating, preventing, or diagnosing cancer or autoimmune diseases; x) sensitizing cancer cells to chemotherapy; xi) inducing apoptosis in cancer cells; xii) stimulating an immune response in a subject; xiii) immunizing a non-human animal; xiv) preparing hybridomas; xv) modifying and / or manipulating and / or disrupting genetic material (e.g., genome and / or transcriptome) or templates (e.g., nucleotide sequences, e.g., RNA or DNA) within a cell; xvi) delivering nucleotides to the interior of a cell without using a viral vector as a delivery means; xvii) targeting antigen presenting cells (e.g., the use does not include attaching a ligand to the polymer-lipid hybrid nanoparticle); xviii) targeting a cell, preferably the cell is not an antigen-presenting cell (e.g., using includes attaching a ligand to the polymer-lipid hybrid nanoparticle); xix) maintaining long-term stability and / or efficacy of polynucleotides (e.g., siRNA, mRNA (e.g., SEQ ID NO:1, 2, or 3), guide RNA, or self-amplifying mRNA (saRNA)); xx) The use according to any one of i) to xix), which is an in vivo and / or ex vivo and / or in vitro use. Use of the polymer-lipid hybrid nanoparticles, compositions, antigen presenting cells, hybridomas, kits and / or vaccines according to any one of the preceding items for / in one or more of the following:

[0129] 43. The method or use according to any one of the preceding items, which is an in vivo and / or ex vivo and / or in vitro method or use, preferably an organ-specific method or use, more preferably suitable for targeting predominantly (e.g. at least 51%, e.g. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, at least 95%, at least 98%, or at least 99%) in or performed in the liver, spleen, and / or lungs (e.g. cells and / or tissues). EXAMPLES

[0130] Examples of the present invention In order that the present invention may be readily understood and reduced to practice, certain aspects of the present invention will be described by the following illustrative, non-limiting examples.

[0131] Example 1: Polymer-lipid hybrid nanoparticles containing lipids and block copolymers, methods for making same, and their characterization and uses material and method material All solvents and chemicals were purchased from Merck and used as received unless otherwise noted. 1.2k -b-PEO 0.6k(PBD-PEO) and PCL 3.3k -b-PEO 1k (PCL-PEO), PLGA (LA:GA=85:15) 1.9K -PEO 1K Block copolymers containing were synthesized in the laboratory (e.g., Table 1). 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Avanti), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), DLin-MC3-DMA (MC3, Avanti), DMG-PEG-2K (DMG-PEG), and cholesterol (Chol) were purchased from Merck. 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200) was purchased from Corden pharm. EZ Cap™ Firefly Luciferase mRNA (Luc mRNA-SEQ ID NO:1) was purchased from APExBIO. CD19 mRNA (SEQ ID NO:3) and OVA mRNA (SEQ ID NO:2) were purchased from Trilink Biotech and stored at -80°C. Quant-iTRiboGreen RNA assay, Lipofectamine™ MessengerMAX™, MultiTox-Fluor™ Multiplex Cytotoxicity Assay, ONE-Glo™ Luciferase Assay were purchased from Thermofisher. Human Embryonic Kidney (HEK293T) cell line (CRL-11268™) was obtained from ATCC, USA and cultured according to ATCC recommendations. Cells were cultured in Roswell Park Memorial Institute (RPMI 1640) medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin (HyClone, USA) at 37°C and 5% CO2.

[0132] method Fabrication of mRNA-loaded polymer-lipid hybrid nanoparticles Solvent dispersion method Polymer-lipid hybrid nanoparticle-encapsulated mRNA was prepared by a solvent dispersion method followed by dialysis. Briefly, polymer, lipid, ionized lipid, and cholesterol were dissolved in ethanol at a predetermined molar ratio at a total concentration of 5 mM (Table 2).

[0133] Table 2. Physiochemical characterization of Luc mRNA encapsulated by PBD-PEO lipid hybrid nanoparticles (BNPs) prepared by the solvent dispersion method. The in vitro and in vivo transfection and encapsulation efficiency of formulations no. 2 and 8 were investigated. PDI: polydispersity; EE: encapsulation efficiency. TIFF2025508798000024.tif89164

[0134] Aqueous solutions were prepared by dissolving mRNA (Luc mRNA or OVA mRNA or CD19 mRNA) in 20 mM acetate buffer (pH 5.0). The ethanol phase was slowly poured into the aqueous phase in a 3:1 ratio while vortexing using a vortex mixer. Nanoparticles formed during vortexing and were then dialyzed overnight at 4°C against buffer (20 mM Tris buffer, 4.5 mM acetate, 5% sucrose, pH 7.4) using a dialysis membrane (300 kDa molecular weight cut-off (MWCO) cellulose ester membrane, Spectrum Laboratories Inc., Cat. No. 131450) to remove organic solvents and non-encapsulated mRNA. The dialyzed solution was sterile filtered using a 0.22 μm sterile filter (Sartorius) and stored at 4°C. mRNA-encapsulated lipid nanoparticles (LNPs) were prepared using a molar composition similar to that reported in the literature and used as a control. Here, the molar ratio between ionized lipid / cholesterol / DSPC / DMG-PEG-2K was set to 49:39:10.5:1.5. In this method, the N / P ratio (N in the ionized cationic lipid and P in the mRNA) was 4-40.

[0135] Precision NanoSystems Incorporation (PNI) NanoAssembl Platform (sometimes called the PNI method) Polymer-lipid hybrid nanoparticles encapsulating mRNA generated by PNI Polymer-lipid hybrid nanoparticles encapsulated mRNA were produced by Precision Nanosystem Incorporation Nanoasemblr system (Ignite™, PNI, Canada) with molar composition similar to that shown above. Briefly, an ethanol phase containing a mixture of polymer and lipid in a predetermined molar ratio (see, e.g., Tables 2 and 3) and an aqueous phase (20 mM acetate buffer, pH 5.0) containing mRNA were co-injected into a Y-shaped staggered herringbone micromixer with a width of 300 μm and a height of 130 μm. Nanoparticles were produced at a polymer / lipid concentration of 5 mM, a water:organic flow rate (FRR) of 3:1, and a total flow rate (TFR) of 12 mL / min. The nanoparticles were dialyzed overnight at 4° C. against buffer (20 mM TRIS, 4.5 mM acetate, 5% sucrose, pH 7.4) using a 300 kDa molecular weight cut-off (MWCO) cellulose ester membrane, Spectrum Laboratories Inc., Cat. No. 131450) with magnetic stirring. The dialyzed solution was sterile filtered using a 0.22 μm sterile filter (Sartorius) and stored at 4° C. for further use.

[0136] Polymer-lipid hybrid nanoparticles encapsulating Cas12a / gRNA generated by the PNI system EnGen® Lba Cas12a(Cpf1)NED(Cas12a) and gRNA were mixed with ASF p52 at 250 nM concentration. The solution was incubated at room temperature (RT) for 10-15 min to allow Cas12a to bind to gRNA. This was further encapsulated in BNP-002 (Table 2) using the PNI system at TFF 12 ml / min and FRR 3:1 at 1 ml scale (Figure 15). After formulation was completed, DLS of the sample was performed and the remainder of the sample was dialyzed using PBS buffer. After dialysis, the sample was collected and DLS was collected before and after sterile filtration.

[0137] Dynamic light scattering analysis The particle size, polydispersity, and zeta potential of the nanoparticles were measured by a ZetasizerNano ZS system (Malvern Instrument Ltd., Malvern, UK) equipped with a He-Ne laser beam at 658 nm (scattering angle: 90°). 50 μL of sample was diluted 10-fold with dialyzed buffer, and the average of triplicate measurements (10 runs per measurement) was collected, and the data were presented as averages.

[0138] Cryogenic-Transmission Electron Microscopy (Cryo-TEM) 4 μL of mRNA-encapsulated nanoparticles (5 mM) were adsorbed onto lacey holey carbon-coated Cu grids, 200 mesh size (Electron Microscopy Sciences). Before use, the grids were surface-treated with glow discharge for 20 s. After surface treatment, 4 μL sample was added and the grids were blotted with Whatman filter paper (GE Healthcare Bio-Sciences) using blot force 1 for 2 s and then submerged in liquid ethane at −178 °C using a Vitrobot (FEI Company). Cryo-grids were imaged using a FEG 200 keV transmission electron microscope (Arctica; FEI Company) equipped with a direct electron detector (Falcon III; Fei Company).

[0139] Quantification of mRNA encapsulated in nanoparticles The mRNA encapsulated in the nanoparticles was quantified using a modified Quant-iTRiboGreen RNA assay. A 20 μL aliquot of mRNA encapsulated nanoparticles or free mRNA of known concentration was added to a 384-well black plate. A 10 μL volume of TE buffer or TE buffer containing 10% Triton-X 100 was added to each well, and the plate was incubated at 37°C for 15 min to dissolve the ACM vesicles, after which 20 μL of 1× Quant-iT RiboGreen (Invitrogen, Thermo Fisher Scientific) was added. Each sample and standard was prepared in triplicate. The plate was incubated at 25°C for 10 min and fluorescence was measured (excitation, 500 nm; emission, 525 nm) using a plate reader (Biotek). The mRNA concentration was calculated according to the standard curve. The encapsulation efficiency (EE) was calculated as (F t -F0) / F i * The calculation was based on 100. t is the amount of mRNA quantified in the presence of 1% triton X-100, and F0 is the amount of mRNA quantified in the absence of 1% triton X-100. i was the initial amount of mRNA used to prepare nanoparticles.

[0140] Gel electrophoresis assay Gel electrophoresis was used to analyze the integrity of the mRNA in the various formulations. Agarose was dissolved in 1x Tris-Acetate-EDTA (TAE) buffer and heated to form a 1% w / v solution, after which SybrSafe Dye (5μl / 50mL) was added. The solution was mixed well and poured into a casting tray where the comb was placed. The gel was allowed to solidify. The comb was removed and the gel was placed in a buffer tank containing 0.5x TAE buffer. 20μL of mRNA encapsulated nanoparticles (equivalent to 500ng of mRNA) were mixed with 2μL of 20% TritonX for 30 minutes, after which 20ul of 2x RNA loading dye was added. Samples were heated at 70°C for 10 minutes in a heat block. Samples were cooled and then loaded onto the gel (20μ / well). Note that an RNA ladder was used as a molecular weight standard. A power supply was connected to the chamber and a voltage of 80 V was applied for 40-45 min. The gel was then visualized using an ImageQuant LAS 500 system.

[0141] RNase protection assay Test samples (20 μL) were challenged with RNase (1 ul of 10ug / ml stock) and then incubated at 37°C for 20 minutes. 0.2% SDS (4 ul of 1% SDS stock) was then added to each sample. Note that 0.2% SDS acts as an RNAse inhibitor. Positive control samples were incubated with 1% tritonX for 30 minutes at room temperature. For the negative control group, samples (20 μL) were challenged with RNase (1 ul of 10ug / ml stock) and then incubated at 37°C for 20 minutes. 2% TritonX (2 ul of 20% TritonX) was then added to each sample. The mixture was incubated at room temperature for 30 minutes. Addition of 20 ul of 2x RNA loading dye. Samples were heated at 70°C for 10 minutes in a heat block. Samples were cooled and then loaded onto the gel (20 μl / well). Note that an RNA ladder was used as a molecular weight standard. A power supply was connected to the chamber and a voltage of 80 V was applied for 40-45 min. The gel was then visualized using an ImageQuant LAS 500 system.

[0142] mRNA transfection HEK293T cells (CRL-11268™) were seeded in 96-well plates at a density of 25000 cells / well. After overnight incubation at 37°C, cells were transfected with Luc mRNA-encapsulated nanoparticles or control Luc mRNA using Lipofectamine™ MessengerMAX™ (MM, Thermo Fisher). After 24 hours of incubation, the cytotoxicity of free mRNA and mRNA-loaded nanoparticles against HEK293 cells was confirmed by MultiTox-Fluor™ Multiplex Cytotoxicity Assay. Luciferase activity was measured using the ONE-Glo™ Luciferase Assay according to the manufacturer's instructions (Promega).

[0143] HEK293T cells were seeded at 250,000 cells / well in 24-well plates. After overnight incubation, cells were transfected with OVA mRNA nanoparticles (OVA mRNA equivalent: 1 μg) or control OVA mRNA (100 ng and 200 ng) using Lipofectamine™ Messenger MAX™ (Thermo Fisher). Cells were harvested 24 hours after transfection. Cells were lysed and protein was quantified using a BCA assay (Thermo Fisher) according to the manufacturer's protocol. 50 μL of sample (containing 150 ng of protein) was mixed with 50 μL of loading buffer, and the mixture was heated at 95°C for 10 min. Samples (20 μL) were loaded for SDS-PAGE. OVA protein was then detected by Western blotting using a monoclonal antibody against OVA protein. The gel was then visualized using an ImageQuant LAS 500 system.

[0144] In vivo Luc mRNA delivery The animal study was approved by the Institutional Animal Care and Use Committee, A-star, Singapore. Female C57BL / 6 mice aged 6–8 weeks were randomly divided into groups. C57BL / 6 mice were injected with Luc mRNA at a dose of 0.35 mg / kg by IM (thigh muscle), SC (flank), and IV (tail vein), respectively. There were six groups for each administration route (three mice per group, a total of 18 mice per administration route). For the intramuscular (IM) administration route, mice were intramuscularly (IM) injected with Luc mRNA encapsulated in LNP-ON, BNP-002, BNP-008, PCL-008, and PCL-012 at a dose of 0.35 mg / kg into the inner thigh. Here, PBS was used as a negative control for comparison, and LNP-ON was used as a positive control. Six hours after injection, mice were anesthetized with 2% isofluorane in oxygen and imaged 10 minutes after intraperitoneal injection of D-luciferin (150 mg / Kg). Bioluminescence imaging was performed using an IVIS Spectrum Imaging System. Organs were harvested for ex vivo imaging. Mice were imaged 10 minutes after administration of D-luciferin. Bioluminescence values ​​were quantified by measuring the photon flux in the region of interest using Living IMAGE Software provided by Caliper.

[0145] Mice injected with ACM-OVA mRNA polymer-lipid hybrid nanoparticles expressed OVA peptide on the surface of dendritic cells Animal studies were approved by the Laboratory Animal Committee, A-star, Singapore. Six- to eight-week-old female C57BL / 6 mice were injected intramuscularly (IM) into the medial thigh with 3–4 μg of OVA mRNA encapsulated in LNP or ACM carrier. Two days later, animals were sacrificed and inguinal lymph nodes draining the injection site were collected. To release DCs for analysis, lymph nodes were cut into small pieces and digested with 0.2 mg / ml collagenase and 0.05 mg / ml DNase I dissolved in complete RPMI medium for 30 min at 37 °C. Cells were passed through a 70 μm cell strainer. To prepare for flow cytometry, cells were stained with the following antibody panel: BUV395-CD45, FITC-CD3, FITC-CD19, FITC-CD49b, BV510-MHC-II, BV650-CD64, PE-CD594-CD11c, PerCP-Cy5.5-XCR1, APC-CD172a, APC-Cy7-CD86, and PE-SIINFEKL-H2kb. Live / dead discrimination was performed using fixable viability dye eFluor 455 UV. Mouse FcR blocking reagent was used to reduce nonspecific staining. Cells were analyzed using an LSR Fortessa (BD) and data were analyzed using FlowJo V10.

[0146] OVA mRNA vaccine adaptive immunity research Mouse vaccination Animal studies were approved by the Laboratory Animal Committee of A-star, Singapore. A total of 5 μg OVA mRNA was injected intramuscularly (IM) into each thigh muscle of 6-8 week-old female C57BL / 6 (n=5 / group) on days 0 and 14. Blood was collected by retro-orbital puncture on days 7 and 21 to assess circulating T cells and on days 14 and 24 for serum IgG titers.

[0147] T cell analysis Blood was collected in 0.1% EDTA. Cells were pelleted at 500g for 5 min at 4°C and red blood cells were lysed using RBD lysis buffer (Thermo Fisher). Leukocytes were surface stained with antibodies and pentamer for analysis by flow cytometry (Table 7). Cells were acquired on an LSR II cytometer (BD) and data were analyzed using FlowJo V10 software.

[0148] Table 7. Antibodies and pentamers used for flow cytometric analysis TIFF2025508798000025.tif65165

[0149] OVA IgG titer 96-well Corning EIA / RIA plates were coated with 2 μg / ml OVA protein overnight at 4°C. The following morning, plates were washed three times with PBS+0.1% v / v Tween-20 and then blocked with 2% w / v BSA in wash buffer at 37°C for 1.5 h. Sera were serially diluted in assay diluent (PBS+0.5% w / v BSA+0.1% v / v Tween-20) and applied to the corresponding wells of the ELISA plate. Samples were incubated for 1 h at 37°C, after which the plate was washed three times. HRP-conjugated goat anti-mouse IgG (H+L) (BioRad) was diluted 1:10,000 and applied to the ELISA plate. Plates were incubated for 1 min at 37°C and then washed three times. To visualize antibody binding, TMB substrate (Sigma Aldrich) was added and incubated for 30 min at room temperature. The reaction was stopped with Stop Solution (Thermo Fisher) and the absorbance at 450 nm was measured. Data were analyzed using 5-parameter nonlinear regression (GraphPad Prism version 9.1.2). Antibody titers, defined as the reciprocal of the highest dilution that produced an OD value three times higher than background, were interpolated from the titration curve.

[0150] statistical analysis Statistical significance was assessed by unpaired two-tailed Student's t-test or two-way ANOVA. A P value of less than 0.05 was considered statistically significant. Data were analyzed using GraphPad Prism 7 software.

[0151] Results and Discussion Physicochemical characterization of mRNA-loaded polymer-lipid hybrid nanoparticles prepared by solvent dispersion method (cosolvent method / nanoprecipitation method) First, BNPs composed of PBD-PEO, MC3, and Chol encapsulating mRNA were prepared by solvent dispersion method. LNP-onpattro (LNP-ON or LNP-ONP) containing DMG-PEG, DSPC, MC3, and Chol (1.6:10.1:49.3:39.0) with mRNA were prepared by the same method and used as a control. The physicochemical properties of the nanoparticles, including particle size, polydispersity, zeta potential, mRNA encapsulation efficiency, and loading concentration, are summarized in Table 2. BNP-002 has an average particle size of 138 nm and a relative small polydispersity (PDI: 0.176). It is noted that the hydrodynamic diameter of LNP-ON was 158 nm, and the PDI of LNP-ON was 0.17. The results showed that the particle size and PDI of BNP-002 were comparable to those of LNP-ON. The surface potential of BNP-002 was 27.8 mV. This was significantly higher than the surface potential of LNP-ON (11.1 mV). This was consistent with the fact that the N / P value of BNP-002 was significantly higher than that of LNP-ON (27). To explore the potential of mRNA-loaded nanoparticles with tumor antigens for cancer immunotherapy, OVA mRNA was loaded into BNP by solvent dispersion method. Interestingly, OVA mRNA BNP was found to exhibit a diameter value of 107 nm and a low PDI value of 0.137. The morphology of Luc mRNA-loaded BNP and OVA mRNA-loaded BNP was analyzed by cryo-TEM. As illustrated in Figure 1, Luc mRNA-loaded BNP formed spherical particles and exhibited a stacked bilayer structure. It is interesting to notice that OVA mRNA-loaded BNP exhibited a similar stacked bilayer structure (Figure 2). At an N / P value of 27, all mRNA molecules formed complexes with positively charged ionized lipids (MC3). This may lead to the formation of a stacked bilayer structure. It was hypothesized that the amphiphilic bilayer forming polymer PBD-PEO would act as the outer layer of the mRNA BNP and stabilize the inner mRNA-ionized lipid stacked bilayer structure.The structure of mRNA BNP was consistent with that of mRNA-containing LNPs, and LNP-mRNA, composed of KC2, DSPC, Chol, and PEG-lipids with a molar composition of 50 / 10 / 38.5 / 1.5, showed a surface bilayer and a stacked bilayer internal structure. The mRNA encapsulation efficiency and loading concentration were evaluated by Ribogreen assay. The results showed that the physiochemical properties of BNP were not significantly affected by different types of mRNA. The encapsulation efficiency was evaluated using Ribogreen assay. As listed in Table 2, BNP-002 showed a significantly higher encapsulation efficiency (67.8%) compared with LNP-ON (37.7%). In contrast, the loading concentration of BNP-002 was significantly lower than that of LNP-ON (20.3 μg / mL vs. 75.5 μg / mL). The lower loading concentration was attributed to incomplete mixing by the solvent dispersion method.

[0152] Ionized lipids have been reported to play a key role in the performance of mRNA nanoparticles. Numerous studies have shown that ionized lipids, including DLin-KC2-DMA and DLin-MC3-DMA, achieve maximum activity for mRNA delivery. Recently, Kauffmann et al. reported that lipid-like material (lipidoid) C12-200 nanoparticles conjugated with DOPE, DMG-PEG, and Chol (35:16:2.5:46.5) (LLNPs) significantly increased serum EPO expression by 7-fold compared to the benchmark formulation LNP-ON. Inspired by Kauffmann's design, we conjugated PBD-PEO with LLNPs and investigated the functionality of the resulting Luc mRNA BNPs. C12-200-based BNPs were prepared using a solvent dispersion method as listed in Table 2. BNP-008 had an average particle size of 121-200 nm, a low PDI value of less than 0.22, and a surface charge of 25-30 mV. BNP-008 had the smallest particle size and the greatest in vitro transfection efficiency (data not shown), and therefore was selected for further studies.

[0153] The integrity of mRNA in BNP was analyzed by gel electrophoresis. As illustrated in Figure 4A, luciferase mRNA remains intact after encapsulation in BNP and LNP-ON. LNPs are known to not only facilitate cellular uptake and expression, but also protect mRNA from exonuclease and endonuclease (RNase) degradation. The ability of BNP to resist RNase degradation was assessed by RNase protection assay using gel electrophoresis. As shown in Figure 4B, the mRNA remained intact in both BNP (lane 6) and LNP-ON (lane 2) after storage at 4°C for 2 weeks. However, the mRNA was completely degraded when exposed to RNase in the presence of triton (lanes 5 and 9). It is noteworthy that the mRNA encapsulated in BNP was protected from degradation in the presence of 0.2% SDS (RNase inhibitor) (lane 8), whereas the mRNA encapsulated in LNP-ON was completely degraded in the presence of RNase inhibitor (lane 4). The results strongly indicated that BNP could protect mRNA from RNase degradation.

[0154] It should be mentioned that the encapsulation efficiency of BNP produced by the solvent dispersion method was small and the loading concentration was low. To improve the encapsulation efficiency and loading level of BNP, mRNA-loaded nanoparticles were further prepared by Precision Nanosystem Incorporation (PNI Nanoasemblr Platform).

[0155] Physicochemical properties of mRNA-loaded nanoparticles prepared by Precision NanoSystem Incorporation (PNI) Nanoasemblr Platform Physicochemical properties of mRNA-loaded nanoparticles prepared by PNI method BNP and PCL were formulated as specified compositions (eg, Tables 2, 3, 4, and 5) with an N / P molar ratio of 10 using the PNI method.

[0156] Table 3. Physiochemical characterization of Luc mRNA-loaded PCL-PEO lipid hybrid nanoparticles prepared by Precision Nanosystem Incorporation (PNI) Nanoasemblr Platform. TIFF2025508798000026.tif73165

[0157] Table 4. Exemplary BNP Compositions TIFF2025508798000027.tif46166

[0158] Table 5: Buffer composition for mRNA-loaded nanoparticles TIFF2025508798000028.tif36165

[0159] Briefly, mRNA diluted in acetate buffer (20 mM, pH 5.0) was quickly mixed with polymer and / or lipid dissolved in ethanol at a water:ethanol volume ratio of 3:1. The water:organic flow rate (FRR) was set at 3:1, and the total flow rate (TFR) was set at 12 mL / min. Interestingly, BNP-002, BNP-008, PCL-008, and PCL-012 with an N / P ratio of 10 showed z-average diameters of 80–130 nm and low polydispersity (Tables 3 and 6A). It is noteworthy that all formulations showed excellent encapsulation efficiency (approximately 100%) (Table 6B). The benchmark LNP-ON with an N / P of 4 prepared by PNI yielded particles with a diameter of 73 nm, low polydispersity (0.11), and a significantly greater encapsulation efficiency (approximately 100%) (Tables 6A and 6B). This is consistent with previous studies. Importantly, all formulations exhibited high loading concentrations (>75 μg / mL) (Table 6B), which were significantly increased compared to those produced by the solvent dispersion method.

[0160] Table 6A: Physiochemical characterization of Luc mRNA-loaded nanoparticles prepared by Precision Nanosystem Incorporation (PNI) Nanoasemblr Platform TIFF2025508798000029.tif159168

[0161] Table 6B: RiboGreen assay of Luc mRNA-loaded nanoparticles prepared by Precision Nanosystem Incorporation (PNI) Nanoasemblr Platform (luciferase mRNA) 1 This is intended to measure any free, non-encapsulated mRNA or mRNA bound to the surface of the vesicles. 2 The aim is to measure the total mRNA present in a sample when Triton is added to break open the vesicles. 3 mRNA loading was calculated by taking the concentration with Triton minus the concentration without Triton. NSF indicates non-sterile filter. SF indicates sterile filter (e.g., PES syringe filter 0.2 μm (Millipore) was used). TIFF2025508798000030.tif158167

[0162] Among these, BNP-008 with N / P 10 showed a significantly higher loading concentration comparable to the benchmark LNP-ON with N / P 4 (Table 6B). The surface charge of BNP-002 and BNP-008 was about 22 mV, whereas the surface charge of PCL-008 and PCL-012 was about 5-8 mV (Table 6A). The morphology of BNP and PCL was analyzed by cryo-TEM. All formulations formed spherical particles with diameters of 50-200 nm, which is consistent with DLS analysis. The nanoparticles had an electrolucent amorphous inner structure surrounded by a peripheral bilayer (Figure 3A-D). This structure is consistent with previous reports that showed that mRNA-loaded nanoparticles had an electron-lucent amorphous core and a peripheral bilayer membrane with high mRNA loading.

[0163] OVA mRNA was also encapsulated in BNP and PCL using PNI. All formulations showed small particle size, low polydispersity, significantly high encapsulation efficiency (about 100%), and high mRNA loading level (data not shown). The integrity of OVA mRNA encapsulated in BNP and PCL was evaluated by gel electrophoresis. As shown in Figure 7, OVA mRNA remained intact for all formulations produced by PNI. Therefore, we concluded that PNI is a more suitable method to produce BNP and PCL with excellent mRNA encapsulation efficiency (about 100%) and high loading level in this study. The optimal BNP and PCL produced by PNI were selected for in vivo Luc mRNA delivery study.

[0164] Physicochemical properties of Cas12a / gRNA-loaded nanoparticles prepared by PNI Cas12a, a CRISPR effector protein, has shown great promise in the treatment of genetic diseases. CRISPR technology based on Cas12a and gRNA has been reported to work well for RNA-based gene regulation. To explore the potential of polymer-lipid hybrid nanoparticles as carriers for Cas12a and gRNA, we developed BNP-002 nanoparticles to encapsulate Cas12a and gRNA. First, Cas12a and gRNA were mixed and incubated with ASF p52 for 10-15 min. This was further encapsulated in BNP-002 using the PNI system. The resulting nanoparticles were 285.2 nm by dynamic light scattering (Figure 16) and had a low PDI (0.164).

[0165] In vitro transfection efficiency of mRNA-loaded nanoparticles prepared by solvent dispersion method The in vitro delivery efficacy and cytotoxicity of BNP were examined by normalized luminescence intensity after introducing Luc mRNA into HEK293T cells by transfection. As shown in Figure 5, BNP-002 showed up to 30% higher luciferase protein expression compared to 25 ng of MM complex in HEK293T cells. Notably, BNP-002 demonstrated comparable in vitro transfection efficacy compared to LNP-ON (p>0.1). It is noteworthy that the cytotoxicity of BNP-002 was lower compared to LNP-ON (data not shown). The time-dependent luciferase activity of BNP was further evaluated over a period of 3 weeks. As evident from Figure 5, BNP-002 showed a significant increase in luciferase protein expression after 1 week of storage at 4°C. The luciferase activity of BNP-002 was maintained after 3 weeks of storage at 4°C. In contrast, in the case of LNP-ON, luciferase protein expression significantly decreased from 33% at week 1 to 11% at week 3 (p<0.005). Results showed that BNP was stable over a period of 3 weeks at 4°C. The long-term storage stability of BNP is being monitored.

[0166] We next validated BNPs as nanovaccine to deliver antigen-encoding OVA mRNA to HEK293 cells. As illustrated in Figure 6, OVA mRNA-loaded BNP-002 produced dose-dependent OVA protein expression. Importantly, compared with OVA protein expression from mRNA formulated with LNP-ON, OVA mRNA-loaded BNP-002 induced significantly higher levels of OVA protein expression. The results showed that BNPs outperformed LNP-ON in delivering Luc mRNA and OVA mRNA to cells and translating proteins efficiently.

[0167] In vitro transfection efficiency of mRNA-loaded nanoparticles prepared by Precision NanoSystem Incorporation (PNI) Nanoasemblr Platform As previously mentioned, all formulations generated by PNI showed significantly higher encapsulation efficiency (approximately 100%) at an N / P ratio of 10. The in vitro delivery potency of these formulations was evaluated for Luc mRNA delivery in HEK293T cells (Figure 8). BNP-002 and BNP-008 induced lower normalized luminescence intensity compared to LNP-ON (p<0.05). However, PCL-008 and PCL-012 at an N / P ratio of 10 promoted Luc mRNA transfection, with potency significantly increased by 2.7- and 4.5-fold, respectively, compared to LNP-ON at an N / P ratio of 4 (p<0.01). Furthermore, ACM formulations (BNP and PCL) showed less cytotoxicity compared to LNP-ON (Figure 9). Next, the long-term stability of the formulations was evaluated over 1 month of storage at 4°C. As can be seen from Figure 8, there is no significant difference in the in vitro transfection efficacy of Luc mRNA between BNP and PCL over 1 month of storage (p>0.05). In contrast, the transfection efficacy of Luc mRNA encapsulated in LNP ONs was significantly decreased after 1 month of storage (p<0.05). The results showed that BNP and PCL produced by PNI exhibited high thermal stability at 4°C.

[0168] HEK293T cells were treated with OVA mRNA formulations, and OVA protein expression was evaluated by Western blot assay. As illustrated in Figure 10, there is no significant difference in OVA protein expression between BNP-002 and LNP-ON. In contrast, PCL-012 induced significantly higher OVA protein levels compared to LNP-ON (p<0.05). On the other hand, BNP-008 produced less OVA protein than LNP-ON (p<0.05).

[0169] In vivo delivery efficacy of Luc mRNA The in vivo delivery efficacy of BNP and PCL was evaluated in C57BL / 6 mice via various administration routes (IM, SC, and IV). For the IM administration route, mice were randomly assigned to six different groups (three mice per group): control group (1×PBS), BNP-002, BNP-008, PCL-008, and PCL-012. All formulations were administered via intramuscular (IM) route in the thigh muscle region at a dose of 0.35 mg / kg. Strong luciferase protein expression was observed at the injection site and upper abdomen of the mice 6 hours after IM injection (Figure 11A). Ex vivo images of luminescence in major organs revealed abundant Luc protein expression in the liver of the mice 6 hours after injection. In contrast, less luminescence signal was detected in the popliteal and inguinal lymph nodes (Figure 11B). Further ex vivo imaging quantitative analysis revealed that there was no significant difference between different groups in terms of bioluminescence signal at the injection site 6 hours after administration (Figure 12A). Moreover, significantly more abundant Luc protein expression was observed in the liver of mice 6 hours after injection for LNP-ON compared to ACM mRNA nanoparticles (Figure 12A). Notably, PCL-008 and PCL-012 produced significantly higher Luc protein expression in the inguinal lymph node 6 hours after administration than LNP-ON (p>0.05) (Figure 12A). The results showed that optimal BNP and PCL showed strong in vivo mRNA delivery efficacy in the inguinal lymph node via IM, but were significantly off target in the liver compared to LNP-ON.

[0170] Encouraged by the comparable luciferase protein expression between Luc mRNA BNP, PCL, and benchmark LNP-ONP administered via IM, we further investigated the effect of the route of administration (subcutaneous (SC) and intravenous (IV)) of BNP and PCL on mRNA delivery. When Luc mRNA was administered SC, the protein was expressed primarily at the injection site (data not shown). For LNP-ONP LNP-ON, a strong bioluminescence signal was also observed in the upper abdomen 6 hours after SC administration (data not shown). Organs / tissues were then excised and further analyzed for bioluminescence. Luciferase protein generated by LNP-ONP LNP-ON was expressed primarily in the liver (74%) and to a lesser extent in the injection site (16%) and lymph nodes. In contrast, luciferase protein generated by the ACM formulation of the present invention was expressed primarily at the injection site (55-83%). It is noteworthy that 6 hours after SC administration, Luc protein expression levels in the inguinal lymph nodes were similar among LNP-ONP, BNP-008, and PCL-008.

[0171] When Luc mRNA nanoparticles were injected intravenously, strong bioluminescence signals were detected in the liver for LNP-ONP, PCL-008, and PCL-012 (Figures 11C and 12C). Luciferase protein produced by LNP-ONP LNP-ON was expressed mainly in the liver (98%) (Figure 12D). This result is consistent with the findings of Weissman, in which IV injection of Luc mRNA LNPs resulted in significant protein production in the liver, likely due to passive ApoE-mediated liver targeting. Notably, BNP-008-mediated protein expression occurred mainly in the spleen (67%), whereas it was significantly less in the liver (26%) compared to LNP-ONP LNP-ON (98%) (Figure 12D). Anderson et al. reported that an optimized C-35 formulation containing C12-200, DOPE, Chol, and C14-PEG2000 (35:16:46.5:2.5) produced luciferase protein primarily in the liver. BNP-008 may be a key factor for tuning spleen specificity. Siegwart's group developed selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery. Here, charge-mediated targeting was achieved using LNPs mixed with permanently cationic lipids (i.e., DOTAP) or anionic lipids (i.e., 18PA) to target the liver, lung, and spleen, respectively. However, BNP-008 had a net positive surface charge with a zeta potential of 22.9 mV. On the other hand, like LNP-ONP, BNP-002, which is composed of MC3, produced luciferase protein primarily in the liver (98%). Thus, in the case of BNP-008, PBD-PEO works in concert with C12-200, DOPE, and Chol to achieve spleen-targeted delivery of mRNA. To our knowledge, this is the first observation of a hybrid lipid nanoparticle that achieves targeted mRNA delivery to the spleen.

[0172] In vivo immunogenicity studies Classical dendritic cells consist of two subsets (cDC1 and cDC2) that are important in initiating adaptive immune responses. Two days after injection of LNP-mRNA or BNP-mRNA vaccine, a strong upregulation of CD86 activation marker was observed in both cDC subsets (Figure 13). Compared to PBS-injected control, the CD86 increase was highly significant (Figure 13). This indicates that both mRNA formulations were immunostimulatory. Next, we examined DCs for surface expression of SIINFEKL peptide (SEQ ID NO:4) presented in the context of MHC-II (H2kb). Surface expression indicates successful translation of mRNA into OVA protein followed by processing for MHC-II presentation. Both BNP-mRNA and LNP-mRNA formulations induced surface presentation of OVA peptide in cDC1 and cDC2 (Figure 14). Furthermore, the peptide expression levels induced by ACM-OVA mRNA were similar to or significantly higher than those of LNP-OVA mRNA (Figure 14). In summary, our results showed that ACM-mRNA formulations potently activated cDC1 and cDC2 in lymph nodes, and mRNA was translated and processed for surface presentation. Since lymph nodes are the main induction site for adaptive immune responses, our findings strongly suggested that ACM-OVA mRNA is likely to induce OVA-specific immunity.

[0173] ACM-OVA mRNA vaccine adaptive immunity research Mice were injected IM twice with LNP, BNP or PCL formulations encapsulating 5 μg / mouse of OVA mRNA (FIG. 17a). + Immunization with any formulation resulted in approximately 1.7% Pent + CD8 +T cells were generated (Figure 17b). A second dose of LNP-ON or BNP-002 did not increase the frequency, whereas BNP-008 or PCL-012 produced near-significant and significant increases in T cell frequency, respectively. At day 21, BNP-008 and PCL-012 produced significantly more Pent cells than the PBS control. + CD8 + T cell induction (Figure 17c). For OVA IgG titers, a single dose of any formulation produced weak or undetectable titers (Figure 17d). A second dose produced highly significant IgG increases across all formulations. At day 24, the most OVA IgG was observed with LNP or BNP-008 (Figure 17e). Among ACM formulations, BNP-008 produced CD8 T cell activation comparable to the LNP control. + T cell and IgG responses were consistently generated.

[0174] conclusion In conclusion, a new class of polymer-lipid hybrid nanoparticles for mRNA delivery was developed. The optimal formulation exhibited favorable physicochemical properties and excellent encapsulation efficiency (approximately 100%). The optimal formulation performed better with improved in vitro transfection potency and long-term thermal stability as evidenced by higher levels of Luc and OVA protein expression compared to benchmark LNP-ONs. Furthermore, the ACM formulation exhibited less cytotoxicity compared to benchmark LNP-ONs. Importantly, the optimal formulation showed potent in vivo mRNA delivery potency comparable to benchmark LNP-ONs. Furthermore, the OVA mRNA formulation was able to potently activate lymph node-resident cDC1 and cDC2 to promote antigen surface presentation. BNP-008 enhanced CD8 expression comparable to LNP controls. + ACM-OVA mRNA consistently generated T cell and IgG responses. This finding strongly suggested that ACM-OVA mRNA is likely to induce OVA-specific adaptive immunity. In summary, our study reports a novel class of polymer-lipid hybrid nanoparticles with efficient protein and antigen expression and improved thermostability, and therefore great potential for therapeutic mRNA delivery across a broad range of diseases.

[0175] Example 2: Further characterization of polymer-lipid hybrid nanoparticles comprising lipids and block copolymers and their use for organ-specific delivery of mRNA to the liver, spleen, and lungs 1,2-Di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA, Avanti) was purchased from Merck. All other chemicals and reagents and methods were previously described. Therefore, unless otherwise noted, materials and methods are as described above for Example 1. In this example, Luc mRNA-loaded nanoparticles were prepared by microfluidizer according to Table 8, and for mRNA-loaded polymer-lipid hybrid nanoparticles, mRNA was generated by microfluidizer with the molar composition as shown in Table 8. In this case, the N / P molar ratio is 10.

[0176] Table 8. Preparation of exemplary Luc-mRNA-loaded nanoparticles by microfluidizer TIFF2025508798000031.tif205166

[0177] The polymer-lipid hybrid nanoparticles were then characterized as shown in Table 9.

[0178] Table 9. Physiochemical characterization of Luc-mRNA-loaded nanoparticles prepared by microfluidizer. TIFF2025508798000032.tif124165

[0179] Thus, BNP-002.2 showed a z-average diameter of 93 nm and low polydispersity. BNP-012 had a particle size of 51 nm and a polydispersity of 0.188. BNP-025 showed a particle size of 52 nm and a polydispersity of 0.198. BNP-012 and BNP-025 show a zeta potential of approximately 45 mV. Finally, all formulations showed high Luc-mRNA encapsulation efficiency (>90%).

[0180] Following this, exemplary cryo-TEM images of BNP-002.2, BNP-012, and BNP-025 (all carrying luciferase mRNA) were generated (Figure 18). Here, BNP-002.2 showed spherical nanoparticles (50-150 nm) with amorphous structure. BNP-012 showed a predominant distribution: multicompartmental structure, with vesicles consisting of heterogeneous structures (i.e., vesicle fusion; vesicles with buds, vesicle bud surrounding by bilayer). BNP-025 showed vesicular structures (30-150 nm) with relatively high polydispersity.

[0181] After this, the Luc mRNA encapsulation efficiency was measured using the Ribogreen assay as shown in Table 10.

[0182] Table 10. Luc mRNA encapsulation efficiency measured using the Ribogreen assay 1 This is intended to measure any free, non-encapsulated mRNA or mRNA bound to the surface of the vesicles. 2 The aim is to measure the total mRNA present in a sample when Triton is added to break open the vesicles. 3 mRNA loading was calculated by taking the concentration with Triton minus the concentration without Triton. 4 %EE was calculated by dividing the loading by the concentration with Triton to give a percentage. TIFF2025508798000033.tif65165

[0183] Thus, the Ribogreen assay showed that all formulations exhibited high encapsulation efficiency (>90%).

[0184] After this, agarose gel images of Luc mRNA-loaded nanoparticles prepared by microfluidizer were performed, indicating that all samples contained intact mRNA, as no degradation was observed in the gel (Figure 19).

[0185] This was followed by in vitro luciferase mRNA nanoparticle transfection efficiency profiling in HEK293T cells, which revealed that all formulations exhibited high luciferase protein expression comparable to LNP-ON, with BNP-002.2 demonstrating significantly higher in vitro transfection efficacy (p<0.05) compared to LNP-ON.

[0186] After this, endotoxin (Lonza LAL Assay) analysis of Luc mRNA nanoparticles was performed as shown in Table 11. Here, the endotoxin levels of Luc mRNA nanoparticles were examined using Lonza Kinetic Chromogenic LAL Assay according to the manufacturer's instructions. It was shown that the spike recovery of all nanoparticles met the acceptable range, and the endotoxin levels of all nanoparticles were around 2EU / mL, which also met the requirement (<10EU / mL).

[0187] Table 11. Endotoxin (Lonza LAL Assay) analysis of Luc mRNA nanoparticles TIFF2025508798000034.tif75162

[0188] Following this, a luciferase protein expression percent biodistribution profile was performed via IV administration (Figure 21). BNP-002.2 showed that luciferase protein accumulated in the liver (54%), spleen (44.5%), and 2.1% in the lung. BNP-012 expressed luciferase protein in the liver (0.9%), while 1.4% was in the spleen and 92% was in the lung. BNP-025 produced luciferase protein in the liver (2.7%), spleen (13%), and 76% in the lung. BNP-012 and BNP-025, which contain cationic lipids (DOTAP and DOTMA), produced luciferase protein primarily in the lung.

[0189] This was followed by tissue expression profiling (raw flux values) of the protein encoded by Luc mRNA in mice 6 hours after IV administration (Figure 22). This shows that in the liver, LNP ONP produced significantly more Luc protein than the other groups. In the spleen, BNP-008 produced significantly more Luc protein than the other groups. The amount of Luc protein expression in the lungs between the different groups was similar.

[0190] Finally, a luciferase protein expression percent biodistribution profile (flux) via IV administration was performed (Figure 23), which revealed that LNP-ON produced luciferase protein in the liver (98%) versus 1.2% in the spleen and 0.5% in the lungs, BNP-002 produced luciferase protein in the liver (98%) versus 0.5% in the spleen and 0.3% in the lungs, BNP-008 facilitated even higher levels of luciferase protein expression in the spleen (67%), liver (26%), and 5% in the lungs, BNP-012 produced luciferase protein in the spleen (1.4%), liver (0.9%), and 92% in the lungs, and BNP-025 produced luciferase protein in the spleen (13%), liver (2.7%), and 76% in the lungs. Additionally, PCL-008, described elsewhere herein, was shown to produce luciferase protein primarily in the liver (86.8%) compared to 10.7% in the spleen and 1.3% in the lungs, and PCL-012, described elsewhere herein, produced luciferase protein primarily in the liver (97.6%) compared to lesser degrees in the spleen (1.3%) and lungs (0.6%).

[0191] conclusion The engineered block copolymer-lipid hybrid nanoparticles of the present invention delivered mRNA in an organ-specific manner to the liver, spleen, and lungs.

[0192] Those skilled in the art will readily understand that the present invention is well adapted to achieve the objects and obtain the stated goals and advantages as well as those inherent therein. Moreover, it will be readily apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules, and specific compounds described herein are currently representative of certain embodiments, are exemplary, and are not intended to be limitations on the scope of the invention. These variations and other uses will occur to those skilled in the art, within the spirit of the invention and as defined by the claims. The listing or discussion of a previously published document in this specification should not necessarily be construed as an acknowledgement that the document is part of the state of the art or is generally common knowledge.

[0193] The present invention illustratively described herein may be appropriately implemented without any element or elements, or limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like are to be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and in using such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, although the present invention has been specifically disclosed by exemplary embodiments and optional features, it should be understood that those skilled in the art can make modifications and changes to the invention embodied herein, and such modifications and changes are considered to be within the scope of the present invention.

[0194] The present invention has been described broadly and generically herein. Any narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of the present invention. This includes the generic description of the invention with any proviso or negative limitation that removes any subject matter from the genus, regardless of whether the removed material is specifically recited herein. All documents, including patent applications and scientific publications, mentioned in this specification are incorporated by reference herein for all purposes.

[0195] Other embodiments are within the scope of the following claims. Furthermore, those skilled in the art will recognize that when features or aspects of the invention are described in terms of a Markush group, the invention also is described thereby in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A polymer-lipid hybrid nanoparticle comprising a lipid and a block copolymer, the amount of lipid present in the polymer-lipid hybrid nanoparticles, expressed in mole percent (mol%), is greater than the amount of block copolymer present in the polymer-lipid hybrid nanoparticles, expressed in mole percent; the mole percentage is based on the total amount of all components forming the polymer-lipid nanoparticle; The block copolymer is a) poly(butadiene)-poly(ethylene oxide) (PBD-PEO) block copolymers, preferably the PBD-PEO diblock copolymers comprising 5 to 50 blocks of PBD and 5 to 50 blocks of PEO; b) poly(caprolactone)-poly(ethylene oxide) (PCL-PEO) block copolymers, preferably the PCL-PEO diblock copolymers comprising 5 to 50 blocks of PCL and 5 to 50 blocks of PEO; c) poly(lactide-co-glycolide)-poly(ethylene oxide) (PLGA-PEO) block copolymers, wherein the PLGA-PEO diblock copolymer comprises 5 to 50 blocks of PLGA and 5 to 50 blocks of PEO; d) polylactic acid-poly(ethylene oxide) (PLA-PEO) block copolymers, wherein the PLA-PEO diblock copolymer comprises 5 to 50 blocks of PLA and 5 to 50 blocks of PEO; e) poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) block copolymer or triblock copolymer poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA), where the PDMS copolymer comprises 5-60 blocks of PDMS, 5-50 blocks of PEO, and 5-50 blocks of PMOXA; f) PIP-PEO (polyisoprene-polyethylene oxide); g) poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymer; h) Poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer selected from the group consisting of The polymer-lipid hybrid nanoparticles.

2. The mole percent ratio of lipid to block copolymer is from about 31.8:12 to about 35:2.5, preferably the mole percent ratio is as follows: a)49:12、 b)35:2.5、 c)31.8:12、 d)35:12、 e)23.8:4.8、 f)45:10、 g)49:8 2. The polymer-lipid hybrid nanoparticle of claim 1, selected from the group consisting of:

3. The following features: a) the polymer-lipid hybrid nanoparticles are synthetic; b) a diameter of greater than 75 nm, preferably the diameter is in the range of about 80 nm to about 450 nm, more preferably the diameter is in the range of about 80 nm to about 140 nm, and most preferably the diameter is in the range of about 100 nm to about 140 nm, the diameter being determined by a dynamic light scattering (DLS) instrument using Z-average; c) a polydispersity index (PDI) greater than about 0.15, preferably greater than 0.17, and more preferably between about 0.175 and about 0.245; and / or d) a zeta potential of about -40 mV to about +40 mV, preferably a zeta potential greater than 12 mV; e) Spherical particles exhibiting stacked bilayer structures; f) having an electro-lucent amorphous internal structure surrounded by a peripheral bilayer; g) spherical particles with an amorphous structure; h) vesicles with heterogeneous structure surrounded by a bilayer (e.g., capable of fusion and budding) 3. The polymer-lipid hybrid nanoparticle of claim 1 or 2, having one or more of:

4. further comprising a stabilizer, Preferably, the stabilizer comprises cholesterol. The polymer-lipid hybrid nanoparticle of claim 1.

5. 5. The polymer-lipid hybrid nanoparticle of claim 4, wherein the stabilizer is selected from the group consisting of cholesterol and hydroxycholesterol.

6. further comprising another lipid, Preferably, the other lipid is one of the following: a) Formula II: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 14:0 PC), b) Formula III: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, 18:0 PC), c) Formula IV: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d) Formula V: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), e) Formula VI: 1,2-dioleoyl-3-trimethylammonium propane (DODAP), f) Formula VII: 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), g) Formula VIII: 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), h) Formula IX: dimethyldioctadecylammonium (DDA), i) Formula X: 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA), j) Formula XI: 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), k) Formula XII: Bis(monooleoylglycero)phosphate (S and / or R isomers) (18:1 BMP) A cationic lipid, an ionizable cationic lipid, and / or an anionic lipid selected from the group consisting of: The polymer-lipid hybrid nanoparticle of claim 1.

7. The lipid (ionized lipid) is one of the following: a) Formula XIII: the ionizable lipid DLin-MC3-DMA (or MC3, i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), b) Formula XIV: ionized lipid C12-200, c) Formula XVIII: Ionized lipid 306O i10 2. The polymer-lipid hybrid nanoparticle of claim 1, selected from the group consisting of:

8. The block copolymer may be: a) a PBD-PEO block copolymer, the PBD-PEO diblock copolymer comprising: Preferably, Formula XV: wherein n=22, b represents a block, and m=12; * =OCH 3 5-50 blocks of PBD and 5-50 blocks of PEO wherein the PBD-PEO block copolymer comprises a PBD having the formula: 1.2k -b-PEO 0.6k the PBD-PEO block copolymer, b) a PCL-PEO block copolymer, wherein the PCL-PEO diblock copolymer is Preferably, Formula XVI: wherein n=22, m=29, * =H, 5-50 blocks of PCL and 5-50 blocks of PEO wherein the PCL-PEO block copolymer has k=1000 Da. 3.3k -b-PEO 1k the PCL-PEO block copolymer, c) a PLGA-PEO block copolymer, wherein the PLGA-PEO diblock copolymer is Preferably, Formula XVI: wherein x=23, y=4, m=26, n=29; 5 to 50 blocks of PLGA and 5 to 50 blocks of PEO; wherein the PLGA-PEO block copolymer has k=1000 Da. 1.9k -b-PEO 1k the PLGA-PEO block copolymer, d) PLA-PEO block copolymers, wherein the PLA-PEO diblock copolymer comprises 5 to 50 blocks of PLA and 5 to 50 blocks of PEO; e) PDMS-PEO block copolymers or PMOXA-PDMS-PMOXA triblock copolymers, wherein the PDMS copolymer comprises 5 to 60 blocks of PDMS, 5 to 50 blocks of PEO, and 5 to 50 blocks of PMOXA; f) PIP-PEO (polyisoprene-polyethylene oxide), g) poly(butadiene)-poly(ethylene oxide) (PB-PEO) diblock copolymers; h) Poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) diblock copolymer 2. The polymer-lipid hybrid nanoparticle of claim 1, selected from the group consisting of:

9. below: a) PBD-PEO, MC3, CHOL, preferably in a molar ratio of about 12:49:39; b) PBD-PEO, C12-200, CHOL, preferably in a molar ratio of about 12:49:39; c) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 2.5:16:35:46.5; d) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 12:12.8:31.8:43.4; e) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 12:6.5:35:46.5; f) PBD-PEO, DOPE, C12-200, CHOL, preferably in a molar ratio of about 4.8:23.8:23.8:47.6; g) DMG-PEG, DSPC, MC3, CHOL, preferably in a molar ratio of about 1.6:10.1:49.3:39.0; h) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 10:10:45.0:35.0; i) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 10:10:45.0:35.0; j) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 8:4:49.0:39.0; k) PCL-PEO, DMPC, MC3, CHOL, preferably in a molar ratio of about 8:4:49.0:39.0; l) DLin-MC3-DMA:cholesterol:PBD-b-PEO, preferably in a molar ratio of about 49:39:12; m) DLin-MC3-DMA:cholesterol:DSPC:PBD-b-PEO, preferably in a molar ratio of about 49.3:39.0:10.1:1.6; n) DOTAP:cholesterol:DSPC:PBD-b-PEO, preferably in a molar ratio of about 40:48:10:2; o) DOTMA:cholesterol:DSPC:PBD-b-PEO, preferably in a molar ratio of about 40:48:10:2 2. The polymer-lipid hybrid nanoparticle of claim 1, comprising or consisting of:

10. further comprising a soluble encapsulated antigen that is a protein and / or a polynucleotide; Preferably, the protein is a nuclease involved in gene editing or RNA editing, and the polynucleotide is selected from an RNA molecule or a DNA molecule. The polymer-lipid hybrid nanoparticle of claim 1.

11. 10. The polymer-lipid hybrid nanoparticle of claim 1, which is not a polymersome.

12. A composition comprising the polymer-lipid hybrid nanoparticles of claim 1.

13. 13. The composition of claim 12, which is a pharmaceutical or diagnostic composition.

14. An isolated antigen-presenting cell or hybridoma cell exposed to the polymer-lipid hybrid nanoparticle of claim 1.

15. 15. The antigen-presenting cell of claim 14, comprising a dendritic cell, a macrophage, and / or a B cell.

16. The composition of claim 13, further comprising a pharmaceutically acceptable excipient or carrier.

17. An in vitro method for delivering nucleotides to the interior of a cell without using a viral vector as a delivery means, comprising the steps of: i) contacting the polymer-lipid hybrid nanoparticles of claim 1 with cells.

18. 10. The polymer-lipid hybrid nanoparticle of claim 1 for use as a medicament.

19. The following methods: i) methods of treating and / or preventing a disease or disorder; ii) methods for discovering and / or screening and / or preparing antibodies; iii) methods of producing or preparing immunogenic or immunostimulatory compositions; iv) a method for targeted delivery of one or more polypeptides encoded by said polynucleotides, and most preferably, said targeted delivery is carried out within a subject; v) a method for stimulating an immune response against one or more polypeptides encoded by said polynucleotides; vi) CD8 (+) Methods for triggering cross-protection induced by T cell-mediated immune responses vii) CD8 (+) T cell-mediated immune response and / or CD4 (+) methods for triggering an immune response, including a T cell-mediated immune response; viii) A method for treating, ameliorating, preventing, and / or diagnosing an infectious disease, preferably wherein the infectious disease is a viral or bacterial infectious disease, more preferably wherein the viral infectious disease is selected from the group consisting of influenza infection, PED virus infection, food and mouth virus infection, respiratory syncytial virus infection, and herpes virus infection; ix) methods for treating, ameliorating, preventing, or diagnosing cancer or autoimmune diseases; x) methods for sensitizing cancer cells to chemotherapy; xi) methods for inducing apoptosis in cancer cells; xii) a method for stimulating an immune response in a subject; xiii) methods for immunizing non-human animals; xiv) methods for preparing hybridomas; xv) methods for modifying and / or manipulating and / or disrupting genetic material (e.g., genome and / or transcriptome) or templates (e.g., nucleotide sequences, e.g., RNA or DNA) inside a cell; xvi) methods for delivering nucleotides to the interior of cells without using viral vectors as a delivery means; xvii) a method for targeting antigen-presenting cells (e.g., the method does not include attaching a ligand to the polymer-lipid hybrid nanoparticle); xviii) a method for targeting a cell, preferably wherein the cell is not an antigen-presenting cell (e.g., the method comprises attaching a ligand to the polymer-lipid hybrid nanoparticle); xix) The method according to any one of i) to xviii), which is an in vivo and / or ex vivo and / or in vitro method.

19. The polymer-lipid hybrid nanoparticle for use according to claim 18 in one or more of the following:

Images