POEGMA-based lipid nanoparticles

Abstract

Disclosed herein are lipid nanoparticles comprising POEGMA-lipid conjugates that can effectively encapsulate and deliver therapeutic agents without the immunological consequences incurred by their PEG-based counterparts. Exemplary lipid nanoparticles include ionizable lipids, phospholipids, sterols, POEGMA-lipid conjugates, and therapeutic agents. Also disclosed herein are pharmaceutical compositions comprising POEGMA-based lipid nanoparticles, methods of treating diseases or disorders, and methods of delivering therapeutic agents to cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 271,595, filed October 25, 2021, which is hereby incorporated by reference in its entirety. Reference to Electronic Sequence Listing The contents of the electronic sequence listing (Name: 028193-9376-WO01_Sequence_Listing.xml; Size: 9,210 bytes; Creation Date: October 25, 2022) are incorporated by reference in their entirety into this specification. Technical Field The present disclosure relates to lipid nanoparticles comprising POEGMA-lipid conjugates and their use in biomedical applications such as drug delivery. [Background technology]

[0002] Introduction Lipid nanoparticles (LNPs) have emerged as potential carriers for nucleic acid delivery (e.g., mRNA) due to their biocompatibility, efficient complexation with payloads, cellular uptake, and successful endosomal escape. LNPs have emerged as a key component of COVID-19 mRNA vaccines due to their role in effectively protecting and transporting mRNA to cells. LNPs usually contain PEGylated lipids that can provide LNPs with stealth properties. LNPs with PEG coating have a much longer plasma half-life than native LNPs due to reduced opsonization and improved solubility. Unfortunately, in vivo administration of PEGylated LNPs has several limitations, including immunogenicity, allergic side reactions, and enhanced clearance by inducible and pre-existing PEG antibodies. Repeated administration of PEG can also lead to the formation of vacuoles in major organs due to its non-biodegradable structure and clearance by the RES. Furthermore, PEG intolerance has led to the early termination of several clinical trials and the withdrawal of several therapeutics from the market. According to the CDC, PEG has been identified as one of the main components responsible for allergic reactions to the LNP-based Pfizer-BioNTech covid vaccine. Summary of the Invention

[0003] In one aspect, a lipid nanoparticle is disclosed that includes an ionizable lipid; a phospholipid; a sterol; a poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugate at less than 10 mol%, wherein the POEGMA has a number average molecular weight of less than 100 kDa; and a therapeutic agent. In another aspect, a lipid nanoparticle is disclosed comprising (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102); DSPC; cholesterol; a POEGMA-lipid conjugate at about 0.25 mol % to about 3 mol %, wherein POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa; and mRNA. In another aspect, a pharmaceutical composition is disclosed, comprising one or more lipid nanoparticles disclosed herein; and a pharma- ceutically acceptable excipient. In another aspect, a method of treating a disease or disorder in a subject in need thereof is disclosed, comprising administering to the subject an effective amount of one or more lipid nanoparticles disclosed herein, optionally in combination with a pharma- ceutically acceptable excipient. In another aspect, a method of delivering a therapeutic agent to a cell is disclosed, comprising contacting the cell with one or more lipid nanoparticles disclosed herein, whereby the therapeutic agent is delivered to the cell. [Brief description of the drawings]

[0004] [Figure 1A] FIG. 1A shows gel permeation chromatography-multi-angle light scattering (GPC-MALS) (FIG. 1B) and reverse-phase high performance liquid chromatography (HPLC) traces for an exemplary azido poly[oligo(ethylene glycol) ether methacrylate] (POEGMA). [Figure 1B] Figure 1A. [Figure 2A] Purification and characterization of exemplary POEGMA-lipid conjugates (POEGMAL). Figure 2A: Schematic of purification of POEGMAL. Figure 2B: Typical TLC trace of POEGMAylated lipids. Figure 2C: Physical appearance of POEGMA10 and POEGMAL10. Figure 2D: Ratio of theoretical number of protons between δ 0.5-2.5 and δ 7.3-7.8 and corresponding values ​​experimentally determined by proton NMR. [Figure 2B] Figure 2A. [Figure 2C] Figure 2A. [Figure 2D] Figure 2A. [Figure 3A]Dynamic light scattering analysis of exemplary LNPs. Figures 3A-C: Blank LNPs. Hydrodynamic radius (Figure 3A), polydispersity (Figure 3B), and size distribution (Figure 3C) of various LNPs without mRNA. Figures 3D-F: LNPs with mRNA. Hydrodynamic radius (Figure 3D), polydispersity (Figure 3E), and size distribution (Figure 3F) of various LNPs with mRNA. [Figure 3B] Figure 3A. [Figure 3C] Figure 3A. [Figure 3D] Figure 3A. [Figure 3E] Figure 3A. [Figure 3F] Figure 3A. [Figure 4A] Characterization and quantification of encapsulated mRNA in exemplary LNPs. Figure 4A: Gel electrophoresis of various LNPs before (-) and after (+) the addition of TritonX-100. Figure 4B: Schematic of the Ribogreen assay. Figure 4C: Percent mRNA encapsulation efficiency measured by Ribogreen assay. *P<0.01, **P<0.001; Two-way ANOVA (Tukey's multiple comparison test). [Figure 4B] Same Figure 4A. [Figure 4C] Same Figure 4A. [Figure 5A] Analysis of lipid ratios to improve luciferase mRNA encapsulation efficiency (EE) of exemplary LNPs. Figure 5A: EE of LNPs at various mol% of POEGMAL10-50 and SM-102. Relationship between EE and hydrodynamic radius of various LNPs at 0.5 (Figure 5B), 1.5 (Figure 5C), and 2.5 (Figure 5D) mol%. Figure 5E: EE of exemplary LNPs after dialysis against PBS. [Figure 5B] Figure 5A. [Figure 5C] Figure 5A. [Figure 5D] Figure 5A. [Figure 5E] Figure 5A. [Figure 6A]Figure 6 shows analysis of luciferase mRNA EE of exemplary LNPs at different mol% of POEGMAL5 after dialysis against PBS: Figure 6A: radius; Figure 6B: % polydispersity; and Figure 6C: % encapsulation. [Figure 6B] Figure 6A. [Figure 6C] Figure 6A. [Figure 7] FIG. 1 shows cryo-transmission electron microscopy (Cryo-TEM) images of LNPPOEGMAL5 (upper panel) and LNPPOEGMAL10 (lower panel) after dialysis against PBS. [Figure 8A] Analysis of parameters to improve EE of LNPPOEGMAL10 to encapsulate therapeutically important mature full-length SARS COV-2 mRNA. EE before (Figure 8A) and after (Figure 8B) dialysis against PBS. Figure 8C: Hydrodynamic radius after dialysis against the indicated buffers. Effect of N:P (Figure 8D), ethanol fraction during LNP preparation (Figure 8E), and lipid mol% (Figure 8F) on mRNA encapsulation. [Figure 8B] Figure 8A. [Figure 8C] Figure 8A. [Figure 8D] Figure 8A. [Figure 8E] Figure 8A. [Figure 8F] Figure 8A. [Figure 9A] Figure 9 shows expression of Cluc mRNA cargo of exemplary LNPs in HEK293T cells: Figure 9A: relative expression to Lipofectamine 2000 at 500 ng mRNA; Figure 9B: relative expression to Lipofectamine 2000 at 300 ng mRNA; Figure 9C: raw AUC values ​​at different N:P at 500 ng; and Figure 9D: raw AUC values ​​at different N:P at 300 ng. [Figure 9B] Figure 9A. [Figure 9C] Figure 9A. [Figure 9D] Figure 9A. [Figure 10]FIG. 1 shows the toxicity of LNPs to HEK293T cells after 48 h of continuous treatment. [Figure 11A] Figure 11 shows expression of various amounts of CLuc mRNA cargo of exemplary LNPs in HEK293T cells at charge ratios (N:P) ranging from 4:1 to 8:1, circles: LNPPOEGMAL5, squares: LNPPOEGMAL10, and triangles: LNPPEG-DMG. Figure 11A: 500 ng of mRNA at 8:1; Figure 11B: 500 ng of mRNA at 6:1; Figure 11C: 500 ng of mRNA at 4:1; Figure 11D: 300 ng of mRNA at 8:1; Figure 11E: 300 ng of mRNA at 6:1; and Figure 11F: 300 ng of mRNA at 4:1. [Figure 11B] Figure 11A. [Figure 11C] Figure 11A. [Figure 11D] Figure 11A. [Figure 11E] Figure 11A. [Figure 11F] Figure 11A. [Figure 12] Figure 1: RNase protection assay. Exemplary LNPs were incubated for 0.5 hours after addition of RNase. The order of addition is shown in the table on the left and the gel is shown on the right. All of the exemplary LNPs protect the Cluc mRNA cargo from RNase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] Disclosed herein are POEGMA-ylated lipids that can be used to craft stealth LNPs for encapsulating therapeutics, such as full-length model luciferase mRNA and therapeutically important SARS COV-2 mRNA, with an EE of over 85%. Parameters of LNP fabrication, such as ethanol fraction, charge ratio (N:P), lipid mol%, and buffer exchange, were investigated to determine how each parameter can affect the EE of the LNPs. POEGMA-ylated LNPs also provided the necessary protection to the mRNA against RNases, a prerequisite for successful delivery in vivo. Furthermore, in reporter mRNA expression assays, LNPs containing 10 kDa POEGMA-ylated lipids outperformed Moderna biosimilar LNP formulations. This is promising as it makes a strong case for the POEGMA-ylated LNP platform for in vivo mRNA vaccine delivery while potentially avoiding the immunogenicity of PEG.

[0006] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, shall control. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or phrases that do not exclude additional acts or structures. The singular forms "a", "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements provided herein, whether or not expressly indicated.

[0007] The modifier "about" used in connection with an amount is inclusive of the recited value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular amount). The modifier "about" should also be considered to disclose the range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4." The term "about" can refer to plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about," such as rounding, may be apparent from the context, so that, for example, "about 1" can mean 0.5 to 1.4.

[0008] For the description of numerical ranges herein, each intervening number therebetween is expressly contemplated with the same precision. For example, for the range from 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range from 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0009] The term "antigen" refers to a molecule that can be bound by an antibody or a T cell receptor. The term "antigen" also encompasses T cell epitopes. Additionally, an antigen can be recognized by the immune system and / or can induce a humoral and / or cellular immune response leading to the activation of B and / or T lymphocytes. In some embodiments, an antigen contains or is linked to a Th cell epitope. An antigen can have one or more epitopes (B and T epitopes). Antigens can include polypeptides, polynucleotides, carbohydrates, lipids, small molecules, polymers, polymer conjugates, and combinations thereof. An antigen can also be a mixture of several different antigens. The term "antigenicity" refers to the ability of an antigen to specifically bind to a T cell receptor or antibody, including the reactivity of an antigen with pre-existing antibodies in a subject. The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desired biological and / or clinical results. The term "immunogenicity" refers to the ability of an antigen to induce an immune response, including the inherent ability of an antigen to produce antibodies in a subject. As used herein, the terms "antigenicity" and "immunogenicity" refer to different aspects of the immune system and are not interchangeable.

[0010] The term "mRNA" as used herein refers to messenger ribonucleic acid. mRNA may be naturally occurring or non-naturally occurring. For example, mRNA may contain modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. mRNA may contain a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. mRNA may have a nucleotide sequence that encodes a polypeptide. A polypeptide may be produced by translation of the mRNA, for example, by in vivo translation of the mRNA inside a mammalian cell. Typically, basic components of an mRNA molecule include at least a coding region, a 5' untranslated region (5'UTR), a 3'UTR, a 5' cap, and a polyA sequence. The term "N:P ratio," as used herein, refers to the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in lipids to phosphate groups in nucleic acids (e.g., RNA).

[0011] As used herein, the term "nucleic acid" is used in its broadest sense and includes any compound and / or substance that comprises a polymer of nucleotides. Such polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with bD-ribo configuration, a-LNA (a diastereomer of LNA) with aL-ribo configuration, 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-a-LNA with 2'-amino functionalization), or hybrids thereof. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0012] The term "phospholipid," as used herein, refers to a lipid that includes a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid can include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). The terms "polypeptide", "peptide" and "protein" as used herein can be used interchangeably to refer to a string of at least three amino acids linked together by peptide bonds. Peptides can contain natural amino acids, unnatural amino acids (i.e., compounds that do not occur in nature but can be incorporated into a polypeptide chain), and / or amino acid analogs. Also, one or more of the amino acids in a peptide can be modified by addition of a chemical entity, such as, for example, a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. Modifications can include cyclization of the peptide, incorporation of D-amino acids, and the like.

[0013] The term "RNA" as used herein refers to a ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, an RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). An RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0014] The term "treatment" or "treating" refers to protecting a subject from a disease, e.g., preventing, suppressing, inhibiting, ameliorating, or completely resolving the disease. Preventing a disease involves administering a conjugate of the present disclosure to a subject prior to the onset of the disease. Suppressing a disease involves administering a conjugate of the present disclosure to a subject after induction of the disease but prior to its clinical appearance. Suppressing or ameliorating a disease involves administering a conjugate of the present disclosure to a subject after clinical appearance of the disease. The term "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Exemplary subjects of the present disclosure may include mammals, particularly primates, and humans. For veterinary applications, suitable subjects may include, for example, livestock animals such as cattle, sheep, goats, dairy cows, swine, etc.; poultry such as chickens, ducks, geese, turkeys, etc., and domesticated animals, particularly pets such as dogs and cats. For research applications, suitable subjects may include mammals, such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs, etc.

[0015] 2. Lipid Nanoparticles Disclosed herein are lipid nanoparticles (LNPs) comprising POEGMA-lipid conjugates. The lipid nanoparticles can include an ionizable lipid, a phospholipid, a sterol, a POEGMA-lipid conjugate, and a therapeutic agent. The lipid nanoparticles can facilitate the introduction of a therapeutic agent, such as a nucleic acid, into a cell, tissue, organ, subject, etc. The lipid nanoparticles can also include a targeting ligand that can facilitate interaction with a target cell. For example, the targeting ligand can specifically interact with a target cell (e.g., specifically interact with an extracellular protein on the surface of the target cell) to improve the localization of the lipid nanoparticles after administration. Examples of targeting ligands include, but are not limited to, aptamers, carbohydrates, proteins, antibodies, single-chain variable fragments, and the like. In some embodiments, the lipid nanoparticles further include a targeting ligand. In addition, the components of the lipid nanoparticles can be pharma- ceutically acceptable salts thereof.

[0016] When the therapeutic agent includes a nucleic acid, the amount of lipid (e.g., ionizable lipid) and the amount of nucleic acid can be selected to provide a particular N:P ratio. The N:P ratio of a lipid nanoparticle refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the nucleic acid. The one or more nucleic acids, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 20:1, for example, about 3:1 to about 19:1, about 4:1 to about 18:1, about 5:1 to about 17:1, about 4:1 to about 16:1, about 6:1 to about 16:1, about 7:1 to about 15:1, about 8:1 to about 14:1, about 9:1 to about 13:1, about 7:1 to about 12:1, about 5:1 to about 14:1, about 8:1 to about 12:1, about 2:1 to about 12:1, or about 3:1 to about 12:1. In some embodiments, the lipid nanoparticles have an N:P ratio of more than 2:1, more than 3:1, more than 4:1, more than 5:1, more than 6:1, more than 7:1, more than 8:1, more than 9:1, more than 10:1, or more than 11:1. In some embodiments, the lipid nanoparticles have an N:P ratio of less than 20:1, less than 19:1, less than 18:1, less than 17:1, less than 16:1, less than 15:1, less than 14:1, less than 13:1, less than 12:1, or less than 11:1. In some embodiments, the lipid nanoparticles have an N:P ratio of about 10:1. In some embodiments, the lipid nanoparticles have an N:P ratio of about 8:1.

[0017] The lipid nanoparticles may have a variety of particle sizes, which may depend on the lipid components contained in the lipid nanoparticles. For example, the lipid nanoparticles may have a diameter of about 30 nm to about 300 nm, such as about 35 nm to about 250 nm, about 40 nm to about 200 nm, about 30 nm to about 150 nm, about 30 nm to about 100 nm, about 35 nm to about 90 nm, about 40 nm to about 80 nm, or about 35 nm to about 125 nm. In some embodiments, the lipid nanoparticles have a diameter of more than 30 nm, more than 35 nm, more than 40 nm, more than 45 nm, or more than 50 nm. In some embodiments, the lipid nanoparticles have a diameter of less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, or less than 100 nm.

[0018] The zeta potential of lipid nanoparticles can be used to indicate the electrokinetic potential of a composition. For example, the zeta potential can describe the surface charge of lipid nanoparticles. Lipid nanoparticles having a relatively low charge, whether positive or negative, are generally desirable because more highly charged species may have undesirable interactions with cells, tissues, and other elements in the body. Lipid nanoparticles can have a zeta potential of about -10 mV to about +20 mV, e.g., about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about 0 mV to about +5 mV, or about -5 mV to about +10 mV. In some embodiments, lipid nanoparticles have a zeta potential of greater than -5 mV, greater than 0 mV, greater than +1 mV, greater than +2 mV, +3 mV, or greater than +4 mV. In some embodiments, the lipid nanoparticles have a zeta potential of less than +20 mV, less than +19 mV, less than +18 mV, less than +17 mV, less than +16 mV, or less than +15 mV.

[0019] The efficiency of encapsulation of a therapeutic agent refers to the amount of therapeutic agent encapsulated or otherwise associated with a lipid nanoparticle after preparation compared to the initial amount provided. It is desirable for the encapsulation efficiency to be high. The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic agent in a solution containing the lipid nanoparticle before and after disrupting the lipid nanoparticle with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., RNA) in the solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a therapeutic agent molecule can be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, or about 100%. In some embodiments, the encapsulation efficiency is 75% or more. In some embodiments, the encapsulation efficiency is 85% or more. In some embodiments, the encapsulation efficiency is from about 70% to about 99%, such as from about 70% to about 90%, from about 75% to about 95%, or from about 75% to about 99%. Lipid nanoparticles can be characterized by various methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of lipid nanoparticles. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can be used to determine particle size as well. Instruments such as Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure several properties of lipid nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0020] The incorporation of POEGMA-lipid conjugates allows the disclosed lipid nanoparticles to have advantageous immune response properties compared to PEG-lipid conjugates. For example, the lipid nanoparticles can have a reduced immune response compared to lipid nanoparticles containing PEG. The reduced or eliminated immune response can include reduced or eliminated antigenicity, reduced or eliminated immunogenicity, or both for the lipid nanoparticles. The beneficial immune interaction of the lipid nanoparticles can also be viewed in that the lipid nanoparticles may not react with the subject's pre-existing anti-PEG antibodies. Thus, the disclosed lipid nanoparticles can have beneficial interactions with the subject's immune system. Analysis of lipid nanoparticle interactions with the subject's immune system can be evaluated as described in PCT / US2022 / 023158 (published as WO2022 / 212911), the entirety of which is incorporated herein by reference.

[0021] Lipid nanoparticles can be made by a number of different techniques. Exemplary techniques include alcohol injection. For example, ionizable lipids, phospholipids, sterols, and POEGMA-lipid conjugates can be added to alcohol to form a first mixture. The alcohol can be ethanol. The first mixture can be injected into a second mixture to prepare a lipid nanoparticle mixture. The second mixture can include a therapeutic agent and a buffer (e.g., a citrate buffer). The lipid nanoparticle mixture may be dialyzed after preparation.

[0022] A. POEGMA-lipid conjugates POEGMA-lipid conjugates include POEGMA and a lipid. POEGMA-lipid conjugates can infuse the conjugate with advantageous stealth and immune system properties. The lipid nanoparticles can include one type of POEGMA-lipid conjugate (e.g., a single type of conjugate) or can include at least two, at least three, at least four, or at least five different types of POEGMA-lipid conjugates. In some embodiments, the lipid nanoparticles include 2-5 different types of POEGMA-lipid conjugates. As an example, the lipid nanoparticles can include at least two different POEGMA-lipid conjugates that vary by the molecular weight of the POEGMA, the hydrocarbon length of the lipid, or both. These variations (e.g., molecular weight and hydrocarbon chain length) are discussed further below. POEGMA and lipid can be included in a 1:1 stoichiometric molar ratio. For example, the conjugate can include one POEGMA molecule attached to one lipid molecule.

[0023] POEGMA has a poly(methyl methacrylate) backbone and multiple side chains covalently attached to the backbone. The side chains are oligomers of ethylene glycol (EG). For example, each side chain can include 2-9 monomers of EG repeated in tandem, such as 2-8 monomers of EG repeated in tandem, 2-7 monomers of EG repeated in tandem, 2-6 monomers of EG repeated in tandem, 2-5 monomers of EG repeated in tandem, or 2-4 monomers of EG repeated in tandem. In some embodiments, each side chain includes 3 monomers of EG repeated in tandem. Adjacent side chains may be the same or different within the same POEGMA molecule, for example, one side chain may have three monomers of EG repeated in tandem, while another side chain (in the same POEGMA molecule) may have four monomers of EG repeated in tandem.

[0024] Each side chain can have a first end and a second end. The first end can be covalently attached to the backbone. The second end can be free. The second end can be modified. In some embodiments, each second end independently comprises an alkyl, ester, amine, amide, or carboxyl group. In some embodiments, each second end comprises an alkyl. In some embodiments, each second end comprises a C1-C4 alkyl. In some embodiments, each second end comprises a methyl group. In some embodiments, each second end does not comprise a hydroxyl group. The second end of each side chain can be the same or different from the second end of an adjacent side chain within the same POEGMA molecule. In some embodiments, the second end of each side chain is the same throughout the POEGMA. In some embodiments, the second end of at least one side chain is different from the second end of at least one adjacent side chain. In addition, the backbone can have a first end and a second end.

[0025] POEGMA can have various molecular weights. For example, POEGMA can have a number average molecular weight of about 1 kDa to about 100 kDa, for example, about 1 kDa to about 85 kDa, about 1 kDa to about 75 kDa, about 1 kDa to about 60 kDa, about 1 kDa to about 50 kDa, about 2 kDa to about 45 kDa, about 3 kDa to about 40 kDa, about 4 kDa to about 35 kDa, about 5 kDa to about 30 kDa, about 1 kDa to about 30 kDa, about 1 kDa to about 25 kDa, about 1 kDa to about 20 kDa, about 1 kDa to about 15 kDa, about 1 kDa to about 12 kDa, or about 1 kDa to about 10 kDa. In some embodiments, the POEGMA has a number average molecular weight of more than 1 kDa, more than 2 kDa, more than 3 kDa, more than 4 kDa, more than 5 kDa, more than 6 kDa, more than 7 kDa, more than 8 kDa, more than 9 kDa, or more than 10 kDa. In some embodiments, the POEGMA has a number average molecular weight of less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 15 kDa, less than 12 kDa, or less than 10 kDa. In some embodiments, the POEGMA has a number average molecular weight of about 10 kDa. The molecular weight of POEGMA can be measured by techniques used in the art, such as SEC, SEC combined with multi-angle light scattering, gel permeation chromatography, etc.

[0026] The lipid of the conjugate can be any suitable lipid that can be conjugated to POEGMA and allow the conjugate to be included in a lipid nanoparticle. The lipid can be saturated or unsaturated. The lipid can include a hydrocarbon chain of various lengths. The number of carbon atoms in the hydrocarbon chain is indicated by the prefix "C". x-y " or "C x -C y -", where x is the minimum number of carbon atoms in the hydrocarbon chain and y is the maximum number. Thus, for example, "C 6-22 Hydrocarbon chain" or "C6-C 22 "Hydrocarbon chain" refers to a hydrocarbon chain containing 6 to 22 carbon atoms. Lipids include 2-40 Hydrocarbon chains, e.g., C 2-35Hydrocarbon chain, C 2-30 Hydrocarbon chain, C 2-25 Hydrocarbon chain, C 2-20 Hydrocarbon chain, C 4-40 Hydrocarbon chain, C 10-40 Hydrocarbon chain, C 6-22 Hydrocarbon chain, C 10-28 Hydrocarbon chain, C 12-20 Hydrocarbon chain, C 10-22 Hydrocarbon chain, C 12-30 Hydrocarbon chain, C 14-40 Hydrocarbon chain, C 12-18 Hydrocarbon chain, or C 8-18 A hydrocarbon chain may be included. In some embodiments, the lipid of the POEGMA-lipid conjugate has a hydrocarbon chain that is more than 4 carbons long, more than 6 carbons long, more than 8 carbons long, more than 10 carbons long, more than 12 carbons long, or more than 14 carbons long. In some embodiments, the lipid of the POEGMA-lipid conjugate has a hydrocarbon chain that is less than 40 carbons long, less than 36 carbons long, less than 32 carbons long, less than 30 carbons long, less than 24 carbons long, or less than 20 carbons long.

[0027] A lipid can include one hydrocarbon chain or multiple hydrocarbon chains. For example, a lipid can include 1-5 individual hydrocarbon chains, e.g., 1-4 individual hydrocarbon chains, 2-5 individual hydrocarbon chains, 1-3 individual hydrocarbon chains, or 2-4 individual hydrocarbon chains. In some embodiments, a lipid can include more than one individual hydrocarbon chain, more than two individual hydrocarbon chains, or more than three individual hydrocarbon chains. In some embodiments, a lipid can include less than five individual hydrocarbon chains, less than four individual hydrocarbon chains, or less than three individual hydrocarbon chains. Lipids with more than one hydrocarbon chain can have hydrocarbon chains of different lengths, as described above. In addition, embodiments that include multiple hydrocarbon chains can include individual hydrocarbon chains that are all the same length, or the lipids can include individual hydrocarbon chains of different lengths.

[0028] The lipids can also include a number of different functional groups that can allow flexibility in conjugating the lipid to POEGMA. For example, the lipids can include triazoles, amides, esters, ethers, hydrocarbon linkers, and other suitable conjugation linkers. In some embodiments, the lipids are conjugated to POEGMA via triazoles, amides, esters, ethers, or hydrocarbon linkers. In some embodiments, the lipids of the POEGMA-lipid conjugates include 1,2-dimyristoyl-sn-glycerol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dimyristyloxlpropyl-3-amine, or combinations thereof. Further discussion of the various linker strategies is discussed below.

[0029] The POEGMA-lipid conjugate can be included in various amounts in the lipid nanoparticles. For example, the lipid nanoparticles can contain the POEGMA-lipid conjugate in an amount of about 0.1 mol% to about 10 mol%, for example, about 0.2 mol% to about 9.5 mol%, about 0.3 mol% to about 9 mol%, about 0.4 mol% to about 8.5 mol%, about 0.1 mol% to about 8 mol%, about 0.1 mol% to about 7.5 mol%, about 0.1 mol% to about 7 mol%, about 0.1 mol% to about 6.5 mol%, about 0.1 mol% to about 6 mol%, about 0.1 mol% to about 5.5 mol%, about 0.1 mol% to about 5 mol%, about 0.2 mol% to about 7 mol%. 1%, about 0.2 mol% to about 6 mol%, about 0.3 mol% to about 6 mol%, about 0.3 mol% to about 5.5 mol%, about 0.1 mol% to about 5 mol%, about 0.1 mol% to about 4.5 mol%, about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3.5 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2.5 mol%, about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1.5 mol%, about 0.1 mol% to about 1 mol%, or about 0.1 mol% to about 0.75 mol%. Here and throughout, mol% refers to the molar percentage of a component, e.g., the POEGMA lipid conjugate, in relation to the total amount of lipid components (e.g., ionizable lipids, phospholipids, sterols, and POEGMA lipid conjugate) of the lipid nanoparticle.

[0030] In some embodiments, the lipid nanoparticles contain POEGMA-lipid conjugates at greater than 0.1 mol%, greater than 0.15 mol%, greater than 0.2 mol%, greater than 0.25 mol%, greater than 0.3 mol%, greater than 0.35 mol%, greater than 0.4 mol%, greater than 0.45 mol%, greater than 0.5 mol%, greater than 1 mol%, greater than 2 mol%, greater than 3 mol%, greater than 4 mol%, or greater than 5 mol%. In some embodiments, the lipid nanoparticles contain POEGMA-lipid conjugates at less than 10 mol%, less than 9.5 mol%, less than 9 mol%, less than 8.5 mol%, less than 8 mol%, less than 7.5 mol%, less than 7 mol%, less than 6.5 mol%, less than 6 mol%, less than 5.5 mol%, less than 5 mol%, less than 4.5 mol%, less than 4 mol%, less than 3.5 mol%, less than 3 mol%, less than 2.5 mol%, less than 2 mol%, less than 1.5 mol%, less than 1 mol%, less than 0.9 mol%, less than 0.8 mol%, less than 0.75 mol%, or less than 0.6 mol%.

[0031] POEGMA can be conjugated to lipids via any suitable conjugation strategy known in the art. For example, lipids and POEGMA can each have complementary functional groups, in that they can form covalent bonds between the functional groups under the proper conditions. Exemplary complementary functional groups that can form covalent bonds include, but are not limited to, amines and activated esters, amines and isocyanates, amines and isothiocyanates, amines and carbonates, thiols for disulfide formation, aldehydes and amines for enamine formation, and azides for amide formation via Staudinger ligation. Functional groups suitable for conjugation also include bioorthogonal functional groups. Bioorthogonal functional groups can selectively react with complementary bioorthogonal functional groups. Bioorthogonal functional groups include, but are not limited to, azides and alkynes for the formation of triazoles via click chemistry, trans-cyclooctene (TCO) and tetrazines (Tz) (e.g., 1,2,4,5-tetrazine), and the like. In some embodiments, the lipid and POEGMA each individually comprise a bioorthogonal functional group. In some embodiments, the lipid is functionalized with dibenzocyclooctyne and the POEGMA is functionalized with azide or both. Depending on the functional group, various bonds or linkages can be formed between the lipid and the POEGMA. The POEGMA can be functionalized at its backbone or at a side chain.

[0032] Further discussion of POEGMA, its synthesis, and its applications can be found in US Pat. No. 8,497,356 and US Pat. No. 10,364,451, both of which are incorporated by reference herein in their entireties.

[0033] B. Ionizable lipids The lipid nanoparticles can include one or more ionizable lipids. An "ionizable lipid" (or alternatively, a cationic lipid) refers to a lipid that has a positive or partial positive charge at physiological pH (e.g., a pH of about 7.4). An ionizable lipid can also be zwitterionic, i.e., a neutral molecule that has both positive and negative charges. The lipid nanoparticles can include one type of ionizable lipid (e.g., a single type of ionizable lipid), or can include at least two, at least three, at least four, or at least five different types of ionizable lipids. In some embodiments, the lipid nanoparticles include between two and five different types of ionizable lipids.

[0034] Examples of ionizable lipids include, but are not limited to, (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbanioyloxy-3-dimethylaniinopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylamino ... imethylammopropane (DLm-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaeonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate ((6Z,9Z,28Z,3lZ)-heptatriaeonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67), bis-guanidinium-spermidine-cholesterol (BGTC), 3b-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (N-(N AN'-dimethylammoethanej-carbam oxl cholesterol) (DC-Chol), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMR1E), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (N-(l-(2,3-dioleyloxy3)propyl)-N-2-(spenninecarboxamido)ethyl)-N,N-dimethylamrnonium trifluoracetate) (DOSPA), 2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), and (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0035] In some embodiments, the ionizable lipid includes (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or a combination thereof. In some embodiments, the ionizable lipid includes (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102).

[0036] Ionizable lipids can be included in various amounts in lipid nanoparticles. For example, lipid nanoparticles can include ionizable lipids at about 20 mol% to about 65 mol%, for example, about 25 mol% to about 60 mol%, about 25 mol% to about 65 mol%, about 30 mol% to about 65 mol%, about 40 mol% to about 65 mol%, about 45 mol% to about 65 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 20 mol% to about 45 mol%, about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 20 mol% to about 65 mol%, about 40 mol% to about 55 mol%, or about 45 mol% to about 55 mol%. In some embodiments, the lipid nanoparticles comprise more than 20 mol%, more than 25 mol%, more than 30 mol%, more than 35 mol%, more than 40 mol%, more than 45 mol%, more than 50 mol%, or more than 55 mol% of ionizable lipids. In some embodiments, the lipid nanoparticles comprise less than 65 mol%, less than 60 mol%, less than 58 mol%, less than 56 mol%, less than 54 mol%, less than 52 mol%, less than 50 mol%, or less than 45 mol% of ionizable lipids.

[0037] C. Phospholipids The lipid nanoparticles can include one or more phospholipids. Generally, the phospholipids can include a phospholipid moiety and one or more fatty acid moieties. The lipid nanoparticles can include one type of phospholipid (e.g., a single type of phospholipid), or can include at least two, at least three, at least four, or at least five different types of phospholipids. In some embodiments, the lipid nanoparticles include 2-5 different types of phospholipids.

[0038] Examples of phospholipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl 1-Stearoyl-2-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine oleoyl phosphatidylamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), ), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, and dilinoleoylphosphatidylcholine.

[0039] In some embodiments, the phospholipid includes DSPC, DPPE, DMPG, DOPC, DPPC, DOPG, or a combination thereof. In some embodiments, the phospholipid includes DSPC, DPPE, DOPC, or a combination thereof. In some embodiments, the phospholipid includes DSPC. In some embodiments, the phospholipid is DSPC.

[0040] Phospholipids can be included in the lipid nanoparticles in various amounts. For example, the lipid nanoparticles can include phospholipids at about 5 mol% to about 25 mol%, for example, about 6 mol% to about 20 mol%, about 7 mol% to about 18 mol%, about 8 mol% to about 16 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 6 mol% to about 15 mol%, about 6 mol% to about 12 mol%, about 8 mol% to about 12 mol%, or about 9 mol% to about 11 mol%. In some embodiments, the lipid nanoparticles include phospholipids at more than 5 mol%, more than 6 mol%, more than 7 mol%, more than 8 mol%, more than 9 mol%, more than 10 mol%, or more than 15 mol%. In some embodiments, the lipid nanoparticles contain less than 20 mol%, less than 19 mol%, less than 18 mol%, less than 17 mol%, less than 16 mol%, less than 15 mol%, less than 14 mol%, less than 13 mol%, less than 12 mol%, less than 11 mol%, or less than 10 mol% phospholipids.

[0041] D. Sterols The lipid nanoparticles can include one or more sterols. The term "sterol" refers to a subgroup of steroids also known as steroid alcohols. Sterols are commonly divided into two classes: (1) plant sterols, also known as "phytosterols," and (2) animal sterols, also known as "zoosterols." The lipid nanoparticles can include one sterol (e.g., a single sterol), or can include at least two, at least three, at least four, or at least five different types of sterols. In some embodiments, the lipid nanoparticles include between two and five different types of sterols. In some embodiments, the sterol includes zoosterol.

[0042] Examples of sterols include, but are not limited to, cholesterol, campesterol, anthrosterol, desmosterol, nicasterol, stigmasterol, sitosterol, oxysterol, C 4-10 Sterols include cholesterol, campesterol, anthrosterol, desmosterol, nicasterol, stigmasterol, sitosterol, oxysterol, C 4-10 Sterols include ergosterol, cholest-4-en-3-one, or combinations thereof. In some embodiments, the sterols include cholesterol, campesterol, stigmasterol, sitosterol, C 4-10 sterol, ergosterol, cholest-4-en-3-one, or combinations thereof.

[0043] In some embodiments, the sterol comprises cholesterol. In some embodiments, the sterol is cholesterol. The cholesterol can be cholesterol itself or its salt or ester, such as cholesterol succinate, cholesterol sulfate, cholesterol hemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterol valerate, cholesterol acetate, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate, or cholesteryl phosphorylcholine.

[0044] Sterols may include derivatives of cholesterol. Examples of derivatives of cholesterol include, but are not limited to, dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 19 ... Cholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, and fecosterol, or a salt or ester thereof.

[0045] Sterols can be included in the lipid nanoparticles in various amounts. For example, lipid nanoparticles can include sterols at about 10 mol% to about 50 mol%, for example, about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 40 mol%, about 35 mol% to about 45 mol%, about 35 mol% to about 40 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 50 mol%, about 15 mol% to about 40 mol%, or about 15 mol% to about 35 mol%. In some embodiments, lipid nanoparticles include sterols at more than 10 mol%, more than 15 mol%, more than 20 mol%, more than 25 mol%, more than 30 mol%, or more than 35 mol%. In some embodiments, the lipid nanoparticles comprise less than 50 mol%, less than 45 mol%, less than 42 mol%, less than 40 mol%, less than 38 mol%, or less than 35 mol% sterol.

[0046] E. Therapeutic drugs The lipid nanoparticles can include one or more therapeutic agents. Examples of therapeutic agents include, but are not limited to, nucleic acids and anionic polypeptides. In some embodiments, the lipid nanoparticles include nucleic acids, anionic polypeptides, or both. In some embodiments, the lipid nanoparticles include nucleic acids or anionic polypeptides.

[0047] The lipid nanoparticles can include one or more nucleic acids. The nucleic acids can be used, for example, to produce a polypeptide in a cell. Examples of nucleic acids include, but are not limited to, siRNA, miRNA, antisense oligonucleotides, shRNA, mRNA, tRNA, rRNA, CircRNA, and DNA. In some embodiments, the nucleic acid includes siRNA, miRNA, antisense oligonucleotides, shRNA, mRNA, tRNA, rRNA, CircRNA, DNA, or a combination thereof. In some embodiments, the nucleic acid includes siRNA, miRNA, antisense oligonucleotides, shRNA, mRNA, tRNA, rRNA, CircRNA, or DNA. In some embodiments, the nucleic acid includes siRNA, miRNA, antisense oligonucleotides, shRNA, mRNA, tRNA, rRNA, or CircRNA. In some embodiments, the nucleic acid includes siRNA, mRNA, or a combination thereof. In some embodiments, the nucleic acid includes siRNA or mRNA. In some embodiments, the nucleic acid includes mRNA. In some embodiments, the nucleic acid is an mRNA. The mRNA can encode a polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity, hi some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0048] The nucleic acid can be RNA. Examples of RNA include, but are not limited to, messenger RNA (mRNA) (e.g., encoding a protein of interest), modified mRNA (mmRNA), mRNA incorporating a microRNA binding site (miR binding site), modified RNA containing a functional RNA element, microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology. In some embodiments, the nucleic acid comprises SEQ ID NO:1, SEQ ID NO:2, or a combination thereof. In some embodiments, the nucleic acid comprises SEQ ID NO:1 or SEQ ID NO:2.

[0049] Nucleic acids and anionic polypeptides can be purchased commercially. Alternatively, nucleic acids can be prepared by in vitro transcription from DNA templates. Techniques and methods for preparing nucleic acids from DNA templates can be performed by techniques known in the art, such as those described in the Examples. Nucleic acids can be modified before application by sequence stabilization, capping, and polyadenylation. In addition, anionic polypeptides can be prepared via chemical synthesis and / or recombinant methods.

[0050] In some embodiments, the lipid nanoparticles comprise (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102); DSPC; cholesterol; poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugate at about 0.25 mol% to about 3 mol%, wherein POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa; and mRNA.

[0051] 3. Pharmaceutical Compositions Further disclosed herein is a pharmaceutical composition comprising one or more lipid nanoparticles. The pharmaceutical composition may further comprise a pharma- ceutically acceptable excipient. The term "pharmaceutically acceptable excipient" as used herein means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation aid. Some examples of materials that can be utilized as pharma- ceutically acceptable excipients include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as, but are not limited to, corn starch and potato starch; cellulose and its derivatives such as, but are not limited to, sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but are not limited to, cocoa butter and suppository wax; oils such as, but are not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil. glycols such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffers such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, citrate buffer, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator. The type of excipient used can be dictated by the route by which the composition is administered and the form of the composition.

[0052] The pharma- ceutically acceptable excipient may comprise more than 50% of the total mass or volume of the pharmaceutical composition comprising the lipid nanoparticles. For example, the pharma- ceutically acceptable excipient may comprise about 50%, about 60%, about 70%, about 80%, about 90%, or more of the pharmaceutical composition. In some embodiments, the pharma- ceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the pharma- ceutically acceptable excipient is approved for human use and for veterinary use. In some embodiments, the pharma- ceutically acceptable excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the pharma- ceutically acceptable excipient is pharmaceutical grade. In some embodiments, the pharma- ceutically acceptable excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0053] General guidance on the formulation and manufacture of pharmaceutical compositions and medicaments is available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006, the entire contents of which are incorporated herein by reference. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, except to the extent that any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the lipid nanoparticles. An excipient or auxiliary ingredient may be incompatible with the components of the lipid nanoparticles if its combination with the components of the lipid nanoparticles may cause any undesirable biological effects or otherwise adverse effects.

[0054] In some embodiments, the pharma- ceutically acceptable excipients include a buffering agent, a solubilizing agent, a solvent, an antimicrobial preservative, an antioxidant, a suspending agent, a tablet or capsule diluent, a tablet disintegrating agent, or a combination thereof. In some embodiments, the pharma-ceutically acceptable excipients include a buffering agent, a solubilizing agent, a solvent, an antimicrobial preservative, an antioxidant, a suspending agent, a tablet or capsule diluent, or a tablet disintegrating agent. In some embodiments, the pharma- ceutically acceptable excipient includes a buffer. In some embodiments, the buffer includes citrate and alcohol. In some embodiments, the buffer is about 70% to about 80% mM citrate-ethanol buffer. In some embodiments, the buffer is dialyzed against another buffer. In some embodiments, the buffer is dialyzed against tris acetate buffer.

[0055] A pharmaceutical composition can be suitable for administration to a subject (e.g., a patient, which may be a human or a veterinary patient, which may be a non-human) as is well known to those skilled in the pharmaceutical arts. A pharmaceutical composition can be prepared for administration to a subject. Such pharmaceutical compositions can be administered in dosages and by techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight and condition of the particular subject, as well as the route of administration. The composition may be administered prophylactically or therapeutically. In prophylactic administration, the composition may be administered in an amount sufficient to induce a response. In therapeutic applications, the composition may be administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount appropriate to achieve this is defined as a "therapeutically effective dose". The amount effective for this use depends, for example, on the specific composition of the conjugate regimen administered, the mode of administration, the stage and severity of the disease, the general health of the patient, and the judgment of the prescribing physician. The compositions may conveniently be administered in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. The sub-dose itself may, for example, be further divided, into a number of discrete loosely spaced administrations.

[0056] As would be readily apparent to one of skill in the art, useful in vivo dosages to be administered and the particular mode of administration will vary depending on the age, weight, severity of the affliction, and subject being treated, the particular compounds used, and the particular application for which these compounds are being used. The determination of effective dosage levels, i.e., the dosage levels necessary to achieve the desired result, can be made by one of skill in the art using routine methods, such as human clinical trials, in vivo studies, and in vitro studies. Dosage and intervals can be adjusted individually to provide plasma levels of the biologically active agent that are sufficient to maintain the modulatory effect or minimal effective concentration (MEC). The MEC varies from agent to agent, but can be estimated from in vivo and / or in vitro data. The dosage required to obtain the MEC depends on individual characteristics and the route of administration. However, plasma concentrations can be determined using assays well known to those skilled in the art. Dosage intervals can also be determined using the MEC value. The composition can be administered using a regimen that maintains plasma levels above the MEC for 10-90% of the time, for example between 30-90% or 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0057] The pharmaceutical composition may be administered in various dosages depending, for example, on various characteristics of the subject and the route of administration. In some embodiments, the pharmaceutical composition of the present disclosure is administered in a given dose in an amount of about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0. The therapeutic nanoparticles or lipid nanoparticles of the present disclosure may be administered at a dosage level sufficient to deliver about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 1 mg / kg, where a 1 mg / kg dose provides 1 mg of therapeutic nanoparticles or lipid nanoparticles per kg of subject body weight. In some embodiments, a dose of about 0.005 mg / kg to about 5 mg / kg of the therapeutic nanoparticles or lipid nanoparticles of the present disclosure may be administered.

[0058] It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity or organ failure. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response was not adequate (excluding toxicity). The amplitude of the administered dose in the management of the disorder of interest will vary according to the severity of the condition being treated and the route of administration. Furthermore, the dose and perhaps the dose frequency will also vary according to the age, weight, and response of the individual patient. A program comparable to that discussed above can be used in veterinary medicine.

[0059] 4. Method Also disclosed herein are methods of using the lipid nanoparticles and pharmaceutical compositions thereof. The descriptions of the lipid nanoparticles, POEGMA-lipid conjugates, ionizable lipids, phospholipids, sterols, therapeutic agents, and pharmaceutical compositions disclosed herein can also be applied to the methods of treating and the methods of delivering therapeutic agents to cells.

[0060] A. Methods of Treating a Disease or Disorder Further provided is a method for treating a disease or disorder in a subject in need thereof. The method can include administering to the subject an effective amount of one or more lipid nanoparticles disclosed herein. The lipid nanoparticles can be administered in combination with a pharma- ceutically acceptable excipient (e.g., as a pharmaceutical composition disclosed). The disclosed lipid nanoparticles can facilitate the production of polypeptides in cells, tissues, organs, or subjects. Thus, several diseases or disorders can benefit from this capability. Exemplary diseases or disorders include, but are not limited to, infectious diseases, Huntington's disease, muscular dystrophy, autoimmune diseases, and cancer. In some embodiments, the disease or disorder is an infectious disease, cancer, or an autoimmune disease. In some embodiments, the disease or disorder is an infectious disease, such as a virus. In some embodiments, the method can modulate the immune response by a subject suffering from an infectious disease, thereby enhancing the immune response to the infectious disease pathogen in the subject. Non-limiting examples of infectious diseases that can be treated include those caused by viruses, bacteria, fungi, yeast, and parasitic pathogens. Examples of viruses include, but are not limited to, influenza A and B viruses, Zika, rabies, RSV, chikungunya, cyclomegalovirus, human metapneumovirus, Ebola hemorrhagic fever, HIV-1, and SARS-CoV-2.

[0061] The method can be used to modulate an immune response, for example in a manner modulated by a vaccine. In some embodiments, the nucleic acid of the lipid nanoparticle can provide a polypeptide that can stimulate activation or activity of an immune cell, such as a dendritic cell or a myeloid cell. For example, the nucleic acid can encode a polypeptide that is an antigen, such as a vaccine antigen (e.g., a viral antigen, a bacterial antigen, a tumor antigen). In some embodiments, the nucleic acid (e.g., mRNA) associated with / encapsulated by the lipid nanoparticle encodes an antigen of interest, such as a cancer antigen or an infectious disease antigen (e.g., a bacterial antigen, a viral antigen, a fungal antigen, a protozoan antigen, or a parasitic antigen).

[0062] In some embodiments, the method is used to stimulate an immune response by a subject suffering from cancer, thereby enhancing the immune response against the cancer in the subject. Non-limiting examples of cancers that can be treated include adrenal cortical carcinoma, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown primary, Castleman's disease, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndromes (including refractory anemia and refractory cytopenia), myeloproliferative neoplasms or diseases (polycythemia vera, essential cancer, including thrombocytosis and primary myelofibrosis), liver cancer (e.g., hepatocellular carcinoma), non-small cell lung cancer, small cell lung cancer, pulmonary carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue sarcoma, basal and squamous cell carcinoma, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and secondary cancers caused by cancer treatment.

[0063] In some embodiments, the methods are used to modulate immune responses by subjects with abnormal immune activity, including those suffering from an autoimmune disease, an allergic disorder, or an inflammatory response. Non-limiting examples of autoimmune diseases that can be treated include rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), type 1 diabetes, multiple sclerosis, psoriasis, Graves' disease, Hashimoto's thyroiditis, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, myasthenia gravis, glomerulonephritis, and vasculitis. As discussed elsewhere, lipid nanoparticles and pharmaceutical compositions thereof can have advantageous immune properties, for example, after administration, lipid nanoparticles and pharmaceutical compositions thereof can have a reduced immune response compared to lipid nanoparticles containing PEG, may not react with pre-existing anti-PEG antibodies in the subject, or a combination thereof.

[0064] B. Methods of Delivering Therapeutics to Cells Also provided is a method of delivering a therapeutic agent to a cell. The method can include delivering a therapeutic agent to a cell by contacting the cell with one or more lipid nanoparticles or pharmaceutical compositions thereof disclosed herein. In some embodiments, the therapeutic agent is a nucleic acid. Contacting the cell with the lipid nanoparticle can cause the particle to be internalized, for example, by endocytosis, and the nucleic acid (e.g., mRNA) can be translated within the cell to produce a polypeptide of interest. The step of contacting the cells can be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticles contacted with the cells and / or the amount of therapeutic agent therein may depend on the type of cell or tissue contacted, the means of administration, the physicochemical characteristics (e.g., size, charge, and chemical composition) of the lipid nanoparticles and the therapeutic agent therein, and other factors. An effective amount of lipid nanoparticles or pharmaceutical compositions thereof can enable efficient polypeptide production in the cells. Metrics of efficiency can include polypeptide translation (indicated by polypeptide expression), levels of mRNA degradation, and / or immune response indicators.

[0065] The types of cells that can be targeted or delivered are generally not limited. Thus, a myriad of cell types can be used in the methods disclosed herein. Exemplary cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells. As described elsewhere, the nucleic acid (e.g., mRNA) contained in the lipid nanoparticle can encode a polypeptide that is an antigen, such as a vaccine antigen (e.g., a viral antigen, a bacterial antigen, a tumor antigen). In some embodiments, the nucleic acid associated with / encapsulated by the lipid nanoparticle encodes an antigen, such as a cancer antigen or an infectious disease antigen.

[0066] In some embodiments, the nucleic acid contained in the lipid nanoparticle can code for a recombinant polypeptide that can replace one or more polypeptides that may be substantially absent in a cell contacted with the lipid nanoparticle. The one or more substantially absent polypeptides may be absent due to genetic mutations in the encoding gene or its regulatory pathway. Alternatively, the recombinant polypeptide produced by translation of the nucleic acid can antagonize the activity of an endogenous protein present in the cell, present on the surface of the cell, or secreted from the cell. Antagonistic recombinant polypeptides may be desirable to combat adverse effects caused by the activity of endogenous proteins, such as altered activity or localization caused by mutations. In some embodiments, the recombinant polypeptide produced by translation of the nucleic acid can indirectly or directly antagonize the activity of a biological moiety present in the cell, present on the surface of the cell, or secreted from the cell. Antagonized biological moieties may include, but are not limited to, lipids (e.g., cholesterol), lipoproteins (e.g., low density lipoprotein), nucleic acids, carbohydrates, and small molecule toxins. The recombinant polypeptide produced by translation of the nucleic acid can be engineered for localization within a specific compartment within the cell, such as the nucleus, or can be engineered for secretion from the cell or for translocation to the plasma membrane of the cell.

[0067] Methods of treating a disease or disorder may include delivering a therapeutic agent to a cell, and thus the descriptions of methods of delivering a therapeutic agent to a cell may also be applied to methods of treating a disease or disorder, and similarly, the descriptions of methods of treating a disease or disorder may also be applied (where applicable) to methods of delivering a therapeutic agent to a cell. The disclosed invention has multiple aspects and is illustrated by the following non-limiting examples. EXAMPLES

[0068] 5. Working Example Example 1 Materials and Methods General Materials. Unless otherwise stated, all chemicals were purchased from Millipore Sigma (St. Louis, MO). All solvents were purchased from VWR International (Radnor, PA). 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl (16DBCO PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol were sourced from Avanti Polar Lipids (Birmingham, AL). (Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) [SM-102] was obtained from SINOPEG (China). pCMV-CLuc2 encoding the Cypridina luciferase (CLuc) gene, HiScribe™ T7 ARCA mRNA Kit, and Monarch® RNA Cleanup Kit were purchased from New England BioLabs (Ipswich, MA). Quant-it™ RiboGreen RNA Assay kit, Pierce™ Cypridina Luciferase Glow Assay Kit, Lipofectamine 2000, and citrate buffer were obtained from Thermo Fisher. LDH-Glo™ Cytotoxicity Assay kit and proteinase K were procured from Promega.

[0069] Azide POEGMA (POEGMA 5-50Synthesis of . Triethylene glycol methyl ether methacrylate was passed through basic alumina to remove free radical inhibitors. Copper(II) bromide and tris(2-pyridylmethyl)amine (TPMA) were mixed in ultrapure water to prepare a catalyst complex with final concentrations of 0.01 M CuBr2 and 0.08 M TPMA. In a Schlenk flask placed on an ice bath, the following was added: triethylene glycol methyl ether methacrylate (10 mmol, 2.26 mL), methanol (5.8 mL), Cu catalyst complex (0.01 mmol, 1 mL), water-NaCl solution (2 mmol, 4 ml from 0.5 M), azidoethyl-2-bromoisobutyrate (0.1 mmol, 16.4 μL), and the flask was sealed with a septum. In another Schlenk flask, a fresh solution of ascorbic acid (64 mM, 2 mL) in ultrapure water was prepared. Both flasks were kept at low temperature in an ice bath and purged with argon for 45 minutes to remove oxygen. After deoxygenation, ascorbic acid solution was continuously pumped into the polymerization flask at a rate of 0.001 mL / min for various times under inert atmosphere using a syringe pump to obtain various molecular weights. The resulting solution was purged with air for 1 hour to quench the reaction and then dialyzed against water for 4 days. The dialyzed solution was then lyophilized to obtain azide POEGMA as a viscous gel, which was stored at -20°C. The conditions for synthesizing azide POEGMA of various MW are given in Table 1 below.

[0070] [Table 1]

[0071] Gel Permeation Chromatography-Multi-Angle Light Scattering (GPC-MALS). GPC-MALS was used to characterize the size and dispersity of the POEGMA polymer. All GPC experiments were performed at room temperature on two in-line columns (Agilent PLgel mixed-C column 105 Å, 7.5×300 mm, 5 μm, part number PL1110-6500) using THF (stabilized with 100 ppm BHT) as the eluent. A flow rate of 1 ml / min was set using an Agilent 1260 Infinity Isocratic pump. Molecular weights were determined using an in-line Wyatt-DAWN TREOS MALS detector and a Wyatt-Optilab DSP refractive index detector. Refractive index increment (dn / dc) values ​​were determined by using the online 100% mass recovery assumption method built into the Wyatt Astra software for known concentrations and masses of the samples. UV absorbance was measured using an in-line Agilent 1260 Infinity UV detector. All samples were prepared for GPC-MALS analysis by dissolving 2 mg of sample in 1 ml of HPLC grade THF and filtering through a 0.2 μm pore size inorganic membrane syringe filter (Whatman, Anotop™ 10).

[0072] POEGMA lipid (POEGMAL) 5-50 Synthesis of azide POEGMA 10-50 100 mg was dissolved in chloroform at a concentration of 20 mg / mL. A 5 molar excess of 16 DBCO PE was added and the solution was incubated on a shaker at 37 °C for 24 h. The reactions were monitored by thin layer chromatography using 10% methanol in chloroform (v / v) as the mobile phase. After 24 h, the reaction mixtures were evaporated and reprecipitated from a 10% chloroform hexane mixture, except for POEGMAL5, for which a 10% methanol hexane mixture was used. Three to four rounds of reprecipitation afforded pure POEGMA-ylated lipids in >90% yield.

[0073] Reversed-phase high performance liquid chromatography (HPLC). POEGMA 5-50The purity of was assessed by reverse-phase HPLC using a Phenomenex C18 column as the stationary phase and methanol at 1 mL / min as the mobile phase. Compounds were detected at 230 nm using an in-line UV detector. NMR spectroscopy. NMR was carried out on a Bruker 16.4 Tesla spectrometer (Bruker, UK) equipped with a BBO room temperature probe. 5-50 was dissolved in CDCl3 and the solution was heated to 1D 1 The compounds were investigated by H spectroscopy. Data were processed using MestReNova x64.

[0074] Synthesis of mRNA and agarose gel electrophoresis. In vitro transcribed mRNA encoding Cypridina luciferase (CLuc) gene was synthesized using HiScribe™ T7 ARCA mRNA Kit (with tailing) (NEB Catalog No.: E2060S) according to the manufacturer's protocol. Briefly, plasmid pCMV-CLuc2 (NEB Catalog No.: N0321) encoding reporter gene luciferase was linearized by XbaI (20 units / μg DNA) for 30 minutes at 37° C. and purified using Oligo Clean & Concentrator spin columns (Zymo Research Catalog No.: D4060). Linearization of the plasmid downstream of the gene avoids the generation of long heterologous transcripts by T7 RNA polymerase. A 20 μL in vitro transcription (IVT) reaction was set up with 1 μg of linearized plasmid, 2 μL of T7 RNA polymerase mix, and 1× ribonucleotide mix (1 mM GTP, 4 mM anti-reverse cap analog, 1.25 mM CTP, 1.25 mM UTP, final >1.25 mM ATP). The reaction was incubated at 37° C. for 30 minutes. The DNA template in the IVT reaction was then digested using DNase I at a concentration of 0.2 U / μL and incubated at 37° C. for 30 minutes. Poly(A) tailing was then performed in 1× poly(A) buffer using 5 μL of poly(A) polymerase in 100 μL of reaction. The reaction was incubated at 37° C. for 30 minutes. The synthesized mature mRNA was purified using the Monarch® RNA Cleanup Kit (NEB Catalog No.: T2050) and eluted in 20 μL of RNA storage buffer (Invitrogen Catalog No.: AM7001).

[0075] A DNA template of SARS-CoV2 spike protein mRNA was assembled in-house using Gibson assembly. A gblock encoding Wuhan strain SARS-CoV-2 spike protein (1-1273, K986P and V987P) including untranslated regions (UTRs) was assembled in pCMV vector with UTRs. Specifically, to improve mRNA stability and translation efficiency, (i) a human alpha-globin 5'UTR with a Kozak sequence was incorporated upstream of the protein coding sequence, (ii) a 3'UTR derived from mitochondrially encoded 12S rRNA (mtRNR1) and a split amino-terminal enhancer (AES) was incorporated in tandem downstream, and (iii) a polyA tail interrupted by a 10 nt linker (A30LA70, L=GCAUAUGACU) was incorporated at the 3' end of the template for co-transcriptional tailing. These elements were selected based on the mRNA sequence accessed from the WHO International Proprietary Names Program. The T7 promoter sequence was also modified for co-transcriptional capping with CleanCap-AG (Cap1) and a BbsI site was introduced for scarless run-off transcription. Template sequences were verified by Sanger sequencing and plasmids were transformed into NEB-5-alpha competent cells. The pCMV-SARS-Cov2s plasmid was purified using Qiagen's plasmid purification kit and linearized using BbsI. A typical IVT reaction contained T7 RNA polymerase, inorganic pyrophosphatase, ribonuclease inhibitors at concentrations recommended by the manufacturer (Aldevron), nucleotide triphosphates (NTPs, 2.5 mM or 5 mM, respectively), Cleancap AG (3'OMe) (80% of GTP) and MgCl2 in 1x transcription buffer (40 mM HEPES-KOH (pH=7.5), 2 mM spermidine, 10 mM DTT) and was incubated at 37°C for 2 or 4 hours. The Mg:NTP ratio, total NTP concentration, and incubation time were optimized to minimize side products. mRNA was purified using NEB's Monarch mRNA purification kit and quantified using Nanodrop.The quality of the mRNA was verified by 1% agarose gel electrophoresis run for 30 min at 130 mV in TAE buffer after adding RNA loading dye and denaturing the samples at 65° C. for 10 min. Gels were imaged with SyBr safe stain.

[0076] Formulation of lipid nanoparticles (LNPs). LNPs were prepared by using the widely used ethanol injection method (see Duong et al., Preparation of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Drug Delivery and the Effects of Preparation Parameters of Solvent Injection Method. Molecules 2020, 25 (20), 1-36 and Ganesan et al., Lipid Nanoparticles: Different Preparation Techniques, Characterization, Hurdles, and Strategies for the Production of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Oral Drug Delivery. Sustain. Chem. Pharm. 2017, 6, 37-56, both of which are incorporated by reference in their entirety). Briefly, ionizable lipid SM-102, DSPC, cholesterol, and stealth lipid (PEG-DMG or POEGMAL) were mixed and mixed with 100 mL of ethanol. 5-50 ) were dissolved in ethanol at 50:10:38.5:0.5-2.5 mol%. The ethanol solution (1 volume) containing the above lipid mixture was rapidly injected into 4 volumes of 10 mM citrate buffer (pH 4) containing mRNA at a nitrogen (from SM-102) to phosphorus (from mRNA) molar ratio (N:P) of 4:1-8:1. Unless otherwise stated, LNPs were POEGMAL5-50 was prepared with N:P 8:1, while LNP PEG-DMGwas prepared at a ratio of 6:1. The resulting solution was dialyzed against PBS for 24 hours and stored at 4°C.

[0077] Buffer Composition. The following buffers were added to the mRNA-loaded LNPs: POEGMAL(n) and LNP PEG-DMG The following buffers were used for dialysis: A) Pfizer: 10 mM Tris buffer, 300 mM sucrose, pH 7.4. B) Moderna: Tris (0.5 mg / mL), Tris-HCl (2.5 mg / mL), glacial acetic acid (0.042 mg / mL), sodium acetate trihydrate (0.2 mg / mL), sucrose (87 mg / mL). Dynamic light scattering (DLS) studies. The hydrodynamic radius (R h ) and polydispersity were determined by dynamic light scattering (DLS) using a temperature-programmed DynaPro microsampler (Wyatt Technology, Santa Barbara, CA). Samples were prepared in PBS and filtered (0.2 μm cellulose filter) into clear, flat-bottom, black 96-well plates. At least 15 acquisitions were performed at 25° C., and collected data were analyzed by a regularized fit of the autocorrelation function using DYNAMICS v7 software (Wyatt technology).

[0078] Ribogreen assay. Ribogreen assay was performed on non-dialyzed samples. LNPs were assessed for mRNA with or without 1% TritonX-100 in Tris-EDTA buffer using Ribogreen assay kit according to manufacturer's protocol. LNP samples were incubated with 1% TritonX-100 at room temperature for 5 minutes to perform LNP destruction. Encapsulation efficiency was calculated by using the formula: encapsulation%=(RFUf-RFUi) / RFUf*100, where RFUi and RFUf are the relative fluorescence units before and after adding Tritonx-100 to LNP, respectively.

[0079] Cell culture. HEK293T cells were obtained from the Duke University Core Culture facility. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (heat-inactivated, Gibco) and 2 mM L-glutamine (Gibco). Cells were maintained at 37°C in a 5% CO2 incubator. Luciferase expression assay. The assay was performed using the Pierce™ Cypridina Luciferase Glow Assay Kit (Thermo Scientific) according to the manufacturer's recommended protocol. Briefly, HEK293T (1 × 10 4 Cells / well) were seeded into 96-well plates and incubated overnight at 37°C in 5% CO2. Media was removed and cells were treated with 300ng or 500ng of mRNA prepared in 100μL / well of Opti-MEM media and incubated at 37°C in 5% CO2. Lipofectamine 2000 was used according to the manufacturer's protocol. Luciferase expression was monitored at the indicated time points by adding 5μL of media from transfected cells to 25μL of 1x Vargulin in assay buffer provided in the kit. After 10 minutes of incubation at room temperature, luminescence was quantified at 463nm using BioTek's Synergy H1 Hybrid Multimode Reader.

[0080] Cytotoxicity assay. HEK293T cells were seeded in 96-well plates at 3000 cells / well 16 hours prior to treatment. Cells were treated with a range of concentrations of various LNPs. After 48 hours of treatment, released lactate dehydrogenase (LDH) from membrane-deficient cells was assessed using the LDH-glo assay kit according to the manufacturer's instructions. RNase protection assay. LNPs containing 300 ng of CLuc mRNA were incubated with 333 pg RNase / μg mRNA for 2 hours at 37° C. before or after the addition of a final concentration of 0.1% Triton-X. Finally, RNase was quenched by incubating the samples with 1× proteinase K in 50 mM TrisHCl / CaCl2 buffer for 20 minutes at 55° C. Samples were run on a 1% agarose gel as described above.

[0081] Example 2 Azide POEGMA (POEGMA 5-50 Synthesis and characterization of POEGMA-ylated lipids (POEGMAL) were synthesized using click conjugation of two components: POEGMA polymer and fatty acid tail. DBCO-modified fatty acid tail (16:0 DBCO PE) was obtained commercially, while azide-modified POEGMA polymer was synthesized in-house (Scheme 1). Briefly, copper(II)-tris(2-pyridylmethyl)amine (TPMA) catalyst complex was incubated with triethylene glycol methyl ether methacrylate and azidoethyl-2-bromoisobutyrate in an ice bath under an inert atmosphere. The reaction was initiated by successive addition of a fresh solution of ascorbic acid to the flask. Upon completion, the reaction was quenched by purging the mixture with air. The azido-POEGMA was purified by dialysis against water and stored as a lyophilized viscous gel.

[0082] Scheme 1: Synthesis of azidoPOEGMA [ka]

[0083] Five variants of azido-POEGMA were synthesized with MW ranging from 5 to 50 kDa. n ), mass average molecular weight (M wThe size, size distribution, and polydispersity (D) of azide-POEGMA were evaluated by gel permeation chromatography-multi-angle light scattering (GPC-MALS). The GPC-MALS results confirmed the size and narrow polydispersity of azide-POEGMA (Table 2 and Figure 1A). The degree of polymerization (n) was calculated based on the M w from the polymerization initiator M w The mass of the resulting monomer unit is the average M w The purity of the azido-POEGMA was also assessed by reversed-phase high performance liquid chromatography (HPLC). The HPLC traces of all POEGMAs at 230 nm confirmed a purity of >95% (Figure 1B). The structures were determined by calculating the integral ratios of the various protons. 1 Further confirmation was achieved using HNMR, which closely matched the expected structure. Finally, the incorporation of the azide was confirmed by reacting azide-POEGMA with an excess of DBCO-PEG4. DBCO has a characteristic absorbance at 308 nm that is proportional to the concentration of DBCO. Since DBCO reacts with the azide groups of the POEGMA polymer with nearly 100% efficiency, it is possible to determine the concentration of azide in the reaction mixture by calculating the concentration of unreacted DBCO. 5-50 The reactions indicated >90% azide content in all POEGMA polymers.

[0084] [Table 2]

[0085] Example 3 POEGMA-Lipid (POEGMA 5-50 Synthesis and characterization of Azide POEGMA 5-50 was click-conjugated with 5 molar excess of 16DBCO PE in chloroform via strain-promoted azide-alkyne cycloaddition (SPAAC) to give the POEGMA-lipid conjugate (POEGMA 5-50After 24 h of reaction, the products were purified by reprecipitation from methanol for 5 kDa POEGMAL or from chloroform for 10-50 kDa POEGMAL with excess hexane (Figure 2A). The pellets were finally redissolved in methanol and subjected to reversed-phase HPLC. A typical thin-layer chromatography plate is shown in Figure 2B. POEGMAL 10 is POEGMA 10 Also, the typical physical appearance of azide-POEGMA is transparent, whereas the physical appearance of POEGMAL is opaque (Figure 2C). 5-50 The absence of the peak indicates that POEGMA 5-50 and 16 DBCO PE are shown to be completely consumed in the respective reactions. As expected, after addition of the hydrophobic lipid residue, the retention time of POEGMAL increases compared to POEGMA.

[0086] To further confirm the conjugation, 1 HNMR analysis was performed. The NMR spectrogram of the final POEGMA-lipid conjugate shows that 5-50 The POEGMAL 16DBCO PE antibody had traces of both POEGMAL and 16DBCO PE, indicating successful conjugation. 5-50 The presence of aromatic protons at δ approximately 7.3–7.8 ppm suggests successful integration of DBCO-modified fatty acid tails into the POEGMA backbone. 5-50 The 16DBCO PE and 16DBCO PE lipids have some overlapping regions that are difficult to deconvolute, and only the aromatic region of 16DBCO PE (chemical shifts δ approximately 7.3-7.8 ppm) is far from this overlapping region. Therefore, the integral ratio between δ approximately 0.5-2.5 ppm and between 7.3-7.8 (I 0.5-2.5 / I 7.3-7.8) was used to calculate the proton ratios (Figure 2D). The discrepancy between the theoretical and experimental ratios is presumed to be due to (i) the number of protons being larger at δ ≈ 0.5-2.5 ppm than at δ ≈ 7.3-7.8 ppm, which may inhibit the sensitivity; and / or (ii) residual solvent peaks in the δ ≈ 7.3-7.8 ppm region may interfere with the calculation. Nevertheless, taken together, all these data strongly suggest the successful synthesis and purification of POEGMA-ylated lipids.

[0087] Example 4 Formulation and characterization of lipid nanoparticles (LNPs) LNPs were formulated using the ethanol injection method with four different co-lipids with the mol% indicated in brackets: (i) an ionizable lipid (SM102), which helps encapsulate the mRNA, provides stability, and aids the particle in successful endosomal escape (50 mol%); (ii) DSPC (10 mol%); (iii) cholesterol (38.5 mol%); and (iv) a stealth lipid (1.5 mol%), either commercially available PEG-DMG or an in-house synthesized POEGMA-ylated lipid. This lipid ratio is specifically tailored for the PEGylated lipid and provides a good starting point for formulating LNPs with POEGMA-ylated lipids. The LNP formulation with PEG-DMG served as a benchmark, and these lipid compositions were also used in Moderna's mRNA COVID19 vaccine. Of note, POEGMAL of 10-50 kDa was used in the initial study on the role of MW and lipid ratio on LNP mRNA encapsulation efficiency, and POEGMAL5 was incorporated in the later part of the study. Lipids solubilized in a small volume of ethanol were rapidly injected into citrate buffer (pH 4) containing luciferase mRNA to obtain LNPs. The LNPs were then buffer exchanged with PBS at 4°C. A series of LNPs without any mRNA was also synthesized. Dynamic light scattering (DLS) was used to characterize the formation of nanoparticles. As summarized in Figures 3A and 3B, stable LNPs were obtained when stealth lipids were used. LNPs without PEGylated or POEGMAylated lipids appeared more turbid, had high polydispersity, and showed low R h In contrast, all other LNPs were semi-transparent and exhibited R values ​​less than 100 nm with narrow polydispersities. h The mRNA was encapsulated using PEG-DMG and POEGMAL (Figures 3A, 3B, and 3C). 10 , and POEGMAL 20 did not significantly change the size of the nanoparticles. 40 and POEGMAL 50 R of LNP including hexpanded 1.3- and 1.9-fold, respectively, after mRNA encapsulation (Figures 3D, 3E, and 3F).

[0088] Example 5 Quantification of mRNA encapsulation efficiency The mRNA encapsulation efficiency of the LNPs was first qualitatively evaluated using an agarose electrophoretic mobility shift assay (EMSA). Since only free mRNA migrates through the agarose gel - while LNP mRNA does not - it is possible to separate free mRNA from LNP mRNA in the formulation. The total amount of mRNA - encapsulated and free - was estimated on an agarose gel by rupturing the LNPs with a detergent such as Triton-X, which releases the encapsulated mRNA. Free mRNA at an equivalent concentration serves as an experimental control. Electrophoretic experiments also show any degradation of mRNA during preparation and storage. As can be seen in Figure 4A, all LNPs encapsulated a significant amount of mRNA. LNPs POEGMAL40 and LNP PEG-DMG significantly reduced the degradation of mRNA during handling and storage. POEGMAL50 In the case of LNP, the mRNA was degraded. POEGMAL10~20 In LNPs, only free mRNA was degraded, but not encapsulated mRNA. POEGMAL40 In this case, the unencapsulated mRNA is not completely free in solution, but rather weakly associated with the LNP, which would normally POEGMAL10-20The percentage of mRNA encapsulation in POEGMA-ylated LNPs was then quantified by Ribogreen assay and benchmarked with the percentage of encapsulation in PEGylated LNPs. To nullify the effects of handling and storage, the formulations were not dialyzed and encapsulation was evaluated immediately after preparation. DLS confirmed that the size of the LNPs remained similar both before and after dialysis. Ribogreen is an organic dye that binds only to free mRNA and not to LNP-associated mRNA or degraded nucleotides. In the unbound state, Ribogreen possesses little or no fluorescence, but exhibits intense fluorescence when bound to free mRNA (Figure 4B). The relative fluorescence units (RFU) of Ribogreen mixed with LNPs are proportional to the amount of free mRNA in the LNP solution, whereas the RFU of Ribogreen in LNPs burst with 1% Triton-X is proportional to the total mRNA (the sum of both free and encapsulated). Therefore, the relative fluorescence absorbance of Ribogreen can be used to calculate the encapsulation percentage. As evidenced in Figure 4C, LNPs POEGMAL50 The encapsulation efficiency of LNP was the lowest at approximately 12%. In contrast, the encapsulation efficiency of LNP was POEGMAL50 Compared to LNP POEGMAL40 About 4.5 times as much as LNP POEGMAL20 4.1 times, and LNP POEGMAL10 The improvement was 3.8 times. 40 LNPs containing PEG-DMG showed comparable encapsulation of 56%, only 1.5-fold lower than PEG-DMG. Such a lower encapsulation efficiency can be expected as the lipid ratios used to prepare the LNPs are tailored for PEGylated LNPs.

[0089] Example 6 Analysis and characterization of LNPs to maximize mRNA encapsulation To obtain improved encapsulation efficiency (EE), an OFAT study was designed to analyze the mole fraction of POEGMAL and SM-102 that can stably form nanoparticles while maximizing the EE. 10-50 The mol% of POEGMAL and SM-102 were varied and mRNA encapsulation was measured before dialysis by EMSA. 10 and POEGMAL 20 presented a significant loading of ≥75%, which was set as the threshold to down-select LNP candidates. Varying the mol% of SM-102 affected the EE. The relationship between EE and nanoparticle size distribution is illustrated in Figures 5B, 5C, and 5D. All POEGMALs formed stable monodisperse nanoparticles at all mol%, regardless of their EE. Selected candidates (circled in Figure 5A) were dialyzed against PBS and characterized by DLS and Ribogreen assays. All selected candidates formed stable nanoparticles, except for POEGMAL at 0.5 mol%. 10 The LNPs of POEGMA5 showed an approximately 2.5-fold expansion in size, and the formulations exhibited a stable and reproducible EE >85% (Figure 5E). Motivated by this fact, OFAT studies were performed with POEGMA5 by varying the mol% to evaluate whether further lowering the MW of POEGMA5 would improve the EE. All formulations exhibited stable nanoparticles with a radius of less than 100 nm and a polydispersity of <30%. The EE was found to be >90% for all mol% before dialysis, while 0.5 mol% exhibited the highest EE after dialysis (Figures 6A and 6B). In summary, the OFAT results of POEGMA5 and POEGMA5 at 0.5 mol% showed a significant improvement in the EE. 10 Both of these formulations formed stable LNPs that were able to retain over 85% of the mRNA even after dialysis. Therefore, we decided to image these two formulations by cryo-TEM. As can be seen in Figure 7, both formed spherical nanoparticles.

[0090] Example 7 Encapsulation of therapeutically important mRNA Next, the LNP system was probed to encapsulate the therapeutically important SARS COV-2 spike protein mRNA. The mRNA encoding the full-length SARS-CoV-2 spike glycoprotein was accessed from the WHO International "Proprietary Names Programme". This mRNA is presumably used in Pfizer's COVID19 vaccine. Parameters of in vitro transcription with T7 RNA polymerase were used to increase mRNA yield and minimize the formation of by-products (long transcripts and dsRNA). The formulation used was tailored for luciferase mRNA - 0.5mol% POEGMAL 5&10 The EE was dialyzed against N:P 8:1, 20% ethanol, and PBS. POEGMAL5 The results were compromised, with the LNP exhibiting only approximately 30% EE. This is not entirely unexpected for three reasons: (i) the size of SARS COV-2 mRNA is two times larger than luciferase mRNA; (ii) the dialysis buffer was not optimal; and (iii) the preparation method, lipids, and N:P ratio may be ineffective. All these challenges may be addressed by LNP. POEGMAL10 The EE was probed using the Moderna LNP vaccine (Figure 8). First, the role of the dialysis buffer was evaluated (Figure 8A and Figure 8B). As this formulation is a Moderna biosimilar, it showed improved EE (approximately 67%) in a buffer tailored for the Moderna LNP vaccine (Figure 8B). Also, the size of the LNP remained unchanged (Figure 8C). Therefore, the Moderna buffer was used for further analysis. Second, the EE was evaluated in a range of N:P and ethanol concentrations. It is found that N:P 10:1 (Figure 8D) and 30% ethanol (Figure 8E) are useful for encapsulating mRNA, pushing the EE to over 80% after dialysis. Third, a similar OFAT study was designed, where 0.5 mol% still showed an improvement in EE in the buffer tailored for the Moderna LNP vaccine (Figure 8C). 10The most effective lipid concentration for LNPs was found to be 0.01 (Figure 8F). Surface potential, which may increase at higher N:P ratios and may contribute to increased toxicity of LNPs, was also evaluated. Interestingly, the surface charge did not increase significantly, indicating that at higher N:P, the mRNA content of LNPs is improved while neutralizing the surface charge. The surface charge of all dialysis formulations was less than 10 mV. Zeta potentials in this range are considered to be approximately neutral. Taken together, these data suggest that LNPs POEGMAL10 We have been able to successfully encapsulate model luciferase mRNA as well as therapeutically important SARS COV-2 mRNA with high EE (approximately 85%).

[0091] Example 8 In vitro performance of LNPs Finally, motivated by the fact that the present LNP system can serve as an alternative method to deliver SARS COV-2 mRNA vaccines, we evaluated: (i) toxicity in HEK293T cells; (ii) efficacy in expressing luciferase mRNA in HEK293T cell lines over a period of time; and (iii) ability to protect mRNA from RNases. Collectively, these experiments serve as a model screening platform that is widely used in mRNA vaccine research to demonstrate potential in vivo utility. Furthermore, the performance of the LNPs was compared with that of the Moderna biosimilar LNPs. PEG-DMG and / or Lipofectamine 2000. The toxicity of LNPs was evaluated against HEK293T cells by LDH assay. All LNPs showed minimal toxicity even after 48 h of continuous treatment (Figure 10). To evaluate luciferase expression, HEK293T cells were treated with various LNPs at N:P ratios ranging from 4:1 to 8:1 and mRNA amounts ranging from 300 to 500 ng. As summarized in Figures 11A, 11B, 11C, 11D, 11E, and 11F, the LNPs POEGMAL5 and LNP POEGMAL10Both LNPs showed significant expression of luciferase over a 96-hour period. POEGMAL10 is LNP POEGMAL5 - LNP POEGMAL10 Regarding 8:1 and LNP PEG-DMG At an N:P ratio of 6:1, both formulations outperformed Lipofectamine 2000 (Figures 9A and 9B). The difference in expression was more pronounced at 300 ng of mRNA, where LNP POEGMAL10 showed over 300% higher luciferase activity than lipofectamine 2000, and Moderna biosimilar LNPs PEG-DMG Luciferase expression was measured over a period of approximately 3 days, and area under the curve (AUC) measurements were performed using the trapezoidal rule. As summarized in Figures 9C and 9D, LNP POEGMAL10 was LNP at all N:P ratios. POEGMAL5 An equally important observation was that when higher amounts of mRNA (500 ng) were used, LNP POEGMAL10 and LNP PEG-DMG However, at the lower mRNA amount (300 ng), LNP showed similar AUC. POEGMAL10 The Moderna biosimilar LNP had approximately 2-fold higher AUC at the same N:P ratio of 6:1. PEG-DMG At N:P 8:1, it is superior to LNP. POEGMAL10 is LNP PEG-DMGThe LNPs showed a 2.3-fold higher AUC than the POEGMA-LNPs. Finally, we investigated the efficacy of LNPs in protecting the mRNA cargo from RNases, which is a key parameter for successful preclinical and clinical translation of LNP-mRNA vaccines. mRNA can be easily digested by extracellular RNases resulting in poor in vivo performance. In-house assays, in which LNP-mRNA vaccine candidates were incubated with high concentrations of RNase. LNP rupturing agents were added before or after the addition of RNase. After the incubation period, excess RNase was quenched with proteinase K. As summarized in Figure 12, all candidate LNPs can confer significant protection from RNases. The control free mRNA and the group in which Triton-X is added before the addition of RNase show complete degradation of the mRNA. Collectively, these data suggest that POEGMA-LNPs can function as an mRNA vaccine delivery platform and have a high potential for successful clinical translation. It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limiting the scope of the invention.

[0092] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those with respect to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof. For completeness, various aspects of the invention are described in the following numbered paragraphs:

[0093] Item 1. A lipid nanoparticle comprising: an ionizable lipid; a phospholipid; a sterol; a poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugate at less than 10 mol %, wherein the POEGMA has a number average molecular weight of less than 100 kDa; and a therapeutic agent. Section 2. POEGMA is a lipid nanoparticle of section 1 having a poly(methyl methacrylate) backbone and multiple side chains covalently attached to the backbone, each side chain containing 2 to 9 monomers of ethylene glycol (EG) repeated in tandem. Item 3. The lipid nanoparticle of item 1 or 2, wherein the lipid nanoparticle has a reduced immune response compared to a lipid nanoparticle comprising polyethylene glycol (PEG). Clause 4. The lipid nanoparticle of any one of clauses 1 to 3, wherein the lipid nanoparticle is not reactive with pre-existing anti-PEG antibodies in the subject. Section 5. The lipids in the POEGMA-lipid conjugates are C 2-40 A lipid nanoparticle according to any one of clauses 1 to 4, comprising a hydrocarbon chain.

[0094] Clause 6. The lipid nanoparticle of any one of clauses 1 to 5, wherein the lipid of the POEGMA-lipid conjugate is conjugated to the POEGMA via a triazole, amide, ester, ether, or hydrocarbon linker. Clause 7. The lipid nanoparticle of any one of clauses 1 to 6, wherein the POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa. Clause 8. The lipid nanoparticle of any one of clauses 1 to 7, comprising about 0.1 mol% to about 10 mol% of the POEGMA-lipid conjugate. Section 9. Ionizable lipids include (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP). Noleoyl-3-dimethylaminopropane (DLm-DAP), 1,2(l,2)-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D Lin-MC3-DMA), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67), bis-guanidinium-spermidine-cholesterol (BGTC), 3b-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), Nt-butyl-N'-tetradecylamino -propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMR1E), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,9. The lipid nanoparticle of any one of clauses 1 to 8, comprising N-dimethylammonium trifluoroacetate (DOSPA), 2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315)), or a combination thereof.

[0095] Clause 10. The lipid nanoparticle of any one of clauses 1 to 9, wherein the ionizable lipid comprises (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102).

[0096] Clause 11. The lipid nanoparticle of any one of clauses 1 to 10, wherein the lipid nanoparticle comprises about 20 mol% to about 65 mol% of an ionizable lipid.

[0097] Section 12. Phospholipids are distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl -ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,12. The lipid nanoparticle of any one of clauses 1 to 11, comprising 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or a combination thereof.

[0098] Item 13. The lipid nanoparticle of any one of items 1 to 12, comprising phospholipids at about 5 mol% to about 25 mol%. Section 14. Sterols include cholesterol, campesterol, anthrosterol, desmosterol, nicasterol, stigmasterol, sitosterol, oxysterols, C 4-10 14. The lipid nanoparticle of any one of clauses 1 to 13, comprising a sterol, ergosterol, cholest-4-en-3-one, or a combination thereof.

[0099] Clause 15. The lipid nanoparticle of any one of clauses 1 to 14, comprising a sterol at about 10 mol% to about 50 mol%. Clause 16. The lipid nanoparticle of any one of clauses 1 to 15, wherein the therapeutic agent is a nucleic acid comprising siRNA, miRNA, an antisense oligonucleotide, shRNA, mRNA, tRNA, rRNA, CircRNA, DNA, or a combination thereof. Clause 17. The lipid nanoparticle of clause 16, wherein the nucleic acid comprises siRNA, mRNA, or a combination thereof. Item 18. A lipid nanoparticle comprising: (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102); DSPC; cholesterol; a poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugate at about 0.25 mol% to about 3 mol%, wherein the POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa; and mRNA. Clause 19. The lipid nanoparticle of any one of clauses 1 to 18, wherein the lipid nanoparticle has an N:P ratio of about 4:1 to about 16:1.

[0100] Clause 20. The lipid nanoparticle of any one of clauses 1 to 19, wherein the lipid nanoparticle has a diameter of about 30 nm to about 300 nm. Clause 21. The lipid nanoparticle of any one of clauses 1 to 20, wherein the lipid nanoparticle has a therapeutic agent encapsulation efficiency of 75% or more as measured by fluorescence. Clause 22. The lipid nanoparticle of any one of clauses 1 to 21, further comprising a targeting ligand. Clause 23. A pharmaceutical composition comprising one or more lipid nanoparticles according to any one of clauses 1 to 22; and a pharma- ceutically acceptable excipient. Clause 24. A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of one or more lipid nanoparticles according to any one of clauses 1 to 22, optionally in combination with a pharma-ceutically acceptable excipient.

[0101] Clause 25. The method of clause 24, wherein the one or more lipid nanoparticles have a reduced immune response compared to lipid nanoparticles comprising polyethylene glycol (PEG); are not reactive with pre-existing anti-PEG antibodies in the subject; or a combination thereof. Item 26. The method of item 24 or 25, wherein the disease or disorder is an infectious disease, Huntington's disease, muscular dystrophy, an autoimmune disease, or cancer. Clause 27. A method for delivering a therapeutic agent to a cell, comprising contacting the cell with one or more lipid nanoparticles according to any one of clauses 1 to 22, whereby the therapeutic agent is delivered to the cell.

[0102] array SEQ ID NO: 1 Cypridina luciferase mRNA: 200-300

[0103] SEQ ID NO:2 SARS-COV2 spike protein mRNA:

Claims

1. ionizable lipids; phospholipids; sterols; Poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugates at less than 10 mol %, wherein the POEGMA has a number average molecular weight of less than 100 kDa; and Treatment drugs A lipid nanoparticle comprising:

2. The lipid nanoparticle of claim 1, wherein POEGMA has a poly(methyl methacrylate) backbone and multiple side chains covalently attached to the backbone, each side chain comprising 2 to 9 tandemly repeated ethylene glycol (EG) monomers.

3. The lipid nanoparticle of claim 1, which has a reduced immune response compared to lipid nanoparticles containing polyethylene glycol (PEG).

4. The lipid nanoparticle of claim 1, which does not react with pre-existing anti-PEG antibodies in a subject.

5. The lipid in the POEGMA-lipid conjugate is C 2-40 The lipid nanoparticle of claim 1, comprising a hydrocarbon chain.

6. The lipid nanoparticle of claim 1, wherein the lipid of the POEGMA-lipid conjugate is conjugated to the POEGMA via a triazole, amide, ester, ether, or hydrocarbon linker.

7. The lipid nanoparticle of claim 1, wherein the POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa.

8. The lipid nanoparticle of claim 1, comprising about 0.1 mol% to about 10 mol% of the POEGMA-lipid conjugate.

9. The ionizable lipids were (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), 3,6-bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2,5-dione (cKK-E12), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP). Leoyl-3-dimethylaminopropane (DLm-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67), bis-guanidinium-spermidine-cholesterol (BGTC), 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-t-butyl-N'-tetradecylamino- Pionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,The lipid nanoparticle of claim 1, comprising N-dimethylammonium trifluoroacetate (DOSPA), 2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or a combination thereof.

10. The lipid nanoparticle of claim 1, wherein the ionizable lipid comprises (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102).

11. The lipid nanoparticle of claim 1, comprising about 20 mol% to about 65 mol% of an ionizable lipid.

12. Phospholipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycero-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-sn-glycero-phosphoethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylethanolamine Sphingomyelin (SM), dimyristoyl phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilinoleoyl-sn-glycerol 2. The lipid nanoparticle of claim 1, comprising 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or a combination thereof.

13. The lipid nanoparticle of claim 1, comprising phospholipids at about 5 mol% to about 25 mol%.

14. Sterols include cholesterol, campesterol, anthrosterol, desmosterol, nicasterol, stigmasterol, sitosterol, oxysterol, C 4-10 The lipid nanoparticle of claim 1, comprising a sterol, ergosterol, cholest-4-en-3-one, or a combination thereof.

15. The lipid nanoparticle of claim 1, comprising a sterol at about 10 mol% to about 50 mol%.

16. The lipid nanoparticle of claim 1, wherein the therapeutic agent is a nucleic acid comprising siRNA, miRNA, antisense oligonucleotide, shRNA, mRNA, tRNA, rRNA, CircRNA, DNA, or a combination thereof.

17. The lipid nanoparticle of claim 16, wherein the nucleic acid comprises siRNA, mRNA, or a combination thereof.

18. (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate) (SM-102); DSPC; cholesterol; a poly[oligo(ethylene glycol) ether methacrylate] (POEGMA)-lipid conjugate at about 0.25 mol % to about 3 mol %, wherein the POEGMA has a number average molecular weight of about 1 kDa to about 50 kDa; and mRNA A lipid nanoparticle comprising:

19. The lipid nanoparticle of claim 1, having an N:P ratio of about 4:1 to about 16:

1.

20. The lipid nanoparticle of claim 1, having a diameter of about 30 nm to about 300 nm.

21. 10. The lipid nanoparticle of claim 1, having a therapeutic agent encapsulation efficiency of 75% or greater as measured by fluorescence.

22. The lipid nanoparticle of claim 1, further comprising a targeting ligand.

23. One or more lipid nanoparticles according to claim 1; and pharmaceutically acceptable excipients A pharmaceutical composition comprising:

24. 24. The pharmaceutical composition of claim 23, wherein the one or more lipid nanoparticles have a reduced immune response compared to lipid nanoparticles comprising polyethylene glycol (PEG); do not react with pre-existing anti-PEG antibodies in the subject; or a combination thereof.

25. The pharmaceutical composition of claim 23 for treating an infectious disease, Huntington's disease, muscular dystrophy, an autoimmune disease, or cancer.

26. A method for delivering a therapeutic agent to a cell, comprising contacting the cell with one or more lipid nanoparticles described in claim 1 ex vivo, in culture, or in vitro, whereby the therapeutic agent is delivered to the cell.

Images