Targeting antigen-presenting cells with nanoparticles containing polyoxazoline-lipid conjugates

Abstract

Methods for targeting antigen-presenting cells and delivering encapsulated payloads, including but not limited to, nucleic acid payloads such as mRNA and modified mRNA, using lipid nanoparticles containing POZ-lipid conjugates. These LNPs are less susceptible to accelerated blood clearance and have a low or reduced immunogenicity profile in vivo.

Description

[Technical Field]

[0001] The present disclosure relates to methods for targeting lipid nanoparticles (LNPs) comprising polyoxazoline-lipid conjugates (or pharmaceutical compositions comprising such LNPs) to antigen-presenting cells. Furthermore, the LNPs of the present disclosure have a low (or reduced) immunogenic profile in vivo compared to their PEG-LNP counterparts. [Background technology]

[0002] Nucleic acid (especially mRNA)-based vaccines offer advantages over other vaccine technologies. Nucleic acid-based vaccines can be produced rapidly, regardless of the encoded antigen, by using a common manufacturing platform and purification method, reducing development time and costs. Furthermore, mRNA-based vaccines only need to be delivered to the cytosol of appropriate antigen-presenting cells to enter the ribosomal translation machinery.

[0003] However, nucleic acid-based vaccines have several drawbacks. For example, mRNA is rapidly degraded by nucleases in the body and is not easily taken up by most cell types. A major obstacle to the development of both DNA and mRNA vaccines is the lack of a potent and well-tolerated delivery system.

[0004] Efforts to address these shortcomings have led to the successful encapsulation of mRNA payloads (and other oligonucleotide payloads) into lipid nanoparticles (LNPs), which protect the mRNA from enzymatic degradation and increase cellular uptake and expression by up to 1000-fold compared to mRNA-polyamine complexes. Such LNPs typically consist of an ionizable lipid (to complex with the oligonucleotide), cholesterol (to provide flexibility to the lipid bilayer), a lipid containing a polyethylene glycol (PEG) moiety (to stabilize the lipid nanoparticle and prevent fusion with other nanoparticles), and a helper lipid, such as distearoylphosphatidylcholine (DSPC), (to provide structural integrity). For example, U.S. Patent Publication No. 2020 / 0230058 discloses liposomes encapsulating RNA encoding an antigen of interest, the liposomes containing at least one PEG-lipid molecule, the PEG being present on the exterior of the liposome and having an average molecular weight of 1 kDa to 3 kDa.

[0005] Early studies with small interfering RNA (siRNA) identified ionized lipids as a key driver of potency, which are crucial for endosomal escape once LNPs traffic through the endosomal compartment within cells.

[0006] LNPs are generally regarded as biocompatible nanocarriers with an excellent safety profile and the ability to transport both lipophilic and hydrophilic payloads. However, as briefly discussed above, concerns exist regarding the immunogenicity of LNPs when administered to humans and animals. Indeed, LNP components, such as PEG, appear to contribute in part to vaccine potency through the influence of anti-PEG responses. There is growing recognition that administration of PEGylated components, including lipids, to patients can lead to the formation of antibodies that specifically recognize and bind to PEG (i.e., anti-PEG antibodies). Anti-PEG antibodies have been detected in patients who have been exposed to PEG-containing products, even if they have never received a PEGylated drug.

[0007] Therefore, administration of LNPs containing PEGylated lipids to patients who have acquired anti-PEG antibodies may result in accelerated blood clearance of the LNPs, reduced efficacy, hypersensitivity, and potentially life-threatening side effects, including an allergic reaction called anaphylaxis. This immunogenicity of PEG can be particularly problematic if subjects receive repeated vaccinations with LNPs containing PEGylated lipids over a period of time or if the subjects have previously been exposed to products containing PEG.

[0008] Thus, there is a need in the art for solutions to address the shortcomings of current LNP technology. Indeed, there is a particular need in the art for LNP formulations that exhibit reduced or no immunogenicity after the first administration of the LNP and after subsequent administrations of the LNP. Furthermore, it would be advantageous to provide LNPs that encapsulate payloads that enable preferential targeting to antigen-presenting cells, as such LNPs are likely to lead to vaccines, particularly for infectious diseases and cancer immunotherapy. The present disclosure provides such a solution. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 0230058 Summary of the Invention [Problem to be solved by the invention]

[0010] The utility of lipid nanoparticles (LNPs) for delivering mRNA to cells has recently been demonstrated in vaccines for Covid-19 (or SARS-CoV-2). Given the millions of deaths from this viral disease and the apparent broad utility of cellular mRNA delivery for a variety of diseases, from cancer to influenza, significant efforts have been devoted to elucidating and improving LNP delivery of mRNA. As part of this research, we have explored polyoxazoline (POZ)-lipid conjugates (POZ-lipids) as an alternative to polyethylene glycol (PEG)-lipid conjugates (PEG-lipids) currently used in commercially available mRNA vaccines. PEG-lipids have numerous drawbacks, including immunogenicity and the associated adverse immune responses in patients, as well as the need for ultra-low temperature storage conditions to maintain stability.

[0011] The present disclosure relates to nanoparticles comprising polyoxazoline (POZ)-lipids used to target antigen-presenting cells. In particular, the present inventors have now surprisingly discovered that LNPs made with POZ-lipids (compared to PEG-lipids) enable preferential uptake by antigen-presenting cells in the spleen, which are essential for the development of an immune response. These antigen-presenting cells are macrophages and dendritic cells, which ultimately lead to antibody production and the generation of cytotoxic T cells, which are crucial for vaccine function. This preferential uptake or targeting by the LNPs of the present disclosure is believed to result in an enhanced vaccine response. As a result, a lower dose of vaccine may be used to mount an optimal protective antibody response. In turn, this lower dose can be expected to result in reduced side effects from vaccine injection.

[0012] Without being bound by any particular theory, this uptake by antigen-presenting cells is formulation-independent. More specifically, the inventors observed uptake with certain LNP compositions containing POZ-lipid LNPs modeled after the commercially available Moderna® Spikevax COVID-19 vaccine (currently delivered using PEG-lipid LNPs). The inventors also observed similarly effective preferential uptake with POZ-lipid LNPs modeled after the commercially available Alnylam® neuropathy drug marketed as Onpattro® (currently delivered using PEG-lipid LNPs). Targeted delivery of POZ-lipid-containing LNPs is believed to be a highly effective method for improving various oligonucleotide therapeutics, including vaccines, cancer immunotherapy, and gene therapy. [Means for solving the problem]

[0013] The present disclosure relates to a method for preferentially delivering mRNA to antigen presenting cells, comprising administering to said cells a nucleic acid sequence comprising a nucleic acid sequence selected from the group consisting of:

[0014] [ka] The method includes preparing lipid nanoparticles comprising a POZ-lipid of the formula: wherein R comprises an initiator group, POZ comprises a poly(oxazoline), L comprises a biodegradable linking group, and Lipid comprises an uncharged lipid comprising at least one hydrophobic moiety. In certain embodiments, POZ is poly(ethyloxazoline).

[0015] In certain embodiments, the antigen-presenting cells comprise macrophage cells, dendritic cells, or a combination thereof. In other embodiments, POZ is [N(COR2)CH2CH2] nwherein R2 is ethyl. In yet another embodiment, R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole group with an attached carboxylic acid, or a substituted or unsubstituted aralkyl group. In yet another embodiment, L comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof. In yet another embodiment, the Lipid comprises two hydrophobic moieties. In another embodiment, the Lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or combinations thereof. In yet another embodiment, the Lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof.

[0016] The present disclosure relates to a method for preferentially delivering mRNA to antigen presenting cells, comprising administering to said cells a compound of formula II:

[0017] [ka] wherein Lipid comprises an uncharged lipid comprising at least one hydrophobic moiety, L1 comprises a biodegradable linking group, POZ comprises a polyoxazoline polymer of the structure [N(COR2)CH2CH2] (wherein R2 is ethyl), n is 1 to 1,000, a is ran representing a random copolymer or block representing a block copolymer, and T comprises a terminating group.

[0018] In certain embodiments, L1 comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof. In other embodiments, L1 comprises a triazole group.

[0019] In yet another embodiment, T comprises ZBQ, where Z comprises S, O, or N, B is an optionally present linking group, and Q is a terminating nucleophile or portion thereof. In yet another embodiment, Lipid comprises two hydrophobic moieties. In another embodiment, Lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof. In yet another embodiment, Lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0020] The present disclosure relates to a method for preferentially delivering mRNA to antigen presenting cells, comprising administering to said cells a compound of formula III:

[0021] [ka] The method includes preparing lipid nanoparticles comprising a POZ-lipid of the formula: wherein R comprises an initiator group, POZ comprises a polyoxazoline polymer of the structure [N(COR2)CH2CH2] (wherein R2 is ethyl), n is 1 to 1,000, a is ran representing a random copolymer or block representing a block copolymer, Z comprises S, O, or N, L2 comprises a biodegradable linking group, and Lipid comprises an uncharged lipid comprising at least one hydrophobic group.

[0022] In certain embodiments, L2 comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof. In other embodiments, the Lipid comprises two hydrophobic moieties. In yet other embodiments, the Lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or combinations thereof. In yet other embodiments, the Lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof. In yet other embodiments, R comprises hydrogen or a substituted or unsubstituted alkyl group, and n is 15 to 35.

[0023] The present disclosure relates to a method for preferentially delivering mRNA to antigen presenting cells, comprising the steps of:

[0024] [ka] wherein R comprises an initiating group, L3 comprises a biodegradable linking group, Lipid comprises an uncharged lipid comprising at least one hydrophobic moiety, n is 1 to 5, R2 is independently selected for each repeating unit from an unsubstituted or substituted alkyl group, alkenyl group, aralkyl group, heterocyclylalkyl group, or active functional group, m is 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T comprises a terminating group.

[0025] In certain embodiments, L3 comprises an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amide, and combinations thereof. In other embodiments, L3 comprises a triazole group. In yet other embodiments, T comprises ZBQ, where Z comprises S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or portion thereof. In yet other embodiments, Lipid comprises two hydrophobic moieties. In yet other embodiments, Lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof. In yet other embodiments, Lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0026] In certain embodiments, the LNPs of the present disclosure encapsulate a payload, and the payload is administered to a subject in an amount effective to treat a pathological condition. In this embodiment, the LNPs of the present disclosure allow delivery of the encapsulated payload to a target tissue of the subject while suppressing an immune response to allow repeated administration of the LNP. The method includes administering an initial dose of LNP to the subject, such that the subsequent dose of LNP produces a suppressed immune response, and administering the subsequent doses of LNP to the subject, such that the subject produces a suppressed ABC response to the subsequent doses of LNP.

[0027] In other embodiments, the methods of the present disclosure include administering to a subject an initial dose of LNPs of the present disclosure encapsulating mRNA encoding an antigen or protein of interest, wherein the initial dose of LNPs induces an inhibited immune response that reduces accelerated blood clearance (ABC) after administration of subsequent doses of LNPs. In certain embodiments, the LNPs of the present disclosure are insensitive to accelerated blood clearance after repeated in vivo administration.

[0028] Other features and advantages can be seen from the following detailed description taken in conjunction with the drawings below. [Brief explanation of the drawings]

[0029] [Figure 1A] Figure 1A is a graphical representation of the hydrodynamic diameter of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 1B is a graphical representation of the hydrodynamic diameter of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 1B] Figure 1A is a graphical representation of the hydrodynamic diameter of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 1B is a graphical representation of the hydrodynamic diameter of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 2A]Figure 2A is a graphical representation of the polydispersity index of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 2B is a graphical representation of the polydispersity index of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 2B] Figure 2A is a graphical representation of the polydispersity index of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 2B is a graphical representation of the polydispersity index of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 3A] Figure 3A is a graphical representation of the polydispersity index of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 3B is a graphical representation of the encapsulation efficiency of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 3B] Figure 3A is a graphical representation of the polydispersity index of SM-102-containing LNPs formulated with PEG2000 and DMG-PEOZ2000. Figure 3B is a graphical representation of the encapsulation efficiency of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 4] 1 is a graphical representation of the uptake and expression of aVHH+ by cells in the liver and spleen following intravenous administration of SM-102-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 5] 1 is a graphical representation of the uptake and expression of aVHH+ by cells in the liver and spleen following intravenous administration of MC3-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 6] 1 is a graphical representation of aVHH+ uptake and expression by cells in muscle and abdominal periaortic (lumbar) lymph nodes following intramuscular administration of SM-102-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 7] 1 is a graphical representation of the uptake and expression of aVHH+ by cells in the liver and spleen following intramuscular administration of DMG-containing LNPs formulated with PEG2000 and PEOZ2000. [Figure 8A]Figure 8A is a graphical representation of bioluminescence in the liver after repeated intramuscular administration of SM-102-containing LNPs formulated with PEG-lipid conjugates and POZ-lipid conjugates, and Figure 8B is a graphical representation of bioluminescence in the spleen after repeated intramuscular administration of SM-102-containing LNPs formulated with PEG-lipid conjugates and POZ-lipid conjugates. [Figure 8B] Figure 8A is a graphical representation of bioluminescence in the liver after repeated intramuscular administration of SM-102-containing LNPs formulated with PEG-lipid conjugates and POZ-lipid conjugates, and Figure 8B is a graphical representation of bioluminescence in the spleen after repeated intramuscular administration of SM-102-containing LNPs formulated with PEG-lipid conjugates and POZ-lipid conjugates. [Figure 9] 1 is a graphical representation of anti-PEG and anti-PEOZ IgM following intramuscular administration of SM-102-containing LNPs formulated with PEG-lipid conjugates and PEOZ-lipid conjugates. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure provides a method for targeting lipid nanoparticles (LNPs) containing POZ-lipid conjugates to antigen-presenting cells. The LNPs can be used to preferentially deliver an encapsulated payload (e.g., a nucleic acid payload, including, but not limited to, mRNA or modified mRNA) to macrophage cells and dendritic cells. Because LNPs containing the POZ-lipid conjugates of the present disclosure are non-immunogenic or have reduced immunogenicity compared to corresponding LNPs containing PEG-lipids, such LNPs are believed to not only exhibit higher efficacy but also provide a safer method for delivering nucleic acid vaccines.

[0031] Prior art LNPs generally contain (i) helper lipids to support bilayer structure and facilitate endocytosis; (ii) sterol lipids (i.e., cholesterol) to stabilize the lipid bilayer of the LNP; and (iii) cationic or ionizable lipids, along with PEG-lipids to provide a hydration layer to the LNP to improve colloidal stability, prevent nascent particle fusion, inhibit protein adsorption and nonspecific uptake, and prevent clearance by the reticuloendothelial system. However, as discussed above, the PEG-lipids in these LNPs pose a risk to patient safety due to the potential for anti-PEG immune responses. The use of POZ-lipid-containing LNPs in accordance with the present disclosure provides a highly effective method for delivering nucleic acids while avoiding the immunogenicity issues commonly associated with PEGylated LNPs.

[0032] definition All patent applications, patents, and printed publications cited herein are incorporated by reference in their entirety, except for any definitions, disclaimers, or disclaimers, and in the event that the incorporated material conflicts with the disclosure set forth in this application, the language of the present disclosure will control.

[0033] The indefinite articles are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or two or more elements.

[0034] The terms "about" and "approximately" generally refer to an acceptable degree of error or variation in the measured quantity, given the type or precision of the measurement. Numerical quantities set forth herein are approximate unless otherwise specified, meaning that the terms "about" and "approximately" can be inferred even when not expressly stated.

[0035] As used herein, the terms "active" or "activated" when used in reference to a particular functional group refers to a functional group that readily reacts with an electrophile or nucleophile on another molecule, as opposed to a group that requires a catalyst or impractical reaction conditions in order to react (i.e., a "nonreactive" or "inert" group).

[0036] As used herein, the term "inhibit," when used in reference to a drug or immune response (including, but not limited to, induction of IgM, induction of IgG, accelerated blood clearance, or a combination thereof), means that the response, abundance, or activity is reduced or attenuated compared to the response, abundance, or activity prior to administration of the compound / composition.

[0037] As used herein, the terms "biodegradable" or "releasable under physiological conditions" refer to a bond that contains a cleavable moiety. The terms degradable and releasable do not imply a particular mechanism for cleaving the linker.

[0038] As used herein, the terms "link," "linked," "bond," or "linker," when used in connection with a POZ polymer, POZ conjugate, drug, or compound described herein, or a component thereof, refer to a bond that is typically formed as a result of a chemical reaction and is generally a covalent bond.

[0039] As used herein, the term "lipid nanoparticle" or "LNP" refers to any of a wide variety of nanoparticles (including liposomes) formed by one or more lipid layers surrounding a core containing molecules to be released into the body. Liposomes generally have one or more continuous lipid bilayers encapsulating an aqueous core. Other forms of liposome-like nanocarriers include those with a lipid monolayer and those with discontinuous bilayers, and may or may not have an aqueous core.

[0040] As used herein, the term "hydrophilic," e.g., hydrophilic group, refers to a compound or molecule, or portion thereof, whose interaction with water is thermodynamically more favorable than its interaction with oil or other hydrophobic solvents. Hydrophilic compounds are capable of dissolving or dispersing in water.

[0041] As used herein, the term "hydrophobic," e.g., hydrophobic moiety, refers to a compound or molecule, or portion thereof, whose interaction with water is thermodynamically less favorable than its interaction with oil or other hydrophobic solvent. A hydrophobic compound is capable of dissolving or dispersing in oil or other hydrophobic solvent.

[0042] As used herein, the terms "inert" or "non-reactive" when used in reference to a particular functional group means a functional group that does not readily react with an electrophile or nucleophile on another molecule, requiring a catalyst or impractical reaction conditions in order to react.

[0043] As used herein, the term "pendant group" refers to a moiety that is part of a POZ polymer and is attached to the POZ polymer.

[0044] As used herein, the term "pendant moiety" refers to a substituent that is connected to the POZ polymer segment via a linking group, an example of a pendant moiety being R2 in Formula IV described herein.

[0045] As used herein, the term "pharmaceutically acceptable" refers to a compound that is compatible with the other ingredients of a composition and not harmful to the subject to which the compound or composition is administered. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for animal use, particularly for human use, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia.

[0046] As used herein, the term "pharmaceutically acceptable form" is intended to include known forms of a compound or POZ conjugate that can be administered to a subject, including, but not limited to, solvates, hydrates, prodrugs, isomorphs, polymorphs, pseudomorphs, neutral forms, or salt forms of the compound. In certain embodiments, pharmaceutically acceptable forms exclude prodrugs, isomorphs, and / or pseudomorphs. In certain embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts, neutral forms, solvates, and hydrates. In certain embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts and neutral forms. In certain embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts.

[0047] As used herein, the term "alkyl," whether used alone or as part of a substituent, is a term of art and refers to a saturated aliphatic group that optionally contains one or more heteroatoms (e.g., O, S, or N), which may be optionally substituted, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl group has about 30 or fewer carbon atoms in its backbone (e.g., C1-C5 for a straight chain). 30 , and in branched chains, C3-C 30 ), or about 20 or less, or 10 or less. In certain embodiments, the term "alkyl" refers to C-C 10 It refers to a straight chain alkyl group or a C1-C3 straight chain alkyl group. In certain embodiments, the term "alkyl" refers to a C3-C 12In certain embodiments, the term "alkyl" refers to a C3-C8 branched chain alkyl group. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. In certain embodiments, the term "alkyl" refers to a C1-C8 branched chain alkyl group that includes one or more heteroatoms (e.g., O, S, or N) in place of a carbon atom. 10 refers to a straight chain alkyl group, wherein the heteroatom may be optionally substituted. In certain embodiments, the term "alkyl" refers to a C-C substituted with up to five groups selected from the group consisting of OH, NH, and =O. 10 It means a straight chain alkyl group.

[0048] The term "alkenyl," as used herein, whether used alone or as part of a substituent, is a term of art and refers to an unsaturated aliphatic group optionally containing one or more heteroatoms (e.g., O, S, or N), which may be optionally substituted, including straight- or branched-chain hydrocarbon groups having 2 to 30 carbon atoms and containing at least one carbon-carbon double bond formed by the removal of two hydrogen atoms. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. The unsaturated bond in an alkenyl group may be located anywhere within the group, and may have a (Z) or (E) configuration centered around the double bond.

[0049] The term "alkynyl," as used herein, whether used alone or as part of a substituent, is a term of art and refers to an unsaturated aliphatic group that optionally contains one or more heteroatoms (e.g., O, S, or N), which may be optionally substituted, including straight or branched chain hydrocarbon groups containing 2 to 30 carbon atoms and at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 4-pentynyl, and 1-butynyl.

[0050] As used herein, the terms "substituted alkyl," "substituted alkenyl," and "substituted alkynyl" refer to alkyl, alkenyl, and alkynyl groups, as defined above, in which one or more bonds to a carbon or hydrogen has been replaced with a bond to a non-hydrogen or non-carbon atom, including, but not limited to, halogen atoms in halides (e.g., F, Cl, Br, and I); oxygen atoms in groups such as carbonyl, carboxyl, hydroxyl, alkoxy, aryloxy, heterocyclyloxy, and ester groups; thiol, alkylsulfide, arylsulfide, sulfone, sulfonyl, and sulfo groups. sulfur atoms in groups such as oxide groups; nitrogen atoms in groups such as amine groups, amide groups, alkylamine groups, dialkylamine groups, arylamine groups, alkylarylamine groups, diarylamine groups, N-oxide groups, imide groups, enamine groups, imine groups, oxime groups, hydrazone groups, heterocyclylamine groups, (alkyl)(heterocyclyl)amine groups, (aryl)(heterocyclyl)amine groups, diheterocyclylamine groups, triazole groups, and nitrile groups; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. In a specific embodiment, "polar alkyl," "polar alkenyl," and "polar alkynyl" refer to alkyl, alkenyl, and alkynyl groups substituted with atoms that result in polar covalent bonds. In another specific embodiment, "polar alkyl," "polar alkenyl," and "polar alkynyl" refer to C1-C5 alkyl, alkenyl, and alkynyl groups substituted with atoms that result in polar covalent bonds. In a specific embodiment, "polar alkyl," "polar alkenyl," and "polar alkynyl" refer to alkyl, alkenyl, and alkynyl groups, such as C1-C5 alkyl, alkenyl, and alkynyl groups substituted with -OH and / or -C(O)-OH groups.

[0051] As used herein, the terms "halo" or "halogen," whether used alone or as part of a substituent, are terms of art and refer to -F, -Cl, -Br, or -I.

[0052] The term "alkoxy," as used herein, whether used alone or as part of a substituent group, is a term of art and refers to an alkyl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0053] As used herein, the terms "aralkyl" or "arylalkyl," whether used alone or as part of a substituent, are terms of art and refer to an alkyl group substituted with an aryl group, where the aryl moiety is attached to the parent molecule via the alkyl group. The arylalkyl group may be optionally substituted. "Substituted aralkyl" has the same meaning with respect to unsubstituted aralkyl groups that substituted aryl groups have with respect to unsubstituted aryl groups. However, substituted aralkyl groups also include groups in which a carbon or hydrogen bond in the alkyl portion of the group is replaced with a bond to an atom other than carbon or hydrogen.

[0054] As used herein, the terms "heteroaralkyl" or "heteroarylalkyl," whether used alone or as part of a substituent, are terms of art and refer to an alkyl group substituted with a heteroaryl group, where the heteroaryl moiety is attached to the parent molecule via the alkyl group. The heteroarylalkyl group may be optionally substituted. The term "substituted heteroarylalkyl" has the same meaning with unsubstituted heteroarylalkyl groups that substituted aryl groups had with unsubstituted aryl groups.

[0055] The term "heterocyclylalkyl," as used herein, whether used alone or as part of a substituent, is a term of art and refers to an unsubstituted or substituted alkyl, alkenyl, or alkynyl group in which a hydrogen or carbon bond of the unsubstituted or substituted alkyl, alkenyl, or alkynyl group has been replaced with a bond to a heterocyclyl group. A heterocyclylalkyl group may be optionally substituted. The term "substituted heterocyclylalkyl" has the same meaning with respect to an unsubstituted heterocyclylalkyl group that the term "substituted aryl" has with respect to an unsubstituted aryl group. However, substituted heterocyclylalkyl groups also include groups in which an atom other than hydrogen is bonded to a heteroatom in the heterocyclyl group of the heterocyclylalkyl group (for example, but not limited to, a nitrogen atom in the piperidine ring of a piperidinylalkyl group).

[0056] The term "aryl," as used herein, whether used alone or as part of a substituent, is a term of art and is intended to include monocyclic, bicyclic, and polycyclic aromatic hydrocarbon groups, such as benzene, naphthalene, anthracene, and pyrene. The aromatic ring may be optionally substituted at one or more ring positions with one or more substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (e.g., trifluoromethyl), cyano, and the like. The term "aryl" also includes polycyclic ring systems containing two or more rings, where two adjacent rings share two or more carbon atoms (the rings are "fused rings"), and at least one of the rings is aromatic hydrocarbon, e.g., the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. In certain embodiments, the term "aryl" refers to a phenyl group. Aryl groups can be optionally substituted.

[0057] The term "cycloalkyl," as used herein, whether used alone or as part of a substituent, is a term of art and refers to a saturated carbocyclic group of 3 to 6 ring carbon atoms, such ring optionally being substituted with a substituted or unsubstituted alkyl group or with substituents such as those described for a substituted alkyl group. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-methylcyclobutyl, and 4-ethylcyclohexyl.

[0058] The term "heteroaryl," as used herein, whether used alone or as part of a substituent group, is a term of art that refers to monocyclic, bicyclic, and polycyclic aromatic groups containing one or more heteroatoms, such as nitrogen, oxygen, or sulfur, in the ring structure and having a total of 3 to 30 atoms. Typical heteroaryl groups include azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl, pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl, thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl, thienyl, thiomorpholinyl, triazolyl, or tropanyl, and the like. A "heteroaryl" may be optionally substituted at one or more ring positions with one or more substituents such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (e.g., trifluoromethyl), cyano, etc. The term "heteroaryl" also includes polycyclic ring systems containing two or more rings, where two adjacent rings share two or more carbon atoms (the rings are "fused rings"), and at least one of the rings is an aromatic group having one or more heteroatoms in the ring structure; for example, the other rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl.

[0059] The term "heterocyclyl," as used herein, whether used alone or as part of a substituent, is a term of art and refers to a radical of a non-aromatic ring system, including, but not limited to, monocyclic, bicyclic, and tricyclic rings containing 3 to 15 member atoms, which may be fully saturated or which may contain one or more units of unsaturation, but for the avoidance of doubt, the degree of unsaturation is such that it does not constitute an aromatic ring system and which contains at least one heteroatom, such as nitrogen, oxygen, or sulfur. Although not to be construed as limiting the scope of the invention, examples of heterocycles, by way of illustration and not limitation, include aziridinyl, azirinyl, oxiranyl, thiiranyl, thiirenyl, dioxiranyl, diazirinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, azetyl, oxetanyl, oxetyl, thietanyl, thienyl, diazetidinyl, dioxetanyl, dioxetenyl, dithietanyl, dithiethyl, dioxalanyl, oxazolyl, thiazolyl, triazinyl, isopropyl, ... Examples include isothiazolyl, isoxazolyl, azepine, azetidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, quinuclidinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.Heterocyclyl groups may be optionally substituted at one or more ring positions with one or more substituents such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (e.g., trifluoromethyl), cyano, and the like.

[0060] As used herein, the terms "treatment," "treating," and "therapeutic" refer to a course of action (e.g., administration of a conjugate described herein or a pharmaceutical composition comprising a conjugate described herein) to prevent, eliminate, or alleviate a symptom, aspect, or characteristic of a disease or condition. Such treatment need not be absolute to be useful. In one embodiment, treatment includes a course of action initiated concurrently with or after the onset of a symptom, aspect, or characteristic of a disease or condition. In another embodiment, treatment includes a course of action initiated prior to the onset of a symptom, aspect, or characteristic of a disease or condition.

[0061] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver that a patient requires or would benefit from treatment. This judgment is based on a variety of factors within the caregiver's realm of expertise, including a recognition that the patient is currently unwell or will become unwell as a result of a disease or condition treatable by the methods or compounds of the present disclosure.

[0062] As used herein, the terms "individual," "subject," or "patient" refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, and humans. These terms may specify or exclude male or female or both sexes. In a preferred embodiment, the terms "individual," "subject," or "patient" refer to a human.

[0063] As used herein, the term "therapeutically effective amount" refers to an amount of a conjugate, alone or as part of a pharmaceutical composition, that is capable of having some detectable positive effect on some symptom, aspect, or characteristic of a disease or condition. Such effect need not be absolute to be beneficial.

[0064] It will be understood that "substituted" or "substituted with" implicitly stipulates that such substitution is in accordance with the allowed valences of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformations such as, for example, rearrangement, fragmentation, decomposition, cyclization, elimination, or other reactions.

[0065] Of course, when a group is specified as part of a compound, substitution of that group can be adjusted to include specific linkages, for example, when an alkyl group is linked to two other groups, the alkyl group is considered an alkylene group.

[0066] The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In one broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described above. For purposes of this disclosure, heteroatoms such as oxygen or nitrogen can have hydrogen substituents and / or any permissible substituents that satisfy the valences of the heteroatoms of organic compounds described herein. Typical substituents include, but are not limited to, hydroxy, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (e.g., trifluoromethyl), cyano, and the like. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0067] Other chemical terms in this application are used according to conventional usage in the art, as specifically set forth in The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill, San Francisco, which is incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from inorganic or organic acids, such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, phosphoric acid, formic acid, acetic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glycolic acid, salicylic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, malonic acid, trifluoroacetic acid, trichloroacetic acid, naphthalene-2-sulfonic acid, and other acids. Pharmaceutically acceptable salt forms can include salts with a ratio of salt molecules other than 1:1. For example, the salt can include more than one inorganic or organic acid molecule per base molecule, such as two hydrochloric acid molecules per conjugate molecule. As another example, the salt can include less than one inorganic or organic acid molecule per base molecule, such as two conjugate molecules per inorganic or organic acid molecule.

[0069] As used herein, the terms "excipient" and "pharmaceutically acceptable excipient" refer to a diluent, adjuvant, additive, or vehicle used in administering a compound or in formulating it for administration. Non-limiting examples of such pharmaceutically acceptable excipients include liquids such as water, saline, and oils, and solids such as gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Additionally, auxiliary substances, stabilizers, thickeners, tonicity agents, cryoprotectants, lubricants, flavoring agents, and coloring agents may be used. Other examples of suitable pharmaceutical excipients can be found in Remington's Science and Practice of Pharmacy (23 rd edition, ISBN 9780128200070) and Handbook of Pharmaceutical Excipients (8 th edition, 978-0-85-711271-2), the disclosures of each of which are incorporated herein by reference in their entirety.

[0070] As used herein, the term "targeting agent molecule" refers to any molecule having a therapeutic or diagnostic use or targeting function, or a base used in administering or formulating a compound for administration, which is capable of forming a bond with an active functional group on a POZ polymer or POZ derivative of the present disclosure, and includes, but is not limited to, a therapeutic agent (e.g., but not limited to, a drug), a diagnostic agent, a targeting agent, a small organic molecule, an oligonucleotide, a polypeptide, an antibody, an antibody fragment, a protein, a carbohydrate such as heparin or hyaluronic acid, or a lipid such as a glycerolipid, glycolipid, or phospholipid.

[0071] As used herein, the term "lipid" or "lipid moiety" refers to (i) organic compounds that contain fatty acid esters or their derivatives and are characterized by being water-insoluble but soluble in many organic solvents (including, but not limited to, simple lipids such as fats, oils, and waxes, complex lipids such as phospholipids, glycolipids, cationic lipids, non-cationic lipids, neutral lipids, and anionic lipids, and derived lipids such as steroids), and (ii) organic compounds that do not contain fatty acid esters but resemble the above-mentioned organic compounds by virtue of their amphiphilic character, i.e., they have both hydrophobic and hydrophilic moieties and are therefore capable of aggregating in specific ways in aqueous environments to form layers, vesicles, and LNPs.

[0072] As used herein, "small interfering RNA (siRNA)" refers to a class of 16-40 nucleotide double-stranded RNA molecules that participate in the RNA interference (RNAi) pathway and disrupt the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways.

[0073] As used herein, "sgRNA" refers to a class of guide RNA molecules involved in CRISPR-Cas9 genome editing, which provides a template for precise genome editing to minimize "off-target" editing and maximize "on-target" editing.

[0074] As used herein, "saRNA" refers to a class of RNA molecules that are "self-amplifying" or "self-replicating" depending on the type of protein (generally a replicase) that is encoded, thereby producing multiple copies of the RNA.

[0075] As used herein, "RNA" refers to a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, self-amplifying RNA, sgRNA, gRNA, and polyvalent RNA. "Ribonucleotide" refers to a nucleotide containing a hydroxyl group at the 2' position of a β-D-ribofuranose moiety, including, but not limited to, modified ribonucleotides. These terms include isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, and substantially pure RNA, synthetic RNA, and recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides.

[0076] LNPs containing POZ-lipid conjugates Any of the POZ-lipid conjugates described below may be used to prepare LNPs according to the present disclosure. In one embodiment, LNPs can be formed using a POZ-lipid conjugate of the present disclosure and at least one cationic or ionizable lipid. For example, LNPs can be formed using a POZ-lipid conjugate and a cationic lipid. In another embodiment, LNPs can be formed using a POZ-lipid conjugate and an ionizable lipid. In yet another embodiment, LNPs comprise a POZ-lipid conjugate, a cationic or ionizable lipid, and other lipid components (underivatized lipids) capable of forming vesicles and / or liposomes (not including polyoxazoline components). Examples of suitable underivatized lipids include, but are not limited to, helper lipids and lipids for stabilizing the composition.

[0077] In one embodiment, an LNP formed in accordance with the present disclosure comprises a POZ-lipid conjugate, a cationic or ionizable lipid, and (i) a helper lipid to provide structural support and facilitate endocytosis, and / or (ii) a sterol lipid for stability.

[0078] LNPs formed according to the present disclosure can further comprise a payload. In this embodiment, the payload can be an oligonucleotide, a protein, or a combination thereof. For example, LNPs of the present disclosure can comprise (i) an ionizable lipid, (ii) a helper lipid, (iii) a sterol lipid, (iii) a POZ-lipid of the present disclosure, and (iv) an oligonucleotide. In another specific embodiment (not shown), LNPs of the present disclosure can comprise a cationic lipid, a helper lipid, a sterol lipid, a POZ-lipid of the present disclosure, and an oligonucleotide. In yet another embodiment, LNPs of the present disclosure can comprise a cationic or ionizable lipid, a helper lipid, a sterol lipid, a POZ-lipid of the present disclosure, and a protein.

[0079] In one embodiment, the oligonucleotide comprises DNA, siRNA, self-replicating mRNA, mRNA containing modified nucleosides, and mRNA containing natural nucleosides. In one embodiment, the oligonucleotide is DNA. In another embodiment, the oligonucleotide is siRNA. In yet another embodiment, the oligonucleotide is self-replicating mRNA, mRNA containing modified nucleosides, or mRNA containing natural nucleosides. In yet another embodiment, the oligonucleotide is an sgRNA used in genome editing.

[0080] In one embodiment, when incorporated into an LNP, the POZ-lipid conjugate is present in the lipid layer of the LNP at a molar ratio of about 0.25 to about 5 mol %, about 0.5 to about 3 mol %, about 0.75 to about 2 mol %, or about 0.8 to about 1.5 mol %. In other embodiments, the POZ-lipid conjugate is present in the lipid layer of the LNP at a molar ratio of about 1 to about 5 mol %, about 1 to about 2.5 mol %, or about 1.5 to about 5 mol %.

[0081] One non-limiting example of a cationic lipid suitable for use in accordance with the present invention is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Suitable ionizable lipids include, but are not limited to, MC3 98, Lipid 319, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Moderna Lipid H, SM-102, Acuitas A9

[59] , Arcturus Lipid 2,2(8,8)4C CH3, and Genevant CL1. In one embodiment, the cationic or ionizable lipid has a pKa in the range of 6-7 as measured by the TNS dye binding assay.

[0082] Helper lipids are amphipathic lipids with a hydrophobic portion and a polar head group that can spontaneously form bilayer vesicles, such as phospholipids, in water, or that can stably incorporate into lipid bilayers, with the hydrophobic portion contacting the inner hydrophobic region of the bilayer membrane and the polar head group facing the outer polar surface of the membrane. Such helper lipids typically contain one or two hydrophobic acyl hydrocarbon chains or steroid groups, and the polar head groups may contain chemically reactive groups such as amines, acids, esters, aldehydes, or alcohols. Non-limiting examples include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), in which the two hydrocarbon chains are typically about 14-22 carbon atoms long and have various degrees of unsaturation. Other suitable helper lipids include, but are not limited to, glycolipids such as cerebrosides and gangliosides. In one embodiment, the helper lipid is at least one of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

[0083] A suitable sterol lipid for use in accordance with the present disclosure is cholesterol. In one particular embodiment, an LNP according to the present disclosure comprises a cationic or ionizable lipid along with (i) DSPC, (ii) cholesterol, (iii) a POZ-lipid of the present disclosure, and (iv) an oligonucleotide.

[0084] It should be noted that even slight modifications or additions to the LNPs of the present disclosure may affect not only the structure of the LNPs but also the delivery of the encapsulated payload. For example, if a sterol lipid, such as cholesterol, is added to the LNP composition along with the ionizable lipid and the POZ-lipids described herein, the resulting LNPs will have a single bilayer. If phytosterols are added, the LNP structure will become more complex, and the manner in which the payload is delivered will likely differ. In this context, the compositions of the present disclosure containing POZ-lipid conjugates may be unilamellar or non-unilamellar.

[0085] The particle size of LNPs produced according to the present disclosure can vary. In one embodiment, LNPs formed according to the present disclosure are amphiphilic spherical vesicles formed by one or more lipid bilayers surrounding an aqueous core and have a particle size of about 10 nm to about 10 microns. In another embodiment, LNPs formed according to the present disclosure have a particle size of about 25 nm to about 8 microns. In yet another embodiment, LNPs formed according to the present disclosure have a particle size of about 30 nm to about 5 microns. In this embodiment, the particle size of the LNP can be about 20 nm to about 3 microns. In other embodiments, the LNP can be about 10 nm to about 1000 nm, about 25 nm to about 500 nm, about 35 nm to about 250 nm, about 40 nm to about 150 nm, or about 45 nm to about 100 nm. Size fractionation methods are disclosed herein. However, in certain embodiments, size fractionation is not required.

[0086] In certain embodiments, LNPs formed in accordance with the present disclosure have a particle size at least about 25% larger than that of their PEG-LNP counterparts (i.e., LNPs identical to POZ-LNPs formed in accordance with the present disclosure, except that POZ (in the lipid conjugate) is replaced with PEG). For example, POZ-LNPs formed in accordance with the present disclosure can have a particle size that is about 25% to about 99% larger than that of their PEG-LNP counterparts. In one embodiment, POZ-LNPs of the present disclosure have a particle size that is at least 50% larger than that of their PEG-LNP counterparts.

[0087] The LNP compositions of the present disclosure can be prepared by various methods. In one embodiment, the liposomes are prepared by reverse-phase evaporation (Szoka et al., PNAS 1978, vol. 75, pp. 4194-4198; Smirnov et al., Byulleten'Experimental'noi Biologii i Meditsiny, 1984, vol. 98, pp. 249-252; U.S. Pat. No. 4,235,871). In this method, an organic solution of liposome-forming lipids, which may include polyoxazoline-lipid conjugates with or without targeting molecules linked thereto, is mixed with a smaller volume of aqueous medium, and the mixture is dispersed, preferably using pyrogen-free ingredients, to form a water-in-oil emulsion. If the targeting molecule to be delivered is a lipophilic targeting molecule, it is added to the lipid solution; if it is a water-soluble targeting molecule, it is added to the aqueous medium. The lipid solvent is removed by evaporation, and the resulting gel is converted into liposomes. Vesicles prepared by reverse phase evaporation (REV) typically have an average particle size of about 0.2-0.4 microns and are primarily oligolamellar, i.e., contain no more than one lipid bilayer shell. REV can be readily sized by extrusion, as described below, to yield oligolamellar vesicles with a selected particle size, preferably about 0.05-0.2 microns.

[0088] Additionally, multilamellar vesicles (MLVs) can be prepared. In this method, a mixture of liposome-forming lipids, which may include polyoxazoline-lipid conjugates as described herein with or without a targeting agent molecule attached, is dissolved in a suitable solvent, and the solvent is evaporated in a container to form a thin film. The thin film is then covered with an aqueous medium. The lipid film hydrates, forming MLVs. MLVs typically have a particle size of about 0.1 to 10 microns. MLVs can be reduced to the desired particle size by extrusion and other methods described herein.

[0089] One effective method for sizing REVs and MLVs involves extruding an aqueous suspension of liposomes through a polycarbonate membrane with a selected uniform pore size, typically 0.05 microns, 0.08 microns, 0.1 microns, 0.2 microns, or 0.4 microns (Szoka et al., PNAS 1978, vol. 75, 4194-4198). The membrane pore size roughly corresponds to the maximum size of liposomes produced by extrusion through this membrane, particularly by extruding a preparation more than once through the same membrane. A method for sizing larger MLVs has been presented by Zhu et al. (PLoS One. 2009;4(4):e5009. Epub 2009 Apr 6).

[0090] If small particle sizes are desired, REV or MLV preparations can be processed to produce small unilamellar vesicles (SUVs), characterized by particle sizes in the 0.04-0.08 micron range. Such particles may be useful for targeting tumor or lung tissue, as they can be absorbed through the capillary walls in these tissues (whereas particles larger than 0.1 microns cannot be absorbed).

[0091] LNPs formed according to the present disclosure can have a polydispersity index (i.e., the ratio of weight-average molecular weight to number-average molecular weight) ranging from about 0.05 to about 0.3. In certain embodiments, LNPs formed according to the present disclosure have a polydispersity index of about 0.08 to about 0.25. In other embodiments, the polydispersity index of POZ-LNPs formed according to the present disclosure is about 0.1 to about 0.2. In still other embodiments, LNPs formed according to the present disclosure have a polydispersity index of about 0.0.1 to about 0.18.

[0092] In certain embodiments, LNPs formed according to the present disclosure have a polydispersity index substantially equivalent to that of the corresponding PEG-LNP. In this context, "substantially equivalent" means that the difference in polydispersity index between the POZ-LNP and the PEG-LNP is about 0.05 or less. For example, a POZ-LNP formed according to the present disclosure may have a polydispersity index that differs from that of its PEG-LNP counterpart by about 0.03 or less.

[0093] Additionally, POZ-lipid conjugates can be introduced into LNPs after liposome formation using the techniques described above. In this approach, preformed liposomes are incubated in the presence of the POZ-lipid conjugate, and the POZ-lipid conjugate is incorporated into the liposomes by diffusion. The concentration of the POZ-lipid conjugate, either free in solution or incorporated into the liposomes, can be monitored, and the process is terminated when the desired concentration of POZ-lipid conjugate in the LNP is reached. Detergents or other substances can be added to the incubation solution to facilitate diffusion of the POZ-lipid conjugate into the LNP.

[0094] LNPs can be treated to remove extraneous components before use. For example, if a surfactant is used as described above, excess surfactant can be removed before use. Furthermore, if a payload, such as an oligonucleotide, is encapsulated in the LNP composition, excess or unencapsulated payload can be removed before use. Separation techniques for accomplishing this task are known in the art, and the particular method selected will depend on the type of component to be removed. Suitable methods include, but are not limited to, centrifugation, dialysis, and molecular sieve chromatography. The composition can be sterilized by filtration through a conventional 0.22 micron depth filter.

[0095] LNPs can be prepared by conventional methods involving the hydration of a lipid film containing a POZ-lipid conjugate, an ionizable lipid, a helper lipid, and cholesterol. In this method, these ingredients are dissolved in an organic solvent such as chloroform or dichloromethane, followed by evaporation of the solvent to form a thin film. The thin film is then hydrated with an aqueous buffer containing a drug or nucleic acid, allowing passive encapsulation of the payload. Typically, LNPs are formed as heterogeneous particles with low encapsulation efficiency, requiring particle size reduction by extrusion or sonication.

[0096] In certain embodiments, the formulations can be prepared by microfluidic synthesis methods. In other embodiments, the formulations can be prepared by any method suitable for LNP production, including but not limited to, a T-mixer, manual pipetting, and a syringe pump connected to a PDMS microfluidic chip.

[0097] For example, one suitable technique uses rapid mixing using a microfluidizer. A lipid stock solution is prepared by dissolving lipids in an organic solvent such as ethanol. Nucleic acid is dissolved in a buffer solution of known pH, ionic strength, and buffer capacity to form an aqueous stock solution. These two stock solutions are passed through a micromixer at a predetermined speed to allow the cationic lipids to interact with the negatively charged nucleic acid, resulting in high encapsulation efficiency (i.e., >80%) and uniform particle size distribution. The ratio of aqueous to organic solvent during the mixing process is important. The organic solvent is removed by dialysis, tangential flow filtration, centrifugation, or other techniques. By controlling microfluidic operating parameters, LNPs with specific particle sizes can be produced, resulting in LNPs with low polydispersity and uniform particle size. The mean particle size, polydispersity, and zeta potential of LNPs are three methods used to characterize preparations. LNPs formed according to the present disclosure can have encapsulation efficiencies ranging from about 75% to about 100%. In certain embodiments, LNPs formed according to the present disclosure have encapsulation efficiencies of about 80% to about 99%. In other embodiments, the encapsulation efficiency of POZ-LNPs formed according to the present disclosure is about 85 to about 95%. In certain embodiments, LNPs formed according to the present disclosure have an encapsulation efficiency substantially equivalent to that of the corresponding PEG-LNP. In this context, "substantially equivalent" means that the encapsulation efficiency of POZ-LNPs and PEG-LNPs differs by about 5% or less. For example, POZ-LNPs formed according to the present disclosure can have an encapsulation efficiency that differs by about 3% or less from that of their PEG-LNP counterparts.

[0098] The ratio of POZ-lipid to ionizable lipid to cholesterol can be varied to optimize the particle size of the hydrated formulation, enhance payload release and transfection, and improve stability. In one embodiment, the molar percentage of POZ-lipid in the LNP is about 0.5 to 60%. In another embodiment, the molar percentage of POZ-lipid is about 1 to about 40%. In yet another embodiment, the POZ-lipid is present in an amount less than about 10% of the total lipid content in the LNP. In this embodiment, the POZ-lipid can be present in an amount of about 0.5 to about 5%, about 1 to about 4%, or about 1.5 to about 3.5%. In this embodiment, the remainder of the LNP can be about 35 to about 50% sterol lipid, about 30 to about 70% cationic lipid, and about 5 to about 15% helper lipid.

[0099] In one embodiment, the LNP comprises a lipid bilayer encapsulating an aqueous core, the lipid bilayer comprising at least one POZ-lipid conjugate molecule, the POZ having an average molecular weight of about 0.5-5 kDa, and the aqueous core comprising an oligonucleotide. In another embodiment, the LNP comprises a lipid bilayer encapsulating an aqueous core, the lipid bilayer comprising at least one POZ-lipid conjugate molecule, the POZ having an average molecular weight of about 2-5 kDa, and the aqueous core comprising an oligonucleotide.

[0100] The oligonucleotide can be encapsulated into the LNP with high efficiency. In one embodiment, the oligonucleotide is encapsulated into the LNP with an efficiency of at least 75%. In another embodiment, the oligonucleotide is encapsulated into the LNP with an efficiency of about 80 to about 99%. In yet another embodiment, the oligonucleotide is encapsulated into the LNP with an efficiency of about 85 to about 95%. In yet another embodiment, the oligonucleotide is encapsulated into the LNP with an efficiency of about 90 to about 95%. In yet another embodiment, the oligonucleotide is encapsulated into the LNP with an efficiency of greater than about 95%.

[0101] POZ-lipid conjugates The POZ-lipid conjugates of the present disclosure comprise a lipid moiety linked to a polyoxazoline (POZ) polymer. In this embodiment, the lipid moiety of the POZ-lipid conjugate comprises at least one hydrophobic moiety. In another embodiment, the lipid moiety comprises two hydrophobic moieties. In this embodiment, the hydrophobic moiety can be an acyl chain, an alkyl chain, or a combination thereof. The acyl chain and alkyl chain can be of various lengths. Furthermore, the acyl chain and alkyl chain can be saturated or contain one or more unsaturated regions (e.g., one or more double bonds).

[0102] Regardless of the number of hydrophobic moieties, the lipid moiety also includes a chemical group capable of forming a bond with a chemical group on the POZ polymer. In this embodiment, the chemical group can be an amine group, a hydroxyl group, an aldehyde group, a carboxylic acid group, or a combination thereof, but other chemical groups are not excluded. In one embodiment, the lipid moiety can include a reactive amino group that can be used to form a bond with the POZ polymer.

[0103] In certain embodiments, the chemical group on the lipid can be located at a hydrophilic head group position, and as noted above, the POZ polymer can be attached to the lipid via a suitable chemical group on the starting or terminal end of the polymer or at a suitable chemical group pendant on the polymer.

[0104] As described in more detail below, the type of linkage varies depending on the chemical groups present on the POZ polymer and the lipid moiety. In certain embodiments, the linkage is degradable in the presence of certain enzymes. In other embodiments, the linkage is stable in the presence of these same enzymes.

[0105] In one embodiment, the lipid moiety of the POZ-lipid conjugate is an uncharged lipid. For example, any uncharged lipid capable of forming a layer, vesicle, and / or LNP composition, alone or with other lipid components, is suitable for use in forming the POZ-lipid conjugates of the present disclosure. In another embodiment, the lipid moiety can be synthetic or natural.

[0106] The lipid moiety of the POZ-lipid conjugate can be selected to impart desired characteristics to the LNPs described herein. For example, the degree of unsaturation of the lipid can be selected to provide desired properties to the LNPs described herein. For example, increasing the degree of unsaturation of the lipid moiety can impart fluidity to the LNP composition. Alternatively, cis configurations around the unsaturated region can also impart increased fluidity to the LNP composition. Similarly, saturated lipid moieties can impart rigidity to the LNP composition. Fluidity and / or rigidity can be selected to control, at least in part, the stability of the LNP and / or the release rate of the POZ-lipid conjugate from the LNP composition.

[0107] In one embodiment, the lipid moiety of the POZ-lipid conjugate is a phospholipid. For example, the lipid moiety of the POZ-lipid conjugate can be phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), or a combination thereof.

[0108] In another embodiment, the lipid moiety of the POZ-lipid conjugate is a glycerolipid. For example, the lipid moiety can be αβ-diacylglycerol.

[0109] In another embodiment, the lipid moiety of the POZ-lipid conjugate is a dialkylamine. For example, the lipid moiety can be dimyristylamine.

[0110] In certain embodiments, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is saturated. In another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each saturated. In yet another embodiment, one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is saturated, and the other acyl or alkyl chain is unsaturated. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each unsaturated. When one or more acyl or alkyl chains of the lipid in the POZ-lipid conjugate are unsaturated, the acyl or alkyl chain may contain 1 to 6, 1 to 4, 1 to 3, or 1 to 2 unsaturated regions. If a double bond is present, it may be in the cis or trans configuration, or a mixture of cis and trans configurations.

[0111] In one embodiment, at least one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 1 to 5 carbons in length, 6 to 10 carbons in length, 11 to 16 carbons in length, or 17 to 21 carbons in length. In another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 1 to 5 carbons in length, 6 to 10 carbons in length, 11 to 16 carbons in length, or 17 to 21 carbons in length. Such acyl or alkyl chains are independent of chain length, including both even and odd chain lengths, and may be saturated or unsaturated as described above.

[0112] In one specific embodiment, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is 6 to 10 carbons in length. In another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 6 to 10 carbons in length. Such acyl or alkyl chains are independent of chain length, including both even and odd chain lengths, and may be saturated or unsaturated as described above.

[0113] In another embodiment, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is 11 to 16 carbons in length. In another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 11 to 16 carbons in length. Such acyl or alkyl chains are independent of chain length, including both even and odd chain lengths, and may be saturated or unsaturated as described above.

[0114] In yet another embodiment, at least one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 17 to 21 carbons in length. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 17 to 21 carbons in length. Such acyl or alkyl chains are independent of chain length, including both even and odd chain lengths, and may be saturated or unsaturated as described above.

[0115] In one embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length, and the acyl or alkyl chains are saturated or unsaturated. In another embodiment, the two alkyl or acyl chains of the lipid in the POZ-lipid conjugate are each 11, 12, 13, 14, 15, or 16 carbons in length, and the acyl chains are saturated or unsaturated. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 12, 13, 14, or 15 carbon atoms in length, and the acyl chains are saturated or unsaturated. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 13 or 14 carbon atoms in length, and the acyl chains are saturated or unsaturated. In yet another embodiment, the two alkyl or acyl chains of the lipid in the POZ-lipid conjugate are each 11, 12, 13, 14, 15, or 16 carbon atoms in length, and the alkyl chains are saturated or unsaturated. In another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 12, 13, 14, or 15 carbon atoms in length, and the alkyl chains are saturated or unsaturated. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each 13 or 14 carbon atoms in length, and the alkyl chains are saturated or unsaturated.

[0116] In yet another embodiment, each acyl chain or alkyl chain of the lipid moiety in the POZ-lipid conjugate is the same length and is unsaturated.For example, the acyl chain or alkyl chain can be 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, 20 carbons, or 21 carbons, or 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, or 16 carbons, or 12 carbons, 13 carbons, 14 carbons, or 15 carbons, or 13 carbons or 14 carbons, and can be unsaturated.

[0117] In another embodiment, the lipid moiety in the POZ-lipid conjugate is 1,2-dimyristoyl-sn-glycerol or 1,2-dilauroyl-sn-glycerol. In yet another embodiment, the lipid moiety in the POZ-lipid conjugate is di(tetradecyl)acetamide or di(dodecyl)acetamide. In yet another embodiment, the lipid moiety in the POZ-lipid conjugate is N,N-di(tetradecyl)acetamide or N,N-di(dodecyl)acetamide. In yet another embodiment, the lipid moiety can be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE).

[0118] Various POZ polymers can be used in the POZ-lipid conjugate. The POZ can be a linear POZ polymer, a branched POZ polymer, a pendant POZ polymer, or a multi-arm POZ polymer. Representative POZ polymers are described in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, and 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated herein by reference in its entirety for such teachings. The polyoxazoline polymer can be a homopolymer, or it can be a random copolymer or block copolymer in which one or more units of a first polyoxazoline polymer are separated by one or more units of a second polyoxazoline polymer. Similarly, the POZ may be poly(methyloxazoline) (PMOZ), which is highly hydrophilic, or poly(ethyloxazoline) (PEOZ), which is less hydrophilic. For example, in certain embodiments, the POZ is PEOZ.

[0119] In one embodiment, the POZ polymer is prepared by living cationic polymerization. Other methods known in the art may also be used to prepare the POZ polymer. As described in more detail below, the POZ can be directly attached to the lipid or linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the POZ to the lipid can be used, including, but not limited to, non-ester-containing linker moieties and ester-containing linker moieties. Also, as described in more detail below, the POZ polymer can be linked to the lipid moiety via a suitable chemical group on the starting or terminal end of the polymer or a suitable chemical group at a pendant position on the polymer.

[0120] Because polymers characterized by low polydispersity (PD) values ​​and / or high purity are useful for pharmaceutical applications, whenever a polyoxazoline derivative or polyoxazoline polymer is referred to in the present disclosure, the polyoxazoline polymer can be considered to have such characteristics. In a specific embodiment, the method of the present disclosure provides a polyoxazoline derivative having a high molecular weight (MW) value and a low PD value. In one embodiment, for example, the POZ portion has a molecular weight of about 500 to about 10,000 daltons. In another embodiment, the POZ portion has a molecular weight of about 500 to about 5,000 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 1,000 daltons to about 2,500 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 2,000 daltons to about 5,000 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 5,000 daltons to about 10,000 daltons. In such embodiments, at least one polyoxazoline polymer chain has a polydispersity value of 1.2 or less, 1.1 or less, or 1.05 or less. Methods for synthesizing polyoxazoline polymers and derivatives thereof having low PD values ​​are described in International Application Nos. PCT / US2008 / 002626 and PCT / US2008 / 078159, the entire disclosures of which are incorporated herein by reference for such teachings.

[0121] Generally, covalent attachment of POZ to lipids is achieved by reaction of an activated chemical group on the POZ polymer with a complementary chemical group on the lipid. Prior to reaction, the chemical groups on the POZ polymer and / or the lipid can be activated (e.g., but not limited to, removal of protecting groups). For coupling, hydroxyl, amine, or carboxyl groups can be activated, among others, with monofunctional activators such as N-hydroxysuccinimide, ethyl chloroformate, DCCD, Woodward's reagent K, cyanuric acid, and trifluoromethanesulfonyl chloride. Many bifunctional crosslinkers containing groups with different reactivities, such as certain diisocyanates, can also be used.

[0122] In one embodiment, the POZ-lipid conjugate has the general formula I:

[0123] [ka] or a pharmaceutically acceptable form thereof (for example, but not limited to, a pharmaceutically acceptable salt), wherein R is an initiating group; POZ is a polyoxazoline polymer; n is 1 to 1,000 and represents the number of monomer units constituting the polyoxazoline polymer; L is a linking group that optionally includes a cleavable moiety that allows for controlled cleavage rate, or represents a direct bond between a reactive group on the lipid and a reactive group on the polymer, where the direct bond can form a cleavable moiety that allows for controllable cleavage rate from highly degradable to stable; Lipid refers to the lipid moiety described herein.

[0124] In one embodiment of Structure I, the POZ polymer comprises at least one reactive group capable of forming a bond with a lipid or a linking group. The bond between the polymer and the lipid (whether direct or via a linking group) can be formed between any reactive group on the polymer backbone, including reactive groups at a terminal or pendant (terminal) position, and a reactive group on the lipid. In one embodiment, the bond between the linking group and the polymer can be formed at the end of the polymer. In another embodiment, the bond between the linking group and the polymer can be formed at a pendant position on the polymer. Furthermore, the bond (whether direct or via a linking group) can include a component of a reactive group originally present on the polymer or the lipid. The bond (whether direct or via a linking group) can be biodegradable. In this embodiment, the bond can include a cleavable moiety. Suitable linking groups include, but are not limited to, ethers, esters, amines, amides, and combinations thereof.

[0125] In one aspect of this embodiment, L is a stable bond. In another aspect of this embodiment, L is biodegradable and comprises a cleavable moiety. For example, in one embodiment, L can be selected from ester, carboxylate ester (-C(O)-O-), carbonate ester (-OC(O)-O-), carbamate (-OC(O)-NH-), and amide (-C(O)-NH-). In yet another aspect of this embodiment, L is a bond that does not comprise a cleavable moiety.

[0126] Exemplary R groups include, but are not limited to, hydrogen, alkyl groups, and substituted alkyl groups. In one embodiment, the initiator group is an alkyl group, such as a C1-C4 alkyl group. In one specific embodiment of the above, the initiator group is a methyl group. In another embodiment, the initiator group is H. In certain embodiments, the initiator group can be selected to lack an active functional group. In other embodiments, the initiator group can be selected to include an active functional group. Other suitable initiator groups are disclosed in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, and 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated herein by reference in its entirety for such teachings.

[0127] In certain embodiments, R is H or CH3.

[0128] In one embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] n wherein R2 is independently selected for each repeat unit of the POZ polymer from unsubstituted or substituted alkyl, alkenyl, aralkyl, and heterocyclylalkyl groups; R is H or CH3; and the degree of polymerization, "n," can be 15-35, 20-30, or 25.

[0129] In another embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] n wherein R2 is independently selected from unsubstituted and substituted alkyl groups for each repeat unit of the POZ polymer; R is H or CH3; and n can be 15-35, 20-30, or 25.

[0130] In yet another embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] nwherein R2 is independently selected from -CH3 and -CH2-CH3 for each repeat unit of the POZ polymer; optionally, R is H or CH3; and n can be 15-35, 20-30, or 25.

[0131] The POZ polymer is [N(COR2)CH2CH2] n When the POZ polymer of the conjugate is a polymer represented by the formula (R2), the POZ polymer is soluble in an aqueous environment. The type of pendant group (R2) can affect the solubility to some extent. For example, when R2 is methyl (as in PMOZ), the polymer is highly water-soluble, and when R2 is ethyl (as in PEOZ), the polymer is water-soluble, but to a lesser extent than PMOZ. The solubility of the POZ polymer allows it to extend beyond the liposome surface into the extraliposomal environment. Thus, the POZ polymer can effectively shield the liposome surface.

[0132] Specific embodiments of structure I above include, but are not limited to, those in which L is an amide cleavable by an amidase, as shown in formulas I(a)(1) and I(a)(2) below.

[0133] [ka]

[0134] In an alternative embodiment of structure I, the same lipid group can be incorporated as a stable amine (rather than an amide), as shown in formula I(b) below.

[0135] [ka]

[0136] In yet another embodiment of structure I, similar lipids can be coupled through relatively cleavable ester bonds, as shown in formulas I(c) and I(d) below.

[0137] [ka]

[0138] In yet another embodiment of structure I, the lipid can be coupled through a relatively stable ether bond, as shown in formulas I(e) and I(f) below.

[0139] [ka]

[0140] In one embodiment, in structures I(a)-I(f), (where appropriate), m is 1 or 2, n is 1-1000, o is 1-5, and p is 1-10.

[0141] As demonstrated in the Examples section below, the above embodiments of structure I (I(a)-I(f)) hydrolyze at different rates in plasma and therefore exemplify one of the key elements of the novel POZ-lipid conjugates of the present disclosure.

[0142] Other specific embodiments of structure I above include, but are not limited to, those shown in formulas I(g) and I(h) below.

[0143] [ka]

[0144] In one embodiment, in structures I(g)-I(h), m is 1 or 2 and n is 1-1000.

[0145] In another embodiment of the invention, the POZ-lipid conjugate has the general formula II: wherein the lipid is attached to the polymer chain at the starting end, rather than the terminal end as in formula I:

[0146] [ka] or a pharmaceutically acceptable form thereof (for example, but not limited to, a pharmaceutically acceptable salt), wherein POZ is a polyoxazoline polymer of the structure [N(COR2)CH2CH2]; L1 is a linking group that optionally includes a cleavable moiety that allows for controllable cleavage rate, or represents a direct bond between a reactive group on the lipid and a reactive group on the polymer, wherein the direct bond can form a cleavable moiety that allows for controllable cleavage rate; R2 is independently selected for each repeat unit of the polyoxazoline polymer from an unsubstituted or substituted alkyl group, an alkenyl group, an alkyne-substituted alkyl group, an aralkyl group, a heterocyclylalkyl group, or an active functional group; T is a terminating end group; a is ran, which represents a random copolymer, or block, which represents a block copolymer; n is an integer from 1 to 1,000.

[0147] The type of pendant group (R2) can affect solubility to some extent. The solubility of the POZ polymer allows it to extend beyond the liposome surface into the extraliposomal environment. Thus, the POZ polymer can effectively shield the liposome surface during LNP formation and prevent aggregation. Furthermore, without being bound by theory, it is believed that the POZ-lipid conjugate must be "shed" from the LNP surface after administration to efficiently deliver the nucleic acid payload. In certain embodiments, a highly hydrophilic group can be added to the POZ-lipid conjugate at the R2 position to aid in shielding during LNP formation and / or administration and / or enhance the shed of the POZ-lipid conjugate from the LNP, facilitating delivery of the payload. In one embodiment, R2 comprises at least one hydrophilic group. In another embodiment, R2 comprises multiple hydrophilic groups.

[0148] L1 (whether a direct bond or a linking group) can comprise a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L1 can optionally comprise a cleavable moiety, including, but not limited to, ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), and amide (-C(O)-NH-).

[0149] Exemplary active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In certain aspects, the active functional group is a hydrophilic group. In certain embodiments, the active functional group is used to add a hydrophilic group to the POZ-lipid conjugate, for example, by click chemistry using an alkyne or azide active functional group.

[0150] In one aspect of this embodiment, L1 is a stable bond. In another aspect of this embodiment, L1 is biodegradable and includes a cleavable moiety. In an alternative aspect of this embodiment, L1 is a bond that does not include a cleavable moiety. Suitable L1 bond(s) are as described herein.

[0151] In one aspect of this embodiment, L1 is a triazole linking group as detailed below.

[0152] In another aspect of this embodiment, T is a terminating nucleophile. For example, T can be ZBQ, where Z is S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a terminating moiety of a nucleophile.

[0153] Groups B include, but are not limited to, alkylene groups. In one particular embodiment, B is -(CH) y In the formula, y is an integer selected from 1 to 16.

[0154] In a specific embodiment, Z is S. POZ-lipid conjugates containing sulfur groups as described herein can be prepared by terminating the POZ cation with a mercaptide agent, such as, but not limited to, a mercaptoester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale purification by ion exchange chromatography (to remove secondary amines), as well as control of polydispersity values ​​(polydispersity values ​​of 1.10 or less) and the preparation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In yet another embodiment, Z is O.

[0155] In certain embodiments, Q is inert (i.e., does not contain a functional group). When Q is an inert group, any inert group can be used, including, but not limited to, a —CH mercaptide group, an alkyl mercaptide group, and an aryl mercaptide group. In alternative embodiments, Q is or contains an active functional group. When Q is or contains an active functional group, suitable functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). When Q is or contains an active functional group, Q may be the same as R2, or Q may be different from R2 (i.e., Q and R2 may be chemically orthogonal to each other).

[0156] In one particular aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl groups for each repeat unit of the polyoxazoline polymer, and optionally, R is H or CH3 and n is 15 to 35, 20 to 30, 22 to 28, or 25. In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of the polyoxazoline polymer, and optionally, R is H or CH3 and n is 15 to 35, 20 to 30, 22 to 28, or 25. In any of the above aspects, T is ZBQ, wherein Z is S and B is -(CH2) y and Q is an inert group such as, but not limited to, a —CH mercaptide group, an alkyl mercaptide group, and an aryl mercaptide group. Alternatively, in any of the above embodiments, T is ZBQ, where Z is S and B is —(CH ). y - and Q is or includes a functional group, including but not limited to, an alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal, thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), activated carbonate, chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS).

[0157] A specific embodiment of Structure II above includes, but is not limited to, Structure II(a) below, in which the lipid is attached to the initiating end and the terminating nucleophile is sulfur.

[0158] [ka]

[0159] In an alternative embodiment of structure II, as shown in formula II(b) below, the lipid is attached to the initiating end and the terminating nucleophile is -OH.

[0160] [ka]

[0161] In an alternative embodiment of structure II, shown in formula II(c) below, the lipid is attached to the initiating end, the terminating nucleophile is nitrogen, and the lipid is attached via a 2-propionate ester.

[0162] [ka]

[0163] Compounds II(a)-II(c) can be generated by a "click" reaction of an azide with a pendant alkyne group as follows:

[0164] [ka]

[0165] In yet another alternative embodiment of structure II, shown in formula II(d) below, the lipid is attached to the initiating end, the terminating nucleophile is sulfur, and the lipid is attached via an acetate ester.

[0166] [ka]

[0167] II(d) is generated by a "click" reaction of an azide with an alkyne starting group as follows:

[0168] [ka]

[0169] In one embodiment, in structures II(a)-II(d), m is 1-2, n is 1-1000, and o is 1-5 (where appropriate).

[0170] In another embodiment, the POZ-lipid conjugate has the general formula III:

[0171] [ka] or a pharmaceutically acceptable form thereof (for example, but not limited to, a pharmaceutically acceptable salt), wherein POZ is a polyoxazoline polymer of the structure [N(COR2)CH2CH2]; L2 is a linking group that optionally includes a cleavable moiety that allows for rate-controllable cleavage, or represents a direct bond between a reactive group on the lipid and a reactive group on the polymer, wherein the direct bond can form a cleavable moiety that allows for rate-controllable cleavage; R is an initiating group; R2 is independently selected for each repeat unit of the polyoxazoline polymer from a substituted or unsubstituted alkyl group, an alkenyl group, an alkyne-substituted alkyl group, an aralkyl group, a heterocyclylalkyl group, or an active functional group; Z is S, O, or N; A is ran, which represents a random copolymer, or block, which represents a block copolymer; n is an integer from 1 to 1,000.

[0172] As with Structure II, the type of pendant group (R2) can influence solubility to some extent. The solubility of the polyoxazoline polymer allows it to extend beyond the liposome surface into the extraliposomal environment. Thus, the polyoxazoline polymer can effectively shield the liposome surface. Furthermore, for efficient delivery of nucleic acid payloads, the POZ-lipid conjugate must be "shed" from the LNP surface after administration. In certain embodiments, the POZ-lipid conjugate can be added with a highly hydrophilic group at the R2 position to aid in shielding during LNP formation and / or administration and / or enhance the shed of the POZ-lipid conjugate from the LNP, facilitating delivery of the payload.

[0173] L2 (whether a direct bond or a linking group) can comprise a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L2 can optionally comprise a cleavable moiety, including, but not limited to, ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), and amide (-C(O)-NH-).

[0174] R groups include, but are not limited to, hydrogen, alkyl groups, and substituted alkyl groups. In one embodiment, the initiator group is an alkyl group, such as a C1-C4 alkyl group. In one specific embodiment of the above, the initiator group is a methyl group. In another embodiment, the initiator group is H. The initiator group can be selected to lack an active functional group. Alternatively, the initiator group can be selected to include an active functional group. Other exemplary initiator groups are disclosed in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, and 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated herein by reference in its entirety for such teachings.

[0175] Active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In certain aspects, the active functional groups are hydrophilic groups. In certain embodiments, the active functional groups are used to add hydrophilic groups to the POZ-lipid conjugates, for example, by click chemistry using an alkyne or azide active functional group.

[0176] In one aspect of this embodiment, L2 is a stable bond. In another aspect of this embodiment, L2 is biodegradable and includes a cleavable moiety. In an alternative aspect of this embodiment, L2 is a bond that does not include a cleavable moiety. Suitable L2 bond(s) are as described herein.

[0177] In one particular embodiment, Z is S. POZ conjugates containing sulfur groups as described herein can be prepared by terminating the POZ cation with a mercaptide agent, such as, but not limited to, a mercapto-ester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale purification by ion exchange chromatography (to remove secondary amines), as well as control of polydispersity values ​​(polydispersity values ​​of 1.10 or less) and the preparation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In yet another embodiment, Z is O.

[0178] In one aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl groups for each repeat unit of the polyoxazoline polymer; optionally, R is H or CH3; and n is 15-35, 20-30, or 25.

[0179] In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of said polyoxazoline polymer; optionally, R is H or CH3; and n is 15-35, 20-30, or 25.

[0180] In another embodiment, the POZ-lipid conjugate has the general formula IV:

[0181] [ka] or a pharmaceutically acceptable form thereof (for example, but not limited to, a pharmaceutically acceptable salt), wherein R is an initiating group; R2 is independently selected for each repeat unit of the polyoxazoline polymer from an unsubstituted or substituted alkyl group, an alkenyl group, an alkyne-substituted alkyl group, an aralkyl group, a heterocyclylalkyl group, or an active functional group; L3 is a linking group that optionally includes a cleavable moiety that allows for rate-controllable cleavage, or represents a direct bond between a reactive group on the lipid and a reactive group on the polymer, wherein the direct bond can form a cleavable moiety that allows for rate-controllable cleavage; Lipid is a lipid; T is a terminating end group; a is ran, which represents a random copolymer, or block, which represents a block copolymer; m is an integer from 1 to 100; n is an integer of 1 to 5.

[0182] In one embodiment, n is 1 and this monomer unit is the initial unit adjacent to R.

[0183] The type of pendant group (R2) can affect solubility to some extent. The solubility of the polyoxazoline polymer allows it to extend beyond the liposome surface into the extraliposomal environment. Thus, the polyoxazoline polymer can effectively shield the liposome surface. Furthermore, to efficiently deliver a nucleic acid payload, the POZ-lipid conjugate must be "shed" from the LNP surface after administration. In certain embodiments, a highly hydrophilic group can be added to the POZ-lipid conjugate at the R2 position to aid in shielding during LNP formation and / or administration and / or enhance the shed of the POZ-lipid conjugate from the LNP, facilitating delivery of the payload.

[0184] L3 (whether a direct bond or a linking group) can comprise a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L3 can optionally comprise a cleavable moiety, including, but not limited to, ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), and amide (-C(O)-NH-).

[0185] R groups include, but are not limited to, hydrogen, alkyl groups, and substituted alkyl groups. In one embodiment, the initiator group is an alkyl group, such as a C1-C4 alkyl group. In one specific embodiment of the above, the initiator group is a methyl group. In another embodiment, the initiator group is H. The initiator group can be selected to lack an active functional group. Alternatively, the initiator group can be selected to include an active functional group. Other exemplary initiator groups are disclosed in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, and 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated herein by reference in its entirety for such teachings.

[0186] Active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In certain aspects, the active functional groups are hydrophilic groups. In certain embodiments, the active functional groups are used to add hydrophilic groups to the POZ-lipid conjugates, for example, by click chemistry using an alkyne or azide active functional group.

[0187] In one aspect of this embodiment, L3 is a stable bond. In another aspect of this embodiment, L3 is biodegradable and includes a cleavable moiety. In an alternative aspect of this embodiment, L3 is a bond that does not include a cleavable moiety. Suitable L3 bond(s) are as described herein.

[0188] In one aspect of this embodiment, T is a terminating nucleophile. In one aspect of this embodiment, T is ZBQ, where Z is S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a terminating moiety of a nucleophile.

[0189] Groups B include, but are not limited to, alkylene groups. In one particular embodiment, B is -(CH) y In the formula, y is an integer selected from 1 to 16.

[0190] In one particular embodiment, Z is S. POZ conjugates containing sulfur groups as described herein can be prepared by terminating the POZ cation with a mercaptide agent, such as, but not limited to, a mercapto-ester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale purification by ion exchange chromatography (to remove secondary amines), as well as control of polydispersity values ​​(polydispersity values ​​of 1.10 or less) and the preparation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In yet another embodiment, Z is O.

[0191] In certain embodiments, Q is inert (i.e., does not contain a functional group). When Q is an inert group, any inert group can be used, including, but not limited to, a —CH mercaptide group, an alkyl mercaptide group, and an aryl mercaptide group. In other embodiments, Q is or contains an active functional group. When Q is or contains an active functional group, exemplary groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). When Q is or contains an active functional group, Q can be the same as R2, or Q can be different from R2 (i.e., Q and R2 are chemically orthogonal to each other).

[0192] In one aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl groups for each repeat unit of the polyoxazoline polymer; optionally, R is H or CH3, m is 15-35, 20-30, or 25, and n is 1. In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of the polyoxazoline polymer; optionally, R is H or CH3, m is 15-35, 20-30, or 25, and n is 1. In any of these aspects, T is ZBQ, wherein Z is S and B is -(CH2) y and Q is an inert group such as, but not limited to, a —CH mercaptide group, an alkyl mercaptide group, and an aryl mercaptide group. In any of these embodiments, T is ZBQ, where Z is S and B is —(CH) y- and Q is or includes a functional group such as, but not limited to, an alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal, thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (e.g., but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), activated carbonate, chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS).

[0193] In one specific embodiment of general formula IV, the POZ-lipid conjugate can be:

[0194] [ka]

[0195] A similar group of compounds is formed by coupling an azide with an initiating alkyne group, as shown below.

[0196] [ka]

[0197] These compounds contain a triazole ring. This triazole ring has been shown to be a privileged scaffold capable of transporting its associated ligands via the 26S protease, an enzyme involved in the intracellular cleavage of polypeptides. These so-called proteolysis-targeted chimeras (PROTACs) are a promising class of drugs that have been shown to conjugate with one end of a PROTAC and "knock down" the level of proteins shuttling by the 26S protease for cleavage. Without being bound by any particular theory, it is believed that POZ polymers containing a triazole ring can shuttling the POZ polymers by the 26S protease, thereby preventing immune presentation of non-proteolytic POZ polymers. In particular, when lipids are released by hydrolysis of the degradable ester bond, pendant acid groups remain attached to the polymer. The inventors have found that soluble POZ conjugated to these remaining pendant groups via the triazole ring is non-immunogenic, despite being taken up by dendritic cells in the subcutaneous compartment. Furthermore, rotigotine-conjugated C 14 Labeling studies of labeled 20 kD POZ polymers showed that the polymer conjugates were taken up through lymphatic vessels draining the subcutaneous injection site. The labeled conjugates ultimately appeared in the spleen and were taken up almost exclusively by the red pulp (macrophage compartment). Without being bound by theory, it is believed that the POZ-lipid LNPs of the present disclosure can also be selectively taken up by dendritic cells. Similarly, without being bound by theory, it is believed that the POZ-lipid LNPs of the present disclosure can also be selectively taken up by macrophages. If so, the oligonucleotide payload in the LNPs can be preferentially expressed in the dendritic cell / macrophage compartment, which is thought to be involved in immune presentation. In one embodiment, a triazole-pendant acid may also be directly attached as an R group in the above structure. Without being bound by theory, it is believed that these groups further contribute to reduced immunogenicity of the POZ-lipids contained in the LNPs.

[0198] Other POZ-lipid conjugates include those represented by Formulas V-VII below, in which the POZ polymer is linked to the lipid by L, L1, L2, or L3:

[0199] [ka] In the above formula, Alkyl1 and Alkyl2 are each independently 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbon atoms in length, 11, 12, 13, 14, 15, or 16 carbon atoms in length, 12, 13, 14, or 15 carbon atoms in length, or a saturated or unsaturated alkyl chain having a length of 13 or 14 carbon atoms, and preferably Alkyl1 and Alkyl2 have the same number of carbon atoms and are each unsaturated; Acyl1 and Acyl2 are each independently 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbon atoms in length, or 11, 12, 13, 14, 15, or 16 carbon atoms in length, or 12, 13, 14, or 15 carbon atoms in length, or a saturated or unsaturated acyl chain having a length of 13 or 14 carbon atoms, and preferably Acyl1 and Acyl2 have the same number of carbon atoms and are each unsaturated; j is an integer from 1 to 8.

[0200] As mentioned above, in some of the above embodiments, the lipid can be linked to the POZ polymer via a cleavable bond. In one aspect, a linking group is inserted between the POZ polymer and the lipid, the linking group comprising a cleavable moiety. In other words, the linking group comprises a biodegradable bond that can be cleaved in a specific environment. For example, the bond can be cleaved in vivo within the body of a subject after administering an LNP comprising a POZ-lipid conjugate of the present disclosure to the subject.

[0201] In one embodiment, the cleavable moiety is cleaved by a chemical reaction. In one aspect of this embodiment, the cleavage is achieved by reduction of an easily reduced group, such as, but not limited to, a disulfide. In another embodiment, the cleavable moiety is cleaved by a substance that is naturally present or induced to be present in the subject's body. In one aspect of this embodiment, such a substance is an enzyme or a polypeptide. Thus, in one embodiment, the cleavable moiety is cleaved by an enzymatic reaction. In yet another embodiment, the cleavable moiety is cleaved by a combination of the above. As described below, certain portions of the POZ polymer and / or certain portions of the lipid react to form the linking group, and therefore the linking group can include such moieties.

[0202] In this aspect, suitable cleavable moieties include, but are not limited to, esters, carboxylates (-C(O)-O-), carbonates (-OC(O)-O-), carbamates (-OC(O)-NH-), and amides (-C(O)-NH-, including amide groups in peptides), and other releasable moieties are described herein. In a specific embodiment, the cleavable moiety is an ester. In another specific embodiment, the cleavable moiety is a carbonate or a carboxylate. Furthermore, the linking group can be a natural amino acid, an unnatural amino acid, or a polymer comprising one or more natural and / or unnatural amino acids. The linking group can include a group from the polymer chain and / or the lipid.

[0203] In certain embodiments of the POZ-lipid conjugates of Formulas I-IV, L, L1, L2, and L3, at each occurrence, are biodegradable linkages comprising a cleavable moiety selected independently from ester, carboxylate ester (-C(O)-O-), carbonate ester (-OC(O)-O-), carbamate (-OC(O)-NH-), amide (-C(O)-NH-), and combinations thereof.

[0204] In certain other embodiments of the POZ-lipid conjugates of Formulas I-IV, L, L, L, and L are independently at each occurrence -(CH) f -(cleavable moiety)-(CH2) g wherein f and g are each independently an integer selected from 0 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. In one embodiment, f and g are each independently an integer selected from 1 to 4. In another embodiment, any of L, L1, L2, and L3 can be a disubstituted triazole group as described herein.

[0205] In certain embodiments of the POZ-lipid conjugates of Formulas I-IV, L, L, L, and L are independently at each occurrence -(CH) f -C(O)-(CH2) g -, -(CH2) f -C(O)-(CH2) h -NHC(O)-(CH2) g -, -(CH2) f -NH(CO)-(CH2) h -C(O)-(CH2) g -, -(CH2) f -NHC(O)-(CH2) g -, -(CH2) f -C(O)NH-(CH2) g -, -(CH2) f -NHOC(O)-(CH2) g -, -(CH2) f -OC(O)NH-(CH2) g -, -(CH2) f -OC(O)ONH-(CH2) g-, -(CH2) f -NHOC(O)O-(CH2) g -, O-(CH2) h , (CH2) h -O or (CH2) h wherein f, g, and h are each independently an integer selected from 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, or 0 to 5. In one aspect of this particular embodiment, f, g, and h can each be 0 to 4.

[0206] In another embodiment of the POZ-lipid conjugates of Formulas I-IV, L, L1, L2, and L3 are independently selected at each occurrence from a di-substituted triazole group containing a cleavable moiety in one of the R3 or R4 groups. Preferably, the cleavable moiety is in the R4 group. In one specific embodiment, the di-substituted triazole group has the following structure:

[0207] [ka] It has.

[0208] In the formula above, R3 is a linker connecting the triazole moiety to the polymer chain. R3 can be defined in part by functional groups on the polymer chain, or in other words, R3 can include a portion of functional groups on the polymer chain. In one embodiment, R3 is -C(O)-R5-, where R5 is absent or a substituted or unsubstituted alkyl group of 1 to 10 carbon atoms in length.

[0209] R4 is a linker connecting the triazole moiety to the lipid. R4 can be partially defined by functional groups on the lipid, or in other words, R4 can include a portion of functional groups on the lipid. In one embodiment, R4 is -R6-R7-R8-, where R6 is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aralkyl group, R7 is a group containing the cleavable moiety or a portion of the cleavable moiety, and R8 is absent or selected from the group consisting of O, S, CR c , or NRc wherein R c is H or a substituted or unsubstituted alkyl group. In certain embodiments, R7 and R8 together may form the cleavable moiety. In one embodiment, R7 is -R a -(O)-R b -, -R a -OC(O)-R b -, -R a -C(O)-NH-cyclic-OC(O)-R b - (wherein cyclic represents a substituted or unsubstituted aryl group, heterocycloalkyl group, heterocyclic group, or cycloalkyl group), -R a -C(O)-NH-(C6H4)-OC(O)-R b -, -R a -C(O)-R b -, -R a -C(O)-OR b -, -R a -OC(O)-OR b -, -R a -OC(O)-NR 15 -R b -(In the formula, R 15 is H or a substituted or unsubstituted C1 to C5 alkyl group, -R a -CH(OH)-OR b -, -R a -SSR b -, -R a -OP(O)(OR 11 )-OR b -(In the formula, R 11 is H or a substituted or unsubstituted C1-C5 alkyl group, or -R a -C(O)-NR 15 -R b -(In the formula, R 15 is H or a substituted or unsubstituted C1-C5 alkyl group, and R a and R b are each independently absent or a substituted or unsubstituted alkyl group. a and R bare each independently absent or a substituted or unsubstituted C2 to C16 alkyl group. In one embodiment of the above, R6 is a straight chain substituted or unsubstituted C1 to C8 alkyl group or a branched substituted or unsubstituted C1 to C8 alkyl group, and R7 is -R a -C(O)-OR b In another embodiment of the above, R6 is a straight chain substituted or unsubstituted C1-C4 alkyl group or a branched substituted or unsubstituted C1-C4 alkyl group, and R7 is -R a -C(O)-OR b In one embodiment of the above, R6 is -CH2-, -CH2-CH2-, or -CH2(CH3)-, R7 is -C(O)-O-, and R8 is absent.

[0210] In one particular embodiment, R3 is -C(O)-(CH2)3 and R4 is -CH2-C(O)-O-, -CH2-CH2-C(O)-O-, or -CH2(CH3)-C(O)-O-.

[0211] In one particular embodiment, R3 is -C(O)-(CH2)3 and R4 is -CH2-CH2-OC(O), -CH2-CH2-CH2-OC(O), -CH2-CH2-CO-NH-(C6H4)-OC(O)-.

[0212] In certain embodiments of the POZ-lipid conjugates of Formulas I-IV, L, L1, L2, and L3, at each occurrence, are independently selected from one or more of the cleavable moieties described above. In a particular embodiment, when two or more L, L1, L2, or L3 bonds are present in a POZ-lipid conjugate, L, L1, L2, and L3 are each identical. In another particular embodiment, when two or more L, L1, L2, or L3 bonds are present in a POZ-lipid conjugate, at least one L, L1, L2, and L3 is different from the other bonds.

[0213] In certain embodiments, the POZ-lipid degrades when exposed to enzymes and amidases in plasma. In particular, the ester bond linking the POZ to the lipid can be controlled to increase or decrease the rate of cleavage to release lipids from the POZ-lipid in plasma. In other embodiments, the POZ-lipid does not degrade in plasma, but degrades when exposed to amidases present in certain tissues. In yet other embodiments, the POZ-lipid is stable in plasma and under amidase exposure. In other words, the LNPs of the present disclosure can include POZ-lipids (e.g., amines, amides) that are stable in plasma, POZ-lipids (e.g., esters) that can be tailored or controlled to degrade at a desired rate in plasma, and POZ-lipids that are stable in plasma but degrade in specific environments. Thus, by carefully selecting the bond between the POZ and the lipid, the degradation rate can be precisely controlled. In this embodiment, the POZ-lipid can have controllable degradability in physiological environments. For example, in one embodiment, the controllable degradability of the linking group results in a POZ-lipid that is stable under physiological conditions. In this embodiment, the hydrolysis rate of the POZ-lipid, i.e., the time required for the POZ-lipid to degrade (usually measured as its half-life), is an indicator of the stability / degradability of the bond between the POZ and the lipid. In one aspect, the linking group between the POZ polymer and the lipid results in a POZ-lipid with a 50% human plasma hydrolysis half-life of at least about 120 hours.

[0214] In another embodiment, the controllable degradability of the linking group results in a POZ-lipid that degrades over time in a physiological environment. In certain embodiments, the linking group between the POZ polymer and the lipid results in a POZ-lipid with a 50% human plasma hydrolysis half-life of about 10 minutes or less. For example, the linking group between the POZ polymer and the lipid can be selected so that the POZ-lipid has a 50% human plasma hydrolysis half-life of about 3 minutes to about 7 minutes. In other embodiments, the linking group between the POZ polymer and the lipid can be selected so that the POZ-lipid has a 50% human plasma hydrolysis half-life of greater than about 10 minutes. For example, the linking group between the POZ polymer and the lipid can be selected so that the POZ-lipid has a 50% human plasma hydrolysis half-life of about 11 minutes to about 8 hours. In one embodiment, the POZ-lipid has a 50% human plasma hydrolysis half-life of about 2 hours to about 5 hours.

[0215] Administration Although the LNPs of the present disclosure target antigen-presenting cells, they can be delivered to any cell type. After in vivo administration of the LNPs, the payload (e.g., oligonucleotides) is released. In this embodiment, the LNPs of the present disclosure can be incorporated into pharmaceutical compositions capable of inducing the treatment of disorders or diseases. For example, pharmaceutical compositions containing LNPs prepared according to the present disclosure can be used to prevent or treat infectious diseases such as, but not limited to, SARS-CoV-2, rabies, and influenza by specifically targeting certain immune cells other than antigen-presenting cells. Furthermore, pharmaceutical compositions containing LNPs prepared according to the present disclosure can be used as therapeutic agents for cancer and genetic diseases. Such pharmaceutical compositions can also contain pharmaceutically acceptable excipients in addition to the LNPs.

[0216] In one embodiment, a pharmaceutical composition comprising an effective amount of LNPs of the present disclosure can be delivered to an animal. In one embodiment, the animal is a human. Delivery of an effective amount of LNPs of the present disclosure can be by subcutaneous, intravenous, intramuscular, intradermal, or aerosol routes. In certain embodiments, a pharmaceutical composition comprising an effective amount of LNPs of the present disclosure can be administered intravenously to deliver an encapsulated payload to the liver and spleen. In certain embodiments, intravenous administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to endothelial cells, dendritic cells, Kupffer cells, and / or hepatocytes in the liver. In other embodiments, intravenous administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to macrophages, B cells, T cells, and / or dendritic cells in the spleen. In yet other embodiments, intravenous administration of LNPs of the present disclosure facilitates enhanced delivery of an encapsulated payload to dendritic cells and / or macrophages in the spleen (compared to their PEG-LNP counterparts). For example, the amount of encapsulated payload in dendritic cells in the spleen after intravenous administration of LNPs of the present disclosure can be increased by at least about 15% compared to the amount of encapsulated payload in dendritic cells in the spleen after administration of the corresponding PEG-LNP. Similarly, the amount of encapsulated payload in macrophages in the spleen after intravenous administration of LNPs of the present disclosure can be increased by at least about 10% compared to the amount of encapsulated payload in macrophage cells in the spleen after administration of the corresponding PEG-LNP. Without being bound by theory, it is believed that POZ-LNPs not only effectively deliver encapsulated payloads after intravenous injection, but are also more effective than their PEG-LNP counterparts in delivering encapsulated payloads to antigen-presenting spleen cells, i.e., macrophages and dendritic cells.

[0217] In other embodiments, pharmaceutical compositions comprising an effective amount of LNPs of the present disclosure can be administered intramuscularly to deliver an encapsulated payload to on-target tissues, such as muscle and para-aortic (lumbar) lymph nodes, in addition to the liver and spleen. In certain embodiments, intramuscular administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to macrophages, dendritic cells, endothelial cells, and / or fibroblasts within the muscle. In other embodiments, intramuscular administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to macrophages, monocytes, B cells, and / or dendritic cells within the muscle. In yet other embodiments, intramuscular administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to endothelial cells, dendritic cells, Kupffer cells, and / or hepatocytes within the liver. In yet other embodiments, intramuscular administration of LNPs of the present disclosure facilitates delivery of an encapsulated payload to macrophages, B cells, T cells, and / or dendritic cells within the spleen.

[0218] In certain embodiments, intramuscular administration of the LNPs of the present disclosure facilitates enhanced delivery of encapsulated payloads to endothelial cells in the liver (compared to their PEG-LNP counterparts). For example, the amount of encapsulated payload in endothelial cells in the liver after intramuscular administration of the LNPs of the present disclosure can be increased by at least about 20% compared to the amount of encapsulated payload in endothelial cells in the liver after intramuscular administration of the corresponding PEG-LNPs. In certain embodiments, the amount of encapsulated payload in endothelial cells in the liver after intramuscular administration of the LNPs of the present disclosure can be increased by at least about 30% compared to the amount of encapsulated payload in endothelial cells in the liver after administration of the corresponding PEG-LNPs.

[0219] In other embodiments, intramuscular administration of LNPs of the present disclosure facilitates enhanced delivery of encapsulated payload to dendritic cells and / or macrophage cells in the spleen (compared to their PEG-LNP counterparts). For example, the amount of encapsulated payload in dendritic cells in the spleen after intramuscular administration of LNPs of the present disclosure can be increased by at least about 5% compared to the amount of encapsulated payload in dendritic cells in the spleen after administration of the corresponding PEG-LNP. In certain embodiments, the amount of encapsulated payload in dendritic cells in the spleen after intramuscular administration of LNPs of the present disclosure can be increased by at least about 10% compared to the amount of encapsulated payload in dendritic cells in the spleen after administration of the corresponding PEG-LNP. Similarly, the amount of encapsulated payload in macrophage cells in the spleen after intramuscular administration of LNPs of the present disclosure can be increased by at least about 5% compared to the amount of encapsulated payload in macrophage cells in the spleen after administration of the corresponding PEG-LNP. Without being bound by any particular theory, it is believed that POZ-LNPs not only effectively deliver encapsulated payloads to tissues near the injection site and / or the abdominal para-aortic (lumbar) lymph nodes draining the injection site after intramuscular injection, but are also more effective than their PEG-LNP counterparts in delivering encapsulated payloads to antigen-presenting spleen cells, i.e., macrophages and dendritic cells, and liver endothelial cells.

[0220] Importantly, administration of the LNPs of the present disclosure does not elicit a significant immune response compared to the immune response elicited by the corresponding PEG-LNPs, including, but not limited to, the production of IgM antibodies specific to the POZ. More specifically, the LNPs described herein elicit a reduced immune response compared to the corresponding PEG-LNPs, including, but not limited to, the production of IgM and / or IgG antibodies specific to the polymer moiety. In certain embodiments, after a second administration of the LNPs of the present disclosure, the LNPs are present in the blood or tissues of a subject at a concentration that is at least 75%, e.g., 80%, 85%, 90%, 95%, or more, compared to the first administration. In other embodiments, the LNPs of the present disclosure exhibit reduced accelerated blood clearance after a second administration.

[0221] Repeated administration of POZ-LNPs formed according to the present disclosure, i.e., three or more times at weekly intervals, results in increased particle delivery to target cell types compared to repeated administration of the corresponding PEG-LNPs. In certain embodiments, after repeated administration of POZ-LNPs, production of IgM antibodies specific to the polymer moiety is reduced by at least about 10% compared to the corresponding PEG-LNPs. In other embodiments, after repeated administration of POZ-LNPs, production of IgM antibodies specific to the polymer moiety is reduced by at least about 15% compared to the corresponding PEG-LNPs. In yet other embodiments, after repeated administration of POZ-LNPs, production of IgM antibodies specific to the polymer moiety is reduced by at least about 20% compared to the corresponding PEG-LNPs.

[0222] Without being bound by any particular theory, it is believed that multiple administrations of POZ-LNPs formed according to the present disclosure at intervals of more than 7 days / weeks can further reduce the production of IgM and / or IgG antibodies specific to the polymer moiety. In this embodiment, when repeatedly administered at intervals of 30 days or more, the production of IgM and / or IgG antibodies specific to the polymer moiety after repeated administration of POZ-LNP is at least about 50% lower than that of the corresponding PEG-LNP. In certain embodiments, when repeatedly administered at intervals of 30 days or more, the production of IgM and / or IgG antibodies specific to the polymer moiety after repeated administration of POZ-LNP is at least about 60% lower than that of the corresponding PEG-LNP. In other embodiments, when repeatedly administered at intervals of 30 days or more, the production of IgM and / or IgG antibodies specific to the polymer moiety after repeated administration of POZ-LNP is at least about 70% lower than that of the corresponding PEG-LNP.

[0223] In certain embodiments, the LNPs of the present disclosure enable delivery of an encapsulated payload to a target tissue of a subject without generating an immune response that promotes accelerated blood clearance (ABC) in response to administration of subsequent doses of the LNP. In particular, an initial dose of the LNPs of the present disclosure generates a suppressed immune response after administration of subsequent doses of the LNP, and the subject does not generate an ABC response when subsequent doses of the LNP are administered to the subject. In other embodiments, after repeated administration, with each subsequent dose administered at a predetermined time interval, the LNPs of the present disclosure are unable to bind to IgM (including, but not limited to, natural IgM). [Example]

[0224] The following examples are not intended to limit the invention or the claimed subject matter, but rather are intended to further illustrate various embodiments of the present disclosure.

[0225] Example 1: Formulation of LNPs of the present disclosure Various formulations, namely: 1a. Spikevax, a Moderna® LNP containing SM-102 (a proprietary ionizable lipid), cholesterol, DSPC (a structural lipid), and PEG-1,2-dimyristoyl-rac-glycerol (DMG); 1b. Spikevax, a Moderna® LNP containing SM-102, cholesterol, DSPC, and poly(ethyloxazoline) (PEOZ)-DMG; 2a. Onpattro®, an Alnylam LNP containing MC3 (a proprietary ionizable lipid optimized for siRNA payloads), cholesterol, DSPC, and PEG-DMG; and 2b. Onpattro®, an Alnylam LNP containing MC3, cholesterol, DSPC, and PEOZ-DMG According to the method described above, LNPs were prepared by microfluidic synthesis using the NanoAssemblr® Ignite™ system.

[0226] Specifically, mRNA was diluted with 10 mM citrate buffer (Teknova). d-Lin-MC3-DMA (MC3) (MedKoo Biosciences, 555308) or SM-102 (Cayman Chemical Company, 33474), DMG-PEOZ2,000 or DMG-PEG2,000 (Avanti, 880151), cholesterol (Avanti, 700100), and distearoylphosphatidylcholine (DSPC) (Avanti, 850365) were diluted with 100% ethanol. Both phases were loaded into separate syringe pumps. The citrate and ethanol phases were introduced into the microfluidic device and mixed at a rate of 600 μL / min and 200 μL / min, respectively.

[0227] The hydrodynamic diameter of the LNPs was measured using high-throughput dynamic light scattering (DLS) (DynaPro Plate Reader II, Wyatt). LNPs were diluted in sterile 1x PBS and analyzed. Fluorescence was read using a VICTOR X4 2030 multilabel reader (PerkinElmer), and encapsulation efficiency was assessed using the RiboGreen assay (Thermo Fisher Scientific, R11490).

[0228] Comparison of the hydrodynamic diameter, polydispersity index, and encapsulation efficiency of the corresponding LNPs (i.e., 1a vs. 1b and 2a vs. 2b) reveals that the POZ-LNPs of the present disclosure have similar biophysical characteristics to their PEG-LNP counterparts. More specifically, as can be seen in Figure 1A, the hydrodynamic diameter of 1b (145 nm) is larger than that of 1a (78 nm). As can be seen in Figure 1B, the hydrodynamic diameter of 2b is slightly larger than that of 2a. As can be seen in Figure 2A, the polydispersity index of 1a and 1b is comparable. As can be seen in Figure 2B, the polydispersity index of 2a is larger than that of 2b. As can be seen in Figure 3A, the encapsulation efficiencies of 1a and 1b are comparable. Similarly, as can be seen in Figure 3B, the encapsulation efficiencies of 2a and 2b are also comparable.

[0229] Example 2: Payload uptake and expression after intravenous administration LNPs 1a to 2b were each formulated to carry mRNA encoding a glycosylphosphatidylinositol (GPI)-anchored camelid-derived VHH antibody (anchored VHH, i.e., aVHH+). aVHH+ is an antibody specifically recognized by a reporter antibody not recognized in any mouse tissue and can be used to quantify LNP delivery. Four C57BL / 6J mice (Jackson Laboratory) were used for each group. LNPs were injected via the lateral tail vein at the following doses: Formulations 1a and 1b: 0.25 mg per kg of mouse body weight Formulations 2a and 2b: 1.00 mg per kg of mouse body weight.

[0230] Mice administered PBS were used as a negative control. 24 hours later, mouse tissues were harvested and the presence of aVHH+ was visualized as a % above background signal. Tissues were then evaluated for the presence of aVHH+ in the liver and spleen using the cell type-specific antibodies in the groups shown in Table 1 below.

[0231] [Table 1]

[0232] As shown in Figure 4, most tissues, such as hepatocytes, B and T cells in the spleen, and resident dendritic cells in the liver, showed no obvious differences in payload expression. On the other hand, as shown in Figure 5, the macrophage and dendritic cell compartments in the spleen showed significant differences when PEG-DMG LNPs and PEG-MC3 LNPs (i.e., Formulations 1a and 2a) were compared with PEOZ-DMG LNPs and PEOZ-MC3 LNPs (i.e., Formulations 1b and 2b). Furthermore, as shown in Figure 5A, the hepatic endothelial cell compartment in the liver showed significant differences when PEG-MC3 LNPs (i.e., Formulation 2a) were compared with PEOZ-MC3 LNPs (i.e., Formulation 2b). Notably, payload expression in these compartments was independent of the formulation.

[0233] The results were unexpected: PEOZ-DMG LNPs prepared according to the present disclosure were preferentially taken up by antigen-presenting cells in the spleen, and payload expression by these cells was formulation-independent. Other cell types present in other tissues (e.g., endothelial cells, Kupffer cells, and hepatocytes) did not show preferential uptake compared to PEG-DMG LNPs. Indeed, other immune cells, including B cells and T cells in the spleen, did not show preferential uptake of PEOZ-DMG LNPs (Figures 4B and 5B). These results were limited to antigen-presenting cells in the macrophage and dendritic cell compartments in the spleen.

[0234] Example 3: Payload uptake and expression after intramuscular administration LNP1a and LNP1b were again formulated to carry mRNA encoding aVHH. Four C57BL / 6J mice (Jackson Laboratory) were used for each group. LNP was injected into the quadriceps muscles of the left and right hind legs at the following doses: Formulations 1a and 1b: 3 μg per hind paw.

[0235] After 24 hours, mouse tissues were harvested and the presence of anchored antibodies (aVHH+) was visualized as a percentage above background signal. Specifically, injected quadriceps muscles were isolated, minced, and transferred to 5 mL of digestion enzyme solution consisting of collagenase B (Sigma Aldrich, #11088831001), dispase II (neutral protease, grade II) (Sigma Aldrich, #04942078001), and RPMI-1640 medium. Lymph nodes were minced in 300 μL of RPMI-1640 medium containing 700 μL of digestion enzyme solution containing collagenase D (Sigma Aldrich, #11088866001) and 10% FBS (Sigma Aldrich, #F2442-500ML). The tissue was incubated at 37°C and 600 rpm for 20 minutes. Spleen tissue was minced and then immersed in 1x PBS. The liver tissue was minced and then immersed in a digestive enzyme solution containing type I collagenase (Sigma Aldrich), type XI collagenase (Sigma Aldrich), and hyaluronidase (Sigma Aldrich) at 37°C and 550 rpm for 45 minutes.

[0236] Utilizing the same panel of cell type-specific antibodies from Example 2, Table 1, tissues were evaluated for the presence of aVHH+ in muscle, abdominal periaortic (lumbar) lymph nodes, liver, and spleen.

[0237] As shown in Figure 6, there was no significant difference between aVHH+ cells in the on-target organ (i.e., the muscle near the abdominal para-aortic (lumbar) lymph nodes draining the injection site). On the other hand, as shown in Figure 7, the endothelial cell compartment in the liver and the dendritic cell compartment in the spleen showed statistically significant differences when PEG-DMG LNPs (i.e., formulation 1a) were compared with PEOZ-DMG LNPs (i.e., formulation 1b). Notably, payload expression in these compartments was formulation-independent.

[0238] It is unexpected that PEOZ-DMG LNPs produced according to the present disclosure are preferentially taken up by liver endothelial cells and splenic dendritic cells, and that payload expression by these cells is formulation independent.

[0239] Example 4: Payload uptake and expression after intramuscular administration LNP1a to 1b were each formulated again to carry mRNA encoding luciferase, and protein expression and immune stimulation were measured after repeated administration.

[0240] Four C57BL / 6J mice (Jackson Laboratory) were used in each group. LNP was injected via the lateral tail vein at the following doses: Group A: 0.25 mg per kg of body weight once every 4 weeks Group B: 0.25 mg per kg of mouse body weight once a week for 4 weeks.

[0241] Tissues were isolated 48 hours after LNP administration. To measure luminescence, mice were euthanized, organs were harvested, and organs were immersed in Nano-Glo luciferase assay substrate (Promega, N1110) for 5 minutes before being mounted on black imaging paper. Luminescence was measured using an IVIS imaging system (PerkinElmer) and quantified using LivingImage software (PerkinElmer). To quantify anti-PEG or anti-PEOZ IgM in serum, 100 μL of carboxy-functionalized latex beads (Life Technologies, C37259) were coupled with 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (TCI Chemicals, D1601-5G) for 15 minutes on a plate shaker at room temperature under maximum rpm. The activated beads were incubated with 12 μg of DMG-PEG2,000 or DMG-PEOZ2,000 for 2 hours. The beads were washed twice with PBS, resuspended in 2% MSD Blocker A (Meso Scale Diagnostics, R93AA), and stored overnight at 4°C. A 10 μL sample of mouse serum isolated 48 hours after LNP administration was used at a 1:100 dilution and incubated with the beads for 1 hour at room temperature on a plate shaker. The beads were then incubated with a mouse IgM monoclonal (primary) antibody in the dark. The sample was washed and incubated with a highly cross-adsorbed goat anti-rat IgG (H+L) secondary antibody conjugated with Alexa Fluor Plus 647 (Thermo Fisher Scientific, A48265) for fluorescence detection. After further washing, the beads were analyzed by flow cytometry.

[0242] As shown in Figures 8A and 8B, the livers and spleens from Group B mice were significantly less luminescent than those from Group A mice. Meanwhile, as shown in Figure 8A, the decrease in liver luminescence in Group B mice injected with Formulation 1b (67%) was less than the decrease in liver luminescence in Group B mice injected with Formulation 1a (91%). Similarly, as shown in Figure 8B, the decrease in spleen luminescence in Group B mice injected with Formulation 1b was less than the decrease in liver luminescence in Group B mice injected with Formulation 1a (71%). The smaller decrease in liver and spleen luminescence with PEOZ-LNP (Formulation 1b) suggests that anti-PEOZ and anti-PEG antibodies were formed in Group B mice repeatedly injected with Formulation 1b and Formulation 1a, respectively, but that the amount of anti-PEOZ antibodies in Group B / Formulation 1b mice was significantly less than the amount of anti-PEG antibodies in Group B / Formulation 1a mice after repeated administration.

[0243] As can be seen from Figure 9, after repeated dosing, Group B / Formulation 1b produced significantly less antibody than Group B / Formulation 1a.

Claims

1. 1. A method for preferentially delivering a payload to antigen-presenting cells in a subject, comprising: Formula I: 【Chemistry 1】 wherein R comprises an initiating group; POZ comprises poly(ethyloxazoline); L comprises a biodegradable linking group; Lipid comprises an uncharged lipid containing at least one hydrophobic moiety; an ionized or cationic lipid; Helper lipids and; sterol lipids providing a lipid nanoparticle comprising the payload encapsulated therein; The method comprises administering an effective amount of the lipid nanoparticles to the subject.

2. The method of claim 1 , wherein the antigen-presenting cells comprise splenic macrophage cells, dendritic cells, or a combination thereof.

3. POZ is [N(COR ２ ) CH ２ CH ２ ] ｎ wherein R ２ The method of claim 1 , wherein is ethyl.

4. 2. The method of claim 1, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole group having a carboxylic acid attached thereto, or a substituted or unsubstituted aralkyl group.

5. The method of claim 1 , wherein L comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof.

6. The method of claim 1 , wherein the lipid comprises two hydrophobic moieties.

7. The method of claim 1 , wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof.

8. 2. The method of claim 1, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

9. The method of claim 1 , wherein the payload comprises an oligonucleotide.

10. 1. A method for preferentially delivering a payload to antigen-presenting cells in a subject, comprising: Formula II: 【Chemistry 2】 wherein Lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; L １ comprises a biodegradable linking group; POZ has the structure [N(COR ２ ) CH ２ CH ２ ] (wherein, R ２ is ethyl. n is 1 to 1,000; a is ran, which represents a random copolymer, or block, which represents a block copolymer; T comprises a terminating group; and an ionized or cationic lipid; Helper lipids and; sterol lipids and preparing lipid nanoparticles comprising the oligonucleotides encapsulated in the lipid nanoparticles; The method comprises administering an effective amount of the lipid nanoparticles to the subject.

11. L １ 11. The method of claim 10, wherein comprises ethers, esters, carboxylic acid esters, carbonate esters, carbamates, amines, amides, urethanes, disulfides, and combinations thereof.

12. L １ The method of claim 10 , wherein comprises a triazole group.

13. 11. The method of claim 10, wherein T comprises Z-B-Q, wherein Z comprises S, O, or N, B is an optionally present linking group, and Q is a terminating nucleophile or portion thereof.

14. The method of claim 10 , wherein the lipid comprises two hydrophobic moieties.

15. The method of claim 10 , wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof.

16. The method of claim 10, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

17. 11. The method of claim 10, wherein the oligonucleotide comprises mRNA and the antigen-presenting cells comprise splenic macrophage cells, dendritic cells, or a combination thereof.

18. 1. A method for preferentially delivering a payload to antigen-presenting cells in a subject, comprising: Formula III: 【Transformation 3】 wherein R comprises an initiating group; POZ has the structure [N(COR ２ ) CH ２ CH ２ ] (wherein, R ２ is ethyl. n is 1 to 1,000; a is ran, which represents a random copolymer, or block, which represents a block copolymer; Z comprises S, O, or N; L ２ comprises a biodegradable linking group; Lipid comprises an uncharged lipid containing at least one hydrophobic group; an ionized or cationic lipid; Helper lipids and; sterol lipids providing a lipid nanoparticle comprising the payload encapsulated therein; The method comprises administering an effective amount of the lipid nanoparticles to the subject.

19. 20. The method of claim 18, wherein the payload comprises mRNA.

20. 19. The method of claim 18, wherein the antigen-presenting cells comprise splenic macrophage cells, dendritic cells, or a combination thereof.

21. L ２ 20. The method of claim 18, wherein comprises ethers, esters, carboxylic acid esters, carbonate esters, carbamates, amines, amides, urethanes, disulfides, and combinations thereof.

22. 19. The method of claim 18, wherein the lipid comprises two hydrophobic moieties.

23. 19. The method of claim 18, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof.

24. 19. The method of claim 18, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

25. 19. The method of claim 18, wherein R comprises hydrogen or a substituted or unsubstituted alkyl group and n is 15 to 35.

26. 1. A method for preferentially delivering a payload to an antigen-presenting cell, comprising: Formula IV: 【Chemistry 4】 wherein R comprises an initiating group; L ３ comprises a biodegradable linking group; Lipid comprises an uncharged lipid containing at least one hydrophobic moiety; n is 1 to 5; R ２ is independently selected for each repeat unit from an unsubstituted or substituted alkyl group, alkenyl group, aralkyl group, heterocyclylalkyl group, or active functional group; m is 1 to 100; a is ran, which represents a random copolymer, or block, which represents a block copolymer; T comprises a terminating group); and an ionized or cationic lipid; Helper lipids and; sterol lipids providing a lipid nanoparticle comprising the payload encapsulated therein; The method comprises administering an effective amount of the lipid nanoparticles to a subject.

27. L ３ 27. The method of claim 26, wherein comprises an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amide, and combinations thereof.

28. L ３ The method of claim 26 , wherein comprises a triazole ring.

29. 27. The method of claim 26, wherein T comprises Z-B-Q, wherein Z comprises S, O, or N, B is an optionally present linking group, and Q is a terminating nucleophile or portion thereof.

30. 27. The method of claim 26, wherein the lipid comprises two hydrophobic moieties.

31. 27. The method of claim 26, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof.

32. 27. The method of claim 26, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

33. 27. The method of claim 26, wherein the payload comprises mRNA and the antigen-presenting cells comprise splenic macrophage cells, dendritic cells, or a combination thereof.

34. 1. A method for delivering a payload to a target tissue in a subject, comprising: Formula I: 【Transformation 5】 wherein R comprises an initiating group; POZ comprises poly(ethyloxazoline); L comprises a biodegradable linking group; Lipid comprises an uncharged lipid containing at least one hydrophobic moiety; an ionized or cationic lipid; Helper lipids and; sterol lipids providing a lipid nanoparticle comprising the payload encapsulated therein; The method comprises administering an effective amount of the lipid nanoparticles to the subject.

35. 35. The method of claim 34, wherein the administering step comprises injecting the subject intramuscularly.

36. 35. The method of claim 34, wherein the administering step comprises intramuscularly injecting multiple doses into the subject, wherein an initial dose is administered followed by each subsequent dose at predetermined time intervals.

37. 37. The method of claim 36, wherein the predetermined time interval is at least 30 days.

38. 35. The method of claim 34, wherein the target tissue comprises the liver, the spleen, or a combination thereof.

39. POZ is [N(COR ２ ) CH ２ CH ２ ] ｎ wherein R ２ The method of claim 34, wherein is ethyl.

40. 35. The method of claim 34, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole group having a carboxylic acid attached thereto, or a substituted or unsubstituted aralkyl group.

41. 35. The method of claim 34, wherein L comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof.

42. 35. The method of claim 34, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylacetamide, or a combination thereof.

43. 35. The method of claim 34, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

44. 35. The method of claim 34, wherein the payload comprises mRNA.

45. 1. A method for delivering therapeutic levels of an immunogenic payload to a target tissue in a subject, comprising: administering to the subject multiple doses of lipid nanoparticles, wherein the lipid nanoparticles encapsulate mRNA encoding an antigen, the lipid nanoparticles comprising an ionizable lipid, a helper lipid, a structural lipid, and a lipid having Formula I: 【Transformation 6】 wherein R comprises an initiating group; POZ comprises poly(ethyloxazoline); L comprises a biodegradable linking group; Lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; The method, wherein the LNP induces a drug response that suppresses the response associated with the LNP.

46. 46. ​​The method of claim 45, wherein the drug response comprises reduced induction of IgM, reduced induction of IgG, reduced accelerated blood clearance, or a combination thereof.

47. 46. ​​The method of claim 45, wherein the target tissue comprises the liver, the spleen, or a combination thereof.

48. POZ is [N(COR ２ ) CH ２ CH ２ ] ｎ wherein R ２ 46. ​​The method of claim 45, wherein is ethyl.

49. 46. ​​The method of claim 45, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole group having a carboxylic acid attached thereto, or a substituted or unsubstituted aralkyl group.

50. 46. ​​The method of claim 45, wherein L comprises an ether, an ester, a carboxylic acid ester, a carbonate ester, a carbamate, an amine, an amide, a urethane, a disulfide, and combinations thereof.

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