Pharmaceutical system

Abstract

Provided are lipid-based systems, compositions, and methods of making and using the same. The lipid-based systems contain lipid nanoparticles and specifically formulated liposomes to reduce or prevent lipid nanoparticle clearance in vivo in subjects towards LNPs or components thereof.

Description

PHARMACEUTICAL SYSTEMRELATED APPLICATIONSThis Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 738068, filed on December 23, 2024, the entire contents of which are incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTINGThe contents of the electronic sequence listing (G069670004WO00-SEQ-ACZ.xml; Size: 13,197 bytes; and Date of Creation: December 9, 2025) are herein incorporated by reference in their entirety.FIELD

[0001] The disclosure provides lipid-based systems for preventing immune-mediated clearance of therapeutic compositions and methods of making and using the same.BACKGROUND

[0002] Lipid nanoparticles (LNP) are attractive for nucleic acid delivery due to their advantages of simple formulations, good biocompatibility, and high capacity for payload. However, certain elements of LNPs, such as polyethylene glycol (PEG)-conjugated lipids, may induce anti-drug antibodies (AD As), which can lead to reduced efficacy of such pharmaceutical compositions. Therefore, there is a need to develop novel pharmaceutical systems and methods to improve the therapeutic utility of such pharmaceutical compositions.SUMMARY

[0003] Provided are lipid-based systems.

[0004] In some embodiments, the pharmaceutical systems contain lipid nanoparticles and specifically formulated nanoparticle (e.g., liposomes, micelles, emulsions) to reduce or prevent lipid nanoparticle clearance in vivo in subjects towards LNPs or components thereof.

[0005] In some aspects, this disclosure provides a pharmaceutical system comprising two particles, wherein the first particle and the second particle share at least one common element, wherein the first particle is a liposome and the second particle is a lipid nanoparticle (LNP), and wherein: (i) both the first particle and the second particle bind to at least one innate immune system opsonin; and / or (ii) the common element is capable of inducing an anti-drug antibody1#14739410v1against the first particle and the second particle in a subject when the pharmaceutical system is administered to the subject.

[0006] In some embodiments, the common element comprises a polymer or a compound comprising a polymer, optionally wherein the polymer comprises a polyethylene glycol (PEG) or the compound comprising the polymer is a PEG-lipid.

[0007] In some embodiments, the anti-drug antibody is an anti-PEG antibody.

[0008] In some embodiments, the liposome comprises a PEG or a compound comprising a PEG, optionally wherein the compound comprising the PEG is a PEG-lipid.

[0009] In some embodiments, the liposome further comprises one or more helper lipids.

[0010] In some embodiments, the liposome comprises the PEG-lipid and the one or more helper lipids at a molar ratio of about 5:95 to about 25:75.

[0011] In some embodiments, the liposome does not comprise a sterol and / or does not comprise an ionizable lipid. In some embodiments, the liposome further comprises a sterol.

[0012] In some embodiments, the liposome comprises the PEG-lipid, the one or more helper lipids, and the sterol at a molar ratio of about 10:45:45.

[0013] In some embodiments, the LNP comprises an ionizable lipid and a PEG-lipid. In some embodiments, the ionizable lipid is, or a salt thereof.

[0014] In some embodiments, the ionizable lipid is present in the LNP at a range of about 10 mol% to about 70 mol%. In some embodiments, the PEG-lipid is present in the LNP at a range of about 0.5 mol% to about 4 mol%.

[0015] In some embodiments, the LNP further comprises a payload compound, a structural lipid, and optionally a neutral phospholipid.

[0016] In some embodiments, the LNP further comprises a conjugate having the formula of [Lipid] - [optional linker] - [antibody], wherein the antibody specifically binds to a type of cell, tissue, or organ. In some embodiments, the LNP comprises two conjugates both having the formula of [Lipid] - [optional linker] - [antibody], and the first conjugate comprises a first antibody that binds to a first antigen of a first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of a second type of immune cell.2#14739410v1

[0017] In some embodiments, the LNP is for delivering a nucleic acid into an immune cell, and wherein LNP comprises a Fab lacking the native interchain disulfide bond.

[0018] In some embodiments, the neutral phospholipid in the LNP has a concentration of about 5% to 30%.

[0019] In some embodiments, the ionizable lipid has a concentration about 10 g / g to about 20 g / g mRNA. In some embodiments, the structural lipid comprises or is a sterol, and the sterol has a concentration about 3.0 g / g to 5.0 g / g mRNA. In some embodiments, the neutral phospholipid in the LNP has a concentration about 2.0 g / g to 5.0 g / g mRNA. In some embodiments, the PEG-lipid in the LNP has a concentration about 1.0 g / g to 1.5 g / g mRNA. In some embodiments, the [Lipid] - [optional linker] - [antibody] conjugate in the LNP has a concentration about 0.05 g / g to 0.1 g / g mRNA.

[0020] In some aspects, this disclosure provides a composition comprising a pharmaceutical system described herein.

[0021] In some aspects, this disclosure provides a kit comprising a pharmaceutical system described herein or a composition described herein.

[0022] In some aspects, this disclosure provides a method of delivering a payload to a subject in need thereof, comprising administering a pharmaceutical system described herein or a composition described herein to the subject. In some embodiments, the second particle comprises the payload.

[0023] In some aspects, this disclosure provides a method of treating a disease in a subject in need thereof, comprising administering a pharmaceutical system described herein or a composition described herein to the subject.

[0024] In some embodiments, the method comprises administering to the subject the first particle and the second particle separately. In some embodiments, the first particle and the second particle are administered sequentially. In some embodiments, the first particle and the second particle are administered to the subject simultaneously. In some embodiments, the administration is by systemic administration, optionally wherein the systemic administration is by intravenous injection or intravenous infusion.

[0025] In some aspects, this disclosure provides a use of a pharmaceutical system described herein, a composition described herein, or a kit described herein in the manufacture of a medicament for delivering a payload to a subject.

[0026] In some aspects, this disclosure provides a use of a pharmaceutical system described herein, a composition described herein, or a kit described herein in the manufacture of a medicament for treating a disease in a subject in need thereof.3#14739410v1

[0027] In some aspects, this disclosure provides a pharmaceutical system described herein, a composition described herein, or a kit described herein for use in delivering a payload to a subject.

[0028] In some aspects, this disclosure provides a pharmaceutical system described herein, a composition described herein, or a kit described herein for use in treating a disease in a subject in need thereof.

[0029] In some aspects, this disclosure provides a method of reducing immunogenicity of a lipid nanoparticle (LNP) in a subject in need thereof, the method comprising administering to the subject a pharmaceutical system described herein or a composition described herein.

[0030] In some aspects, this disclosure provides a method of reducing toxicity of a particle comprising polyethylene glycol (PEG), reducing anti -PEG antibody production in a subject caused by a lipid nanoparticle (LNP) comprising PEG or PEG-lipid, or eliminating anti-PEG antibody production in a subject caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof, the method comprising administering to the subject a pharmaceutical system described herein or a composition described herein.

[0031] In some aspects, this disclosure provides a use of a pharmaceutical system described herein, a composition described herein, or a kit described herein in the manufacture of a medicament for reducing toxicity of a particle comprising polyethylene glycol (PEG), reducing anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid, or eliminating anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof

[0032] In some aspects, this disclosure provides a pharmaceutical system described herein, a composition described herein, or a kit described herein

[0033] In some aspects, this disclosure provides a pharmaceutical system described herein, a composition described herein, or a kit described herein for use in reducing toxicity of a particle comprising polyethylene glycol (PEG), reducing anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid, or eliminating anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof.BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1A depicts the administration strategy. Doses were administered by intravenous tail vein injection once weekly for three weeks (three total doses). Blood collection was performed to determine lipid pharmacokinetics and the presence of anti-PEG antibodies. Blood4#14739410v1was collected at 15 minutes, 1 hour, 6 hours and 24 hours post dose. These timepoints were collected after both the 1stand the 3rddose.

[0035] FIG. IB depicts compositions comprising LNP alone or in combination with a liposome formulation that were administered to mice.

[0036] FIG.2A and FIG.2B show the concentration of anti-IgM antibodies in mice following a single dose and three doses of LNPs alone and LNP / liposome co-administration. FIG. 2A shows the concentration of anti-IgM antibodies 24 hours after a first dose. FIG. 2B shows the concentration of anti-IgM antibodies 24 hours after a third dose.

[0037] FIG.3A and FIG.3B show the concentration of anti-IgG antibodies in mice following a single dose and three doses of LNPs alone and LNP / liposome co-administration. FIG. 3A shows the concentration of anti-IgG antibodies 24 hours after a first dose. FIG. 3B shows the concentration of anti-IgG antibodies 24 hours after a third dose.

[0038] FIG.4A depicts Lipid 15 lipid plasma concentration in mice after a single dose of LNP alone or in combination with liposome. FIG. 4B depicts Lipid 15 lipid plasma concentration in mice after three doses of LNP alone or in combination with liposome.

[0039] FIG. 5A depicts Lipid 15 lipid plasma concentration in 7 mouse groups and a control group (no LNP) after a single dose of LNP alone or in combination with liposome. FIG. 5B depicts Lipid 15 lipid plasma concentration in 7 mouse groups and a control group (no LNP) after three doses of LNP alone or in combination with liposome.

[0040] FIG. 6 summarizes Lipid 15 lipid plasma concentration in mice that received a single dose of LNP alone or a single dose of LNP + liposome, or three doses of LNP alone LNP + liposome.

[0041] FIG. 7A depicts CAR expression in human donor blood cells after aCD8 targeted CD22 CAR mRNA LNPs were dosed to human venous whole blood with and without Liposomes, 24 hours after incubation following the administration, measured as CAR mean fluorescence intensity (MFI).

[0042] FIG.7B depicts CAR expression in human donor blood cells after aCD8 targeted CD22 CAR mRNA LNPs were dosed to human venous whole blood with and without Liposomes, measured as %CAR+. Data is arranged according to the level of pre-existing anti-PEG antibody levels of the donors.

[0043] FIG. 7C depicts CAR expression in human donor blood cells after aCD8 targeted CD22 CAR mRNA LNPs were dosed to human venous whole blood with and without Liposomes, measured as CAR MFI. Data is arranged according to the level of pre-existing anti-PEG antibody levels of the donors.5#14739410v1DETAILED DESCRIPTION

[0044] Provided herein is a novel pharmaceutical compositions for cargo delivery and method of using the same. The pharmaceutical compositions of the present disclosure can reduce multiple clearance mechanisms thus improving the activity, tolerability, specificity, and target engagement. In some embodiments, the novel strategy includes the use of two unique lipid-based nanoparticles in a single therapeutic product. In some embodiments, the two nanoparticles are either co-administered or administered consecutively to a subject in need. In some embodiments, the first particle and the second particle share at least one common element, and wherein both the first particle and the second particle bind to at least one opsonin; and the common element is capable of inducing an anti-drug antibody against the first particle and the second particle in a subject when the pharmaceutical system is administered to the subject. In some embodiments, the first particle plays as a decoy to reduce or deplete the antidrug antibody in the subject against the common element, enabling the second particle to implement pharmaceutical effect in the subject with reduced immunogenicity.

[0045] In some embodiments, the first particle and the second particle are lipid-based nanoparticles. In some embodiments, the first particle and the second particle are liposomes. In some embodiments, the first particle is a lipid-based nanoparticle and the second particle is a liposome. In some embodiments, the first particle is a liposomal composition. In some embodiments, the second particle is a lipid nanoparticle (LNP). In some embodiments, the first particle is a liposome and the second particle is a lipid-based nanoparticle. For example, in some embodiments, both the liposome and the LNP comprise a common element, such as a PEG-lipid. The LNP may contain a molecule comprising an antibody for targeted delivery to a certain tissue or cell type. In some embodiments, the LNP comprises a nucleic acid payload. The nucleic acid payload when delivered would lead to desired efficacy in the subject.

[0046] In some aspects, the dose of the liposomal composition is sufficiently high to tolerize and prevent a significant anti-drug immune response and / or does not significantly increase the clearance of a subsequent administration of LNPs at a later time or simultaneously (i.e. days or weeks later).Definitions

[0047] The term “and / or,” as used herein is a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended 6#14739410v1to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The use of the term “or” means “and / or” unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive.

[0048] The expression “at least one of,” as used herein, includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0049] The term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value and within a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). When the term “approximately” or “about” is applied herein to a particular value, the value without the term “approximately” or “about” is also disclosed herein. Further, although not always explicitly stated, all numerical designations may be preceded by the term “about.”

[0050] As described herein, any concentration range, percentage range, ratio range, or integer range includes the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The description of an interval of values should be considered as specifically disclosing all possible intermediate intervals as well as each of the values within this interval. For example, the description of an interval from 1 to 6 should be considered as specifically describing each of the intervals that it comprises, such as the intervals from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this interval, for example 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This definition is valid independently of the scope of the interval.

[0051] The terms “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” “comprise,” “comprises,” or “comprising,” including grammatical equivalents thereof, as used herein, should be understood generally as open-ended and nonlimiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0052] The term “pharmaceutical system,” as used herein, refers to a combination of two or more particles that share at least one common element. For example, the two or more particles can share at least one lipid, at least one polymer (e.g., a polyethylene glycol (PEG)), and / or at least one compound comprising a conjugate (e.g., a PEG-lipid).7#14739410v1

[0053] The term “particle,” as used herein, refers to a structure that is composed of various elements including elements of similar origin and elements of different origin. Particles include, but are not limited to, lipid-based particles and liposomes and can carry a payload or be free of a payload.

[0054] The terms “opsonin” or “opsonins,” as used herein refer to a protein that binds to a molecule and induces phagocytosis of the molecule by a phagocyte. Opsonins include, but are not limited to, complement, antibodies, and lipoproteins. Many proteins are known that act as opsonins for pathogens or other targets. (See e.g., Lockram, Tom O. J.; Dundee, Jacob M.; Popescu, Alma S.; Brown, Guy C. (2021). "The Phagocytic Code Regulating Phagocytosis of Mammalian Cells". Frontiers in Immunology. 12: 629979; PMC 8220072. PMID 34177884).

[0055] The term “phagocyte,” as used herein, refers to a cell capable of engulfing and consuming foreign material, such as bacteria and particles and non-foreign materials such as other cells. Phagocytes include, but are not limited to, myeloid cells including monocytes, macrophages, and neutrophils.

[0056] The term “opsonin receptor,” as used herein refers to a molecule that binds an opsonin. For example, an opsonin receptor is present on an immune cell, e.g., a phagocyte (e.g., a monocyte or macrophage), and when binding to an opsonin, e.g., an antibody or complement brings a molecule bound by the opsonin in close proximity to the phagocyte such that engulfment of the molecule by the phagocyte (phagocytosis) is induced.

[0057] The phrase “share at least one common element,” as used herein refers to two or more particles, e.g., two or more lipid particles that contain at least one element that is similar between the two or more particles. For example, a common element can be a polymer or a compound comprising a polymer.

[0058] The term “polymer,” as used herein refers to a material consisting of repeating structural subunits derived from one or more species of monomers. Polymers can be natural or synthetic and include, but are not limited to, polyethylene glycol (PEG), poly(2-methyl-2-oxazoline, polysarcosine, Zwitterionic Polymers (such as poly(carboxybetaine) and poly(sulfobetaine)), poly(amino acid) based lipopolymers, xtenylated polymers, poly(thioglycidyl glycerol), hydrophilic polymers (such as polysaccharides and polyvinylpyrrolidone (PVP)), etc., or a mixture thereof. The term “a compound comprising a polymer,” as used herein, refers, e.g., to a lipid comprising a polymer. Nonlimiting examples of such compounds are a PEG-lipid.

[0059] The phrase “capable of inducing an anti-drug antibody,” as used herein refers to the structural characteristics of a molecule or compound, e.g., a drug, which structural 8#14739410v1characteristics cause the molecule or compound, e.g., the drug, to be recognized by an immune cell of a subject to which the molecule or compound is given, where the immune cell induces a humoral immune response, e.g., the generation of antibodies that bind the molecule or compound, e.g., the drug, in the subject.

[0060] The term “anti-drug antibody,” as used herein refers to an antibody that specifically binds to a drug molecule or drug compound. For example, anti-drug antibodies are observed in subjects administered a drug molecule or drug compound, and can be part of an immune reaction to a drug molecule or drug compound. In some instances, anti-drug antibodies can prevent a re-administration of the drug molecule or drug compound to the subject.

[0061] The term “specifically binds,” as used herein, refers to an affinity ligand, in particular, an antibody, that recognizes and binds a specific antigen, but does not substantially recognize or bind other molecules in a sample. In some aspects, the terms “specific binding” or “specifically binding,” refers to an interaction of an antibody, a protein, or a peptide with a second protein or peptide where the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second protein or peptide. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. In some aspects, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0062] The term “liposome,” as used herein, refers to a lipid vesicle composed of one or more lipid bilayers. In some aspects, a liposome is a spherical vesicle. In some aspects, a liposome has a mean diameter of about 500 nm to about 50 nm; or about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, about 100 nm or less, or about 50 nm or less. In some aspects, a liposome has a diameter of about 200 nm to about 50 nm. In some aspects, a liposome has a diameter of about 80 nm. Liposomes can have a unilamellar structure and form small and large unilamellar vesicles. Liposomes can also have an onion-like multilamellar structure and form multilamellar vesicles and multivesicular vesicles. In some embodiments, liposomes of the present disclosure are mostly composed of phospholipids (e.g., phosphatidylcholine) and sterol (e.g., cholesterol). In 9#14739410v1some embodiments, liposomes of the present disclosure do not contain cholesterol or other sterols. In some embodiments, liposomes may also include other lipids as long as they are compatible with lipid bilayer structure. In some embodiments, liposomes of the present disclosure do not include an ionizable lipid. In some embodiments, liposomes of the present disclosure may contain surface ligands for attaching to desired cells or tissues. In some embodiments, liposomes may be selected from multilamellar large vesicle (MLV), oligolamellar vesicle (OLV), small unilamellar vesicle (SUV), medium-sized unilamellar vesicle (MUV), large unilamellar vesicle (LUV), giant unilamellar vesicle (GUV) and multivesicular vesicles (MW). In some aspects, the size of the liposome is selected based on the tissue or cell types being targeted.

[0063] The term “lipid-based nanoparticle,” “lipid nanoparticle,” or “LNP,” as used interchangeably herein, refers to a nanoparticle comprising a single layer of one or more types of lipids (e.g., a lipid blend comprising one or more types of lipids, for example, selected from one or more ionizable lipids, helper lipids, PEG-lipids, and / or structural lipids). In some embodiments, a lipid-based nanoparticle includes an ionizable lipid. In some aspects, the lipid-based nanoparticle has a mean diameter of about 400 nm to about 50 nm; or about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, about 100 nm or less, or about 50 nm or less. In some aspects, the lipid-based nanoparticle has a diameter of about 200 nm to about 50 nm. In some aspects, the lipid-based nanoparticle has a diameter of 80 nm. In some aspects, the size of the LNP is selected based on the tissue or cell types being targeted.

[0064] The term “total lipids,” as used herein, refers to the collection of lipids present in a lipid blend of a LNP and / or a liposome including ionizable lipids, structural lipids, helper lipids, and PEG-lipids.

[0065] The term “ionizable lipid,” as used herein, refers to a lipid that is capable of modulating its charge depending on the environment it is present in. Ionizable lipids include cationic lipids. Ionizable lipids also include, but are not limited to, unsaturated ionizable lipids, multi-tail ionizable lipids, polymeric ionizable lipids, biodegradable ionizable lipids, and branched-tail ionizable lipids. In some aspects, an ionizable lipid includes a lipid of Formula I (Lipid 15), a lipid of Formula II, Formula III (KC2), Formula IV (KC3). Other ionizable lipids can be used in the LNPs and / or liposomes described herein.

[0066] The terms “helper lipid,” “neutral lipid,” “phospholipid,” or “neutral phospholipid,” as used herein, refer to a non-polar lipid of a lipid-based nanoparticle which provides the lipid-10#14739410v1based nanoparticle with stability, blood compatibility, and, if the lipid-based nanoparticle carries a payload, enhances payload delivery.

[0067] The term “PEG-lipid,” “lipid-PEG,” or “PEGylated lipid” are used herein interchangeably and refer to one or more lipids that are modified with polyethylene glycol. As described herein, PEG-lipids can be free PEG-lipids or PEG-lipids can be part of a lipid-cell targeting conjugate. Any suitable chemistry may be used to conjugate a polypeptide to the PEG of the PEG-lipid, see Parhiz et al., Journal of Controlled Release 291:106-115, 2018; Kolb et al., Angewandte Chemie International Edition 40(11):2004-2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007. For example, lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, PEG-dibenzocyclooctyne (DBCO), lipid-PEG-bromo maleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid-PEG-azide can be used to produce a PEG-lipid-polypeptide conjugate.

[0068] The term “free PEG-lipid,” as used herein, refers to a PEG-lipid that is not part of a cell targeting group conjugate. For example, a free PEG-lipid can be part of a lipid blend of a LNP and / or liposome.

[0069] The term “structural lipid,” as used herein, refers to sterols and also to lipids containing sterol moieties.

[0070] The term “mol%” or “mol percent,” as used herein, refers to the mol fraction of a mixture multiplied by 100; where the mol fraction is the number of moles of one ingredient in a mixture divided by the total number of moles in the mixture.

[0071] The term “cell targeting group conjugate,” as used herein, refers to a conjugate of the general formula: [Lipid] - [optional linker] - [cell targeting group], wherein the lipid can be any lipid described herein and the cell targeting group can be any cell surface molecule-binding group described herein. A cell targeting group includes an antibody or fragment thereof, including a Fab, a scFv, an immunoglobulin single variable domain including but not limited to a VHH domain, humanized VHH domain, or camelized VH domain. A lipid of a cell targeting group conjugate preferably is a PEG-lipid.

[0072] The term “reference liposome” as used herein refers to a liposome that has a different lipid component or does not have one lipid component but is otherwise the same as a tested liposome. In some aspects, a reference liposome is a liposome that has a different phospholipid but is otherwise the same as a tested liposome.

[0073] The term “reference LNP” as used herein refers to an LNP that does not have, e.g., a cell targeting group but is otherwise the same as the tested LNP. In some aspects, a reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise the same as the tested LNP.11#14739410v1

[0074] The term “linker” or “spacer,” as used herein, denotes a molecule that fuses together a lipid and a polypeptides, e.g., antibody. In some aspects, the linker is a peptide. For example, a class of peptide linkers are known as the “Gly-Ser” or “GS” linkers and essentially consist of glycine (G) and serine (S) residues. A linker can also be an alanine linker.

[0075] The term “coupled,” as used herein refers to a covalent interaction between two molecules. Therefore, “coupled” includes “covalently coupled.”

[0076] The terms “peptide,” “polypeptide,” and “protein” are used herein interchangeably and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, and to longer chains, which generally are referred to in the art as proteins. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0077] The term “antibody,” as used herein, refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. It is understood that the term encompasses an intact antibody, antigen-binding fragment thereof, or an Fc fragment that optionally has been modified or engineered. Examples of antigen binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, immunoglobulin single variable domains (ISVDs), e.g., ISVD, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multi-specific antibodies (e.g., bispecific antibodies). The term also encompasses an immunoglobulin single variable domain, such as an ISVD (e.g., a variable heavy chain domain, VHH).

[0078] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full length light chain (typically having a molecular weight of about 25 kDa) and one full length heavy chain (typically having a molecular weight of about 50-70 kDa). The terms “heavy chain” and “light chain,” as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino- 12#14739410v1terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus. Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgGhas several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety herein. The variable regions of each light / heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0079] The term “CDR set,” as used herein, refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. In some aspects, an immunoglobulin single variable domain comprises three CDRs. The exact boundaries of CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs.13#14739410v1Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2, and L3 or Hl, H2, and H3 where the “L” and the “H” designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the mentioned systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain aspects use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A.C. Protein sequence and structure analysis of antibody variable domains, In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer-Verlag, Berlin, p. 33-51, 2010. The amino acid sequence of the heavy and / or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, Nucleic Acids Res. 22: 4673-80, 1994. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.

[0080] The term “Fc,” as used herein, refers to a fragment crystallizable region, that is a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl and IgG2 are preferred. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3,14#14739410v1IgAl, and IgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0081] The term “Fab,” as used herein, refers to a F(ab) fragment of an antibody and typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CHI domain. In some aspects, a Fab is engineered to replace one or both cysteine on the native constant light and the native constant heavy chain that form the native interchain disulfide with a non-cysteine amino acid to remove the native interchain disulfide bond in the Fab.

[0082] The term “F(ab’)2 fragment” typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.

[0083] The term “immunoglobulin single variable domain” or “ISVD,” as used herein, refers to an immunoglobulin domain capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain. Therefore, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. The single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (e.g., a functional antigen binding unit that essentially consists of the CDRs, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). For example, an immunoglobulin single variable domain can be a heavy chain immunoglobulin single variable domain, such as a VH, VHH, including a camelized VH or humanized VHH or a domain antibody (“dAb”) or an amino acid sequence that is suitable for use as a dAb. In some aspects, an immunoglobulin single variable domain is a VHH, including a camelized VH or humanized VHH. In some aspects, single variable domains are derived from certain species of shark (for example, the so-called “IgNAR domains.” In some aspects, an immunoglobulin single variable domain is an ISVD. For example, an ISVD can be obtained (1) by isolating the 15#14739410v1VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human, or by expression of a nucleic acid encoding a camelized VH domain; (5) by “camelization” of a “domain antibody” or “dAb” or by expression of a nucleic acid encoding a camelized VH domain; (6) using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding an ISVD using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of the foregoing.

[0084] The term “scFv,” as used herein, refers to a single chain antibody comprising a heavy chain variable domain and a light chain variable domain linked by a linker.

[0085] The term “humanized antibody,” as used herein, refers to an antibody which is wholly or partially of non-human origin and whose protein sequence has been modified to replace certain amino acids, for instance that occur at the corresponding position(s) in the framework regions of the VH and VL domains in a sequence of an antibody from a human being, to increase its similarity to antibodies produced naturally in humans, in order to avoid or minimize an immune response in humans. For example, using techniques of genetic engineering, the variable domains of a non-human antibodies of interest may be combined with the constant domains of human antibodies. In some aspects, the constant domains of a humanized antibody are human CH and CL domains.

[0086] The term “humanized VHH,” as used herein, refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional four-chain antibody from a human.

[0087] The term “camelized VH,” as used herein, refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized,” i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a (camelid) heavy chain antibody. In some aspects, such “camelizing” substitutions are inserted 16#14739410v1at amino acid positions that form and / or are present at the VH-VL interface. In some aspects, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human, such as, e.g., a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner and is not strictly limited to a polypeptide that has been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material. For example, cam elid immunoglobulin sequences and humanized cam elid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAb can be used herein. In some aspects, immunoglobulin single variable domains are fused forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains).

[0088] The term “multivalent,” as used herein, refers to the presence of multiple immunoglobulin single variable domains in a polypeptide. In some aspects, the polypeptide is “bivalent,” i.e., comprises or consists of two immunoglobulin single variable domains. In some aspects, the polypeptide is “trivalent,” i.e., comprises or consists of three immunoglobulin single variable domains. In some aspects, the polypeptide is “tetravalent,” i.e. comprises or consists of four immunoglobulin single variable domains. The polypeptide can thus be “bivalent,” “trivalent,” “tetravalent,” “pentavalent,” “hexavalent,” “heptavalent,” “octavalent,” “nonavalent,” etc., i.e., the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine, etc., immunoglobulin single variable domains, respectively. In some aspects, a multivalent immunoglobulin single variable domain polypeptide is trivalent. In some aspects, a multivalent immunoglobulin single variable domain polypeptide is tetravalent. In some aspects, the multivalent immunoglobulin single variable domain polypeptide is pentavalent. In some aspects, a multivalent immunoglobulin single variable domain polypeptide can also be multispecific.

[0089] The term “multispecific,” as used herein, refers to binding to multiple different target molecules (also referred to as antigens). In some aspects, a multivalent immunoglobulin single variable domain polypeptide is “bispecific,” “trispecific,” “tetraspecific,” etc., i.e., can bind to two, three, four, etc., different target molecules, respectively. For example, a polypeptide may be bispecific-trivalent, such as a polypeptide comprises or consists of three immunoglobulin single variable domains, wherein two immunoglobulin single variable domains bind to a first target and one immunoglobulin single variable domain binds to a second target different from the first target. In some aspects, a polypeptide is trispecific-tetravalent, such as a polypeptide comprises or consists of four immunoglobulin single variable domains,17#14739410v1wherein, e.g., one immunoglobulin single variable domain binds to a first target, two immunoglobulin single variable domains bind to a second target different from the first target and one immunoglobulin single variable domain binds to a third target different from the first and the second target. In some aspects, a polypeptide is trispecific-pentavalent, such as a polypeptide comprises or consists of five immunoglobulin single variable domains, wherein, e.g., two immunoglobulin single variable domains bind to a first target, two immunoglobulin single variable domains bind to a second target different from the first target and one immunoglobulin single variable domain binds to a third target different from the first and the second target. In some aspects, a multispecific immunoglobulin polypeptide is multiparatopic.

[0090] The term “multiparatopic,” as used herein, refers to binding to multiple different epitopes on the same target molecules (also referred to as antigens). In some aspects, a multivalent immunoglobulin single variable domain polypeptide can thus be “biparatopic,” “triparatopic,” etc., i.e., can bind to two, three, etc., different epitopes on the same target molecules, respectively.

[0091] The term “alkyl,” as used, herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 atoms, referred to herein as C1-C12 alkyl, C1-C10 alkyl, or Ci-Ce alkyl, respectively. In some aspects, alkyl is substituted. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-m ethyl- 1 -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3 -methyl- 1 -butyl, 2-methyl-3 -butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1 -pentyl, 4-methyl-l -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-l-butyl, 2-ethyl-1 -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc. In some aspects, an alkyl is a methyl.

[0092] The term “alkenyl,” as used herein, refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8, or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The group may be in either the cis or trans configuration (Z or E configuration, respectively) about the double bond(s). Alkenyl groups include, but are not limited to, ethenyl, propenyl (e.g., prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl), and butenyl (e.g., but-l-en-l-yl, but-l-en-2-yl, 2-methyl-prop-1-en-l-yl, but-2-en-l-yl, but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl) etc.

[0093] The terms “amine” and “amino,” as used herein, refer to both unsubstituted and substituted amines, e.g., a moiety represented by the general formula -N(R3)(R3B), wherein R3represents a bond, hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, or aryl. In some aspects,18#14739410v1R3taken together with the N atom to which it is attached completes a heterocycle having from4 to 8 atoms in the ring structure. In some aspects, R3Brepresent. In some aspects, R3B1is Ci-6 alkylene. In some aspects, R3B2and R3B3are each independently H, unsubstituted Ci-6 alkyl, or Ci-6 alkyl substituted with 1 or 2 -OH. In some aspects, R3B1is Ci-6 alkylene and R3B2and R3B3are each independently H, unsubstituted Ci-6 alkyl, or Ci-6 alkyl substituted with 1 or 2 -OH. In some aspects, R3B2and R3B3are each methyl. In some aspects, R3B1is -(CH2)3-.

[0094] The terms “alkoxyl” or “alkoxy,” as used herein, refer to an alkyl group, as defined above, having an oxygen radical attached thereto. In some aspects, alkoxyl is optionally substituted. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders the alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2)m-R12, where m and R12are described above. The term “haloalkoxyl” refers to an alkoxyl group that is substituted with at least one halogen. For example, -O-CH2F, -O-CHF2, -O-CF3, and the like. In some aspects, the haloalkoxyl is an alkoxyl group that is substituted with at least one fluoro group. In some aspects, the haloalkoxyl is an alkoxyl group that is substituted with from 1-6, 1-5, 1-4, 2-4, or 3 fluoro groups.

[0095] The term “oxo,” as used herein, refers to a “=O” substituent. For example, a cyclopentane substituted with an oxo group is cyclopentanone.

[0096] The term “substituted”, whether preceded by the term “optionally” or not, as used herein, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group. In some aspects, when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. In some aspects, combinations of substituents are those that result in the formation of stable or chemically feasible compounds. In some aspects, “optionally substituted” is equivalent to “unsubstituted or substituted.” In some aspects, “optionally substituted” indicates that the designated atom or group is optionally substituted with one or more substituents independently selected from optional substituents provided herein.19#14739410v1

[0097] The term “pseudouridine,” as used herein, refers to the natural product which is a C-glycosyl pyrimidine that consists of uracil having a beta-D-ribofuranosyl residue attached at position 5 (i.e., 5-(beta-D-Ribofuranosyl)uracil). In some aspects, the term refers to 1- methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine. In some aspects, the term refers to 1-methylpseudouridine. In some aspects, the term refer’ to 2'-O- methylpseudouridine. In some aspects, the term refers to 5- methyldihydrouridine. In some aspects, the term refers to 3-methylpseudouridine. In some aspects, the term refers to a pseudouridine moiety that is not further modified. In some aspects, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In some aspects, the term refers to any other pseudouridine known in the art.

[0098] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” as used herein refers to all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. In some aspects, a composition comprises various stereoisomers of a compound and / or mixture of compounds described herein. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Individual stereoisomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Further, enantiomers can be separated using supercritical fluid chromatographic (SFC) techniques described in the literature. Still further, stereoisomers can be obtained from stereomerically-pure intermediates, reagents, and catalysts by well- 20#14739410v1known asymmetric synthetic methods. Geometric isomers can also exist in the compounds of the present invention.

[0099] The symbol “ ” denotes a bond that may be a single, double or triple bond as described herein. The present invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carboncarbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E’ are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis / trans.”

[0100] As is known to those of skill in the art, “salts” of the compounds described herein may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0101] Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWU, wherein W is Ci-4 alkyl, and the like.

[0102] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the 21#14739410v1like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NWf (wherein W is a Ci-4 alkyl group), and the like.

[0103] The terms “carrier” or “excipient,” as used herein, refer to a diluent, adjuvant, or vehicle with which a particle, lipid nanoparticle, viral vector particle and / or a liposome is comprised or administered to a subject. In some aspects, a carrier is a pharmaceutical carrier. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases), and solutions or compositions thereof. Other examples of carriers or excipients include phosphate buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. Other examples of carriers or excipients that might be used include saline (e.g., sterilized, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly useful for delivery of a particle, lipid nanoparticle, viral vector particle and / or liposome to human subjects.

[0104] The term “pharmaceutically acceptable carrier,” as used herein, refers to any of the standard pharmaceutical excipients, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents.

[0105] The term “specificity of the targeted delivery” by an LNP is defined by the ratio between % of a desired cell type that receives the delivered nucleic acid (e.g., on-target delivery), and % of an undesired cell type that is not meant to be the destination of the delivery,22#14739410v1but receives the delivered nucleic acid (e.g., off-target delivery). For example, the specificity is higher when more desired cells receive the delivered nucleic acid, while less undesired cells receive the delivered nucleic acid. Specificity of the targeted delivery by an LNP can also be defined by the ratio of amount of nucleic acid being delivered to the desired cells (e.g., on-target delivery) and amount of nucleic acid being delivered to the undesired cells (e.g., off-target delivery). Specificity of the delivery can be determined using any suitable method. As a non-limiting example, expression level of the nucleic acid in the desired cell type can be measured and compared to that of a different cell type that is not meant to be the destination of the delivery.

[0106] The terms “subject” and “patient,” as used herein, refer to organisms that are preferably mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably humans.

[0107] The term “pharmaceutical composition,” as used herein, refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0108] The term “effective amount,” as used herein, refers to the amount of a compound (e.g., a nucleic acid) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. The term effective amount can be considered to include therapeutically and / or prophylactically effective amounts of a compound.

[0109] The phrase “therapeutically effective amount,” as used herein, refers to an amount of a lipid nanoparticle comprising, e.g., a nucleic acid or a composition comprising a lipid nanoparticle, which is effective for producing a desired therapeutic effect in at least a subpopulation of cells in a mammal, for example, a human, or a subject (e.g., a human subject) at a reasonable benefit / risk ratio applicable to any medical treatment.

[0110] The phrase “prophylactically effective amount,” as used herein, refers to an amount of a lipid nanoparticle comprising, e.g., a nucleic acid or composition comprising a lipid nanoparticle, which is effective for producing a desired prophylactic effect in at least a subpopulation of cells in a mammal, for example, a human, or a subject (e.g., a human subject) by reducing, minimizing or eliminating the risk of developing a condition or by reducing or minimizing severity of a condition at a reasonable benefit / risk ratio applicable to any medical treatment.23#14739410v1

[0111] The terms “treat,” “treating,” and “treatment” include any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of a condition, disease, disorder, and the like, or ameliorating a symptom thereof.PHARMACEUTICAL SYSTEM

[0112] Lipid nanoparticles (LNPs) are cleared from the circulation through a variety of mechanisms. One of those is through a process of opsonization where serum proteins bind the surface of the nanoparticle, and are in turn recognized by receptors on the surface of myeloid cells, like liver or spleen macrophages that make up part of the Mononuclear Phagocyte System (MPS). The most well-known members of the MPS are proteins of the complement family, such as C3a. LNPs may also be recognized due to their surface charge by oppositely charged glycans on the surface of endothelial cells, such as the negatively charged proteoglycans on the surface of the lung endothelium that bind to positively charged LNPs imparted by cationic lipids present in the formulation. LNPs may also bind other proteins in the circulation, such as ApoE that subsequently causes uptake by cells expressing receptors for lipoproteins, either endothelial cells or liver hepatocytes. Finally, anti-drug antibodies can be induced against components on the surface of the LNP, including antibodies or other targeting ligands, immunogenic linkers or aromatic residues, or most notably poly(ethylene glycol) or PEG. These immune responses can result in rapid clearance of the LNPs upon subsequent administration of the LNPs and can even cause rapid clearance of an initial injection if preexisting anti-PEG antibodies are present or will be induced in the blood when LNPs are administered to a subject.

[0113] The prevalence of anti-PEG antibodies in the general population is estimated to be between roughly 20-30%, with a slightly elevated presence in females compared to males, presumably due to their use of pegylated cosmetics and thus increased exposure to the molecule (Chen et al. 2021 ACS Nano 15, 14022-14048). The induction of anti-PEG antibodies can be influenced by the dose of pegylated nanoparticle being administered and the schedule of administration. Lower concentrations and more frequent dosing are consistent with higher levels of anti-PEG IgG and IgM responses and increased clearance of subsequent administrations. The inclusion of immunogenic nucleic acid sequences, such as those that activate toll receptor (TLR) responses, can act as an adjuvant and increase the anti-PEG response against the nanoparticle. The response can also be impacted by the stability of association of the PEGylated lipids. Many LNPs employ short chained PEG-diacylglycerols, such as dimyristoylglycerol-PEG2000 (DMG-PEG) where the pegylated lipid is rapidly shed 24#14739410v1upon entering the circulation to increase the transfection potential of the LNP and the LNP’s ability to engage its target cell population when dependent on the natural tropism of the LNP. The rapid shedding of the pegylated lipid significantly reduces the anti-PEG immune response, and thus allows for repeated dosing. Unfortunately, this rapid shedding also leads to increased recognition of the LNP through the opsonization process and increased clearance of the LNP via one of the alternative clearance mechanisms described above. More stable anchors, such as di stearoylglycerol or dipalmitoylglycerol disassociate more slowly from the LNP, allowing for slower clearance from the circulation and more time to engage their target. The use of more stable anchors combined with attachment of targeting moieties to the surface of the LNP allows for increased specificity of the LNPs for transfection of the target cell of interest, but at the increased risk of developing anti-PEG immune responses and rapid clearance upon repeat administration, negating one of the important advantages of LNPs over viral vector delivery which is consistently plagued by immunogenicity concerns.

[0114] The present disclosure provides a novel solution to address the issue associated with pharmaceutical compositions comprising particles that could be rapidly cleared in a subject receiving the pharmaceutical compositions.

[0115] Provided herein are pharmaceutical systems that comprises at least two particles, wherein the first particle is used as a decoy to reduce rapid clearance of the second particle comprising a pharmaceutically active component when such a pharmaceutical system is administered to a subject.

[0116] In order to achieve the desired result, the first particle can reduce rapid clearance of the second particle through one or more mechanisms: (1) the first particle can play as a decoy to reduce clearance of the second particle through clogging process of opsonization. For example, the process may involve either or both of the adaptive immune system and the innate immune system. For the adaptive immune system, the first particle may play a decoy to reduce clearance of the second particle through depleting anti-drug antibodies that would bind to the second particle, wherein such anti-drug antibodies are induced or already exist in the subject receiving the pharmaceutical systems. For the innate immune system, the first particle may play a decoy to reduce clearance of the second particle through depleting components including but not limited to complement proteins (e.g., C4b, C3b and iC3b), alternative pathway of complement activation (e.g., C3b that is deposited directly on to antigens with particular PAMPs), or other components (e.g., mannose-binding lectins, or ficolins, along with pentraxins and collectins) that would bind to the second particle; (2) the first particle may play as a decoy to reduce clearance of the second particle through occupying cell surface molecules that can recognize 25#14739410v1the second particle because such surface molecules are oppositely charged compared to the surface charge of the second particle; against components on the surface of the second particle, or already pre-existing in the subject. At least 50 proteins are known to act as opsonins for pathogens or other targets. [Cockram, Tom O. J.; Dundee, Jacob M.; Popescu, Alma S.; Brown, Guy C. (2021). "The Phagocytic Code Regulating Phagocytosis of Mammalian Cells". Frontiers in Immunology. 12: 629979. doi:10.3389 / fimmu.2021.629979. PMC 8220072. PMID 34177884.].Decoys clogging process of innate opsonization

[0117] In some aspects, the two or more particles of the pharmaceutical system bind to at least one opsonin. In some aspects, the two or more particles of the pharmaceutical system bind to a same opsonin. In some aspects, the two or more particles of the pharmaceutical system bind to different opsonins that share a binding affinity for a common element present in the two or more particles.

[0118] In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to an opsonin, wherein the opsonin is recognized by a receptor on the surface of myeloid cells, such as liver or spleen macrophages that make up part of Mononuclear Phagocyte System (MPS). In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to a same opsonin. In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to different opsonins, wherein the different opsonins are capable of being recognized through the same or different receptors on the surface of myeloid cells. In some aspects, the different opsonins share a binding affinity for the common element present in the two or more particles of the pharmaceutical system.

[0119] In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to an opsonin that is a member of the complement family. In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to more than one opsonin, wherein the more than one opsonin is a member of the complement family. In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to an opsonin that is complement C3a.

[0120] In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to an opsonin that is a lipoprotein. In some aspects, the two or more particles of the pharmaceutical system comprise a common element that binds to an opsonin26#14739410v1that is Apolipoprotein E (Apo E), which can be subsequently recognized by cells expressing receptors for lipoproteins, such as endothelial cells or liver hepatocytes.Decoys binding to anti-drug antibodies (clogging process of adaptive opsonization)

[0121] Antibodies are synthesized by B cells and are secreted in response to recognition of specific antigenic epitopes, and bind only to specific epitopes (regions) on an antigen. They comprise the adaptive opsonization pathway. Accordingly, when the first particle and the second particle comprise a same element, the first particle may play a decoy to reduce clearance of the second particle also through depleting anti-drug antibodies that would bind to the second particle, wherein such anti-drug antibodies are induced against the same element on the surface of the first particle and the second particle, or already pre-existing in the subject.

[0122] In some aspects, pharmaceutical systems comprise at least two particles that share at least one common element. In some aspects, the pharmaceutical systems comprise two particles that share at least one common element. In some aspects, the pharmaceutical systems comprise two or more particles that share at least one common element. In some aspects, the two or more particles of the pharmaceutical system comprise a common element that can be recognized by an antibody in the subject binding to the element - in such a situation, the antibody in the subject would be an anti-drug antibody against the second particle, and the first particle when presented in sufficient amount would bind to the anti-drug antibody, thus reducing the clearance of the second particle in the subject. In some aspects, the anti-drug antibody that binds the common element of the two or more particles of the pharmaceutical system is an IgG antibody. In some aspects, the anti-drug antibody that binds the common element of the two or more particles of the pharmaceutical system is an IgM antibody. In some aspects, the IgM antibody and the IgG antibody share a binding affinity for a common element present in the two or more particles of the pharmaceutical system.Decoys occupying oppositely charged cell surface molecules

[0123] In some aspects, the first particle may play as a decoy to reduce clearance of the second particle through occupying cell surface molecules that can recognize the second particle because such surface molecules are oppositely charged compared to the surface charge of the first and second particle.

[0124] For example, when the second particle is positively charged due to one or more elements forming the particle, such as a cationic ionizable lipid in an LNP, the first particle can also be designed to be positively charged through formulation. Or, when the second particle is 27#14739410v1negatively charged due to one or more elements forming the particle, the first particle can also be designed to be negatively charged through formulation. Accordingly, when the first particle is administered to a subject in need ahead of time before the second particle is administered to the subject, or when the first particle and the second particle are administered to the subject in need at the same time, the first particle, when presented in sufficient amount, would occupy the cell surface molecules that are oppositely charged compared to the surface charge of the first and the second particle.Formulation of the pharmaceutical system

[0125] Different formulations are made in view of the general concept as explained above.I. Basic Compositions

[0126] In some aspects, when the first particle of the pharmaceutical system is used as a decoy to reduce clearance of the second particle, the first particle does not comprise a payload, while the second particle comprises a payload. In some aspects, the payload in the second particle when delivered into a subject is able to induce preferred biologic activity, including but not limited to, gene expression, gene editing, gene silencing, transcriptional regulation, post-transcriptional regulation, etc.

[0127] In some aspects, the first particle and the second particle of the pharmaceutical system comprise the same set of elements, except for that the first particle does not comprise any payload while the second particle comprises a payload.

[0128] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition. In some aspects, a pharmaceutical system comprises at least two particles that do not differ in their particle composition.

[0129] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise at least one common element. In some aspects, the common element comprises or is a common lipid. In some aspects, the common element comprises or is a common polymer. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0130] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element that is a polymer, wherein the polymer comprises or is PEG. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.28#14739410v1

[0131] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a PEG-lipid. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0132] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a positively charged PEG-lipid. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0133] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a negatively charged PEG-lipid. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0134] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a neutral PEG-lipid. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0135] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a PEG-lipid selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols, a derivative thereof, or any combination thereof. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0136] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is a PEG-lipid selected from a PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero- 29#14739410v1phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid, PEG-l,2-distearoyl-sn-glycero-3-phosphocholine (PEG-DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),PEG-dilinoleoyl-glycero-phosphocholine (PEG-DLPC), PEG-dimyristoyl-glycero-phosphocholine (PEG-DMPC), PEG-dipalmitoyl-glycero-phosphocholine (PEG-DPPC), PEG-diundecanoyl-glycero-phosphocholine (PEG-DUPC), PEG-palmitoyl-oleoyl-glycero-phosphocholine (PEG-POPC), PEG-ceramide, PEG-2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG-dioctadecenyl-glycero-phosphocholine, PEG-oleoyl-cholesterylhemisuccinoyl-glycero-phosphocholine, PEG-hexadecyl-glycero-phosphocholine, PEG-dilinolenoyl-glycero-phosphocholine, PEG-diarachidonoyl-glycero-3-phosphocholine, PEG-didocosahexaenoyl-glycero-phosphocholine, PEG-sphingomyelin or a derivative thereof, or any combination thereof. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0137] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of particles comprises a PEG-lipid selected from a PEG- dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid, PEG-l,2-distearoyl-sn-glycero-3-phosphocholine (PEG-DSPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),PEG-dilinoleoyl-glycero-phosphocholine (PEG-DLPC), PEG-dimyristoyl-glycero-phosphocholine (PEG-DMPC), PEG-dipalmitoyl-glycero-phosphocholine (PEG-DPPC), PEG-diundecanoyl-glycero-phosphocholine (PEG-DUPC), PEG-palmitoyl-oleoyl-glycero-phosphocholine (PEG-POPC), PEG-ceramide, PEG-2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, PEG-dioctadecenyl-glycero-phosphocholine, PEG-oleoyl-cholesterylhemisuccinoyl-glycero-phosphocholine, PEG-hexadecyl-glycero-phosphocholine, PEG-dilinolenoyl-glycero-phosphocholine, PEG-diarachidonoyl-glycero-3-phosphocholine, PEG-didocosahexaenoyl-glycero-phosphocholine,30#14739410v1PEG-sphingomyelin or a derivative thereof, or any combination thereof. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0138] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprising or is PEG-DSPE. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0139] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the common element is a part of two different chemical compounds, each of which is a component of the first and the second particle; or the common element is a part of, or is the same chemical compound that both the first particle and the second particle comprise. For example, the common element can be polyethylene glycol (PEG), so in some aspects, each of the first particle and the second particle comprises a chemical compound that comprises PEG, wherein the compound of the first particle and the compound of the second particle may be the same or different but at least share the PEG moiety. For another example, the first particle may comprises a chemical compound comprising PEG or PEG-lipid, while the second particle In some aspects, the PEG lipid in the first particle is present at a range of about 0.01 mol% to about 30 mol%, for example, about 30 mol%, about 29 mol%, about 28 mol%, about 27 mol%, about 26 mol%, about 25 mol%, 24 mol%, about 23 mol%, about 22 mol%, about 21 mol%, 20 mol%, about 19 mol%, about 18 mol%, about 17 mol%, about 16 mol%, about 15 mol%, about 14 mol%, about 13 mol%, about 12 mol%, about 11 mol%, about 10 mol%, about 9 mol%, about 8 mol%, about 7 mol%, about 6 mol%, about 5 mol%, about 4 mol%, about 3 mol%, about 2 mol%, about 1 mol%, about 0.9 mol%, about 0.8 mol%, about 0.7 mol%, about 0.6 mol%, about 0.5 mol%, about 0.4 mol%, about 0.3 mol%, about 0.2 mol%, about 0.1 mol%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload. In some aspects, the PEG lipid in the first particle and the PEG lipid in the second particle are the same or different. For example, the PEG lipid in the first particle comprises or is DSPG-PEG-DSPE, and the PEG lipid in the second particle comprises or is also DSPE-PEG, or the PEG lipid in the first particle comprises or is DSPE-PEG, and the PEG lipid in the second particle comprises or is also DPG-PEG. In some aspects, the first particle may contain a mixture of two or more different PEG lipids. For example, in some aspects, the first particle may contain a mixture of two different PEG lipids, such as DSPE-PEG and DSPC-PEG, while the second particle may contain the 31#14739410v1same or different PEG lipid or PEG lipid mixture. For example, the second particle may contain DPG-PEG. In some other aspects, the first particle may contain a mixture of two different PEG lipids, such as DSPE-PEG and DOPC-PEG, while the second particle may contain the same or different PEG lipid or PEG lipid mixture. For example, the second particle may contain DPG-PEG.

[0140] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles comprises a PEG-lipid, wherein the PEG lipid is present at a range of about 0.1 mol% to about 30 mol%, for example, about 30 mol%, about 29 mol%, about 28 mol%, about 27 mol%, about 26 mol%, about 25 mol%, 24 mol%, about 23 mol%, about 22 mol%, about 21 mol%, 20 mol%, about 19 mol%, about 18 mol%, about 17 mol%, about 16 mol%, about 15 mol%, about 14 mol%, about 13 mol%, about 12 mol%, about 11 mol%, about 10 mol%, about 9 mol%, about 8 mol%, about 7 mol%, about 6 mol%, about 5 mol%, about 4 mol%, about 3 mol%, about 2 mol%, about 1 mol%, about 0.9 mol%, about 0.8 mol%, about 0.7 mol%, about 0.6 mol%, about 0.5 mol%, about 0.4 mol%, about 0.3 mol%, about 0.2 mol%, about 0.1 mol%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02%, about 0.01%. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.

[0141] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least two particles are lipid-based nanoparticles. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle share at least one common element, and wherein the first particle and the second particle are lipid-based nanoparticles. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least two particles are liposomes. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle share at least one common element, and wherein the first particle and the second particle are liposomes. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a lipid-based nanoparticle and wherein at least one of the at least two particles is a liposome.32#14739410v1In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle share at least one common element, and wherein the first particle is a lipid-based nanoparticle and the second particle is a liposome. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle share at least one common element, and wherein the first particle is a liposome and the second particle is a lipid-based nanoparticle.

[0142] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles does not comprise a structural lipid, e.g., a sterol. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload. For example, in some aspects, the first particle as the decoy is a liposome, a micelle, an emulsion, or a mixture thereof and the second particle for payload delivery is a lipid nanoparticle (LNP), and the liposome, the micelle, the emulsion, or a mixture thereof does not contain a sterol (e.g., a cholesterol), while the LNP may or may not contain a sterol, such as a cholesterol (e.g., a cholesterol).

[0143] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles comprises a structural lipid, e.g., a sterol. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload. For example, in some aspects, the first particle as the decoy is a liposome and the second particle for payload delivery is a lipid nanoparticle (LNP), and the liposome contains a sterol (e.g., a cholesterol), while the LNP may or may not contain a sterol, such as a cholesterol (e.g., a cholesterol).

[0144] A sterol as used in the present disclosure may be selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol or any combination thereof. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles comprises is cholesterol. For example, in some aspects, the first particle as the decoy is a liposome and the second particle for payload delivery is a lipid nanoparticle (LNP), and the liposome does not contain cholesterol, while the LNP for payload delivery contains cholesterol. In further aspects, the first particle does not comprise any payload while the second particle comprises a payload.33#14739410v1

[0145] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles comprises an ionizable lipid in a range of about 10 mol% to about 70 mol%.

[0146] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the particles comprises an ionizable lipid in a range of about 10 mol% to about 50 mol%. In some aspects, an ionizable lipid is present in at least one of the at least two particles of the pharmaceutical system at about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, or about 70 mol%.

[0147] In some aspects, an ionizable lipid is present in at least one of the at least two particles of the pharmaceutical system at range of about 25 mol% to about 65 mol%; about 30 mol% to about 60 mol%; about 35 mol% to about 55 mol%; about 40 mol% to about 60 mol%, or about 40 mol% to about 50 mol%. In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle at range of about 40 mol% to about 60 mol%; about 45 mol% to about 55 mol%; or about 47 mol% to about 50 mol%. In some aspects, the ionizable lipid is present in the lipid blend of a lipid nanoparticle at about 49.2 mol%.

[0148] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element a compound comprising a PEG-lipid and at least one of the two particles comprises a helper lipid. In some aspects, at least one of the two particles comprises a PEG-lipid and a helper lipid. In some aspects, the PEG-lipid and the helper lipid in the particle has a ratio of about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, about 11:89, about 12:88, about 13:87, about 14:86,34#14739410v1about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, about 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, about 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, about 40:60, about 41:59, about 42:58, about 43:57, about 44:56, about 45:55, about 46:54, about 47:53, about 48:52, about 49:51, about 50:50, about 51:49, about 52:48, about 53:47, about 54:46, about 55:45, about 56:44, about 57:43, about 58:42, about 59:41, about 60:40, about 61:39, about 62:38, about 63:37, about 64:36, about 65:35, about 66:34, about 67:33, about 68:32, about 69:31, about 70:30, about 71:29, about 72:28, about 73:27, about 74:26, about 75:25, about 76:24, about 77:23, about 78:22, about 79:21, about 80:20, about 81:19, about 82:18, about 83:17, about 84:16, about 85:15, about 86: 14, about 87: 13, about 88: 12, about 89: 11, about 90: 10, about 91:9, about 92:8, about 93:7, about 94:6, about 95:5, about 96:4, about 97:3, about 98:2 or about 99:1.

[0149] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element a compound comprising a PEG-lipid and the at least two particles comprise a helper lipid, wherein the two particles comprise a PEG-lipid and a helper lipid at a ratio of about 5:95 to about 25:75.

[0150] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element a compound comprising a PEG-lipid and the at least two particles comprise a helper lipid, wherein the two particles comprise a PEG-lipid and a helper lipid at a ratio of about 10:90.

[0151] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least two particles are lipid-based nanoparticles. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle are different from each other in their particle composition except that the at least two particles comprise a common element, and wherein the first particle and the second particle are lipid-based nanoparticles.

[0152] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome.

[0153] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles 35#14739410v1comprise a common element and at least one of the at least two particles is a lipid nanoparticle (LNP).

[0154] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles is a liposome and at least one of the at least two particles is a lipid nanoparticle (LNP). In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle are different from each other in their particle composition except that the at least two particles comprise a common element, and wherein the first particle is a liposome and the second particle is a lipid-based nanoparticle. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle are different from each other in their particle composition except that the at least two particles comprise a common element, and wherein the first particle is a lipid-based nanoparticle and the second particle is a liposome.

[0155] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least two particles are liposomes. In some embodiments, a pharmaceutical system comprises a first particle and a second particle, wherein the first particle and the second particle are different from each other in their particle composition except that the at least two particles comprise a common element, and wherein the first particle and the second particle are liposomes.

[0156] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles that is a liposome comprises PEG or a compound comprising PEG. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a compound comprising PEG comprises a PEG-lipid.

[0157] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a compound comprising PEG, wherein the compound comprising PEG comprises a positively charged PEG-lipid.36#14739410v1

[0158] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a compound comprising PEG, wherein the compound comprising PEG comprises a negatively charged PEG-lipid.

[0159] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a compound comprising PEG, wherein the compound comprising PEG comprises a neutral PEG-lipid.

[0160] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a PEG-lipid, wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

[0161] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a PEG-lipid, wherein the PEG-lipid is selected from the group consisting of PEG- dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

[0162] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome and comprises a PEG-lipid, wherein the PEG-lipid is PEG-DSPE.37#14739410v1

[0163] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome, wherein the liposome comprises a helper lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), succinyl-distearoyl-sn-glycero-3-phosphoethanolamine (Suc-DSPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), di stearoylglycerol (DSG), dioleoylgylcerol (DOG), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), dilinoleoyl-glycero-phosphatidyl ethanolamine (DLPE), diacylglycerol (DAG), ceramide 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain and combinations thereof.

[0164] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome, wherein the liposome comprises a helper lipid that is DSPC, DOPC, or a mixture thereof.

[0165] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome, wherein the liposome comprises DSPC and a PEG-lipid. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles is a liposome, wherein the liposome comprises a higher amount of PEG-lipid than a second particle of the at least two particles, wherein the second particle is a lipid nanoparticle of the pharmaceutical system.II. Targeting group

[0166] In some aspects, a pharmaceutical system of the present disclosure optionally comprises at least two particles described herein, wherein at least one of the particles comprises a targeting group. In some aspects, both particles comprise targeting group. In some aspects, the targeting group is a conjugate. In some aspects, the targeting group is conjugated to a lipid of at least 38#14739410v1one of the at least two particles. In some aspects, a particle of a pharmaceutical system comprises more than one cell targeting group, wherein some or all targeting groups are conjugated to a lipid of at least one of the particles of the pharmaceutical system comprises.

[0167] In some aspects, a targeting group comprises an antibody. In some aspects, the antibody is a human or humanized antibody. In some aspects, a targeting group comprises an antibody fragment without an Fc component. In some aspects, a cell targeting group comprises an antigen-binding fragment selected from the group consisting of a Fab, F(ab’)2, Fab’-SH, Fv, scFv fragment, or ISVD. In some aspects, a targeting group comprises an ISVD. In some aspects, a targeting group comprises an Fab. In some aspects, a targeting group comprises one or more antibody fragments such as ISVD, Fab, scFv, or combinations thereof.

[0168] In some aspects, a targeting group comprises immunoglobulin sequences of different origin, comprising, e.g., mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. In some aspects, a targeting group comprises fully human, humanized or chimeric sequences.

[0169] In some aspects, a targeting group is a monovalent, multivalent, or multispecific polypeptide. In some aspects, a targeting group comprises a multivalent and multispecific polypeptide that contains one or more antibodies, one or more ISVDs, e.g., one or more VHH domain, and / or one or more Fab, and / or one or more scFv.

[0170] In some aspects, a multivalent targeting group comprises two or more ISVD, Fab, or scFv that target a same antigen, e.g., the same part or epitope of said antigen or two or more different parts or epitopes of said antigen. In some aspects, a multivalent targeting group comprises two or more ISVD, Fab, or scFv that are directed to different antigens. In some aspects, a multivalent targeting group comprises a combination of ISVD, Fab, or scFv, some of which target the same antigen and some of which target different antigens.

[0171] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles comprises a targeting group conjugated to a lipid.

[0172] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles comprises a targeting group having the formula of [Lipid] - [optional linker] - [antibody], wherein the antibody specifically binds to one type of cell, tissue, or organ.39#14739410v1

[0173] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprises at least one particle that comprise a targeting group conjugated to a lipid that is a bispecific antibody. In some aspects, the bispecific antibody of the at least one particle of the pharmaceutical system binds a first type of immune cell and a second type of immune cell.

[0174] In some aspects, the bispecific antibody of the at least one particle of the pharmaceutical system binds a first type of immune cell that is a macrophage and a second type of immune cell that is a macrophage, a T cell or an NK cell.

[0175] In some aspects, a first conjugate of a particle of the pharmaceutical system comprises a first antibody that binds a first antigen and a second conjugate of a particle of the pharmaceutical system comprises a second antibody that binds a second antigen. In some aspects, a first antibody of the at least one particle of the pharmaceutical system binds a first type of immune cell that is a macrophage and a second antibody of the at least one particle of the pharmaceutical system binds a second type of immune cell that is a macrophage, a T cell or an NK cell.

[0176] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprises a bivalent cell targeting group.

[0177] In some aspects, a bivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a first part or epitope of an antigen and a second ISVD, Fab, or scFv targeting the same part or epitope of said antigen or another part or epitope of said antigen. In some aspects, a bivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a first antigen and a second ISVD, Fab, or scFv targeting a second antigen different from said first antigen.

[0178] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprises a trivalent cell targeting group. In some aspects, a trivalent cell targeting group comprises three identical or different ISVD, Fab, or scFv targeting the same or different parts or epitopes of the same antigen. In some aspects, a trivalent cell targeting group comprises two identical or different ISVD, Fab, or scFv targeting the same or different parts or epitopes on a first antigen and a third ISVD targeting a second antigen different from said first antigen.

[0179] In some aspects, a trivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a first antigen, a second ISVD, Fab, or scFv targeting a second antigen different from 40#14739410v1said first antigen, and a third ISVD, Fab, or scFv targeting a third antigen different from said first and second antigen.

[0180] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprises a cell targeting group that comprises a Fab lacking the native interchain disulfide bond.

[0181] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element comprises a cell targeting group that comprises a Fab that is engineered to replace one or both cysteine on the native constant light chain and the native constant heavy chain that form the native interchain disulfide bond with a non-cysteine amino acid, thereby removing the native interchain disulfide bond in the Fab.

[0182] In some aspects, a cell targeting group comprises one or more ISVD, Fab, or scFv and optionally further comprises one or more further amino acid sequences (all optionally linked via one or more suitable linkers). In some aspects, a cell targeting group further comprises one or more other groups, residues, moieties or binding units. Such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the ISVD, Fab, or scFv (and / or to the polypeptide in which they are present) and may or may not modify the properties of the ISVD, Fab, or scFv.

[0183] In some aspects, such further groups, residues, moieties or binding units may be one or more additional amino acids, such that the cell targeting group is a (fusion) protein or (fusion) polypeptide. In some aspects, said one or more other groups, residues, moieties or binding units are immunoglobulins. In some aspects, said one or more other groups, residues, moieties or binding units are domain antibodies (dAbs), amino acids that are suitable for use as a domain antibody, ISVD, amino acids that are suitable as ISVD, Fab, amino acids that are suitable as Fab, scFv, or amino acids that are suitable as scFv.

[0184] In some aspects, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and / or pharmacologically active. In some aspects, such groups are linked to an ISVD, Fab, or scFv so as to provide a “derivative” of the ISVD, Fab, or scFv.

[0185] In some aspects, said further residues may be effective in preventing or reducing binding of so-called “pre-existing antibodies” to the polypeptides. For this purpose, the polypeptides and constructs may contain a C-terminal extension (X)n in which n is 1 to 10, 1 to 5, 2 or 1; and each X is an (preferably naturally occurring) amino acid residue that is 41#14739410v1independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). In some aspects, X is not cysteine.

[0186] In some aspects, the one or more ISVD, Fab, or scFv and the one or more groups, residues, moieties or binding units may be linked directly to each other and / or via one or more suitable linkers. For example, when the one or more groups, residues, moieties or binding units are amino acids, the linkers may also be an amino acid, so that the resulting polypeptide is a fusion protein or fusion polypeptide.

[0187] In some aspect, a linker comprises one or more repeats of a peptide motif such as, e.g., a GS (Glycine-Serine) motif. In some aspects, a linker comprises a GGGGS (SEQ ID NO: 1) motif (for example, having the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more (SEQ ID NO: 13)). In some aspects, a linker is a 9GS linker (GGGGSGGGS) (SEQ ID NO: 4), 15GS linker (n=3) and 35GS linker (n=7). Reference is, e.g., made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325-330). In some aspects, a linker is a 3A linker, i.e. comprising three alanines. In some aspects, a linker is a 5GS linker, a 7GS linker, a 8GS linker, a 9GS linker, alOGS linker, a 15GS linker, a 18GS linker, a 20GS linker, a 25GS linker, a 30GS linker, a 35GS linker or a 40GS linker as shown in TABLE 11 below.TABLE 1142#14739410v1

[0188] In some aspects, a cell targeting group or combination of targeting groups can be selected based on the desired localization, function, or structural features of a given target cell. For example, a cell targeting group can target a monocyte, macrophage, T cell or NK cell.

[0189] In some aspects, a cell targeting group that targets a monocyte cell comprises CD 14, CCR2, CCR5, CD62L, HLA, CD68, CXCR1, CXCR3, and CDllc, or any combination thereof.

[0190] In some aspects, a cell targeting group that targets a macrophage cell comprises CDIIB, CD68, CD80, CD86, TRL-2, TRL-4, iNOS, MHC-II, CD163, CD206, CD209, FIZZ1, or Yml / 2, or any combination thereof.

[0191] In some aspects, a cell targeting group that targets a T cell comprises one or more antibodies, ISVD, Fab, scFv, or derivatives thereof that target a T cell surface antigen. Exemplary T cell surface antigens include, but are not limited to, CD3, CD4, CD7, and CD8.

[0192] An exemplary CD3 binding agent (CD3y / 6 / s, CD3y, CD36, CD3y / s, CD36 / s, or CD3s) can be an antibody selected from the group consisting of MEM-57 (CD3y / 6 / s, EnzoLife Sciences), MAB100 (CD3s, R&D Systems), CD3-H5 (CD3s, Abnova Corporation), CD3-12 (CD3s, Cell Signaling Technology), LE-CD3 (CD3s, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3y, Novus Biologicals), 16669-1-AP (CD36, Invitrogen) and antigen binding fragments thereof. In some aspects, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of MEM-57 (CD3y / 6 / s, EnzoLife Sciences), MAB100 (CD3s, R&D Systems), CD3-H5 (CD3s, Abnova Corporation), CD3-12 (CD3s, Cell Signaling Technology), LE-CD3 (CD3s, Santa Cruz Biotechnology, Inc.), NBP1- 31250 (CD3y, Novus Biologicals), and 16669-1-AP (CD36, Invitrogen). In some aspects, the binding agent comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antibody selected from the group consisting of MEM-57 (CD3y / 6 / s, EnzoLife Sciences), MAB100 (CD3s, R&D Systems), CD3-H5 (CD3s, Abnova Corporation), CD3-12 (CD3s, Cell Signaling Technology), LE-CD3 (CD3s, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3y, Novus Biologicals), and 16669-1-AP (CD36,43#14739410v1Invitrogen). An exemplary CD3 binding agent can also be selected from antibodies or antibody fragments employing CDRs of clones hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, Catumaxomab, teplizumab, 3-106, 3-114, 3-148, 3-190, 3-271, 3-550, 4-10, 4-48, H2C, F12Q, I2C, SP7, 3F3A1, CD3-12, 301, RIV9, JB38-29, JE17-74, GT0013, 4E2, 7A4, 4D10A6, SPV-T3b, M2AB, ICO-90, 30A1 or Hu38E4.vl (US Patent Application 20200299409A1), REGN5458 (US Patent Application 20200024356A1), or Blinatumomab

[0193] An exemplary CD4 binding agent can be an antibody selected from the group consisting of Ibalizumab (genome.jp / dbget-bin / www_bget?D09575), AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), CAL4 (Abeam), and antigen binding fragments thereof. In some aspects, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abeam). In some aspects, the binding agent comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antibody selected from the group consisting of AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abeam). An exemplary CD4 binding agent can also be selected from antibodies or antibody fragments employing CDRs of clones Ibalizumab, OKT4, RPA-T4, S3.5, SK3, N1UG0, RIV6, OTI18E3, MEM-241, B486A1, RFT-4g, 7E14, MDX.2, MEM-115, MEM- 16, ICO-86, Edu-2, or ilbalizumab.

[0194] An exemplary CD7 binding agent can be an antibody selected from the group consisting of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abeam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), NBP2-38440 (Novus Biologicals), and antigen binding fragments thereof. In some aspects, the binding agent comprises a VH domain and a VL of an antibody selected from the group consisting of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abeam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), and NBP2-38440 (Novus Biologicals). In some aspects, the binding agent comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia 44#14739410v1C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antibody selected from the group consisting of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abeam), lG10D8(Proteintech), NBP2-32097 (Novus Biologicals), and NBP2-38440 (Novus Biologicals). An exemplary CD7 binding agent can also be selected from antibodies or antibody fragments employing CDRs of clones TH-69, 3Afll, T3-3A1, 124-1D1, 3Alf, CD7-6B7, or VHH6.

[0195] An exemplary CD8 (CD8a, CD8a / a, CD8a / p or CD8P) binding agent can be an antibody selected from the group consisting of 15C01v8, 2.43 (Invitrogen), Du CD8-1 (CD8a, Invitrogen), 9358-CD (CD8a / p, R&D Systems), MAB116 (CD8a, R&D Systems), ab4055 (CD8a, Abeam), C8 / 144B (CD8a, Novus Biologicals), YTS105.18 (CD8a, Novus Biologicals), TRX2 (US 2017-0198045 Al or US Pat. No. 10,647768), and antigen binding fragments thereof. In some aspects, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of 15C01v8, 2.43 (Invitrogen), 51.1 (ATCC HB-230), Du CD8-1 (CD8a, Invitrogen), 9358-CD (CD8a / p, R&D Systems), MAB116 (CD8a, R&D Systems), ab4055 (CD8a, Abeam), C8 / 144B (CD8a, Novus Biologicals), and YTS105.18 (CD8a, Novus Biologicals). In some aspects, the binding agent comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3, determined under Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745), or any other CDR determination method known in the art, of the VH and VL sequences of an antibody selected from the group consisting of 15C01v8, 2.43 (Invitrogen), Du CD8- 1 (CD8a, Invitrogen), 9358-CD (CD8a / p, R&D Systems), MAB116 (CD8a, R&D Systems), ab4055 (CD8a, Abeam), C8 / 144B (CD8a, Novus Biologicals), and YTS105.18 (CD8a, Novus Biologicals). An exemplary CD8 binding agent can also be selected from antibodies or antibody fragments employing CDRs of clones OKT-8, 51.1, S6F1, TRX2, and UCHT4, SP16, 3B5, C8-144B, HIT8a, RAVB3, LT8, 17D8, MEM-31, MEM-87, RIV11, DK-25, YTC141.1HL, orYTC182.20.

[0196] In some aspects, a cell targeting group targets an NK cell, or NK cell population. In some aspects, an NK cell targeting group comprises one or more antibodies, antigen binding fragments or antigen binding derivatives thereof that target an NK cell surface antigen. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a,45#14739410v1CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, CD337 / NKP30.

[0197] In some aspects, an immune cell targeting group comprises a C-terminal cysteine residue. In some aspects, an immune cell targeting group comprises a Fab that comprises a cysteine at the C-terminus of the heavy or light chain fragment. In some aspects, the Fab further comprises one or more amino acids between the heavy chain of the Fab and the C- terminal cysteine. For example, in some aspects, an Fab comprises two or more amino acids derived from an antibody hinge region (e.g., a partial hinge sequence) between the C- terminus of the Fab and the C-terminal cysteine. In some aspects, an Fab comprises a heavy chain variable domain linked to an antibody CHI domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CHI domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single chain variable fragment, e.g., a scFv or an ISVD linked to the C-terminus of the light chain constant domain by an amino acid linker.

[0198] In some aspects, an immune cell targeting group comprises an ISVD (e.g., a VHH). In some aspects, the ISVD comprises a cysteine at the C-terminus. In some aspects, the ISVD further comprises a linker comprising one or more amino acids between the VHH domain and the C-terminal cysteine. In some aspects, the linker comprises one or more glycine residues, e.g., two glycine residues. In some aspects, the immune cell targeting group comprises two or more VHH domains. In some aspects, the two or more VHH domains are linked by an amino acid linker. In some aspects, the amino acid linker comprises one or more glycine and / or serine residues (e.g., one or more repeats of the sequence GGGGS (SEQ ID NO: 1)). In some aspects, the immune cell targeting group comprises a first ISVD linked to an antibody CHI domain and a second ISVD linked to an antibody light chain constant domain, and wherein the antibody CHI domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some aspects, the immune cell targeting group comprises an ISVD linked to an antibody CHI domain, and wherein the antibody CHI domain is linked to an antibody light chain constant domain by one or more disulfide bonds.III. Cell Targeting Group Conjugates

[0199] In some aspects, a pharmaceutical system comprising at least two particles as described herein comprises one or more cell targeting group conjugates. In some aspects, a cell targeting group conjugate comprises a cell targeting polypeptide, or fragment thereof, and a lipid. In 46#14739410v1some aspects, a cell targeting group is covalently coupled to a lipid either directly or via a linker. In some aspects, a cell targeting group is covalently coupled to a lipid via a polyethylene glycol (PEG) containing linker. PEG is a polymer comprising multiple repeating units each having the structure -(OCH2CH2)-), e.g., H-(O-CH2-CH2)n-OH. In some aspects, a PEG-cell targeting group conjugate is an insertable cell targeting group conjugate.

[0200] In some aspects, the lipid linked to a cell-targeting group via a PEG linker is di stearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

[0201] In some aspects, a PEG-cell targeting group conjugate comprises a DSG-PEG, DSPE-PEG, DMPE-PEG, DSPG-PEG, DMG-PEG, DPPE-PEG, DPG-PEG or ceramide-PEG. In some aspects, a PEG-cell targeting group conjugate comprises a DPG-PEG. In some aspects, a PEG-cell targeting group conjugate comprises a DMG-PEG.

[0202] In some aspects, the PEG in a PEG-cell targeting conjugate is PEG 500, PEG 1000, PEG 2000, PEG 3000, PEG 3400, PEG 3450, PEG 4000, or PEG 5000. In some aspects, the PEG is PEG 2000 or PEG 3400. In some aspects, the PEG is PEG 2000 (PEG2K).

[0203] In some aspects, a cell targeting group conjugate comprises a PEG2K-antibody. In some aspects, a cell targeting group conjugate comprises a PEG2K-Fab. In some aspects, a cell targeting group conjugate comprises a PEG2K-scFv. In some aspects, a cell targeting group conjugate comprises a PEG2K-ISVD.

[0204] In some aspects, a cell targeting group conjugate of a particle of a pharmaceutical system comprises between about 1 g and about 50 g of cell-targeting group per mol lipid. In some aspects, a cell-targeting group conjugate of a particle of a pharmaceutical system comprises between about 10 g and about 20 g of cell-targeting group per mol lipid. In some aspects, an antibody conjugate of a particle of a pharmaceutical system comprises about 12 g of cell-targeting group per mol lipid.

[0205] In some aspects, a cell targeting group conjugate comprises about 1 g to about 10 g of cell targeting group per mole of lipid. In some aspects, a cell targeting group conjugate comprises about 2 g to about 9 g of cell targeting group per mole of lipid. In some aspects, a cell targeting group conjugate comprises about 3 g to about 9 g of cell targeting group per mole of lipid.47#14739410v1

[0206] In some aspects, a cell targeting group conjugate is presented in the LNP at a density from about 0.4 to 11.4, about 1.9 to 9.5, about 3.8 to 7.6, about 4.6 to 6.5, about 5.3 to 6.1, about 3.8, or about 5.7 micromoles of conjugate per gram of mRNA in the LNP.

[0207] In some aspects, a cell targeting group conjugate is presented in the LNP at a density from about 0.01 to about 1.0 g / g mRNA in the LNP, such as about 0.01 to about 0.05, about 0.05 to about 0.1, about 0.1 to about 0.5, about 0.5 to about 1 g / g mRNA in the LNP.

[0208] In some aspects, a cell targeting group conjugate is present in a particle of a pharmaceutical system in a range of about 0.001 mol% to about 0.5 mol%. In some aspects, a cell targeting group conjugate is present in a particle of a pharmaceutical system in a range of about 0.002 mol% to about 0.2 mol%. In some aspects, a cell targeting group conjugate is present in a particle of a pharmaceutical system in a range of about 0.001 mol% to about 0.4 mol%; about 0.001 mol% to about 0.3 mol%; about 0.001 mol% to about 0.2 mol%; about 0.001 mol% to about 0.1 mol%; or about 0.001 mol% to about 0.08 mol%; about 0.001 mol% to about 0.06 mol%; about 0.001 mol% to about 0.04 mol%; about 0.001 mol% to about 0.02 mol%; or about 0.001 mol% to about 0.01 mol%. In some aspects, a cell targeting group conjugate is present in a particle of a pharmaceutical system in a range of about 0.002 mol% to about 0.5 mol%; about 0.003 mol% to about 0.5 mol%; about 0.004 mol% to about 0.5 mol%; about 0.005 mol% to about 0.5 mol%; about 0.006 mol% to about 0.5 mol%; about 0.008 mol% to about 0.5 mol%; about 0.01 mol% to about 0.5 mol%; about 0.02 mol% to about 0.5 mol%; about 0.03 mol% to about 0.5 mol%; about 0.04 mol% to about 0.5 mol%; about 0.05 mol% to about 0.5 mol%; about 0.06 mol% to about 0.5 mol%; about 0.07 mol% to about 0.5 mol%; about 0.08 mol% to about 0.5 mol%; about 0.09 mol% to about 0.5 mol%; about 0.1 mol% to about 0.5 mol%; about 0.15 mol% to about 0.5 mol%; about 0.2 mol% to about 0.5 mol%; about 0.25 mol% to about 0.5 mol%; about 0.3 mol% to about 0.5 mol%; about 0.35 mol% to about 0.5 mol%; or about 0.4 mol% to about 0.5 mol%.IV. Payload

[0209] In some aspects, at least one particle of a pharmaceutical system comprises a payload, e.g., a nucleic acid molecule disposed in the particle for delivery to a cell (e.g., an immune cell or hematopoietic stem cell) or tissue in a subject.

[0210] In some aspects, a particle of a pharmaceutical system comprises a nucleic acid, e.g., a DNA or RNA, such as an mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozymes, decoy RNA or dicer substrate siRNA. In some aspects, a nucleic acid comprises naturally occurring components, such as, naturally occurring bases, sugars or linkage groups (e.g., phosphodiester linkage groups). In some aspects, a nucleic acid comprises 48#14739410v1a pseudouridine. In some aspects, a nucleic acid comprises an Nl-methyl-pseudouridine. In some aspects, a nucleic acid comprises non-naturally occurring components or modifications, (e.g. , thioester linkage groups). For example, the nucleic acid can be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Furthermore, the nucleic acids can be linear or circular, or have any desired configuration. In some aspects, a particle of a pharmaceutical system includes multiple nucleic acid molecules, e.g., multiple RNA molecules, which can be the same or different.

[0211] In some aspects, one or more particles of a pharmaceutical system including one or more different mRNAs are combined, and / or simultaneously contacted with a cell. In some aspects, an mRNA may include one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure. In some aspects, an mRNA may encode a receptor, such as a chimeric antigen receptor (CAR), for transfection of an immune cell. In some aspects, an mRNA may encode a receptor, such as a synthetic T cell receptor (synTCR), for transfection of an immune cell, e.g., a T cell.

[0212] In some aspects, a mRNA comprises a marker gene. In some aspects, the marker gene is mCherry.

[0213] In some aspects, the mRNA may encode a Cas nuclease. In some aspects, the RNA may comprise a guide RNA.

[0214] In some aspects, a pharmaceutical system comprises at least two particles, wherein at least one particle comprises mRNA. In some aspects, the at least one particle of the pharmaceutical system comprises an ionizable lipid at a concentration of about 10 g / g mRNA to about 18 g / g mRNA; or about 10.1 g / g mRNA, 10.2 g / g mRNA, 10.3 g / g mRNA, about 10.4 g / g mRNA, 10.5 g / g mRNA, 10.6 g / g mRNA, about 10.7 g / g mRNA, about 10.8 g / g mRNA, about 10.9 g / g mRNA, about 11.0 g / g mRNA, about 11.1 g / g mRNA, about 11.2 g / g mRNA, about 11.3 g / g mRNA, about 11.4 g / g mRNA, about 11.5 g / g mRNA, about 11.6 g / g mRNA, about 11.7 g / g mRNA, about 11.8 g / g mRNA, about 11.9 g / g mRNA, about 12.0 g / g mRNA, about 12.1 g / g mRNA, about 12.2 g / g mRNA, about 12.3 g / g mRNA, about 12.4 g / g mRNA, about 12.5 g / g mRNA, about 12.6 g / g mRNA, about 12.7 g / g mRNA, about 12.8 g / g mRNA, about 12.9 g / g mRNA, about 13.0 g / g mRNA, about 13.1 g / g mRNA, about 13.2 g / g mRNA, about 13.3 g / g mRNA, about 13.4 g / g mRNA, about 13.5 g / g mRNA, about 13.6 g / g mRNA, about 13.7 g / g mRNA, about 13.8 g / g mRNA, about 13.9 g / g mRNA, about 14.0 g / g mRNA, about 14.1 g / g mRNA, about 14.2 g / g mRNA, about 14.3 g / g mRNA, about 14.4 g / g mRNA, about 14.5 g / g mRNA, about 14.6 g / g mRNA, about 14.7 g / g mRNA, about 14.8 g / g mRNA, about 14.9 g / g mRNA, about 15.0 g / g mRNA, about 15.1 g / g mRNA, about 15.2 g / g mRNA,49#14739410v1about 15.3 g / g mRNA, about 15.4 g / g mRNA, about 15.5 g / g mRNA, about 15.6 g / g mRNA, about 15.7 g / g mRNA, about 15.8 g / g mRNA, about 15.9 g / g mRNA, about 16.0 g / g mRNA, about 16.1 g / g mRNA, about 16.2 g / g mRNA, about 16.3 g / g mRNA, about 16.4 g / g mRNA, about 16.5 g / g mRNA, about 16.6 g / g mRNA, about 16.7 g / g mRNA, about 16.8 g / g mRNA, about 16.9 g / g mRNA, about 17.0 g / g, about 17.1 g / g mRNA, about 17.2 g / g mRNA, about 17.3 g / g mRNA, about 17.4 g / g mRNA, about 17.5 g / g mRNA, about 17.6 g / g mRNA, about 17.7 g / g mRNA, about 17.8 g / g mRNA, about 17.9 g / g mRNA, or about 18.0 g / g mRNA In some aspects, the at least one particle of the pharmaceutical system comprises an ionizable lipid at a concentration of about 14.2 g / g mRNA. In some aspects, a pharmaceutical system comprises at least two particles, wherein at least one particle comprises mRNA. In some aspects, the at least one particle of the pharmaceutical system comprises an ionizable lipid at a concentration of about 40 mol% to about 60 mol% of the LNP, such as about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%. In some aspects, the ionizable lipid at a concentration of about 48% to about 50%, such as about 48.1%, about 48.2%, about 48.3%, about 48.4%, about 48.5%, about 48.6%, about 48.7%, about 48.8%, about 48.9%, about 49.0%, about 49.1%, about 49.2%, about 49.3%, about 49.4%, about 49.5%, about 49.6%, about 49.7%, about 49.8%, or about 49.9% mol% of the LNP.

[0215] In some aspects, the at least one particle of the pharmaceutical system comprises a sterol at a concentration of about 2.0 g / g mRNA to about 6.0 g / g mRNA; or about 2.1 g / g mRNA, about 2.2 g / g mRNA, about 2.3 g / g mRNA, about 2.4 g / g mRNA, about 2.5 g / g mRNA, about 2.6 g / g mRNA, about 2.7 g / g mRNA, about 2.8 g / g mRNA, about 2.9 g / g mRNA, about 3.0 g / g mRNA, about 3.1 g / g mRNA, about 3.2 g / g mRNA, about 3.3 g / g mRNA, about 3.4 g / g mRNA, about 3.5 g / g mRNA, about 3.6 g / g mRNA, about 3.7 g / g mRNA, about 3.8 g / g mRNA, about 3.9 g / g mRNA, about 4.0 g / g mRNA, about 4.1 g / g mRNA, about 4.2 g / g mRNA, about 4.3 g / g mRNA, about 4.4 g / g mRNA, about 4.5 g / g mRNA, about 4.6 g / g mRNA, about 4.7 g / g mRNA, about 4.8 g / g mRNA, about 4.9 g / g mRNA, about 5.0 g / g mRNA, about 5.1 g / g mRNA, about 5.2 g / g mRNA, about 5.3 g / g mRNA, about 5.4 g / g mRNA, about 5.5 g / g mRNA, about 5.6 g / g mRNA, about 5.7 g / g mRNA, about 5.8 g / g mRNA, about 5.9 g / g mRNA, or about 6.0 g / g mRNA.

[0216] In some aspects, the at least one particle of the pharmaceutical system comprises a sterol at a concentration of about 4.64 g / g mRNA.#14739410v1

[0217] In some aspects, the at least one particle of the pharmaceutical system comprises a neutral phospholipid at a concentration of about 1.0 g / g mRNA to about 4.0 g / g mRNA; or about 1.1 g / g mRNA, about 1.2 g / g mRNA, about 1.3 g / g mRNA, about 1.4 g / g mRNA, about 1.5 g / g mRNA, about 1.6 g / g mRNA, about 1.7 g / g mRNA, about 1.8 g / g mRNA, about 1.9 g / g mRNA, about 2.0 g / g mRNA, about 2.1 g / g mRNA, about 2.2 g / g mRNA, about 2.3 g / g mRNA, about 2.4 g / g mRNA, about 2.5 g / g mRNA, about 2.6 g / g mRNA, about 2.7 g / g mRNA, about 2.8 g / g mRNA, about 2.9 g / g mRNA, or about 3.0 g / g mRNA, about 3.1 g / g mRNA, about 3.2 g / g mRNA, about 3.3 g / g mRNA, about 3.4 g / g mRNA, about 3.5 g / g mRNA, about 3.6 g / g mRNA, about 3.7 g / g mRNA, about 3.8 g / g mRNA, about 3.9 g / g mRNA, or about 4.0 g / g mRNA.

[0218] In some aspects, the at least one particle of the pharmaceutical system comprises a neutral phospholipid at a concentration of about 2.37 g / g mRNA.

[0219] In some aspects, the at least one particle of the pharmaceutical system comprises a PEG-lipid at a concentration of about 0.5 g / g mRNA to about 2.0 g / g mRNA; about 0.6 g / g mRNA, about 0.7 g / g mRNA, about 0.8 g / g mRNA, about 0.9 g / g mRNA, about 1.0 g / g mRNA, about 1.1 g / g mRNA, about 1.2 g / g mRNA, about 1.3 g / g mRNA, about 1.4 g / g mRNA, about 1.5 g / g mRNA, about 1.6 g / g mRNA, about 1.7 g / g mRNA, about 1.8 g / g mRNA, about 1.9 g / g mRNA, or about 2.0 g / g mRNA.

[0220] In some aspects, the at least one particle of the pharmaceutical system comprises a PEG-lipid at a concentration of about 1.15 g / g mRNA.

[0221] In some aspects, the at least one particle of the pharmaceutical system comprises a targeting group at a concentration of about 0.05 g / g mRNA to about 2.0 g / g mRNA; about 0.055 g / g mRNA, about 0.06 g / g mRNA, about 0.065 g / g mRNA, about 0.07 g / g mRNA, about 0.075 g / g mRNA, about 0.08 g / g mRNA, about 0.085 g / g mRNA, about 0.09 g / g mRNA, about 0.095 g / g mRNA, about 0.1 g / g mRNA, about 0.15 g / g mRNA, about 0.2 g / g mRNA, about 0.25 g / g mRNA, about 0.3 g / g mRNA, about 0.35 g / g mRNA, about 0.4 g / g mRNA, about 0.45 g / g mRNA, about 0.5 g / g mRNA, about 0.55 g / g mRNA, about 0.6 g / g mRNA, about 0.65 g / g mRNA, about 0.7 g / g mRNA, about 0.75 g / g mRNA, about 0.8 g / g mRNA, about 0.85 g / g mRNA, about 0.9 g / g mRNA, about 0.95 g / g mRNA, about 1.0 g / g mRNA, about 1.1 g / g mRNA, about 1.15 g / g mRNA, about 1.2 g / g mRNA, about 1.25 g / g mRNA, about 1.3 g / g mRNA, about 1.35 g / g mRNA, about 1.4 g / g mRNA, about 1.45g / g mRNA, about 1.5 g / g mRNA, about 1.55 g / g mRNA, about 1.6 g / g mRNA, about 165 g / g mRNA, about 1.7 g / g mRNA, about 1.75 g / g mRNA, about 1.8 g / g mRNA, about 1.85 g / g mRNA, about 1.9 g / g mRNA, about 1.95 g / g mRNA, or about 2.0 g / g mRNA.51#14739410v1

[0222] In some aspects, the at least one particle of the pharmaceutical system comprises a targeting group at a concentration of about 0.084 g / g mRNA.

[0223] In some aspects, at least one particle of the pharmaceutical system is an LNP and comprises a [Lipid] - [optimal linker] - [antibody] conjugate at a concentration of about 0.05 g / g mRNA to about 2.0 g / g mRNA; about 0.055 g / g mRNA, about 0.06 g / g mRNA, about 0.065 g / g mRNA, about 0.07 g / g mRNA, about 0.075 g / g mRNA, about 0.08 g / g mRNA, about 0.085 g / g mRNA, about 0.09 g / g mRNA, about 0.095 g / g mRNA, about 0.1 g / g mRNA, about 0.15 g / g mRNA, about 0.2 g / g mRNA, about 0.25 g / g mRNA, about 0.3 g / g mRNA, about 0.35 g / g mRNA, about 0.4 g / g mRNA, about 0.45 g / g mRNA, about 0.5 g / g mRNA, about 0.55 g / g mRNA, about 0.6 g / g mRNA, about 0.65 g / g mRNA, about 0.7 g / g mRNA, about 0.75 g / g mRNA, about 0.8 g / g mRNA, about 0.85 g / g mRNA, about 0.9 g / g mRNA, about 0.95 g / g mRNA, about 1.0 g / g mRNA, about 1.1 g / g mRNA, about 1.15 g / g mRNA, about 1.2 g / g mRNA, about 1.25 g / g mRNA, about 1.3 g / g mRNA, about 1.35 g / g mRNA, about 1.4 g / g mRNA, about 1.45g / g mRNA, about 1.5 g / g mRNA, about 1.55 g / g mRNA, about 1.6 g / g mRNA, about 165 g / g mRNA, about 1.7 g / g mRNA, about 1.75 g / g mRNA, about 1.8 g / g mRNA, about 1.85 g / g mRNA, about 1.9 g / g mRNA, about 1.95 g / g mRNA, or about 2.0 g / g mRNA.

[0224] In some aspects, the encapsulation efficiency of the payload (e.g., mRNA) in a particle of the pharmaceutical system is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 99%.

[0225] In some aspects, in a particle of the pharmaceutical system as described herein exhibits dye accessible RNA of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7.5%, less than 5%, less than 2.5%, or less than 1%.V. Liposome

[0226] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a PEG-lipid and a helper lipid at a ratio of about 1:99, about 2:98, about 3:97, about4:96, about 5:95, about6:94, about7:93, about8:92, about 9:91, about 10:90, about 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, about 21:79, about 22:78, about 23:77, about 24:76, about 25:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, about 31:69, about 52#14739410v132:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, about 40:60, about 41:59, about 42:58, about 43:57, about 44:56, about 45:55, about 46:54, about 47:53, about 48:52, about 49:51, or about 50:50.

[0227] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a PEG-lipid and a helper lipid at a ratio of about 5:95 to about 25:75.

[0228] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a PEG-lipid and a helper lipid at a ratio of about 10:90.

[0229] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a helper lipid, wherein the helper lipid is present in the liposome at about 20 mol% to about 100 mol%. In some aspects, a helper lipid is present in the liposome at about 25 mol% to about 95 mol%. In some aspects, a helper lipid is present in the liposome at about 30 mol% to about 90 mol%. In some aspects, a helper lipid is present in the liposome at about 35 mol% to about 85 mol%. In some aspects, a helper lipid is present in the liposome at about 40 mol% to about 80 mol%. In some aspects, a helper lipid is present in the liposome at about 45 mol% to about 75 mol%. In some aspects, a helper lipid is present in the liposome at about 50 mol% to about 70 mol%. In some aspects, a helper lipid is present in the liposome at about 55 mol% to about 65 mol%.

[0230] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a helper lipid, wherein the helper lipid is present at about 20 mol% to about 25 mol%. In some aspects, a helper lipid is present in the liposome at about 26 mol% to about 30 mol%. In some aspects, a helper lipid is present in the liposome at about 31 mol% to about 35 mol%. In some aspects, a helper lipid is present in the liposome at about 36 mol% to about 40 mol%. In some aspects, a helper lipid is present in the liposome at about 41 mol% to about 45 mol%. In some aspects, a helper lipid is present in the liposome at about 46 mol% to about 50 mol%. In some aspects, a helper lipid is present in the liposome at about 51 mol% to about 55 mol%. In some aspects, a helper lipid is present in the liposome at about 56 mol% to about 53#14739410v160 mol%. In some aspects, a helper lipid is present in the liposome at about 61 mol% to about 65 mol%. In some aspects, a helper lipid is present in the liposome at about 66 mol% to about 70 mol%. In some aspects, a helper lipid is present in the liposome at about 71 mol% to about 75 mol%. In some aspects, a helper lipid is present in the liposome at about 76 mol% to about 80 mol%. In some aspects, a helper lipid is present in the liposome at about 81 mol% to about 85 mol%. In some aspects, a helper lipid is present in the liposome at about 86 mol% to about 90 mol%. In some aspects, a helper lipid is present in the liposome at about 91 mol% to about 95 mol%. In some aspects, a helper lipid is present in the liposome at about 96 mol% to about 100 mol%.

[0231] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a helper lipid, wherein the helper lipid is present at about 20 mol%, at about 22 mol%, about 24 mol%, about 26 mol%, about 28 mol%, about 30 mol%, about 32 mol%, about 34 mol%, about 36 mol%, about 38 mol%, about 40 mol%, about 42 mol%, about 44 mol%, about 46 mol%, about 48 mol%, about 50 mol%, about 52 mol%, about 54 mol%, about 56 mol%, about 58 mol%, about 60 mol%, about 62 mol%, about 64 mol%, about 66 mol%, about 68 mol%, about 70 mol%, about 72 mol%, about 74 mol%, about 76 mol%, about 78 mol%, about 80 mol%, about 82 mol%, about 84 mol%, about 86 mol%, about 88 mol%, about 90 mol%, about 92 mol%, about 94 mol%, about 96 mol%, about 98 mol%, about 99 mol%, or 100 mol%.

[0232] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome does not comprise a structural lipid, e.g., a sterol (such as cholesterol).

[0233] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise as a common element, wherein the at least one of the at least two particles that is a liposome comprises a structural lipid that is selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol or any combination thereof.

[0234] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles54#14739410v1comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises is cholesterol.

[0235] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a structural lipid that is present at a range of about 20 mol% to about 70 mol%. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles that is a liposome comprises a structural lipid that is present at about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, or about 70 mol%.

[0236] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome comprises a DOPC and / or DSPC helper lipid, a PEG-lipid and a structural lipid, e.g., cholesterol. In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein the at least one of the at least two particles that is a liposome and comprises a DOPC, DSPC helper lipid, PEG-lipid and a structural lipid binds lower levels of, e.g., opsonins compared to a particle of the pharmaceutical system that is a liposome and comprises a DOPC and / or DSPC helper lipid but lacks a structural lipid.

[0237] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element, wherein at least one of the at least two particles comprises an ionizable lipid that is present at about 49.2 mol%, a steroid that is present at a concentration of#14739410v1about 39.4 mol%, a neutral phospholipid that is present at a concentration of about 0.9 mol%, and a PEG-lipid that is present at about 1.5 mol%.

[0238] Various liposomes can be used according to the present disclosure. In some embodiments, liposomes of the present disclosure are mostly composed of phospholipids (e.g., phosphatidylcholine) and sterol (e.g., cholesterol). In some embodiments, liposomes of the present disclosure do not contain cholesterol or other sterols. In some embodiments, liposomes may also include other lipids as long as they are compatible with lipid bilayer structure. In some embodiments, liposomes of the present disclosure may contain surface ligands for attaching to desired cells or tissues. In some embodiments, liposomes may be selected from multilamellar large vesicle (MLV), oligolamellar vesicle (OLV), small unilamellar vesicle (SUV), medium-sized unilamellar vesicle (MUV), large unilamellar vesicle (LUV), giant unilamellar vesicle (GUV) and multivesicular vesicles (MW).

[0239] In some aspects, liposomes of the present disclosure comprise a polymer, such as polyethylene glycol (PEG). In some aspects, liposomes of the present disclosure do not comprise a polymer, such as non-PEGylated liposomes.

[0240] In some aspects, liposomes of the present disclosure are used as decoys and do not contain any pharmaceutically active agent.

[0241] Depending on the charging status of the second particle which contains a pharmaceutically active agent, such as a nucleic acid sequence, liposomes used as decoys of the present disclosure may be cationic, anionic, or neutral, as long as the charging status of liposomes are as the same as the charging status of the second particle, such as an LNP.

[0242] In some aspects, liposomes of the present disclosure are cationic liposomes. In some aspects, cationic liposomes may contain a cationic lipid, such as DOTMA, DOSPA, DOGS, DOTAP, CD-Chol, DODMA, DDAB, octadecylamine, or a mixture thereof.

[0243] In some aspects, liposomes of the present disclosure are anionic liposomes. In some aspects, anionic liposomes may contain Phosphatidyl-choline (PC), Phosphatidylethanolamine (PE), L-a-phosphatidic acid (PA), l,2-dimyristoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (PG), l,2-dimyristoyl-sn-glycero-3-phospho-L-serine (PS), or any combination thereof.

[0244] In some aspects, liposomes of the present disclosure are neutral liposomes. In some aspects, neutral liposomes may contain cholesterol, DOPE, EPC, DPPC, DSPC, DOPC, HSPC, or any combination thereof.

[0245] In some aspects, liposomes of the present disclosure may further comprise a PEGylated lipid, such as PEG-DSPE, PEG-DSG, PEG-DSPC, PEG-DOPC, PEG-DOPE, PEG-DMPE,56#14739410v1PEG-DSPG, PEG-DMG, PEG-DAG, PEG-DOG, PEG-DPPE, PEG-DLPE, PEG-DPG, PEG-ceramide, PEG-2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidyl-ethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain and combinations thereof.

[0246] In some aspects, liposomes of the present disclosure comprise a neutral lipid and a PEGylated lipid. In some aspects, liposomes of the present disclosure consists of a neutral lipid and a PEGylated lipid. In some aspects, liposomes of the present disclosure consist essentially of a neutral lipid and a PEGylated lipid.

[0247] In some aspects, liposomes of the present disclosure may be selected from the following: HSPC:Cholesterol:PEG 2000-DSPE; DSPC and Cholesterol; DOPC, DPPG, Cholesterol and Triolein; EPGCholesterol; DOPS:POPC; SM:Cholesterol; DSPGMPEG-2000:DSPE; DMPGDMPG; HSPC:DSPG:Cholesterol:Amphotericin B; Cholesteryl sulphate:Amphotericin B; VerteporphimDMPC and EPG; DOPC, DPPG, Cholesterol and Triolein; DEPC, DPPG, Cholesterol and Tricaprylin; DOPC:DOPE; DOPGDOPE; DSPE:DSPC; DSPE / DOPC, or any combination of at least two or more elements selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), succinyl-distearoyl-sn-glycero-3-phosphoethanolamine (Suc-DSPE), 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), di stearoylglycerol (DSG), dioleoylgylcerol (DOG), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), dilinoleoyl-glycero-phosphatidyl ethanolamine (DLPE), diacylglycerol (DAG), ceramide 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain.

[0248] For example, a particle of the pharmaceutical system that is a liposome can comprise a PEGylated lipid and a non-PEGylated lipid, such as a combination of PEG-DSPE and DSPC, or a combination of PEG-DSPE and DOPC. In some aspects, the liposomes can comprise PEGylated lipid and a non-PEGylated lipid at a ratio of about 1: 99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, about 10:90, about 11:89, about 12:88, about 13:87, about 14:86, about 15:85, about 16:84, about 17:83, about 18:82, about 19:81, about 20:80, about 21:79, about 22:78, about 23:77, about 24:76, about 57#14739410v125:75, about 26:74, about 27:73, about 28:72, about 29:71, about 30:70, about 31:69, about 32:68, about 33:67, about 34:66, about 35:65, about 36:64, about 37:63, about 38:62, about 39:61, about 40:60, about 41:59, about 42:58, about 43:57, about 44:56, about 45:55, about 46:54, about 47:53, about 48:52, about 49:51, about 50:50, about 51:49, about 52:48, about 53:47, about 54:46, about 55:45, about 56:44, about 57:43, about 58:42, about 59:41, about 60:40, about 61:39, about 62:38, about 63:37, about 64:36, about 65:35, about 66:34, about 67:33, about 68:32, about 69:31, about 70:30, about 71:29, about 72:28, about 73:27, about 74:26, about 75:25, about 76:24, about 77:23, about 78:22, about 79:21, about 80:20, about 81:19, about 82:18, about 83:17, about 84:16, about 85:15, about 86:14, about 87:13, about 88:12, about 89:11, about 90:10, about 91:9, about 92:8, about 93:7, about 94:6, about 95:5, about 96:4, about 97:3, about 98:2, about 99:1.

[0249] In some aspects, a particle of a pharmaceutical system that is a liposome and comprising PEG-DSPE and DOPC at a ratio of 10:90.VI. Lipid nanoparticles (LNP)

[0250] In one aspect, a lipid nanoparticle (LNP) is used as the second particle for payload delivery. In some embodiments, the LNP does not contain any targeting moiety. In some embodiments, the LNP comprises (i) a targeting moiety, such as a compound comprising an antibody targeting a desired cell type, a desired tissue type, and / or a desired organ type.

[0251] In some embodiments, the LNPs also comprise (ii) an ionizable cationic lipid.

[0252] In some embodiments, the LNPs further comprise (iii) a structural lipid.

[0253] In some embodiments, the LNP further comprise (iv) a neutral phospholipid.

[0254] In some embodiments, the LNPs further comprise (v) a free PEG-lipid.

[0255] In some embodiments, the LNPs further comprise (vi) a payload.

[0256] In some embodiments, the LNPs comprises (i) to (vi) or (ii) to (vi) and any combination thereof.A. Targeting moiety

[0257] In some aspects, the LNPs may contain a targeting moiety for specific cell / tissue / organ delivery as described herein. In some aspects, the conjugate comprises the compound of Formula (V): [Lipid] - [optional linker] - [antibody]. In some aspects, the Lipid of Formula (V) is a phospholipid. In some embodiments, the optional linker of Formula (V) is PEG. In some aspects, the antibody in the Formula (V) is an immunoglobulin single variable domain (ISVD, such as a VHH). In some aspects, the Formula contains a bioconjugation linker, such as maleimide.58#14739410v1

[0258] In some aspects, the conjugate is present in the LNP in a range of 0.001-0.5 mol percent, 0.001-0.1 mol%, 0.01-0.5 mol%, 0.05-0.5 mol%, 0.1-0.5 mol%, 0.1-0.3 mol%, 0.1-0.2 mol%, 0.2-0.3 mol%, of about 0.01 mol%, about 0.05 mol%, about 0.1 mol%, about 0.15 mol%, about 0.2 mol%, about 0.25 mol%, about 0.3 mol%, about 0.35 mol%, about 0.4 mol%, about 0.45 mol%, or about 0.5 mol%. In some aspects, the conjugate is present in the LNP in a range of 0.01 to 0.03 mol percent. In some aspects, the conjugate is present in the LNP in about 0.015 to about 0.016 mol percent.

[0259] In some embodiments, the conjugate is presented in the LNP at a density from about 0.4 to 11.4, about 1.9 to 9.5, about 3.8 to 7.6, about 4.6 to 6.5, about 5.3 to 6.1, about 3.8, or about 5.7 micromoles of conjugate per gram of payload in the LNP.B. Ionizable Cationic Lipids

[0260] Provided herein are ionizable cationic lipids that can be used to produce lipid nanoparticle compositions to facilitate the delivery of a payload (e.g., a nucleic acid, such as a DNA orRNA, such as an mRNA) disposed thereinto cells, e.g., mammalian cells, e.g., immune cells. In some embodiments, an ionizable cationic lipid of the present disclosure is used to produce LNPs as described herein. The ionizable cationic lipids have been designed to enable intracellular delivery of a nucleic acid, e.g, mRNA, to the cytosolic compartment of a target cell type and rapidly degrade into non-toxic components. The complex functionalities of the ionizable cationic lipids are facilitated by the interplay between the chemistry and geometry of the ionizable lipid head group, the hydrophobic “acyl-tail” groups and the linkers connecting the head group and the acyl tail groups. Typically, the substituents of the ionizable amine head group are chosen to tune the apparent pKa of amine head group in the LNP formulation to fall in the range of 6-8, such as between 6.2-7.4, or between 6.7-7.2. As such, the amine head groups remains strongly cationic under acidic formulation conditions (e.g., pH 4 - pH 5.5), neutral or slightly anionic or slightly cationic (typical zeta potential of 0 ± 5 mV) in physiological pH (7.4), but strongly cationic in the early and late endosomal compartments e.g., pH 5.5 - pH 7). The acyl-tail groups play a key role in fusion of the lipid nanoparticle with endosomal membranes and membrane destabilization through structural perturbation. The three-dimensional structure of the acyl-tail (determined by its length, and degree and site of unsaturation) along with the relative sizes of the head group and tail group are thought to play a role in promoting membrane fusion, and hence lipid nanoparticle endosomal escape (a key requirement for cytosolic delivery of a nucleic acid payload). The linker connecting the head59#14739410v1group and acyl tail groups is designed to degrade by physiologically prevalent enzymes (e.g., esterases, or proteases) or by acid catalyzed hydrolysis.

[0261] In some embodiments, the cationic lipid in a targeted LNP of the present disclosure has a concentration between about 10 mol% to about 60 mol% of the LNP, such as about 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or 60 mol%. In some embodiments, the cationic lipid in a targeted LNP of the present disclosure has a concentration between about 48.0 mol% to about 50.0 mol%, such as 48.0 mol%, 48.1 mol%, 48.2 mol%, 48.3 mol%, 48.4 mol%, 48.5 mol%, 48.6 mol%, 48.7 mol%, 48.8 mol%, 48.9 mol%, 49.0 mol%, 49.1 mol%, 49.2 mol%, 49.3 mol%, 49.4 mol%, 49.5 mol%, 49.6 mol%, 49.7 mol%, 49.8 mol%, 49.9 mol%, or 50.0 mol%.

[0262] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles is a LNP and comprises an ionizable lipid of a structure of Formula I (Lipid 15) and a salt thereof:Formula (I).

[0263] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles is a LNP and comprises an ionizable lipid of a structure of Formula IIFormula II (KC2), or a salt thereof.#14739410v1

[0264] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles is a LNP and comprises an ionizable lipid of a structure of Formula III"<"Formula III (KC3), or a salt thereof.

[0265] In some embodiments, the ionizable lipid is Formula IV (DLin-MC3-DMA):

[0266] In some aspects, a pharmaceutical system comprises at least two particles that are different from each other in their particle composition except that the at least two particles comprise a common element and at least one of the at least two particles is a LNP and comprises an ionizable lipid selected from DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DLin-K-DMA, SM-102, ALC-0315, DOTAP, DOTMA, OF-02, A6, DLin-MC3-DMA, A18-Iso5- 2DC18, 98N12-5, , C12-200, 9A1P9, cKK-E12, 7C1, G0-C14, OF-Deg-Lin, L319, 304013, 306-O12B, 3060iio, FTT5, 2,2(8, 8) 4C CH3, CL1, or LP01 (See, e.g., Han et al., Nat. Comm., 12: 7233, 2021).

[0267] In some aspects, the ionizable lipid comprises one or more lipids as described in International PCT Publication Nos. W02009028824A2, WO2011043913A2, WO2012091523A2, W02015074085A1, W02016081029A1, WO2017117530A1, WO2018118102A1, WO2018119163A1, WO2019045897A1, W02020069445A1, W02020106903 Al, WO2020219876A1, WO2021055892A1, WO2021163339A1, WO2021188389A2, WO2021202694 Al, W02022016070 Al, WO2022119883 A2, WO2022120388A2, WO2022159475 Al, WO2022207938A1, WO2022218957A1, WO2022246555A1, W02023023410A2, W02023091490A1, WO2023091787A1, WO2023114937A2, WO2023114944A1, WO2023115221 Al, WO2023121970A1, WO2023121975A1, WO2023141624A1, WO2023144798A1, WO2023183616A1, WO2023196444 Al, WO2023196527 A2, W02023207101A1, WO2023235589A1,#14739410v1WO2023240156A1, W02024008967A1, W02024010330A1, W02024019770 Al, and W02024035710A2.C. Structural lipid

[0268] In certain embodiments, the lipid blend of the lipid nanoparticle may comprise a structural lipid, such as a sterol component, for example, one or more sterols selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol. In certain embodiments, the sterol is cholesterol.

[0269] The sterol (e.g., cholesterol) may be present in the lipid blend in a range of 20-70 mole percent, 20-60 mole percent, 20-50 mole percent, 30-70 mole percent, 30-60 mole percent, 30-50 mole percent, 40-70 mole percent, 40-60 mole percent, 40-50 mole percent, 50-70 mole percent, 50-60 mole percent, or about 20 mole percent, about 25 mole percent, about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent or about 65 mole percent.D. Neutral Phospholipid

[0270] In certain embodiments, the lipid blend of the lipid nanoparticle may contain one or more neutral phospholipids. The neutral phospholipid can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), sphingomyelin (SM).

[0271] Other neutral phospholipids can be selected from the group consisting of distearoylphosphatidylethanolamine (DSPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dioleoyl-glycero-phosphoethanolamine (DOPE), dilinoleoyl-glycero-phosphocholine (DLPC), dimyristoyl-glycero-phosphocholine (DMPC), dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC), diundecanoyl-glycero-phosphocholine (DUPC), palmitoyl-oleoyl-glycero-phosphocholine (POPC), dioctadecenyl-glycero-phosphocholine, oleoyl-cholesterylhemisuccinoyl-glycero-phosphocholine, hexadecyl-glycero-phosphocholine, dilinolenoyl-glycero-phosphocholine, diarachidonoyl-glycero-3 -phosphocholine, didocosahexaenoyl-glycero-phosphocholine, or sphingomyelin.

[0272] The neutral phospholipid may be present in the lipid blend in a range of 1-10 mole percent, 1-15 mole percent, 1-20 mole percent, 1-30 mole percent, such as 1-12 mole percent, 1-10 mole percent, 3-15 mole percent, 15 - 25 mole percent, 3-12 mole percent, 3-10 mole 62#14739410v1percent, 4-15 mole percent, 4-12 mole percent, 4-10 mole percent, 4-8 mole percent, 5-15 mole percent, 5-12 mole percent, 5-10 mole percent, 6-15 mole percent, 6-12 mole percent, 6-10 more percent, or about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, about 5 mole percent, about 6 mole percent, about 7 mole percent, about 8 mole percent, about 9 mole percent, about 10 mole percent, about 11 mole percent, about 12 mole percent, about 13 mole percent, about 14 mole percent, about 15 mole percent, about 16 mole percent, about 17 mole percent, about 18 mole percent, about 19 mole percent, about 20 mole percent, about 21 mole percent, about 22 mole percent, about 23 mole percent, about 24 mole percent, about 25 mole percent, about 26 mole percent, about 27 mole percent, about 28 mole percent, about 29 mole percent, or about 30 mole percent.E. PEG-lipid

[0273] The lipid blend of the lipid nanoparticle may include one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. As noted above, free PEG-lipids can be included in the lipid blend to reduce or eliminate non-specific binding via a targeting group when a lipid-immune cell targeting group is included in the lipid blend.

[0274] A PEG lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, a PEG lipid may be PEG- dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl- phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

[0275] In certain embodiments, the blend may contain a free PEG-lipid that can be selected from the group consisting of PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEGDAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) and PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE). In some embodiments, the free PEG-lipid63#14739410v1comprises a diacylphosphatidylcholines comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain.

[0276] The PEG-lipid may be present in the lipid blend in a range of 1-10 mole percent, 1-8 mole percent, 1-7 mole percent, 1-6 mole percent, 1-5 mole percent, 1-4 mole percent, 1-3 mole percent, 2-8 mole percent, 2-7 mole percent, 2-6 mole percent, 2-5 mole percent, 2-4 mole percent, 2-3 mole percent, or about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, or about 5 mole percent. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0277] In some embodiments, the PEG-lipid may be present in the lipid blend in the range of 0.01-10 mole percent, 0.01-5 mole percent, 0.01-4 mole percent, 0.01-3 mole percent, 0.01-2 mole percent, 0.01-1 mole percent, 0.1-10 mole percent, 0.1-5 mole percent, 0.1-4 mole percent, 0.1-3 mole percent, 0.1-2 mole percent, 0.1-1 mole percent, 0.5-10 mole percent, 0.5-5 mole percent, 0.5-4 mole percent, 0.5-3 mole percent, 0.5-2 mole percent, 0.5-1 mole percent, 1-2 mole percent, 3-4 mole percent, 4-5 mole percent, 5-6 mole percent, or 1.25-1.75 mole percent. In some embodiments, the PET-lipid may be about 0.5 mole percent, about 1 mole percent, about 1.5 mole percent, about 2 mole percent, about 2.5 mole percent, about 3 mole percent, about 3.5 mole percent, about 4 mole percent, about 4.5 mole percent, about 5 mole percent, or about 5.5 mole percent of the lipid blend. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0278] In some embodiments, the lipid anchor length of PEG-lipid is C14 (as in PEG-DMG). In some embodiments, the lipid anchor length of PEG-lipid is C16 (as in DPG). In some embodiments, the lipid anchor length of PEG-lipid is Cl 8 (as in PEG-DSG). In some embodiments, the backbone or head group of PEG-lipid is diacyl glycerol or phosphoethanolamine. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0279] A LNP of the present disclosure may comprise one or more free PEG-lipid that is not conjugated to a targeting group, and a PEG-lipid that is conjugated to the targeting group. In some embodiments, the free PEG-lipid comprises the same or a different lipid as the lipid in the targeting group conjugate.F. Payload

[0280] The LNP compositions may comprise an agent, for example, a nucleic acid molecule for delivery to a cell (e.g., an immune cell) or tissue, for example, a cell (e.g., an immune cell) or tissue in a subject.

[0281] The LNP compositions of the present invention may include a nucleic acid, for example, a DNA or RNA, such as an mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA),64#14739410v1circRNA (circular RNA), ribozymes, decoy RNA or dicer substrate siRNA. It is contemplated that nucleic acids can contain naturally occurring components, such as, naturally occurring bases, sugars or linkage groups (e.g, phosphodiester linkage groups) or may contain non-naturally occurring components or modifications, (e.g, thioester linkage groups). For example, the nucleic acid can be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Furthermore, the nucleic acids can be linear or circular, or have any desired configuration. The LNP compositions can include multiple nucleic acid molecules, for example, multiple RNA molecules, which can be the same or different.

[0282] In certain embodiments, the payload is an mRNA. In certain embodiments, a particular LNP composition may contain a number of mRNA molecules that can be the same or different. In certain embodiments, one or more LNP compositions including one or more different mRNAs may be combined, and / or simultaneously contacted, with a cell. It is contemplated that an mRNA may include one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure. The mRNA may encode a receptor, such as a chimeric antigen receptor (CAR), for use in for example, an immune disorder, inflammatory disorder or cancer. In addition, the mRNA may encode an antigen for use in a therapeutic or prophylactic vaccine, for example, for treating or preventing an infection by a pathogen, for example, a microbial or viral pathogen, or for reducing or ameliorating the side effects caused directly or indirectly by such an infection.

[0283] In certain embodiments, the LNP composition may include one or more other components including, but not limited to, one or more pharmaceutically acceptable excipients, small hydrophobic molecules, therapeutic agents, carbohydrates, polymers, permeability enhancing molecules, and surface altering agents.

[0284] In some embodiments, the wt / wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting LNP composition is from about 1:1 to about 50:1. In certain embodiments, the wt / wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting composition is from about 5:1 to about 50:1. In certain embodiments, the wt / wt ratio is from about 5:1 to about 40:1. In certain embodiments, the wt / wt ratio is from about 10:1 to about 40: 1. In certain embodiments, the wt / wt ratio is from about 15 : 1 to about 25 : 1.

[0285] In certain embodiments, the RNA payload is an mRNA, tRNA, microRNA, or siRNA payload.

[0286] In certain embodiments, the lipid nanoparticle compositions are optimized for the delivery of RNA, e.g., mRNA, to a target cell for translation within the cell. An mRNA may65#14739410v1be a naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides.

[0287] The nucleobases may be selected from the non-limiting group consisting of adenine, guanine, uracil, cytosine, 7-methylguanine, 5 -methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, N1 -methylpseudouracil, hypoxanthine, and xanthine. In some embodiments, nucleobase is N1 -methylpseudouracil.

[0288] A nucleoside of an mRNA is a compound including a sugar molecule (e.g., a 5-carbon or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) in combination with a nucleobase. A nucleoside may be a canonical nucleoside (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or an analog thereof and may include one or more substitutions or modifications.

[0289] A nucleotide of an mRNA is a compound containing a nucleoside and a phosphate group or alternative group (e.g., boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl group, amidate, and glycerol). A nucleotide may be a canonical nucleotide (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine monophosphates) or an analog thereof and may include one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction of the nucleobase, sugar, and / or phosphate or alternative component. A nucleotide may include one or more phosphate or alternative groups. For example, a nucleotide may include a nucleoside and a triphosphate group. A "nucleoside triphosphate" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) may refer to the canonical nucleoside triphosphate or an analog or derivative thereof and may include one or more substitutions or modifications as described herein.

[0290] An mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translating sequence. An mRNA may include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosine in an mRNA may be 5-methylcytosine. In certain embodiments, one or more or all uridine bases may be Nl-methylpseudouridines.66#14739410v1

[0291] In certain embodiments, an mRNA may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0292] A cap structure or cap species is a compound including two nucleoside moi eties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or a cap analog. A cap species may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g., m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an antireverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m7Gpppm7G, m73'dGpppG, and m2702'GppppG.

[0293] Alternatively, or in addition, an mRNA may include a chain terminating nucleoside. For example, a chain terminating nucleoside may include those nucleosides deoxygenated at the 2' and / or 3' positions of their sugar group. Such species may include 3'-deoxyadenosine (cordycepin), 3 '-deoxyuridine, 3 '-deoxy cytosine, 3 '-deoxy guanosine, 3 '-deoxythymine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2', 3'-dideoxycytosine, 2', 3 '-dideoxy guanosine, and 2',3'-dideoxythymine.

[0294] Alternatively, or in addition, an mRNA may include a stem loop, such as a histone stem loop. A stem loop may include 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' untranslated region or a 3' untranslated region), a coding region, or a polyA sequence or tail.

[0295] Alternatively, or in addition, an mRNA may include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3' untranslated region of an mRNA.

[0296] An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell. In some embodiments, the mRNA may encode an antibody, enzyme, growth factor, hormone, cytokine, viral protein (e.g., a viral capsid protein), antigen, vaccine, or receptor. In some embodiments, the mRNA may encode an engineered receptor such as a CAR or an antigen for use in a therapeutic vaccine (e.g., a cancer vaccine) or a prophylactic vaccine (e.g., a vaccine 67#14739410v1for minimizing the risk or severity of an infection by a microbial or viral pathogen). In some embodiments, the mRNA encodes a polypeptide capable of regulating immune response in the immune cell. In some embodiments, the mRNA encodes a polypeptide capable of reprogramming the immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a Chimeric Antigen Receptor (CAR).

[0297] A lipid composition may be designed for one or more specific applications or targets. For example, an LNP composition may be designed to deliver mRNA to a particular cell, tissue, organ, or system or group thereof in a mammal's body, such as the renal system. Physiochemical properties of LNP compositions may be altered in order to increase selectivity for particular target site within a subject. For instance, particle sizes may be adjusted based on the fenestration sizes of different organs. The mRNA included in an LNP composition may also depend on the desired delivery target or targets. For example, an mRNA may be selected for a particular indication, condition, disease, or disorder and / or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery).

[0298] The amount of mRNA in a lipid composition may depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in an LNP may also depend on the size, composition, desired target, and other characteristics of the LNP composition. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. The amount of mRNA in an LNP composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0299] In some embodiments, the one or more mRNAs, lipids, and polymers and amounts thereof may be selected to provide a specific N:P ratio (the ratio of positively chargeable lipid or polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups). The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an mRNA. In general, a lower N:P ratio is preferred. A N:P ratio may be dependent on a specific lipid and its pKa. In certain embodiments, the mRNA and LNP composition, and / or their relative amounts may be selected to provide an N :P ratio from about 1 : 1 to about 30 : 1 , or from about 1 : 1 to about 20 : 1. In certain embodiments, the N:P ratio can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In certain embodiments, the N :P ratio may be from about 2: 1 to about 5 : 1. In certain embodiments, the N:P ratio may be about 4: 1. In other embodiments, the N:P ratio is from about 4: 1 to about 8:1. For example, the N:P ratio may be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1, or about 7.0:1.68#14739410v1

[0300] The amount of mRNA in a nanoparticle composition may depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in a nanoparticle composition may also depend on the size, composition, desired target, and other characteristics of the nanoparticle composition. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the wt / wt ratio of the lipid component to an mRNA in a nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50: 1. For example, the wt / wt ratio of the lipid component to an mRNA may be from about 10:1 to about 40:1. The amount of mRNA in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0301] The efficiency of encapsulation of an mRNA describes the amount of mRNA that is encapsulated or otherwise associated with a lipid composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of mRNA in a solution containing the lipid composition before and after breaking up the LNP composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free mRNA in a solution. For the LNP compositions of the invention, the encapsulation efficiency of an mRNA may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the encapsulation efficiency may be at least 80%.COMPOSITIONS

[0302] Provided herein are compositions. In some aspects, a composition comprises a pharmaceutical system described herein. In some aspects, a composition comprises a pharmaceutical system described herein and a carrier or an excipient.

[0303] In some aspects, a composition comprises a pharmaceutical system described herein and one or more pharmaceutically acceptable excipient.

[0304] In some aspects, a pharmaceutical composition comprises a conventional excipient and accessory ingredients, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a particle of the pharmaceutical system. For example, an excipient or accessory ingredient may be incompatible with a component of a particle of the pharmaceutical system and / or may result in any undesirable biological effect or otherwise deleterious effect.69#14739410v1

[0305] In some aspects, one or more excipients may make up greater than 50% of the total mass or volume of a pharmaceutical composition comprising a pharmaceutical system. For example, the one or more excipients may make up 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition.

[0306] In some aspects, a composition may contain at least about 0.1% of a pharmaceutical system, although the percentage of the particle may, of course, be varied. In some aspects, a composition may contain between about 1 or 2% and about 70% or 80% or more of the weight or volume of a particle of a pharmaceutical system. Naturally, the amount of a particle of a pharmaceutical system in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the particle of the pharmaceutical system. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art when preparing a pharmaceutical composition as described herein, and as such, a variety of dosages and treatment regimens may be designed. Pharmaceutical compositions as described herein, e.g., comprising a particle of a pharmaceutical system can be prepared by one of ordinary skill in the art by known methods.

[0307] In some aspects, a composition of the present disclosure comprises a plurality of a first particle as a decoy as described herein, and a plurality of a second particle for payload delivery as described herein. In some aspects, the first particle is a liposome as described herein and the second particle is an LNP as described herein. In some embodiments, the molar ratio between the first particle and the second particle in the composition is about 1:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, 104:1, 105:1, 106:1, 107:1, 108:1, 109:1, 110:1, 111:1, 112:1, 113:1, 114:1, 115:1, 116:1, 117:1, 118:1, 119:1, 120:1, 121:1, 122:1, 123:1, 124:1, 125:1, 126:1, 127:1, 128:1, 129:1, 130:1, 131:1, 132:1, 133:1, 134:1, 135:1, 136:1, 137:1, 138:1, 139:1, 140:1, 141:1, 142:1, 143:1, 144:1, 145:1, 146:1, 147:1, 148:1, 149:1, 150:1, 151:1, 152:1, 153:1, 154:1, 155:1, 156:1, 157:1, 158:1, 159:1, 160:1, 161:1, 162:1, 163:1, 164:1, 165:1, 166:1, 167:1, 168:1, 169:1, 170:1, 171:1, 172:1, 173:1, 174:1, 175:1, 176:1, 177:1, 178:1, 179:1, 180:1, 181:1, 182:1, 183:1, 184:1, 185:1, 186:1, 187:1, 188:1, 189:1, 190:1, 191:1, 192:1, 193:1,70#14739410v1194:1, 195:1, 196:1, 197:1, 198:1, 199:1, 200: 1, 201:1, 202:1, 203:1, 204:1, 205:1, 206:1, 207:1, 208:1, 209:1, 210:1, 211:1, 212:1, 213: 1, 214:1, 215:1, 216:1, 217:1, 218:1, 219:1, 220:1, 221:1, 222:1, 223:1, 224:1, 225:1, 226: 1, 227:1, 228:1, 229:1, 230:1, 231:1, 232:1, 233:1, 234:1, 235:1, 236:1, 237:1, 238:1, 239: 1, 240:1, 241:1, 242:1, 243:1, 244:1, 245:1, 246:1, 247:1, 248:1, 249:1, 250:1, 251:1, 252: 1, 253:1, 254:1, 255:1, 256:1, 257:1, 258:1, 259:1, 260:1, 261:1, 262:1, 263:1, 264:1, 265: 1, 266:1, 267:1, 268:1, 269:1, 270:1, 271:1, 272:1, 273:1, 274:1, 275:1, 276:1, 277:1, 278: 1, 279:1, 280:1, 281:1, 282:1, 283:1, 284:1, 285:1, 286:1, 287:1, 288:1, 289:1, 290:1, 291: 1, 292:1, 293:1, 294:1, 295:1, 296:1, 297:1, 298:1, 299:1, 300:1, 350:1, 400:1, 450:1, 500: 1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, or more.

[0308] In some aspects, the composition comprises more liposome over LNP as measured by molar percentage. For example, in some aspects, the composition comprises at least more than 50 mol% of liposome and less than 50 mol% of LNP. In some aspects, the composition comprises at least 60 mol% of the liposome and no more than 40 mol% of LNP; at least 70 mol% of the liposome and no more than 30 mol% of LNP; at least 80 mol% of the liposome and no more than 20 mol% of LNP; at least 90 mol% of the liposome and no more than 10 mol% of LNP; at least 90 mol% of the liposome and no more than 10 mol% of LNP; at least 90 mol% of the liposome and no more than 10 mol% of LNP; at least 91 mol% of the liposome and no more than 9 mol% of LNP; at least 92 mol% of the liposome and no more than 8 mol% of LNP; at least 93 mol% of the liposome and no more than 7 mol% of LNP; at least 94 mol% of the liposome and no more than 6 mol% of LNP; at least 95 mol% of the liposome and no more than 5 mol% of LNP; at least 96 mol% of the liposome and no more than 4 mol% of LNP; at least 97 mol% of the liposome and no more than 3 mol% of LNP; at least 98 mol% of the liposome and no more than 2 mol% of LNP; at least 99 mol% of the liposome and no more than 1 mol% of LNP; at least 99.9 mol% of the liposome and no more than 0.1 mol% of LNP; at least 99.999 mol% of the liposome and no more than 0.001 mol% of LNP. In some aspects, the molar ratio of the liposome and the LNP is about 1:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, 104:1, 105:1, 106:1, 107:1, 108:1, 109:1, 110:1, 111:1, 112:1, 113:1, 114:1, 115:1, 116:1,71#14739410v1117:1, 118:1, 119:1, 120:1, 121:1, 122:1, 123:1, 124:1, 125:1, 126:1, 127:1, 128:1, 129:1, 130:1, 131:1, 132:1, 133:1, 134:1, 135:1, 136:1, 137:1, 138:1, 139:1, 140:1, 141:1, 142:1, 143:1, 144:1, 145:1, 146:1, 147:1, 148:1, 149:1, 150:1, 151:1, 152:1, 153:1, 154:1, 155:1, 156:1, 157:1, 158:1, 159:1, 160:1, 161:1, 162:1, 163:1, 164:1, 165:1, 166:1, 167:1, 168:1, 169:1, 170:1, 171:1, 172:1, 173:1, 174:1, 175:1, 176:1, 177:1, 178:1, 179:1, 180:1, 181:1, 182:1, 183:1, 184:1, 185:1, 186:1, 187:1, 188:1, 189:1, 190:1, 191:1, 192:1, 193:1, 194:1, 195:1, 196:1, 197:1, 198:1, 199:1, 200:1, 201:1, 202:1, 203:1, 204:1, 205:1, 206:1, 207:1, 208:1, 209:1, 210:1, 211:1, 212:1, 213:1, 214:1, 215:1, 216:1, 217:1, 218:1, 219:1, 220:1, 221:1, 222:1, 223:1, 224:1, 225:1, 226:1, 227:1, 228:1, 229:1, 230:1, 231:1, 232:1, 233:1, 234:1, 235:1, 236:1, 237:1, 238:1, 239:1, 240:1, 241:1, 242:1, 243:1, 244:1, 245:1, 246:1, 247:1, 248:1, 249:1, 250:1, 251:1, 252:1, 253:1, 254:1, 255:1, 256:1, 257:1, 258:1, 259:1, 260:1, 261:1, 262:1, 263:1, 264:1, 265:1, 266:1, 267:1, 268:1, 269:1, 270:1, 271:1, 272:1, 273:1, 274:1, 275:1, 276:1, 277:1, 278:1, 279:1, 280:1, 281:1, 282:1, 283:1, 284:1, 285:1, 286:1, 287:1, 288:1, 289:1, 290:1, 291:1, 292:1, 293:1, 294:1, 295:1, 296:1, 297:1, 298:1, 299:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, or more. In some aspects, the ratio between liposome and LNP in the composition is about 10: 1 to about 100: 1, such as about 10: 1 to 20: 1, about 20: 1 to 30:1, about 30:1 to 40:1, about 40:1 to 50:1, about 50:1 to 60:1, about 60:1 to 70:1, about 70:1 to 80:1, about 80:1 to 90:1, about 90:1 to 100:1.METHODSMethods of Making Particles of the Pharmaceutical System

[0309] Liposomes and LNPs of the present disclosure can be made using a method available. In some aspects, lipid nanoparticles are produced by using either rapid mixing by an orbital vortexer or by microfluidic mixing. Orbital vortexer mixing is accomplished by rapid addition of a lipid solution in ethanol to an aqueous solution of a nucleic acid followed immediately by vortexing at 2,500 rpm. In some aspects, a lipid nanoparticle is produced using a microfluidic mixing step. In some aspects, microfluidic mixing is achieved mixing aqueous and organic streams at a controlled flow rate in a microfluidic channel using, e.g., a NanoAssemblr device and microfluidic chips featuring optimized mixing chamber geometry (Precision Nanosystems, Vancouver, BC). In some aspects, a lipid nanoparticle is produced using a microfluidic mixing step to rapidly mix an ethanolic lipid solution and an aqueous nucleic acid solution, resulting in encapsulation of the nucleic acid in the solid lipid nanoparticles.72#14739410v1

[0310] In some aspects, LNPs encapsulating an mRNA payload are prepared by mixing an aqueous mRNA solution and a lipid blend solution (e.g., containing cationic ionizable lipid, neutral lipid, PEG-lipid, and helper lipid) using an in-line T-mixing process. In some aspects, the mRNA diluted in a buffer and lipid components dissolved are mixed.

[0311] In some aspects, a high-energy method, also known as top-down method, such as high-pressure homogenization (e.g., ultrasonic homogenization), is used to make the LNPs.

[0312] In some aspects, a low-energy method, where nanoparticles will precipitate from homogeneous systems, is used to make the LNPs. In some aspects, the method comprises a microemulsion and / or a supercritical fluid step (SCFs).

[0313] In some aspects, a solvent-based method, also known as bottom-up, such as microfluidics, is used to make the LNPs.

[0314] In some aspects, a nanoprecipitation-based method is used to produce the LNPs. The method is based on self-assembly / nanoprecipitation. The underlying principle is mixing an aqueous phase, containing the hydrophilic API or oligonucleotide to be encapsulated, with a water-miscible solvent, such as ethanol, containing the lipids.

[0315] In some aspects, a solvent evaporation method, such as ethanol injection method or thin film hydration / Bangham method is used to produce the LNPs. Solvent evaporation methods are very well adapted for the generation of lipid nanoparticle-trapping molecules with low miscibility in water.

[0316] In some aspects, an impingement jet mixing / T -junction method is used to produced the LNPs.

[0317] The above-mentioned methods can be modified or combined as needed.Method of Use

[0318] Provided herein is a method of delivering a payload into a cell using a pharmaceutical system or the pharmaceutical composition of the present disclosure, comprising contacting the cell with a pharmaceutical system or the pharmaceutical composition as described herein. In some aspects, the cell is an animal cell. In some aspects, the cell is a mammal cell. In some aspects, the mammal is a human cell. In some aspects, the payload is a nucleic acid sequence. In some aspects, the payload is a DNA or an RNA. In some aspects, the payload is an mRNA.

[0319] Also provided herein is a method of delivering a payload into a subject using a pharmaceutical system or the pharmaceutical composition of the present disclosure, comprising administering a pharmaceutical system or the pharmaceutical composition as described herein to the subject. In some aspects, the subject is an animal. In some aspects, the subject is a 73#14739410v1mammal. In some aspects, the mammal is a human. In some aspects, the payload is a nucleic acid sequence. In some aspects, the payload is a DNA or an RNA. In some aspects, the payload is an mRNA.

[0320] Also provided herein is a method of expressing an mRNA in a cell using a pharmaceutical system or the pharmaceutical composition of the present disclosure, comprising contacting the cell with a pharmaceutical system or the pharmaceutical composition as described herein, wherein a nanoparticle of the pharmaceutical system or the pharmaceutical composition. In some aspects, the cell is an animal cell. In some aspects, the cell is a mammal cell. In some aspects, the mammal is a human cell. In some aspects, the payload is a nucleic acid sequence. In some aspects, the payload is a DNA or an RNA. In some aspects, the payload is an mRNA.

[0321] Also provided herein is a method of increasing expression of a nucleic acid in a cell or in a subject using a pharmaceutical system or the pharmaceutical composition of the present disclosure, comprising contacting the cell or the subject with a pharmaceutical system or the pharmaceutical composition as described herein, wherein the pharmaceutical system or the pharmaceutical composition comprises two particles, and the first particle is used as a decoy and the second particle comprises the nucleic acid. In some embodiments, expression of the nucleic acid is increased compared to the expression of the same nucleic acid when delivered using the same second particle but without the first particle or with a different first particle. In some aspects, the cell is an animal cell. In some aspects, the cell is a mammal cell. In some aspects, the mammal is a human cell. In some aspects, the subject is an animal. In some aspects, the subject is a mammal. In some aspects, the mammal is a human. In some aspects, the payload is a nucleic acid sequence. In some aspects, the payload is a DNA or an RNA. In some aspects, the payload is an mRNA.

[0322] Also provided herein is a method of reducing immunogenicity of a composition comprising a therapeutic agent in a subject using a pharmaceutical system or the pharmaceutical composition of the present disclosure, comprising administering a pharmaceutical system or the pharmaceutical composition as described herein to the subject, wherein the pharmaceutical system or the pharmaceutical composition comprises two particles, and the first particle is used as a decoy and the second particle comprises the therapeutic agent. In some embodiments, immunogenicity of the second particle comprising the therapeutic agent when administered using the pharmaceutical system or the pharmaceutical composition is reduced compared immunogenicity of the same particle when the same second particle is delivered to the subject without the first particle or with a different first particle. In some 74#14739410v1aspects, the subject is an animal. In some aspects, the subject is a mammal. In some aspects, the mammal is a human. In some aspects, the payload is a nucleic acid sequence. In some aspects, the payload is a DNA or an RNA. In some aspects, the payload is an mRNA.

[0323] Provided herein is a novel strategy of using a first and a second particles together in a pharmaceutical system described herein to improve the activity, tolerability, specificity, and / or target engagement of a particle, e.g., a therapeutic particle of the pharmaceutical system comprising a therapeutic agent.

[0324] In some aspects, provided is a method of delivering two unique lipid-based nanoparticles to a subject in need thereof, wherein the two lipid-based nanoparticles are delivered together in a composition comprising the pharmaceutical system described herein or separately in the context of a pharmaceutical system described herein.

[0325] In some aspects, a method of the present disclosure described above comprises delivering a payload to a subject, the method comprising administering a pharmaceutical system as described herein or a composition as described herein to a subject.

[0326] In some aspects, a method of the present disclosure described above comprises delivering a pharmaceutical system as described herein to a subject in need thereof. In some aspects, the pharmaceutical system comprises at least two particles. In some aspects, the pharmaceutical system comprises a first particle and a second particle that share at least one common element.

[0327] In some aspects, a method of the present disclosure described above comprises delivering a pharmaceutical system comprising a first particle and a second particle that share at least one common element, wherein both the first particle and second particle bind at least one opsonin.

[0328] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first particle is, e.g., a liposome and the second particle is, e.g., an LNP, wherein the liposome and the LNP share at least one common element. In some aspects, a method comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first particle is, e.g., a liposome and the second particle is, e.g., an LNP, wherein the liposome and the LNP share at least one common element and both bind at least one opsonin.

[0329] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, where the first particle is, e.g., a liposome and the second particle is, e.g., an LNP, wherein the liposome and the LNP share at least one common element and both bind the same opsonin.75#14739410v1

[0330] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, where the first particle is, e.g., a liposome and the second particle is, e.g., an LNP, wherein the liposome and the LNP share at least one common element and both bind a different opsonin. In some aspects, a method comprises administering a pharmaceutical system that comprises a first and a second particle, where the first particle is, e.g., a liposome and the second particle is, e.g., an LNP, wherein the liposome and the LNP share at least one common element and both bind a different opsonin, wherein the different opsonins share a common structural element.

[0331] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a liposome and an LNP, wherein the liposome and the LNP share at least one common element and the liposome and LNP both bind the same opsonin. In some aspects, a method comprises administering a pharmaceutical system that comprises a liposome and an LNP, wherein the liposome and the LNP share at least one common element and the liposome and LNP both bind a different opsonin, wherein the different opsonins share a common structural element.

[0332] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered simultaneously. In some aspects, a method comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered simultaneously in one composition.

[0333] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered but in two separate compositions.

[0334] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered but in two separate compositions, wherein the first and second particle are co-administered with a time interval of about 0.5 minutes to about 4.9 minutes between the administration of the first particle and the administration of the second particle.

[0335] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered sequentially.

[0336] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the 76#14739410v1first and second particle are administered sequentially with a time interval of about 5 minutes to about 30 minutes between the administration of the first particle and the administration of the second particle.

[0337] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered sequentially with a time interval of about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks, 3 weeks or 4 weeks.

[0338] In some aspects, a method of the present disclosure described herein comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered sequentially, wherein the sequential administration is by intravenous (IV) infusion (e.g., continuous intravenous infusion).

[0339] In some embodiments, where a pharmaceutical composition is administered by intravenous infusion (e.g., continuous intravenous infusion), the first particle and the second particle are administered sequentially with a time interval of about 0 minutes to about 60 minutes between the end of the infusion of the first particle and the beginning of the infusion of the second particle. In some embodiments, where a pharmaceutical composition is administered by intravenous infusion (e.g., continuous intravenous infusion), the first particle and the second particle are administered sequentially with a time interval of about 0-5 minutes, 0-10 minutes, 0-20 minutes, 0-30 minutes, 0-60 minutes, 5-10 minutes, 5-20 minutes, 5-30 minutes, 5-60 minutes, 10-20 minutes, 10-30 minutes, 10-60 minutes, 30-60 minutes, or 0 minutes, 1 minute, 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes between the end of the infusion of the first particle and the beginning of the infusion of the second particle.77#14739410v1

[0340] In some embodiments, where a pharmaceutical composition is administered by intravenous infusion (e.g., continuous intravenous infusion), the first particle and the second particle are administered sequentially with a time interval of about 5 minutes to about 60 minutes between the end of the infusion of the first particle and the beginning of the infusion of the second particle, wherein the intravenous line is flushed during the time interval between the end of the infusion of the first particle and the beginning of the infusion of the second particle. In some embodiments, the intravenous line is flushed with normal saline.

[0341] In some embodiments, where a pharmaceutical composition is administered by intravenous infusion (e.g., continuous intravenous infusion), the first particle and the second particle are administered sequentially with a time interval of about 0 minutes to about 60 minutes between the end of the infusion of the first particle and the beginning of the infusion of the second particle, wherein the intravenous line is not flushed during the time interval between the end of the infusion of the first particle and the beginning of the infusion of the second particle.

[0342] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered sequentially, wherein the composition comprising the first particle is administered before the composition comprising the second particle.

[0343] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered sequentially, wherein the composition comprising the second particle is administered before the composition comprising the first particle.

[0344] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered by intravenous (IV) (e.g., intravenous infusion, such as continuous intravenous infusion), subcutaneous, intramuscular, intraperitoneal, intraocular, intravitreal, subretinal, intracerebro-ventricular, intrathecal, intracistemal administration, and / or via inhalation. In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered by intravenous infusion. In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered by continuous intravenous infusion.78#14739410v1

[0345] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered by local administration including topically, or by direct injection to one or more cells, tissues, or organs.

[0346] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered in separate compositions and wherein the first and second particle are administered by intravenous injection.

[0347] In some aspects, a method of the present disclosure described above comprises coadministering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered in separate compositions and wherein the first and second particle are administered by intravenous injection. In some aspects, a method comprises co-administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered in a same composition and wherein the first and second particle are administered by intravenous injection.

[0348] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered in separate compositions and wherein the first and second particle are administered by intravenous infusion. In some aspects, a method comprises co-administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered in separate compositions and wherein the first and second particle are administered by intravenous infusion. In some aspects, a method comprises co-administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are co-administered in a same composition and wherein the first and second particle are administered by intravenous infusion.

[0349] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first and second particle are administered in separate compositions and wherein the first and second particle are administered via different routes of administration. For example, in some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the first particle is administered by intravenous injection and the second particle is administered by intramuscular injection. Or, in some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system that comprises a first and a second particle, wherein the 79#14739410v1first particle is administered by intramuscular injection and the second particle is administered by intravenous injection.

[0350] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein that comprises a particle that induces an immune reaction in the subject upon administration of the particle to the subject. In some aspects, a method of the present disclosure described above comprises administering a particle that induces a humoral immune response. In some aspects, a method comprises administering a particle that induces an IgM antibody response. In some aspects, a method comprises administering a particle that induces an IgG antibody response. In some aspects, a method comprises administering a particle that binds to an opsonin in the subject. In some aspects, the opsonin is an antibody. In some aspects, the opsonin is a complement factor. In some aspects, the opsonin is a lipoprotein.

[0351] In some aspects, a method of the present disclosure described above comprises administering a first particle that comprises PEG to a subject. In some aspects, a method comprises administering a first PEG containing particle that induces an IgM antibody response. In some aspects, a method comprises administering a first PEG containing particle that induces an IgG antibody response. In some aspects, the method further comprises administering a second particle that comprise PEG to the subject. In some aspects, the second particle comprises a payload, such as a DNA or an RNA. In some aspects, administration of the first particle reduces immunogenicity against the second particle in the subject.

[0352] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject that has a pre-existing immune response against a component of the pharmaceutical system.

[0353] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject that has been determined to have a pre-existing immune response against a component of the pharmaceutical system.

[0354] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject that may have a preexisting immune response against a component of the pharmaceutical system.

[0355] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system binds to an opsonin in the subject. In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system 80#14739410v1has been configured to bind an opsonin in the subject. In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject determined to have a pre-existing immune response to a component of the pharmaceutical system, wherein the pre-existing immune response comprises an opsonin, wherein at least one of the at least two particles of the pharmaceutical system has been configured to bind to the pre-existing opsonin in the subject.

[0356] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject determined to have a preexisting immune response to a component of the pharmaceutical system, wherein the preexisting immune response comprises an antibody, wherein at least one of the at least two particles of the pharmaceutical system has been configured to bind the pre-existing antibody in the subject.

[0357] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject determined to have a preexisting immune response to a component of the pharmaceutical system, wherein the preexisting immune response comprises a complement factor, wherein at least one of the at least two particles of the pharmaceutical system has been configured to bind the pre-existing complement factor in the subject.

[0358] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject determined to have a preexisting immune response to a component of the pharmaceutical system, wherein the preexisting immune response comprises a lipoprotein, wherein at least one of the at least two particles of the pharmaceutical system has been configured to bind the pre-existing lipoprotein in the subject.

[0359] In some aspects, a method of the present disclosure described above comprises administering a first particle of a pharmaceutical system described herein to a subject determined to have a pre-existing immune response to a component of the pharmaceutical system, wherein said component is a shared element present in the first and second particle of the pharmaceutical system. In some aspects, the method further comprises administering a second particle of the pharmaceutical system to the subject, wherein the pre-existing immune response determined to be present in the subject comprises an opsonin, and the pharmaceutical system has been configured such that the common element bind the pre-existing opsonin when administered to the subject.81#14739410v1

[0360] In some aspects, a method of the present disclosure described above comprises administering a first particle of a pharmaceutical system described herein to a subject determined to have a pre-existing immune response to a component of the pharmaceutical system, wherein said component is a shared element present in the first and second particle of the pharmaceutical system. In some aspects, the method further comprises administering a second particle of the pharmaceutical system to the subject, wherein the pre-existing immune response determined to be present in the subject comprises an antibody, and the pharmaceutical system has been configured such that the common element bind the pre-existing antibody when administered to the subject. In some aspects, the pre-existing immune response determined to be present in the subject comprises anti -PEG antibodies. In some aspects, the antibodies are either IgM or IgG subtype, or a mixture thereof.

[0361] In some aspects, a method of the present disclosure described above comprises administering a first particle of a pharmaceutical system described herein to a subject determined to have a pre-existing immune response to a component of the pharmaceutical system, wherein said component is a shared element present in the first and second particle of the pharmaceutical system. In some aspects, the method further comprises administering a second particle of the pharmaceutical system to the subject, wherein the pre-existing immune response determined to be present in the subject comprises a complement factor, and the pharmaceutical system has been configured such that the common element bind the preexisting complement factor when administered to the subject.

[0362] In some aspects, a method of the present disclosure described above comprises administering a first particle of a pharmaceutical system described herein to a subject determined to have a pre-existing immune response to a component of the pharmaceutical system, wherein said component is a shared element present in the first and second particle of the pharmaceutical system. In some aspects, the method further comprises administering a second particle of the pharmaceutical system to the subject, wherein the pre-existing immune response determined to be present in the subject comprises a lipoprotein, and the pharmaceutical system has been configured such that the common element bind the preexisting lipoprotein when administered to the subject

[0363] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system has been configured to bind an opsonin in the subject. In some aspects, the binding can be specific or unspecific.82#14739410v1

[0364] In some aspects, a method comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system has been configured to bind an antibody in the subject.

[0365] In some aspects, a method comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system has been configured to bind a complement factor in the subject.

[0366] In some aspects, a method comprises administering a pharmaceutical system described herein to a subject wherein at least one particle of the pharmaceutical system has been configured to bind a lipoprotein in the subject.

[0367] In some aspects, the method of the present disclosure described above comprises administering a liposome first and thereafter administering an LNP. In some aspects, the method comprises co-administering a liposome first and thereafter administering an LNP. In some aspects, the method comprises administering an LNP first and thereafter administering the liposome.

[0368] In some aspects, the method of the present disclosure described above comprises administering a liposome that is designed to bind an opsonin that would otherwise bind to an LNP that is co-administered with the liposome. In some aspects, the method comprises administering to a subject a liposome comprising a PEG, which liposome binds a PEG antibody in the subject, and further administering a LNP to the subject, which LNP comprises a PEG, wherein the LNP does not bind a PEG antibody.

[0369] In some aspects provided is a method of reducing immunogenicity of a first particle in a subject in need thereof, the method comprising administering to the subject a second particle that is effective in reducing the immunogenicity of the first particle administered to the subject.

[0370] In some aspects provided is a method of reducing immunogenicity of a lipid nanoparticle (LNP) in a subject in need thereof, the method comprising administering to the subject a liposome that is effective in reducing the immunogenicity of the LNP administered to the subject.

[0371] In some aspects, the method of the present disclosure described above comprises administering a liposome that is designed to bind an opsonin to a subject, which liposome binds an opsonin in the subject, and further administering a LNP to the subject, which LNP does not bind an opsonin in the subject. In some aspects, the method comprises administering a liposome that is designed to bind a complement protein in a subject. In some aspects, the method comprises administering a liposome that is designed to bind a lipoprotein in a subject.83#14739410v1

[0372] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system comprising a first particle that is designed to have membrane defects that bind proteins, including opsonin proteins.

[0373] In some aspects, a liposome is designed to have membrane defects that bind proteins, including opsonin proteins.

[0374] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system comprising a first particle that is designed to have a low melting temperature and binds proteins, including opsonin proteins.

[0375] In some aspects, a liposome is designed to have a low melting temperature and binds proteins, including opsonin proteins.

[0376] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system comprising a first particle that is designed to comprise a large number of PEG moieties on its surface and binds PEG proteins. In some aspects, the method further comprises administering a second particle of the pharmaceutical system, wherein the second particle is designed to comprise a lower number of PEG moieties on its surface compared to the first particle.

[0377] In some aspects, a liposome is designed to comprise a large amount of PEG on its surface such that the liposome binds PEG antibodies present in a subject.

[0378] In some aspects, the method of the present disclosure described above comprises administering a liposome that is designed to comprise a large amount of PEG on its surface and a LNP that comprises a lower amount of PEG than the liposome.

[0379] In some aspects, the method of the present disclosure described above comprises administering a pharmaceutical system to a subject, wherein the pharmaceutical system comprises a first particle, e.g., a liposome that is administered at a higher concentration to the subject than a second particle, e.g., a LNP. In some aspects, the method comprises administering a pharmaceutical system to a subject, wherein a liposome of the pharmaceutical system is administered at a higher concentration to the subject prior to, with, or after the LNP of the pharmaceutical system is administered. In some aspects, the second particle comprises a therapeutic agent. In some aspects, the molar ratio of the first particle and the second particle is about 1:1, 2:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1,84#14739410v180:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 101:1, 102:1, 103:1, 104:1, 105:1, 106:1, 107:1, 108:1, 109:1, 110:1, 111:1, 112:1, 113:1, 114:1, 115:1, 116:1, 117:1, 118:1, 119:1, 120:1, 121:1, 122:1, 123:1, 124:1, 125:1, 126:1, 127:1, 128:1, 129:1, 130:1, 131:1, 132:1, 133:1, 134:1, 135:1, 136:1, 137:1, 138:1, 139:1, 140:1, 141:1, 142:1, 143:1, 144:1, 145:1, 146:1, 147:1, 148:1, 149:1, 150:1, 151:1, 152:1, 153:1, 154:1, 155:1, 156:1, 157:1, 158:1, 159:1, 160:1, 161:1, 162:1, 163:1, 164:1, 165:1, 166:1, 167:1, 168:1, 169:1, 170:1, 171:1, 172:1, 173:1, 174:1, 175:1, 176:1, 177:1, 178:1, 179:1, 180:1, 181:1, 182:1, 183:1, 184:1, 185:1, 186:1, 187:1, 188:1, 189:1, 190:1, 191:1, 192:1, 193:1, 194:1, 195:1, 196:1, 197:1, 198:1, 199:1, 200:1, 201:1, 202:1, 203:1, 204:1, 205:1, 206:1, 207:1, 208:1, 209:1, 210:1, 211:1, 212:1, 213:1, 214:1, 215:1, 216:1, 217:1, 218:1, 219:1, 220:1, 221:1, 222:1, 223:1, 224:1, 225:1, 226:1, 227:1, 228:1, 229:1, 230:1, 231:1, 232:1, 233:1, 234:1, 235:1, 236:1, 237:1, 238:1, 239:1, 240:1, 241:1, 242:1, 243:1, 244:1, 245:1, 246:1, 247:1, 248:1, 249:1, 250:1, 251:1, 252:1, 253:1, 254:1, 255:1, 256:1, 257:1, 258:1, 259:1, 260:1, 261:1, 262:1, 263:1, 264:1, 265:1, 266:1, 267:1, 268:1, 269:1, 270:1, 271:1, 272:1, 273:1, 274:1, 275:1, 276:1, 277:1, 278:1, 279:1, 280:1, 281:1, 282:1, 283:1, 284:1, 285:1, 286:1, 287:1, 288:1, 289:1, 290:1, 291:1, 292:1, 293:1, 294:1, 295:1, 296:1, 297:1, 298:1, 299:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, or more. In some aspects, the ratio between liposome and LNP in the composition is about 10:1 to about 100:1, such as about 10:1 to 20:1, about 20:1 to 30:1, about 30:1 to 40:1, about 40:1 to 50:1, about 50:1 to 60:1, about 60:1 to 70:1, about 70:1 to 80:1, about 80:1 to 90:1, about 90:1 to 100:1

[0380] In some aspects, the method of the present disclosure described above comprises administering a pharmaceutical system to a subject, wherein a liposome of the pharmaceutical system is administered at a dose sufficiently high to tolerize and prevent an anti-PEG immune response in a subject. In some aspects, the method comprises administering a pharmaceutical system to a subject, wherein a liposome of the pharmaceutical system is administered at a dose sufficiently high prevent the clearance of a subsequently administered therapeutic.

[0381] In aspects, provided is a method for tolerizing a subject to an LNP. In some aspects, a method comprises administering to a subject that comprises pre-existing immunity to a component of a pharmaceutical system or composition described herein a liposome of a pharmaceutical system described herein. In some aspects, a subject that comprises pre-existing immunity to a molecule that shares immunogenic similarity with a component of a pharmaceutical system or composition described herein is administered a liposome of a system or composition described herein. The term “immunogenic similarity,” as used herein refers to 85#14739410v1structural similarity between two molecules that causes an immune response against one molecule to also target the other molecule. In some aspects, a structural similarity between two molecules that causes an immune response against one molecule to also target the other molecule is limited to a portion of a molecule. For example, in some aspects, a subject is treated with a system or composition described herein, which subject has antibodies that bind a PEG molecule, which antibodies were generated in the subject after the subject received a therapeutic and / or cosmetic that contained PEG.

[0382] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject that has previously received a particle comprising PEG or a lipid nanoparticle comprising PEG and has developed anti-PEG antibodies. In some aspects, a method comprises administering a pharmaceutical system described herein to a subject that has not previously received a particle comprising PEG or lipid nanoparticle comprising PEG described herein but the subject has nevertheless anti-PEG antibodies.

[0383] In some aspects, a method of the present disclosure described above comprises administering a pharmaceutical system described herein to a subject that has anti-PEG IgM antibodies. In some aspects, a method comprises administering a pharmaceutical system described herein to a subject that has anti-PEG IgG antibodies. In some aspects, a method comprises administering a pharmaceutical system described herein to a subject that has anti-PEG IgM and anti-PEG IgG antibodies.

[0384] In some aspects, a subject is administered a pharmaceutical system described herein and / or a composition described herein once. In some aspects, a subject is administered a pharmaceutical system described herein and / or a composition described herein more than once, e.g., between about 2 times and about 10 times. In some aspects, a subject is administered a pharmaceutical system described herein and / or a composition described herein three times.

[0385] In some aspects, a liposome of a pharmaceutical system and / or composition described herein comprises a positively charged helper lipid. In some aspects, a liposome comprising a positively charged helper lipid binds negatively charged proteins present, e.g., in a plasma of a subject. In some embodiments, the positive charge and negative charge referred to herein are net charge at physiological pH.

[0386] In some aspects, a liposome of a pharmaceutical system and / or composition described herein comprises a negatively charged helper lipid. In some aspects, a liposome comprising a negatively charged helper lipid binds positively charged proteins present, e.g., in a plasma of a86#14739410v1subject. In some embodiments, the positive charge and negative charge referred to herein are net charge at physiological pH.

[0387] Further provided is a method of reducing immunogenicity of a particle, e.g., lipid nanoparticle in a subject in need thereof. In some aspects, the method comprises coadministering with a particle, e.g., a lipid nanoparticle a liposome described herein, wherein the liposome binds proteins that are involved in inducing an immune response to a particle, lipid nanoparticle and / or viral vector particle.

[0388] Further provided is a method of delivering a payload to a target cell or tissue, e.g., a target cell or tissue in a subject.

[0389] In some aspects, provided is a method of treating a disease or disorder in subject. In some aspects, the disease or disorder is treated by delivering the payload of a particle, e.g., a lipid nanoparticle to a cell of a subject.

[0390] In some aspects, the method comprises contacting a cell with a particle, e.g., a lipid nanoparticle, described herein, e.g., a particle and / or lipid nanoparticle comprising an mRNA encoding a polypeptide of interest. The amount of a particle, e.g., lipid nanoparticle contacted with a cell, and / or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the particle, lipid nanoparticle, viral vector particle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors.Uses of the Pharmaceutical System

[0391] In some aspects, the disclosure provides a pharmaceutical system and / or a composition as described above for use in the manufacture of a medicament for treating a disease in a subj ect in need thereof.

[0392] In some aspects, disclosure provides a pharmaceutical system and / or a composition as described above for use in delivering a payload to a cell or a subject.

[0393] In some aspects, disclosure provides a pharmaceutical system and / or a composition as described above for use in expressing a nucleic acid sequence in a cell or a subject.

[0394] In some aspects, disclosure provides a pharmaceutical system and / or a composition as described above for use in reducing immunogenicity of a composition in a cell or a subject.KITS

[0395] In some aspects, provided is a kit for treating a disorder. In some aspects, a kit comprises a pharmaceutical system comprising at least two particles, wherein a first and a 87#14739410v1second particle share at least one common element as described herein. In some aspects, a kit comprises a system comprising a liposome and a therapeutic, and instructions for preparing and delivering the pharmaceutical system and / or composition to a subject in need thereof.EXAMPLES EXAMPLE 1. Preparation of LNPs by T-Mixing using Cationic Lipid 15

[0396] LNPs were produced using cationic Lipid 15 as described previously in WO / 2023 / 240156, which is hereby incorporated by reference in its entirety for all purposes. LNPs encapsulating an mRNA payload were prepared by mixing an aqueous mRNA solution and an ethanolic lipid blend solution (containing ionizable Lipid 15, DSPC, PEG-lipid, and, optionally, cholesterol) using an in-line T-mixing process. The mRNA (mCherry mRNA or CAR mRNA) stock solution was diluted in pH 4.5 acetate buffer. The lipid components were dissolved in anhydrous ethanol at the relative ratios set forth in TABLE 1 below. The mRNA and lipid solutions were mixed at a 3 : 1 v / v ratio of mRNA solution to lipid solution using inline T-mixing to provide an overall LNP composition of 30.46 mmol of total lipids per g of mRNA. The resulting suspension was diluted with aqueous buffer to reduce the total ethanol concentration to 8% v / v or less before proceeding to ethanol removal and buffer exchange. Following dilution, ethanol removal and buffer exchange was performed on the resulting LNP suspension using discontinuous diafiltration with centrifugal ultrafiltration devices (CUF) or continuous diafiltration by TFF. T cell targeting LNPs were prepared using DSPE-PEG- VHH conjugate , and non-targeting LNPs were prepared by performing a similar insertion procedure using micelles lacking the VHH.TABLE 188#14739410v1

[0397] The resulting LNP batches were characterized using methods described in WO / 2023 / 240156, and the results are given in TABLE 2 below.TABLE 2EXAMPLE 2. Preparation of Liposomes by Extruder using Exemplary Lipid Combination

[0398] Exemplary liposomes were produced using different lipid combinations described in Table 3 and Table 4.TABLE 3TABLE 489#14739410v1

[0399] Each batch of liposomes was prepared using an ethanol injection method. Briefly, the lipid components were dissolved in anhydrous ethanol at a total lipid concentration of 616 mM in the molar ratio given in TABLE 4. The lipid solution was pre-heated to 70°C, loaded in a glass syringe, and then injected rapidly into MBS buffer (25 mM pH 6.5 MES buffer with 150 mMNaCl) stirred at 800 rpm and held at 70°C. The two solutions were mixed at a 12.33:1 v / v ratio of MBS buffer to lipid solution. The resulting suspension was held at room temperature under mixing at 500 rpm for a minimum of 30 minutes before proceeding to the extrusion step.

[0400] After the ethanol injection process, the solution was reheated at 70°C for 20 mins and passed through an extruder (LIPEX 10 mL Thermobarrel extruder, part no. 002282) at 70°C and under 200 psi pressure. A polyester filter drain disk (Whatman, Part No. 2300600) and a single 100 nm pore-size polycarbonate extrusion membrane (Whatman, Part No. 110605) were used to perform the extrusion process. The temperature of the extruder was maintained at 70°C during the process using hot water circulation through the jacket. The extrusion process was repeated three to four times to reduce the particle size in the range of -80-120 nm and PDI <0.1.

[0401] Following the extrusion process, ethanol removal was performed on the resulting liposome solution using a size exclusion PD 10 column (Cytiva Disposable PD-10 Desalting Columns, part no. 45-000-148). Each column was washed thoroughly with 20 mL MBS buffer and 2.5 mL of liposome solution was loaded in each column. The elute was collected by adding 2.5 mL of MBS buffer to the column and another 1 mL of buffer to collect the remaining liposome from the column. Collected liposome solutions from each column were combined and stored at 4 °C until further use.90#14739410v1

[0402] Liposomes were characterized to determine the average hydrodynamic diameter and PDI. The hydrodynamic diameter and poly dispersity were determined by dynamic light scattering (DLS) using a Zetasizer model ZEN3600 (Malvern Pananalytical, UK). For measurement of hydrodynamic diameter and zeta potential, 25 pL of each LNP solution was diluted 100-fold in lx PBS or nuclease-free water (Invitrogen). For size measurement, 1 mL of diluted LNP samples was loaded in a 1.5 mL clear cuvette (Fisher Scientific), and for zeta potential measurement, 0.8 mL of each sample was transferred into the capillary cuvette (Malvern Panalytical). A Malvern Nano Zetasizer (Malvern Panalytical) was utilized to measure both parameters. The results are set forth in TABLE 5.TABLE 5

[0403] Subsequently, phosphate content was determined colorimetrically using the method of Fiske and Subbarow. In brief: the sample was diluted to a nominal target concentration of 1 pM total phosphate. 10 pL of sample was mixed with 100 pL of 6 M sulfuric acid and heated to between 180-200°C for 20 min. The sample was cooled to room temperature, 20 pL of 30% hydrogen peroxide was added and then the sample was heated at 150°C for 30 min. The sample was again cooled to room temperature. 950 pL of a 2.2 g / L aqueous solution of ammonium molybdate was added, followed by the addition of 50 pL of a 10% w / v aqueous solution of ascorbic acid. The sample was then heated at 100°C for 12 min and cooled to room temperature. VU absorbance was then read at 823 nm using a spectrophotometer. Phosphate content in the sample was determined relative to a standard curve made from contemporaneously run phosphate standards as shown in Table 6.TABLE 691#14739410v1EXAMPLE 3. Conjugation of Targeting Ligands to PEG-Lipid prior to Incorporation into the LNP

[0404] In order to incorporate targeting ligands (ISVD, scFv or Fab) into the LNP surface, first the targeting ligands were conjugated to a PEG-lipid. An ISVD-PEG-lipid conjugate was prepared and incorporated into an LNP to obtain the final targeted LNP. The ISVD was engineered with a free cysteine group in the C-terminal of the heavy chain. The PEG-lipid with a headgroup of Maleimide was added to a thiolated ISVD solution for conjugation. To perform conjugation, the ISVD solution was prepared in lx PBS, containing 5mM EDTA (Millipore Sigma) at a target concentration of 3 mg / mL. To reduce the cysteine group on C terminal, TCEP reducing agent (Thermo Scientific) was added to the solution at a final concentration of 0.1 mM, and samples were incubated at room temperature for 90 minutes. After completion of the reduction reaction, the sample was washed through a Zeba desalting column (Thermo Scientific) to remove the TCEP reducing agent. A 7 kDa Zeba column was used for ISVD; a 40 kD Zeba columns can be used for Fabs. For ISVD conjugation, DSPE-PEG3400-Mal eimide (NOF America Corporation) was mixed with 18:0 PEG2000 PE (Avanti Polar Lipids) at a molar ratio of 1:4 to form micelles. For Fab conjugation, DSPE-PEG2000-Maleimide (Avanti Polar Lipids) can be used and the molar ratio would be 2:3. The resulting ISVD-lipid solution was incubated at 37°C for 2 hours. Following the incubation, a thorough buffer exchange into lx PBS was performed to remove free ISVD antibodies (10 diafiltration volume). The resulting solution was stored at 2-8°C for up to 2 weeks.

[0405] The conjugated ISVD micelles were added to LNP solutions at a specific density (grams of ISVD antibody per mole of total lipid, referred to as “grafting density herein”). The solution was mixed at 300 rpm and incubated at 37°C for 4 hours using Thermomixer C (Eppendorf). The resulting LNP solutions were used for in vitro and in vivo screenings. Grafting density for ISVD varied in a range of 0.5-15 g / mol.EXAMPLE 4. Protocol for multi-dosed LNP with liposome co-administration in immunocompetent (Balb / c mice)92#14739410v1Mouse Strains

[0406] The mouse model was purchased from The Jackson Laboratory. 8-10 week-old naive female mice were enrolled in the study with an initial body weight ranging from 19-23 grams.Stimulating anti-PEG Immune Response In Vivo

[0407] At time zero, mice (n=3 per group per blood collection timepoint) were injected with either LNP alone or in combination with a liposome formulation (FIG. IB). LNPs were thawed 30 minutes prior to dosing and kept on wet ice until administered. Liposomes were kept on wet ice until ready to be mixed and administered. Immediately prior to dosing, LNPs and liposomes were separately mixed via inversion to ensure even distribution, and then either dosed directly (LNP only) or mixed at appropriate ratios. Doses were administered by intravenous tail vein injection once weekly for three weeks (three total doses) (FIG. 1A). Blood collection was performed to determine lipid pharmacokinetics and the presence of anti-PEG antibodies. Blood was collected at 15 minutes, 1 hour, 6 hours and 24 hours post dose. These timepoints were collected after both the 1stand the 3rddose (FIG. 1A).Blood Sample Collection and Processing

[0408] At above specified timepoints, mice were anesthetized with isoflurane and bled retro-orbitally before being sacrificed via CO2 inhalation. 200-500 pL blood was collected in a K3EDTA mini collection tube (Microvette #20.1341.102). Blood was kept on wet ice until all samples were collected. Plasma from whole blood was separated via centrifugation (lOOOxg for 10 minutes), and carefully transferred into separate tubes. Plasma was immediately frozen down at -20°C and kept frozen until future analyses.Lipid Determination and Pharmacokinetics in Mouse Plasma

[0409] For the analysis of mouse plasma, 10.0 pL of plasma was mixed with 100 pL of an internal standard solution composed of 50.0 ng / mL DOTMA (N-[l-(2,3-Dioleyloxy) propyl]-N,N,N-trimethylammonium chloride) in 50% isopropyl alcohol-50% acetonitrile. The sample was vortexed for 1 minute and then centrifuged at 5500 rpm for 10 minutes. 50 pL of the supernatant was transferred into an autosampler vial and mixed with 100 pL of 50% isopropyl alcohol in water with 2 mM of EDTA sodium salt for analysis by LC-MS / MS.

[0410] For the analysis of mouse tissue samples, each of the tissue samples was first homogenized with 50% isopropyl alcohol in water with 2 mM of EDTA sodium salt at a ratio of 1:4 (1.00 g of the tissue with 4.00 mL of the solution). Following homogenization, 25.0 pL of tissue homogenate sample was mixed with 25.0 pL of 50% isopropyl alcohol-50% acetonitrile and 100 pL of internal standard solution (50.0 ng / mL DOTMA in 50% isopropyl alcohol-50% acetonitrile). The samples were vortexed for 1 minute and centrifuged at 550093#14739410v1rpm for 10 minutes. An aliquot of the supernatant (50.0 pL) was transferred into an autosampler vial and mixed with 200 pL of 50% isopropyl alcohol in water with 2 mM of EDTA sodium salt for analysis by LC-MS / MS.

[0411] LC-MS / MS was performed using a Shimadzu Nexera UHPLC system with a Sciex API 6500 mass spectrometer. Chromatographic separation employed an Acquity BEH Phenyl, 1.7 pm (2.1 x 30 mm) HPLC column maintained at 50°C. Solvent A was 0.1% formic acid in acetonitrile / isopropyl alcohol (80 / 20). Solvent B was 0.1% formic acid in water. The gradient was as shown in Table 7.TABLE 7

[0412] Mass Spectrometry was performed using electrospray ionization in positive ion mode. The source voltage was 5.5 kV, and the source temperature was 550°C. LIPID 15 was detected using selective reaction monitoring of the 945.0 — 900.0 m / z transition (retention time 1.21 min). The internal standard (DOTMA) was monitored using the 634.5 — 55.1 m / z transition (retention time 1.19 minutes). LIPID 15 content was determined relative to contemporaneously analyzed LIPID 15 standards spiked into naive plasma or tissue.EXAMPLE 5. Mouse anti-PEG IgM, IgG ELISA Method

[0413] Mouse anti-PEG IgM and anti-PEG IgG were assayed using an ELISA kit from Life Diagnostics (mouse anti-PEG IgM ELISA; Catalog #PEGM-1 and mouse anti-PEG IgG ELISA; Catalog #PEGG-1). Mouse plasma was diluted either 50-fold (IgG) or 500-fold (IgM) in reagent diluent (Life Diagnostics, PEGD50-1), then 100 pL of diluted samples were added to separate PEG-BSA coated plates (Life Diagnostics, PBSA20PL). IgG or IgM standards were prepared on the same individual plate as the associated samples and were serially diluted. Plates 94#14739410v1were incubated on a shaker for 1 hour at room temperature, and then washed five times with wash buffer (Life Diagnostics, PEGW50-20). 100 pL of anti-PEG IgG or IgM HRP conjugate (diluted in reagent diluent) was added to each well of the appropriate plate. Plates were incubated on a shaker for either 45 minutes (IgG) or 30 minutes (IgM) at room temperature, then washed five times with wash buffer. 100 pL of TMB reagent (Life Diagnostics, TMB 11-1) was added to each plate, after which both plates were incubated on a shaker for 20 minutes at room temperature. The reaction was quenched by adding 100 pL of Stop Solution (Life Diagnostics, SSI 1-1) to each well. Optical density at 450nm was read on a SpectraMax M5 plate reader. Anti-PEG IgG or IgM concentration was quantified relative to a standard curve (fitted to a second order polynomial model) based on contemporaneously analyzed anti-PEG IgG or IgM standards (FIG. 2A, FIG. 2B, FIG. 3A and FIG. 3B).EXAMPLE 6. In Vitro Experimental Protocol for LNP CAR with Liposome Transfection in Whole Blood

[0414] This Example describes the method used to transfect immune cells in whole blood using targeted mRNA LNPs in combination with liposomes.

[0415] Venous blood from healthy volunteers was anti-coagulated in sodium heparin tubes (BD Biosciences #367874) and seeded at 160 pL in a 96-well round-bottom plate. Whole blood was pre-treated with either PBS or titrated concentrations of liposomes (PEG-DSPE / DSPC (ratio 10:90)), starting at 100 pg / mL. Titrated liposomes or PBS were mixed well with the whole blood and incubated for 1 hour at 37°C until time for LNP transfection. Transfection of whole blood was carried out by adding nanoparticles containing 1 pg / mL mRNA to the blood (+ / - liposomes) and co-culturing at 37°C until the time of analysis. A subset of blood samples were incubated with liposomes and LNP at the same time (no pre-incubation).

[0416] To assess transfection efficiency, cells were analyzed 24-hours post-transfection by flow cytometry. Prior to the analysis of whole blood transfection efficiency, red blood cells were lysed three times with VersaLyse Lysing Solution (Beckman Coulter #A09777) for 10 minutes each at room temperature. Post red blood cell lysis, cells were washed in PBS and dead cells were labeled with Fixable Viability Dye eFluor780 (eBiosciences #65-0865-14) for 10 minutes at room temperature. Cells were then Fc-blocked (BD Biosciences #564219) for 5 minutes at room temperature, followed by surface staining for 30 minutes at room temperature with specific antibodies.

[0417] Primary antibodies applied in the flow cytometry analysis of whole blood included the following: CD4-BUV395 (1:200) (BD Biosciences #564107), CD14-BV650 (1:200) (BD 95#14739410v1Biosciences #563419), CD8-BV421 (1:200) (Biolegend #344748), CD3-AF700 (1:300) (BD Biosciences #557917), CD20-AF488 (1:300) (Biolegend #302316), CD159a-APC (1:300) (Beckman Coulter #A60797). aCD22 CAR expression was evaluated by utilizing an antibody against the G4S linker in the CAR: G4S Linker-PE (1 : 100) (Cell Signaling #38907S).

[0418] Compensation for each fluorochrome was performed in the multicolor flow panels using positive and negative compensation beads (Invitrogen #01-2222-42). Fluorescence minus one (FMO) samples and unstained controls were included to determine the level of background fluorescence and to set the gates for the negative cell populations versus the positive cell populations.

[0419] All samples were acquired on a NovoCyte Penteon (Agilent Technologies) running NovoExpress software (Agilent Technologies). All data collected were analyzed using FlowJo 10.8.2 software and GraphPad Prism version 10.0.EXAMPLE 7. In Vitro Experimental Protocol for LNP CAR with Liposome Transfection in PBMC

[0420] This Example describes the method used to transfect immune cells in whole blood using targeted mRNA LNPs in combination with liposomes.

[0421] Human PBMC from healthy donors were plated at 250,000 cells / well. Cells were pretreated with either PBS or titrated concentrations of liposomes (PEG-DSPE / DSPC (ratio 10:90)), starting at 100 pg / mL. Titrated liposomes or PBS were mixed well with the cells and incubated for 15 minutes at 37°C until time for LNP transfection. Transfection of PBMC was carried out by adding nanoparticles containing 1 pg / mL mRNA to the blood (+ / - liposomes) and co-culturing at 37°C until the time of analysis. A subset of samples was incubated with liposomes and LNP at the same time (no pre-incubation).

[0422] To assess transfection efficiency, cells were analyzed 24-hours post-transfection by flow cytometry. Cells were washed in PBS and dead cells were labeled with Fixable Viability Dye eFluor780 (eBiosciences #65-0865-14) for 10 minutes at room temperature. Cells were then Fc-blocked (BD Biosciences #564219) for 5 minutes at room temperature, followed by surface staining for 30 minutes at room temperature with specific antibodies.

[0423] Primary antibodies applied in the flow cytometry analysis of PBMC included the following: CD4-BUV395 (1:200) (BD Biosciences #564107), CD14-BV650 (1:200) (BD Biosciences #563419), CD8-BV421 (1:200) (Biolegend #344748), CD3-AF700 (1:300) (BD Biosciences #557917), CD20-AF488 (1:300) (Biolegend #302316), CD159a-APC (1:300)96#14739410v1(Beckman Coulter #A60797). aCD22 CAR expression was evaluated by utilizing an antibody against the G4S linker in the CAR: G4S Linker-PE (1 : 100) (Cell Signaling #38907S).

[0424] Compensation for each fluorochrome was performed in the multicolor flow panels using positive and negative compensation beads (Invitrogen #01-2222-42). Fluorescence minus one (FMO) samples and unstained controls were included to determine the level of background fluorescence and to set the gates for the negative cell populations versus the positive cell populations.

[0425] All samples were acquired on a NovoCyte Penteon (Agilent Technologies) running NovoExpress software (Agilent Technologies). All data collected were analyzed using Flow Jo 10.8.2 software and GraphPad Prism version 10.0.EXAMPLE 8. In vitro CAR Expression in T cells with Targeted LNPs and Liposomes in Human Whole Blood

[0426] This Example compared aCD22 CAR expression (visualized by the scFv G4S linker in the CAR) in T cells when targeted LNPs were incubated in human whole blood, with or without the co-incubation of PEG-DSPE / DSPC liposomes. Nanoparticles bearing an aCD22 CAR encoding mRNA (TTR-102) were produced using the microfluidic mixing and buffer exchange processes described in Example 1. An aCD8 VHH-conjugate (15C01v8) was incorporated into the parent LNP to obtain the final targeted LNP formulation, the process of which is described in Example 3. Liposomes were produced using the mixing and extrusion processes described in Example 2. LNPs and liposomes thus produced were tested in vitro in human whole blood to assess protein (CAR) expression via detection of the G4S linker on the scFv of the CAR. Liposomes were serially diluted and either incubated 1 hour prior to LNP incubation, or comixed with LNP and incubated concurrently, as described in Example 6. Transfected cells were gated as CD4- (CD8 population) from the live CD3+ population, and levels of protein expression evaluated (as %G4S+ and G4S MFI for CAR expression) in this population were used to assess the transfection impact of mixing liposomes with LNP. CAR protein expression observed in the CD4- population demonstrated elevated CAR expression when LNP were mixed with liposomes (either pre-incubated or co-incubated). CAR protein expression in CD4-T cells increased with the increasing concentration of liposomes.EXAMPLE 9. In vitro CAR Expression in T cells with Targeted LNPs and Liposomes in Human PBMCs97#14739410v1

[0427] This Example compared aCD22 CAR expression (visualized by the scFv G4S linker in the CAR) in T cells when targeted LNPs were incubated in human PBMCs, with or without the co-incubation of PEG-DSPE / DSPC liposomes. LNPs bearing an aCD22 CAR encoding mRNA (TTR-102) were produced using the microfluidic mixing and buffer exchange processes described in Example 1. An aCD8 ISVD-conjugate (15C01v8) was incorporated into the parent LNP to obtain the final targeted LNP formulation, the process of which is described in Example 3. Liposomes were produced using the mixing and extrusion processes described in Examples 1 and 2. LNPs and liposomes thus produced were tested in vitro in human PBMCs to assess protein (CAR) expression via detection of the G4S linker on the scFv of the CAR. Liposomes were serially diluted and either incubated 1 hour prior to LNP incubation, or comixed with LNP and incubated concurrently, as described in Examples 6 and 7.

[0428] Transfected cells were gated as CD4- (CD8 population) from the live CD3+ population, and levels of protein expression evaluated (as %G4S+ and G4S MFI for CAR expression) in this population were used to assess the transfection impact of mixing liposomes with LNP. CAR protein expression observed in the CD4- population (CD8+ cells, as illustrated by the CAR% and CAR MFI values) demonstrated no significant difference in CAR expression when LNP were mixed with liposomes (either pre-incubated or coincubated).EXAMPLE 10. Lipid 15 pharmacokinetics after single and multi-dosing LNPs (alone or co-administered) with liposomes

[0429] Balb / c mice were dosed with LNP and / or liposomes using protocols described in Example 4. Six mice were used in each Group. Plasma samples were drawn at the specified timepoints post the first and third injection, following the study design shown in Table 8.98#14739410v1TABLE 8 - Balb / c Mice Immunogenicity Study Design

[0430] Plasma was analyzed for Lipid 15 lipid content using the processes described in Example 4. After a single dose, LNP alone (both DPG-PEG and DMG-PEG) Groups 1 and 7 showed lower concentrations of Lipid 15 lipid in the plasma than in Groups 2-5 with liposomes co-administered with LNP, as shown in FIG. 4A, FIG. 4B, FIG. 5A and FIG. 5B. Of the liposome formulations, Group 6 (PEG-DSPE / Suc-DSPE / DSPC) had higher plasma lipid concentrations than LNP alone at 1 hour post administration, but was not superior to DPG-PEG LNP alone at 6 hours post administration (FIG. 4A and FIG. 5A). After 3 doses, all liposome 99#14739410v1Groups (9-13) had higher plasma concentrations of Lipid 15 lipid than either LNP alone Groups (8 and 14) at 1 and 6 hours post administration (FIG. 4B and FIG. 5B). Overall, the groups with liposomes co-administered with LNP reduced the accelerated blood clearance seen in DPG-PEG LNP alone after the 3rddose, as shown in FIGs. 4B and 5B.EXAMPLE 11. Anti-PEG IgM and IgG in mouse plasma after single and multi-dosing LNPs (alone or co-administered) with liposomes

[0431] Balb / c mice were dosed with LNP and / or liposomes using protocols described in Example 5. Plasma samples were drawn at the specified timepoints post the first and third injection, following the study design shown in Table 8.

[0432] Plasma was analyzed for the presence of anti-PEG antibodies (IgM and IgG) 24 hours after either a single dose or the third dose of LNP alone or co-administered with liposomes using the protocol described in Example 5. As shown in FIG.2A and FIG. 2B, anti-PEG IgM was not detected over baseline in any group 24 hours after a single dose (FIG. 2A) but was significantly elevated in the LNP-alone group (Group 8; DPG-PEG) after the third dose (FIG.2B). Anti-PEG IgM was reduced to baseline levels in liposome co-administered Groups 9-12 after the third dose (FIG. 2B). After the third dose, Group 13 (PEG-DSPE / Suc-DSPE / DSPC co-administered with LNP) and Group 14 (LNP alone; DMG-PEG) had lower concentrations of plasma anti-PEG IgM than Group 8 (DPG-PEG LNP alone), but higher than the other coadministered liposome groups (FIG.2B). As shown in FIG.3, anti-PEG IgG was not detected over baseline in any group after a single dose (FIG. 3A) but was elevated in the LNP-alone group (Group 8; DPG-PEG) after the third dose (FIG.3B). Anti-PEG IgG was reduced to near baseline levels in liposome co-administered groups, as well as in DMG-PEG LNP alone (Group 14), with the exception of Group 11 (PEG-DSPE / DOPC / Chol liposome co-administered with LNP) (FIG. 3B).

[0433] Comparison of Groups 1 and 7 without liposomes and Group 2 with liposome coadministration demonstrated that liposome co-administration reduced loss of LNP lipid in plasma after both a single dose and a third dose compared to LNPs administered alone (FIG.6).EXAMPLE 12. In vitro experimental protocol for whole blood transfection

[0434] This Example describes the method used to transfect immune cells in whole blood using VHH-targeted mRNA LNPs and Liposomes.100#14739410v1

[0435] Venous blood from healthy volunteers was anti-coagulated in heparin tubes (BD Biosciences #367526) and seeded at 100 pL in a 96-well round-bottom plate. Transfection of whole blood was carried out simply by adding nanoparticles containing 0.33 pg / mL mRNA to the cells co-dosing with Liposomes at 100 ug / mL. To assess transfection efficiency, cells were analyzed 24-hours post-transfection by flow cytometry. Prior to the analysis of whole blood transfection efficiency, red blood cells were lysed twice with ACK lysis buffer for 5 minutes at room temperature. Primary antibodies applied in the flow cytometry analysis of whole blood included the following: CD4-BV786 (1:100) (BD Biosciences #563914), CD20-BUV395 (1:100) (BD Biosciences #563782), CD56-APC (1:100) (BD Biosciences #341027), CD8-BV421 (1 : 100) (Biolegend #344748), CD14-BV650 (1 : 100) (BD Biosciences #563419), CD3-AF700 (1:100) (BD Biosciences #557917), G4S-PE (1:50) (Cell Signaling Technology #38907). Fixable Viability Dye eFluor780 (eBiosciences #65-0865-14) was used to assess viability for all samples. For flow analysis, cells were Fc-blocked (BD Biosciences #564219) for 5 minutes on ice, followed by labeling dead cells with fixable viability dye eFluor780 and surface staining for 30 minutes on ice with specific antibodies.

[0436] Compensation for each fluorochrome was performed in the multicolor flow panels using positive and negative compensation beads. All samples were acquired on a Novocyte Penteon (Agilent). All data collected were analyzed using OMIQ (Dotmatics) and GraphPad Prism version 9.0.EXAMPLE 13. Experimental protocol for anti-PEG IgG and IgM ELISA on human plasma

[0437] This example describes the method used to measure anti-PEG IgG and IgM antibody levels in plasma collected from human whole blood from healthy donors. 2 mL of blood was transferred to Eppendorf tubes and spun down at 2000 x g at 4C for 10 minutes. The separated plasma was then transferred to a clean Eppendorf tube and stored at -80C until analysis.

[0438] Human anti-PEG IgG (Life Diagnostics #PEGG-20) and human anti-PEG IgM (Life Diagnostics #PEGM-20) were analyzed according to manufacturer’s instructions. The plates were read within five minutes of adding the stop solution at 450 nm (absorbance) with a plate reader.101#14739410v1EXAMPLE 14. CD22 CAR expression in CD8 T-cells with Lipid 15 «CD8 (15C01) targeted LNPs co-dosed with and without Liposomes in human whole blood

[0439] aCD8 targeted CD22 CAR mRNA LNPs were dosed to human venous whole blood with and without Liposomes, incubated for 24 hours and analyzed using the protocol described in Example 12 for whole blood transfections. Subsequently, plasma from each donor was analyzed for pre-existing anti PEG IgG antibodies as described in Example 13.

[0440] As seen in FIG. 7A, FIG. 7B, FIG. 7C, Table 9, and Table 10, donors with high levels of pre-existing anti -PEG IgG did not show CAR expression when only LNPs were added, while all donors showed high levels of CAR expression (measured as %CAR positive cells and CAR MFI) when LNPs were co-dosed with liposomes. This indicates that co-dosing LNPs with liposomes rescues the transfection in human donors with high levels of pre-existing anti-PEG IgG antibodies.TABLE 9. Donor pre-existing anti-PEG IgG levels and CAR expression (%CAR+) after administration of LNPs only or LNPs and liposomes102#14739410v1TABLE 10. Donor pre-existing anti -PEG IgG levels and CAR expression (MFI) after administration of LNPs only or LNPs and liposomes103#14739410v1Embodiments1. A pharmaceutical system comprising two particles, wherein the first particle and the second particle share at least one common element, and wherein(i) both the first particle and the second particle bind to at least one innate immune system opsonin; and / or(ii) the common element is capable of inducing an anti-drug antibody against the first particle and the second particle in a subject when the pharmaceutical system is administered to the subject.2. A pharmaceutical system comprising a first particle and a second particle, wherein the first particle and the second particle share at least one common element, and wherein the first particle is a liposome and the second particle is a lipid nanoparticle (LNP).3. The pharmaceutical system of embodiment 1, wherein the opsonin binds to an opsonin receptor on the surface of a myeloid cell.4. The pharmaceutical system of embodiment 1 or embodiment 3, wherein the opsonin is a member of the Complement family.5. The pharmaceutical system of embodiment 3, wherein the opsonin is C3a.6. The pharmaceutical system of embodiment 1 or embodiment 3, wherein the opsonin is Apolipoprotein E (Apo E).7. The pharmaceutical system of any one of embodiments 3 to 6, wherein the myeloid cell is a phagocyte.8. The pharmaceutical system of embodiment 7, wherein the phagocyte is a monocyte or a macrophage.9. The pharmaceutical system of any one of embodiments 1 to 8, wherein the common element comprises a polymer or a compound comprising the polymer.10. The pharmaceutical system of embodiment 9, wherein the polymer comprises a polyethylene glycol (PEG).11. The pharmaceutical system of embodiment 9, wherein the compound comprising the polymer is a PEG-lipid.104#14739410v112. The pharmaceutical system of embodiment 11, wherein the anti-drug antibody is an anti -PEG antibody.13. The pharmaceutical system of any one of embodiments 1 or 3-12, wherein:(a) both the first particle and the second particle are lipid-based nanoparticles;(b) both the first particle and the second particle are liposomes;(c) the first particle is a liposome and the second particle is a lipid-based nanoparticle; or(d) the first particle is a lipid-based nanoparticle and the second particle is a liposome.14. The pharmaceutical system of embodiment 13, wherein the first particle is a liposome.15. The pharmaceutical system of embodiment 14, wherein the liposome comprises a PEG or a compound comprising a PEG.16. The pharmaceutical system of embodiment 15, wherein the compound comprising a PEG is a PEG-lipid.17. The pharmaceutical system of embodiment 16, wherein the PEG-lipid is positively charged, neutral, or negatively charged.18. The pharmaceutical system of embodiment 17, wherein the PEG-lipid is positively charged, and the opsonin that binds to the first particle and the second particle is negatively charged; or wherein the PEG-lipid is negatively charged, and the opsonin that binds to the first particle and the second particle is positively charged.19. The pharmaceutical system of any one of embodiments 16 to 18, wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols, optionally wherein the PEG-lipid is PEG- dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-105#14739410v1phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.20. The pharmaceutical system of claim 16, wherein the PEG-lipid is PEG-DSPE.21. The pharmaceutical system of any one of embodiments 14 to 20, wherein the liposome further comprises one or more helper lipids.22. The pharmaceutical system of embodiment 21, wherein the one or more helper lipids is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), succinyl-distearoyl-sn-glycero-3-phosphoethanolamine (Suc-DSPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), di stearoylglycerol (DSG), dioleoylgylcerol (DOG), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), dilinoleoyl-glycero-phosphatidyl ethanolamine (DLPE), diacylglycerol (DAG), ceramide 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or diacylphosphatidylethanolamine comprising Dipalmitoyl (C16) chain or Distearoyl (C18) chain, and a combination thereof.23. The pharmaceutical system of embodiment 22, wherein the one or more helper lipids is DSPC, DOPC, Suc-DSPE, or a combination thereof.24. The pharmaceutical system of embodiment 22 or embodiment 23, wherein the one or more helper lipids is DSPC or DOPC.25. The pharmaceutical system of any one of embodiments 21-24, wherein the liposome comprises a PEG-lipid and a helper lipid at a molar ratio of about 5:95 to about 25:75.26. The pharmaceutical system of embodiment 25, wherein the liposome comprises the PEG-lipid and the helper lipid at a molar ratio of about 10:90.27. The pharmaceutical system of embodiment 22 or embodiment 23, wherein the one or more helper lipids is a combination of Suc-DSPE and DSPC.106#14739410v128. The pharmaceutical system of embodiment 27, wherein the liposome comprises the PEG-lipid, the Suc-DSPE helper lipid, and the DSPC helper lipid at a molar ratio of about 10:45:45.29. The pharmaceutical system of any one of embodiments 14-28, wherein the liposome does not comprise a sterol and / or does not comprise an ionizable lipid.30. The pharmaceutical system of any one of embodiments 14-28, wherein the liposome further comprises a sterol.31. The pharmaceutical system of embodiment 30, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol, and any combination thereof.32. The pharmaceutical system of embodiment 31, wherein the sterol is cholesterol.33. The pharmaceutical system of any one of embodiments 30 to 32, wherein the liposome comprises the PEG-lipid, the one or more helper lipids, and the sterol at a molar ratio of about 10:45:45.34. The pharmaceutical system of any one of embodiments 13-33, wherein the second particle is a lipid nanoparticle (LNP).35. The pharmaceutical system of embodiment 34, wherein the LNP comprises an ionizable lipid and a PEG-lipid.36. The pharmaceutical system of embodiment 35, wherein the ionizable lipid is, or a salt thereof.37. The pharmaceutical system of embodiment 35 or 36, wherein the ionizable lipid is present in the LNP at a range of about 10 mol% to about 70 mol%.38. The pharmaceutical system of embodiment 37, wherein the ionizable lipid is present in the LNP at a range of about 40 mol% to about 60 mol%.107#14739410v139. The pharmaceutical system of any one of embodiments 37 to 38, wherein the PEG-lipid of the LNP is selected from the group consisting of PEG-DSPE, PEG-DSG, PEG-DSPC, PEG-DOPC, PEG-DOPE, PEG-DMPE, PEG-DSPG, PEG-DMG, PEG-DAG, PEGDOG, PEG-DPPE, PEG-DLPE, PEG-DPG, PEG-ceramide, PEG-2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain, and a combination thereof.40. The pharmaceutical system of embodiment 39, wherein the PEG-lipid in both the first particle and the second particle is DPG-PEG or DMG-PEG.41. The pharmaceutical system of any one of embodiments 35 to 40, wherein the PEG-lipid is present in the LNP at a range of about 0.5 mol% to about 4 mol%.42. The pharmaceutical system of embodiment 41, wherein the PEG-lipid is present in the LNP at about 1.5 mol%.43. The pharmaceutical system of any one of embodiments 35 to 42, wherein the LNP further comprises a payload compound, a structural lipid, and optionally a neutral phospholipid.44. The pharmaceutical system of embodiment 43, wherein the payload compound comprises nucleic acid.45. The pharmaceutical system of embodiment 44, wherein the nucleic acid comprises or is RNA.46. The pharmaceutical system of embodiment 45, wherein the RNA comprises or is mRNA.47. The pharmaceutical system of embodiment 46, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a Chimeric Antigen Receptor (CAR).48. The pharmaceutical system of embodiment 45, wherein the RNA comprises guide RNA (gRNA) for gene editing.49. The pharmaceutical system of any one of embodiments 44 to 48, wherein the nucleic acid comprises pseudouridine.108#14739410v150. The pharmaceutical system of embodiment 49, wherein the pseudouridine is Nl-methyl-pseudouridine.51. The pharmaceutical system of any one of embodiments 43 to 50, wherein the structural lipid comprises or is sterol.52. The pharmaceutical system of embodiment 51, wherein the sterol comprises or is cholesterol.53. The pharmaceutical system of any one of embodiments 43 to 52, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), and sphingomyelin.54. The pharmaceutical system of any one of embodiments 43 to 53, wherein the neutral phospholipid comprises or is DSPC.55. The pharmaceutical system of any one of embodiments 35 to 54, wherein the PEG-lipid in the LNP comprises PEG-DPG.56. The pharmaceutical system of embodiment 55, wherein the PEG-DPG is PEG 2000-DPG.57. The pharmaceutical system of any one of embodiments 34 to 56, wherein the LNP further comprises a conjugate having the formula of [Lipid] - [optional linker] - [antibody], wherein the antibody specifically binds to one type of cell, tissue, or organ.58. The pharmaceutical system of embodiment 57, wherein the antibody is an immunoglobulin single variable domain (ISVD).59. The pharmaceutical system of embodiment 58, wherein the ISVD is a VHH.60. The pharmaceutical system of any one of embodiments 57 to 59, wherein the cell is an immune cell.61. The pharmaceutical system of embodiment 57, wherein the antibody is a Fab, Fab’, Fab'-SH, F(ab’)2, Fv, single-chain Fab (scFab), or single-chain variable fragments (scFv).109#14739410v162. The pharmaceutical system of embodiment 57, wherein the antibody is a bispecific antibody.63. The pharmaceutical system of embodiment 62, wherein the bispecific antibody binds both a first macrophage antigen and a second macrophage antigen.64. The pharmaceutical system of embodiment 62, wherein the bispecific antibody binds both a first type of immune cell and a second type of immune cell.65. The pharmaceutical system of embodiment 64, wherein the first type of immune cell and the second type of immune cell are CD4+ T cells and CD8+ T cells.66. The pharmaceutical system of embodiment 65, wherein the first type of immune cell is a first macrophage, and the second type of immune cell is a second macrophage, a T-cell, or an NK cell.67. The pharmaceutical system of any one of embodiments 57 to 66, wherein the LNP comprises two conjugates both having the formula of [Lipid] - [optional linker] - [antibody], and the first conjugate comprises a first antibody that binds to a first antigen of a first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of a second type of immune cell.68. The pharmaceutical system of any one of embodiments 57 to 67, wherein the LNP is for delivering a nucleic acid into an immune cell, and wherein the LNP comprises a Fab lacking the native interchain disulfide bond.69. The pharmaceutical system of embodiment 61, wherein the antibody is a Fab, and the Fab is engineered to replace one or both cysteines on the native constant light chain and the native constant heavy chain that form the native interchain disulfide with a non-cysteine amino acid, therefor to remove the native interchain disulfide bond in the Fab.70. The pharmaceutical system of any one of embodiments 43 to 69, wherein the neutral phospholipid in the LNP has a concentration of about 5% to 30%.71. The pharmaceutical system of embodiment 70, wherein the neutral phospholipid in the LNP has a concentration of about 10% to about 20%.110#14739410v172. The pharmaceutical system of embodiment 71, wherein the neutral phospholipid in the LNP has a concentration of about 20%.73. The pharmaceutical system of any one of embodiments 46 to 72, wherein the ionizable lipid has a concentration about 10 g / g to about 20 g / g mRNA.74. The pharmaceutical system of any one of embodiments 46 to 73, wherein the ionizable lipid has a concentration about 15 g / g mRNA.75. The pharmaceutical system of any one of embodiments 46 to 74, wherein the structural lipid comprises or is sterol, and the sterol has a concentration about 3.0 g / g to 5.0 g / g mRNA.76. The pharmaceutical system of any one of embodiments 46 to 75, wherein the neutral phospholipid in the LNP has a concentration about 2.0 g / g to 5.0 g / g mRNA.77. The pharmaceutical system of any one of embodiments 46 to 76, wherein the PEG-lipid in the LNP has a concentration about 1.0 g / g to 1.5 g / g mRNA.78. The pharmaceutical system of any one of embodiments 46 to 77, wherein the [Lipid] - [optional linker] - [antibody] conjugate in the LNP has a concentration about 0.05 g / g to 0.1 g / g mRNA.79. The pharmaceutical system of any one of embodiments 51 to 78, wherein in the LNP:(i) the ionizable lipid has a concentration about 49.2 mol% of the LNP;(ii) the sterol has a concentration about 39.4 mol% of the LNP;(iii) the neutral phospholipid has a concentration about 0.9 mol% of the LNP; and (iv) the PEG-lipid has a concentration about 1.5% of the LNP.80. The pharmaceutical system of any one of claims 57 to 79, wherein in the LNP:(i) the ionizable lipid has a concentration about 14.2 g / g mRNA in the LNP;(ii) the sterol has a concentration about 4.64 g / g mRNA in the LNP;(iii) the neutral has a concentration about 2.37 g / g mRNA in the LNP;(iv) the PEG-lipid has a concentration about 1.15 g / g mRNA in the LNP; and(v) the [Lipid] - [optional linker] - [antibody] conjugate has a concentration about 0.084 g / g mRNA in the LNP.Ill#14739410v181. A composition comprising the pharmaceutical system of any one of embodiments 1-80.82. The composition of embodiment 81, wherein the first particle and the second particle are in a same container.83. The composition of embodiment 81, wherein the first particle and the second particle are in different containers.84. The composition of any one of embodiments 81 to 83, further comprising one or more carrier or excipient.85. The composition of embodiment 84, wherein the one or more carrier or excipient is a pharmaceutically acceptable carrier or excipient.86. A kit comprising the pharmaceutical system of any one of embodiments 1 to 80, or the composition of any one of embodiments 81 to 85.87. A method comprising delivering a payload to a subject in need thereof, comprising administering the pharmaceutical system of any one of embodiments 1-80 or a composition of any one of embodiments 81 to 85 to the subject, wherein the second particle comprises the payload.88. The method of embodiment 87, wherein the method comprises administering to the subject the first particle and the second particle separately.89. The method of embodiment 88, wherein the first particle and the second particle are administered sequentially.90. The method of embodiment 89, wherein the first particle is administered first to the subject followed by administering the second particle to the subject.91. The method of embodiment 89, wherein the second particle is administered to the subject followed by administering the first particle to the subject.92. The method of any one of embodiments 88 to 91, wherein first particle and the second particle are administered at a time interval of about 1 hour to about 5 minutes.112#14739410v193. The method of embodiment 92, wherein the first particle and the second particle are administered at a time interval of about 15 minutes.94. The method of embodiment 87, wherein the first particle and the second particle are administered to the subject simultaneously.95. The method of any one of embodiments 87 to 94, wherein the administration is by systemic administration.96. The method of embodiment 95, wherein the systemic administration is by intravenous injection or intravenous infusion.97. Use of a pharmaceutical system of any one of embodiments 1-80, a composition of any one of embodiments 81-85, or a kit of embodiment 86 in the manufacture of a medicament for delivering a payload to a subject.98. The use of embodiment 97, wherein the first particle and the second particle in the pharmaceutical system are administered to the subject separately.99. The use of embodiment 97, wherein the first particle and the second particle in the pharmaceutical system are administered to the subject sequentially.100. The use of embodiment 97, wherein the first particle and the second particle in the pharmaceutical system are administered to the subject simultaneously.101. The use of embodiment 98 or embodiment 99, wherein the first particle is administered first to the subject followed by administering the second particle to the subject.102. The use of embodiment 98 or embodiment 99, wherein the second particle of the pharmaceutical system is administered first to the subject followed by administering the first particle.103. The use of any one of embodiments 98-99, wherein the first particle and the second particle are administered at a time interval of about 1 hour to about 5 minutes.104. The use of embodiment 103, wherein the first particle and the second particle are administered at a time interval of about 15 minutes.113#14739410v1105. The use of any one of embodiments 98-104, wherein the administration is by systemic administration.106. The use of embodiment 105, wherein the systemic administration is by intravenous injection or intravenous infusion.107. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a system of any one of embodiments 1-80, a composition of any one of embodiments 81-85, or a kit of embodiment 86.108. The method of embodiment 107, wherein the pharmaceutical system of any one of embodiments 1-80 is administered to the subject by separately administering the first particle and the second particle.109. The method of embodiment 108, wherein the first particle and the second particle are administered sequentially.110. The method of embodiment 107, wherein the first particle and the second particle are administered simultaneously.111. The method of embodiment 107 or embodiment 108, wherein the first particle is administered first to the subject followed by administering the second particle.112. The method of any one of embodiments 107-108, wherein the second particle is administered first to the subject followed by administering the first particle.113. The method of any one of embodiments 107-108 or 111-112, wherein the first particle and the second particle are administered at a time interval of about 1 hour to about 5 minutes.114. The method of embodiment 113, wherein the first particle and the second particle are administered at a time interval of about 15 minutes.115. The method of any one of embodiments 107-114, wherein the administration is by systemic administration.116. The method of embodiment 115, wherein the systemic administration is by intravenous injection or intravenous infusion.114#14739410v1117. Use of a system of any one of embodiments 1-80, a composition of any one of embodiments 81-85, or a kit of embodiment 86 in the manufacture of a medicament for treating a disease in a subject in need thereof.118. The use of embodiment 117, wherein the first particle and the second particle are administered to the subject separately.119. The use of embodiment 118, wherein the first particle and the second particle are administered sequentially.120. The use of embodiment 117, wherein the first particle and the second particle are administered to the subject simultaneously.121. The use of embodiment 119, wherein the first particle is administered first to the subject followed by administering the second particle.122. The use of any one of embodiments 118-119, wherein the second particle is administered first to the subject followed by administering the second particle.123. The use of any one of embodiments 118-119 and 121-122, wherein the first particle and the second particle are administered at a time interval of about 1 hour to about 5 minutes.124. The use of embodiment 123, wherein the first particle and the second particle are administered at a time interval of about 15 minutes.125. The use of any one of embodiments 118 to 124, wherein the administration is by systemic administration.126. The use of embodiment 125, wherein the systemic administration is by intravenous injection or intravenous infusion.127. A method of reducing immunogenicity of a lipid nanoparticle (LNP) in a subject in need thereof, the method comprising administering to the subject a liposome that is effective in reducing the immunogenicity of the LNP administered to the subject.128. The method of embodiment 127, wherein the LNP and the liposome share at least one common element, and(i) both the liposome and LNP bind to at least one opsonin; and115#14739410v1(ii) the common element is a capable of inducing anti-drug antibody against the LNP and the liposome in a subject when the pharmaceutical system is administered to the subject.129. The method of embodiment 128, wherein the opsonin binds to an opsonin receptor on the surface of a myeloid cell.130. The method of embodiment 128-129, wherein the opsonin is a member of the Complement family.131. The method of embodiment 130, wherein the opsonin is C3a.132. The method of embodiment 129 or embodiment 130, wherein the opsonin is Apolipoprotein E (Apo E).133. The method of any one of embodiments 129-132, wherein the myeloid cell is a phagocyte.134. The method of embodiment 133, wherein the phagocyte is a monocyte or a macrophage.135. The method of any one of embodiments 128-134, wherein the common element comprises a polymer or a compound comprising the polymer.136. The method of embodiment 135, wherein the polymer comprises polyethylene glycol (PEG).137. The method of embodiment 136, wherein the compound comprising the polymer is a PEG-lipid.138. The method of embodiment 137, wherein the anti-drug antibody is an anti-PEG antibody.139. The method of any one of embodiments 127-138, wherein the liposome comprises PEG or a compound comprising PEG.140. The method of embodiment 139, wherein the compound comprising PEG is a PEG-lipid.141. The method of embodiment 140, wherein the PEG-lipid is positively charged, neutral, or negatively charged.116#14739410v1142. The method of embodiment 141, wherein the PEG-lipid is positively charged, and the opsonin that binds to the LNP and the liposome is negatively charged; or wherein the PEG-lipid is negatively charged, and the opsonin that binds to the LNP and the liposome is positively charged.143. The method of embodiments 140-142, wherein the PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols, optionally the PEG-lipid is PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.144. The method of embodiment 140, wherein the PEG-lipid is PEG-DSPE.145. The method of any one of embodiments 127-144, wherein the liposome further comprises one or more helper lipid.146. The method of embodiment 145, wherein the helper lipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), succinyl-distearoyl-sn-glycero-3-phosphoethanolamine (Suc-DSPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), di stearoylglycerol (DSG), dioleoylgylcerol (DOG), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), dilinoleoyl-glycero-phosphatidyl ethanolamine (DLPE), diacylglycerol (DAG), ceramide 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain and combinations thereof.117#14739410v1147. The method of embodiment 146, wherein the helper lipid is DSPC, DOPC, or a mixture thereof.148. The method of any one of embodiments 127-147, wherein the liposome comprises a PEG-lipid and a helper lipid at a molar ratio of about 5:95 to 25:75.149. The method of embodiment 148, wherein the liposome comprises the PEG-lipid and the helper lipid at a molar ratio of about 10:90.150. The method of any one of embodiments 127-149, wherein the liposome does not comprise a sterol and / or does not comprise an ionizable lipid.151. The method of any one of embodiments 127-149, wherein the liposome further comprises a sterol.152. The method of embodiment 151, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol, and any combination thereof.153. The method of embodiment 152, wherein the sterol is cholesterol.154. The method of any one of embodiments 151-153, wherein the PEG-lipid, the helper lipid, and the sterol are present in the liposome at a molar ratio of 10:45:45.155. The method of any one of embodiments 127-154, wherein the liposome comprises a second helper lipid.156. The method of embodiment 155, wherein the second helper lipid is selected from the group consisting of DSPC, DSPE, Suc-DSPE, DOPC, DOPE, DOG, DAG, DPG, DSG, DMG, DMPE, DPPE, DLPE, DSPG, ceramide, 2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain and combinations thereof.157. The method of any one of embodiments 127-156, wherein the liposome comprises Suc-DSPE and DSPC.158. The method of embodiment 157, wherein the liposome comprises the PEG-lipid, the Suc-DSPE helper lipid, and the DSPC helper lipid at a molar ratio of 10:45:45.118#14739410v1159. The method of any one of embodiments 127 to 158, wherein the LNP comprises an ionizable lipid and a PEG-lipid.160. The method of embodiment 159, wherein the ionizable lipid of the LNP is, or a salt thereof.161. The method of embodiment 159 or 160, wherein the ionizable lipid is present in the LNP at a range of about 10 mol% to about 70 mol%.162. The method of embodiment 161, wherein the ionizable lipid is present in the LNP at a range of about 40 mol% to about 60 mol%.163. The method any one of embodiments 159-162, wherein the PEG-lipid of the LNP is selected from the group consisting of PEG-DSPE, PEG-DSG, PEG-DSPC, PEG-DOPC, PEG-DOPE, PEG-DMPE, PEG-DSPG, PEG-DMG, PEG-DAG, PEG-DOG, PEG-DPPE, PEG-DLPE, PEG-DPG, PEG-ceramide, PEG-2-[(polyethylene glycol)-2000]-N,N-ditetradecyl acetamide, diacylphosphatidyl ethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain and combinations thereof.164. The method of embodiment 153, wherein the PEG-lipid in both the LNP and the liposome is DPG-PEG or DMG-PEG.165. The method of any one of embodiments 159-164, wherein the PEG-lipid is present in the LNP at a range of about 0.5 mol% to about 4 mol%.166. The method of embodiment 165, wherein the PEG-lipid is present in the LNP at about 1.5 mol%.167. The method of any one of embodiments 159-166, wherein the LNP further compr...

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical system comprising two particles, wherein the first particle and the second particle share at least one common element, wherein the first particle is a liposome and the second particle is a lipid nanoparticle (LNP), and wherein:(i) both the first particle and the second particle bind to at least one innate immune system opsonin; and / or(ii) the common element is capable of inducing an anti-drug antibody against the first particle and the second particle in a subject when the pharmaceutical system is administered to the subject.

2. The pharmaceutical system of claim 1, wherein the common element comprises a polymer or a compound comprising a polymer, optionally wherein the polymer comprises a polyethylene glycol (PEG) or the compound comprising the polymer is a PEG-lipid.

3. The pharmaceutical system of claim 1 or claim 2, wherein the anti-drug antibody is an anti -PEG antibody.

4. The pharmaceutical system of any one of claims 1-3, wherein the liposome comprises a PEG or a compound comprising a PEG, optionally wherein the compound comprising the PEG is a PEG-lipid.

5. The pharmaceutical system of any one of claims 1-4, wherein the liposome further comprises one or more helper lipids.

6. The pharmaceutical system of claim 5, wherein the liposome comprises the PEG-lipid and the one or more helper lipids at a molar ratio of about 5:95 to about 25:75.

7. The pharmaceutical system of any one of claims 1-6, wherein the liposome does not comprise a sterol and / or does not comprise an ionizable lipid.

8. The pharmaceutical system of any one of claims 1-6, wherein the liposome further comprises a sterol.

9. The pharmaceutical system of claim 8, wherein the liposome comprises the PEG-lipid, the one or more helper lipids, and the sterol at a molar ratio of about 10:45:45.125#14739410v110. The pharmaceutical system of any one of claims 1-9, wherein the LNP comprises an ionizable lipid and a PEG-lipid.

11. The pharmaceutical system of claim 10, wherein the ionizable lipid is, or a salt thereof.

12. The pharmaceutical system of claim 10 or 11, wherein the ionizable lipid is present in the LNP at a range of about 10 mol% to about 70 mol%.

13. The pharmaceutical system of any one of claims 10-12, wherein the PEG-lipid is present in the LNP at a range of about 0.5 mol% to about 4 mol%.

14. The pharmaceutical system of any one of claims 10-13, wherein the LNP further comprises a payload compound, a structural lipid, and optionally a neutral phospholipid.

15. The pharmaceutical system of any one of claims 10-14, wherein the LNP further comprises a conjugate having the formula of [Lipid] - [optional linker] - [antibody], wherein the antibody specifically binds to a type of cell, tissue, or organ.

16. The pharmaceutical system of any one of claims 10-15, wherein the LNP comprises two conjugates both having the formula of [Lipid] - [optional linker] - [antibody], and the first conjugate comprises a first antibody that binds to a first antigen of a first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of a second type of immune cell.

17. The pharmaceutical system of any one of claims 1-16, wherein the LNP is for delivering a nucleic acid into an immune cell, and wherein LNP comprises a Fab lacking the native interchain disulfide bond.

18. The pharmaceutical system of any one of claims 14-17, wherein the neutral phospholipid in the LNP has a concentration of about 5% to 30%.126#14739410v119. The pharmaceutical system of any one of claims 10-18, wherein the ionizable lipid has a concentration about 10 g / g to about 20 g / g mRNA.

20. The pharmaceutical system of any one of claims 14-19, wherein the structural lipid comprises or is a sterol, and the sterol has a concentration about 3.0 g / g to 5.0 g / g mRNA.

21. The pharmaceutical system of any one of claims 14-20, wherein the neutral phospholipid in the LNP has a concentration about 2.0 g / g to 5.0 g / g mRNA.

22. The pharmaceutical system of any one of claims 10-21, wherein the PEG-lipid in the LNP has a concentration about 1.0 g / g to 1.5 g / g mRNA.

23. The pharmaceutical system of any one of claims 15-22, wherein the [Lipid] -[optional linker] - [antibody] conjugate in the LNP has a concentration about 0.05 g / g to 0.1 g / g mRNA.

24. A composition comprising the pharmaceutical system of any one of claims 1-23.

25. A kit comprising the pharmaceutical system of any one of claims 1 to 23, or the composition of claim 24.

26. A method of delivering a payload to a subject in need thereof, comprising administering the pharmaceutical system of any one of claims 1-23 or the composition of claim 24 to the subject.

27. The method of claim 26, wherein the second particle comprises the payload.

28. A method of treating a disease in a subject in need thereof, comprising administering the pharmaceutical system of any one of claims 1-23 or the composition of claim 24 to the subject.

29. The method of any one of claims 26-28, wherein the method comprises administering to the subject the first particle and the second particle separately.

30. The method of any one of claims 26-29, wherein the first particle and the second particle are administered sequentially.

31. The method of any one of claims 26-28, wherein the first particle and the second particle are administered to the subject simultaneously.127#14739410v132. The method of any one of claims 26 to 31, wherein the administration is by systemic administration, optionally wherein the systemic administration is by intravenous injection or intravenous infusion.

33. Use of the pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 in the manufacture of a medicament for delivering a payload to a subject.

34. Use of the pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 in the manufacture of a medicament for treating a disease in a subject in need thereof.

35. The pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 for use in delivering a payload to a subject.

36. The pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 for use in treating a disease in a subject in need thereof.

37. A method of reducing immunogenicity of a lipid nanoparticle (LNP) in a subject in need thereof, the method comprising administering to the subject the pharmaceutical system of any one of claims 1-23 or the composition of claim 24.

38. A method of reducing toxicity of a particle comprising polyethylene glycol (PEG), reducing anti -PEG antibody production in a subject caused by a lipid nanoparticle (LNP) comprising PEG or PEG-lipid, or eliminating anti -PEG antibody production in a subject caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof, the method comprising administering to the subject the pharmaceutical system of any one of claims 1 to 23 or the composition of claim 24.

39. Use of the pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 in the manufacture of a medicament for reducing toxicity of a particle comprising polyethylene glycol (PEG), reducing anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid, or eliminating anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof40. The pharmaceutical system of any one of claims 1-23, the composition of claim 24, or the kit of claim 25 for use in reducing toxicity of a particle comprising polyethylene glycol128#14739410v1(PEG), reducing anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid, or eliminating anti-PEG antibody production caused by a LNP comprising PEG or PEG-lipid in a subject in need thereof.129#14739410v1

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