Lipid nanoparticle

Abstract

The disclosure provides a lipid nanoparticle (LNP) composition comprising a polynucleotide, an ionizable lipids, a quaternary ammonium lipid at a molar percentage from about 20% to about 65%, a phospholipid, a cholesterol, and a PEG-lipid and method of using the LNP.

Description

DESCRIPTIONLIPID NANOPARTICLE

[0001] This application claims the benefit of priority to United States Provisional Application No. 63 / 516,777, filed on July 31, 2023, the entire contents of which are incorporated by reference.BACKGROUND

[0002] There is an unmet need for lipid nanoparticle compositions for specific delivery of polynucleotides. The present disclosure provides lipid nanoparticle compositions, as well as pharmaceutical compositions, methods of using them, and further related compositions and methods.SUMMARY

[0003] In one aspect, the disclosure provides a lipid nanoparticle (LNP) composition including (i) a payload, optionally a polynucleotide, (ii) an ionizable lipid, (iii) a quaternary ammonium lipid at a molar percentage from about 20% to about 65%, (iv) a phospholipid, and (vi) a polyethylene glycol-conjugated lipid (PEG-lipid).

[0004] In some embodiments, the quaternary ammonium lipid is a trimethylammonium propane (TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-dimyristoyl-3- trimethylammonium-propane (14:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-stearoyl-3 -trimethylammoniumpropane (18:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is 1,2- dioleoyl-3-trimethylammonium-propane (18: 1 TAP). In some embodiments, the quaternary ammonium lipid is 18: 1 l,2-dioleoyl-3-dimethyl(ethanolyl)ammonium-propane (RI). In some embodiments, the trimethylammonium propane (TAP) is 18:1 RI.

[0005] In some embodiments, the quaternary ammonium lipid is an ethylphosphocholine (EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC). In some embodiments, ethylphosphocholine (EPC) is 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC). In some embodiments, the1ethylphosphocholine (EPC) is l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-distearoyl-sn-glycero-3- ethylphosphocholine (18:0 EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC). In some embodiments, the ethylphosphocholine (EPC) is l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0- 18:0 EPC).

[0006] In some embodiments, the quaternary ammonium lipid is present in the composition at a molar percentage from about 20% to about 65%, from about 20% to about 55%, from about 20% to about 50%, from about 30% to about 65%, from about 30% to about 60%, or from about 30% to about 55%. In some embodiments, the quaternary ammonium lipid is present in the composition at a molar percentage from about 30% to about 50%.

[0007] In some embodiments, the phospholipid iiss l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE). In some embodiments, the phospholipid is 1,2-distearoyl-sn- glycero- 3 -phosphocholine (DSPC). In some embodiments, the phospholipid is present in the composition at a molar percentage from about 7.5% to about 60%, from about 7.5% to about 50%, from about 7.5% to about 40%, from about 7.5% to about 30%, from about 7.5% to about20%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about40%, from about 10% to about 30%, or from about 10% to about 20%.

[0008] In some embodiments, the cholesterol is present in the composition at a molar percentage from about 15% to about 46%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 20% to about 46%, from about 20% to about 40%, from about 20% to about 35%, or from about 20% to about 30%.

[0009] In some embodiments, the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%, from about 0.5% to about 7.5%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 1% to about 10%, from about 1% to about 7.5%, from about 1% to about 5% from about 1% to about 4% from about 1% to about 3%.

[0010] In some embodiments, the ionizable lipid is a compound of Formula (D-I):Core-Repeating Unit-Terminating Group (D-I), or a pharmaceutically acceptable salt thereof, wherein the core is linked to four to six repeating units and each repeating unit is linked to a nitrogen of the core, wherein: the core has the formula:wherein:X3 is -N R6-, wherein R6is hydrogen or alkyl,C≤8); R3and R4 are each independently amino, alkylamino(c≤12), dialkylamimrc 12,,wherein: each e independently is 1, 2, or 3;Rc, Rd, and Rf are each independently hydrogen or alkyl(c<6); c and d are each independently 1, 2, 3, 4, 5, or 6; the repeating unit is a degradable diacyl group having the formula:wherein:A1and A2are eachY3 is alkanediyl(c≤12); andRg is alkyl(c<8), and the terminating group has the formula:wherein:Y4 is alkanediyl(C≤18); andRio is hydrogen.

[0011] In some embodiments, the ionizable lipid is present in the composition at a molar percentage from about 5% to about 30%.

[0012] In some embodiments, the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof:Y3is -CH2CH2-;Rg is -CH3; and the terminating group is selected from the group consisting of:

[0013] In some embodiments, the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four repeating units and the core has the following structure:

[0014] In some embodiments, the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four or five repeating units and the core has the following structure:

[0015] In some embodiments, the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the ccoorree has the following structure:

[0016] In some embodiments, the ionizable lipid is a dendrimer. In some embodiments, the dendrimer is 4A3-SC7. In some embodiments, the dendrimer is 4A3-SC8. In some embodiments, the dendrimer is 5A2-SC8.

[0017] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 30%, a quaternary ammonium lipid at a molar percentage from about 20% to about 60%, DOPE at a molar percentage from about 7.5% to about 66%, the cholesterol at a molar percentage from about 15% to about 46%, and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

[0018] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 30%, a quaternary ammonium lipid at a molar percentage from about 20% to about 55%, DOPE at a molar percentage from about 7.5% to about 66%,the cholesterol at a molar percentage from about 15% to about 46%, and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

[0019] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 25%, a quaternary ammonium lipid at a molar percentage from about 30% to about 50%, the phospholipid at a molar percentage from about 7.5% to about 20%, the cholesterol at a molar percentage from about 15% to about 40%, and the PEG- lipid at a molar percentage from about 1 % to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an trimethylammonium propane (TAP).

[0020] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 25%, a quaternary ammonium lipid at a molar percentage from about 30% to about 50%, the phospholipid at a molar percentage from about 7.5% to about 20%, the cholesterol at a molar percentage from about 15% to about 40%, and the PEG- lipid at a molar percentage from about 1 % to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an trimethylammonium propane (TAP) or 18: 1 RI.

[0021] In some embodiments , The composition of any one of claim 1 to 31, wherein the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 25%, a quaternary ammonium lipid at a molar percentage from about 30% to about 50%, the phospholipid at a molar percentage from about 7.5% to about 20%, the cholesterol at a molar percentage from about 15% to about 40%, and the PEG-lipid at a molar percentage from about 1% to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an ethylphosphocholine (EPC).

[0022] In some embodiments, the polynucleotide comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or microRNA. In some embodiments, the polynucleotide comprises a messenger RNA (mRNA). In some embodiments, the mRNA comprises Nl- methylpseudouridine-5'-triphosphate. In some embodiments, the mRNA comprises Capl cap structure. In some embodiments, the mRNA encodes a gene-editing system or component thereof.

[0023] In some embodiments, the LNP includes a protein corona. In some embodiments, the protein corona includes at least one plasma protein. In some embodiments, the quaternary ammonium lipid controls causes association of the at least one plasma protein. In some embodiments, the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin. In some embodiments, the protein coronacomprises vitronectin. In some embodiments, the protein corona comprises prothrombin. In some embodiments, the protein corona comprises lower levels of component Clq subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid.

[0024] In some embodiments, the composition is an intravenous composition.

[0025] In another aspect, the disclosure provides a pharmaceutical composition including the LNP composition described herein. In another aspect, the disclosure provides a kit comprising the LNP compositionor the pharmaceutical composition described herein, and an instructions for use thereof.

[0026] In another aspect, the disclosure provides a method for treating or preventing a disease in a subject in need thereof, including administering the LNP composition or the pharmaceutical composition described herein.

[0027] In some embodiments, the method of administering comprises administration by intravenous injection.

[0028] In some embodiments, the LNP acquires a protein corona after adaministration. In some embodiments, the protein corona comprises at least one plasma protein. In some embodiments, the quaternary ammonium lipid controls causes association of the at least one plasma protein. In some embodiments, the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin. In some embodiments, the protein corona comprises vitronectin. In some embodiments, the protein corona comprises prothrombin. In some embodiments, the protein corona comprises lower levels of component Clq subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid. In some embodiments, the protein corona increases specific delivery of the pay load to the lung of the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIGs. 1A & IB show addition of a quaternary ammonium lipid to an LNP formulation in a suitable quantity enables mRNA delivery to the lungs via an endogenous targeting mechanism.

[0031] FIGs 2A-2D show incorporation of one of eleven quaternary ammonium lipids as a SORT molecule to mDLNP reduces mRNA delivery to the liver and spleen while enhancing mRNA delivery to the lungs with varying degrees of potency and selectivity. FIG. 2A shows ex vivo luminescence images of major organs excised from C57BL6 / J mice intravenously injected with four-component mDLNP or five-component TAP series SORT LNPs encapsulating FLuc mRNA. Beneath the images, pie charts profile the tissue-specificity of FLuc expression in the lungs, liver, and spleen and quantify the percent expression detected in the lungs (0.1 mg / kg, 6 h post- injection, n = 3). FIG. 2B shows quantification of the total luminescent flux produced by FLuc expressed in the lungs, liver, and spleens of mice treated with mDLNP or TAP series SORT LNPs (0. 1 mg / kg, 6 h post-injection, n = 3). FIG. 2C shows ex vivo luminescence images of major organs excised from C57BL6 / J mice intravenously injected with five-component EPC series SORT LNPs encapsulating FLuc mRNA. Beneath the images, pie charts profile the tissue-specificity of FLuc expression in the lungs, liver, and spleen and quantify the percent expression detected in the lungs (0.1 mg / kg, 6 h post-injection, n = 3). FIG. 2D shows quantification of the total luminescent flux produced by FLuc expressed in the lungs, liver, and spleens of mice treated with mDLNP or EPC series SORT LNPs (0.1 mg / kg, 6 h post-injection, n = 3). Data represented as mean + SEM.

[0032] FIGs. 3A-3D show lead SORT molecules enabing transfection and genome editing of therapeutically-relevant cells of the lungs. FIG. 3A shows depiction of genetic induction of tdTom expression following intracellular delivery of Cre mRNA by lung-targeting SORT LNPs. Translation of Cre from LNP-delivered mRNA removes the stop cassette that represses tdTom expression, yielding fluorescence in cells where functional mRNA delivery occurs. FIG. 3B shows fluorescence imaging of lungs excised from tdTom mice two days following treatment with a single dose of Cre mRNA encapsulated in lung-targeting SORT LNPs that employ either 18:1 TAP, 18:1 Rl, or 18:0 EPC as SORT molecules (0.3 mg / kg). FIG. 3C shows ex vivo fluorescence imaging measurement of mean tdTom fluorescence in major organs removed from tdTom mice treated with Cre mRNA (0.3 mg / kg, 2 days post-injection, n = 3). FIG. 3D shows FACS quantification of the percent of tdTom+ endothelial, epithelial, and immune cells in the lungs of tdTom mice treated with a single dose of Cre mRNA (0.3 mg / kg, 2 days post-injection, n = 3). Data represented as mean ± SEM.

[0033] FIG. 4 shows SDS-PAGE of the protein corona of four-component liver-targeting mDLNP or five-component lung-targeting SORT LNPs. While all lung-targeting SORT LNPs bind to similar plasma proteins, there are subtle differences in the identities and quantities of bound proteins that may affect in vivo delivery outcomes.

[0034] FIGs. 5A-5C show SORT molecule chemical structure impacting the identities and quantities of protein corona constituents in a manner that correlates with the specificity of mRNA delivery to the lungs. FIG. 5A shows protein corona fingerprint of four-component liver-targeting mDLNP or five-component lung-targeting SORT LNPs (n = 3). FIG. 5B shows univariate analysis of the lung specificity of FLuc mRNA delivery as a function of relative protein abundance. Vitronectin, clusterin, albumin, and prothrombin are positively correlated with lung delivery in a significant manner whereas complement component Clq subunits and immunoglobulin chains are negatively correlated with lung delivery. FIG. 5C shows univariate analysis of the liver specificity of FLuc mRNA delivery as a function of relative protein abundance. Vitronectin is negatively correlated with liver delivery in a significant manner whereas complement component Clq subunits and immunoglobulin chains are positively correlated with liver delivery. (*; p < 0.05)

[0035] FIGS. 6A-6C show physicochemical characterization of mDLNP and five-component LNPs incorporating various quaternary ammonium lipids as SORT molecules. (A) Dynamic light scattering measurement of LNP hydrodynamic diameter and polydispersity index (n = 3). (B) LNP ^-potential (n = 3). (C) LNP encapsulation efficiency determined by RiboGreen Assay (n = 3).

[0036] FIG. 7 shows lead SORT molecules promote sustained production of a therapeutically- relevant protein, hEPO, with 18:1 RI yielding greatest protein production. ELISA quantification of serum hEPO protein at 6, 24, 48, and 72 h after injecting C57BL6 / J mice with lead lung-targeting SORT LNPs carrying hEPO mRNA (0.3 mg / kg, n = 3).

[0037] FIG. 8 shows non-lung major organs excised from tdTom mice treated with five- component lung-targeting SORT LNPs (0.3 mg / kg, 2 days post-injection). As a negative control, three tdTom mice were treated with an equivalent volume of IX PBS.

[0038] FIGS. 9 A & 9B show flow cytometry gating for lung cells recovered from tdTom mice treated with either PBS or five-component lung-targeting SORT LNPs encapsulating Cre mRNA (0.3 mg / kg, 2 days post-injection).

[0039] FIG. 10 shows SDS-PAGE of plasma proteins that bind to three independently formulated batches of either four-component mDLNP or five-component lung-targeting SORT LNPs incorporating various quaternary ammonium lipids as SORT molecules. Reproducible protein binding patterns can be identified for each formulation. The same samples were submitted for mass spectrometry proteomics analysis.

[0040] FIGS. 11A & 11B show SDS-PAGE of C57BL / 6 mouse plasma that has either undergone the protein corona isolation protocol or is in its native state (diluted 10-fold in IX PBS). The first lane is the ladder, the second lane (labeled A) is plasma put through the protein corona purification process, and the third lane (labeled B) is plasma diluted 10-fold in IX PBS. No discernible pellet was formed from plasma not incubated with LNPs nor were protein bands stained on the gel, suggesting that the protein corona samples are free of contaminants that simply pellet out during the centrifugation protocol.

[0041] FIGS. 12A & 12B show serum stability of mDLNP and lung-targeting SORT LNPs in 10 % fetal bovine serum (FBS). All formulations tested demonstrate small changes in hydrodynamic diameter, in part likely due to protein corona formation, up to 48 hr.DETAILED DESCRIPTIONDefinitions

[0042] Reference is made herein to particular features (including method steps) It is to be understood that the disclosure in this specification includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments.

[0043] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes that possibility).

[0044] The practice of the technology will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that arewithin the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those of skill in the art.

[0045] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Specifically, features described in one section may be combined with features in any other section of the description.

[0047] While illustrative embodiments are described and depicted, it will be appreciated that various changes can be made to these illustrative embodiments without departing from the spirit and scope of the invention.

[0048] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole.

[0049] The singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0050] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In some embodiments, about means within a standard deviation using measurements generally acceptable in the art. In some embodiments, about means a range extending to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0051] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.

[0052] The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)- (a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0053] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and "comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. For example, a composition “comprising” (or “which comprises”) components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components.

[0054] As used herein, the term “consisting of’ refers to including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0055] As used herein, the terms “specific”, “specifically”, “specificity”, or the like of a composition refers to the composition’s ability to cause a particular action, such as inhibition, to a particular molecular target with minimal or no action to other proteins in the cell.

[0056] Reference throughout this specification to, for example, “one embodiment”, “an embodiment”, “another embodiment", “a particular embodiment”, “a related embodiment”, “a certain some embodiments”, “an additional embodiment”, or “a further embodiment" orcombinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0057] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0058] The term “recombinant” indicates that the material (e.g., a nucleic acid or protein) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. Specifically, e.g., a Coronavirus is recombinant when it is produced by the expression of a recombinant nucleic acid. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis; a “recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid; and a “recombinant virus,” e.g., a recombinant Coronavirus, is produced by the expression of a recombinant nucleic acid. A “fusion protein” or “tetramers” described here are recombinant and do not occur in nature.

[0059] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids, such as a glycan or other post-translational modification, or a non-natural chemical moeity. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single polymer molecule.

[0060] The term “recombinant polypeptide” refers a polypeptide that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segmentsof sequence. In several embodiments, a recombinant polypeptide is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell.

[0061] The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. In some aspects, expression can be detected using conventional techniques for detecting protein (e.g. , ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0062] A “cell” as used herein, refers to a membrane-bound biological unit capable of carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0063] The term “host cell” refers to any cell capable of use in expression of a recombinant polypeptide or polynucleotide. In some embodiments, the host cell is a bacterial cell. In some embodiments, the bacterial cell is an E. colt cell.

[0064] The terms “culturing” and “culture medium” refers to standard cell culture and recombinant protein expression techniques.

[0065] The term “manufacturing” refers to production of a recombinant polypeptide or protein nanostructure or polynucleotide at any scale, including at least 25-mL, 50-mL, 1-L, 1 ,000-L, 50,000-L, or greater scale.

[0066] The term “payload” refers to any chemical entity, pharmaceutical, drug (such drug can be, but not limited to, a small molecule, an inorganic solid, a polymer, or a biopolymer), small molecule, nucleic acid (e.g., DNA, RNA, siRNA, etc.), protein, peptide and the like that is complexed with a lipid nanoparticle formulation described in the present disclosure. A payload also encompasses a candidate (e.g., of unknown structure and / or function) for use in treating or preventing a disease, illness, sickness, or disorder of bodily function and includes, but is notlimited to, test compounds that are both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.

[0067] As may be used herein, the terms “nucleic acid,” “ 'nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0068] The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence, followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166-?1554 * 100=75.0). As the terms are used herein, gaps in the alignment do not decrease the percent sequence identity. Unless otherwise specificed, optimal alignment of sequences for comparison is conducted by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) as implemented by EMBOSS Needle (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(Wl):W276-W279 (2022)). In embodiments, other alignment methods may be used, including without limitationthose described in Devereux, et al, Nucleic Acids Res. 12:387-95 (1984) ; Atschul et al. J. Mo. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appt. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994); Sequence Analysis in Molecular Biology (von Heinje, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). In embodiments, sequence identity is calclated using the implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln)

[0069] For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) that can be used in conjunction with the computer program.

[0070] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0071] As used herein, the term “shRNA” or “shor hairpin RNA” refer to a short sequence of RNA which makes a tight hairpin turn and can be used to silence gene expression.

[0072] As used herein, the term “microRNA” refers noncoding RNA consisting of about 22 ribonucleotides which regulates gene expression in the post transcriptional stage by silencing messenger RNA by base-pairing with omplementary sequence in its targeted mRNA.

[0073] As used herein, the term “gene-editing system” refers to a DNA or RNA editing system that comprises one or more guide RNA elements and one or more RNA-guided endonuclease elements. The guide RNA element comprises a target RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence at the one or more target genomic regions or a nucleic acid comprising a nucleotide sequence(s) encoding the target RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or brought to a target genomic region(s) by a guide RNA element or a nucleic acid comprising a nucleotide sequence(s) encoding such endonuclease.

[0074] The term “pharmaceutically acceptable excipients” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient and can mean excipients approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0075] The term “subject” refers to a human or non-human animal to which a composition may be administered for vaccination, treatment, or other purpose. In some embodiments, the non- human animal is a non-human primate, rabbit, hamster, gerbil, pig, cow, sheep, goat, guinea pig, rat, mouse, squirrel, wolf, fox, horse, zebra, giraffe, hyena, elephant, cat, dog, llama, or ferret.

[0076] As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to aninfection, disease, disorder, and / or condition, to treat, improve signs and symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0077] For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5 -fold, or more effect over a control.

[0078] As used herein, the term “delivering” means causing, through chemical or biophysical properties of a composition (e.g., an LNP composition), a pay load (e.g., a polynucleotide) to pass from a site of administration to a subject to a target organ, target tissue, or target cell.. As used herein, the term “selectively delivering” refers delivery to a target organ, tissue, or cell at a greater rate or in a great amount than to a reference, non-target organ, tissue, or cell, or that a greater fraction of total amount administered to a subject is delivered to a target organ, tissue, or cell by the composition than by a reference composition. For example, selective delivery may mean that at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the total amount administered is delivered to the target organ, tissue, or cell. In embodiments, “selective delivery” is determined by comparing the fraction of an LNP composition or pay load that is delivery to a target organ (e.g., the lung) by an LNP composition comprises a selected lipid (e.g. a quatenary ammonium lipid) compared to a reference LNP composition in which the selected lipid is replace by a control lipid.

[0079] “Prevention” or “preventing” refers to inhibiting the onset of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and / or slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and / or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease. The prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.

[0080] “Treating” or “treatment” as used herein (and as well-understood in the art) broadly refers to any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of theextent of a disease, stabilizing (z.e. , not worsening) the state of disease, prevention of a disease’ s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, “treatment” as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0081] The term “administering” refers to providing a composition to a subject in a manner that permits the composition to have its intended effect. Administration for vaccination or postexposure prophylaxis may be performed by intramuscular injection, intravenous injection, intraperitoneal injection, or any other suitable route.

[0082] “Co-administer” means that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compositions provided herein can be administered alone or can be coadministered to the subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation).

[0083] As used herein, the term “contacting" refers to allowing two species to interact, such as by chemical interactions including ionic, non-ionic, polar, hydrophobic, or hydrophilic interactions, or physically touch as accepted in the art, where the two species may be lipid nanoparticle and a cell, mucus, or lining of tissue. In cell culture, an LNP may be contacted with a cell by mixing an LNP composition with a suitable cell culture media or liquid or mucus surrounding the cells, thereby allowing the LNP to contact the cell.

[0084] As used herein, the term “lipid nanoparticle” refers to a vesicle formed by one or more lipid components typically used as carriers for nucleic acid, protein, or oligonucleotide delivery in the context of pharmaceutical development. Generally, lipid nanoparticle compositions for delivery are composed of synthetic ionizable or cationic lipids, phospholipids, cholesterol, and a polyethylene glycol (PEG) lipid. However, these compositions may also include other lipids.

[0085] As used herein, the term “ionizable lipid” refers to lipid and lipid-like molecules with atoms that can acquire charge (e.g., nitrogen) in some conditions.

[0086] As used herein, the term “neutral phospholipid” refers to phospholipids that have little or no net charge at physiological pH. In many embodiments, neutral phospholipids are zwitterions, although other types of net neutral phospholipids are known and may be used.

[0087] As used herein, the term “PEG-lipid” refers to a lipid modified with a polyethylene glycol unit. In some embodiments, the PEG-lipid comprises DMG. In some embodiments, the PEG-lipid comprises DSPE.

[0088] As used herein, the term “cholesterol” refers a sterol molecule found in the cell membranes of all animal tissues. Cholesterol may be transported in an animal's blood plasma by lipoproteins including very low density lipoprotein (VLDL), intermediate density lipoprotein (IDE), low density lipoprotein (LDL), and high density lipoprotein (HDL).

[0089] As used herein, the term “apparent pKa” refers to the overall dissociation constant of all titratable groups in the lipid nanoparticles.

[0090] As used herein, the term “N / P ratio” refers to a molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide.

[0091] As used herein, the term “lipid:RNA ratio” refers to mg of lipid for each mg of mRNA drug substance which influence the encapsulation efficiency of lipid nanoparticles.

[0092] The terms “lung disease,” “pulmonary disease,” “pulmonary disorder,” broadly refer to diseases or disorders of the lung. Lung diseases may be characterized by symptoms including but not limited to difficulty breathing, coughing, airway discomfort and inflammation, increased mucus, and / or pulmonary fibrosis. Examples of lung diseases include Primary Ciliary Dyskinesia (PCD) (also referred to as Kartageners Syndrome, or Immotile Cilia Syndrome), cystic fibrosis, asthma, lung cancer, Chronic Obstructive Pulmonary Disease (COPD), bronchitis, emphysema, bronchiectasis, pulmonary edema, pulmonary fibrosis, sarcoidosis, pulmonary hypertension, pneumonia, tuberculosis, Interstitial Pulmonary Fibrosis (IFF), Interstitial Lung Disease (ILD), Acute Interstitial Pneumonia (A1P), Respiratory Bronchiolitis-associated Interstitial Lung Disease (RBILD), Desquamative Interstitial Pneumonia (DIP), Non-Specific Interstitial Pneumonia (NSIP), Idiopathic Interstitial Pneumonia (IIP), Bronchiolitis obliterans, with Organizing Pneumonia (BOOP), restrictive lung disease, or pleurisy.

[0093] The term “quaternary amine” as used herein refers to a nitrogen atom having four organic substituents. For example, the nitrogen atom in tetramethylammonium chloride or a trialkyl ammonium chloride is a quaternary amine. Examples of cationic lipids comprising aquaternary amine include, but are not limited to, N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (“DOTAP”), N,N,-Distearyl-N,N-dimethylammonium bromide (“DDBA”), l-methyl-4-(cis-9-dioleyl)-methylpyridinium-chloride (“SAINT-solid”), N-(2,3- dioleyloxy)propyl)-N,N,N-triethylammonium chloride (“DOTMA”), N,N-dioleyl-N,N- dimethylammonium chloride (“DODAC”), (l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (“DIMRIE”), l,2-dimyristoyl-3-trimethylammonium- propane (14:0 TAP), l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2- stearoyl-3-trimethylammonium-propane (18:0 TAP), 1 ,2-dioleoyl-3-trimethylammonium- propane (18:1 TAP), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l- ammonium (DORI, 18:1 RI), l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), l,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine (16:0 EPC), l,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18: 1 EPC), and l-palmitoyl-2-oleoyl- sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC).

[0094] The term “protein corona,” as used herein, refers to the formation of a layer of proteins, lipids and other plasma component on the surface of a nanoparticle after the particle has been contacted with biological samples or biological system (e.g., plasma).

[0095] “Plasma protein” as used herein refers to the soluble proteins found in the plasma of normal humans or animals. These include but are not limited to coagulation proteins, antibodies, albumin, lipoproteins, complement proteins, and the like.Lipid Nanoparticle Compositions

[0096] Provided herein are compositions and methods related to lipid nanoparticle compositions. In one aspect, the disclosure provides a lipid nanoparticle (LNP) composition including (i) a payload, optionally a polynucleotide, (ii) an ionizable lipid, (iii) a quaternary ammonium lipid at a molar percentage from about 20% to about 65%, (iv) a phospholipid, and (vi) a polyethylene glycol-conjugated lipid (PEG-lipid).

[0097] In some embodiments, the lipid nanoparticles described herein may be prepared according to the methods described in International Publication Nos. WO2016094342, WO2017048789, W02017201091, WO2017205767, WO2019246203, W02020051220,WO2022169508, W02022204053 and WO2022204215; the contents of each of which are incorporated herein by reference in their entireties.Ionizable Lipids

[0098] In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable lipid. In some embodiments, the ionizable lipid is an ionizable lipid. In some embodiments, the ionizable lipids contain one or more groups which is protonated at physiological pH but may deprotonated and has no charge at a pH above 8, 9, 10, 11, or 12. The ionizable group may contain one or more protonatable amines which are able to form a cationic group at physiological pH. The ionizable lipid compound may also further comprise one or more lipid components such as two or more fatty acids with C6-C24alkyl or alkenyl carbon groups. These lipid groups may be attached through an ester linkage or may be further added through a Michael addition to a sulfur atom. In some embodiments, these compounds may be a dendrimer, a dendron, a polymer, or a combination thereof.

[0099] As used herein, the term “ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. For instance, an ionizable lipid may be positively charged at lower pHs, in which case it could be referred to as “cationic lipid.” In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or - 2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (z.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidazolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

[0100] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule. The terms “partial negative charge” and “partial positive charge” are given its ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partialnegative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[0101] A lipid nanoparticle composition may include one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH). Ionizable lipids may he selected from the non-limiting group consisting of 3- (didodecylamino)-N 1 ,N1 ,4-tridodecy 1-1 -piperazineethanamine (KL 10), N1 - [2-(didodecylamino)ethyl] Nl,N4,N4-tridodecyl-l,4- piperazinediethanamine (KL22), 14,25- ditridecy 1-15,18,21 ,24-tetraaza-octatriacontane (KL25) , 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoley 1 -4-dimethylaminomethyl-[ 1 ,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31 -tetraen- 19-yl -4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)- [l,3]-dioxolane (DLin-KC2-DMA), l,2-dioleyloxy-N,Ndimethylaminopropane (DODMA), 2-({ 8 [(3(3)-cholest-5-en-3- yloxy]octyl!oxy)N,N dimethyl -3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({ 8-[(3(3)-cholest-5-en-3-yloxy]octylIoxy)-N,N-dimethyl -3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA (2R)), and (2S) 2- ({ 8-[(3(3)-chol e st-5-en-3 -yloxy] octyl } oxy)-N,N-dimethyl-3 -[(9Z,12Z)-octadeca-9,12-di en-1 -yloxy]propan-l-amine (Octyl-CLinDMA (2S)), 4-hydroxybutyl ) azanediyl)bis (hexane- 6, 1 -diyl)bis(2 -hexyldecanoate (ALC-0315), or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate (SM-102). In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group.

[0102] Ionizable lipids can also be the compounds disclosed in International Publication No. WO 2017 / 075531 Al, hereby incorporated by reference in its entirety. Ionizable lipids can also be the compounds disclosed in International Publication No. WO 2015 / 199952 Al, hereby incorporated by reference in its entirety. In one embodiment, the ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Publication Nos. W02012040184, W02011153120, WO2011149733, W02011090965, W02011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724,W0201021865, W02008103276, WO2013086373 and WO2013086354, US Patent Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No.US20100036115, US20120202871, US20130064894, US20130129785, US20130150625,US20130178541 and S20130225836; the contents of each of which are herein incorporated by reference in their entirety.

[0103] As a non-limiting example, a ionizable lipid may be selected from (20Z,23Z)-N,N- dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)-N,Ndimemylhexacosa-17,20-dien-9- amine,(lZ,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)-N,N- dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa- 15,18-dien-7-amine, (18Z,21Z)-N,Ndimethylheptacosa- 18,21 -dien- 10-amine, (15Z, 18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4- amine, (19Z,22Z)-N,Ndimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N- dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7- amine, (16Z,19Z)-N,Ndimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N- dimethylhentriaconta-22,25-dien- 10-amine, (21Z ,24Z)-N,N-dimethyltriaconsa-21,24-dien-9- amine, (18Z)-N,Ndimetylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9- amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10- amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien- 10-amine, 1- [( 11Z, 14Z)- 1 - nonylicosa-l l,14-dien-l-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en- 10-amine, (15Z)- N,N-dimethyleptacos-15-en- 10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)- N,N-dimethylnonacos-17-en- 10-amine, (24Z)-N,N-dimethyltritriacont-24-en- 10-amine, (20Z)-N,N-dimethylnonacos-20-en- 10-amine, (22Z)-N,Ndimethylhentriacont-22-en-10- amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2- nonylhenicosa- 12, 15-dien- 1 -amine, ( 13Z, 16Z)-N,Ndimethy 1 -3-nonyldocosa- 13,16-dien-l- amine, N,N-dimethyl-l-[(lS,2R)-2-oetyleyclopropyl]eptadecan-8-amine, l-[(lS,2R)-2- hexylcyclopropy 1 ] -N,Ndimethylnonadecan- 10-amine, N,N-dimethy 1 - 1 - [( 1 S,2R)-2- oety Icyclopropy 11 non adecan - 10-amine, N,N-dimethy 1 -21 - [( 1 S ,2R)-2- octy Icyclopropy I |hen i cosari- 10-amine, N,N-dimethy 1 - 1 - [( 1 S ,2S)-2- { [(lR,2R)-2- pentylcyclopropyl]methyl)cyclopropyl]nonadecan- 10-amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(lR,2S)-2 undecyIcyclopropyl]tetradecan-5-amine, N,N-dimethy 1 -3- { 7- [( 1 S,2R)-2- octylcyclopropyl]heptylldodecan-l-amine, l-[(lR,2S)-2-heptylcyclopropyl]-N,Ndimethyloctadecan-9-amine, l-[(lS,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan- 6-amine, N,N-dimethy 1 - 1 - [( 1 S ,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,Ndimethy 1-1 - [ (9Z , 12Z)-octadeca-9, 12-dien-l-yloxy ] -3-(octyloxy)propan-2-amine, S- N,Ndimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2- [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy ] - 1 - [(octy loxy )methy 1 |e thy 11 pyrrol idi ne, (2S)-N,Ndimethy 1 - 1 - [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy ] -3- [(5Z)-oct-5-en-l-loxy ]propan-2-amine, 1 - { 2-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy ]- 1 -[(octyloxy)methyl]ethyllazetidine, (2S)-l-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2S)-l-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-lyloxy]propan-2-amine, N,N-dimethy 1 - 1 -(nonyloxy)-3- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]propan-2-amine, N,N- dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyl oxy)prop an-2-amine; (2 S)-N,N-dimethyl- l-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1- [(HZ,14Z)-icosa-l l,14-dien-l-yloxy]-N,Ndimethyl-3-(pentyloxy)propan-2-amine, (2S)-1- (hexyloxy)-3-[(l lZ,14Z)-icosa-l 1 ,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine, 1 - [( 11 Z, 14Z)-icosa- 11 , 14-dien- 1 -yloxy ]-N,N-dimethy 1 -3 -(octyloxy)propan-2-amine, 1- [( 13Z, 16Z)-docosa- 13,16-dien-l-yloxy ]-N,N-dimethy 1 -3-(octyloxy )propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-l-yloxy]-3-(hexyl oxy)-N,N-dimethylpropan-2-amine, (2S)-1- [(13Z)-docos-13-en-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, l-[(13Z)-docos-13- en- 1 -yloxy]-N,Ndimethy 1 -3-(octyl oxy)propan-2-amine, 1 -[(9Z)-hexadec-9-en-l-yloxy]-N,N- dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l-metoyloctyl)oxy]-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2R)- 1 - [(3 ,7 -dimethyloctyl)oxy ] -N,Ndimethyl-3-|(9Z,12Z)-octadeca-9,12-dien-l-yloxy |propan-2-amine, N, N -dimethyl -1 - (octyloxy)-3-({8-[(l S,2 S)-2-[(lR,2R)-2- pentylcyclopropyl]methylIcyclopropyl]octylIoxy)propan-2-amine, N,N-dimethyl-l-[8-(2- oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,2OZ,23Z)- N,Ndimethylnonacosa-l l,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0104] ln some embodiments of the lipid composition of the present application, the ionizable lipids refer to lipid and lipid-like molecules with nitrogen atoms that can acquire charge (pKa). These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl such as C6-C24alkyl or alkenyl groups, but may have at least 1 or more that 6 tails. In some embodiments, these -ionizable lipids are dendrimers, which are a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core and are characterized by a core, at least one interior branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994.) In other embodiments, the term “dendrimer” as used herein is intended to include, but is not limited to, a molecular architecture with an interior core, interior layers (or “generations”) of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation. A “dendron” is aspecies of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a larger dendrimer. In some embodiments, the dendrimer structures have radiating repeating groups from a central core which doubles with each repeating unit for each branch. In some embodiments, the dendrimers described herein may be described as a small molecule, medium-sized molecules, lipids, or lipid-like material. These terms may be used to describe compounds described herein which have a dendron like appearance (e.g. molecules which radiate from a single focal point).

[0105] While dendrimers are polymers, dendrimers may be preferable to traditional polymers because they have a controllable structure, a single molecular weight, numerous and controllable surface functionalities, and traditionally adopt a globular conformation after reaching a specific generation. Dendrimers can be prepared by sequentially reactions of each repeating unit to produce monodisperse, tree-like and / or generational structure polymeric structures. Individual dendrimers consist of a central core molecule, with a dendritic wedge attached to one or more functional sites on that central core. The dendrimeric surface layer can have a variety of functional groups disposed thereon including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during the preparation.

[0106] Modifying the functional groups and / or the chemical properties of the core, repeating units, and the surface or terminating groups, their physical properties can be modulated. Some properties which can be varied include, but are not limited to, solubility, toxicity, immunogenicity and bioattachment capability. Dendrimers are often described by their generation or number of repeating units in the branches. A dendrimer consisting of only the core molecule is referred to as Generation 0, while each consecutive repeating unit along all branches is Generation 1, Generation 2, and so on until the terminating or surface group. In some embodiments, half generations are possible resulting from only the first condensation reaction with the amine and not the second condensation reaction with the thiol.

[0107] Preparation of dendrimers requires a level of synthetic control achieved through series of stepwise reactions comprising building the dendrimer by each consecutive group. Dendrimer synthesis can be of the convergent or divergent type. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous and then changing functional groups for the next stage of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would lead to a highly branched molecule that is not monodisperse and is otherwise known as a hyperbranched polymer. Due to steric effects,continuing to react dendrimer repeat units leads to a sphere shaped or globular molecule, until steric overcrowding prevents complete reaction at a specific generation and destroys the molecule's monodispersity. Thus, in some embodiments, the dendrimers of G1-G10 generation are specifically contemplated. In some embodiments, the dendrimers comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. In some embodiments, the dendrimers used herein are GO, Gl, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by reducing the spacing units in the branching polymer.

[0108] Additionally, dendrimers have two major chemical environments: the environment created by the specific surface groups on the termination generation and the interior of the dendritic structure which due to the higher order structure can be shielded from the bulk media and the surface groups. Because of these different chemical environments, dendrimers have found numerous different potential uses including in therapeutic applications.

[0109] In some embodiments of the lipid composition of the present application, the dendrimers are assembled using the differential reactivity of the acrylate and methacrylate groups with amines and thiols. The dendrimers may include secondary or tertiary amines and thioethers formed by the reaction of an acrylate group with a primary or secondary amine and a methacrylate with a mercapto group. Additionally, the repeating units of the dendrimers may contain groups which are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diethers, esters, amides, or disulfides groups. In some embodiments, the core molecule is a monoamine which allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches which each may comprise one or more repeating units. The dendrimer may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom. In some embodiments, the terminating group is a lipophilic groups such as a long chain alkyl or alkenyl group. In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In still other embodiments, the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0110] The ionizable lipids of the present application may contain one or more asymmetrically- substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form.Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Ionizable lipids may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the ionizable lipids of the present application can have the .S' or the R configuration. Furthermore, it is contemplated that one or more of the ionizable lipids may be present as constitutional isomers. In some embodiments, the compounds have the same formula but different connectivity to the nitrogen atoms of the core. Without wishing to be bound by any theory, it is believed that such ionizable lipids exist because the starting monomers react first with the primary amines and then statistically with any secondary amines present. Thus, the constitutional isomers may present the fully reacted primary amines and then a mixture of reacted secondary amines.

[0111] Chemical formulas used to represent ionizable lipids of the present application will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.

[0112] The ionizable lipids of the present disclosure may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance) than, and / or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.

[0113] In addition, atoms making up the ionizable lipids of the present application are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C.

[0114] It should be recognized that the particular anion or cation forming a part of any salt form of a ionizable lipids provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts andtheir methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0115] In some embodiments of the lipid composition of the present application, the ionizable clipid is a dendrimer or dendron. In some embodiments, the ionizable lipid comprises an ammonium group which is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH from about 6 to about 8. In some embodiments, the ionizable lipid is a dendrimer or dendron. In some embodiments, the ionizable lipid comprises at least two C6-C24alkyl or alkenyl groups.

[0116] In some embodiments of the lipid composition, the ionizable lipid comprises at least two C8-C24alkyl groups.

[0117] In some embodiments, the ionizable lipid is a dendrimer further defined by the formula:Core-(Repeating Unit)n-Terminating Group (D-I) wherein one or more hydrogen atoms of the core are replaced with a repeating unit and wherein: the core has the formula:wherein:Xi is amino or C1-C12alkylamino, C1-C12dialkylamino, C3-C12heterocycloalkyl, C5-C12heteroaryl, or a substituted version thereof;Ri is amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the formula: wherein:X2is N(R5)y;Rs is hydrogen, C1-Cis alkyl, or substituted C1-C1s alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3;R2is amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1 , 2, or 3 ; provided that the sum of z and y is 3 ; or the core has the formula:wherein:X3 is -NR6-, wherein R6is hydrogen, C1-C8 alkyl, or C1-C8 substituted alkyl, -O-, or C1-C8alkylaminodiyl, C1-C88lkoxydiyl, C6-C8 arenediyl, C5-C8 heteroarenediyl, C3-C8heterocycloalkanediyl, or a substituted version of any of these groups; R3and R4are each independently amino, hydroxy, mercapto, C1-C12alkylamino, or C1-C12dialkylamino, or a substituted version of either of these groups; or a group of the formula: -N(Rf)f(CH2CH2N(Rc))eRd,wherein: e and f are each independently 1 , 2, or 3 ; provided that the sum of e and f is 3;Rc, Rd, and Rfare each independently hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is C1-C18alkylamine, C1-C36dialkylamine, C3-C12heterocycloalkane, or a substituted version of any of these groups; wherein the repeating unit comprises a degradable diacyl or a degradable diacyl and a linker; the degradable diacyl group has the formula:Owherein:A; and A2 are each independently -O- , -S-, or -NRa-, wherein: Rais hydrogen, C1-C6alkyl, or substituted C1-C6alkyl;¥3 is C1-C12alkanediyl, C1-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups; or a group of the formula:wherein:X3 and X4 are C1-C12alkanediyl, C2-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups;Y5 is a covalent bond, C1-C12alkanediyl, C1-C12alkenediyl, C6- C12arenediyl, or a substituted version of any of these groups; andR9is C1-C8alkyl or substituted C1-C8alkyl; the linker group has the formula:wherein:Yi is C1-C12alkanediyl, C1-C12alkenediyl, C6-C12arenediyl, or a substituted version of any of these groups; and* wherein each independently denotes a point of attachment to another repeating unit or a terminating group; and the terminating group has the formula:wherein:¥4 is alkanediyl or an C1-C1s alkanediyl wherein one or more of the hydrogen atoms on the C1-C1s alkanediyl has been replaced with -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3;Rio is hydrogen, carboxy, hydroxy,wherein the final degradable diacyl in the chain is attached to a terminating group; n is 0, 1, 2, 3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.

[0118] In some embodiments, the terminating group is further defined by the formula: wherein:Y4is C1-C18alkanediyl; andRio is hydrogen. In some embodiments, Ai and A2are each independently -O- or -NRa-.

[0119] In some embodiments of the dendrimer of formula (D-I), the terminating group is a structure selected from the structures in Table 1.

[0120] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:wherein:X2is N(R5)y;R5is hydrogen or C1-C8alkyl, or substituted C1-C18alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3;R2is amino, hydroxy, or mercapto, or C1-C12alkylamino, C1-C12dialkylamino, or a substituted version of either of these groups;b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3; provided that the sum of z and y is 3.

[0121] In some embodiments of the dendrimer of formula (D-I), the core is further defined by the formula:wherein:X3 is -NRo-, wherein R6is hydrogen, C1-C8alkyl, or substituted C1-C8alkyl, -O-, or C1-C8alkylaminodiyl, C1-C8alkoxydiyl, C1-C8arenediyl, C1-C8heteroarenediyl, C1-Csheterocycloalkanediyl, or a substituted version of any of these groups; R3and R4 are each independently amino, hydroxy, or mercapto, or C1-C12alkylamino, dialkylamino, or a substituted version of either of these groups; or a group ofwherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;Rc, Rd, and Rf are each independently hydrogen, C1-C6alkyl, or substituted C1-C6alkyl; c and d are each independently 1, 2, 3, 4, 5, or 6.

[0122] In some embodiments of the dendrimer of formula (I), the terminating group is represented by the formula:wherein:Y4 is alkanediyl(c<i8); and Rio is hydrogen.

[0123] In some embodiments of the dendrimer of formula (D-I), a core of the structure of formula (D-IV) is:

[0124] In some embodiments of the dendrimer of formula (D-I), the core comprises a structural formula set forth in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a repeating unit (i.e. , where a hydrogen of the core is relaced with a repeating unit).

[0125] In some embodiments of the dendrimer of formula (D-I), the degradable diacyl is further defined as:O MeO'O

[0126] In some embodiments of the dendrimer of formula (D-I), the linker is further defined aswherein Y i is C1-C8alkanediyl or substituted C1-C12alkanediyl.

[0127] In some embodiments, in the core of formula (D-IV), Re is H. In some embodiments, in the core of formula (D-IV), Re is C1-C8alkyl. In some embodiments, in the core of formula (D- IV), Re is substituted alkyl (e.g., alkyl substituted with -NH2, alkyl substituted with -NHCH3, or alkyl substituted with -NHCH2CH3).

[0128] In some embodiments one or two hydrogen atoms of the core are replaced with a repeating unit. In some embodiments three or four hydrogen atoms of the core is replaced with a repeating unit. In some embodiments five hydrogen atoms of the core is replaced with a repeating unit. In some embodiments six hydrogen atoms of the core is replaced with a repeating unit.

[0129] In some embodiments of the dendrimer of formula (D-I), the dendrimer is selected from the group consisting of:and pharmaceutically acceptable salts thereof.Dendrimers of Formula (X)

[0130] In some embodiments of the lipid composition, the ionizable lipid is a dendrimer of the formulaIn some embodiments, the ionizable lipid is a dendrimer of the formula

[0131] In some embodiments of the lipid composition, the ionizable lipid is a dendrimer of a generation (g) having a structural formula:or a pharmaceutically acceptable salt thereof, wherein:(a)the core comprises a structural formula (Xcore):wherein:Q is independently at each occurrence a covalent bond, -O-, -S-, -NR2-, or - CR3aR3b-;R2is independently at each occurrence R1gor -L2-NR1eR1f;R3aand R3bare each independently at each occurrence hydrogen or an optionally substituted (e.g., C1-C6, such as C1-C3) alkyl;R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted (e.g., C1-C12) alkyl;L°, L1, and L2are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, | alkylene |-| heterocycloalkyl |-| alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L1form a (e.g., C4-C6) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of R1cand R1d; and x1is 0, 1, 2, 3, 4, 5, or 6; and(b)each branch of the plurality (N) of branches independently comprises a structural formula ( XBranch):wherein:* indicates a point of attachment of the branch to the core;G-0, when g-1 ; or G =221, when g^l;(c) each diacyl group independently comprises a structural formula, wherein:* indicates a point of attachment of the diacyl group at the proximal end thereof;** indicates a point of attachment of the diacyl group at the distal end thereof;Y3is independently at each occurrence an optionally substituted (e.g. , C1-C12); alkylene, an optionally substituted (e.g., C1-C12) alkenylene, or an optionally substituted (e.g., C1-C12) arenylene;A1and A2are each independently at each occurrence -O-, -S-, or - NR4-, wherein:R4is hydrogen or optionally substituted (e.g., C1-C6) alkyl; m1and m2are each independently at each occurrence 1, 2, or 3; andR3C, R3d, R3e, and R3fare each independently at each occurrence hydrogen or an optionally substituted (e.g., Ci-Cg) alkyl; and(d) each linker group independently comprises a structural formulawherein:** indicates a point of attachment of the linker to a proximal diacyl group;*** indicates a point of attachment of the linker to a distal diacyl group; andYi is independently at each occurrence an optionally substituted (e.g. , C1-C12) alkylene, an optionally substituted (e.g., C1-C12)alkenylene, or an optionally substituted (e.g., C1-C12) arenylene; and(e) each terminating group is independently selected from optionally substituted (e.g., C1-C1s, such as Cr-C1s) alkylthiol, and optionally substituted (e.g., C1- C18, such as C4-C18) alkenylthiol.

[0132] In some embodiments of Xcore, Q is independently at each occurrence a covalent bond, -O-, -S-, -NR2-, or -CR3aR3b. In some embodiments of Xcore Q is independently at each occurrence a covalent bond. In some embodiments of Xcore Q is independently at each occurrence an -O-. In some embodiments of Xcore Q is independently at each occurrence a -S- . In some embodiments of Xcore Q is independently at each occurrence a -NR2and R2is independently at each occurrence Rlgor -L2-NRleRlf. In some embodiments of Xcore Q is independently at each occurrence a -CR3aR3bR3a, and R3aand R3bare each independently at each occurrence hydrogen or an optionally substituted alkyl (e.g., C1-C6, such as C1-C3).

[0133] In some embodiments of Xcore, R1a, R1b, R1c, R , R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen, or an optionally substituted alkyl. In some embodiments of Xcore, R1a, R1b, R1c, R1d, R1e, R1f, and R1g(if present) are each independently at each occurrence a point of connection to a branch, hydrogen. In some embodiments of Xcore, Rla, Rlb, Rlc, Rld, Rle, Rlf, and Rlg(if present) are each independently at each occurrence a point of connection to a branch an optionally substituted alkyl (e.g., C1- C12).

[0134] In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]- [heterocycloalkyl] - [alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, part of L1form a heterocycloalkyl (e.g., C4-C6, and containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur) with one of Rlcand Rld. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a covalent bond. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a hydrogen. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be an alkylene (e.g., C1-C12, such as C1-C6or C1-C3). In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a heteroalkylene (e.g. , C1-C12, such as C1-C8or C1-C6). In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a heteroalkylene (e.g., C2-C8alkyleneoxide, such as oligo(ethyleneoxide)). In some embodiments of Xcore, L°, L1, and L2areeach independently at each occurrence can be a [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g. , C1-C6) alkylene] -[(e.g., C4-C6) heterocycloalkyl] -[(e.g., C1-C6) alkylene]. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] [(e.g., C1-Ce) alkylene]-(arylene)-[(e.g. , C1-C6) alkylene]. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a [alkylene]-(arylene)-[alkylene] (e.g., [(e.g. , C1-C6) alkylene] -phenylene- [(e.g. , C1-C6) alkylene]). In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be a heterocycloalkyl (e.g., C4-C6heterocycloalkyl). In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence can be an arylene (e.g. , phenylene). In some embodiments of Xcore, part of L1form a heterocycloalkyl with one of Rlcand R1d. In some embodiments of Xcore, part of L1form a heterocycloalkyl (e.g., C4-C6heterocycloalkyl) with one of R1cand R1dand the heterocycloalkyl can contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.

[0135] In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence selected from a covalent bond, C1-Cr, alkylene (e.g., C1-C3alkylene), C2-C12(e.g., C2-Cs) alkyleneoxide (e.g., oligo(ethyleneoxide), such as -(CH2CH2O)I-4-(CH2CH2)-), [(C1-C4)N alkylene]- [(C4-C6) heterocycloalkyl]- [(C1-C4) alkylene] (e.g., *5), and [(C1-C4) alkyleneJ-phenylene-[(C1-C4) alkylene] (e.g. ,). In some embodiments ofXcore, L°, L1, and L2are each independently at each occurrence selected from C1-C6alkylene (e.g., C1-C3alkylene), -(C1-C3alkylene-O)i-4-(C1-C3alkylene), -(C1-C3alkylene)-phenylene- (C1-C3alkylene)-, and -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-. In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence C1-C6alkylene (e.g., C1-C3alkylene). In some embodiments, L°, L1, and L2are each independently at each occurrence C2- C12(e.g., C2-C8) alkyleneoxide (e.g., -(C1-C3alkylene-O)1-4-(C1-C3alkylene)). In some embodiments of Xcore, L°, L1, and L2are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl] -[(C1-C4) alkylene] (e.g., -(C1-C3alkylene)- phenylene-(C1-C3alkylene)-) and [(C1-C4) alkylene]- [(C4-Ce) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-).

[0136] In some embodiments of Xcore, x1is 0, 1, 2, 3, 4, 5, or 6. In some embodiments of Xcore, x1is 0. In some embodiments of Xcore, x1is 1. In some embodiments of Xcore, x1is 2. In some embodiments of Xcore, x1is 0, 3. In some embodiments of Xcore x1is 4. In some embodiments of Xcore x1iiss 55.. IInn ssoommee eemmbbooddiimmeennttss ooff XXcc™ore, x1is 6.

[0137] Tn some embodiments of Xcoi the core comprises a structural formula:). In some embodiments of Xcore, the core comprises a structural formula: In some embodiments of Xcore, thecore comprises a structural formula:In someembodiments of Xcore, the core comprises a structural formula:In some embodiments of Xcore, the core comprises a structural formula:. In some embodiments of Xcore, the core comprises a structural formula:some embodiments of Xcore,the core comprises a structural formula:(e.g.,some embodiments ofXcore, the core comprises a structural formula:, wherein Q’ is -NR2- or -CR3aR3b-; q1and q2are each independently 1 or 2. In some >re, the core comprises a structural formula:some embodiments of Xcoi the core comprises a structural formulaorwherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C3-C12, such as C3-C5) heteroaryl. In some embodiments of Xcore, the core comprises has a structural formula

[0138] In some embodiments of Xcore, the core comprises a structural formula set forth in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.

[0139] In some embodiments, the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality (N) of branches comprises at least 3 branches. In some embodiments, the plurality (N) of branches comprises at least 4 branches. In some embodiments, the plurality (N) of branches comprises at least 5 branches.

[0140] In some embodiments of XBranch, g is 1, 2, 3, or 4. In some embodiments of XBranch, g is 1. In some embodiments of XBranch, g is 2. In some embodiments of XBranch, g is 3. In some embodiments of XBranch, g is 4.

[0141] In some embodiments of XBranch, Z = 2(g-1) and when g=1, G=0. In some embodiments of

[0142] In some embodiments of XBranch, g=l, G=0, Z=1, and each branch of the plurality of branches comprises a structural formula each branch of the plurality of branches comprises a structural formula

[0143] In some embodiments of XBranch, g=2, G=1, Z=2, and each branch of the plurality of branches comprises a structural formula

[0144] In some embodiments of XBranch,g=3, G=3, Z=4, and each branch of the plurality of branches comprises a structural formula

[0145] In some embodiments of XBranch, g=4, G=7 , Z=8, and each branch of the plurality of branches comprises a structural formula

[0146] In some embodiments, the dendrimers described herein with a generation (g) = 1 has thestructure:

[0147] In some embodiments, the dendrimers described herein with a generation (g) = 1 has the structure:

[0148] An example formulation of the dendrimers described herein for generations 1-4 is shown in Table 3. The number of diacyl groups, linker groups, and terminating groups can be calculated based on g.Table 3. Formulation of Dendrimer Groups Based on Generation (g)

[0149] In some embodiments, the diacyl group independently comprises a structural formula* indicates a point of attachment of the diacyl group at theproximal end thereof, and ** indicates a point of attachment of the diacyl group at the distal end thereof.

[0150] In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted; alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted alkylene (e.g., C1-C12). In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C12). In some embodiments of the diacyl group of XBranch, Y3is independently at each occurrence an optionally substituted arenylene (e.g., C1- C12).

[0151] In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -O-, -S-, or -NR4-. In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -O-. In some embodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -S-. In someembodiments of the diacyl group of XBranch, A1and A2are each independently at each occurrence -NR4- and R4is hydrogen or optionally substituted alkyl (e.g., C1-Ce). In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 1, 2, or 3. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 1. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 2. In some embodiments of the diacyl group of XBranch, m1and m2are each independently at each occurrence 3. In some embodiments of the diacyl group of XBranch, R3C, R3d, R3e, and R3fare each independently at each occurrence hydrogen or an optionally substituted alkyl. In some embodiments of the diacyl group of XBranch, R3c, R3d, R3e, and R3fare each independently at each occurrence hydrogen. In some embodiments of the diacyl group of XBranch, R3c, R3d, R3e, and R3fare each independently at each occurrence an optionally substituted (e.g., C1-C8) alkyl.

[0152] In some embodiments of the diacyl group, A1is -O- or -NH-. In some embodiments of the diacyl group, A1is -O-. In some embodiments of the diacyl group, A2is -O- or -NH-. In some embodiments of the diacyl group, A2is -O-. In some embodiments of the diacyl group, Y3is C1-C12(e.g., C1-C6, such as C1-C3) alkylene.

[0153] In some embodiments of the diacyl group, the diacyl group independently at each occurrence comprises a structural formula(e.g., and optionally R3c, R3d,R3e, and R3fare each independently at each occurrence hydrogen or C1-C3alkyl.

[0154] In some embodiments, linker group independently comprises a structural formula ** indicates a point of attachment of the linker to a proximal diacyl group,and *** indicates a point of attachment of the linker to a distal diacyl group.

[0155] In some embodiments of the linker group of XBranchif present, Y 1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arenylene. In some embodiments of the linker group of XBranchif present, Yi is independently at each occurrence an optionally substituted alkylene (e.g., C1-C12). Insome embodiments of the linker group of XBranchif present, Yi is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C12). In some embodiments of the linker group of XBranchif present, Yi is independently at each occurrence an optionally substituted arenylene (e.g., C1-C12).

[0156] In some embodiments of the terminating group of XBranch, each terminating group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol. In some embodiments of the terminating group of XBranch, each terminating group is an optionally substituted alkylthiol (e.g. , C1-C18, such as C4-C18). In some embodiments of the terminating group of XBranch, each terminating group is optionally substituted alkenylthiol (e.g., C1-C18, such as C4-C18).

[0157] In some embodiments of the terminating group of Xsranch, each terminating group is independently C1-C1s alkenylthiol or C1-C18alkylthiol, and the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C6-C12aryl, C1-C12alkylamino, C4-C6X-helerocycloalkyl , -OH, -C(O)OH, -C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C1-C12alkylamino), -C(O)N(CI-C3alkyl)-(C1-C6alkylene)-(C4-C6X-heterocycloalkyl), -C(O)-(C1-C12alkylamino), and -C(O)-(C4-Ce X-heterocycloalkyl), and the C4-C6Mheterocycloalkyl moiety of any of the preceding substituents is optionally substituted with C1-C3alkyl or C1-C3hydroxyalkyl.

[0158] In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkenylthiol or C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents each independently selected from halogen, C6-C12aryl (e.g., phenyl), C1-C12(e.g., C1-C8) alkylamino (e.g., C1-C8mono- alkylamino (such as -NHCH2CH2CH2CH3or C1-C8di- alkylamino (such as)), C4-C6N -helerocycloalkyl (e.g. ,N-pyrrolidinyl ( ), N-piperidinyl , N -azepanyl ( )), -OH, -C(O)OH,-C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C1-C12alkylamino (e.g., mono- or di-alkylamino))-C(O)N(C1-C3alkyl)-(C1-C6alkylene)-(C4-C6N-heterocycloalkyl) , -C(O)-(C1-C12alkylamino (e.g., mono- or di-alkylamino)), and -C(O)-(C4-C6A-heterocycloalkyl) wherein theC4-C6A-helerocycloalkyl moiety of any of the preceding substituents is optionally substituted with C1-C3alkyl or C1-C3hydroxyalkyl. In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent -OH. In some embodiments of the terminating group of XBranch, each terminating group is independently C1-C18(e.g., C4-C18) alkylthiol, wherein the alkyl moiety is optionally substituted with one substituent selected from C1-C12(e.g., C1-C8) alkylamino (e.g., C1-C6mono-alkylamino (such as -NHCH2CH2CH2CH3) or C1-C8di-alkylamino (such as)) and C4-C6N- heterocycloalkyl (e.g. , N -pyrrolidinyl (), N -piperidinyl), N-azepanyl)). In some embodiments of the terminating group of XBranch. each terminating group is independently C1-C18(e.g., C4-C18) alkenylthiol or C1-C18(e.g., C4-C18) alkylthiol. In some embodiments of the terminating group of XBranch, each terminating group is independently C1- C18(e.g., C4-C18) alkylthiol.

[0159] In some embodiments of Xcore, the core comprises a structural formula selected from the, and pharmaceutically acceptable salts thereof, wherein * indicates a point of attachment of the core to a branch of the plurality of branches.

[0160] In some embodiments of the terminating group of XBranch, each terminating group is independently a structure selected from the structurea in Table 1. In some embodiments, the dendrimers described herein can comprise a terminating group or pharmaceutically acceptable salt, or thereof selected in Table 1.

[0161] In some embodiments, the dendrimer of Formula (X) is selected from those set forth inTable 4 and pharmaceutically acceptable salts thereof.Table 4. Example ionizable cationic lipo-dendrimersOther Ionizable lipids

[0162] In some embodiments of the lipid composition, the ionizable lipid comprises a structural formula (D-L):wherein: a is 1 and b is 2, 3, or 4; or, alternatively, b is 1 and a is 2, 3, or 4; m is 1 and n is 1; or, alternatively, m is 2 and n is 0; or, alternatively, m is 2 and n is 1; andR1, R2, R3, R4, R5, and R6are each independently selected from the group consisting of H, -CH2CH(OH)R7, -CH(R7)CH2OH, -CH2CH2C(=O)OR7, -CH2CH2C(=O)NHR7, and - CH2R7, wherein R7is independently selected from C3-C18alkyl, C3-C18alkenyl having one C=C double bond, a protecting group for an amino group, -C(=NH)NH2, a poly(ethylene glycol) chain, and a receptor ligand;provided that at least two moieties among R1to R6are independently selected from - CH2CH(OH)R7, -CH(R7)CH2OH, -CH2CH2C(=O)OR7, -CH2CH2C(=O)NHR7, or -CH2R7, wherein R7is independently selected from C3-C18alkyl or C3-C18alkenyl having one C=C double bond; and wherein one or more of the nitrogen atoms indicated in formula (D-F) may be protonated to provide a ionizable lipid.

[0163] In some embodiments of the ionizable lipid of formula (D-F), a is 1. In some embodiments of the ionizable lipid of formula (D-F), b is 2. Tn some embodiments of the ionizable lipid of formula (D-F), m is 1. In some embodiments of the ionizable lipid of formula (D-F), n is 1. In some embodiments of the ionizable lipid of formula (D-F), R1, R2, R3, R4, R5, and R6are each independently H or -CH2CH(OH)R7. In some embodiments of the ionizable lipid of formula (D-F), R1, R2, R3, R4, R5, and R6are each independently H orIn some embodiments of the ionizable lipid of formula (D-F), R1, R2, R3, R4, R5, and R6are eachOH independently H or -'R7. in some embodiments of the ionizable lipid of formula (D-F), R7is C3-C18alkyl (e.g., C6-C12alkyl).

[0164] In some embodiments, the ionizable lipid of formula (D-F) is 13,16,20-tris(2- hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-l l,25-diol:

[0165] In some embodiments, the ionizable lipid of formula (D-F) is (11R, 257?)-13, 16,20- tris((7?)-2-hydroxydodecyl)- 13, 16,20,23-tetraazapentatricontane- 11 ,25-diol:

[0166] Additional ionizable lipids that can be used in the compositions and methods of the present application include those ionizable lipids as described in International PatentPublication W02010144740, WO2013149140, WO2016118725, WO2016118724,WO2013063468, WO2016205691, WO2015184256, W02016004202, WO2015199952,W02017004143, WO2017075531, WO2017117528, WO2017049245, WO2017173054 andWO2015095340, which are incorporated herein by reference for all purposes. Examples of those ionizable lipids include but are not limited to those as shown in Table 5.Table 5. Example Ionizable Lipids

[0167] In some embodiments of the lipid composition of the present application, the ionizable lipid is present in an amount of from about from about 20 mol% to about 23 mol%. In some embodiments, the ionizable lipid is present in an amount of about 20 mol%, about 20.5 mol%, about 21 mol%, about 21.5 mol%, about 22 mol%, about 22.5 mol%, or about 23 mol%. In other embodiments, the ionizable lipid is present in an amount of from about 7.5 mol% to about 20 mol%. In some embodiments, the ionizable lipid is present in an amount of about 7.5 mol%, about 8 mol%, about 9 mol%, 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%, or about 20 mol%.

[0168] In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of from about 5 mol% to about 30 mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of from about 10 mol% to about 25 mol%. In some embodiments of the lipid composition of the present application, the lipidcomposition comprises the ionizable lipid in an amount of from about 15 mol% to about 20 mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of from about 10 mol% to about 20 mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of from about 20mol% to about 30mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of of at least (about) 5 mol%, at least (about) 10 mol%, at least (about) 15 mol%, at least (about) 20 mol%, at least (about) 25 mol%, or at least (about) 30 mol%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the ionizable lipid in an amount of of at most (about) 5 mol%, at most (about) 10 mol%, at most (about) 15 mol%, at most (about) 20 mol%, at most (about) 25 mol%, or at most (about) 30mol%.Helper Lipids

[0169] In some embodiments, helper lipids are phospholipids. Phospholipids, as defined herein, are any lipids that comprise a phosphate group. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0170] Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g. , an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellularrecognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0171] In some embodiments, the phospholipid is present in the composition at a molar percentage from about 7.5% to about 60%, from about 7.5% to about 50%, from about 7.5% to about 40%, from about 7.5% to about 30%, from about 7.5% to about 20%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.

[0172] In some embodiments, the LNP described herein comprises about 5 mol% to about 30 mol% of phospholipid. In some embodiments, the LNP comprises about 10 mol% to about 30 mol%, or about 12 mol% to about 30 mol%, or about 14 mol% to about 30 mol%, or about 16 mol% to about 30 mol%, or about 18 mol% to about 30 mol%, or about 20 mol% to about 30 mol%, or about 22 mol% to about 30 mol%, or about 24 mol% to about 30 mol%, or about 26 mol% to about 30 mol%, or about 28 mol% to about 30 mol%. In some embodiments, the LNP comprises about 10 mol%, or about 11 mol%, or about 12 mol%, or about 13 mol%, or about 14 mol%, or about 15 mol%, or about 16 mol%, or about 17 mol%, or about 18 mol%, or about19 mol%, or about 20 mol%, or about 21 mol%, or about 22 mol%, or about 23 mol%, or about24 mol%, or about 25 mol%, or about 26 mol%, or about 27 mo%, or about 28 mol%, or about29 mol%, or about 30 mol%.

[0173] In some embodiments, the LNP comprises about 5% to about 30% weight of phoshpolipid. In some embodiments, the LNP comprises about 5% weight, or 10%, or 12%, or 15%, or 18%, or 20%, or 25%, or 30% weight of phospholipid.

[0174] In some embodiments of the lipid composition of the present application, the compositions may further comprise a molar percentage of the phospholipid to the total lipid composition from about 20 to about 23. In some embodiments, the molar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5, to about 23 or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 60. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any range derivable therein.

[0175] In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 20%. In someembodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage from about 12% to about 18%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage of at least (about) 8%, at least (about) 10%, at least (about) 12%, at least (about) 15%, at least (about) 18%, at least (about) 20%, or at least (about) 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the phospholipid at a molar percentage of at most (about) 8%, at most (about) 10%, at most (about) 12%, at most (about) 15%, at most (about) 18%, at most (about) 20%, or at most (about) 23%.

[0176] Phospholipids useful or potentially useful in the compositions and methods may be selected from: l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn- glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero- 3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1 ,2-diphytanoyl-sn-glycero-3- phospho-(l'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1 ,2-diphytanoyl-sn-glycero-3- phospho-L-serine (sodium salt) (4ME 16:0 PS), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dilinolenoyl- sn-glycero-3 -phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and 1 ,2-dioleoyl-sn-glycero-3- phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0177] In some embodiments, the phospholipid may contain one or two long chain (e.g., G>- C24) alkyl or alkenyl groups, a glycerol or a sphingosine, one or two phosphate groups, and, optionally, a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.

[0178] In some embodiments of the lipid compositions, the phospholipid is not an ethylphosphocholine.Sterol

[0179] In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms. In one aspect, the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula: . In some embodiments, a steroid derivative comprises the ring structure above with one or more nonalkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as: HO' . In some embodiments of the present application, the steroid or steroid derivative is a cholestane or cholestane derivative. In aUHHA A cholestane, the ring structure is further defined by the formula: . As described above, a cholestane derivative includes one or more non-alkyl substitution of the above ring system. In some embodiments, the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both a cholestere and a sterol or a derivative thereof.

[0180] Sterol useful or potentially useful in the compositions and methods may be selected from: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

[0181] In some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage from about 20% to about 50%.

[0182] In some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage about 10%, about 15%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about41%, about 42%, about 43%, about 44%, about 45%, about 50%, about 55%, or about 60%.

[0183] ln some embodiments of the lipid composition of the present application, the sterol is present in the composition at a molar percentage from about 10% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%.

[0184] In some embodiments of the lipid composition of the present application, the sterol is present at a molar percentage of at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, or at least (about) 50%. In some embodiments of the lipid composition of the present application, the ioizable lipid is present at a molar percentageof at most (about) 60%, at most (about) 15%, at most (about) 45%, at most (about) 40%, at most (about) 35%, at most (about) 30%, at most (about) 25%, or at most (about) 20%.Polymer conjugated lipids

[0185] The lipid compositions of the disclosure may include lipids conjugated to polymers, such as lipids conjugated to polyethylene glycol (“PEG-lipid”). Illustrative methods for making and using PEG-lipids are described for example in Int’l Pat. Pub. No. WO 2012 / 099755 and U.S. Pat. Pub No. 2014 / 0200257.

[0186] A PEG-lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG-lipid may be PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0187] In one embodiment, PEG-lipids useful in the present invention can be PEG-lipids described in Int’l Pat. Pub. No. WO 2012 / 099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG-lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG- lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” is a PEG-lipid having one or more hydroxyl ( — OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEG-lipid comprises an — OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0188] In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG-lipid. In some embodiments, the PEG-lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group. In other embodiments, the PEG- lipid is a compound which contains one or more C6-C24long chain alkyl or alkenyl group or a C6-C24fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG-lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified 1,2- diacyloxypropan-3-amines, PEG modified diacylglycerols and dialkylglycerols. In some embodiments, PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by themolecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750,3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000.Some non- limiting examples of lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.

[0189] In some embodiments of the lipid composition of the present application, the PEG-lipid has a structural formula:, wherein: R12and R13are each independently alkyl ,( 24), alkenyl <( 24), or a substituted version of either of these groups; Rcis hydrogen, alkyl(c<8), or substituted alkyl(c<s); and x is 1-250. In some embodiments, Reis alkyl(c<8) such as methyl. R12and R13are each independently alkyl<c<4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 orx is 100-250. In some embodiments, the PEG-lipid is 1 ,2-di myristoyl -.vn-glycerol, methoxypolyethylene glycol.

[0190] In some embodiments of the lipid composition of the present application, the PEG-lipidMU0 ,0-V o oN has a structural formula:rn is an integer between 1 and 100 and ng and ng are each independently selected from an integer between 1 and 29. In some embodiments, m is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, m is from about 30 to about 50. In some embodiments, m is from 5 to 23. In some embodiments, m is 11 to about 17. In some embodiments, ng is from 5 to 23. In some embodiments, ng is 11 to about 17.

[0191] In some embodiments of the lipid composition of the present application, the compositions may further comprise a molar percentage of the PEG-lipid to the total lipid composition from about 4.0 to about 4.6. In some embodiments, the molar percentage is from about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, to about 4.6 or any range derivable therein. In other embodiments, the molar percentage is from about 1.5 to about 4.0. In some embodiments, the molar percentage is from about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, to about 4.0 or any range derivable therein.

[0192] In some embodiments, the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%, from about 0.5% to about 7.5%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 1% to about 10%, from about 1% to about 7.5%, from about 1% to about 5% from about 1% to about 4% from about 1% to about 3%.

[0193] In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1 % to about 8%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 2% to about 7%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most(about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10%.Additional Lipid (e.g., SORT Lipid)

[0194] The lipid composition may include an additional anionic lipid, ionizable (cationic) lipid, or permanently cationic lipid. In some embodiments of the lipid composition of the present application, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ. An additional lipid which supports selective delivery of the composition to a particular organ is termed herein an “additional lipid (e.g., SORT lipid).”

[0195] ln some embodiments of the lipid compositions, the additional lipid comprises a permanently positively charged moiety (i.e. , is a permanently cationic lipid). The permanently positively charged moiety may be positively charged at a physiological pH such that the additional lipid (e.g., SORT lipid) comprises a positive charge upon delivery of a polynucleotide to a cell. In some embodiments, the positively charged moiety is quaternary amine or quaternary ammonium ion. In some embodiments, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) comprises, or is otherwise complexed to or interacting with, a counterion.

[0196] In some embodiments of the lipid compositions, the additional lipid is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group). The permanently cationic lipid may contain a group which has a positive charge regardless of the pH. One permanently cationic group that may be used in the permanently cationic lipid is a quaternary ammonium group. The permanently cationic lipid may comprise a structural formula:(S-I), wherein:Y1, Y2, or Y3 are each independently XiC(O)Ri or X2N+R3R4Rs; provided at least one of Yi, ¥2, and ¥3 is X2N+R3R4Rs;Ri is C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl;Xi is O or NRa, wherein Rais hydrogen, C1-C4alkyl, or C1-C4substituted alkyl;X2 is C1-C6alkanediyl or C1-C6substituted alkanediyl; R3, R4, and R5 are each independently C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl; andAi is an anion with a charge equal to the number of X2N+R3R4Rs groups in the compound.

[0197] In some embodiments, the permanently cationic additonal lipid (e.g., SORT lipid) has a structural formula:(S-II), wherein:R6-R9are each independently C1-C24alkyl, C1-C24substituted alkyl, C1-C24alkenyl, C1-C24substituted alkenyl; provided at least one of R6-R9is a group of C8-C24; andA2is a monovalent anion.

[0198] In some embodiments of the lipid compositions, the SORT (additional) lipid is an ionizable lipid (i.e., comprising one or more hydrophobic components and an ionizable group, e.g. a tertiary amino group). The ionizable positively charged moiety may be positively charged at a physiological pH. One ionizable group that may be used in the ionizable lipid is a tertiary ammine group. In some embodiments of the lipid compositions disclosed herein, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) has a structural formula:(S-I’a), wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group; and R3and R3' are each independently C1-C6alkyl or substituted C1-C6alkyl.

[0199] In some embodiments of formula (S-I’a) Ri and R2 are each independently C8-C24alkenyl (e.g. hexadecane, heptadecene, or octadecene). In some embodiments of formula (S- I’a), R3and R3' are each independently C1-C6alkyl (e.g., methyl or ethyl). In some embodiments of formula (S-I’a) Ri and R2are each independently C8-C24alkenyl, (e.g. hexadecane, heptadecene, or octadecene) and R3and R3' are each independently C1-C6alkyl (e.g., methyl or ethyl).

[0200] In some embodiments of the lipid compositions, the additional ionizable lipid or permanently cationic lipid comprises a head group of a particular structure. In some embodiments, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) comprises a headgroup having a structural formula:, wherein L is a linker; Z+is positively charged moiety and X- is a counterion. In some embodiment, the linker is a biodegradablelinker. The biodegradable linker may be degradable under physiological pH and temperature. The biodegradable linker may be degraded by proteins or enzymes from a subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or quaternary amine.

[0201] In some embodiments of the lipid compositions, the SORT (additional ionizable lipid or permanently cationic) lipid has a structural formula:, wherein R1and R2are each independently an optionally substituted C6-C24alkyl, or an optionally substituted Ce- C24alkenyl.

[0202] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid(e.g., SORT lipid)) has a structural formula:

[0203] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid(e.g., SORT lipid)) comprises a Linker (L). In some embodiments, L iswherein: p and q are each independently 1, 2, or 3; andR4is an optionally substituted C -G, alkyl

[0204] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid(e.g., SORT lipid)) has a structural formula:wherein:Ri and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group; R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-C6alkyl;R4 is C1-C6alkyl or substituted C1-C6alkyl; andX is a monovalent anion.

[0205] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is further defined as:wherein:R1and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group; R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-C6alkyl; and X" is a monovalent anion.

[0206] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) is a phosphocholine lipid. In some embodiments, the additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) is an ethylphosphocholine. The ethylphosphocholine may be, by way of example, without being limited to, l,2-dimyristoleoyl-sn-glycero-3- ethylphosphocholine, 1 ,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1 ,2-distearoyl-sn- glycero-3-ethylphosphocholine, 1 ,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1 ,2- dimyristoyl-sn-glycero-3-ethylphosphocholine, l,2-dilauroyl-sn-glycero-3- ethylphosphocholine, l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

[0207] In some embodiments of the lipid compositions, the lipid is a trialkylammonium propane(TAP). In some embodiments, the TAP or lipid has a structural formula:(S-F), wherein:Ri and R2are each independently C8-C24alkyl, C8-C24alkenyl, or a substituted version of either group; R3, R3', and R3" are each independently C1-C6alkyl or substituted C1-C6alkyl;X" is a monovalent anion.

[0208] By way of example, and without being limited thereto, a additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is l,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

[0209] In some embodiments of the lipid compositions, the additonal lipid (e.g., additonal lipid(e.g., SORT lipid)) has a structural formula: (S-IF), wherein:R4 and R4' are each independently alkykc6-c24), alkenyl(c6-c24), or a substituted version of either group;R4" is alkyl(c<24), alkenyl (c<24), or a substituted version of either group;R4"' is alkyl(C1-C8), alkenyl(C2-C8), or a substituted version of either group; andX2 is a monovalent anion.

[0210] By way of example, and without being limited thereto, an additonal lipid (e.g., additonal lipid (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is dimethy Idioctadecylammonium (DD AB ) .

[0211] In some embodiments, the additional SORT lipid is l,2-dioleoyl-sn-glycero-3 phosphate (18: 1 PA).

[0212] In some embodiments of the lipid compositions, the additional lipid is selected from the lipids set forth in Table 6.Table 6. Example additonal lipid (e.g., SORT lipids)X is a counterion (e.g., Cl", Br", etc.)

[0213] Numerous cationic lipids with quaternary ammonium head groups have been developed for the delivery of DNA and RNA, such as DOTAP, DMRIE, and DODAB, which were reported to be effective for RNA delivery. The cationic head groups are preferentially formed via quatemization of the corresponding tertiary amines in the final step of the synthesis (Zhang et al. Chem. Rev. (2021)).

[0214] In some embodiments, the permanently cationic lipid comprises a quaternary ammonium ion. In some embodiments, the permanently cationic lipid is a lipid according to Formula (I):wherein:R1 and R2 are each independently alkyl(C8-C24)), alkenyl(C8-C24)), or a substituted version of either group; R3, R3', and R3" are each independently alkyl(C<6) or substituted alkyl(C<6);X- is a monovalent anion.

[0215] In some embodiments, R1 is an alkenyl (C8-C24) or substituted alkenyl(C8-C24)). In some embodiments, R2 is an alkenyl(C8-C24)) or substituted alkenyl(C8-C24)). In other embodiments, R1 is an alkyl(C8-C24)) or substituted alkyl(C8-C24)). In other embodiments, R2 is an alkyl(C8-C24)) or substituted alkyl(C8-C24)). In some embodiments, R1 and R2 are both the same. In some embodiments, R3, R3', and R3" are each identical. In some embodiments, R3, R3’, and R3" are each methyl. In other embodiments, one of R3, R3', and R3" is substituted alkyl(C<6). In some embodiments, one of R3, R3', and R3" is alkyl(C<6) substituted with hydroxyl. In some embodiments, one of R3, R3’, and R3" is ethanolyl. In some embodiments, X- is halide anion such as bromide or chloride. In some embodiments, the permanently cationic lipid is further defined as:

[0216] In some embodiments, the permanently cationic lipid is further defined as:wherein:R4 and R4' are each independently alkyl(C6-C24)), alkenyl(C6-C24)), or a substituted version of either group;R4" is alkyl(C<24), alkenyl(C<24), or a substituted version of either group;R4'" is alkyl(Cl-CS), alkenyl(C2-C8), or a substituted version of either group; andX2 is a monovalent anion.

[0217] In some embodiments, R4 is alkyl(C6-C24)) or substituted alkyl(C6-C24)) such as octadecyl. In some embodiments, R4’ is alkyl(C6-C24)) or substituted alkyl(C6-C24)) such as octadecyl. In some embodiments, R4" is alkyl(C<24) or substituted alkyl(C<24). In some embodiments, R4" is alkyl(C<8) or substituted alkyl (C<8) such as methyl. In some embodiments, R4'" is alkyl(Cl -C8) or substituted alkyl(Cl-CS) such as methyl. In some embodiments, X2 is a halide such as chloride or bromide. In some embodiments, the permanently cationic lipid is further defined as:

[0218] In some embodiments, the permanently cationic lipid is further defined as:wherein:R1 and R2 are each independently alkyl(C8-C24)), alkenyl(C8-C24)), or a substituted version of either group; R3, R3', and R3" are each independently alkyl(C<6) or substituted alkyl(C<6);R4 is alkyl(C<6) or substituted alkyl(C<6); andX- is a monovalent anion.

[0219] In some embodiments, RI is an alkenyl (C8-C24)) or substituted alkenyl(C8-C24)). In some embodiments, R2 is an alkenyl(C8-C24)) or substituted alkenyl(C8-C24)). In other embodiments, RI is an alkyl(C8-C24)) or substituted alkyl(C8-C24)). In other embodiments, R2 is an alkyl(C8-C24)) or substituted alkyl(C8-C24)). In some embodiments, RI and R2 are both the same.

[0220] In some embodiments, R3, R3’, and R3" are each identical such as R3, R3’, and R3" are each methyl. In some embodiments, R4 is alkyl(C<6) such as ethyl. In some embodiments, X- is halide anion such as bromide or chloride.

[0221] In some embodiments, the permanently cationic lipid is further defined as:

[0222] In some embodiments, the quaternary ammonium lipid is a trimethylammonium propane (TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-dimyristoyl-3- trimethylammonium-propane (14:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is l,2-stearoyl-3 -trimethylammoniumpropane (18:0 TAP). In some embodiments, the trimethylammonium propane (TAP) is 1,2- dioleoyl-3-trimethylammonium-propane (18: 1 TAP). In some embodiments, the quaternary ammonium lipid is 18:1 RI. In some embodiments, the trimethylammonium propane (TAP) is 18:1 RI. The 18:1 RI may be supplied as a bromide salt and is available, e.g., from Cayman Chemicals® as N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan- 1 -aminium bromide (DORI).

[0223] In some embodiments, the quaternary ammonium lipids is an ethylphosphocholine (EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC). In some embodiments, ethylphosphocholine (EPC) is 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC).In some embodiments, the ethylphosphocholine (EPC) is l,2-distearoyl-sn-glycero-3- ethylphosphocholine (18:0 EPC). In some embodiments, the ethylphosphocholine (EPC) is l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC). In some embodiments, the ethylphosphocholine (EPC) is l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0- 18:0 EPC).

[0224] In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 20% to about 65%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 20% to about 55%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 20% to about 50%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 30% to about 65%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 30% to about 60%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 30% to about 55%. In some embodiments, the quaternary ammonium lipids is present in the composition at a molar percentage from about 30% to about 50%.

[0225] In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 20% to about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage from about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, at least (about) 60%, or at least (about) 65%. In someembodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at least (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises the additonal lipid (e.g., SORT lipid) at a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or of a range between (inclusive) any two of the foregoing values.

[0226] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 30%, a quaternary ammonium lipid at a molar percentage from about 20% to about 60%, DOPE at a molar percentage from about 7.5% to about 66%, the cholesterol at a molar percentage from about 15% to about 46%, and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

[0227] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 30%, a quaternary ammonium lipid at a molar percentage from about 20% to about 55%, DOPE at a molar percentage from about 7.5% to about 66%, the cholesterol at a molar percentage from about 15% to about 46%, and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

[0228] In some embodiments, the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 25%, a quaternary ammonium lipid at a molar percentage from about 30% to about 50%, the phospholipid at a molar percentage from about 7.5% to about 20%, the cholesterol at a molar percentage from about 15% to about 40%, and the PEG- lipid at a molar percentage from about 1 % to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an trimethylammonium propane (TAP).

[0229] In some embodiments , The composition of any one of claim 1 to 31, wherein the lipid composition includes an ionizable lipid at molar percentage from about 5% to about 25%, a quaternary ammonium lipid at a molar percentage from about 30% to about 50%, the phospholipid at a molar percentage from about 7.5% to about 20%, the cholesterol at a molar percentage from about 15% to about 40%, and the PEG-lipid at a molar percentage from about 1% to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an ethylphosphocholine (EPC).Polynucleotides

[0230] In some embodiments, the LNP includes a polynucleotide. In some embodiments, the polynucleotide comprises a small inte

[0231] rfering RNA (siRNA), a short hairpin RNA (shRNA), or microRNA.

[0232] In some embodiments, the polynucleotide comprises a messenger RNA (mRNA). In some embodiments, the mRNA molecule is greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides in length. In some embodiments, the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 2500 nucleotides in length. In some embodiments, the mRNA molecule is about 3000 nucleotides in length. In some embodiments, the mRNA molecule is about 3500 nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length. In some embodiments, the mRNA molecule is about 4500 nucleotides in length. In some embodiments, the mRNA molecule is about 5000 nucleotides in length.

[0233] In some embodiments, the polynucleotide is from 2000 nucleotides to 5000 nucleotides in length. In some embodiments, polynucleotide is from 2500 to 5000 nucleotides in length. In some embodiments, polynucleotide is from 3000 to 5000 nucleotides in length. In some embodiments, polynucleotide is from 3500 to 5000 nucleotides in length. In some embodiments, polynucleotide is from 4000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length.

[0234] In some embodiments, the polynucleotide has a concentration of 0.5-3.0 mg / mL, of 1.0- 3.0 mg / mL, of 2.0-3.0 mg / mL of 1.0 mg / mL. In some embodiments, the polynucleotide has a concentration of 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL. In some embodiments, the polynucleotide has a concentration of 1.0 mg / mL.

[0235] In some embodiments, the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is from 4000 to 5000nucleotides in length and at a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 1.0 mg / mL.

[0236] In some embodiments, the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.9 mg / mL.

[0237] In some embodiments, the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.8 mg / mL.

[0238] In some embodiments, the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is from 4000 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.7 mg / mL.

[0239] In some embodiments, the polynucleotide is from 2000 to 5000 nucleotides in length and at a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is from 2500 to 5000 nucleotides in length and at a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is from 3000 to 5000 nucleotides in length and at a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is from 3500 to 5000 nucleotides in length and at a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is from 4000 to 5000nucleotides in length and at a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is from 4500 to 5000 nucleotides in length and at a concentration of 0.6 mg / mL.

[0240] In some embodiments, the mRNA encodes dynein axonemal intermediate chain 1(DNAI1) protein. In other embodiments, the mRNA encodes for cystic fibrosis transmembrane conductance regulator (CFTR).

[0241] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 1.

[0242] In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence at least 99% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence identical to SEQ ID NO: 1.

[0243] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 4.

[0244] In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence at least 85% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence at least 90% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence at least 99% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence identical to SEQ ID NO: 4.

[0245] In some embodiments, the mRNA encoding the CFTR protein is incorporated into an LNP. In some embodiments, the mRNA encoding the DNAI1 protein is incorporated into an LNP.

[0246] In some embodiments, the LNP comprises messenger RNA (mRNA).

[0247] In some embodiments, the mRNA comprises between 100 bases and 8 kilobases (kb).

[0248] In some embodiments, the mRNA comprises between 1 Ikb and 8 kb, between 2 kb and8 kb, between 3 kb and 8 kb, between 4 kb and 8 kb, between 5 kb and 8 kb, between 6 kb and8 kb, between 7 kb and 8 kb, between 1 kb and 7 kb, between 2 kb and 7 kb, between 3 kb and7 kb, between 4 kb and 7 kb, between 5 kb and 7 kb, or between 6 kb and 7 kb, between 1 kb and 6 kb, between 2 kb and 6 kb, between 3 kb and 6 kb, between 4 kb and 6 kb, or between 5 kb and 6 kb. In some embodiments, the mRNA comprises (about) 2 kb. In some embodiments, the mRNA comprises (about) 4.6 kb.

[0249] In some embodiments, the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments, the mRNA encodes a Dynein axonemal intermediate chain 1 (DNAI1) protein.

[0250] In some embodiments, the mRNA encodes a gene-editing system or component thereof.

[0251] ln some embodiments, the composition is a pharmaceutical composition.

[0252] In some embodiments, the mRNA comprises Nl-methylpseudouridine-5'-triphosphate.In some embodiments, the mRNA comprises Capl cap structure.

[0253] In some embodiments, the present application provides polynucleotide comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and / or modified linkages. The specific modifications are selected such that the resulting polynucleotide possesses desirable characteristics. In ssoommee embodiments, polynucleotide comprises one or more RNA-like nucleosides. In some embodiments, polynucleotide comprises one or more DNA-like nucleotides.

[0254] In some embodiments, nucleosides of the present application comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present application comprise one or more modified nucleobases.

[0255] In some embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3- deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4- 13][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido| 3',2’:4,5 |pyrrolo| 2,3-d|pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2- pyridone.

[0256] In some embodiments, the present application provides poylnucleotide comprising linked nucleosides. In such embodiments, nucleosides may be linked together using anyinternucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P=0), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non phosphorus containing intemucleoside linking groups include, but are not limited to, methylenemethylimino ( — CTh — N(CH3) — O — CTh — ), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(0)(NH) — S — ); siloxane ( — O — Si(H)2 — O — ); and N,N'-dimethylhydrazine ( — CTh — N( Ü1 / 4~ N(CH3) — ). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In some embodiments, intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are well known to those skilled in the art.Physical properties and characteristics of lipid nanoparticle compositionsEncapsulation Efficiency

[0257] The term “encapsulation efficiency” refers the fraction of a polynucleotides that is encapsulated within or otherwise incorporated into a lipid nanoparticle composition when LNPs are formed. Encapsulation efficiency may be determined by comparing the amount of input polynucleotides to the amount of polynucleotides in a sample of LNPs, or by compairing the amount of polynucleotides in the LNPs to the free excess polynucleotides in the sample. In some embodiments, a flourescence detection assay (e.g., RiboGreen™) is used to determine encapsulation efficiency to measuring the free RNA in a sample with intact LNPs with the total RNA is a sample treated to disrupt the LNPs.

[0258] In various embodiments “encapsulation", as used herein refers to the process of confining a polynucleotides within an LNP described herein. In some embodiments, “encapsulation" refers to confining an mRNA molecule within an LNP described herein.

[0259] In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNPs are formed with an average encapsulationefficiency ranging from about 90% to about 100%. In some embodiments, the LNPs are formed with an average encapsulation efficiency ranging from about 75% to about 95%.

[0260] In various embodiments, the LNPs have an encapsulation efficiency of greater than 50%. For example, the EE of the LNPs is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the LNPs have an EE of50-95%, of 60-95%, of 70-95%, of 80-95%, or of 90-95%. In some embodiments, the LNPs have an EE of 91%, 92%, 93%, 94% or 95%. In some embodiments, the LNPs have an EE of95%.

[0261] In various embodiments, the disclosure provides a composition having a polynucleotide (e.g., an mRNA molecule) incorporated into the LNPs described herein. In some embodiments, majority of the LNPs incorporateare incorporated into the mRNA molecule. In some embodiments, great-er than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the LNPs incorpo-rate are incorporated into the mRNA molecule. In some embodiments, at least 70% of the LNPs incorporateof the composition are incorporated into an mRNA molecule encoding for CFTR. In various embodiments, at least 70% of the LNPs incorporateof the composition are incorporated into an mRNA molecule encoding for DNAI1.LNP Diameter

[0262] In various embodiments, the LNPs have a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 1 10 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about80 nm). In some embodiments, the LNPs have a diameter of about 80 to 150 nm. In some embodiments, the LNPs have a diameter of about 20 to 600 nm, about 40 to 600 nm, about 60 to 600 nm, about 80 to 600 nm, about 100 to 600 nm, or 150 to 600 nm, or 200 to 600 nm, or250 to 600 nm, or 300 to 600 nm, or 350 to 600 nm. In some embodiments, the LNPs have a diameter of about 20 to 400 nm, about 40 to 400 nm, about 60 to 400 nm, about 80 to 400 nm, about 100 to 400 nm, or 150 to 400 nm, or 200 to 400 nm, or 250 to 400 nm, or 300 to 400 nm, or 350 to 400 nm.

[0263] In some embodiments, the lipid nanoparticle preparation has a size distribution in which the mean size (e.g. , diameter) is about 70 nm to about 200 nms. In some embodiments, the LNP has a diameter of 100-155 nm.

[0264] In some embodiments, all of the nanoparticles have a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNPs have an average size of about 150 nm or less, e.g., between 75 nm and 150 nm, in particular between 100 nm and 150 nm.

[0265] In various embodiments, the LNPs have a diameter of 20 to 180 nm, 30 to 180 nm, 40 to 180 nm, 50 to 180 nm, 60 to 180 nm, 70 to 180 nm, 80 to 180 nm, 90 to 180 nm, 100 to 180 nm, or 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, or 180 nm,.

[0266] In some embodiments, the LNPs have a diameter of 100 to 400 nm, 120 to 400 nm, 140 to 400 nm, 160 to 400 nm, 180 to 400 nm, 200 to 400 nm, 220 to 400 nm, 240 to 400 nm, 260 to 400 nm, 280 to 400 nm, 300 to 400 nm, 320 to 400 nm, 340 to 400 nm, 360 to 400, or 380 to 400 nm,.

[0267] In various embodiments, the diameter of the LNP is determined techniques known in the art, including but not limited to dynamic light scattering (DLS).Polydispersity Index (PDI)

[0268] In some embodiments, the LNPs are characterized having a polydispersity (or polydispersity index) of less than 0.5. The polydispersity index is the standard deviation of the particle diameter distribution divided by the mean particle diameter. The PDI is often used as an indication of the quality of the LNP with respect to the size distribution.

[0269] In various embodiments, dynamic light scattering (“DLS”) can be used to characterize the polydispersity index (“PDI”) and size of the LNPs of the present disclosure. DLS measures the scattering of light that results from subjecting a sample to a light source. PDI, as determined from DLS measurements, represents the distribution of particle size (at or around the mean particle diameter) in a population, with a perfectly uniform population having a PDI of zero.

[0270] In various embodiments, the LNPs have a polydispersity of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1. In some embodiments, the LNPs have a polydispersity of 0.5. In some embodiments, the LNPs have a polydispersity of 0.4. In some embodiments, the LNPs have a polydispersity of 0.3. In some embodiments, the LNPs have a polydispersity of 0.2. In some embodiments, the LNPs have a poly dispersity of 0.1.Protein corona

[0271] In some embodiments, the LNP includes a protein corona. In some embodiments, the protein corona includes at least one plasma protein. In some embodiments, the LNP interactswith plasma proteins. The interaction between LNP and plasma proteins can be determined by SDS-polyacrylamide gel electroophoreses (PAGE) and mass spectrometry proteomics.

[0272] In some embodiments, the SORT molecule chemical structure impacts the identities and quantities of protein corona constituents. In some embodiments, the quaternary ammonium lipid controls causes association of the at least one plasma protein. In some embodiments, the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin. In some embodiments, the protein corona comprises vitronectin. In some embodiments, the protein corona comprises prothrombin. In some embodiments, the protein corona comprises lower levels of component Clq subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid.Pharmaceutical compositions

[0273] In another apsect, the disclosure provides a pharmaceutical composition including the LNP composition described herein. In another apsect, the disclosure provides a kit comprising the LNP compositionor the pharmaceutical composition described herein, and an instructions for use thereof.

[0274] In some embodiments, pharmaceutical compositions can also include excipients and / or additives. Examples of these are surfactants, stabilizers, complexing agents, antioxidants, or preservatives which prolong the duration of use of the finished pharmaceutical formulation, flavorings, vitamins, or other additives known in the art. Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. In some embodiments, preservatives include, but are not limited to, those that protect the solution from contamination with pathogenic particles, including benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate. Antioxidants include, but are not limited to, vitamins, provitamins, ascorbic acid, vitamin E, salts or esters thereof.Methods of use and treatment

[0275] In another apsect, the disclosure provides a method for treating or preventing a disease in a subject in need thereof, including administering the LNP composition or the pharmaceutical composition described herein.

[0276] In some embodiments, the LNP acquires a protein corona after adaministration. In some embodiments, the protein corona comprises at least one plasma protein. In some embodiments, the quaternary ammonium lipid controls causes association of the at least one plasma protein. In some embodiments, the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin. In some embodiments, the protein corona comprises vitronectin. In some embodiments, the protein corona comprises prothrombin. In some embodiments, the protein corona comprises lower levels of component Cl q subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid.

[0277] In some embodiments of the method, the protein corona increases specific delivery of the payload to the lung of the subject.

[0278] In some embodiments, the LNP delivers its payload >1.1 fold more, >1.2 fold more, >1.3 fold more, >1.4 fold more, >1.5 fold more, >1.6 fold more, >1.7 fold more, >1.8 fold more, >1.9 fold more, >2.0 fold more, >2.5 fold more, or >3.0 fold more.

[0279] In some embodiments of the method, the pharmaceutical composition of the present application can be administrated through any suitable routes including, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.EXAMPLESExample 1: Development and characterization of alternative lung-targeting SORT LNPs

[0280] Four-component LNPs for liver delivery are formulated from ionizable lipid, phospholipid, cholesterol, and polyethylene glycol-conjugated lipid (PEG- lipid molecules. We used an ionizable amino lipid from a chemical library, 5 A2-SC8, the phospholipid 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Choi), and the PEG-lipid 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). These components were combined at relative molar ratios of 23.8 / 23.8 / 47.6 / 4.8 of 5A2- SC8 / DOPE / Chol / DMG-PEG2k for optimal mRNA delivery in vivo.

[0281] We prepared LNPs that each incorporate 50 mole percent of one of eleven additional permanently cationic quaternary ammonium lipids as SORT molecules (FIG. IB). Two lipid series were considered: the trimethylammonium propane (TAP) series and the ethylphosphocholine (EPC) series. Common to all of these molecules is 1) a hydrophobic domain consisting of two alkyl tails of variable carbon length and degree of unsaturation and 2) a quaternary ammonium headgroup that features a permanent positive charge. For the TAP series, there is a short alkyl linker connecting the quaternary ammonium headgroup to the diester hydrophobic domains. For the EPC series, there is an ethylated phosphate group in the linker region. The ethyl substitution maintains the positive charge on the ammonium headgroup. While the TAP series are fully synthetic and have no biological analogue, the EPC series is based upon naturally-occurring phosphatidylcholine lipids found in cellular membranes whose negatively-charged phosphate group has been neutralized through ethylation.

[0282] The size, zeta potential, and RNA encapsulation efficiency for all 12 formulations was assessed (FIG. 6). Dynamic light scattering (DLS) measurements showed that LNPs had hydrodynamic diameters within the range of 108 to 151 nm (FIG. 6A). Additionally, LNP carried a slightly negative, near neutral ζ-potential of -4.5 mV, while the five-component LNPs carried a slightly positive, near neutral ζ-potential, between 0.323 and 7.06 mV (FIG. 6B). Lastly, all formulations efficiently encapsulated mRNA, although the inclusion of a permanently cationic quaternary ammonium lipid as a SORT molecule led to greater encapsulation efficiency compared to LNP (FIG. 6C). No notable trends were observed between the chemical structures of the chosen SORT molecules and the physicochemical properties of the formulated LNPs. Given that all SORT molecules could be successfully incorporated into LNPs with similar attributes, all formulations were evaluated for in vivo delivery and protein corona formation.Example 2: SORT molecule structure impacts the efficacy of mRNA delivery to the lungs

[0283] To examine the organ-targeting properties of LNPs prepared with different SORT molecules, we intravenously injected C57BL6 / J mice with five-component SORT formulations encapsulating firefly luciferase (FLuc) mRNA. Six hours later, the mice were euthanized and organs excised for bioluminescence imaging. Measurements of total flux produced by the organs are proportional to the total amount of FLuc protein translated from the exogenouslyadministered mRNA, allowing us to estimate the potency and tissue-selectivity of the eleven SORT formulations. As a control, mice were treated with the original liver-targeted formulation, mDLNP, to demonstrate how the incorporation of any quaternary ammonium lipid as a SORT molecule is a driver of mRNA delivery to the lungs.

[0284] The base four-component formulation, mDLNP, has previously demonstrated effective delivery of mRNA to the liver in an ApoE-dependent manner. Further engineering of the formulation through the incorporation of 18:1 TAP as a SORT molecule led to highly selective and potent mRNA delivery to the lungs for protein expression. As alternatives to 18:1 TAP, other TAP lipids were employed as SORT molecules, all of which resulted in highly potent and specific expression of FLuc in the lungs (FIG. 2A). Compared to 18: 1 TAP, 14:0 and 16:0 TAP yielded a lower lung bioluminescence when used as SORT molecules despite similar tissue-specificity (FIG. 2B). Although 18:0 TAP is equally potent as 18: 1 TAP for FLuc mRNA delivery to the lungs, there is a larger degree of off-target delivery to the spleen and the liver, reducing the lung-selectivity of 18:0 TAP SORT LNPs to 80%, the lowest among the TAP series (FIG. 2A). Meanwhile, 18:1 RI, which nearly shares the same chemical structure as 18: 1 TAP but substitutes one of the methyl groups in the headgroup with an ethanol motif, outperforms 18: 1 TAP as a SORT molecule, yielding >1.5 fold more potent mRNA delivery to the lungs while maintaining the same level of selectivity (FIG. 2B).

[0285] The members of the EPC series were also investigated for their capacity to engineer lung-targeting LNPs. Compared to mDLNP, SORT LNPs incorporating EPC molecules could all deliver mRNA to the lungs (FIG. 2C). Within this series, 12:0 and 18:0 EPC produced the most potent mRNA delivery to the lungs (FIG. 2D). However, compared to 18:0 EPC, which had 83% of the total luminescent flux produced in the lungs, the use of 12:0 EPC as a SORT molecule resulted in significantly greater off-target delivery to the spleen and liver, reducing the lung- selectivity of mRNA delivery to 40%. Other lipids from the EPC series had between 2- to 10-fold lower activity than 12:0 EPC and 18:0 EPC SORT LNPs as well as lungselectivity of 72% or less (FIG. 2D).

[0286] Collectively, all eleven quaternary ammonium molecules could be incorporated into mDLNP as SORT molecules and shift mRNA delivery to the lungs. Interestingly, the potency and selectivity of lung delivery depended on the chemical structure of the SORT molecule within and between the two lipid series, aligning with previous work demonstrating how changes to lipid chemistry can affect the efficacy of both lipoplexes and LNPs. Overall, 18:1 RI and 18:0 EPC were identified as the lead SORT molecules within their respective lipid seriesfor potent, lung-specific delivery of FLuc mRNA. However, the EPC showed slightly less potent and selective delivery of FLuc mRNA in lungs when compared with the TAP series (FIGs. 2B and 2D). Notably, the EPC SORT molecules with most potent lung delivery, 12:0 and 18:0 EPC, had comparable delivery efficacy as other potent TAP SORT molecules, 14:0 and 16:0 TAP. To further verify that these findings were consistent across multiple mRNA cargoes, human erythropoietin (hEPO) mRNA using leading formulations were delivered and found 18: 1 RI facilitated the highest levels of protein translation (FIG. 7). hEPO could be accurately quantified in the blood as a secreted protein to provide an additional readout on LNP efficacy.Example 3: Lead SORT molecules enable similar cell transfection profiles

[0287] Given the importance of understanding the intra-organ tropism of mRNA delivery for therapeutic applications, we characterized which cells are transfected by lung-targeting LNPs that incorporate leading SORT molecules. Even further, differences in the protein corona composition may affect which cells are targeted by an LNP, providing further justification for characterizing the relationship between LNP chemistry and cellular transfection.

[0288] To assess the lungs cells targeted by LNPs based on alternative SORT molecules, we treated a genetically-engineered tdTomato (tdTom) reporter mouse model with mRNA encoding Cre recombinase. In this model, a stop cassette is bookended by loxP sites, thereby preventing expression of the fluorescent tdTom protein. Following translation of Cre from the exogenously administered mRNA, the stop cassette is removed to induce expression of tdTom (FIG. 3A). Using the tdTom model, the total amount of fluorescence measured is proportional to the number of cells in which a Cre recombination event occurs, providing an estimate of how many cells are targeted by a given formulation rather than the levels of mRNA activity in the target organ. Additionally, the transfection efficiency of specific cell populations can be elucidated by fluorescence-activated cell sorting (FACS).

[0289] The top-performing SORT molecules from the TAP series and EPC series were compared to the current gold standard of 18:1 TAP for the delivery of Cre mRNA. To account for autofluorescence from the tissue, tdTom mice injected with PBS were used as a control. Two days after intravenous injection of LNPs encapsulating Cre mRNA, the tdTom mice were sacrificed and organs excised for ex vivo fluorescence imaging. Specific organs were imaged together for each group, allowing for ready comparison of tdTom fluorescence induced by the different formulations (FIG. 3B, S3). All formulations induced a high level of tdTomfluorescence in the lungs ranging between 2.0x108 and 3.5x108 p / s / cm2 / sr (FIG. 3B). The greatest tdTom fluorescence was detected in the lungs of mice treated with 18: 1 TAP- containing LNPs.

[0290] When considering the tissue- specificity of tdTom fluorescence, the use of 18:0 EPC as a SORT molecule produced more off-target signal in the liver compared to the other SORT molecules (FIG. 3C). Notably, the use of 18: 1 TAP and 18:1 RI as SORT molecules yielded an average lung:liver tdTom fluorescence ratio of -12 and -9, respectively, whereas the use of 18:0 EPC as a SORT molecule yielded a ratio of -2.5 (FIG. 3C & FIG. 8). Greater delivery to off-target organs by 18:0 EPC SORT LNPs may result from differential interactions with plasma proteins compared to 18:1 TAP and 18:1 RI SORT LNPs that either promote liver targeting or reduce lung selectivity.

[0291] To pinpoint the individual lung cells successfully reached by the leading lung-targeting SORT LNPs, we employed FACS and an antibody panel capable of individually labelling endothelial (CD31+), immune (CD45+), and epithelial (EpCam+) cells (FIG. 9). For all formulations, endothelial cells were most efficiently transfected, followed by epithelial cells, then immune cells (FIG. 3D). The ratio of epithelial to endothelial tdTom positivity was similar for all formulations, ranging between 0.36 and 0.39, suggesting that all three SORT molecules can efficaciously deliver mRNA to the deeper tissues of the lung.

[0292] Collectively, these results indicate that SORT molecules from both the TAP and EPC series are capable of transfecting multiple cell populations in the lungs with high efficiency. The relative proportions of each cell population targeted by these LNPs was consistent between the three formulations, suggesting all have similar intra-organ tropisms. Delivery to epithelial cells is of particular interest for therapeutic applications, due to their involvement in the pathology of multiple diseases including cystic fibrosis, as well as for increasing basic knowledge regarding molecular mechanisms involved in nanoparticle transport in vivo. Unlike the fenestrated endothelium of the liver, which provides LNPs with access to the hepatic parenchyma via passive diffusion out of the vasculature, the pulmonary endothelium is continuous. Presumably, the continuous endothelial barrier would prevent delivery to the deeper tissues of the lungs. Thus, it remains necessary to characterize the mechanism(s) responsible for the functional delivery of Cre mRNA to the epithelium we observed here (FIG. 3D).Example 4: SORT molecule structure alters protein corona composition in a manner correlative with delivery outcomes

[0293] After intravenous administration, PEG-lipid on the LNP surface will spontaneously desorb, allowing distinct proteins to recognize the underlying SORT molecules now exposed on the LNP surface. Cells highly expressing the cognate receptors of the surface-bound proteins can interact and uptake the LNPs through receptor- mediated mechanisms, promoting functional mRNA delivery within specific organs. Additionally, interactions between plasma proteins and nanoparticles can be impacted by a nanoparticle’s molecular composition and surface properties. Thus, we hypothesized that changing the chemical structure of a SORT molecule within a specific biophysical class could lead to subtle changes in the identities and quantities of the constituent of the LNP protein corona, resulting in the differences we observed for tissue-specificity when using different quaternary ammonium lipids as SORT molecules (FIGs. 2 and 3).

[0294] To examine the biological interactions between lung-targeting SORT LNPs and plasma proteins, we implemented a protocol to isolate protein-LNP complexes from bulk plasma after an ex vivo incubation at physiological temperature. Following isolation, LNPs were disrupted using 2 weight % sodium dodecyl sulfate (SDS) and excess lipids were removed from the samples to provide a mixture of plasma proteins suitable for analysis using SDS- polyacrylamide gel electrophoresis (PAGE) and mass spectrometry proteomics (FIGs. 4 and 5). For each of the eleven possible SORT molecules, three independent batches of LNPs were formulated and characterized to ensure the reproducibility and reliability of the isolated protein corona (FIG. 10). LNPs was employed as a relevant control to define how the addition of a SORT molecule to a four-component liver-targeting formulation yields a distinct protein corona composition to drive extrahepatic delivery. To ensure that the proteins detected in the protein corona samples were truly associated with the LNPs, rather than plasma components that co-precipitated during the centrifugation protocol, native plasma was subject to the same isolation protocol. After undergoing the protein-LNP isolation protocol, native plasma did not form a pellet discernible to the naked eye nor were any bands stained on an SDS gel, suggesting that all proteins detected in the LNP protein corona samples are a component of the corona (FIG. 11).

[0295] To begin, we used SDS-PAGE to qualitatively characterize and compare the protein corona of mDLNP and the different lung- targeting SORT LNPs (FIG. 4). The addition of a fifth component to mDLNP leads to notable differences in the composition and quantity ofplasma proteins that bind. Namely, there is increased enrichment of bands between 20 and 25 kDa, around 40 to 50 kDa and near 65 kDa. Between the different lung-targeting LNPs, there was differential enrichment of bands around 30, 34, 75, and 125 kDa, among others (FIG. 4). These differences in protein corona were reproducible with three independently-formulated batches of LNPs incubated with separate aliquots of plasma, supporting the validity of the observed interactions (FIG. 10). All formulations tested were found to be stable in serum for at least 48 hours, suggesting that the protein corona does not destabilize the LNPs within their expected time in the systemic circulation (FIG. 12) (Mui et al. , 2013).

[0296] To identify and quantify the observed differences in protein corona composition between the different LNPs, we analyzed all 36 samples using mass spectrometry proteomics (FIG. 5). We anticipate that the most abundant proteins are those most likely to be functionally relevant, so we focused our analysis on proteins which are present at a level of 2.5% of the total abundance or greater for at least one sample. For all quaternary ammonium lipids incorporated into mDLNP as SORT molecules, there was an increased enrichment of vitronectin (Vtn), a factor previously identified as most important for lung targeting that was further validated as functional in multiple cell lines expressing its cognate receptor av[33 integrin. We also previously showed that 18: 1 TAP Lung SORT LNPs target the lungs in an ApoE independent manner in vivo. Here, with this expanded collection of Lung SORT lipids, we further noted binding of other proteins including prothrombin, clusterin, serum paraoxonase 1 , fibrinogen, and albumin (FIG. 5A). Additionally, there was a decrease in the abundance of factors like complement component Clq subunits and immunoglobulin chains (FIG. 5A). Notably, there were differences in the rank order of most highly enriched proteins as well as the abundances of each protein based on the choice of SORT molecule, highlighting how LNP chemistry can influence the subset and quantities of plasma proteins that associate with lung-targeting SORT LNPs after intravenous injection.

[0297] Through an analysis of the relationship between SORT molecule structure and protein enrichment, we can gain a deeper understanding of how LNP chemistry influences the protein corona. In comparison to TAP SORT molecules, EPC SORT molecules generally facilitated increased adsorption of ApoE, complement component Clq subunits, serum paraoxonase 1, and immunoglobulin chains (FIG. 5A). These findings suggest that lipid series chemistry can bias an LNP to interact with specific proteins that may drive differences in the specificity of organ-specific mRNA delivery. Additionally, while there did not appear to be any correlations between saturated alkyl tail length and protein binding for the TAP series, increasing the alkyltail length of an EPC SORT molecule increased binding of albumin, fibrinogen, prothrombin, and serum paraoxonase 1 (FIG. 5A). Compared to SORT molecules that had fully saturated alkyl tails, SORT molecules that featured at least one alkyl tail with an unsaturated bond led to notably reduced ApoE enrichment but increased binding of other proteins to the LNP surface, such as clusterin, serum paraoxonase 1, and fibrinogen (FIG. 5A). Finally, in comparison to 18:1 TAP, the use of 18:1 RI as a SORT molecule, which substitutes one of the methyl groups found in the headgroup of 18: 1 TAP with an ethanol group, increased binding of clusterin and fibrinogen while slightly reducing Vtn binding (FIG. 5A). These findings demonstrate that the chemistry of individual LNP components can alter protein corona composition.

[0298] Since the protein corona may impact the endogenous mechanisms responsible for both targeting of the lungs as well as de-targeting of the liver, univariate analyses of the relationship between protein abundances and FLuc mRNA delivery to the lungs and the liver were performed the most highly enriched proteins (FIGs. 5B-5C). Significant positive correlations between protein abundance and lung-selectivity were identified for Vtn, clusterin, albumin, and prothrombin while significant negative correlations were seen for complement component Clq subunits and immunoglobulin chains (FIG. 5B). Similarly, Vtn was negatively correlated with liver delivery whereas enrichment of both complement component Clq subunits and immunoglobulin chains was found to be positively correlated with liver delivery in a significant manner (FIG. 5C). Interestingly, proteins like complement components and immunoglobulins are typically associated with detrimental delivery outcomes because they tag nanoparticles for sequestration by cells of the mononuclear phagocytic system (MPS), some of which reside within the liver and the spleen. Thus, it is plausible these proteins could reduce the lungspecificity of SORT LNP delivery by promoting increased MPS interactions. It is also possible that the increased off-target transfection of liver cells in the tdTom mouse model observed when using 18:0 EPC as a SORT molecule, rather than 18: 1 TAP or 18: 1 RI, could be driven by a corresponding -10-fold higher enrichment in ApoE binding, a crucial factor known to promote liver accumulation of LNPs (FIG. 3C & FIG. 8).

[0299] Generally, LNPs incorporating EPC SORT molecules tended to have protein coronas enriched in factors negatively correlated with lung-specificity compared to LNPs incorporating TAP SORT molecules, which may explain the superior in vivo performance of the TAP series (FIG. 5). Notably, no single protein could fully account for the total variation in lung specificity observed between SORT molecules, suggesting that the ensemble of corona proteins may determine delivery outcomes. Altogether, these observations reveal a nuanced relationshipexists between SORT molecule structure and protein corona that can be manipulated by tuning the chemistry of a lipid’s hydrophobic domain or headgroup. Further, these changes in protein corona composition and abundances may have a tangible impact on the tissue-specificity of lung-targeting SORT LNPs (FIG. 2). The tissue-specificity of mRNA delivery could be impacted by the presence of proteins that promote delivery to the lungs, like Vtn and clusterin, as well as proteins that are implicated in delivery to off-target sites, such as immunoglobulins and complement components. The implementation of more complex approaches, like multivariate analysis or machine learning, could provide greater insight into the interplay between the protein corona and organ-targeting properties of SORT LNPs to further clarify the corona proteins most crucial for lung delivery and the associated LNP compositions that generate a protein corona that yields a desired delivery outcome. This would facilitate rational design of improved delivery systems by identifying suitable LNP compositions which enrich for proteins that drive delivery to the target site while reducing binding of proteins responsible for non-specific delivery.Example 5: Conclusions

[0300] The targeted and potent delivery of mRNA is essential to the success of protein replacement and genome editing therapies seeking to remedy genetic diseases of the lungs. Whereas conventional LNPs typically accumulate in the liver following intravenous administration, thereby limiting their scope of applications, SORT LNPs enable site-specific mRNA delivery to therapeutically-relevant cells within the liver, spleen, or lungs of mice. Crucially, LNPs that incorporate different classes of SORT molecules bind to distinct subsets of plasma proteins that likely drive the unique and specific organ-targeting outcomes of SORT LNPs. Building on that foundation, we explicated how changes to the chemical structure within the same class of SORT molecule affect lung-specific delivery efficacy and the biological interactions involved in endogenous targeting to better understand the impact of the protein corona on organ-targeted drug delivery.

[0301] Through our studies of the TAP and EPC lipid series as SORT molecules, we found that SORT molecule chemistry can affect the potency and selectivity of mRNA delivery to the lungs. Notably, the TAP series outperformed the EPC series by yielding higher protein production and tissue- specificity across multiple mRNA cargoes. The top-performing molecules identified from both series, 18: 1 RI and 18:0 EPC, are able to access lung endothelial cells as well as epithelial cells in the deeper tissues of the lungs making them useful fordeveloping SORT formulations for therapeutic protein expression and genome editing. Improved SORT molecules could be designed by tailoring alkyl tail and headgroup functionalities based on structure-property relationships derived from this dataset to promote interactions with the proteins most essential for delivery to the target site and transfection of the desired cell type(s).

[0302] Additionally, we discovered that SORT molecule chemical structure impacts plasma protein interactions in a manner that appears to be related to the lung-specificity of mRNA delivery. As we had previously observed, Vtn was a key protein associated with all lungtargeting SORT LNPs and is correlated with the tissue-specificity of mRNA delivery to the lungs. However, lung targeting appears to be driven by binding of an ensemble of proteins that could play different roles in determining the potency and selectivity of mRNA delivery, such as de-targeting the liver or binding to receptors highly expressed on the luminal side of pulmonary endothelial cells to promote transfection of lung tissue, rather than a single factor. Given the complexity of the relationship between SORT molecule chemical structure and protein corona composition, more intricate modelling approaches could further clarify how individual proteins impact delivery. This would be supported by collection of a larger dataset to profile the protein corona more comprehensively across different SORT formulations. Further mechanistic experiments that clarify the functional effect of major proteins identified as important for delivery within these datasets, such as how genetic knockout of key proteins impacts delivery or profiling of the cellular receptors that facilitate nanoparticle uptake, would deepen our understanding of the intersection between LNP chemistry, plasma protein interactions, and organ-targeting outcomes. Ultimately, this would provide a foundation for rational design and optimization of formulation components that minimize delivery to off- target tissues and further reveal how to harness endogenous proteins for organ-targeted nanoparticle drug delivery.Example 6: Methods

[0303] In vitro transcription of mRNA: mRNAs encoding FLuc, hEPO, and Cre recombinase were synthesized by in vitro transcription. The coding fragments of each protein were prepared using PCR and cloned into a pCS2+MT plasmid backbone featuring customized 5’ and 3’ untranslated regions as well as poly A sequences. In vitro transcription was conducted using the Invitrogen MEGAscript SP6 transcription kit (ThermoFisher) but with Nl- methylpseudouridine-5'-triphosphate replacing the typical uridine triphosphate. Next, a Caplcap structure was installed to the 5’ end of the mRNA using the ScriptCap Capl Capping System (CellScript).

[0304] LNP preparation: the ethanol dilution method was used to formulate mRNA LNPs. To prepare mDLNP, the base four component formulation, a mixture of 23.8 mol % 5A2-SC8, 23.8 mol % DOPE, 47.6 mol % cholesterol, and 4.8 mol % DMG-PEG2K was diluted in ethanol and rapidly mixed with 10 mM, pH = 4.0 citrate buffer containing mRNA using a pipette at a volume ratio of 3:1 (aqueous: organic phases). For all lung-targeting SORT formulations, a mixture of 11.9 mol % 5A2-SC8, 11.9 mol % DOPE, 23.8 mol % cholesterol,2.4 mol % C14-PEG2K, and 50 mol % of the chosen SORT molecule was prepared in ethanol. Once again, this lipid mixture was rapidly mixed with 10 mM, pH = 4.0 citrate buffer containing mRNA using a pipette at a volume ratio of 3:1. Ten minutes after mixing, the LNP formulations were diluted to a desired concentration using lx phosphate buffered saline (PBS). For in vivo experiments, LNPs were dialyzed against lx PBS for 2 h using the Pur-A-Lyzer Midi Dialysis Kit (MWCO 3.5 kDa).

[0305] LNP characterization: LNP size, polydispersity index, and ^-potential were measured using DLS (Malvern Micro V model; He-Ne laser, X = 632 nm). After formulation, LNPs were diluted in lx PBS to a final concentration of 1.0 ng mRNA / pL PBS before measurement of hydrodynamic diameter by DLS. After DLS, the LNPs were diluted 10-fold in DI water to measure ^-potential. To measure mRNA encapsulation, a RiboGreen assay was performed. LNPs were diluted to a final concentration of 0.5 ng mRNA / pL PBS and mRNA standards were prepared at concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0, and 2.0 ng / pL. All samples and standards were added to a 96-well black opaque polystyrene microplate, followed by addition of RiboGreen reagent that had been diluted 200x in PBS. Then the amount of free mRNA was measured in the Infinite F / M200 Pro microplate reader (Tecan) for fluorescence using an excitation of 485 nm and an emission 535 nm. The total mRNA concentration (free and encapsulated) was determined by adding 0.5% Triton X-100 to each well before conducting a second measurement of fluorescence using an excitation of 485 nm and an emission 535 nm. The encapsulation efficiency was calculated as:EE — 1

[0306] In vivo FLuc mRNA delivery and quantification: C57BL6 / J mice between 6 to 8 weeks old were injected with mDLNP or a lung-targeting SORT LNPs that incorporate one of eleven quaternary ammonium lipids at a dosage of 0. 1 mg FLuc mRNA / kg bodyweight (n = 3). After 6 h, mice were injected with D-Luciferin (150 mg / kg, IP) and bioluminescence imaging was conducted with an AMI-HTX in vivo imaging system (Spectral Instruments Imaging). Total luminescence of target organs was quantified using Aura Imaging Software (Spectral Instruments Imaging). Tissue-selectivity was quantified as:

[0307] In vivo hEPO mRNA delivery and quantification: C57BL6 / J mice between 6 to 8 weeks old were injected with lung-targeting SORT LNPs that employ either 18:1 TAP, 18:1 RI, or 18:0 EPC as a SORT molecule at an mRNA dosage of 0.3 mg hEPO mRNA / kg bodyweight. Blood was collected retro-orbitally at 6, 24, 48, and 72 h after injection to assess serum levels of hEPO protein. To prepare serum, whole blood was allowed to clot at room temperature for 30 minutes prior to centrifugation at 2,000 g for 15 minutes at 4 °C. Samples were maintained at -80 °C before analysis using ELISA. Quantification of serum hEPO concentration was performed using a sandwich ELISA developed in-house with the Human Erythropoietin / EPO DuoSet ELISA and DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems).

[0308] In vivo Cre mRNA delivery and quantification: LNP formulations encapsulating Cre mRNA were intravenously injected into tdTom mice at a dosage of 0.3 mg mRNA / kg bodyweight (n - 3). Two days later, mice were sacrificed and organs were excised for fluorescence imaging using an AMI-HTX in vivo imaging system (Spectral Instruments Imaging). To account for background fluorescence, tdTom mice injected with PBS were included as a cohort for this experiment. Mean organ fluorescence was measured as:

[0309] Lung cell isolation: lungs excised from tdTomato mice were placed onto ice cold PBS following resection. IX lung digestion media was prepared from RPMI media supplemented with 2% wt / vol BSA, 300 U / mL collagenase, and 100 U / mL hyaluronidase. The lung tissuewas cut into small pieces using a razor blade, then transferred into a 50 mL tube containing 10 mL of digestion media. The tube was incubated at 37 °C for 1 hr while shaking at 180 rpm. After incubation, the homogenized lung cell solution was pipetted up and down several times to remove cell clumps and finally filtered through a 70-micron cell strainer into a fresh 50 mL tube. The filter was washed with a wash buffer composed of cold PBS and 2% fetal bovine serum prior centrifuging the sample at 1200 rpm for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 10 mL of cold wash buffer. Next, the red blood cells (RBCs) were lysed by resuspending the cell pellet in 5 mL of 1 X RBC lysis buffer (BioLegend) and incubating at room temperature for 5 minutes. Following the incubation, 10 mL of wash media was added to the sample. Once again, the sample was centrifuged at 1200 rpm for 5 minutes. Finally, the RBC free cell pellet was resuspended in 5 mL of cell staining buffer (BioLegend) prior to antibody staining for flow cytometry.

[0310] Lung cell staining and flow cytometry: single-cell suspensions obtained from lungs excised from tdTomato mice were pre-blocked with TruStain FcX™ antibody (BioLegend) for 15 minutes. Next, 100 μL of cell suspension was incubated with 1 μL of an antibody cocktail composed of Alexa Fluor 488-conjugated anti- mouse CD31, Pacific Blue-conjugated antimouse CD45, and Alexa Fluor 647-conjugated anti-mouse EpCAM antibodies (BioLegend) for 15 minutes on ice. Ghost Dye Red 780 (BioLegend) was used to identify the dead cells. Next, excess antibodies were removed by washing the cell pellet with cell staining buffer three times. Finally, the cell pellet was resuspended in 500 μL of cold cell staining buffer and kept on ice until analysis by flow cytometer. Cells were then analyzed by Becton Dickenson (BD) LSR Fortessa flow cytometer. The resulting data were analyzed by FlowJo software (BD) to assess the percentage of tdTomato positive cells within each population stained for in the lungs.

[0311] Isolation of LNP protein corona: to isolate the LNP protein corona, the method we previously developed was employed. Briefly, C57BL6 / J mouse plasma was added to each LNP solution, diluted to a lipid concentration of 1 g / L, at a 1:1 volume ratio and incubated for 15 min at 37 °C. A 0.7 M sucrose solution was prepared by dissolving solid sucrose in MilliQ water. The LNP / plasma mixture was loaded onto a 0.7 M sucrose cushion of equal volume to the mixture and centrifuged at 15,300 g and 4 °C for 1 h. The supernatant was removed, and the pellet was washed with lx PBS. Next, the pellet was centrifuged at 15,300 g and 4 °C for 5 min, and the supernatant was removed. Washing was performed twice more for a total of three washes. Following the final wash, the pellet was resuspended in 2 wt % SDS. Excess lipids were removed from each using the ReadyPrep 2-D Cleanup (Bio-Rad). The resultingpellet from the cleanup step was resuspended in 2x Laemmli buffer. The same procedure was repeated with plasma diluted directly in lx PBS at a 1 : 1 volume ratio to verify that no plasma components were precipitating on their own due to the centrifugation protocol.

[0312] SDS-PAGE characterization of protein corona: plasma proteins isolated from the surface of LNPs were loaded onto a 4-20% TGX Precast Protein Gel. Samples were allowed to stack for 10 minutes at 90 V before increasing the running voltage to 150 V until the dye front reached the bottom edge of the gel. Proteins were visualized by staining the gel for 1 hr in Bio-Safe Coomassie Stain followed by a 1 hr destain in water at room temperature. The gel was then imaged using a Licor Scanner.

[0313] LNP serum stability: The serum stability of SORT LNPs was investigated by incubating the LNPs with a serum solution at 37 °C. Four-component mDLNP and five- component SORT LNPs were suspended with PBS buffer containing 10% of FBS and incubated at 37 °C. At time points of 0, 8, 24, 48, 72, 96, 120, 144, and 168 hours, LNP hydrodynamic diameter was measured using DLS (Malvern Micro V model; He-Ne laser, X = 632 nm).

[0314] Mass spectrometry proteomics analysis of plasma proteins: to prepare samples for mass spectrometry proteomics, 10 uL of the plasma protein mixture isolated from the LNPs was loaded on to a 4-20% TGX gel. Samples were run at 90 V for 10 minutes, allowing them to stack. Then, the gel was stained using Bio-Safe Coomassie Stain to fix and visualize the proteins. After destaining for 1 hr, the protein bands were cut out using a razor blade and diced into small cubes with a volume of approximately 1 mm3. The cubes were added to a 1.5-mL Eppendorf tube and stored at 4 °C until being submitted to the University of Texas Southwestern Proteomics Core for mass spectrometry analysis. A Thermo QExactive HF mass spectrometer was used to identify and quantify the constituents of the protein samples. The identified proteins were ordered based on their abundance / MW. 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Claims

CLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) composition, wherein the LNP comprises:(i) a payload, optionally a polynucleotide;(ii) an ionizable lipid;(iii) a quaternary ammonium lipid at a molar percentage from about 20% to about 65%;(iv) a phospholipid;(v) a cholesterol; and(vi) a polyethylene glycol-conjugated lipid (PEG-lipid).

2. The composition of claim 1 , wherein the quaternary ammonium lipid is a trialkylammonium propane (TAP).

3. The composition of claim 2, wherein the trialkylammonium propane (TAP) is 1,2- dimyristoyl-3-trimethylammonium-propane (14:0 TAP).

4. The composition of claim 2, wherein the trialkylammonium propane (TAP) is 1,2- dipalmitoyl-3-trimethylammonium-propane (16:0 TAP).

5. The composition of claim 2, wherein the trialkylammonium propane (TAP) is 1,2- stearoyl-3-trimethylammonium-propane (18:0 TAP).

6. The composition of claim 2, wherein the trialkylammonium propane (TAP) is 1,2- dioleoyl-3-trimethylammonium-propane (18:1 TAP).

7. The composition of claim 1, wherein the quaternary ammonium lipid is 18:1 RI.

8. The composition of claim 1 , wherein the quaternary ammonium lipid is an ethylphosphocholine (EPC).

9. The composition of claim 8, wherein the ethylphosphocholine (EPC) is 1 ,2-dilauroyl- sn-glycero-3-ethylphosphocholine (12:0 EPC).

10. The composition of claim 8, wherein the ethylphosphocholine (EPC) is 1,2-dioleoyl- sn-glycero-3-ethylphosphocholine (14:0 EPC).15811. The composition of claim 8, wherein the ethylphosphocholine (EPC) is 1,2- dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC).

12. The composition of claim 8, wherein the ethylphosphocholine (EPC) is 1 ,2-distearoyl- sn-glycero-3-ethylphosphocholine (18:0 EPC).

13. The composition of claim 8, wherein the ethylphosphocholine (EPC) is 1 ,2-dioleoyl- sn-glycero-3 -ethylphosphocholine (18:1 EPC).

14. The composition of claim 8, wherein the ethylphosphocholine (EPC) is l-palmitoyl-2- oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC).

15. The composition of any one of claims 1 to 14, wherein the quaternary ammonium lipid is present in the composition at a molar percentage from about 20% to about 65%, from about 20% to about 55%, from about 20% to about 50%, from about 30% to about 65%, from about 30% to about 60%, or from about 30% to about 55%.

16. The composition of any one of claims 1 to 15, wherein the quaternary ammonium lipid is present in the composition at a molar percentage from about 30% to about 50%.

17. The composition of any one of claims 1 to 16, wherein the phospholipid is 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

18. The composition of any one of claims 1 to 16, wherein the phospholipid is 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).

19. The composition of any one of claims 1 to 18, wherein the phospholipid is present in the composition at a molar percentage from about 7.5% to about 60%, from about 7.5% to about 50%, from about 7.5% to about 40%, from about 7.5% to about 30%, from about 7.5% to about 20%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.

20. The composition of any one of claims 1 to 19, wherein the cholesterol is present in the composition at a molar percentage from about 15% to about 46%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 20% to about46%, from about 20% to about 40%, from about 20% to about 35%, or from about 20% to about 30%.

21. The composition of any one of claims 1 to 20, wherein the PEG-lipid is present in the composition at a molar percentage from about 0.5% to about 10%, from about 0.5% to about 7.5%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about3%, from about 1% to about 10%, from about 1% to about 7.5%, from about 1% to about 5% from about 1% to about 4% from about 1% to about 3%.

22. The composition of any one of claims 1 to 21, wherein the ionizable lipid is a compound of Formula (D-I):Core-Repeating Unit-Terminating Group (D-I), or a pharmaceutically acceptable salt thereof, wherein the core is linked to four to six repeating units and each repeating unit is linked to a nitrogen of the core, wherein: the core has the formula:wherein:X3 is -NRe-, wherein Re is hydrogen or alkyl(c≤8 ) R3and R4 are each independently amino, alkylamino(c≤12), dialkylamino(c≤12),wherein: each e independently is 1, 2, or 3;Rc, Rd, and Rf are each independently hydrogen or alkyl(c<6i; c and d are each independently 1, 2, 3, 4, 5, or 6; the repeating unit is a degradable diacyl group having the formula:wherein:A1and A2are each -O-;Y3is alkanediyl(c≤12); andR9is alkyl(c≤8 ,) and the terminating group has the formula:wherein:Y4 is alkanediyl(c<i8); and Rio is hydrogen.

23. The composition of any one of claims 1 to 22, wherein the ionizable lipid is present in the composition at a molar percentage from about 5% to about 30%.

24. The composition of any one of claims 1-23, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof:Y3 is -CH2CH2-;R9is -CH3; and the terminating group is selected from the group consisting of:

25. The composition of any one of claims 1 to 24, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four repeating units and the core has the following structure:

26. The composition of any one of claims 1 to 25, wherein in the compound of Formula (D-I) or the pharmaceutically acceptable salt thereof, the core is linked to four or five repeating units and the core has the following structure:

27. The composition of any one of claims 1 to 26, wherein in the compound of Formula(D-I) or the pharmaceutically acceptable salt thereof, the core has the following structure:

28. The composition of any one of claims 1 to 27, wherein the ionizable lipid is a dendrimer.

29. The composition of claim 28, wherein the dendrimer is(4A3-SC7).

30. The composition of claim 28, wherein the dendrimer is(4A3-SC8).

31. The composition of claim 28, wherein the dendrimer is(5A2-SC8).

32. The composition of any one of claim 1 to 31 , wherein the lipid composition comprises: an ionizable lipid at molar percentage from about 5% to about 30%; a quaternary ammonium lipid at a molar percentage from about 20% to about 60%;DOPE at a molar percentage from about 7.5% to about 66%; the cholesterol at a molar percentage from about 15% to about 46%; and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

33. The composition of any one of claim 1 to 31 , wherein the lipid composition comprises: an ionizable lipid at molar percentage from about 5% to about 30%; a quaternary ammonium lipid at a molar percentage from about 20% to about 55%;DOPE at a molar percentage from about 7.5% to about 66%; the cholesterol at a molar percentage from about 15% to about 46%; and the PEG-lipid at a molar percentage from about 0.5% to about 10%.

34. The composition of any one of claim 1 to 31 , wherein the lipid composition comprises: an ionizable lipid at molar percentage from about 5% to about 25%;a quaternary ammonium lipid at a molar percentage from about 30% to about 50%; the phospholipid at a molar percentage from about 7.5% to about 20%; the cholesterol at a molar percentage from about 15% to about 40%; and the PEG-lipid at a molar percentage from about 1% to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an trimethylammonium propane (TAP).

35. The composition of any one of claim 1 to 31 , wherein the lipid composition comprises: an ionizable lipid at molar percentage from about 5% to about 25%; a quaternary ammonium lipid at a molar percentage from about 30% to about 50%; the phospholipid at a molar percentage from about 7.5% to about 20%; the cholesterol at a molar percentage from about 15% to about 40%; and the PEG-lipid at a molar percentage from about 1% to about 4%, wherein the ionizable lipid is 4A3-SC7 and the quaternary ammonium lipid is an ethylphosphocholine (EPC).

36. The composition of any one of claims 1 to 35, wherein the polynucleotide comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or microRNA.

37. The composition of any one of claims 1 to 36, wherein the polynucleotide comprises a messenger RNA (mRNA).

38. The composition of any one of claims 1 to 37, wherein the mRNA comprises Nl- methylpseudouridine- 5 ' -triphosphate.

39. The composition of any one of claims 1 to 38, wherein the mRNA comprises Capl cap structure.

40. The composition of any one of claims 1 to 39, wherein the mRNA encodes a geneediting system or component thereof.

41. The composition of any one of claims 1 to 40, wherein the LNP comprises a protein corona.

42. The composition of claim 41, wherein the protein corona comprises at least one plasma protein.

43. The composition of any one of claims 1 to 42, wherein the quaternary ammonium lipid controls causes association of the at least one plasma protein.

44. The composition of any one of claims 1 to 43, wherein the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin.

45. The composition of any one of claims 1 to 43, wherein the protein corona comprises vitronectin.

46. The composition of any one of claims 1 to 43, wherein the protein corona comprises prothrombin.

47. The composition of any one of claims 1 to 43, wherein the protein corona comprises lower levels of component Clq subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid.

48. The composition of any one of claims 1 to 47, wherein the composition is an intravenous composition.

49. A pharmaceutical composition comprising the LNP composition of any one of claims 1 to 48.

50. A kit comprising the LNP composition of any one of claims 1 to 48 or the pharmaceutical composition of claim 49, and an instructions for use thereof.

51. A method for treating or preventing a disease in a subject in need thereof, comprising administering the LNP composition of any one of claims 1-48 or the pharmaceutical composition of claim 49.

52. The method of any one of claim 51, wherein the administering comprises administration by intravenous injection.

53. The method of any one of claims 51 to 52, wherein the LNP acquires a protein corona after adaministration.

54. The method of any one of claims 51 to 53, wherein the protein corona comprises at least one plasma protein.

55. The method of any one of claims 51 to 54, wherein the quaternary ammonium lipid controls causes association of the at least one plasma protein.

56. The method of any one of claims 51 to 55, wherein the protein corona comprises one or more of vitronectin, prothrombin, clusterin, serum paraoxonase 1, fibrinogen, and albumin.

57. The method of any one of claims 51 to 55, wherein the protein corona comprises vitronectin.

58. The method of any one of claims 51 to 55, wherein the protein corona comprises prothrombin.

59. The method of any one of claims 51 to 55, wherein the protein corona comprises lower levels of component Clq subunits and immunoglobulin chains than a reference LNP without the quaternary ammonium lipid.

60. The method of any one of claims 53 to 59, wherein the protein corona increases specific delivery of the payload to the lung of the subject.

61. The method of any one of claims 51 to 60, wherein the protein corona increases specific delivery of the payload to the lung of the subject.

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