WO2025080867A1

WO2025080867A1 - Ionizable lipids for improved mrna delivery

Info

Publication number: WO2025080867A1
Application number: PCT/US2024/050817
Authority: WO
Inventors: Daniel SIEGWART; Hu XIONG
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2023-10-10
Filing date: 2024-10-10
Publication date: 2025-04-17
Anticipated expiration: 2026-04-10

Abstract

Provided herein are ionizable cationic lipids and lipid nanoparticle compositions comprising a lipid component comprising the same. Also, provided herein is a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering an effective amount of the lipid nanoparticle composition disclosed herein.

Description

DESCRIPTION IONIZABLE LIPIDS FOR IMPROVED MRNA DELIVERY This application claims the benefit of United States Provisional Application No. 63/589,257, filed on October 10, 2023, the entire contents of which are hereby incorporated by reference. REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on October 10, 2024, is named UTFDP4336WO.xml and is 16,008 bytes in size. BACKGROUND [1] Lipid nanoparticles (LNPs) represent the most clinically advanced non-viral mRNA delivery vehicles, however, the full potential of LNP platform is greatly hampered by inadequate endosomal escape capability. There is a need for lipids designed for improved mRNA delivery in vivo which may be used for broad applications such as gene editing and cancer immunotherapy. SUMMARY [2] In a first aspect, disclosed herein is a compound of Formula (I): wherein: R^D1 is H or C₁-C₄ alkyl; x1 and x2 are each independently 1, 2, or 3; and _{R is wherein RSCC, RSSC, and} R^SC are each a linear or branched C₄-C₂₀ alkyl; and nSC is an integer from 1-10. [3] For example, in some embodiments, the compound of Formula (I) is a compound, having the Formula (I-a), (I-b), or (I-c):

[4] In a further aspect, disclosed herein is a lipid nanoparticle composition comprising a lipid component comprising a compound of Formula (I) (e.g., a compound of Formula (I-a), (I-b), or (I-c), e.g., a compound of Table 1 or 2). [5] In some embodiments, the lipid nanoparticle composition further comprises a phospholipid, a PEG lipid and/or a sterol. BRIEF DESCRIPTION OF THE DRAWINGS [6] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [7] FIG.1 shows the structure of 4A3-SCC-PH and a schematic illustration of mRNA delivery to cells, with lipid nanoparticle compositions of the disclosure, which can significantly facilitate endosomal escape and improve mRNA delivery in vivo. [8] FIG. 2A-2B show heat maps of luciferase expression and cell viability. FIG. 2A shows a heat map of luciferase expression in ovarian cancer IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). A relative luminescence intensity of >105 was counted for in vitro hit rate calculation. FIG.2B shows a heat map of cell viability IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). [9] FIGS.3A-3B show the relative in vitro hit rate of ionizable cationic lipids described herein. FIG.3A shows the relative in vitro hit rate of ionizable cationic lipids of the disclosure with different alkyl chain lengths. FIG. 3B shows the relative in vitro hit rate of ionizable cationic lipids of the disclosure for with different amino headgroups of 2A1−6A1. [10] FIG.4A-4B show analysis of in vivo screening of 4A1 and 4A3 ionizable cationic lipids of the disclosure at a dose of 0.1 mg/kg Fluc mRNA (n = 2 for initial screening) using bioluminescence (FIG.4A) and quantified radiance (FIG.4A) of exemplary ionizable cationic lipids described herein. FIG.4A shows bioluminescence images from in vivo screening of 4A1 and 4A3 ionizable cationic lipids of the disclosure at a dose of 0.1 mg/kg Fluc mRNA (n = 2 for initial screening). Images were obtained 6 h after i.v. injection of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure into C57BL/6 mice. H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; exposure time: 15 s. An average luminescence radiance of >2,500,000 was counted for in vivo hit rate calculation. FIG. 4B shows quantification measured by average radiance [p/s/cm²/sr] in vivo luciferase expression in liver obtained from in vivo screening of 4A1 and 4A3 ionizable cationic lipids as shown in FIG.4A. [11] FIG.5A-5C show graphical representations of in vivo hit rate of ionizable cationic lipids described herein. FIG. 5A shows relative hit rates for ionizable cationic lipids of the disclosure with amino headgroups of 4A1 and 4A3. FIG. 5B shows relative hit rates for ionizable cationic lipids of the disclosure with different lengths of tails (4C−14C). FIG. 5C shows relative hit rates for 4A3 ionizable cationic lipids of the disclosure with linear and branched alkyl chains. [12] FIG. 6A-6G show in vitro evaluation of six LNPs, 4A3-SCC-10/PH, 4A3-SC- 10/PH, and 4A3-SSC-10/PH, for Fluc mRNA delivery (25 ng/well, n = 4) in ovarian cancer IGROV1 cells treated with or without 20 μmol of a GSH-depleting agent N-ethylmaleimide (NEM); DLin-MC3-DMA was used as the positive control. FIG.6A shows luciferase activity for ovarian cancer IGROV1 cells treated with or without 20 μmol of a GSH-depleting agent N- ethylmaleimide (NEM) FIG. 6B-6D shows Z-average size (FIG. 6B), Zeta potential (FIG. 6C), and PDI value (FIG. 6D) of the exemplary six A43 lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure measured by Zetasizer Nano Series Nano- ZS. FIG. 6E-6F shows electrophoretic retardation analysis of six 4A3 LNPs for mRNA encapsulation ability (FIG. 6E) and release behavior after the treatment with 20 mM TCEP (FIG.6F). FIG.6G shows TEM images of 4A3-SCC-PH LNPs and DLin-MC3-DMA LNPs, scale bar = 100 μm. [13] FIG. 7A-7B show mCherry expression intensity of IGROV1 cells after treatment with six 4A3 LNPs for mCherry mRNA delivery. FIG 7A shows cellular fluorescence images. FIG.7B shows flow cytometry analysis of mCherry expression intensity of IGROV1 cells after treatment with six 4A3 LNPs for mCherry mRNA delivery after 24 h (80 ng mRNA/well). [14] FIG. 8A-8C. shows fluorescence analysis in vivo of mice subjects injected with Cy5-RNA-loaded 4A3 LNPs. FIG. 8A shows fluorescence images of major organs 6 h after i.v. injection of Cy5-RNA-loaded 4A3 LNPs into C57BL/6 mice (0.1 mg/kg). H, heart; Li, liver; S, spleen; Lu, lung; K, kidney. FIG.8B shows Cy5 fluorescence intensities of exemplay ionizable cationic lipids described herein. FIG 8C shows flow cytometry analysis showing fluorescence intensity distribution of Cy5-RNA in livers from mice in FIG.8A. [15] FIG. 9A-9B. shows qualitative and qualitative measures of radiance in mice subjects injected with LDIL compositions described herein. FIG.9A shows bioluminescence images of C57BL/6 mice and major organs 6 hours after i.v. injection of six 4A3 LNPs with different Fluc mRNA (0.1 mg/kg, n = 3, exposure time = 15 s) and FIG. 9B shows quantification of in vivo luciferase expression in livers of the mice subjects. [16] FIG. 10A-10B. shows qualitative and qualitative measures of radiance in mice subjects injected with Fluc mRNA-loaded 4A3-SCC-PH LNPs and 4A3-CCC-PH LNPs. FIG. 10A shows bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of Fluc mRNA-loaded 4A3-SCC-PH LNPs and 4A3-CCC-PH LNPs (0.1 mg/kg, exposure time = 15 s) and FIG. 10B shows quantification of in vivo luciferase expression in livers of mice from FIG.10A. [17] FIG.11 shows normalized pH titration profiles of Fluc mRNA-loaded LNPs. [18] FIG. 12A-12B show fluorescence (in vitro) and radiance (in vivo) of exemplar LDIL compositions decribed herein. FIG. 12A shows correlation between TNS fluorescence intensity in pH 5.0 buffer of different LNPs and in vivo mRNA delivery efficacy measured by bioluminescence. FIG. 12B shows non-correlation between TNS fluorescence intensity of different LNPs in pH 7.4 buffer and in vivo mRNA delivery efficacy (0.1 mg/kg mRNA, n=3 biologically independent mice). [19] FIG. 13A-13C show the process and analysis of induced endosomal rupture and membrane fusion with LNP compositions described herein. FIG.13A provides an illustration of lipid fusion and membrane rupture of 4A3-SCC-10/PH and 4A3 LNPs by a FRET assay at pH 5.5 conducted using a pair of DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3- diazole (DOPE-NBD as the donor) and lissamine rhodamine B (DOPE-Rho as the acceptor) incorporated into the single endosomal mimicking liposome. Comparison of membrane fusion with different lipids at pH 5.5 is shown in FIG.13B, and the comparison of membrane fusion with different at pH 5.5 is shown in FIG.13C. [20] FIG. 14A-14D show the process and analysis of induced endosomal rupture and membrane fusion with LNP compositions described herein. FIG.14A provides an illustration of dissociation of 4A3-SCC-10/PH formulated LNPs by FRET characterization after mixing with anionic endosomal mimics for 30 min at pH 5.5, where a pair of DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3-diazole (DOPE-NBD as the donor) and lissamine rhodamine B (DOPE-Rho as the acceptor) are incorporated into the single endosomal mimicking liposome. Comparison of different LNPs' dissociation at pH 5.5 is shown in FIG. 14B, Comparison of different LNPs dissociation at pH 5.5 after a 45 min at 37°C treatment with 10 mM GSH is shown in FIG.14C, or TCEP in shown FIG.14D. [21] FIG.15A-15B show the extent of hemolysis of six 4A3 lipids at pH 5.5 (FIG.15A) and pH 7.4 (FIG.15B). [22] FIG. 16 shows colocalization analysis (yellow) of endo/lysosomal escape (green) and Cy5-RNA cellular uptake (red) after HeLa cells were treated with different 4A3 LNPs for 4 and 8 h, scale bars = 10 μm. [23] FIG. 17A-17B shows Adsorption of endogenous apolipoprotein E (ApoE) on the surface of four 4A3 LNPs as validated by Western blot is shown in FIG.17A and quantitation of ApoE on the surface of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure is shown in FIG.17B. [24] FIG.18A-18C show dose response analyses using bioluminescense mesurements. FIG.18A shows dose-dependent bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of top-performing 4A3-SCC-10 and 4A3-SCC-PH formulated LNPs containing different doses of Fluc mRNA from 0.025 to 0.3 mg/kg, exposure time = 15 s. H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; and FIG. 18B shows quantification of in vivo luciferase expression in livers. FIG.18C shows bioluminescence imaging showing improved temporal resolution upon injection of 4A3-SCC-10/PH LNPs with 0.1 mg/kg Fluc mRNA, exposure time of 0.1 s. [25] FIG. 19A-19D. FIG. 19A shows bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of Fluc mRNA-loaded 4A3-SCC-PH LNPs (0.1 mg/kg, n = 2, exposure time: 15 s) under different conditions. FIG. 19B shows Z-average size, FIG. 19C shows Zeta potential, and FIG.19D shows quantification of in vivo luciferase expression in livers from mice in FIG.19A injected with 4A3-SCC-PH LNPs under different conditions. [26] FIG. 20A-20B. FIG.20A shows the body weight of mice treated with 4A3-SCC- 10 and 4A3-SCC-PH LNPs at a dose of 0.3 mg/kg (i.v.) for 15 days (n=3 biologically independent mice), and FIG. 20B shows the H&E staining of tissue sections of heart, liver, spleen, lung, and kidney after 15 days post injection (scale bar = 200 μm). [27] FIG. 21A-21E show liver specific mRNA delivery. FIG. 21A is a schematic illustration of Cre mRNA delivery and Cre-mediated genetic deletion of the stop cassette to activate tdTomato expression in tdTomato transgenic mice. FIG. 21B and FIG. 21C shows 4A3-SCC-PH LNPs mediated tdTomato expression and quantification of tdTomato protein fluorescence intensity in the liver. The tdTomato fluorescence was recorded 2 days after i.v. injection of Cre mRNA-loaded LNPs (0.2 mg/kg, n = 3). FIG. 21D shows flow cytometry analysis of the percentage of tdTomato positive cells in the liver, and FIG.21E shows confocal microscopy images verifying the efficiency of liver-specific gene editing in this model. [28] FIG.22A-22D show in vitro evaluation of six 4A3 LNPs for Fluc mRNA delivery (25 ng/well, n = 4) in normal cells (3T3 and AML-12) and tumor cells (4T1 and B16F10) treated with or without 20 μmol of NEM. [29] FIG.23 shows co-localization analysis (yellow) of endo/lysosomal escape (green) and Cy5-RNA cellular uptake (red) after different cells treated with 4A3-SCC-PH LNPs for 6 h, Scale bars = 10 μm. [30] FIG. 24A-24D show cell viability of normal cells (3T3 and AML-12) and tumor cells (4T1 and B16F10) after the treatment of six 4A3 LNPs with or without 20 μmol NEM. [31] FIG.25A-G. FIG.25A-25B show cancer metastases delineation by 4A3-SCC-PH LNPs mediated bioluminescence imaging. FIG 25A is a schematic showing the establishment of the 4T1 breast cancer metastasis model in BALB/cmice and the B16F10 melanoma metastasis model in C57BL/6 mice. FIG.25B is an illustration of cancer metastasis detection via bioluminescence imaging. FIG.25C shows bioluminescence images, white-light photos of mouse (i), or ex vivo organs (ii) of 4T1 breast cancer metastasis-bearing BALB/c mice and B16F10 melanoma metastasis-bearing C57BL/6 mice 6 h after injection of PBS or Fluc mRNA-loaded 4A3-SCC-PH LNPs (0.1 mg/kg), exposure time: 15 s. H: heart; Li: liver; S: spleen; Lu: lung; K: kidney; In: intestine. FIG 25D shows bioluminescence images and white- light photos of isolated melanoma metastases from intestine, and FIG.25E shows Signal-to- Noise Ratio (SNR) of relative metastasis in different organs in mice generated as described in FIG.25A-25C. FIG.25F-25G shows H&E staining analysis of harvested tissue sections from mice generated as described in FIG.25A-25C. T: tumor, N: normal, scale bar = 250 μm. DETAILED DESCRIPTION [32] Provided herein are biodegradable ionizable cationic lipids for improved mRNA delivery. In some embodiments, the lipids can be used for cancer metastases delineation in vivo. Also provided herein are methods of use of compositions comprising the biodegradable ionizable cationic lipids, and pharmaceutical compositions. Definitions [33] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. [34] 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. [35] The singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. [36] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. In some cases, the term “about” refers to ±10% of a stated number or value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. [37] 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. [38] 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. [39] As used herein, the phrases “at least one”, “one or more”, and “and/or” are open- ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together. [40] As used herein, “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning. [41] As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps. As also used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of” and/or “consisting of” used herein, in any instance or embodiment described. [42] The terms “increased”, “increasing”, “increase”, “improved”, “improvement”, “improving” and the like, are used herein to generally means an increase by a statically significant amount. In some aspects, the terms “increased” or “improved” means an increase or improvement of at least 10% as compared to a reference level, for example an increase or improvement of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any improvement between 10- 100% as compared to a reference level, standard, or control. Other examples of “increase” or “improvement” includes an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level. [43] The terms “decreased”, “decreasing”, “decrease”, “reduced”, “reducing”, “reduce” and the like, are used herein generally to mean a decrease or reduction by a statistically significant amount. In some aspects, “decreased” or “reduced” means a reduction by at least 10% as compared to a reference level, for example a decrease or reduction by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease or reduction (e.g., absent level or non-detectable level as compared to a reference level), or any decrease or reduction between 10-100% as compared to a reference level. [44] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [45] As used herein, the term “lipid nanoparticle”, “LNP,” “lipid nanoparticle composition,” or “LNP composition” refers to a carrier or vehicle, formed by one or more lipid components, for payload (e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide, or oligonucleotide) delivery in the context of pharmaceuticals. Further, as used herein the term “formulation” refers to a specific lipid nanoparticle composition of the disclosure. In other words the terms “formulation,” lipid nanoparticle”, “LNP,” “lipid nanoparticle composition,” or “LNP composition” are used herein interchangeably. Lipid nanoparticles can have one or more lipids with at least one dimension on the order of nanometers (e.g., 1-1000 nm). Generally, lipid nanoparticle compositions for delivery are composed of one or more lipids, such as, but not limited to, a synthetic ionizable or cationic lipid, a phospholipid, a structural lipid (e.g., a sterol), and a polyethylene glycol (PEG) lipid. These compositions may also include other lipids. In various embodiments, the lipid nanoparticle composition comprises five components: (i) an ionizable cationic lipid; (ii) a phospholipid; (iii) a steroid or steroid derivative; and (iv) a polymer-conjugated lipid; and (v) an additional cationic ionizable cationic lipid, a permanently cationic lipid or an anionic lipid (“SORT lipid”) separate from said ionizable cationic lipid. In some embodiments, at least one therapeutic agent (e.g., mRNA) can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation of other undesirable effects induced by the biological mechanism of a target subject, tissue, and/or cell, e.g., an adverse immune response. In some embodiments, lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) that is either organized within inverse lipid micelles and encased within a lipid monolayer envelop or intercalated between adjacent lipid bilayers. In some embodiments, the morphology of lipid nanoparticles is not like a traditional liposome, which are characterized by a lipid bilayer surrounding an aqueous core. In some embodiments, lipid nanoparticles are substantially non-toxic. In some embodiments, the therapeutic agent (e.g., mRNA) is resistant in aqueous solution to degradation by intracellular or intercellular enzymes by virtue of the lipid nanoparticle. [46] The term Selective Organ Targeting (SORT) lipid, as used herein, refers to a component of a lipid nanoparticle (LNP) composition that provides predictable cell-, tissue-, and/or organ-specific targeting of the LNP (for example as described in Cheng et al. Nat. Nanotechnol.15:313-320 (2020); Wang et al. Nat. Protoc.18(1):265-291; and US 11,766,408 and US 11,229,609, the entire contents of each of which is incorporated herein by reference). A selected SORT lipid provides accurate and specific delivery of the cargo from a rationally- designed LNP based, in part, on the biophysical properties of the selected SORT lipid and its prevalence in the LNP. In some cases, specificity is modulated by a LNP’s surface's acid dissociation constant (pKa), which may be affected by the proportion of charged and uncharged ionizable cationic lipids at the LNP surface and may depend on the type of SORT used in the LNP formulation. Without wishing to be bound by theory, the SORT lipid directs tissue specificity of a rationally-design LNP by adjusting surface properties and/or physicochemical characteristics of the LNP. Illustrative SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwitterionic lipids, and ionizable cationic lipids. See, e.g., Table 6 and Table 7. In some embodiments, anionic SORT lipids generally favor delivery to the spleen, at least when administered intravenously; ionizable cationic SORT lipids or ionizable amino SORT lipids generally favor delivery to the liver; permanently cationic SORT lipids generally favor delivery to the lungs; and zwitterionic SORT lipids favor delivery to the spleen. [47] As used herein, the term “ionizable cationic lipid” refers to lipid and lipid-like molecules having at least one pKa in the range of about 4.5-8, such that, without being bound by theory, they may facilitate release of LNP payloads upon uptake into the endosomal compartment of a cell. The ionizable cationic lipid may maintain a neutral charge in pH above the pKa of the lipid; it becomes positively charged in a pH lower than its pKa which facilitates membrane fusion and subsequent cytosolic release of an LNP. Illustrative ionizable cationic lipids have one or more nitrogen atoms having pKa’s in the range of about 4.5-8, such are tertiary amine groups. [48] As used herein, the term “phospholipid” refers to 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. [49] As used herein, the term “sterol” refers to a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring of a gonane ringsystem. “Cholesterol” is an illustrative sterol that has a structure of four fused hydrocarbon rings (gonane ringsystem) with a polar hydroxyl group at one end and an eight-carbon branched aliphatic tail at the other end. The sterol component of an LNP, e.g., cholesterol influences the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers, for example in LNPs. For example, “sterol” can be cholesterol or sitosterol. [50] As used herein, the term “PEG-lipid” refers to a lipid modified with a polyethylene glycol (PEG) unit. In some embodiments, the PEG-lipid comprises dimyristoyl glycerol (DMG), and is referred to as PEG-DMG. In some embodiments, the PEG-lipid comprises 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE). [51] As used herein, the phrase “N/P ratio” refers to a molar ratio of nitrogen in the lipid composition to phosphate in the payload, e.g., a polynucleotide payload. [52] As used herein, the term “apparent pKa” refers to the overall dissociation constant of all titratable groups in the lipid nanoparticles of an LNP. Apparent pKa is an experimentally determined value of molecules or nanoparticles. Apparent pKa can be expressed as the pH at which the number of ionized (protonated) and deionized groups are equal in a system. The surface charge and ionic interaction of assembled nanomaterials in nanoparticles can be estimated according to apparent pKa. The apparent pKa of a nanoparticle can be the result of the average ratio of all the ionized to deionized groups in the nanoparticle. Thus, apparent pKa is not the intrinsic pKa value for any individual molecule. The apparent pKa of nanoparticles can be measured by various techniques. For example, acid-base titration of 2-(p-toluidino)-6- naphthalene sulfonic acid (TNS) fluorescent methods are widely used in determination of apparent pKa of blank nanoparticles. [53] As used herein, the phrase “lipid:RNA ratio” refers to milligram of lipid for each milligram of RNA payload. This ratio influences the encapsulation efficiency of RNA- containing lipid nanoparticles. [54] The term “encapsulation,” as used herein refers to the process of confining a payload within an LNP. For example, “encapsulation” refers to confining an mRNA molecule within an LNP. The term “encapsulation efficiency” refers to the fraction of a payload that is encapsulated within or otherwise coupled with a lipid nanoparticle composition when LNPs are formed. Encapsulation efficiency may be determined by comparing the amount of input payload to the amount of payload in a sample of LNPs, or by comparing the amount of payload in the LNPs to the free excess payload in the sample. For example, a fluorescence detection assay (e.g., RiboGreen™) is used to determine encapsulation efficiency by measuring the free RNA in a sample with intact LNPs compared with the total RNA in a sample treated to disrupt the LNPs. [55] The term “payload” refers to a bioactive molecule or molecules, such as a small molecule, biomolecule, nucleic acid (e.g., DNA, RNA, siRNA, shRNA), protein, polypeptide, or peptide, which is associated with an LNP composition. For example, the payload can be bound covalently or non-covalently to the LNP, encapsulated in the LNP, coupled to the LNP, or complexed with the LNP within the LNP composition. [56] 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), small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), transfer RNA, ribosomal RNA, a ribozyme, an antisense RNA, a guide RNA (gRNA), cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, 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. When the polynucleotides are chemically and/or structurally modified the polynucleotides may be referred to as “modified polynucleotides.” [57] 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, or chemically synthesized. Where appropriate, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, or backbone modifications. An mRNA sequence is presented in the 5′ to 3′ direction unless otherwise indicated. [58] As used herein, the term “shRNA” or “short hairpin RNA” refers to a short sequence of RNA, which can make a tight hairpin turn and can be used to silence gene expression. [59] As used herein, the term “microRNA” refers to noncoding RNA consisting of about 22 ribonucleotides which regulates gene expression in the post transcriptional stage by silencing messenger RNA by base-pairing with a complementary sequence in its targeted mRNA. [60] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and optionally one or more post-translational modifications (e.g., glycosylation) and/or other modifications known in the art. [61] 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. [62] The terms “identity,” “identical,” and “sequence identity” refer to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can readily be calculated by known methods, including, but not limited to, those described in Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), as such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. The term “percent sequence identity”, “percent identity”, or “identical to” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence. For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. Methods of sequence alignment for comparison and determination of percent sequence identity are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443. [63] The term “isolated” when applied to a polynucleotide or polypeptide, denotes that the polynucleotide or polypeptide is essentially free of other cellular components with which it is associated in the natural state or components present during chemical synthesis. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polynucleotide or polypeptide that is the predominant species present in a preparation is substantially purified. [64] The term “variant” refers to a polypeptide or polynucleotide having one or more insertions, deletions, or amino acid substitutions relative to a reference polypeptide or polynucleotide. [65] The terms “subject” refers to a living organism to which any of the compositions as described herein may be administered. The subject may be suffering from or be at risk for a disease or condition that can be treated by administration of pharmaceutical composition as provided herein or by a therapeutic method disclosed herein. Non-limiting examples of subjects include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the subject is a primate, e.g., a human. [66] The term “therapeutically effective amount,” as used herein, refers to an amount of an LNP and/or an LNP comprising a therapeutic agent sufficient to treat a disease, a disorder, or a condition. 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% of symptoms in a subject in need. 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. The “therapeutically effective amount” can vary depending, for example, but not limited to, on the compound, the disease, or the condition and/or symptoms thereof, severity of the disease or the condition and/or symptoms thereof, the age, weight, and/or health of the subject to be treated, and the judgment of the prescribing physician. An appropriate amount in any given instance can be ascertained by those skilled in the art or capable of determination by routine experimentation. [67] The term “administering” refers to providing a composition to a subject in a manner that permits the composition to have its intended effect. Administration may be performed by intramuscular injection, intravenous injection, intraperitoneal injection, inhalation, or any other suitable route. [68] “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 co-administered to the subject. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce degradation of an LNP or the payload of the LNP). [69] As used herein, the term “delivering” means causing, through chemical or biophysical properties of a composition (e.g., an LNP composition) and/or the payload (e.g., a polynucleotide) of an LNP to pass from a site of administration to a subject to a target organ (e.g., the lung, liver, heart, or spleen), target tissue, or target cell. In some cases, “delivering” is equivalent to “administering”, e.g., to a subject in need thereof. As used herein, the term “selectively delivering” refers to the delivery to a target organ, tissue, or cell at a greater rate or in a greater amount than delivered to a reference, non-target organ, tissue, or cell, or that a greater fraction of total the amount of LNP or payload administered to a subject is delivered to a target organ, tissue, or cell by the composition than delivered 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. “Selective delivery” is determined by comparing the fraction of an LNP composition or payload that is delivered to a target organ (e.g., the lung, liver, heart, or spleen) by an LNP composition comprises a selected lipid (e.g., SORT lipid) compared to a reference LNP composition in which the selected lipid is replaced by a control lipid. [70] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes 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 the extent of a disease, stabilizing (i.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. [71] “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 delaying 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 has not yet experienced or displayed any of the pathology or symptomatology of the disease. Prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment. [72] The term “pharmaceutically acceptable excipients” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of a herein-disclosed composition and absorption by a subject of the same. A pharmaceutically acceptable excipients do not cause a significant adverse toxicological effect on the subject. These excipients are usually 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, a sodium chloride (NaCl) solution, 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 pharmaceutically acceptable excipients are useful in the present disclosure. [73] 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. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, PCR, and immunohistochemistry). [74] Chemical moieties referred to as univalent chemical moieties (e.g., alkyl, aryl, etc.) also encompass structurally permissible multivalent moieties, as understood by those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g., CH₃CH₂-), in appropriate circumstances an “alkyl” moiety can also refer to a divalent radical (e.g., -CH2CH2-, which is equivalent to an “alkylene” group). Similarly, under circumstances where a divalent moiety is required, those skilled in the art will understand that the term “aryl” refers to the corresponding divalent arylene group. [75] As used herein, “Alkyl” refers to optionally substituted, straight and branched chain aliphatic groups having from 1 to 30 carbon atoms. For example, “C1, C2, C3, C4, C5 or C6 alkyl”, “C₁-C₆ alkyl”, “alkyl(C≤6)”, or “alkyl(C1-C6)”, is intended to include C₁, C₂, C₃, C₄, C5 or C6 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5 or C6 branched saturated aliphatic hydrocarbon groups. Examples of alkyl include, moieties having from one to six carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n- butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has six or fewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), and in another embodiment, a straight chain or branched alkyl has four or fewer carbon atoms. Analogously, for example “C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃ or C₂₄ alkyl”, “C₁₈-C₂₄ alkyl”, “alkyl(C≤24)”, or “alkyl(C8-C24)” is intended to include C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃ or C₂₄ straight chain (linear) saturated aliphatic hydrocarbon groups and C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃ or C₂₄ branched saturated aliphatic hydrocarbon groups. Examples of “C₁₈-C₂₄ alkyl” include octadecyl, nonadecyl, didecyl, henicosyl, docosyl, tricosyl, tetracosyl, 5-butylpentadecanyl, 4- methyl-5-(pentan-2-yl)hexadecanyl, 7-methylhenicosanyl, 2,15,15-trimethylhenicosanyl, 8,9- dimethyldocosanyl, 6-ethyl-8-methylnonadecanyl, and 6,7-dimethyl-8-propyltridecanyl. [76] “Alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenyl groups. The term “C₂, C₃, C₄, C₅ or C₆ alkenyl,” “C₂-C₆ alkenyl,” “alkenyl(C≤6)”, or “alkenyl(C2-C6)” includes alkenyl groups containing two to six carbon atoms. The term “C8, C9, C10, C11, C12, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃ or C₂₄ alkenyl,” “C₈-C₂₄ alkenyl,” or “alkenyl(C8-C24)” includes alkenyl groups containing eight to twenty-four carbon atoms. Examples of “C₈-C₂₄ alkenyl” include 2,6-dimethylhept-2-enyl, 2,6-dimethylhept-2-enyl, 2,8- dimethylnon-2-enyl, 2,7-dimethyldec-2-ene, 3-ethyl-8-methylundec-3-ene, and 2,9,9- trimethyltridec-2-ene. [77] The term “optionally substituted alkyl” or “optionally substituted alkenyl” refers to an alkyl or alkenyl having designated substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. [78] Any composition or method disclosed herein is applicable to any herein-disclosed composition or method. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein. Lipid nanoparticle composition [79] A lipid nanoparticle composition comprising a therapeutic polypeptide or a polynucleotide encoding a therapeutic polypeptide, a helper lipid, a sterol, and/or a polyethylene glycol-conjugated lipid (PEG-lipid), an ionizable cationic lipid compound (i.e., a compound of the disclosure, e.g., a compound of Formula (I), (Ia), (Ib), (Ic), or a compound of Table 1 or 2), and a selective organ targeting (SORT) lipid. For example, in some embodiments, a lipid nanoparticle comprising a lipid component comprising an ionizable cationic lipid compound of the disclosure, exhibits high stability, showing outstanding mRNA expression in vivo even after storage at 4 ℃ and -20 ℃ for one month. Further, as demonstrated in the Examples herein, in some embodiments, formulating a payload (e.g., an mRNA) in lipid nanoparticle composition comprising a lipid component comprising an ionizable cationic lipid of the disclosure may provide lipid nanoparticles with superior endosomal escape and rapid mRNA release abilities. Ionizable cationic lipids [80] Chemical formulas used to represent ionizable cationic 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. [81] The ionizable cationic 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. In some embodiments, the ionizable cationic lipid exhibits low toxicity in vitro. In some embodiments, the ionizable cationic lipid exhibits low toxicity in vivo. In some embodiments, the ionizable cationic lipid exhibits liver-specific delivery in vivo. [82] In addition, atoms making up the ionizable cationic 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 include ¹³C and ¹⁴C. [83] It should be recognized that the particular anion or cation forming a part of any salt form of an ionizable cationic lipids provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference. [84] In some embodiments, the ionizable cationic 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 cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C24 alkyl or alkenyl groups. [85] Exemplary compounds of the disclosure are summarized in Table 1 and 2 Table 1. Compounds of the disclosure

Table 2. Further compounds of the disclosure

34. 35. 36. 37.

[86] The letter codes in Table 2 represent the structures of the compounds therein by identifying the headgroup and tail structures for each compound. The structures of the headgroups and tails are summarized in Tables 3 and 4. In each of the structures of Table 3 (i.e., the headgroups), denotes a point of attachment to a lipid tail (structure selected from Table 4). Conversely, in each of the structures of Table 4 (i.e., the tails), denotes a point of attachment to the headgroup. Further, it is understood that in each compound of Table 2 that comprises more than one tail, each of the tail structures in the compound are the same. For example, “4A3-SCC-10” means a compound having a headgroup of structure 4A3 and 4 tails of the structure SSC10. [87] In some embodiments, the ionizable cationic lipid is further defined by the formula: Core-Repeating Unit-Terminating Group (D-I) wherein the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atom with the repeating unit and wherein: the core has the formula:

wherein: X1 is amino or alkylamino(C≤12), dialkylamino(C≤12), heterocycloalkyl(C≤12), heteroaryl(C≤12), or a substituted version thereof; R₁ is amino, hydroxy, or mercapto, or alkylamino_(C≤12), dialkylamino_(C≤12), 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: (D-III) wherein: X2 is N(R5)y; R5 is hydrogen, alkyl(C≤18), or substituted alkyl(C≤18); and y is 0, 1, or 2, provided that the sum of y and z is 3; R₂ is amino, hydroxy, or mercapto, or alkylamino_(C≤12), dialkylamino_(C≤12), 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; or the core has the formula:

wherein: X3 is −NR6−, wherein R6 is hydrogen, alkyl(C≤8), or substituted alkyl(C≤8), −O−, or alkylaminodiyl(C≤8), alkoxydiyl(C≤8), arenediyl(C≤8), heteroarenediyl(C≤8), heterocycloalkanediyl(C≤8), or a substituted version of any of these groups; R₃ and R₄ are each independently amino, hydroxy, or mercapto, or alkylamino_(C≤12), dialkylamino_(C≤12), or a substituted version of either of these groups; or a group of the

wherein: e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3; R_c, R_d, and R_f are each independently hydrogen, alkyl_(C≤6), or substituted alkyl_(C≤6); and c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is alkylamine(C≤18), dialkylamine(C≤36), heterocycloalkane(C≤12), or a substituted version of any of these groups; wherein the repeating unit comprises a degradable diacyl and a linker; the degradable diacyl group has the formula:

A₁ and A₂ are each independently −O− , -S-, or −NR_a−, wherein: Ra is hydrogen, alkyl(C≤6), or substituted alkyl(C≤6); Y3 is a group of the formula:

wherein: X₃ and X₄ are alkanediyl_(C≤12), alkenediyl_(C≤12), arenediyl_(C≤12), or a substituted version of any of these groups; and R9 is alkyl(C≤8) or substituted alkyl(C≤8); the linker group has the formula:

wherein: Y1 is alkanediyl(C≤12), alkenediyl(C≤12), arenediyl(C≤12), or a substituted version of any of these groups; and wherein when the repeating unit comprises a linker group, then the linker group comprises an independent degradable diacyl group attached to both the nitrogen and the sulfur atoms of the linker group if n is greater than 1, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group, the next repeating unit comprises two degradable diacyl groups attached to the nitrogen atom of the linker group; and wherein n is the number of linker groups present in the repeating unit; and the terminating group has the formula:

wherein: R10 is alkyl(C≤6), substituted alkyl(C≤6); alkenyl(C≤6), or substituted alkenyl(C≤6); 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. [88] For example, the structure of compound 72 in Table 2 (4A3-SCC-10) is as follows:

. Table 3. Headgroups of ionizable cationic lipid compounds of the disclosure

Table 4. Tails of ionizable cationic lipid compounds of the disclosure

[89] In some embodiments, an ionizable cationic lipid compound of the disclosure is selected from Table 5. Table 5. Ionizable lipid compounds of the disclosure

[90] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in the composition at a molar percentage about 5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. [91] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%. [92] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30%. In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30%. Selective organ targeting (SORT) lipids [93] 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. [94] In 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 additional lipid (e.g., additional lipid (e.g., SORT lipid)) comprises, or is otherwise complexed to or interacting with, a counterion. In some embodiments, the additional lipid is a SORT lipid. [95] 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 X₁C(O)R₁ or X₂N⁺R₃R₄R₅; provided at least one of Y1, Y2, and Y3 is X2N⁺R3R4R5; R₁ is C₁-C₂₄ alkyl, C₁-C₂₄ substituted alkyl, C₁-C₂₄ alkenyl, C₁-C₂₄ substituted alkenyl; X₁ is O or NR_a, wherein R_a is hydrogen, C₁-C₄ alkyl, or C₁-C₄ substituted alkyl; X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl; R₃, R₄, and R₅ are each independently C₁-C₂₄ alkyl, C₁-C₂₄ substituted alkyl, C₁-C₂₄ alkenyl, C1-C24 substituted alkenyl; and A₁ is an anion with a charge equal to the number of X₂N⁺R₃R₄R₅ groups in the compound. [96] In some embodiments, the permanently cationic additional lipid (e.g., SORT lipid) has a structural formula: (S-II), wherein: R6-R9 are each independently C1-C24 alkyl, C1-C24 substituted alkyl, C1-C24 alkenyl, C₁-C₂₄ substituted alkenyl; provided at least one of R₆-R₉ is a group of C₈-C₂₄; and A2 is a monovalent anion. [97] In some embodiments, the permanently cationic lipid is 1,2-dilauroyl-sn-glycero-3- ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn- glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0 EPC), 1,2- dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), Dimethyldioctadecylammonium (18:0 DDAB), 1,2-dimyristoyl-3-trimethylammonium- propane(14:0 TAP), 1,2-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, DOTAP), or 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). [98] In some embodiments of the lipid compositions, the SORT (additional) lipid is an ionizable cationic lipid (e.g., 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 cationic lipid is a tertiary ammine group. In some embodiments of the lipid compositions disclosed herein, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) has a structural _formula:

_wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; and R3 and R3′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl. [99] In some embodiments of formula (S-I’a) R₁ and R₂ are each independently C₈-C₂₄ alkenyl (e.g., hexadecane, heptadecene, or octadecene). In some embodiments of formula (S- I’a), R₃ and R₃′ are each independently C₁-C₆ alkyl (e.g., methyl or ethyl). In some embodiments of formula (S-I’a) R1 and R2 are each independently C8-C24 alkenyl, (e.g., hexadecane, heptadecene, or octadecene) and R₃ and R₃′ are each independently C₁-C₆ alkyl (e.g., methyl or ethyl). [100] In some embodiments, the ionizable cationic lipid is 1,2-distearoyl-3- dimethylammonium-propane (18:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dioleoyl-3- dimethylammonium-propane (18:1 DAP, DODAP), or 1,2-dioleyloxy-3- dimethylaminopropane (DODMA). [101] In some embodiments of the lipid compositions, the additional ionizable cationic lipid or permanently cationic lipid comprises a head group of a particular structure. In some embodiments, the additional lipid (e.g., additional 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 biodegradable linker. 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. [102] In some embodiments of the lipid compositions, the SORT (additional ionizable cationic lipid or permanently cationic) lipid has a structural formula:

, wherein R¹ and R² are each independently an optionally substituted C₆-C₂₄ alkyl, or an optionally substituted C6-C24 alkenyl. [103] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) has a structural formula:

. [104] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) comprises a Linker (L). In some embodiments, L is , wherein: p and q are each independently 1, 2, or 3; and R⁴ is an optionally substituted C1-C6 alkyl [105] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) has a structural formula:

wherein: R₁ and R₂ are each independently C₈-C₂₄ alkyl, C₈-C₂₄ alkenyl, or a substituted version of either group; R₃, R₃′, and R₃′′ are each independently C₁-C₆ alkyl or substituted C₁-C₆ alkyl; R4 is C1-C6 alkyl or substituted C1-C6 alkyl; and X⁻ is a monovalent anion. [106] In some embodiments of the lipid compositions, the additional lipid (e.g., SORT lipid) is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is further defined as:

wherein: R₁ and R₂ are each independently C₈-C₂₄ alkyl, C₈-C₂₄ alkenyl, or a substituted version of either group; R₃, R₃′, and R₃′′ are each independently C₁-C₆ alkyl or substituted C₁-C₆ alkyl; and X⁻ is a monovalent anion. [107] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) is a phosphocholine lipid. In some embodiments, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) is an ethylphosphocholine. The ethylphosphocholine may be, by way of example, without being limited to, 1,2-dimyristoleoyl-sn-glycero-3- ethylphosphocholine (14:1 EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-dipalmitoyl-sn-glycero-3- ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), 1-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine (16:0-18:0 EPC). [108] In some embodiments of the lipid compositions, the lipid has a structural formula:

wherein: R1 and R2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R3, R3′, and R3′′ are each independently C1-C6 alkyl or substituted C1-C6 alkyl; X⁻ is a monovalent anion. [109] By way of example, and without being limited thereto, a additional lipid (e.g., additional lipid (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt). [110] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) has a structural formula:

(S-II’), 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(C1-C8), alkenyl(C2-C8), or a substituted version of either group; and X₂ is a monovalent anion. [111] By way of example, and without being limited thereto, an additional lipid (e.g., additional lipid (e.g., SORT lipid)) of the structural formula of the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB). [112] In some embodiments of the lipid compositions, the additional lipid (e.g., additional lipid (e.g., SORT lipid)) is

1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA). [113] In some embodiments of the lipid compositions, the additional lipid is selected from the lipids set forth in Table 6. Table 6. Example additional lipid (e.g., SORT lipids)

X- is a counterion (e.g., Cl-, Br-, etc.) Table 7. Example additional lipid (e.g., SORT lipids)

[114] In some embodiments of the lipid composition of the present application, the SORT lipid is present in the composition at a molar percentage from about 5% to about 50%. [115] In some embodiments of the lipid composition of the present application, the SORT lipid is present in the composition at a molar percentage about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. [116] In some embodiments of the lipid composition of the present application, the SORT lipid is present in the composition at a molar percentage from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 20% to about 25%. [117] In some embodiments of the lipid composition of the present application, the SORT lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, or at least (about) 55%. In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present at a molar percentage of at most (about) 60%, at most (about) 55%, at most (about) 50%, at most (about) 45%, at most (about) 40%, at most (about) 35%, at most (about) 30%, or at most (about) 25%. Helper lipids [118] In some embodiments, lipid compositions described herein comprise a helper lipid. In some embodiments, the helper lipid is a phospholipid. Phospholipids, as defined herein, are any lipid 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. [119] 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 cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). [120] Phospholipids useful or potentially useful in the compositions and methods described herein may comprise a: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, or a derivative or analog thereof. [121] Phospholipids useful or potentially useful in the compositions and methods described herein may be selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-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-(1'-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-(1-glycerol) sodium salt (DOPG), and sphingomyelin. [122] As described herein, phosphatidylcholine and phosphocholine may be used interchangeably. In some embodiments, the phosphatidylcholine is selected from: 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC). [123] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-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-(1'-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-(1-glycerol) sodium salt (DOPG), and sphingomyelin. [124] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC). [125] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE). [126] In some embodiments, the lipid composition comprises 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC). In some embodiments, the lipid composition comprises 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the lipid composition comprises 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). In some embodiments, the lipid composition comprises 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC). In some embodiments, the lipid composition comprises 1,2-Dimyristoyl-sn-glycero- 3-phosphoethanolamine (DMPE). In some embodiments, the lipid composition comprises 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE). [127] In some embodiments, the lipid composition comprises a phospholipid selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-distearoyl-sn- glycero-3-phosphorylethanolamine (DSPE). [128] In some embodiments, the lipid composition comprises a phospholipid selected from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2- distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE). [129] In some embodiments, the phospholipid may contain one or two long chain (e.g., C6-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. [130] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage from about 7.5% to about 30%. [131] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage about 5%, about 7.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. [132] In some embodiments of the lipid composition of the present application, the helper lipid is present in the composition at a molar percentage from about 5% to about 25%, from about 5% to about 50%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 12% to about 30%, from about 12% to about 25%, from about 12% to about 20%, from about 14% to about 30%, from about 14% to about 25%, from about 14% to about 20%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 15% to about 20%, from about 16% to about 30%, from about 16% to about 20%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, or from about 10% to about 25%. [133] In some embodiments of the lipid composition of the present application, the helper lipid is present at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30%. In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30%. [134] In some embodiments, the helper lipid is present in an amount of about 10 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 11 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 12 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 13 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 14 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 15 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 16 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 17 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 18 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 19 mol % of the total lipids in the lipid component. In some embodiments, the helper lipid is present in an amount of about 20 mol % of the total lipids in the lipid component. Structural lipids [135] The lipid nanoparticle may include one or more structural lipids. Structural lipids can be steroids or steroid derivatives. 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 non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol wherein the formula is further defined as:

some embodiments of the present application, the steroid or steroid derivative is a cholestane or cholestane derivative. In a cholestane, the ring structure is further defined by the formula:

. As described above, a cholestane derivative comprises 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 cholesterol and a sterol or a derivative thereof. [136] 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. [137] 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%. [138] 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%, about 41%, about 42%, about 43%, about 44%, about 45%, about 50%, about 55%, or about 60%. [139] In 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 about 20% to about 25%, from about 25% to about 50%, from about 25% to about 40%, from about 25% to about 30%, from about 30% to about 50%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 50%, from about 35% to about 45%, from about 35% to about 40%, from about 40% to about 50%, from about 40% to about 45%, or from about 45% to about 50%. [140] 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 ionizable cationic lipid is present at a molar percentage of 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%. Polyethylene glycol-conjugated lipid (PEG-lipid) [141] 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. WO2012099755 and U.S. Pat. Pub No.2014/0200257. [142] 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. [143] 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 comprises 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. [144] 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-C₂₄ long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain. Some non-limiting examples of a PEG-lipid comprises 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. [145] In some embodiments, the PEG modification is measured by the molecular 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, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, up to about 25,000. 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. [146] In some embodiments, the PEG lipid is present in an amount of from about 0.5 mol % to about 5 mol % of the total lipids in the lipid component. In some embodiments, the PEG lipid is present in an amount of from about 1.0 mol % to about 2 mol % of the total lipids in the lipid component. In some embodiments, the PEG lipid is present in an amount of from about 2 mol % to about 3 mol % of the total lipids in the lipid component. In some embodiments, the PEG lipid is present in an amount of from about 2 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the PEG lipid is present in an amount of from about 3 mol % to about 4 mol % of the total lipids in the lipid component. In some embodiments, the PEG lipid is present in an amount of from about 4 mol % to about 5 mol % of the total lipids in the lipid component. [147] In some embodiments, the PEG lipid comprises a phosphoglyceride PEG lipid. In some embodiments, the PEG lipid comprises a diglyceride PEG lipid. In some embodiments, the PEG lipid comprises a PEG-ceramide. [148] In some embodiments, the PEG lipid is a PEG-ceramide. In some embodiments the PEG-ceramide is a C8 PEG-ceramide. In some embodiments the PEG-ceramide is a C12 PEG- ceramide. In some embodiments the PEG-ceramide is a C14 PEG-ceramide. In some embodiments the PEG-ceramide is a C16 PEG-ceramide. In some embodiments the PEG- ceramide is a C18 PEG-ceramide. [149] In some embodiments, the PEG-ceramide comprises N-octanoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000 ceramide), N-octanoyl- sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000 ceramide), N- octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750 ceramide), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000]} (C16 PEG5000 ceramide), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG200 ceramide), or N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750 ceramide), [150] In some embodiments, the PEG-ceramide is selected from the group consisting of N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)5000]} (C8 PEG5000 ceramide), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000 ceramide), N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (C8 PEG750 ceramide), N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)5000]} (C16 PEG5000 ceramide), N-palmitoyl- sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C16 PEG200 ceramide), or N- palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (C16 PEG750 ceramide). [151] In some embodiments, the PEG-ceramide is C8-PEG-750-ceramide. In some embodiments, the PEG-ceramide is C8-PEG-2000-ceramide. [152] In some embodiments, the lipid composition comprises more than one PEG lipid. In some embodiments, the lipid composition comprises a first PEG lipid and a second PEG lipid. In some embodiments, the PEG lipid comprises a first PEG ceramide and a second PEG ceramide. In some embodiments, the PEG lipid comprises a first PEG ceramide and a second PEG ceramide, wherein the first PEG ceramide and the second PEG ceramide are not the same. In some embodiments, the first PEG ceramide and the second PEG ceramide are each independently selected from: a PEG750-ceramide, a PEG2000-ceramide, and a PEG5000- ceramide. In some embodiments, the first PEG ceramide and the second PEG ceramide are each independently a C8 PEG-ceramide (i.e., the lipid composition comprises a dual C8- Ceramide). In some embodiments, the first PEG lipid and the second PEG lipid are in a ratio of about: 1:1, 1:2, 1:3, 2:1, 2:3, 1:4, 1:5, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, or 5:1. [153] In some embodiments, the first PEG lipid or the second PEG lipid is a DMG PEG lipid. In some embodiments, the first PEG lipid or the second PEG lipid is a PEG-ceramide lipid. In some embodiments, the first PEG lipid or the second PEG lipid is a diglyceride PEG lipid. In some embodiments, the first PEG lipid or the second PEG lipid is a polyglyceride PEG lipid. [154] In some embodiments the first PEG lipid and the second PEG lipid are both DMG PEG lipids. In some embodiments the first PEG lipid and the second PEG lipid are both PEG- ceramide lipids. In some embodiments the first PEG lipid and the second PEG lipid are both diglyceride PEG lipids. In some embodiments the first PEG lipid and the second PEG lipid are both polyglyceride PEG lipids. [155] [156] In some embodiments of the lipid composition of the present application, the PEG- lipid has a structural formula:

, wherein: R₁₂ and R₁₃ are each independently alkyl(C≤24), alkenyl(C≤24), or a substituted version of either of these groups; Re is hydrogen, alkyl_(C≤8), or substituted alkyl_(C≤8); and x is 1-250. In some embodiments, R_e is alkyl(C≤8) such as methyl. R12 and R13 are each independently alkyl(C≤4-20). In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol. [157] In some embodiments of the lipid composition of the present application, the PEG- lipid has a structural formula:

, wherein: n1 is an integer between 1 and 100 and n2 and n3 are each independently selected from an integer between 1 and 29. In some embodiments, n1 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, n1 is from about 30 to about 50. In some embodiments, n₂ is from 5 to 23. In some embodiments, n₂ is 11 to about 17. In some embodiments, n3 is from 5 to 23. In some embodiments, n3 is 11 to about 17. [158] In some embodiments of the lipid composition of the present application, the PEG- lipid is present in the composition at a molar percentage from about 0.5% to about 10%. [159] In some embodiments of the lipid composition of the present application, the PEG- lipid is present in the composition at a molar percentage about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. [160] In some embodiments of the lipid composition of the present application, 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 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1% to about 5%, from about 1% to about 4.5%, from about 1% to about 4%, from about 1% to about 3.5%, from about 1% to about 3%, from about 1% to about 2%, from about 2% to about 5%, from about 2% to about 4.5%, from about 2% to about 4%, from about 2% to about 3.5%, from about 2% to about 3%, from about 3% to about 5%, from about 3% to about 4.5%, from about 3% to about 4%, from about 3% to about 3.5%, from about 4% to about 5%, or from about 4% to about 4.5%. [161] In some embodiments of the lipid composition of the present application, the PEG- lipid is present at a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, or at least (about) 3.5%. In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present at a molar percentage of at most (about) 10%, at most (about) 9%, at most (about) 8%, at most (about) 7%, at most (about) 6%, or at most (about) 5%. Payloads [162] The present disclosure contemplates delivery of various payloads useful in the treatment of a liver disease. Payloads comprise therapeutic polypeptides or polynucleotides encoding polypeptides. For example, the payload may be a polynucleotide encoding a gene related to liver disease, or a polynucleotide encoding a gene editor for editing a gene related to liver disease. [163] In some embodiments, lipid nanoparticle compositions described herein further comprise a payload. In some embodiments, the payload comprises a polypeptide or a protein. In some embodiments, the payload comprises a small interfering RNA (siRNA). In some embodiments, the payload comprises an mRNA. In some embodiments, the mRNA encodes a gene editing system of component thereof. In some embodiments the gene editing system of component thereof comprises a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), and a guide RNA.Polypeptides. [164] In some embodiments, the disclosure provides polypeptides comprising one or more therapeutic proteins. Therapeutic proteins comprise, but are not limited to cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anti-coagulants, blood factors, bone morphogenic proteins, immunoglobulins, or enzymes. Some non-limiting examples of particular therapeutic proteins include Erythropoietin (EPO), Granulocyte colony- stimulating factor (G-CSF), Alpha-galactosidase A, Alpha-L-iduronidase, Thyrotropin a, N- acetylgalactosamine-4-sulfatase (rhASB), Dornase alfa, Tissue plasminogen activator (TP A) Activase, Glucocerebrosidase, Interferon (IF) b-la, Interferon b-lb, Interferon gamma, Interferon alpha, TNF-alpha, IL-1 through IL-36, Human growth hormone (rHGH), Human insulin (BHI), Human chorionic gonadotropin a, Darbepoetin a, Follicle-stimulating hormone (FSH), and Factor VIII. In some embodiments, the cytokine is a proinflammatory cytokine. In some embodiments, the cytokine is IL-1β, MIP-1α, MCP-1, TNF-α, G-CSG, or a related cytokine thereof. [165] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject. For example, the polynucleotide encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein, Dynein axonemal heavy chain 5, Dynein axonemal heavy chain 11, Bone morphogenetic protein receptor type 2, Fumarylacetoacetate hydrolase, Phenylalanine hydroxylase, Alpha-L-iduronidase, Collagen type IV alpha 3 chain, Collagen type IV alpha 4 chain, Collagen type IV alpha 5 chain, Poly cystin 1, Polycystin 2, Fibrocystin (or polyductin), Solute carrier family 3 member 1, Solute carrier family 7 member 9, Paired box gene 9, Myosin VIIA, Cadherin related 23, Usherin, Clarin 1, Gap junction beta-2 protein, Gap junction beta-6 protein, Rhodopsin, dystrophia myotonica protein kinase , Dystrophin, Sodium voltage-gated channel alpha subunit 1, Sodium voltage-gated channel beta subunit 1, Coagulation factor VIII, Coagulation factor IX ,N- glycanase 1, Palmitoyl-protein thioesterase 1, Tripeptidyl peptidase l,Kvl 1.1 (alpha subunit of potassium ion channel), Palmitoyl-protein thioesterase 1, ATM serine/threonine kinase, or Fibrillin 1. Polynucleotides [166] In some embodiments, the lipid composition described herein comprises one or more polynucleotides. In some embodiments, the polynucleotides encode for one or more polypeptides described herein. [167] Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof. [168] In addition, it should be clear that the present disclosure is not limited to the specific polynucleotides disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of polynucleotides, however, as one of ordinary skill in the art could readily identify related homologs in various other sources of the polynucleotides including polynucleotides from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species). It is contemplated that the polynucleotides used in the present disclosure can comprise a sequence based upon a naturally-occurring sequence. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the naturally-occurring sequence. In some embodiments, the polynucleotide is a complementary sequence to a naturally occurring sequence, or complementary to at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein. [169] In some embodiments, the polynucleotide used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In some embodiments, the polynucleotide comprises complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as “mini–genes”. The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy. [170] In some embodiments, the polynucleotide comprises one or more segments comprising a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a long non- coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, deoxyribonucleic acid (DNA), a double stranded deoxyribonucleic acid (dsDNA), a single stranded deoxyribonucleic acid (ssDNA), a single stranded ribonucleic acid (ssRNA), a or double stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic agents (or prophylactic agents) described herein. [171] In some embodiments, the polynucleotide is greater than 30 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, greater than 200 nucleotides, greater than 300 nucleotides, greater than 400 nucleotides, greater than 500 nucleotides, greater than 600 nucleotides, greater than 700 nucleotides, greater than 800 nucleotides, greater than 900 nucleotides, greater than 1000 nucleotides, greater than 1500 nucleotides, 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. [172] In some embodiments, the mRNA is about 50 nucleotides in length. In some embodiments, the mRNA molecule is about 100 nucleotides in length. In some embodiments, the mRNA molecule is about 200 nucleotides in length. In some embodiments, the mRNA molecule is about 300 nucleotides in length. In some embodiments, the mRNA molecule is about 400 nucleotides in length. In some embodiments, the mRNA molecule is about 500 nucleotides in length. In some embodiments, the mRNA molecule is about 600 nucleotides in length. In some embodiments, the mRNA molecule is about 700 nucleotides in length. In some embodiments, the mRNA molecule is about 800 nucleotides in length. In some embodiments, the mRNA molecule is about 900 nucleotides in length. In some embodiments, the mRNA molecule is about 1000 nucleotides in length. In some embodiments, the mRNA molecule is about 2000 nucleotides in length. In some embodiments, the mRNA molecule is about 3000 nucleotides in length. In some embodiments, the mRNA molecule is about 4000 nucleotides in length. In some embodiments, the mRNA molecule is about 5000 nucleotides in length. [173] In some embodiments, the polynucleotide comprises about 50 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 50 to about 100 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 500 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 300 nucleotides. In some embodiments, the polynucleotide comprises about 100 to about 200 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 500 to about 1000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 100000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 5000 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2500 nucleotides. In some embodiments, the polynucleotide comprises about 1000 to about 2000 nucleotides. [174] In some embodiments, the LNP composition comprises mRNA at a lipid:mRNA (weight/weight) ratio is between 5:1 and 40:1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio between 10:1 and 40:1, between 15:1 and 40:1, between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, between 25:1 and 36:1, between 5:1 and 45:1, between 20:1 and 40:1, between 25:1 and 40:1, between 35:1 and 40:1, or between 30:1 and 40:1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio of 30:1. In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio of 40:1. [175] In some embodiments, the mRNA encodes a gene or a portion of a gene related to liver disease shown in Table 8. Exemplary sequences of genes related to liver diseases are shown in Table 9. [176] It is understood that T is T in DNA and T is U in RNA polynucleotide sequences. Table 8. Exemplary genes related to liver diseases

Table 9. Exemplary sequences of genes related to liver diseases

[177] In some embodiments, the mRNA encoding ASL comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding AAT comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the mRNA encoding SLC25A13 comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 3. In some embodiments, the mRNA encoding SERPINA1 comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding ALDOB comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 5. In some embodiments, the mRNA encoding CFTR comprises a polynucleotide sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 6. [178] In some embodiments, the mRNA encodes a protein selected from the group consisting of ALDOB, GBE1, FAH, ATP7B, ASL, SLC25A13, LIPA, SERPONA1, CFTR, HFE. [179] In some embodiments, the mRNA encodes a protein selected from the group consisting of alpha-1-antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- -phosphatase (GPase), glucose -phosphate translocase, lysosomal glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose-1-phosphate uridylyltransferase, galactokinase, UDP-galactose 4- epimerase, transthyretin, a complement regulatory protein, phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N- acetylgalactosamine--sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β- hexosaminidase B, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α- glucosaminide N-acetyltransferase, N-acetylglucosamine--sulfate sulfatase, alpha-N- acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A. In some embodiments, the polynucleotide is a DNA, such as, for example, a DNA encoding a functional protein associated with a protein deficiency disease (e.g., a protein selected from the proteins listed above). [180] Polynucleotide sequences can be optimized for expression in various cells and tissues by adjusting codon usage. Codon usage optimization is known in the art, for example at world wide web owpgenomes.urv.es/OPTIMIZER/. In some embodiments, the codon usage of the polynucleotide is optimized for expression in a cell, for example a human cell. Modified polynucleotides [181] In some embodiments, the polynucleotide comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio-l-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1- deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine, and combinations thereof. [182] In some embodiments, a polynucleotide of the disclosure comprises a modified pyrimidine, such as a modified uridine. In some cases, a uridine analogue is selected from pseudouridine (Ψ), 1-methylpseudouridine (m^lP), 2-thiouridine (s²U), 5-methyluridine (m⁵U), 5-methoxyuridine (mo⁵U), 4-thiouridine (s⁴U), 5-bromouridine (Br⁵U), 2'O-methyluridine (U2'm), 2'-amino-2'-deoxyuridine (U2'NH₂), 2'-azido-2'-deoxyuridine (U2'N₃), and 2'-fluoro- 2'-deoxyuridine (U2'F). Modification in untranslated regions [183] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, or a polyribonucleotide of the disclosure can comprise one or more untranslated regions. An untranslated region can comprise any number of modified or unmodified nucleotides. Untranslated regions (UTRs) of a gene are transcribed but not translated into a polypeptide. In some cases, an untranslated sequence can increase the stability of the polynucleotide and the efficiency of translation. The regulatory features of a UTR can be incorporated into the modified mRNA molecules of the present disclosure, for instance, to increase the stability of the molecule. The specific features can also be incorporated to ensure controlled down- regulation of the transcript in case they are misdirected to undesired organ sites. Some 5' UTRs play roles in translation initiation. A 5' UTR can comprise a Kozak sequence which is involved in the process by which the ribosome initiates translation of many genes. Kozak sequences can have the consensus GCC(R)CCAUGG, where R is a purine (adenine or guanine) that is located three bases upstream of the start codon (AUG).5 ' UTRs may form secondary structures which are involved in binding of translation elongation factor. In some cases, one can increase the stability and protein production of the polynucleotide molecule of the disclosure, by engineering the features typically found in abundantly expressed genes of specific target organs. For example, introduction of 5 'UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can be used to increase expression of a polynucleotide in a liver. Likewise, use of 5' UTR from muscle proteins (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie- 1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D) can be used to increase expression of a polynucleotide in a desired cell or tissue. [184] Other non-UTR sequences can be incorporated into the 5' (or 3' UTR) UTRs of the polynucleotides of the present disclosure. The 5' and/or 3' UTRs can provide stability and/or translation efficiency of polynucleotides. For example, introns or portions of intron sequences can be incorporated into the flanking regions of a polynucleotide. Incorporation of intronic sequences can also increase the rate of translation of the polynucleotide. [185] In some embodiments, 3' UTRs may have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into classes: Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and T F-α. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif c-Jun and Myogenin are two well-studied examples of this class. Proteins binding to the AREs may destabilize the messengerRNA (mRNA), whereas members of the ELAV family, such as HuR, may increase the stability of mRNA. HuR may bind to AREs of all the three classes. Engineering the HuR specific binding sites into the 3 ' UTR of polynucleotide molecules can lead to HuR binding and thus, stabilization of the message in vivo. Engineering of 3' UTR AU rich elements (AREs) can be used to modulate the stability of a polynucleotide. One or more copies of an ARE can be engineered into a polynucleotide to modulate the stability of a polynucleotide. AREs can be identified, removed, or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using polynucleotides and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE- engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection. [186] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, a polyribonucleotide, or compositions of the disclosure can comprise an engineered 5' cap structure, or a 5 '-cap can be added to a polynucleotide intracellularly. The 5 'cap structure of an mRNA can be involved in binding to the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature pseudo-circular mRNA species. The 5 'cap structure can also be involved in nuclear export, increases in mRNA stability, and in assisting the removal of 5' proximal introns during mRNA splicing. [187] In some embodiments, a polynucleotide such as a nucleic acid construct, a vector, or a polynucleotide can be 5 '-end capped generating a 5 '-GpppN-3 ' -triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. The cap-structure can comprise a modified or unmodified 7- methylguanosine linked to the first nucleotide via a 5 '-5 ' triphosphate bridge. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue (Cap-0 structure). The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5'end of the mRNA may optionally also be 2'-O-methylated (Cap-1 structure). 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a polynucleotide molecule, such as an mRNA molecule, for degradation. In some cases, a cap can comprise further modifications, including the methylation of the 2' hydroxy-groups of the first 2 ribose sugars of the 5' end of the mRNA. For instance, a eukaryotic cap-1 has a methylated 2'-hydroxy group on the first ribose sugar, while a cap-2 has methylated 2 '-hydroxy groups on the first two ribose sugars. The 5' cap can be chemically similar to the 3 ' end of an RNA molecule (the 5 ' carbon of the cap ribose is bonded, and the free 3'-hydroxyls on both 5'- and 3 '- ends of the capped transcripts. Such double modification can provide significant resistance to 5' exonucleases. Non-limiting examples of 5 ' cap structures that can be used with a polynucleotide include, but are not limited to, m⁷G(5')ppp(5')N (Cap-0), m⁷G(5')ppp(5')N1mpNp (Cap-1), and m⁷G(5')- ppp(5 ')N1mpN2mp (Cap-2). [188] Modifications to the modified mRNA of the present disclosure may generate a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life while facilitating efficient translation. Because cap structure hydrolysis requires cleavage of 5'-ppp- 5' triphosphate linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with guanosine a-thiophosphate nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides. Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the mRNA on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a polynucleotide. [189] The modified mRNA may be capped post-transcriptionally. According to the present disclosure, 5' terminal caps may include endogenous caps or cap analogues. According to the present disclosure, a 5' terminal cap may comprise a guanine analogue. Useful guanine analogues include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido- guanosine. [190] In some embodiments, an untranslated region can comprise any number of nucleotides. An untranslated region can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length. An untranslated region can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length. [191] In some embodiments, a polynucleotide of the disclosure can comprise a polyA sequence. A polyA sequence (e.g., polyA tail) can comprise any number of nucleotides. A polyA sequence can comprise a length of about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length. In some examples, a polyA sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length. A polyA sequence can comprise a length of for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length. A polyA sequence can comprise a length of at most 100 bases or base pairs, 90 bases or base pairs, 80 bases or base pairs, 70 bases or base pairs, 60 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 30 bases or base pairs, 20 bases or base pairs, 10 bases or base pairs, or 5 bases or base pairs. Gene Editing Payload [192] The LNPs of the present disclosure can comprise one or more components for gene editing, such as, but not limited to, a guide RNA, a tracrRNA, a sgRNA, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease (e.g., Cas9), a DNA template for gene editing, or a combination thereof. In some embodiments, the payload of the LNPs can be suitable for a genome editing technique. In some embodiments, the genome editing technique can be CRISPR or TALEN. In some embodiments, the LNPs can comprise one or more mRNAs, which can encode a gene editing or base editing protein. In some embodiments, the LNPs can comprises both a gene- or base- editing protein encoding mRNA and one or more guide RNAs. In some embodiments, the LNPs can comprise at least one nucleic acid suitable for a genome editing technique, such as a CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA), a guide RNA (gRNA), and a DNA repair template. In some embodiments, CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it can be a nickase instead of making double-strand cuts or so that it can bind the sequence specified by the guide RNA but has no enzymatic activity. In some embodiments, the base editing protein can be a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain, such as an inactive CRISPR nuclease. Gene Editing Methods [193] The presently described LNPs or pharmaceutical composition can comprise a payload of any conventional gene editing methods. In some embodiments, gene editing components can be selectively delivered to the cells of target organ. In some embodiments, the target organ can be liver. In some embodiments, the cells of target organ can be liver cells. In some embodiments, the cells can be ciliated cells, goblet cells, secretory cells, club cells, basal cells or ionocytes. [194] In some embodiments, the gene editing can be targeted editing. Targeted editing can be achieved either through a nuclease-independent approach or through a nuclease- dependent approach. [195] The nuclease-independent targeted editing, such as base-editing and/or prime editing, can involve precise modifications to DNA sequences without creating double-strand breaks. Homologous recombination can be guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the cells of target organ. [196] Base editing can allow for the conversion of one DNA base pair into another at a specific target site. In some embodiments, the nuclease can be a fusion of a deaminase enzyme to a modified Cas9 protein (dCas9) or other engineered Cas variants. In some embodiments, base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in the endogenous DNA. A guide RNA can be designed to target the specific genomic location of interest in the cells of target organ. [197] Prime editing can allow for more complex and precise DNA modifications, including insertions, deletions, and all 12 possible base-to-base conversions (A, C, G, T) without double-strand breaks. A prime editing guide RNA, which can consist of a guide sequence and a template for the desired edit, can be designed. The prime editor protein (PE2), which can combine a reverse transcriptase and a Cas9 variant, can be guided to the target site by the prime editing guide RNA. The Cas9 variant can generate a single-strand break (nick) in the DNA. The reverse transcriptase then can use the prime editing guide RNA’s template sequence to copy the desired changes into the nicked strand of DNA. Subsequently, the cellular repair machinery of the cells of target organ can repair the nick, incorporating the edited sequence, via homology-directed repair (HDR). [198] The nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare- cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing can also utilize DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which can occur in response to DSBs. In some embodiments, DNA repair by NHEJ can lead to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which can result in targeted integration of the exogenous genetic material. In some embodiments, a nuclease of the nuclease-dependent targeted editing can include, but not limited to, CRISPR- Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo, and/or TALEN. [199] Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are well-known techniques. See for example, Bauer et al., J Vis Exp. 95:e52118 (2015). Available endonucleases capable of introducing specific and targeted DSBs can include, but not limited to, ZFN, TALEN, and CRISPR/Cas9. [200] In some embodiments, targeted gene editing can be achieved via dual integrase cassette exchange (DICE) system utilizing phiC31 and Bxb1 integrases. CRISPR-Cas9 Gene Editing System [201] The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing. It can rely on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (crRNA) and transactivating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA can be used by the prokaryote to detect and destroy similar foreign DNA upon re-introduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus can result in the formation of an RNA molecule comprising the spacer sequence, which can associate with and target Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described in e.g., Koonin et al., Curr Opin Microbiol 37:67-78 (2017). [202] crRNA can drive sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with about 20 nucleotide sequence in the target DNA. Changing the sequence of the 5’ 20 nucleotides in the crRNA can allow targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex can only bind DNA sequences that contain a sequence match to the first 20 nucleotides of the crRNA, if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM). [203] tracrRNA can hybridize with the 3’ end of crRNA to form an RNA-duplex structure that can be bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA. [204] Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end). [205] After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, cells can use two main DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is a repair mechanism that is highly active in the majority of cell types, including non-dividing cells. NHEJ can be error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications can typically be less than 20 nucleotides. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR can use a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells and can occur at a relatively low frequency in most cell types. [206] CRISPR Endonucleases [207] In some embodiments, Cas9 endonuclease can be used in a CRISPR method for genetically engineering cells of the target organ of the LNPs described herein. In some embodiments, Cas9 enzyme can be from Streptococcus pyogenes, although other Cas9 homologs can also be used. In some embodiments, the Cas9 enzyme can be wild-type Cas9. In some embodiments, the Cas9 enzyme can be a modified version of Cas9 (e.g., evolved versions of Cas9, or Cas9 orthologues or variants). In some embodiments, Cas9 can be substituted with another RNA-guided endonuclease, such as Cpf1 (class II CRISPR/Cas system). [208] In some embodiments, the CRISPR/Cas system can comprise components derived from a Type-I, Type-II, or Type-III system. In some embodiments, the CRISPR/Cas system can comprise components derived from Class 1 and Class 2 CRISPR/Cas systems, having Types I to V or Types II, V, and VI, respectively (Makarova et al., Nat Rev Microbiol 13(11):722-36 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)). [209] Class 2 CRISPR/Cas systems can have single protein effectors. Cas proteins of Types II, V, and VI can be single-protein, RNA-guided endonucleases, herein called Class 2 Cas nucleases. Class 2 Cas nucleases can include, for example, but not limited to, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain. [210] In some embodiments, the Cas nuclease can be from a Type-II CRISPR/Cas system (e.g., a Cas9 protein from a CRISPR/Cas9 system). In some embodiments, the Cas nuclease can be from a Class 2 CRISPR/Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 family of proteins are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing an appropriate guide RNA, which is further explained infra. [211] In some embodiments, a Cas nuclease can comprise more than one nuclease domain. In some embodiments, a Cas9 nuclease can comprise at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease can introduce a DSB in the target sequence. In some embodiments, the Cas9 nuclease can be modified to contain only one functional nuclease domain. For example, the Cas9 nuclease can be modified such that one of the nuclease domains can be mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease can be modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease can be modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one of the nuclease domains can be functional, the Cas9 nuclease can be a nickase that can introduce a single-stranded break (nick) into the target sequence. In some embodiments, a conserved amino acid within a Cas9 nuclease domain can be substituted to reduce or alter a nuclease activity. In some embodiments, the Cas nuclease nickase can comprise an amino acid substitution in the RuvC- like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain can include D10A (based on the S. pyogenes Cas9 nuclease). In some embodiments, the nickase can comprise an amino acid substitution in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain can include, but not limited to, E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 nuclease). [212] In some embodiments, the Cas nuclease can be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease can be a component of the Cascade complex of a Type- I CRISPR/Cas system. For example, the Cas nuclease can be a Cas3 nuclease. In some embodiments, the Cas nuclease can be derived from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from Type-IV CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from a Type-V CRISPR/Cas system. In some embodiments, the Cas nuclease can be derived from a Type-VI CRISPR/Cas system. [213] A Type I CRISPR/Cas system can utilize a large effector complex known as Cascade (CRISPR-associated complex for antiviral defense) for target binding and interference. The Cascade complex can contain multiple Cas proteins, including Cas3, which can be responsible for the destruction of the target DNA. A Type II CRISPR/Cas system, particularly the CRISPR-Cas9 system, can utilize a single Cas9 protein, guided by a synthetic guide RNA (sgRNA), to introduce double-strand breaks in target DNA for subsequent repair or modification. A Type III CRISPR/Cas system can utilize a Csm (CRISPR-Cas subtype multiprotein) or Cmr (CRISPR-Cas subtype ribonucleoprotein) complex for interference. Type III CRISPR/Cas system can target RNA molecules in addition to DNA. A Type V CRISPR/Cas system, including Cpf1 (also known as Cas12) and C2c2 (also known as Cas13), can utilize a single effector protein to perform interference. A Type VI CRISPR/Cas system can utilize a single Cas protein, such as C2c2 (also known as Cas13), to target and cleave RNA molecules, making it useful for RNA editing and manipulation. [214] Guide RNAs (gRNAs): The CRISPR technology can involves the use of a genome- targeting nucleic acid that can direct one or more endonucleases to a specific target sequence within a target gene for gene editing at the specific target sequence. The genome-targeting nucleic acid can be an RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. A guide RNA can comprise at least one spacer sequence that can hybridize to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence. [215] In Type II systems, the gRNA can also comprise a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence can hybridize to each other to form a duplex. In the Type V gRNA, the crRNA can form a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide can form a complex. In some embodiments, the genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid can thus direct the activity of the site-directed polypeptide. [216] As is understood by the person of ordinary skill in the art, each guide RNA can be designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science 337:816-821 (2012); Deltcheva et al., Nature 471:602-607 (2011). [217] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) can be a double-stranded guide RNA, comprising two strands of RNA molecules. The first strand can comprise in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence. The second strand can comprise a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence. [218] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) can be a single-molecule guide RNA (sgRNA). sgRNA in a Type II system can comprise, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that can contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. A single-molecule guide RNA in a Type V system can comprise, in the 5’ to 3’ direction, a minimum CRISPR repeat sequence and a spacer sequence. [219] A spacer sequence in a gRNA is a sequence (e.g., a 20-nucleotide sequence) that can define the target sequence (e.g., a DNA target sequences, such as a genomic target sequence) of a target gene of interest (e.g., DNAI1 or CFTR). In some embodiments, the spacer sequence can range from 15 to 30 nucleotides. For example, the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, a spacer sequence can contain 20 nucleotides. [220] The target sequence is in a target gene (e.g., DNAI1 or CFTR) that can be adjacent to a PAM sequence and can be the sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The target sequence is on the PAM strand in a target nucleic acid, which is a double- stranded molecule containing the PAM strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence can hybridize to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence can be the RNA equivalent of the target sequence. The spacer of a gRNA can interact with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus can vary depending on the target sequence of the target nucleic acid of interest. [221] In a CRISPR/Cas system, the spacer sequence can be designed to hybridize to a region of the target nucleic acid that is located 5’ of a PAM recognizable by a Cas9 enzyme used in the system. The spacer can perfectly match the target sequence or can have mismatches. Each Cas9 enzyme can have a particular PAM sequence that it can recognize in a target DNA. For example, S. pyogenes can recognize in a target nucleic acid a PAM that comprises the sequence 5’-NRG-3’, where R can comprise either A or G, where N can be any nucleotide and N can be immediately 3’ of the target nucleic acid sequence targeted by the spacer sequence. [222] In some embodiments, the target nucleic acid sequence can have about 20 nucleotides in length. In some embodiments, the target nucleic acid can have less than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have more than about 20 nucleotides in length. In some embodiments, the target nucleic acid can have at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid can have at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence can have 20 bases immediately 5’ of the first nucleotide of the PAM. For example, in a sequence comprising 5′-NNNNNNNNNNNNNNNNNNNNNRG-3′, the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence can be the S. pyogenes PAM. [223] The guide RNA can target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can be 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch. [224] The length of the spacer sequence in gRNAs can depend on the CRISPR/Cas9 system and components used for editing any of the target genes (e.g., DNAI1 or CFTR). For example, different Cas9 proteins from different bacterial species can have varying optimal spacer sequence lengths. Accordingly, the spacer sequence can have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence can have 18-24 nucleotides in length. In some embodiments, the targeting sequence can have 19-21 nucleotides in length. In some embodiments, the spacer sequence can comprise 20 nucleotides in length. [225] In some embodiments, the gRNA can be an sgRNA, which can comprise a 20- nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a less than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a more than 20 nucleotide spacer sequence at the 5’ end of the sgRNA sequence. In some embodiments, the sgRNA can comprise a variable length spacer sequence with about 17-30 nucleotides at the 5’ end of the sgRNA sequence. [226] In some embodiments, the gRNAs can comprise unmodified ribonucleic acid. In some embodiments, the gRNAs can comprise modified ribonucleic acid. Various types of RNA modifications can be introduced during or after chemical synthesis and/or enzymatic generation of RNAs, e.g., modifications that can enhance stability, reduce the likelihood or degree of innate immune response, and/or enhance other attributes, as described in the art. In some embodiments, non-natural modified nucleobases can be introduced into any of the gRNAs during synthesis or post-synthesis. In some embodiments, modifications can be on internucleoside linkages, purine or pyrimidine bases, or sugar. In some embodiments, a modification can be introduced at the terminal of a gRNA with chemical synthesis or with a polymerase enzyme. [227] In some embodiments, more than one guide RNAs can be used with a CRISPR/Cas nuclease system. Each guide RNA can contain a different targeting sequence, such that the CRISPR/Cas system can cleave more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or differing properties, such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA can be used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA can be the same or different. [228] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, and the like. [229] In some embodiments, the CRISPR/Cas nuclease system can contain multiple gRNAs, for example, 2, 3, or 4 gRNAs. Such multiple gRNAs can target different sites in a same target gene. Alternatively, the multiple gRNAs can target different genes. In some embodiments, the guide RNA(s) and the Cas protein can form a ribonucleoprotein (RNP), e.g., a CRISPR/Cas complex. The guide RNAs can guide the Cas protein to a target sequence(s) on one or more target genes (e.g., DNAI1 and CFTR), where the Cas protein can cleave the target gene at the target site. In some embodiments, the CRISPR/Cas complex can be a Cpf1/guide RNA complex. In some embodiments, the CRISPR complex can be a Type-II CRISPR/Cas9 complex. In some embodiments, the Cas protein can be a Cas9 protein. In some embodiments, the CRISPR/Cas9 complex can be a Cas9/guide RNA complex. [230] In some embodiments, the indel frequency (editing frequency) of a particular CRISPR/Cas nuclease system, comprising one or more specific gRNAs, can be determined using a TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing a target gene. In some embodiments, a highly efficient gRNA can yield a gene editing frequency of higher than 80%. For example, a gRNA can be considered to be highly efficient if it can yield a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%. Other Gene Editing Methods [231] Besides the CRISPR system disclosed herein, additional gene editing systems as known in the art can also be used as a payload of the LNPs described herein. In some embodiments, the additional gene editing system can comprise zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), restriction endonucleases, meganucleases homing endonucleases, or the like. [232] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which can be a polypeptide domain that can bind DNA in a sequence- specific manner through one or more zinc fingers. A zinc finger can be a domain of about 30 amino acids within the zinc finger binding domain whose structure can be stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain can be a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A selected zinc finger domain can be a domain not found in nature whose production can result primarily from an empirical process such as phage display, interaction trap or hybrid selection. In some embodiments, a ZFN can be a fusion of the FokI nuclease with a zinc finger DNA binding domain. [233] A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. A “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” is a polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins can be secreted by plant pathogens of the genus Xanthomonas during infection. These proteins can enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity can depend on an effector-variable number of imperfect 34 amino acid repeats, which can comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). In some embodiments, a TALEN can be a fusion polypeptide of the FokI nuclease to a TAL effector DNA binding domain. [234] Additional examples of targeted nucleases suitable for use can include, but not limited to, Bxb1, phiC31, PhiBT1, and Wβ/SPBc/TP901-1, whether used individually or in combination. The Bxb1 nuclease, also known as the Bxb1 integrase, is a site-specific recombinase enzyme derived from the mycobacteriophase Bxb1. The Bxb1 integrase can catalyze site-specific recombination between two specific DNA sequences, referred to as attachment (att) sites. The Bxb1 integrase can recognize a specific 48 base-pair sequence within the attachment sites. The phiC31 nuclease, also known as the phiC31 integrase, is derived from the bacteriophage phiC31. The phiC31 nuclease can catalyze site-specific recombination between two specific DNA sequences, referred to as attB (attachment site in bacteriophage) and attP (attachment site in the phage). The phiC31 nuclease can promote integration of a DNA fragment flanked by attB and attP into the genome in cells of target organ. The phiBT1 nuclease can integrate into a different attachment site than phiC31. The Wβ/SPBc/TP901-1 nuclease, also known as bacteriophage P2 Bxb1 Cre nuclease, is a site- specific recombination enzyme derived from the temperate bacteriophage P2. [235] In some embodiments, the polynucleotide encodes a gene-editing system or component thereof. In some embldiments, the gene-editing system selected from the group consisting of alpha-1-antitrypsin (A1AT), carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl CoA carboxylase beta subunit (PCCB), a subunit of branched-chain ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin, glucose- -phosphatase (GPase), glucose -phosphate translocase, lysosomal glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1), Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor IX, galactose-1-phosphate uridylyltransferase, galactokinase, UDP-galactose 4- epimerase, transthyretin, a complement regulatory protein, phenylalanine hydroxylase (PAH), homogentisate 1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1, alpha-galactosidase A, acid ceramidase, acid α-L-fucosidase, acid β-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid α-mannosidase, β-mannosidase, arylsulfatase B, arylsulfatase A, N- acetylgalactosamine--sulfate sulfatase, acid β-galactosidase, acid α-glucosidase, β- hexosaminidase B, heparan-N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA:α- glucosaminide N-acetyltransferase, N-acetylglucosamine--sulfate sulfatase, alpha-N- acetylgalactosaminidase, sialidase, β-glucuronidase, β-hexosaminidase A. In some embodiments, the polynucleotide is a DNA, such as, for example, a DNA encoding a functional protein associated with a protein deficiency disease (e.g., a protein selected from the proteins listed above). Pharmaceutical composition [236] The disclosure also provides pharmaceutical compositions comprising the LNP composition described herein and a pharmaceutically acceptable excipient and/or diluent. Such compositions can be used for the treatment of a liver disease as described herein in a patient or subject. The pharmaceutical compositions of the disclosure may include a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in Chapter 30 of Remington: The Science and Practice of Pharmacy (23^rd ed., 2021). [237] In some embodiments, the composition comprises Tris buffer, optionally at a pH from 6-9. In some embodiments, the composition comprises sucrose, optionally at 5-15%. In some embodiments, the composition comprises citrate buffer, optionally at a pH 4-6. In some embodiments, the composition comprises 15 mM Tris buffer, optionally at a pH from 6-9, and/or 5-15% sucrose. In some embodiments, the composition comprises 10mM citrate buffer, optionally at a pH from 4-6. [238] In some embodiments, the pharmaceutical compositions include one or more of a poloxamer (e.g., Poloxamer 188), polyethylene glycol (“PEG”), sucrose, and a buffer, wherein the buffer comprises a citrate buffer, an acetate buffer, or a Tris buffer. [239] In some embodiments, the composition comprises a citrate buffer. For example, the citrate buffer is at a pH from 4 to 8. In some embodiments, the buffer is an acetate buffer and has a pH from 4 to 8. In another embodiments, the composition comprises a Tris buffer, and the Tris buffer has a pH from 4 to 8. [240] In some embodiments, the composition comprises sucrose. In some embodiments, the sucrose is at a concentration from 1% to 15% w/v, 5% to 15% w/v, 1% to 10% w/v, or 5% to 10% w/v. [241] 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. [242] In some embodiments, one or more tonicity agents may be added to provide the desired ionic strength. Tonicity agents for use herein include those which display no or only negligible pharmacological activity after administration. Both inorganic and organic tonicity adjusting agents may be used. Method of treatment [243] In some embodiments, the LNP compositions and pharmaceutical compositions described herein can be employed to treat or prevent a liver disease or disorder, including but not limited to a disease or disorder from the following: Glycogen Storage Disease Type IV, Hereditary Fructose Intolerance, Wilson Disease, Type I Tyrosinemia, Hereditary Hemochromatosis, Alpha-1 Antitrypsin Deficiency, Cystic fibrosis. [244] In some embodiments, the LNP compositions can be employed to treat Glycogen Storage Disease Type IV. In some embodiments, the LNP compositions can be employed to treat Hereditary Fructose Intolerance. In some embodiments, the LNP compositions can be employed to treat Wilson Disease. In some embodiments, the LNP compositions can be employed to treat Type I Tyrosinemia. In some embodiments, the LNP compositions can be employed to treat Hereditary Hemochromatosis. In some embodiments, the LNP compositions can be employed to treat Alpha-1 Antitrypsin Deficiency. In some embodiments, the LNP compositions can be employed to treat Cystic fibrosis. [245] In some embodiments, the LNP composition can be delivered to liver, wherein the composition delivers a payload preferentially in a liver cell. In some embodiments, the LNP composition can be delivered to liver, wherein the composition delivers a payload preferentially to both a liver cell and a lung cell. [246] In another aspect, the disclosure provides a method for treating and/or preventing a liver disease in a subject in need thereof, wherein the method comprises administering the composition described herein to the subject by intravenous injection. [247] In some embodiments, the payload is a messenger RNA (mRNA) and the method results in delivery of the payload to the liver in an amount effective to increase expression and/or function of a gene encoded by the mRNA. [248] In some embodiments, the method results in expression of a polypeptide in a liver of the subject, wherein the expression is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased compared to the polypeptide expression prior to delivery. [249] In some embodiments, the method results in expression of the polypeptide in the liver of the subject between 10 min and 24 hours after administration of the composition to the subject. [250] In another aspect, the disclosure provides a method of delivering a payload to a cell in a liver of a subject, wherein the method comprising administering to the subject, by intravenous injection, the LNP composition described herein. [251] In another aspect, the disclosure provides a kit including the composition described herein. In another aspect, the disclosure provides use of the LNP composition described herein for treatment of a liver disease by intravenous injection. Liver diseases [1] Glycogen Storage Disease Type IV is an autosomal recessive disease due to mutations in the gene encoding the glycogen branching enzyme (GBE1) that catalyzes the alpha 1, bond of the first glucose in the side chains of glycogen. The altered glycogen branching reduces its solubility, thus impairing the osmotic pressure within the hepatocyte. Hereditary Fructose Intolerance is an autosomal recessive disease due to the deficiency of fructose 1-phosphate aldolase (aldolase B) involved in the metabolism of fructose-1-phosphate into dihydroxyacetone phosphate and D-glyceraldehyde. Wilson Disease is an autosomal recessive disorder which depends on mutations in the gene encoding the ATP7B Cu translocase. ATP7B, mainly expressed by the hepatocyte, regulates the levels of copper in the liver. When the activity of ATP7B is reduced, copper accumulates within the hepatocyte. Furthermore, ATP7B modulates the synthesis of ceruloplsmin. Type I Tyrosinemia is an autosomal recessive disease which is the most severe form of genetic tyrosinemia and is the only one that causes a severe liver involvement. Type I tyrosinemia is due to the altered activity of fumarylacetoacetate hydrolase, which causes the elevation of plasma and urine succinylacetone and high plasma concentration of tyrosine, methionine, and phenylalanine. Hereditary Hemochromatosis is an autosomal recessive disease characterized by iron overload that my cause liver cirrhosis, cardiomyopathy, diabetes, and arthritis. Molecular analysis in HFE gene confirm hereditary hemochromatosis. Homozygous patients for .Cys22Tyr have a higher risk for iron overload. Alpha-1 Antitrypsin Deficiency is an autosomal recessive disease due to mutations in the SERPINA1 gene which encodes the serine protease inhibitor AAT. The protein is mainly synthesized by liver cells, and inhibits proinflamattory proteases. The liver damage is due to the accumulation of AAT mutant polymers and not to the lack of circulating AAT. Cystic fibrosis is a progressive, genetic disease that affects the lungs, pancreas, liver, kidneys, and other organs. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the CFTR protein to become dysfunctional, resulting in thick and sticky mucus that blocks airways and leads to lung damage and traps germs and makes infections more likely. Method of administration [252] In another aspect, the disclosure provides a method of delivering a payload to a cell in a liver of a subject, wherein the method comprising administering to the subject, by intravenous injection, the composition disclosed herein. In another aspect, the disclosure provides a kit comprising the composition disclosed herein. [253] In some embodiments, the administering to the subject is done by intravenous (I.V.) delivery. In some embodiments, the administering to the subject is done by intrathecal (I.T.) delivery. In some embodiments the administering to the subject is done by intramuscular (I.M.) delivery. In some embodiments, the administering to the subject is done by intradermal (I.D.) delivery. In some embodiments of the method, the administering to the subject is done by intranasal delivery. [254] In some embodiments, the administration is single administration. In some embodiments, the administration is a multiple administration. In some embodiments, the multiple administrations occur three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every three weeks, every four weeks, or monthly. EXAMPLES [255] The following examples provide general procedures for the synthesis and uses of various ionizable cationic lipids described herein. Example 1: Synthesis of 4A3-SCC-PH

[256] Step 1: Synthesis of biodegradable disulfide bond linker 1

[257] Bis(2-hydroxyethyl) disulfide (15.40 g, 100.00 mmol) and triethylamine (11.13 g, 110.00 mmol) were added to dry THF (180 mL) in a 250 mL round-bottom flask equipped with a stir bar and cooled to 0 °C. Acryloyl chloride (8.23 g, 90.90 mmol) was added dropwise via an addition funnel. The solution was stirred overnight under nitrogen at room temperature. Upon completion, the precipitate was filtered off and the residue was purified by silica gel column (50% ethyl acetate in petroleum ether). The resulting product, Intermediate 1, was concentrated under reduced pressure to yield biodegradable disulfide bond linker as yellow oil (11.82 g, 63%).¹H NMR (400 MHz, CDCl3) δ 6.43 (d, J = 17.2 Hz, 1H), 6.12 (dd, J = 17.2, 10.4 Hz, 1H), 5.86 (d, J = 10.4 Hz, 1H), 4.43 (t, J = 6.8 Hz, 2H), 3.88 (t, J = 6.0 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.88 (t, J = 5.6 Hz, 2H). [258] Step 2

[259] 2-propylheptanol (5.00 g, 31.60 mmol) and triphenylphosphine (11.61 g, 44.25 mmol) were added in 60 mL DCM to a 250 mL round bottom flask and cooled to 0°C. N- bromosuccinimide (7.32 g, 41.05 mmol) dissolved in 30 mL DCM was added dropwise to above mixture. The solution was stirred at room temperature under nitrogen overnight. Upon completion, the solvent removed under reduced pressure and the residue was purified by silica gel column (petroleum ether). The product was concentrated under reduced pressure to yield Intermediate 2 as colorless oil (3.50 g, 50%). [260] Step 3

[261] Intermediate 2 (3.50 g, 15.91 mmol) and potassium thioacetate (2.73 g, 23.86 mmol) were dissolved in 25 mL DMF and stirred at room temperature for 24 h. Then, the mixture was diluted with dichloromethane (25 mL) and washed with water two times. The organic phase was separated and dried with anhydrous MgSO₄. The desired product was purified on a column of silica gel (petroleum ether) to obtain Intermediate 3 as colorless oil (3.20 g, 93%). [262] Step 4: Synthesis of SC-PH

[263] Intermediate 3 (3.20 g, 14.81 mmol) and sodium hydroxide (1.80 g, 45.00 mmol) were dissolved in 35 mL methanol. The solution was stirred at room temperature under nitrogen overnight.1% hydrochloric acid was used to adjust pH < 7.0 and the solvent was washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure and SC-PH was obtained as pale-yellow oil (2.40 g, 93%). [264] Step 5

[265] SC-PH (4.21 g, 24.19 mmol) and methyl methacrylate (2.63 g, 26.30 mmol) were added in 10 mL round bottom flask with addition of 5 mol% dimethylphenylphosphine (DMPP) as catalyst at 60°C for 24 h. The residue was purified by silica gel column (5-10% ethyl acetate in petroleum ether) to afford Intermediate 4 as pale-yellow oil (4.52 g, 68%). [266] Step 6

[267] Intermediate 4 (4.52 g, 16.50 mmol) was dissolved in 24 mL methanol and added 15% sodium hydroxide aqueous stirring at room temperature under nitrogen overnight. Upon completion, 1% hydrochloric acid was used to adjust pH < 7.0 and the solvent was removed under reduced pressure. The desired acrylate purified on a column of silica gel (50% ethyl acetate in petroleum ether) to obtain Intermediate 5 as pale-yellow oil (3.56 g, 83%). [268] Step 7

[269] For the syntheses of Intermediate 6 synthesis, Intermediate 5 (3.56 g, 13.69 mmol) was dissolved in 3 mL DCM and cooled to 0°C. Oxalyl chloride (9.26 g, 73.27 mmol) was added dropwise to the solution with two drops of dimethylformamide (DMF) and stirred 16 overnight under nitrogen at room temperature. The product Intermediate 6 was yielded under reduced pressure to remove solvent as orange oil (3.35 g, 88%). [270] Step 8

[271] Biodegradable linker Intermediate 1 (2.76 g, 13.25 mmol) and triethylamine (1.46 g, 14.46 mmol) were added in dry THF (40 mL) to a 250 mL round-bottom flask and cooled to 0°C.6 (3.35 g, 12.05 mmol) was added dropwise via an addition funnel. The solution was stirred overnight under nitrogen at room temperature. Upon completion, the solvent was removed under reduced pressure and the residue was purified by silica gel column (10% ethyl acetate in petroleum ether). The product was concentrated under reduced pressure to yield Intermediate 7 (SCC-PH) as yellow oil (4.06 g, 75%).¹H NMR (400 MHz, CDCl3) δ 6.43 (d, J = 17.2, 1H), 6.13(dd, J = 17.2, 10.4 Hz, 1H), 5.86 (d, J = 10.4 Hz, 1H), 4.42 (t, J = 6.8 Hz, 2H), 4.36 (t, J = 6.8 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.94 (t, J = 5.2 Hz, 2H), 2.80 (dd, J = 12.8, 7.2 Hz, 1H), 2.72-2.64 (m, 1H), 2.57-2.52 (m, 1H), 2.49 (d, J = 6.4 Hz, 2H), 1.54-1.50 (m, 1H), 1.34-1.24 (m, 15H), 0.90-0.86 (m, 6H). [272] Step 9

[273] 4A3-SCC-PH was synthesized via a Michael addition reaction. Intermediate 7 (500 mg, 1.11 mmol) and amino headgroup 4A3 (36 mg, 0.25 mmol) were equipped to a 5 mL vial with a stir bar in the presence of 10 mol% of butylated hydroxyltoluene (BHT) at 60 °C for 24 h. The crude product was purified by silica gel column (15% menthol in dichloromethane) to yield 4A3-SCC-PH as pale-yellow oil (150 mg, 30.83%).¹H NMR (400 MHz, CDCl3) δ 4.39-4.32 (m, 16H), 3.06-2.92 (m, 16H), 2.84-2.65 (m, 20H), 2.57-2.44(m, 24H), 2.01 (s, 3H), 1.60-1.53 (m, 8H), 1.31- 1.25 (m, 60H), 0.89-0.87 (m, 24H).13C NMR (400 MHz, CDCl3) δ 174.98, 172.24, 62.27, 51.19, 48.90, 48.84, 40.22, 37.73, 37.13, 36.16, 35.40, 33.02, 32.38, 32.18, 26.19, 22.68, 19.72, 16.82, 14.36, 14.08. HRMS (MALDI): m/z calculated for C91H171N3O16S12 [M+H]⁺ 1946.94. Example 2: Synthesis of 4A3-SCC-10

[274] Step 1

[275] 1-Decanethiol (4.21 g, 24.19 mmol), methyl methacrylate (2.63 g, 26.30 mmol) and 5 mol% dimethylphenylphosphine (DMPP) were added in 10 mL round bottom flask at 60°C for 24 h. Upon completion, the residue was purified by silica gel column (5-10% ethyl acetate in petroleum ether) as colorless oil Intermediate 8 (4.2 g, 63%). [276] Step 2

[277] Intermediate 8 (4.21 g, 15.33 mmol) was dissolved in mixed solvent of 20 mL methanol and 15% sodium hydroxide aqueous at room temperature under nitrogen overnight. Upon completion, pH of solution was adjusted to < 7 with 1% hydrochloric acid and the solvent was removed under reduced pressure to obtain crude Intermediate 9 as colorless oil (2.99 g, 75%). [278] Step 3

[279] Intermediate 9 (2.99 g, 11.54 mmol) was dissolved in 2 mL DCM and cooled to 0°C in 250 mL round bottom flask. Oxalyl chloride (8.73 g, 68.8 mmol) was added dropwise and two drops of dimethylformamide (DMF) were added as catalyst stirring overnight under nitrogen at room temperature. The unreacted oxalyl chloride was removed by vacuum drying to obtain Intermediate 10 as orange oil (2.78 g, 87%). [280] Step 4: Synthesis of SCC-10

[281] Intermediate 1 (3.7 g, 15.05 mmol) and triethylamine (2.03 g, 20.06 mmol) were dissolved in dry THF (30 mL) and cooled to 0°C. Intermediate 10 (2.78 g, 10.01 mmol) was added dropwise and the solution was stirred overnight under nitrogen at room temperature. The residue was purified by silica gel column (10% ethyl acetate in petroleum ether). The product was concentrated under reduced pressure to yield Intermediate 11 as yellow oil (3.42 g, 76%). ¹H NMR (400 MHz, CDCl₃) δ 6.44 (d, J = 17.2, 1H), 6.13 (dd, J = 17.2, 10.4 Hz, 1H), 5.86 (d, J = 10.4 Hz), 4.42 (t, J = 6.8 Hz, 2H), 4.37 (t, J = 6.4 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 2.94 (t, J = 4.8 Hz, 2H), 2.83 (dd, J = 12.8, 7.2 Hz, 1H), 2.73-2.64 (m, 1H), 2.60-2.49 (m, 3H), 1.60- 1.53 (m, 4H), 1.37-1.25 (m, 15H), 0.88 (t, J = 6.8 Hz, 3H). [282] Step 5

[283] 4A3-SCC-10 was made in a manner analogous to 4A3-SCC-PH. Intermediate 11 (500 mg, 1.11 mmol) and amino headgroup 4A3 (36 mg, 0.25 mmol) were equipped to a 5 mL vial with a stir bar with addition of 10 mol% butylated hydroxyltoluene (BHT) at 60°C for 24 h. The crude product was purified by silica gel column (12-15% menthol in dichloromethane) to yield 4A3-SCC-10 as pale- yellow oil (165 mg, 33.9%).¹H NMR (400 MHz, CDCl3) δ 4.38- 4.31 (m, 16H), 2.96- 2.91 (m, 16H), 2.85-2.75 (m, 12H), 2.71-2.64 (m, 4H), 2.60-2.44 (m, 24H), 2.32-2.18 (m, 4H), 2.00 (s, 3H), 1.60-1.53 (m, 12H), 1.38-1.34 (m, 8H), 1.32-1.25 (m, 60H), 0.88 (t, J = 6.8 Hz, 12H). ¹³C NMR (400 MHz, CDCl₃) δ 174.90, 172.19, 62.27, 48.96, 40.19, 37.15, 37,11, 35.42, 32.68, 32.40, 31.88, 29.65, 29.64, 29.61, 29.58, 29.51, 29.32, 29.22, 28.86, 22.65, 16.84, 14.09. Mass spectrometry of 4A3-SCC-10. HRMS (MALDI): m/z calculated for C91H171N3O16S12 [M+H]⁺ 1946.9386; Found 1948.9362. [284] Other ionizable cationic lipid compounds of the disclosure comprising SCC-PH or SCC-10 tails were made in a manner similar to the syntheses of 4A3-SCC-PH and 4A3-SCC- 10 described herein. Example 3: Synthesis of 4A3-SSC-PH

[285] Step 1

[286] SC-PH (3 g, 17.2 mmol) and 1,2-di (pyridin-2-yl) disulfane (7.58 g, 34.41 mmol) were added in 100 mL round bottom flask and dissolved in 30 mL methanol and 12 mL dichloromethane mixture solvent.472 μL acetic acid were added as catalyst and stirred at room temperature under nitrogen overnight. The residue was purified by silica gel column (10% ethyl acetate in petroleum ether) to afford Intermediate 12 as colorless oil (4.87 g, 100%). [287] Step 2

[288] Intermediate 12 (4.87 g, 17.2 mmol) and 390 μL acetic acid were dissolved in 60 mL methanol and stirred at room temperature under nitrogen.2-mercaptoethanol (2.02 g, 25.8 mmol) was added dropwise. Upon completion, the desired product Intermediate 13 was obtained by a column of silica gel (20% ethyl acetate in petroleum ether) as colorless oil (2 g, 46%). [289] Step 3: Synthesis of SSC-PH

[290] Intermediate 13 (2 g, 7.99 mmol) was dissolved in dry dichloromethane (40 mL) and cooled to 0°C. Acryloyl chloride (1.08 g, 12 mmol) was added dropwise and the solution was stirred overnight under nitrogen at room temperature. The residue was purified by silica gel column (10% ethyl acetate in petroleum ether) to yield SSC-PH (Intermediate 14) as pale- yellow oil (3.42 g, 76%).¹H NMR (400 MHz, CDCl3) δ 6.43 (d, J = 17.6 Hz, 1H), 6.13 (dd, J = 17.2, 10.4 Hz, 1H), 5.85 (d, J = 10.4 Hz, 1H), 4.42 (t, J = 6.4 Hz, 2H), 2.93 (t, J = 6.8 Hz, 2H), 2.73 (d, J = 6.4 Hz, 2H), 1.69-1.64 (m, 1H), 1.36-1.26 (m, 12H), 0.91-0.87(m, 6H). [291] Step 4

[292] Amino headgroup 4A3 (48 mg, 0.24 mmol) was mixed with Intermediate 14 (300 mg, 0.99 mmol) in 5 mL vial with a stir bar in the presence of 10 mol% of butylated hydroxyltoluene (BHT) and heated at 60°C for 24 h. The crude product was purified by flash chromatography on silica gel to yield product 4A3-SSC-PH as yellow oil (182 mg, 56%).¹H NMR (400 MHz, CDCl₃) δ 4.35-4.33 (m, 8H), 2.90-2.87 (m, 8H), 2.78-2.71 (m, 16H), 2.47- 2.22 (m, 16H), 2.00 (s, 3H), 1.68-1.64 (m, 8H), 1.37-1.25 (m, 48H), 0.92-0.87 (m, 24H). ¹³C NMR (400 MHz, CDCl₃) δ 172.22, 62.48, 55.31, 51.35, 48.97, 44.86, 37.45, 36.78, 35.11, 32.74, 32.40, 32.07, 26.10, 22.59, 19.65, 14.32, 14.07. HRMS (ESI): m/z calculated for C₆₇H₁₃1N₃O₈S₈ [M+H]⁺ 1362.7775; Found 1362.7781. Example 4: Synthesis of 4A3-SC-PH

[293] 4A3-SC-PH was synthesized according to the synthetic methods reported by Zhou et. al. (Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. USA 2016, 113, 520-525) and Xiong et. al. (Theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo. J. Control. Release 2020, 325, 198-205) incorporated by reference herein in their entirety.¹H NMR (400 MHz, CDCl3) δ 4.34-4.25 (m, 16H), 2.81-2.70 (m, 16H), 2.69-2.63 (m, 4H), 2.55-2.50 (m, 4H), 2.48-2.43 (m, 24H), 1.93 (s, 3H), 1.53-1.51 (m, 4H), 1.36-1.23 (m, 60H), 0.89-0.85 (m, 24H).¹³C NMR (400 MHz, CDCl3) δ 174.91, 172.16, 62.15, 62.09, 48.93, 40.16, 39.27, 39.26, 37.29, 36.09, 36.07, 32.29, 28.79, 25.43, 22.93, 16.79, 14.06, 10.74, 10.73. HRMS (ESI): m/z calculated for C83H155N3O16S4 [M+H]⁺ 1579.0363; Found 1579.0368. Example 5: Synthesis of 4A3-SC-10

[294] 4A3-SC-10 was synthesized according to the synthetic methods reported by Zhou et. al. (Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. USA 2016, 113, 520-525) and Xiong et. al. (Theranostic dendrimer-based lipid nanoparticles containing PEGylated BODIPY dyes for tumor imaging and systemic mRNA delivery in vivo. J. Control. Release 2020, 325, 198-205) incorporated by reference herein in their entirety.¹H NMR (400 MHz, CDCl₃) δ 4.32-4.25 (m, 16H), 2.83-2.64 (m, 16H), 2.58-2.53 (m, 4H), 2.51-2.29 (m, 24H), 2.19 (s, 3H), 1.58- 1.51 (m, 12H), 1.36-1.24 (m, 68H), 0.87 (t, J = 6.4 Hz, 12H). ¹³C NMR (400 MHz, CDCl₃) δ 174.90, 172.17, 62.17, 62.08, 48.92, 40.13, 35.37, 32.69, 32.32, 31.85, 29.61, 29.53, 29.50, 29.27, 29.21, 28.85, 22.64, 16.83, 14.08. HRMS (ESI): m/z calculated for C₈₃H₁₅₅N₃O₁₆S₄ [M+H]+ 1579.0363; Found 1579.0340. Example 6: Synthesis of 4A3-SSC-10

[295] 4A3-SSC-10 was synthesized according to the synthetic method reported by Wang et. Al. (Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. USA 2016, 113, 2868-2873), incorporated by reference herein in its entirety.¹H NMR (400 MHz, CDCl3) δ 4.34-4.31 (m, 8H), 2.91-2.88 (m, 8H), 2.77-2.70 (m, 16H), 2.68-2.43 (m, 16H), 2.29 (s, 3H), 1.70- 1.63 (m, 12H), 1.39-1.36 (m, 8H), 1.30-1.26 (m, 48H), 0.88 (t, J = 6.4 Hz, 12H).¹³C NMR (400 MHz, CDCl₃) δ 172.24, 62.49, 55.34, 51.46, 49.01, 39.17, 36.98, 32.45, 31.85, 29.52, 29.48, 29.27, 29.21, 29.12, 28.49, 22.64, 14.08. HRMS (ESI): m/z calculated for C₆₇H₁₃₁N₃O₈S₈ [M+H]⁺ 1362.7775; Found 1362.7768. Example 7: Preparation and characterization of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure [296] mRNA-loaded LNP formulations were prepared using the ethanol dilution method as described by Zhou et. al. (Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. USA 2016, 113, 520-525) and Wang et. al. (CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery. Chem. Rev. 2017, 117, 9874–9906), incorporated by reference herein in their entirety. Unless otherwise stated, all lipids were dissolved in ethanol (or DMF) at molar ratios of 15: 15: 30: 3 and mRNA was dissolved in 10 mM citrate buffer (pH 4.2) with weight molar of 40: 1. The two solutions were pipette mixed rapidly with aqueous/ethanol volume ratio of 3/1, then incubated for 20 min at room temperature. Subsequently, the lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure were diluted with 1 × DPBS to 0.5 ng/μL mRNA for in vitro assays and size detection. For in vivo experiments, lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure were dialyzed against 1 × DPBS for 2 h, then diluted with DPBS to 20 ng/μL mRNA to perform intravenous (i.v.) injection. [297] The Zetasizer Nano Series Nano-ZS instrument was used to measure particle sizes and zeta potentials of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure with a 4.0 mW He–Ne laser at the scattering angle of 90° producing a wavelength of 633 nm, using 1 mL nanoparticles. [298] mRNA condensation ability of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure was quantified by 1% agarose gel electrophoresis with 20 ng/μL mRNA in PBS. Example 8: Cell culture and Animal experiments [299] Human ovarian adenocarcinoma cells (IGROV1), human cervical cancer cells (HeLa), human hepatoellular carcinomas (HepG2), mouse embryonic fibroblasts (3T3), and mouse skin melanoma cells (B16F10) were cultured in DMEM with 10% FBS. Mouse breast cancer cells (4T1) was cultured in RPMI-1640 with 10% FBS. Mouse normal liver cells (AML- 12) was cultured in DMEM-F12 with 10% FBS. All medium solutions containing 1% Penicillin/Streptomycin (P/S) were incubated at 37°C with 5% CO2 under standard cell culture conditions. [300] FIG. 2A-2B show heat maps of luciferase expression and cell viability. FIG. 2A shows a heat map of luciferase expression in ovarian cancer IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). A relative luminescence intensity of >105 was counted for in vitro hit rate calculation. FIG.2B shows a heat map of cell viability IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). [301] All animal experiments were approved by the Ethical Committee of Nankai University (2021-SYDWLL-000091) and were conducted in accordance with the guidelines for animal experiments. Female C57BL/6 mice (6-8 weeks, 18-20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. B6.ROSA26-CAG-LSL- tdTOMATO reporter mice with LoxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent tdTomato protein were purchased from Beijing Vitalstar Biotechnology Co., Ltd. Following Cre-mediated recombination, ROSA26 mice could express tdTomato fluorescence. ROSA26 mice were congenic on C57BL/6 genetic background. [302] Example 9: in vitro mRNA delivery and cell viability measurement [303] To screen lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure in vitro, IGROV1 cells were seeded into white 96-well plates with 1×104 cells each well and incubated overnight. The medium was replaced with fresh DMEM medium with 10% FBS at volume of 150 μL, and Fluc-mRNA LNPs formulated with ionizable cationic lipids of the disclosure were added with 25 ng/well mRNA (80 ng mRNA per well for mCherry mRNA). After 24 h incubation, ONE-Glo + Tox kits were used for mRNA expression and cytotoxicity evaluation using standard protocol. And inverted fluorescence microscope (Nikon, TE2000) was used to capture fluorescence images of mCherry mRNA expression at different time-points. [304] FIGS. 3A and 3B show the relative in vitro hit rate of ionizable cationic lipids described herein with different alkyl chain lengths in FIG.3A, and for ionizable cationic lipids described herein with different amino headgroups of 2A1−6A1 in FIG.3B. [305] ionizable cationic lipids of the disclosure containing 3A1, 3A2, 4A1, and 4A3 headgroups exhibited higher mRNA delivery efficacy, especially for 3A1 and 4A3 with a small amine head, the hit rates reached up to 100% (FIG. 3A). Among all ionizable cationic lipids of the disclosure, the hydrophobic tail length was also crucial for cone conformation and the hit rates of 10-14 chain lengths exceeded 60% (FIG.3B). [306] FIG. 6A-6G shows in vitro evaluation of six LNPs, 4A3-SCC-10/PH, 4A3-SC- 10/PH, and 4A3-SSC-10/PH, for Fluc mRNA delivery (25 ng/well, n = 4) in ovarian cancer IGROV1 cells treated with or without 20 μmol of a GSH-depleting agent N-ethylmaleimide (NEM); DLin-MC3-DMA was used as the positive control. FIG.6A shows luciferase activity for ovarian cancer IGROV1 cells treated with or without 20 μmol of a GSH-depleting agent N- ethylmaleimide (NEM) FIG. 6B-6D shows Z-average size (FIG. 6B), Zeta potential (FIG. 6C), and PDI value (FIG. 6D) of the exemplary six A43 lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure measured by Zetasizer Nano Series Nano- ZS. [307] FIG.6E-6F shows electrophoretic retardation analysis of six 4A3 LNPs for mRNA encapsulation ability (FIG. 6E) and release behavior after the treatment with 20 mM TCEP (FIG.6F). FIG.6G shows TEM images of 4A3-SCC-PH LNPs and DLin-MC3-DMA LNPs, scale bar = 100 μm. [308] FIG. 17A-17B shows Adsorption of endogenous apolipoprotein E (ApoE) on the surface of four 4A3 LNPs as validated by Western blot is shown in FIG.17A and quantitation of ApoE on the surface of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure is shown in FIG.17B. Example 10: in vivo mRNA delivery [309] FIG.1 shows the structure of 4A3-SCC-PH and a schematic illustration of mRNA delivery to cells, with lipid nanoparticle compositions of the disclosure, which can significantly facilitate endosomal escape and improve mRNA delivery in vivo. [310] Fluc mRNA lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure were prepared as above-mentioned method at a dose of 0.1 mg/kg. Afterward, the lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure were administered to C57BL/6 mice via i.v. injection. 6 h later, D-luciferin DPBS solution (150 mg/kg) was injected subcutaneously to image using an IVIS-Lumina II imaging system. Following, mice were sacrificed, and the organs were isolated and imaged on the IVIS-Lumina II imaging system. To further quantify Fluc mRNA delivery in vivo, the relative luciferase expression (average bioluminescence intensity) was analyzed using region-of-interest analysis across the whole liver. [311] Cy5-RNA formulations were prepared at a dose of 0.1 mg/kg. After 6 h, mice were killed and the organs were isolated and imaged on an IVIS-Lumina II imaging system. [312] Cre mRNA formulations were prepared at a dose of 0.2 mg/kg. After 2 d, mice were sacrificed and the major organs were imaged on an IVIS-Lumina II imaging system. [313] FIG.4A-4B. shows analysis of in vivo screening of 4A1 and 4A3 ionizable cationic lipids of the disclosure at a dose of 0.1 mg/kg Fluc mRNA (n = 2 for initial screening) using bioluminescence (FIG. 4A) and quantified radiance of exemplary ionizable cationic lipids described herein. FIG.4A shows bioluminescence images from in vivo screening of 4A1 and 4A3 ionizable cationic lipids of the disclosure at a dose of 0.1 mg/kg Fluc mRNA (n = 2 for initial screening). Images were obtained 6 h after i.v. injection of lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure into C57BL/6 mice. H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; exposure time: 15 s. An average luminescence radiance of >2,500,000 was counted for in vivo hit rate calculation. FIG. 4B shows quantification measured by average radiance [p/s/cm²/sr] in vivo luciferase expression in liver obtained from in vivo screening of 4A1 and 4A3 ionizable cationic lipids of the disclosure as shown in FIG.4A. [314] FIG.5A-5C. shows graphical representations of in vivo hit rate of ionizable cationic lipids described herein. FIG. 5A shows relative hit rates for ionizable cationic lipids of the disclosure with amino headgroups of 4A1 and 4A3. FIG. 5B shows relative hit rates for ionizable cationic lipids of the disclosure with different lengths of tails (4C−14C). FIG. 5C shows relative hit rates for 4A3 ionizable cationic lipids of the disclosure with linear and branched alkyl chains. [315] FIG. 8A-8C. shows fluorescence analysis in vivo of mice subjects injected with Cy5-RNA-loaded 4A3 LNPs. FIG. 8A shows fluorescence images of major organs 6 h after i.v. injection of Cy5-RNA-loaded 4A3 LNPs into C57BL/6 mice (0.1 mg/kg). H, heart; Li, liver; S, spleen; Lu, lung; K, kidney. FIG.8B shows Cy5 fluorescence intensities of exemplay ionizable cationic lipids described herein. FIG 8C shows flow cytometry analysis showing fluorescence intensity distribution of Cy5-RNA in livers from mice in FIG.8A. [316] FIG. 9A-9B. shows qualitative and qualitative measures of radiance in mice subjects injected with LDIL compositions described herein. FIG.9A shows bioluminescence images of C57BL/6 mice and major organs 6 hours after i.v. injection of six 4A3 LNPs with different Fluc mRNA (0.1 mg/kg, n = 3, exposure time = 15 s) and FIG. 9B shows quantification of in vivo luciferase expression in livers of the mice subjects. [317] FIG. 10A-10B. shows qualitative and qualitative measures of radiance in mice subjects injected with Fluc mRNA-loaded 4A3-SCC-PH LNPs and 4A3-CCC-PH LNPs. FIG. 10A shows bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of Fluc mRNA-loaded 4A3-SCC-PH LNPs and 4A3-CCC-PH LNPs (0.1 mg/kg, exposure time = 15 s) and FIG. 10B shows quantification of in vivo luciferase expression in livers of mice from FIG.10A. [318] FIG.18A-18C show dose response analyses using bioluminescense mesurements. FIG.18A shows dose-dependent bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of top-performing 4A3-SCC-10 and 4A3-SCC-PH formulated LNPs containing different doses of Fluc mRNA from 0.025 to 0.3 mg/kg, exposure time = 15 s. H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; and FIG. 18B shows quantification of in vivo luciferase expression in livers. FIG.18C shows bioluminescence imaging showing improved temporal resolution upon injection of 4A3-SCC-10/PH LNPs with 0.1 mg/kg Fluc mRNA, exposure time of 0.1 s. [319] FIG. 19A-19D. FIG. 19A shows bioluminescence images of C57BL/6 mice and major organs 6 h after i.v. injection of Fluc mRNA-loaded 4A3-SCC-PH LNPs (0.1 mg/kg, n = 2, exposure time: 15 s) under different conditions. FIG. 19B shows Z-average size, FIG. 19C shows Zeta potential, and FIG.19D shows quantification of in vivo luciferase expression in livers from mice in FIG.19A injected with 4A3-SCC-PH LNPs under different conditions. [320] FIG. 20A-20B. FIG.20A shows the body weight of mice treated with 4A3-SCC- 10 and 4A3-SCC-PH LNPs at a dose of 0.3 mg/kg (i.v.) for 15 days (n=3 biologically independent mice), and FIG. 20B shows the H&E staining of tissue sections of heart, liver, spleen, lung, and kidney after 15 days post injection (scale bar = 200 μm). [321] FIG. 2A-2B show heat maps of luciferase expression and cell viability. FIG. 2A shows a heat map of luciferase expression in ovarian cancer IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). A relative luminescence intensity of >105 was counted for in vitro hit rate calculation. FIG.2B shows a heat map of cell viability IGROV1 cells after treatment with lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (25 ng/well mRNA, n = 4). [322] Cytotoxicity evaluated as described above and as shown in FIG. 2B showed that the top-performing ionizable cationic lipids of the disclosure showed negligible toxicity. Example 11: pKa determination using the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay [323] The pKa of lipid nanoparticle compositions comprising the ionizable cationic lipid compounds of the disclosure (e.g. compounds of Formula (I), (I-a), (I-b), (I-c), e.g., compounds of Tables 1 and 2), were determined with TNS assay following the procedure reported by Jayaraman et. al. (Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo. Angew. Chem. Int. Ed. 2012, 51, 8529-8533), incorporated by reference herein in its entirety. The lipid nanoparticles comprised of an ionizable cationic lipid compound of Formula (I), (I-a), (I-b), (I-c), e.g., a compound of Tables 1 and 2), : DOPE: Chol: DMG-PEG2000 (15:15:30:3) were formulated in DPBS at a concentration of 1.5 mM total lipid. TNS was prepared as a 100 μM stock solution in milliQ water. The nanoparticles were diluted to 30 μM total lipid in 100 μL volume per well in 96-well plates with buffer solutions containing 10 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), 10 mM 4-morpholineethanesulfonic acid (MES), 10 mM ammonium acetate and 130 mM sodium chloride (NaCl), where the pH ranged from 2.5 to 11. The TNS stock solution was added into each well to give a final concentration of 2 μM. After 5 min slight shaking, the Tecan plate reader (Ex/Em = 321/445 nm) was used to read the fluorescence intensity. A sigmoidal fit analysis was applied to the fluorescence data and the pKa was measured as the pH giving rise to half-maximal fluorescence intensity. [324] FIG.11 shows normalized pH titration profiles of Fluc mRNA-loaded LNPs. [325] FIG. 12A-12B show fluorescence (in vitro) and radiance (in vivo) of exemplar LDIL compositions decribed herein. FIG. 12A shows correlation between TNS fluorescence intensity in pH 5.0 buffer of different LNPs and in vivo mRNA delivery efficacy measured by bioluminescence. FIG. 12B shows non-correlation between TNS fluorescence intensity of different LNPs in pH 7.4 buffer and in vivo mRNA delivery efficacy (0.1 mg/kg mRNA, n=3 biologically independent mice). Example 12: LNP Dissociation and membrane fusion of compounds of the disclosure measured by fluorescence resonance energy transfer (FRET) assay. [326] Endosomal mimicking anionic liposomes were used to measure the dissociations of lipid nanoparticles comprising an ionizable cationic lipid compound of the disclosure. The DOPE- conjugated FRET probes DOPE-NBD and DOPE-Rho were added into the lipid nanoparticle formulation at molar ratio 15: 15: 30: 3: 1: 1 (Ionizable lipid compound: DOPE: Chol: DMG- PEG2000: DOPE-NBD: DOPE-Rho) and total lipid final concentration of 3 mM. Other procedures were the same as above mentioned protocol. Endosomal mimicking anionic liposomes were prepared by mixing DOPC: DOPE: Chol (molar ratio = 60: 20: 20) in chloroform followed by rotary evaporation to obtain a thin lipid film. The dried film was subsequently hydrated in PBS (pH 7.4) and sonicated for 20 min at final total lipid concentration of 10 mM. PBS (pH 5.5) was added to black 96-well plates (100 μL/well), and 1 μL lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure were added to each well. Upon incubating at 37°C for 45 min, fluorescence measurements (F) were determined on Tecan plate reader (Ex = 465 nm, Em = 520 nm). lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (with DOPE-NBD and DOPE-Rho inside) incubated in PBS (pH 7.4) were used as negative control (F^min). lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure (with DOPE-NBD and DOPE-Rho inside) in Triton X-100 solutions (2 wt.%) were set as positive control (Fmax). The lipid nanoparticle compositions comprising ionizable cationic lipids of the disclosure dissociation (%) was calculated as (F-F^min) /(F^max-F^min) × 100%. [327] The membrane fusion of ionizable cationic lipid compound of the disclosure was measured by mixing a lipid nanoparticles comprising ionizable cationic lipid compound of the disclosure and endosomal mimicking anionic liposomes containing DOPE-NBD and DOPE- Rho at molar ratio 60: 20: 20: 2: 2 (LDIL: DOPE: Chol: DMG-PEG2000: DOPE-NBD: DOPE- Rho). Other procedures were the simliar to that described above. [328] FIG. 13A-13C show the process and analysis of induced endosomal rupture and membrane fusion with LNP compositions described herein. FIG.13A provides an illustration of lipid fusion and membrane rupture of 4A3-SCC-10/PH and 4A3 LNPs by a FRET assay at pH 5.5 conducted using a pair of DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3- diazole (DOPE-NBD as the donor) and lissamine rhodamine B (DOPE-Rho as the acceptor) incorporated into the single endosomal mimicking liposome. Comparison of membrane fusion with different lipids at pH 5.5 is shown in FIG.13B, and the comparison of membrane fusion with different at pH 5.5 is shown in FIG.13C. [329] 4A3-SCC-10/PH and related LNPs, for example, showed higher membrane fusion efficacy up to 75%, demonstrating stronger trend to fuse and destroy endosomal membranes than their parent lipids and control lipids. 4A3-SCC-PH LNPs and 4A3-SCC-10 LNPs exhibited easier dissociation and release of the mRNA once mixing with endosomal mimics, especially after the treatment with GSH (FIG. 13A-13C). The dissociation efficacy of 4A3- SCC-PH LNPs increased from 30% to 46% after incubated with GSH for 45 min. The observed data indicated that GSH contributes to mRNA release of LDILs formulated LNPs. [330] FIG. 14A-14D show the process and analysis of induced endosomal rupture and membrane fusion with LNP compositions described herein. FIG.14A provides an illustration of dissociation of 4A3-SCC-10/PH formulated LNPs by FRET characterization after mixing with anionic endosomal mimics for 30 min at pH 5.5, where a pair of DOPE-conjugated FRET probes, 7-nitrobenzo-2-oxa-1,3-diazole (DOPE-NBD as the donor) and lissamine rhodamine B (DOPE-Rho as the acceptor) are incorporated into the single endosomal mimicking liposome. Comparison of different LNPs' dissociation at pH 5.5 is shown in FIG. 14B, Comparison of different LNPs dissociation at pH 5.5 after a 45 min at 37°C treatment with 10 mM GSH is shown in FIG.14C, or TCEP in shown FIG.14D. Example 13: Hemolysis assay [331] Membrane-disruptive activity of six 4A3 lipids and related LNPs was evaluated via a hemolysis assay. [332] Sheep erythrocytes were washed and centrifuged several times until the supernatant became colorless before ten times dilution with PBS (pH 5.5 or 7.4). Then erythrocytes were treated with six lipid nanoparticles comprising ionizable cationic lipid compounds of the disclosure at final concentrations of 0.01 mM, 0.25 mM, 0.50 mM, 1.00 mM. After 2 h of incubation, the cells were centrifuged for 10 min (3500 rpm). The absorbance of the supernatant was measured using a Tecan plate reader at the wavelength of 540 nm.1 × PBS was used as the negative control, and 1% Triton X-100 was used as the positive control. [333] 4A3-SCC-10/PH and their LNPs exhibited low hemolytic activity at physiological pH (7.4), while as the increase of concentration, they displayed higher membrane-disruptive ability at acidic endosomal pH (FIG.15A-15B). [334] FIG.15A-15B show the extent of hemolysis of six 4A3 lipids at pH 5.5 (FIG.15A) and pH 7.4 (FIG.15B). Example 14: Co-localization analysis and cellular uptake kinetics study by confocal laser scanning microscope [335] To further verify the higher endosomal escape ability of ionizable cationic lipid compound of the disclosure, co-localization experiment of endo/lysosomes with Cy5-RNA- loaded LNPs was performed. HeLa cells were seeded in 2 cm diameter glass bottom cell culture dishes at a density of 1×10⁴ cells.24 h later, six 4A3-LNPs loaded with Cy5-RNA were prepared following the preparation procedure described above and added to dishes. Upon 4-8 h incubation, cells were washed with PBS three times. The Lysotracker Green DND-26 or Hoechest 33342 was used to stain the endosome/lysosome organelles or nucleus for 30 min. Afterwards, cells were observed with confocal laser scanning microscope (CLSM) with λE^x/λE^m = 504/511 nm (endosome/lysosome organelles), 346/460 nm (nucleus), and 649/670 nm (Cy5-RNA). [336] FIG. 16 shows colocalization analysis (yellow) of endo/lysosomal escape (green) and Cy5-RNA cellular uptake (red) after HeLa cells were treated with different 4A3 LNPs for 4 and 8 h, scale bars = 10 μm. Example 15: Cell isolation for flow cytometry [337] To further quantify the transfection efficiency, hepatocytes were isolated from the injected mice and analyzed them via flow cytometry. To test the tdTomato or Cy5-RNA positive cells in hepatocyte of liver, hepatocytes were isolated using a two-step collagenase perfusion as reported by Cheng et. al. (Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 2020, 15, 313-320) and Liu et. al. (Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing. Nat. Mat.2021, 20, 701-710), incorporated by reference herein in their entirety. The mice were anesthetized by isofluorane and fixed. Then liver was perfused with perfusion medium (Hanks balanced Salt Solution, pH = 7.4) for 8-10 min, and followed by liver digestion medium (Type IV Collagenase) for another 8-10 min. Liver was cut to release the hepatocytes in liver digestion medium (10 mL), and the hepatocytes were washed with cold wash medium and collected by centrifugation (low speed, 50 g, 5 min). The supernatant was decanted and cell pellet were resuspended in PBS and passed through a 100 μm filter. Then, the hepatocytes were washed twice more using cold wash medium, once with 1× PBS and passed through the 100 μm filter again. The hepatocytes were collected by centrifugation (50 g, 5 min) followed by analyzing with the fluorescence-activated cell sorting (FACS, BD LSRFortessa X-20). [338] FIG. 7A-7B. show mCherry expression intensity of IGROV1 cells after treatment with six 4A3 LNPs for mCherry mRNA delivery. FIG 7A shows cellular fluorescence images. FIG.7B shows flow cytometry analysis of mCherry expression intensity of IGROV1 cells after treatment with six 4A3 LNPs for mCherry mRNA delivery after 24 h (80 ng mRNA/well). [339] FIG 8C shows flow cytometry analysis showing fluorescence intensity distribution of Cy5-RNA in livers from mice in FIG.8A. [340] As shown, 99% of all hepatocytes were positive and strongly expressing tdTomato protein at a dose of 0.2 mg/kg Cre mRNA (FIG.21A-21E and FIG.24A-24D). The tdTomato positive cells were readily seen using confocal imaging of liver sections (FIG. 21E). These results demonstrated that 4A3-SCC-PH is an efficient ionizable cationic lipid and could act as a delivery vehicle to treat diverse liver diseases. [341] FIG. 21A-21E show liver specific mRNA delivery. FIG. 21A is a schematic illustration of Cre mRNA delivery and Cre-mediated genetic deletion of the stop cassette to activate tdTomato expression in tdTomato transgenic mice. FIG. 21B and FIG. 21C shows 4A3-SCC-PH LNPs mediated tdTomato expression and quantification of tdTomato protein fluorescence intensity in the liver. The tdTomato fluorescence was recorded 2 days after i.v. injection of Cre mRNA-loaded LNPs (0.2 mg/kg, n = 3). FIG. 21D shows flow cytometry analysis of the percentage of tdTomato positive cells in the liver, and FIG.21E shows confocal microscopy images verifying the efficiency of liver-specific gene editing in this model. [342] FIG. 24A-24D show cell viability of normal cells (3T3 and AML-12) and tumor cells (4T1 and B16F10) after the treatment of six 4A3 LNPs with or without 20 μmol NEM. Example 16: Statistical analysis [343] Statistical analysis was performed based on the one-way analysis of variance (ANOVA) to assess the significance of differences among groups using GraphPad Prism 8.4.0 software. The statistical significance was concluded as follow: NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Example 17. Precise cancer metastases delineation by 4A3-SCC-PH LNPs mediated bioluminescence imaging. [2] GSH is one of the most abundant biological thiols with millimolar cellular concentrations in living organisms, which can behave as a biomarker for cancer detection. There are bioreductive enzymes such as cell surface oxidoreductases that can reduce the disulfide bonds. Because these ionizable cationic lipids harbor a GSH-responsive disulfide bond-bridged linker, they may have an intrinsic advantage in promoting mRNA delivery and release inside cancer cells. Translating Fluc mRNA to luciferase can catalyze D-luciferin to generate bioluminescence signals in tumor cells, thereby improving background-free cancer detection via specific bioluminescence imaging. To demonstrate this proof of concept, we first examined their abilities of 4A3-SCC-10/PH, 4A3-SC-10/PH, and 4A3-SSC-10/PH to deliver Fluc mRNA in cancer cells and normal cells. As shown in Figure 6a and S20, 4A3-SCC-10/PH enabled Fluc mRNA delivery more efficiently than their parent lipids 4A3-SC-10/PH and control lipids 4A3-SSC-10/PH in 4T1 breast cancer cells and B16F10 melanoma cells. On the other hand, 4A3-SC-10/PH LNPs could differentiate mRNA delivery in cancer cells from that in normal cells (AML-12, normal mouse liver cells; 3T3, mouse embryo fibroblast cells), exhibiting much higher bioluminescence. [3] FIG. 25A-25B show cancer metastases delineation by 4A3-SCC-PH LNPs mediated bioluminescence imaging. FIG 25A is a schematic showing the establishment of the 4T1 breast cancer metastasis model in BALB/cmice and the B16F10 melanoma metastasis model in C57BL/6 mice. FIG.25B is an illustration of cancer metastasis detection via bioluminescence imaging. FIG.25C shows bioluminescence images, white-light photos of mouse (i), or ex vivo organs (ii) of 4T1 breast cancer metastasis-bearing BALB/c mice and B16F10 melanoma metastasis-bearing C57BL/6 mice 6 h after injection of PBS or Fluc mRNA-loaded 4A3-SCC- PH LNPs (0.1 mg/kg), exposure time: 15 s. H: heart; Li: liver; S: spleen; Lu: lung; K: kidney; In: intestine. FIG 25D shows bioluminescence images and white-light photos of isolated melanoma metastases from intestine, and FIG. 25E shows Signal-to-Noise Ratio (SNR) of relative metastasis in different organs in mice generated as described in FIG.25A-25C. FIG. 25F-25G shows H&E staining analysis of harvested tissue sections from mice generated as described in FIG.25A-25C. T: tumor, N: normal, scale bar = 250 μm. ABBREVIATIONS LNP lipid nanoparticle NHP non-human primates RIPA buffer Radioimmunoprecipitation assay buffer BCA assay Bicinchoninic acid assay CV coefficient of variance HPLC High performance liquid chromatography DFA Difluoroacetic acid MS mass spectrometry PBS phosphate-buffered saline FBS Fetal bovine serum IVIS in vivo imaging system INCORPORATION BY REFERENCE [344] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS [345] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS What is claimed is: 1. A compound of Formula (I):

wherein: R^D1 is H or C₁-C₄ alkyl; x1 and x2 are each independently 1, 2, or 3; and R is

wherein R^SCC, R^SSC and R^SC are each a linear or branched C₄-C₂₀ alkyl; and nSC is an integer from 1-10. 2. The compound of claim 1, wherein x1 and x2 are each 2. 3. The compound of claim 1 or 2, wherein R^D1 is methyl. 4. The compound of any one of claims 1-3, wherein nSC is 4, 5, or 6. 5. The compound of any one of claims 1-4, wherein R^SCC, R^SSC, and R^SC are selected from:

6. The compound of any one of claims 1-4, wherein R^SCC, R^SSC, and R^SC are selected from:

7. The compound of claim 1, having the Formula (I-a):

9. The compound of claim 1 or 4, having the Formula (I-b):

10. The compound of claim 0, selected from:

12. The compound of claim 1, selected from:

. 13. A compound selected from Table 2. 14. A lipid nanoparticle composition comprising a lipid component comprising the compound of any one of the preceding claims. 15. The lipid nanoparticle composition of claim 14, wherein the lipid component comprises the compound in an amount of from about 10 mol% to about 20 mol % of the total lipids in the lipid component. 16. The lipid nanoparticle composition of claim 15, wherein the lipid component comprises the compound in an amount of about 15 mol% of the total lipids in the lipid component. 17. The lipid nanoparticle composition of any one of claims 14-16, wherein the lipid component further comprises a phospholipid. 18. The lipid nanoparticle composition of 17, wherein the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn- glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl- sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3- phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), 1-stearoyl-2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, and lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) sphingomyelin. 19. The lipid nanoparticle composition of claim 17, wherein the phospholipid is DOPE or DSPC. 20. The lipid nanoparticle composition of any one of claims 17-19, wherein the lipid component comprises the phospholipid in an amount of about 15 mol% of the total lipids in the lipid component. 21. The lipid nanoparticle composition of any one of claims 17-19, wherein the lipid component comprises the phospholipid in an amount of from about 10 mol% to about 20 mol% of the total lipids in the lipid component. 22. The lipid nanoparticle composition of any one of claims 14-21, wherein the lipid component further comprises a PEG lipid. 23. The lipid nanoparticle composition of claim 22, wherein the PEG lipid is selected from: 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG- DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG- distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). 24. The lipid nanoparticle composition of claim 22, wherein the PEG lipid is 1,2- dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG). 25. The lipid nanoparticle composition of any one of claims 22-24, wherein the lipid component comprises the PEG lipid in an amount of from about 20 mol% to about 40 mol % of the total lipids in the lipid component. 26. The lipid nanoparticle composition of any one of claims 22-25, wherein the lipid component comprises the PEG lipid in an amount of about 30 mol % of the total lipids in the lipid component. 27. The lipid nanoparticle composition of any one of claims 14-26, wherein the lipid component further comprises a sterol. 28. The lipid nanoparticle composition of claim 27, wherein the sterol is cholesterol.

29. The lipid nanoparticle composition of claim 27 or 28, wherein the lipid component comprises the sterol in an amount of from about 0.5 mol% to about 5 mol % of the total lipids in the lipid component. 30. The lipid nanoparticle composition of claim 27 or 28, wherein the lipid component comprises the sterol in an amount of about 3 mol % of the total lipids in the lipid component. 31. The lipid nanoparticle composition of any one of claims 14-30, wherein the lipid component comprises further comprises an additional cationic ionizable cationic lipid, a permanently cationic lipid, or an anionic lipid. 32. The lipid nanoparticle composition of any one of claims 14-31, wherein the lipid component further comprises a payload. 33. The lipid nanoparticle composition of claim 32, wherein the payload comprises a polypeptide or a protein. 34. The lipid nanoparticle composition of claim 32, wherein the payload comprises a nucleic acid. 35. The lipid nanoparticle composition of claim 34, wherein the nucleic acid is selected from an siRNA, a miRNA, a pri-miRNA, a messenger RNA (mRNA), a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), a guide RNA, a double stranded DNA (dsDNA), a single stranded DNA (ssDNA), a single stranded RNA (ssRNA), and a double stranded RNA (dsRNA). 36. The lipid nanoparticle composition of claim 35, wherein the payload comprises a small interfering RNA (siRNA). 37. The lipid nanoparticle composition of claim 35, wherein the payload comprises an mRNA. 38. The lipid nanoparticle composition of claim 37, wherein the mRNA encodes a gene- editing system or component thereof. 39. The lipid nanoparticle composition of claim 37, wherein the gene-editing system or component thereof comprises a cluster regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), and a guide RNA.

40. A pharmaceutical composition comprising a lipid nanoparticle composition of any one of claims 14-39 and a pharmaceutically acceptable excipient. 41. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 32-39 or the pharmaceutical composition of claim 40. 42. A method of delivering a payload to a target organ of a subject in need thereof, the method comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of claims 32-39 or the pharmaceutical composition of claim 0. 43. The method of claim 41 or 42, wherein the subject is a primate. 44. The method of claim 41 or 42, wherein the subject is a human. 45. The method of any one of claims 42-44, wherein the method comprises selectively delivering the payload to a target organ. 46. The method of any one of claims 42-44, wherein the method comprises selectively delivering the payload to a target cell. 47. The method of any one of claims 42-46, wherein the target organ is the liver, lung, heart, or spleen. 48. The method of any one of claims 42-47, wherein the target organ is the liver. 49. The method of any one of claims 42-47, wherein the target organ is the lung. 50. The method of any one of claims 42-49, wherein the payload is an mRNA and wherein the selectively delivering results in expression of a protein encoded by the mRNA in a cell of the target organ. 51. The method of any one of claims 42-49, wherein the payload is a polynucleotide encoding a gene product and wherein the selectively delivering results in expression of the gene product in a cell of the target organ and optionally wherein the gene product is functional in the cell. 52. The method of any one of claims 42-49, wherein the payload comprises an mRNA encoding a gene-editing system or component thereof and wherein the selectively delivering results in altered expression of a protein targeted by the gene-editing system in a cell of the target organ.