Substituted amidine ionizable lipid compounds, methods of preparation thereof, lipid nanoparticles (LNPS) comprising the same, and methods of use thereof

Abstract

In one aspect, the present disclosure relates to certain ionizable lipid compounds and methods of preparation thereof. In another aspect, the present disclosure relates to lipid nanoparticles (LNPs) comprising certain ionizable lipid compounds of the present disclosure and methods of use thereof.

Description

[0001]Attorney Docket No.046483-7437WO1(03698) TITLE OF THE INVENTION Substituted Amidine Ionizable Lipid Compounds, Methods of Preparation Thereof, Lipid Nanoparticles (LNPs) Comprising the Same, and Methods of Use Thereof CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.63 / 589,051, filed October 10, 2023, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under TR002776 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The XML file named “046483-7437WO1 - Sequence Listing.xml” created on October 7, 2024, comprising 3.0 Kbytes, is hereby incorporated by reference in its entirety. BACKGROUND Messenger RNA (mRNA)-based therapeutics hold great promise to prevent and treat a variety of diseases. However, safe and efficient delivery of mRNA-based vaccines and therapeutics to target tissues remains challenging. Lipid nanoparticles (LNPs) represent the most clinically advanced non-viral gene delivery system with one approved small interfering RNA (siRNA)-delivering DLin-MC3-DMA (MC3) LNP (Onpattro) and two approved mRNA vaccine-delivering SM-102 and ALC-0315 LNPs (BNT162b2 and mRNA-1273. LNPs generally comprise four components, including at least one cationic lipid, at least one phospholipid, at least one polymer-conjugated (e.g., PEGylated) lipid (PEG-lipid) and cholesterol. Among which, the cationic lipid plays an essential role in protecting and transporting nucleic acids. Cationic lipids typically comprise a cationic head and several lipophilic tails. The common headgroups include amines (from primary to quaternary), guanidine, and N- heterocyclic groups, which are involved in binding with negatively charged mRNAs during LNP formulation and interacting with anionic membrane phospholipids during endosome trafficking. While permanently charged cationic lipids (e.g., DOTAP) were once widely used - 1 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) in LNPs, they are now largely replaced by the state-of-the-art ionizable cationic lipids (e.g., MC3 and SM-102) due to their pH-dependent protonation property, enhanced delivery efficiency, and improved biocompatibility. The lipid tails impact lipophilicity, fluidity, fusogenicity and biodegradability, thereby influencing the endosomal escape, potency, and toxicity of LNPs. Previous studies have suggested that branched tails that enable lipids to adopt a more cone-shaped structure are favorable for mRNA delivery. Additionally, ester bond-bearing tails are highly recommended for in vivo applications owing to the degradability. The structure of the cationic lipid is critical as subtle changes (e.g., one atom or group) greatly affect the cellular uptake, endosomal escape, tissue targeting, cellular tropism, and potency of LNPs. Therefore, to discover efficacious cationic lipids and study structure-activity relationships (SARs), dozens or even hundreds of chemically divergent candidates are usually needed for comparison. For example, over 60 rationally designed cationic lipids were synthesized by standard organic chemistry methods and tested in vivo for years before elucidating valuable structural criteria and identifying the DLin-MC3-DMA (MC3) lipid that was later approved by the Food and Drug Administration (FDA) for siRNA delivery. Alternatively, combinatorial chemistries, such as aza-Michael addition, epoxide- mediated ring-opening reaction (e.g., SN2), and Ugi multi-component reaction (MCR) have been utilized to accelerate these processes. Despite the successful discovery of some efficacious lipids, these combinatorial chemistries are generally performed under harsh conditions for several days and yield non-degradable lipids. Hence, there is still vast interest to develop new combinatorial chemistries to rapidly generate structurally diverse cationic lipids in order to study SARs for guiding the design of the next-generation delivery materials and fulfill the broader applications of mRNA therapeutics. Thus, there is a need in the art for synthetic methods which enable rapid and facile synthesis of degradable cationic lipids without the requirement for harsh conditions and / or specialized instruments. Further, there is a need in the art for lipid nanoparticles (LNPs) comprising said lipids, and methods of use thereof for the treatment, prevention, and / or amelioration of one or more diseases and / or disorders. The present disclosure addresses these needs. BRIEF SUMMARY In one aspect, the disclosure provides a compound of formula (I), or a salt or solvate - 2 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) thereof: (I), are defined In (Ia): R2N T1R1a. In certain embodiments, the (I) is a compound of formula (Ib): . In certain is a compound of formula (Ic): . In certain a compound of formula (Id): . In another (LNP) composition. In certain embodiments, the LNP comprises at least one ionizable lipid compound of the disclosure. In certain embodiments, the LNP comprises at least one helper lipid. In certain embodiments, the LNP comprises at least one cholesterol lipid. In certain embodiments, the LNP comprises at least one polymer-conjugated lipid and / or a modified derivative thereof. In another aspect, the disclosure provides a method of preparing an ionizable lipid of formula (II): (II), are defined In certain embodiments, the method comprises: (a) contacting an amine compound of formula (A1) or (A2): - 3 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) , wherein Z, R2, R3a, and a , wherein R5a, R5b, R5c, R5d, R5e, and herein; to form a thiolate (b) contacting the thiolate intermediate and a compound of formula compound of formula (C1), (C2), or (C3): , wherein R6a, In a comprising the lipid nanoparticle of the disclosure and at least one pharmaceutically acceptable carrier. In another aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the disclosure. In another aspect, the disclosure provides a method of delivering at least one cargo molecule to the lung and / or spleen of a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the disclosure. In certain embodiments, the LNP of the disclosure comprises at least one cargo molecule. BRIEF DESCRIPTION OF THE FIGURES The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application. FIGs.1A-1B: fast and facile synthesis of AID-lipids via a tandem multi-component reaction (T-MCR) for mRNA delivery. FIG.1A: T-MCR based on amine-thiol-acrylate conjugation between amines, Traut’s reagent and alkyl acrylates. The reaction scheme and mechanism of T-MCR as well as chemical structures of amines (1-25) and alkyl acrylates (O9-O16) are shown. FIG.1B: scheme of the formulation and application of mRNA-loaded - 4 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) LNPs. AID-lipid formulated LNPs can aid the delivery of mRNA-based vaccines as well as protein replacement and gene editing therapeutics to target tissues. FIGs.2A-2B: optimization of amine–thiol–acrylate conjugation. FIG.2A: model reaction between amine 19, Traut’s reagent and acrylate O12. FIG.2B: reaction kinetics under different conditions. The reaction was monitored by LC-MS (0-2 min, 0% ACN; 2-10 min, 0-100% ACN; 10-11 min, 100% ACN). FIGs.3A-3H: high-throughput synthesis and screening of AID-lipids. FIG.3A: fast and high-throughput synthesis of 100 AID-lipids. All reactions were performed separately and simultaneously in 1.5 mL Eppendorf tubes using ultrasound-mediated mixing for 1 h. FIG.3B: AID-LNPs mediated mLuc transfection in vitro (n= 2, initial screening). mLuc- loaded AID-LNPs were formulated by pipette mixing and used to transfect HepG2 cells at a dose of 15 ng mRNA / well for 24 h. The performance was evaluated based on luminescence readout (>100,000 units, outstanding; 1,000 - 100,000 units, acceptable). FIG.3C: Luciferase expression shown using a heat map. FIG.3D: distribution of outstanding AID-lipids based on amines. FIG.3E: analysis of relative hit rates (percentage in the group with >100,000 units – percentage in library) based on amine subclasses. FIG.3F: distribution of outstanding AID- lipids and analysis of relative hit rates based on acrylate tails. FIG.3G: top 8 AID-LNPs mediated dose-dependent mLuc transfection in vitro (n= 2, initial screening). HepG2 cells were treated with 3.7, 7.5 or 15 ng mRNA / well for 24 h. FIG.3H: the top 8 AID-LNPs mediated mRNA delivery in vivo (n= 2, initial screening). AID-LNPs were i.m. injected into mice (2 µg mLuc / injection) and total flux of luminescence at the injection site was quantified at 4 h post-treatment. A representative in vivo bioluminescence image is shown. Data are presented as mean ± SD. FIG.4: cell viability. HepG2 cells were treated with mLuc-loaded AID-LNPs at a dose of 15 ng mRNA / well for 24 h (n= 2, initial screening). The dash line indicates 80% cell viability. Data are presented as mean ± SD. FIG.5: the impact of alkyl substituent on mRNA delivery. The elongation of alkyl substituent on the aniline generally increased mRNA delivery efficiency. HepG2 cells were treated with mLuc-loaded AID-LNPs at a dose of 15 ng mRNA / well for 24 h (n= 2, initial screening). Data were adapted from FIG.3B. Data are presented as mean ± SD. ns, no significance; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. FIG.6: the impact of atom polarity on mRNA delivery. Replacement of one carbon with oxygen atom in the alkyl substituent dramatically decreased mRNA delivery efficiency. HepG2 cells were treated with mLuc-loaded AID-LNPs at a dose of 15 ng mRNA / well for 24 - 5 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) h (n= 2, initial screening). Data were adapted from FIG.3B. Data are presented as mean ± SD. ns, no significance; *p<0.05; ***p<0.001; ****p<0.0001. FIGs.7A-7E: stepwise optimization of 12T-O14 LNP formulation. HepG2 cells were treated with 12T-O14 LNP formulated with different weight ratio of excipients at a dose of 15 ng mLuc / well for 24 h (n= 2, initial screening). Mice were i.v. injected with 12T-O14 LNP at a dose of 2 μg mLuc / mouse (n= 2, initial screening). The best group in each graph (indicated by the rectangle) was chosen for subsequent optimization. When optimizing DMG- PEG, a lead group from in vivo screening was chosen, as PEGylation dramatically affects LNP circulation and distribution, which may not be recapitulated in vitro.12T-O14 LNP without DMG-PEG was not tested in mice due to its rapid precipitation. Data are presented as mean ± SD. FIG.8: TNS assay to determine pKa values of 12T-O14 LNP and DOTAP LNP. TNS fluorescent signal corresponds to ionization. pKa is calculated as the pH corresponding to half of the maximum TNS fluorescence value. FIG.9: comparing the immunostimulatory effect of 12T-O14 LNP with other benchmark LNPs. RAW264.7 cells were treated with various LNPs for 24 h at a dose of 50 ng mRNA / 10,000 cells. The concentration of TNF-α and IL-6 in the supernatant was determined by ELISA. Only C12-200 LNP triggered detectable level of IL-6. Data are presented as mean ± SD (n = 3). FIGs.10A-10C: Flow cytometry analysis of GFP knockdown in HepG2-GFP cells. FIGs.10A-10B: gating strategies and representative results. HepG2-GFP cells were treated with LNP-formulated GFP siRNA (20 nM) for 48 h before flow cytometry analysis. FIG. 10C: statistical analysis of mean fluorescence intensity (MFI) of HepG2-GFP cells. Data are presented as mean ± SD (n = 3). FIGs.11A-11G: characterization and in vitro evaluation of 12T-O14 LNP. FIG.11A: a representative cryo-EM image of 12T-O14 LNP. Scale bar, 50 nm. FIG.11B: cytotoxicity of LNPs (n=3). HepG2 cells were treated with the indicated mRNA dose for 24 h. FIG.11C: LNPs mediated mLuc transfection in vitro (n=3). HepG2 cells were treated with the indicated mRNA dose for 24 h. FIG.11D: representative confocal laser scanning microscope (CLSM) images of cellular uptake. HepG2 cells were treated with DiO-labelled LNPs for 2 h before staining with LysoTracker Deep Red and Hoechst 33342 for imaging. Scale bars, 20 μm. FIG.11E: chemical structures of cationic lipids and proposed mechanism of benzene ring- mediated cone shape formation.12T-O14 adopts a more conic shape due to the existence of the bulky benzene ring that acts as a wedge to increase the distance of the two tails. - 6 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Consequently, 12T-O14 forms a more cone-shaped ion pair with the anionic membrane phospholipid that can enhance endosome disruption. FIG.11F: structural illustration of cationic lipids. FIG.11G: hemolysis of LNPs at pH 7.4 or 6.0 (n=3). RBCs were incubated with LNPs at 37 °C for 1 h before the supernatant was transferred into a clear 96-well plate (left) to determine the absorption at 540 nm (right). Data are presented as mean ± SD. FIG.12A-12C: liver function analysis. FIG.12A: serum alanine aminotransferase (ALT). FIG.12B: serum aspartate aminotransferase (AST). FGF21 mRNA-loaded LNPs were i.v. injected into mice (5 μg mRNA / mouse) and serum was collected before or 24 h after treatment for analysis. Data are presented as mean ± SD (n = 3). FIG.12C: H&E staining of major organs; scale bar: 150 µm; major organs collected 24 h after treatment. FIG.13: in vivo and ex vivo bioluminescence imaging of mice treated with mLuc- loaded MC3 / 12T-O14 LNP at a total lipids:mRNA weight ratio of 5:1. LNPs were i.v. injected into mice (2 μg mLuc / mouse) and the total flux of the spleen was quantified at 4 h post treatment. Data are presented as mean ± SD (n = 3). FIGs.14A-14B: representative CLSM images of lung sections. mLuc (FIG.14A) or Cre mRNA (FIG.14B) (5 μg / mouse) was delivered into Ai6 mice by lung-tropic MC3 / 12T- O14 LNP via i.v. injection. On day 2, mice were euthanized and lungs were harvested for analysis. Cryosections (7 μm) were prepared, probed with anti-CD31 antibody (R&D, AF3628) overnight at 4 °C and then incubated with Alexa Fluor™ 647-conjugated donkey anti-goat secondary antibody (Thermo, Cat#A-21447) at RT for 1 h. Nuclei were stained by DAPI (1 μM) for 5 min. Images were taken using a confocal laser scanning microscope. Scale bars, 100 μm. FIG.15: representative CLSM images of spleen sections. Cre or mLuc mRNA (5 μg / mouse) was delivered into Ai6 mice by spleen-tropic MC3 / 12T-O14 LNP via i.v. injection. On day 2, mice were euthanized and spleens were harvested for analysis. Cryosections (7 μm) were prepared and nuclei were stained by DAPI (1 μM) for 5 min. Images were taken using a confocal laser scanning microscope. Scale bars, 100 μm. FIG.16: Gating strategy and antibody panel for lung cells. Gating scheme for the analysis of ZsGreen1 expression in all live cells, endothelial cells (PECAM+), macrophages (CD11b+), monocytes (CD64+), neutrophils (Ly6G+) and DCs (CD11c+). FIGs.17A-17E: 12T-O14 aids local mRNA vaccine and systemic mRNA drug delivery. FIG.17A: in vivo bioluminescence imaging of mice after LNP-mediated local mLuc delivery (n=3). LNPs were i.m. injected into mice (2 µg mLuc / injection) and the total flux of luminescence at the injection site was quantified at 4 h post-treatment. FIG.17B: LNP- - 7 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) mediated SARS-CoV-2 Spike mRNA vaccine delivery (n=5). Mice were i.m. vaccinated twice using a prime-boost strategy at a three-week interval. Anti-Spike IgG titers were determined by enzyme-linked immunosorbent assay (ELISA). FIGs.17C-17D: In vivo and ex vivo bioluminescence imaging of mice after LNP-mediated systemic mLuc delivery (n=3). LNPs were i.v. injected into mice (2 µg mLuc / mouse) and total flux of whole body or excised organs was quantified at 4 h post-treatment. FIGs.17E: LNP-mediated FGF21 mRNA delivery (n=3). LNPs were i.v. injected into mice (5 µg mRNA / mouse) and serum FGF21 concentration was determined by ELISA. Data are presented as mean ± SD. FIG.18: Hematoxylin and Eosin (H&E) staining of lung tissues. Cas9 mRNA and sgTTR (4:1, wt:wt) were co-delivered into C57BL / 6 mice by lung-tropic MC3 / 12T-O14 LNP via four i.v. injections at a total RNA dose of 1 mg / kg. On day 7, lungs were harvested for H&E staining. No histological abnormality was observed in lung-tropic MC3 / 12T-O14 LNP treated mice compared to untreated control mice. Scale bars, 200 μm. FIG.19: MC3 LNP-mediated gene editing. Cas9 mRNA and sgTTR (4:1, wt:wt) were co-delivered into C57BL / 6 mice by MC3 LNP at a total RNA dose of 1 mg / kg. On day 7, Livers and lungs were harvested to determine the indels of TTR by Next-Generation Sequencing (NGS). MC3 LNP induced roughly 6% indels in the liver but no editing in the lung (indels < 0.05%). The indels in naive C57BL / 6 mice are below 0.05% in the database. Data are presented as mean ± SD (n = 3). FIGs.20A-20E: 12T-O14 aids liver-tropic LNPs to selectively target lung or spleen. FIG.20A: in vivo and ex vivo bioluminescence imaging of mice treated with mLuc-loaded MC3 / 12T-O14 LNP or MC3 / DOTAP LNP (n=3). LNPs with a total lipids:mRNA weight ratio of 40:1 were i.v. injected into mice (2 µg mLuc / mouse) and the total flux of the lung was quantified at 4 h post-treatment. FIGs.20B-20D: in vivo and ex vivo bioluminescence imaging of mice treated with mLuc-loaded MC3 / 12T-O14 LNP (n=3). LNPs with different total lipids:mRNA weight ratios were i.v. injected into mice (2 µg mLuc / mouse) and the total flux of the region of interest (ROI) was quantified at 4 h post-treatment. Total flux at the liver region for MC3 LNP, lung region for MC3 / 12T-O14 LNP (40:1), lung plus spleen region for MC3 / 12T-O14 LNP (20:1) and spleen region for MC3 / 12T-O14 LNP (10:1) was plotted in c. MC3 LNP was used as a positive control for liver transfection. The dash lines indicate total flux of 1×108p / s. FIG.20E: a schematic illustration of 12T-O14 supplementation and lipids / mRNA weight ratio manipulation enabled selective lung and / or spleen transfection. Data are presented as mean ± SD. FIG.21: Gating strategy and antibody panel for spleen cells. Gating scheme for the - 8 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) analysis of ZsGreen1 expression in leukocytes (CD45+), macrophages (CD11b+), monocytes (CD64+), neutrophils (Ly6G+) and DCs (CD11c+). FIGs.22A-22G: 12T-O14 aids liver-tropic LNPs to deliver functional mRNAs to non-liver cells. FIG.22A: a schematic illustration of Cre-mediated genetic recombination to turn on ZsGreen1 in Ai6 mice. FIG.22B: flow cytometry analysis of ZsGreen1+cells within defined cell type populations of the lung after lung-tropic MC3 / 12T-O14 LNP-mediated Cre mRNA delivery (n=3). Cre mRNA (5 µg / mouse) was delivered into Ai6 mice by lung-tropic MC3 / 12T-O14 LNP via i.v. injection. On day 2, mice were euthanized and lungs were harvested for analysis. FIG.22C: CRISPR / Cas9-mediated gene editing in the lung (n=3). Cas9 mRNA and sgTTR (4:1, wt:wt) were co-delivered into C57BL / 6 mice by lung-tropic MC3 / 12T-O14 LNP via four i.v. injections at a total RNA dose of 1 mg / kg. On day 7, lungs were harvested to determine the indels of TTR by Next-Generation Sequencing (NGS). FIG. 22D: flow cytometry analysis of ZsGreen1+cells within defined cell type populations of the spleen after spleen-tropic MC3 / 12T-O14 LNP-mediated Cre mRNA delivery (n=3). Cre mRNA (5 µg / mouse) was delivered into Ai6 mice by spleen-tropic MC3 / 12T-O14 LNP via i.v. injection. On day 2, mice were euthanized and spleens were harvested for analysis. FIG. 22E: spleen-tropic MC3 / 12T-O14 LNP-mediated SARS-CoV-2 Spike mRNA vaccine delivery (n=3). C57BL / 6 mice were i.v. vaccinated twice using a prime-boost strategy at a three-week interval. FIGs.22F-22G: C57BL / 6 mice were i.v. vaccinated twice with spleen- tropic MC3 / 12T-O14 LNP or spleen-SORT LNP using a prime–boost strategy with a three- week interval. Anti-Spike IgG titres were determined by ELISA (FIG.22F). NAb titres were determined by a lentivirus-based pseudovirus neutralization assay (FIG.22G). The neutralization titre 50% (NT50) was defined as the greatest serum dilution at which the luminescence was reduced by 50% relative to control cells treated with the pseudovirus in the absence of serumAnti-Spike IgG titers were determined by ELISA. Data are presented as mean ± SD. FIG.23: Cell viability. HepG2 cells were treated with mLuc-loaded AID-LNPs at a dose of 15 ng mRNA / well for 24 h (n= 2 biologically independent samples, initial screening). The dash line indicates 80% cell viability. Data are presented as mean ± SD. FIG.24: Comparing in vivo mRNA delivery efficiency between unpurified and purified AID-lipids (n= 2 biologically independent samples). Selected AID-lipids were purified using a CombiFlash NextGen 300+ chromatography system and target products were confirmed by mass spectrometry. AID-LNPs were i.m. injected into mice (2 µg mLuc / injection) and total flux of luminescence at the injection site was quantified at 4 h post- - 9 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) treatment. A representative in vivo bioluminescence image is shown. Data are presented as mean. FIGs.25A-25E: Synthesis of 12T-O18a and 12T-O18b and comparison of their mRNA delivery efficiency with 12T-O14. FIG.25A: Synthetic route of 12T-O18a. FIG. 25AB: Synthetic route of 12T-O18b. FIG.25C: AID-LNPs mediated mLuc transfection in vitro (n= 3 biologically independent samples). mLuc-loaded AID-LNPs were formulated by pipette mixing and used to transfect HepG2 cells at a dose of 15 ng mRNA / well for 24 h. FIG.25D: Cell viability. HepG2 cells were treated with mLuc-loaded AID-LNPs at a dose of 15 ng mRNA / well for 24 h (n= 3 biologically independent samples). The dash line indicates 80% cell viability. FIG.25E: AID-LNPs mediated mRNA delivery in vivo (n= 2 biologically independent samples). AID-LNPs were i.m. injected into mice (2 µg mLuc / injection) and total flux of luminescence at the injection site was quantified at 4 h post-treatment. Data are presented as mean ± SD. FIG.26: Size distribution of 12T-O14 LNP. Hydrodynamic size of 12T-O14 LNP was analyzed by dynamic light scattering using a Zetasizer Nano ZS90. The refractive index was set as 1.33 when data we collected. FIG.27: Hemolytic activity of 12T-O14 LNP in the presence of 10% serum. RBCs were incubated with 12T-O14 LNP in PBS buffer (pH 7.4) supplemented with or without 10% serum at 37 °C for 1 h before the supernatant was transferred into a clear 96-well plate to determine the absorption at 540 nm. The adsorption of serum proteins onto 12T-O14 LNP reduced its hemolytic activity. Data are presented as mean ± SD (n=3 biologically independent samples). unpaired two-tailed Student’s t-test. FIG.28: NAbs in SARS-CoV-2 mRNA-loaded LNP-vaccinated mice. Mice were i.m. vaccinated twice using a prime-boost strategy at a three-week interval. Serum was collected from mice two weeks post boost vaccination. NAb titers were determined by a lentivirus- based pseudovirus neutralization assay. The neutralization titer 50% (NT50) was determined as the greatest serum dilution at which the luminescence was reduced by 50% relative to control cells treated with the pseudovirus in the absence of serum. Data are presented as mean ± SD (n=5 biologically independent samples). one-way ANOVA with Tukey’s correction. FIG.29: In vivo and ex vivo bioluminescence imaging of mice treated with mLuc- loaded spleen-SORT LNP. LNPs were i.v. injected into mice (2 μg mLuc / mouse) and images were taken at 4 h post-treatment. Strong off-target expression in the liver was observed. Data are presented as mean ± SD (n = 3 biologically independent samples). - 10 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) DETAILED DESCRIPTION OF THE INVENTION Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter. Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. Description - 11 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) In one aspect, the present disclosure relates to a one-pot tandem multi-component reaction (T-MCR). In certain aspects, this T-MCR enables fast and facile synthesis of Amidine-Incorporated Degradable (AID) lipids. This T-MCR is based on rationally designed amine-thiol-acrylate conjugation between amines, Traut’s reagent (i.e., 2-iminothiolane hydrochloride) and alkyl acrylates (FIGs.1A-1B). Mechanistically, in certain embodiments, a primary amine (i.e., a weak nucleophile) is rapidly converted to a basic amidine through the nucleophilic ring-opening of 2-iminothiolane and generates a thiol (a strong nucleophile) in situ, which immediately reacts with acrylate via Michael addition to afford the AID-lipid. The simplicity of T-MCR enabled the synthesis of 100 chemically diverse AID-lipids in approximately one hour at room temperature (RT). Through investigating the SARs, a new tail-like amino head-ring-alkyl aniline was identified which generally provides efficacious AID-lipids. Mechanistic studies demonstrated that the bulk benzene ring could greatly enhance membrane disruption and endosomal escape by aiding the AID-lipid to adopt a more conical shape. This lead AID-lipid 12T-O14 efficiently aided intramuscular delivery of mRNA vaccines and systemic delivery of mRNA therapeutics without noticeable toxicity. Moreover, it has been demonstrated herein that 12T- O14 serves as a superior supplementary lipid to redirect liver-tropic LNPs to selectively target the lung or spleen via simple adjustment of the formulation, which enabled CRISPR / Cas9-mediated gene editing in the lung and mRNA vaccine delivery to the spleen. Definitions The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “adjuvant” as used herein is defined as any molecule to enhance an antigen- specific adaptive immune response. The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others. The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl - 12 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith. The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to – C ^CH, -C ^C(CH3), -C ^C(CH2CH3), -CH2C ^CH, -CH2C ^C(CH3), and -CH2C ^C(CH2CH3) among others. The term “alkylene” or “alkylenyl” as used herein refers to a bivalent saturated aliphatic radical (e.g., -CH2-, -CH2CH2-, and -CH2CH2CH2-, inter alia). In certain embodiments, the term may be regarded as a moiety derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from the same (e.g., - CH2-) different (e.g., -CH2CH2-) carbon atoms. Similarly, the terms “heteroalkylenyl”, “cycloalkylenyl”, “heterocycloalkylenyl”, and the like, as used herein, refer to a divalent radical of the moiety corresponding to the base group (e.g., heteroalkyl, cycloalkyl, and / or - 13 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) heterocycloalkyl). A divalent radical possesses two open valencies at any position(s) of the group, wherein each radical may be on a carbon atom or heteroatom. Thus, the divalent radical may form a single bond to two distinct atoms or groups, or may form a double bond with one atom. The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein. The term “amino group” as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group. - 14 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids. The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof. The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). It has been found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos.5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cat-ionic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, C18alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA. The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, - 15 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group. A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced. As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In particular, in the case of a mRNA, and “effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid as relating to a mRNA is an amount sufficient to produce the desired effect, e.g., mRNA-directed expression of an amount of a protein that causes a desirable biological effect in the organism within which the protein is expressed. For example, in some embodiments, the expressed protein is an active form of a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the protein that is normally expressed in the cell type of a healthy individual. For example, in some embodiments, the expressed protein is a protein that is normally expressed in a cell type - 16 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) within the body, and the therapeutically effective amount of the mRNA is an amount that produces a similar level of expression as observed in a healthy individual in an individual with aberrant expression of the protein (i.e., protein deficient individual). Suitable assays for measuring the expression of an mRNA or protein include, but are not limited to dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art. The term “encode” as used herein refers to the product specified (e.g., protein and RNA) by a given sequence of nucleotides in a nucleic acid (i.e., DNA and / or RNA), upon transcription or translation of the DNA or RNA, respectively. In certain embodiments, the term “encode” refers to the RNA sequence specified by transcription of a DNA sequence. In certain embodiments, the term “encode” refers to the amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In certain embodiments, the term “encode” refers to the amino acid sequence specified by transcription of DNA to mRNA and subsequent translation of the mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may comprise a direct transcription or translation product. In certain embodiments, the encoded product may comprise post-translational modifications understood or reasonably expected by one skilled in the art. The term “fully encapsulated” indicates that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particle is degraded in a treatment that would normally degrade 100% of free active agent or therapeutic agent, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particle is degraded. In the context of nucleic acid therapeutic agents, full encapsulation may be determined by an OLIGREEN® assay. OLIGREEN® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). RiboGreen is an ultra-sensitive fluorescent nucleic acid stain for quantitating RNA in solution. “Fully encapsulated” also indicates that the lipid particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration. The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. - 17 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly- halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like. The term “helper lipid” as used herein refers to a lipid capable of increasing the effectiveness of delivery of lipid-based particles such as cationic lipid-based particles to a target, preferably into a cell. The helper lipid can be neutral, positively charged, or negatively charged. In certain embodiments, the helper lipid is neutral or negatively charged. Non- limiting examples of helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl- 2-oleoyl-sn-glycero-3phosphocholin (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The term “heteroalkyl” as used herein by itself or in combination with another term, means, unless otherwise stated, a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., O, N, P, and S) may be placed at any interior position of the heteroalkyl group or at either terminal position at which the group is attached to the remainder of the molecule. The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, - 18 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein. Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N- hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3- dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), - 19 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like. The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl can include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited, to the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, - 20 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein. The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups. As used herein, the term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (C1-C4)hydrocarbyl means the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group. The term “immune cell,” as used herein refers to any cell involved in the mounting of an immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells (e.g., dendritic cells and macrophages), monocytes, neutrophils, eosinophils, basophils, and the like. The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations. The term “ionizable lipid” as used herein refers to a lipid (e.g., a cationic lipid) or lipidoid having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. - 21 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. The term “local delivery,” as used herein, refers to delivery of an active agent or therapeutic agent such as a messenger RNA directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like. The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. The terms “polymer-conjugated lipid” and “conjugated lipid” are used interchangeably herein to refer to a lipid which is conjugated to one or more polymeric groups, which inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (e.g., U.S. Pat. No.5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used. As used herein, “lipid encapsulated” can refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a protein cargo), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle). The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids and / or additional agents. The term “lipid particle” is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a - 22 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) target site of interest. In the lipid particle of the disclosure, which is typically formed from a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle, the active agent or therapeutic agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation. The term “monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond. The term “mRNA” or “messenger RNA” as used herein refers to a ribonucleic acid sequences which encodes a peptide or protein. In certain embodiments, the mRNA may comprise a “transcript” that is produced by using a DNA template and encodes a peptide or protein. Typically, mRNA comprises 5’-UTR, protein coding region and 3’-UTR. mRNA can be produced by in vitro transcription from a DNA template. Methods of in vitro transcription are known to those of skill in the art. For example, various in vitro transfer kits are commercially available. According to the present invention, mRNA can be modified by further stabilizing modifications and cap formation in addition to the modifications according to the invention. The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols. The term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’- - 23 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)). As used herein, the term “nucleic acid” includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides. In particular embodiments, oligonucleotides of the disclosure are from about 15 to about 60 nucleotides in length. Nucleic acid may be administered alone in the lipid particles of the disclosure, or in combination (e.g., co-administered) with lipid particles of the disclosure comprising peptides, polypeptides, or small molecules such as conventional drugs. In other embodiments, the nucleic acid may be administered in a viral vector. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The terms “patient,” “subject,” or “individual” are used interchangeably herein, and - 24 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human. As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N’-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, - 25 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are - 26 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG), DSPE-PEG- DBCO, DOPE-PEG-Azide, DSPE-PEG-Azide, DPPE-PEG-Azide, DSPE-PEG-Carboxy- NHS, DOPE-PEG-Carboxylic Acid, DSPE-PEG-Carboxylic acid and the like. The term “room temperature” as used herein refers to a temperature of about 15 °C to 28 °C. The term “solvent” as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids. By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at - 27 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) least about 99.999% or more, or 100%. The term “substantially free of” as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term “substantially free of” can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%. The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1- C100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl. A “therapeutic” treatment is a treatment administered to a subject who exhibits signs - 28 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) of pathology, for the purpose of diminishing or eliminating those signs. The term “therapeutic protein” as used herein refers to a protein or peptide which has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In some embodiments, a therapeutic protein or peptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein or peptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “therapeutic protein” includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Exemplary therapeutic proteins include, but are not limited to, an analgesic protein, an anti-inflammatory protein, an anti-proliferative protein, an proapoptotic protein, an anti-angiogenic protein, a cytotoxic protein, a cytostatic protein, a cytokine, a chemokine, a growth factor, a wound healing protein, a pharmaceutical protein, or a pro-drug activating protein. Therapeutic proteins may include growth factors (EGF, TGF-α, TGF- β, TNF, HGF, IGF, and IL-1-8, inter alia) cytokines, paratopes, Fabs (fragments, antigen binding), and antibodies. The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject. Ionizable Lipid (Lipidoid) Compounds In one aspect, the present disclosure provides an ionizable lipid compound of formula (I), or a salt or solvate thereof: (I), wherein: A is selected from the group consisting ; L comprises at least one divalent moiety of - (optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, - (optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2-C8heterocycloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, and -(optionally substituted C2-C10heteroarylenyl)-, - 29 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each ; each occurrence of T1, T2, and T3is ; each occurrence of R1a, R1b, and R1cis ; R2is selected from the group consisting of optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, optionally substituted C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3 alkylenyl)C(=O)OR8, wherein the C3-C8cycloalkyl, C3-C8heterocycloalkyl, C6-C10aryl, and C2- C10 heteroaryl in R2are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R7is selected from the group consisting of is selected from the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; R8is selected from the group consisting of optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20heteroalkynyl; RAand RBare each independently selected from the group consisting of H, optionally - 30 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10heteroaryl; X1is O; and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In certain embodiments, the compound of formula (I) is a compound of formula (Ia): R2N T1R1a. In certain embodiments, the (I) is a compound of formula (Ib): , wherein: L1is selected from the - substituted C2-C6 alkylenyl)-, - (optionally substituted C1-C6 heteroalkylenyl)-, -(optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-, and -(optionally substituted C1-C6heteroalkylenyl)(optionally substituted C2-C8heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-. In certain embodiments, the compound of formula (I) is a compound of formula (Ic): (Ic). wherein: L1and L2are each independently selected from the group consisting of -(optionally substituted C2-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-, and -(optionally substituted C1-C6 heteroalkylenyl)(optionally substituted C2-C8heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-. In certain embodiments, the compound of formula (I) is a compound of formula (Id): . wherein: L1, L2, and L3are each independently selected from the group consisting of - (optionally substituted C2-C6 alkylenyl)-, -(optionally substituted C1-C6 heteroalkylenyl)-, - - 31 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) (optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-, and -(optionally substituted C1-C6 heteroalkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-. In certain embodiments, R2is C1-C20 alkyl. In certain embodiments, R2is C2-C20 alkenyl. In certain embodiments, R2is -NHC(=O)(C1-C20alkyl). In certain embodiments, R2is -(C1-C3 alkylenyl)C(=O)O(C1-C20 alkyl). In certain embodiments, R2is phenyl, wherein the phenyl is optionally substituted with at least one C1-C20alkyl or C1-C20alkoxy. In certain embodiments, R2is phenyl substituted with a C1-C20 alkyl. In certain embodiments, R2is phenyl substituted with a C6-C16alkyl. In certain embodiments, R2is phenyl substituted with a C6-C16 alkoxy. In certain embodiments, R2is -(CH2)9CH3. In certain embodiments, R2is - (CH2)11CH3. In certain embodiments, R2is -(CH2)13CH3. In certain embodiments, R2is - (CH2)15CH3. In certain embodiments, R2is -(CH2)17CH3. In certain embodiments, R2is In certain embodiments, R2is . In certain embodiments, R2is 14CH3. In certain (=O)(CH2)16CH3 . In certain embodiments, R2is -(CH2)C(=O)O(CH2)7CH3. In certain embodiments, R2is . In certain embodiments, R2is . In certain embodiments, R2 . L1is -(CH2)3-. In certain embodiments, L1is -(CH2)4-. In certain embodiments, L1is -(CH2)5-. In certain embodiments, L1is -(CH2)2NH(CH2)2-. In certain embodiments, L1is -(CH2)2NCH3(CH2)2-. In certain embodiments, L1is -(CH2)3NCH3(CH2)3-. In certain embodiments, L1is . . In certain embodiments, L2is -(CH2)4-. In certain embodiments, L2is -(CH2)5-. In certain embodiments, L2is -(CH2)2NH(CH2)2-. In certain embodiments, L2is -(CH2)2NCH3(CH2)2-. In certain embodiments, L2is -(CH2)3NCH3(CH2)3-. In certain embodiments, L2is - 32 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) . . In . . embodiments, L3is -(CH2)2NH(CH2)2-. In certain embodiments, L3is -(CH2)2NCH3(CH2)2-. In certain embodiments, L3is -(CH2)3NCH3(CH2)3-. In certain embodiments, L3is . 2 or 3. one certain embodiments, at least two of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least three R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least four of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least five of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, each of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, T1is . In certain embodiments, T2is . and R6dis H. In certain embodiments, at least two of R6a, R6b, R6c, and R6dare H. In certain embodiments, at least three of R6a, R6b, R6c, and R6dare H. In certain embodiments, each of R6a, R6b, R6c, and R6dare H. In certain embodiments, R1a. In certain embodiments, R1bis . R7is decyl. In certain embodiments, R7is undecyl. In certain embodiments, R7is dodecyl. In certain embodiments, R7is tridecyl. In certain embodiments, R7is tetradecyl. In certain embodiments, R7is pentadecyl. In certain embodiments, R7is hexadecyl. In certain embodiments, R7is heptadecyl. In certain embodiments, R7is octadecyl. In certain embodiments, R1ais -(CH2)2C(=O)O(CH2)8CH3. In certain embodiments, - 33 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) R1ais -(CH2)2C(=O)O(CH2)11CH3. In certain embodiments, R1ais - (CH2)2C(=O)O(CH2)13CH3. In certain embodiments, R1ais -(CH2)2C(=O)O(CH2)15CH3. In certain embodiments, R1bis -(CH2)2C(=O)O(CH2)8CH3. In certain embodiments, R1bis -(CH2)2C(=O)O(CH2)11CH3. In certain embodiments, R1bis - (CH2)2C(=O)O(CH2)13CH3. In certain embodiments, R1bis -(CH2)2C(=O)O(CH2)15CH3. In certain embodiments, R1cis -(CH2)2C(=O)O(CH2)8CH3. In certain embodiments, R1cis -(CH2)2C(=O)O(CH2)11CH3. In certain embodiments, R1cis - (CH2)2C(=O)O(CH2)13CH3. In certain embodiments, R1cis -(CH2)2C(=O)O(CH2)15CH3. In another aspect, the present disclosure provides an ionizable lipid compound of formula (II): (II), wherein: A is selected from the group consisting ; L comprises at least one divalent moiety of - (optionally substituted C1-C12alkylenyl)-, -(optionally substituted C1-C12heteroalkylenyl)-, - (optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocycloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, and -(optionally substituted C2-C10heteroarylenyl)-, wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each occurrence of G is ; each occurrence of T1, T2, and T3is ; each occurrence of R9a, R9b, and R9cis selected group of of optionally substituted C1-C20 alkyl, optionally substituted C1-C20heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted - 34 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3alkylenyl)C(=O)OR8, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, and C2- C10heteroaryl are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, optionally substituted C1-C6alkyl; the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; R10is selected from the group consisting of is selected from the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; RAand RBare each independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10heteroaryl; X2is selected from the group consisting of a bond, O, S, and N(RA); X3is selected from the group consisting of O, S, and N(RA); and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In certain embodiments, the compound of formula (II) is a compound of formula (IIa): (IIa). In certain embodiments, the compound of formula (I) is a compound of formula (IIb): , - 35 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) wherein: L1is selected from the group consisting of -(optionally substituted C2-C6alkylenyl)-, - (optionally substituted C1-C6 heteroalkylenyl)-, -(optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-, and -(optionally substituted C1-C6 heteroalkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-. In certain embodiments, the compound of formula (II) is a compound of formula (IIc): R9bT2G R3bN L2N L1N R3bT1R9a(IIc). wherein: L1and L2are each independently selected from the group consisting of -(optionally substituted C2-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-, and -(optionally substituted C1-C6heteroalkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-. In certain embodiments, the compound of formula (II) is a compound of formula (IId): wherein: L1, L2, and L3are each independently selected from the group consisting of - (optionally substituted C2-C6 alkylenyl)-, -(optionally substituted C1-C6 heteroalkylenyl)-, - (optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-, and -(optionally substituted C1-C6 heteroalkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-. In certain embodiments, R2is C1-C20 alkyl. In certain embodiments, R2is C2-C20 alkenyl. In certain embodiments, R2is -NHC(=O)(C1-C20alkyl). In certain embodiments, R2is -(C1-C3 alkylenyl)C(=O)O(C1-C20 alkyl). In certain embodiments, R2is phenyl, wherein the phenyl is optionally substituted with at least one C1-C20alkyl or C1-C20alkoxy. In certain embodiments, R2is phenyl substituted with a C1-C20 alkyl. In certain embodiments, R2is - 36 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) phenyl substituted with a C6-C16 alkyl. In certain embodiments, R2is phenyl substituted with a C6-C16alkoxy. In certain embodiments, R2is -(CH2)9CH3. In certain embodiments, R2is - (CH2)11CH3. In certain embodiments, R2is -(CH2)13CH3. In certain embodiments, R2is - (CH2)15CH3. In certain embodiments, R2is -(CH2)17CH3. In certain embodiments, R2is In certain embodiments, R2is . In certain embodiments, R2is 14CH3. In certain (=O)(CH2)16CH3. In certain 2 R is -(CH2)C(=O)O(CH2) R2is . In certain embodiments, R2. In certain embodiments, R2 . L1is -(CH2)3-. In certain embodiments, L1is -(CH2)4-. In certain embodiments, L1is -(CH2)5-. In certain embodiments, L1is -(CH2)2NH(CH2)2-. In certain embodiments, L1is -(CH2)2NCH3(CH2)2-. In certain embodiments, L1is -(CH2)3NCH3(CH2)3-. In certain embodiments, L1is . . In certain embodiments, L2is -(CH2)4-. In certain embodiments, L2is -(CH2)5-. In certain embodiments, L2is -(CH2)2NH(CH2)2-. In certain embodiments, L2is -(CH2)2NCH3(CH2)2-. In certain embodiments, L2is -(CH2)3NCH3(CH2)3-. In certain embodiments, L2is . . In certain embodiments, L3is -(CH2)4-. In certain embodiments, L3is -(CH2)5-. In certain embodiments, L3is -(CH2)2NH(CH2)2-. In certain embodiments, L3is -(CH2)2NCH3(CH2)2-. In certain embodiments, L3is -(CH2)3NCH3(CH2)3-. In certain embodiments, L3is . - 37 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) In certain embodiments, R4aand R4bare both H. In certain embodiments, m is 2 or 3. In certain embodiments, at least one of R5a, R5b, R5c, R5d, R5e, and R5fis H. In certain embodiments, at least two of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least three R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least four of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, at least five of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, each of R5a, R5b, R5c, R5d, R5e, and R5fare H. In certain embodiments, T1is . In certain embodiments, T2is . In T3. and R6dis H. In certain are In certain embodiments, at least three of R6a, R6b, R6c, and R6dare H. In certain embodiments, each of R6a, R6b, R6c, and R6dare H. In certain embodiments, R9ais . In certain embodiments, R9bis . R10is decyl. In certain embodiments, R10is undecyl. In certain embodiments, R10is dodecyl. In certain embodiments, R10is tridecyl. In certain embodiments, R10is tetradecyl. In certain embodiments, R10is pentadecyl. In certain embodiments, R10is hexadecyl. In certain embodiments, R10is heptadecyl. In certain embodiments, R10is octadecyl. In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, optionally substituted heterocycloalkylenyl, optionally substituted arylenyl, and optionally substituted heteroarylenyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C3haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O)2N(R’)(R’’), N(R’)C(=O)R’’, - 38 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) N(R’)S(=O)2R’’, C2-C8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl. In certain embodiments, the compound of formula (I) or (II) is selected from the group consisting of: nonyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; nonyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; nonyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; dodecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; tetradecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; hexadecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; - 39 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) dodecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; nonyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; nonyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate hexadecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dinonyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; didodecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; ditetradecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dihexadecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dinonyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; didodecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; ditetradecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; - 40 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) dihexadecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 12-(2-(4-((3-(dodecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)-8,16- diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 12-(2-(4-((3-(hexadecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)- 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; didodecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dihexadecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dinonyl 8,18-diimino-13-(3-(3-((3-(nonyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17-triazapentacosanedioate; didodecyl 13-(3-(3-((3-(dodecyloxy)-3-oxopropyl)thio)propanimidamido)propyl)- 8,18-diimino-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-(3-(3-((3-oxo-3- (tetradecyloxy)propyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17- triazapentacosanedioate; dihexadecyl 13-(3-(3-((3-(hexadecyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-8,18-diimino-4,22-dithia-9,13,17- - 41 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) triazapentacosanedioate; dinonyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; didodecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; ditetradecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; dihexadecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; nonyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-((2-((2-(4-(2-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-((2-((2-(4-(2-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; dinonyl 8,23-diimino-13,18-bis(3-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22-tetraazatriacontanedioate; didodecyl 13,18-bis(3-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; ditetradecyl 8,23-diimino-13,18-bis(3-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; and dihexadecyl 13,18-bis(3-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate. Ionizable Lipids and / or Cationic Lipids or Lipidoids - 42 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) The scope of ionizable lipids contemplated for use in the present disclosure is not limited to ionizable lipids of formula (I). In the lipid nanoparticles of the disclosure, the cationic lipid or ionizable lipid may comprise, e.g., one or more of the following: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLinMC3DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate (SM-102), 1,1′-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1- piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3- 45 dimethylaminopropyl)- 1,3]-dioxolane (D Lin-K-C3-D MA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2- dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N- methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dili-noleyl-4-dimethylaminomethyl- [1,3]-dioxolane (DLin-KDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (D Lin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylaminoacetoxypropane (DLin-DAC), 1- 2dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy- 3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N- methylpiperazino)propane (D Lin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (D LinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (D Lin-EG-D MA), N,N-dioleyl-N,N-dimethylanrmonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2- distearyloxy-N,N-dimethylaminopropane (DSD MA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N, N-trimethylammonium chloride (DOTAP), 3- (N-(N’,N’dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl anrmonium bromide (DMRIE), 2,3- dioleyloxy-N-[2 (spermine-carboxamidoethyl]-N,N-dimethy 1-1- propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3- - 43 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) dimethyl-1-(cis,cis-9’,1-2’-octadecadienoxy) propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1,2-N,N’dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N’-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof. The ionizable lipids are not limited to those recited herein, and can further include ionizable lipids known to those skilled in the art, or described in PCT Application No. PCT / US2020 / 056255 and / or PCT Application No. PCT / US2020 / 056252, the disclosures of which are herein incorporated by reference in its entirety. The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3- DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U.S. Application Publication No. US 2011 / 0256175, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Non-cationic Lipid In the nucleic acid-lipid particles of the present disclosure, the non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. In some embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2’-hydroxyethyl ether, cholesteryl-4’- hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2’-hydroxyethyl ether is known to one skilled in the art and described in U.S. Patent Nos.8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378, all of which are hereby incorporated herein in their entireties for all purposes. Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, - 44 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), ioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids can be, for example, acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl- 2’-hydroxyethyl ether, cholesteryl-4’-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof. Conjugated Lipid In the nucleic acid-lipid particles of the present disclosure, the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG) lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof. In some embodiments, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate. PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEGOH), monomethoxypolyethylene glycolsuccinate (MePEGS), monomethoxypolyethylene glycolsuccinimidyl succinate (MePEG-S-NHS), - 45 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) monomethoxypolyethylene glycolamine (MePEG-NH2), monomethoxypolyethylene glycoltresylate (MePEG-TRES), and monomethoxypolyethylene glycolimidazolylcarbonyl (MePEG-IM). Other PEGs such as those described in U.S. Patent Nos.6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates. In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. The conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEGDAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18), or mixtures thereof. Additional PEG-lipid conjugates suitable for use in the disclosure include, but are not limited to, mPEG2000-l,2-diO-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8’-(l,2-dimyristoyl-3-propanoxy)-carboxamido-3’,6’- dioxaoctanyl] carbamoyl-methyl-poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S. Patent No.7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In some embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. In addition to the foregoing, it will be readily apparent to those of skill in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, - 46 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose. In addition to the foregoing components, the particles (e.g., LNP) of the present disclosure can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in the present disclosure, and methods of making and using SPLPs and SPLP-CPLs, are disclosed, e.g., in U.S. Patent No.6,852,334 and PCT Publication No. WO 00 / 62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes. In certain instances, the conjugated lipid that inhibits aggregation of particles (e.g., PEG-lipid conjugate) may comprise from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 1 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, from about 1.4 mol% to about 1.5 mol%, or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol% (or any fraction thereof or range therein) of the total lipid present in the particle. In the lipid nanoparticles of the present disclosure, the active agent or therapeutic agent may be fully encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation. In some embodiments, a nucleic acid-lipid particle comprising a nucleic acid such as a messenger RNA (i.e., mRNA) is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37° C. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agent or therapeutic agent (e.g., nucleic acid such as siRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulations of the present disclosure is that the lipid particle compositions are substantially non-toxic to mammals such as humans. Lipid Nanoparticles (LNPs) In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition. In certain embodiments, the composition comprises (a) at least one ionizable - 47 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) lipid compound of formula (I). In certain embodiments, the composition comprises (b) at least one helper lipid. In certain embodiments, the composition comprises (c) at least one cholesterol lipid. In certain embodiments, the composition comprises (d) at least one polymer-conjugated lipid and / or a modified derivative thereof. In certain embodiments, the at least one helper lipid comprises about 1, 2, 3, 4, 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, or about 30 mol% of the LNP. In certain embodiments, the at least one helper lipid comprises less than about 1, 2, 3, 4, 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, or about 30 mol% of the LNP. In certain embodiments, the at least one helper lipid comprises more than about 1, 2, 3, 4, 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, or about 30 mol% of the LNP. In certain embodiments, the at least one helper lipid comprises about 5 mol% of the LNP. In certain embodiments, the at least one helper lipid comprises about 17 mol% of the LNP. In certain embodiments, the at least one helper lipid is dioleoylphosphatidylethanolamine (DOPE). In certain embodiments, the at least one helper lipid is diastereoylphosphatidylcholine (DSPC). In certain embodiments, the cholesterol comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the cholesterol comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the cholesterol comprises more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the cholesterol comprises about 19.25 mol% of the LNP. In certain embodiments, the cholesterol comprises about 48.5 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7.6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, - 48 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.9, or about 10.0 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7.6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.9, or about 10.0 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7.6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.9, or about 10.0 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 0.75 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 1.5 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG-2000). In certain embodiments, the at least one ionizable lipid comprises about 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99 mol% of the LNP. In certain embodiments, the at least one ionizable lipid comprises less than about 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99 mol% of the LNP. In certain embodiments, the at least one ionizable lipid comprises more than about 1, 2, 3, 4, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, - 49 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99 mol% of the LNP. In certain embodiments, the at least one ionizable lipid consists essentially of the compound of formula (I). In certain embodiments, the at least one ionizable lipid comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the at least one ionizable lipid comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the at least one ionizable lipid comprises more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the LNP has a molar ratio of (a):(b):(c):(d) of about 33:17:48.5:1.5. In certain embodiments, the at least one ionizable lipid comprises at least one compound of formula (I) and at least one additional ionizable lipid. In certain embodiments, the at least one additional ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3). In certain embodiments, the at least one ionizable lipid of formula (I) comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of formula (I) comprises less than about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of formula (I) comprises more than about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of formula (I) comprises about 50 mol% of the LNP. In certain embodiments, the at least one additional ionizable lipid comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one additional ionizable lipid comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, - 50 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one additional ionizable lipid comprises more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one additional ionizable lipid comprises about 25 mol% of the LNP. In certain embodiments, the LNP has a ratio of the compound of formula (I) to the at least one additional ionizable lipid of about 2:1. In certain embodiments, the LNP has a molar ratio of (a):(b):(c):(d) of about 75:5:19.25:0.75. In certain embodiments, the LNP further comprises at least one cargo molecule. In certain embodiments, the cargo molecule is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof. In certain embodiments, the cargo comprises a nucleic acid. In certain embodiments, the nucleic acid cargo is DNA or RNA. In certain embodiments, the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof. In certain embodiments, the cargo is at least partially encapsulated in the LNP. In certain embodiments, the LNP has a weight ratio of total lipid ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, or about 1:1 (total lipid:cargo (w / w)). Methods Synthetic Methods In another aspect, the present disclosure provides a method of preparing the ionizable lipid compound of formula (II): (II), the method comprising: (a) contacting an amine compound of formula (A1) or (A2): , - 51 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) wherein Z comprises at least one divalent moiety selected from the group consisting of -(optionally substituted C1-C12alkylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8heterocycloalkylenyl)-, - (optionally substituted C6-C10 arylenyl)-, and -(optionally substituted C2-C10 heteroarylenyl)-, and wherein each divalent moiety in Z is optionally substituted with at least one occurrence of Y; each occurrence of Y is ; and a compound of formula (B): ; to form a thiolate (b) contacting the thiolate intermediate and a compound of formula compound of formula (C1), (C2), or (C3): , In certain embodiments, compound of formula (A1) or (A2) is selected from the group consisting of decan-1-amine, dodecan-1-amine, tetradecan-1-amine, hexadecan-1- amine, octadecan-1-amine, (Z)-octadec-9-en-1-amine, palmitoylhydrazide, stearoylhydrazide, 2-hexyldecan-1-amine, octyl glycinate, 4-octylaniline, 4-octylaniline, 4-decylaniline, 4- dodecylaniline, 4-(nonyloxy)aniline, ethane-1,2-diamine, propane-1,3-diamine, pentane-1,5- diamine, N1-(2-aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2- diamine, N1,N1-bis(2-aminoethyl)ethane-1,2-diamine, N1-(3-aminopropyl)- N1- methylpropane-1,3-diamine, N1,N1-bis(3-aminopropyl)propane-1,3-diamine, 3,3’-(piperazine- 1,4-diyl)bis(propan-1-amine), N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2- diamine, and N1,N1’-(butane-1,4-diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine). In certain embodiments, the compound of formula (B) is dihydrothiophen-2(3H)- imine. - 52 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) In certain embodiments, the compound of formula (C1), (C2), or (C3) is selected from the group consisting of nonyl acrylate, dodecyl acrylate, tetradecyl acrylate, and hexadecyl acrylate. Therapeutic Methods In another aspect, the present disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure. In certain embodiments, the LNP is formulated as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. In certain embodiments, the disease is cancer. In certain embodiments, the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. In certain embodiments, the subject is further administered at least one additional agent or therapy useful for treating, preventing, and / or ameliorating cancer in the subject. In another aspect, the present disclosure provides a method of delivering at least one cargo molecule to the lung and / or spleen of a subject. In certain embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP). In certain embodiments, the LNP comprises (a) at least one ionizable lipid compound of formula (I). In certain embodiments, the LNP comprises (b) at least one helper lipid. In certain embodiments, the LNP comprises (c) at least one cholesterol lipid. In certain embodiments, the LNP comprises (d) at least one polymer-conjugated lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises (e) at least one cargo molecule. In certain embodiments, the wherein the cargo comprises a nucleic acid. In certain embodiments, the nucleic acid cargo is DNA or RNA. In certain embodiments, the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof. In certain embodiments, the cargo is at least partially encapsulated in the LNP. In certain embodiments, the LNP has a weight ratio of total lipids ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, - 53 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 20:1, 15:1, 10:1, 5:1, or about 1:1 (total lipid:cargo (w / w)). In certain embodiments, the nucleic caid cargo is RNA. In certain embodiments, the RNA is mRNA. In certain embodiments, the LNP has a weight ratio of total lipids to nucleic acid cargo of about 40:1 (w / w). In certain embodiments, the nucleic acid cargo is selectively delivered to the lung. In certain embodiments, the nucleic caid cargo is RNA. In certain embodiments, the RNA is mRNA. In certain embodiments, the LNP has a weight ratio of total lipids to nucleic acid cargo of about 10:1 (w / w). In certain embodiments, the nucleic acid cargo is selectively delivered to the spleen. In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human. Anti-Cancer Agents In some embodiments, the at least one additional agent is an anti-cancer agent. Any suitable anti-cancer agent may be used in the compositions and methods of the present disclosure. The selection of a suitable anti-cancer agent may depend upon, among other things, the type of cancer to be treated and the nanoparticle compositions of the present disclosure. In certain embodiments, the anti-cancer agent may be effective for treating one or more of pancreatic cancer, esophageal cancer, rectal cancer, colon cancer, prostate cancer, kidney cancer, liver cancer, breast cancer, ovarian cancer, and stomach cancer. Examples of anti-cancer agents include, but are not limited to, chemotherapeutic agents, antiproliferative agents, anti-tumor agents, checkpoint inhibitors, and anti-angiogenic agents. For example, In some embodiments, the anti-cancer agent is gemcitabine, doxorubicin, 5-Fu, tyrosine kinase inhibitors, sorafenib, trametinib, rapamycin, fulvestrant, ezalutamide, or paclitaxel. Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, - 54 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, Halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p’-DDD, dacarbazine, CCNU, BCNU, cis- diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all- trans retinoic acid, gliadel and porfimer sodium). Antiproliferative agents are compounds that decrease the proliferation of cells. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and analogs thereof, toremifene, droloxifene and roloxifene), Additional examples of specific antiproliferative agents include, but are not limited to levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron. The inhibitors of the invention can be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / anti-neoplastic agents are defined as agents which attack and kill cancer cells. Some cytotoxic / anti-neoplastic agents are alkylating agents, which alkylate the genetic material in tumor cells, e.g., cis-platin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belustine, uracil mustard, chlomaphazin, and dacabazine. Other cytotoxic / anti-neoplastic agents are antimetabolites for tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate, mercaptopuirine, azathioprime, and procarbazine. Other cytotoxic / anti-neoplastic agents are antibiotics, e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin. There are numerous liposomal formulations commercially available for - 55 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) these compounds. Still other cytotoxic / anti-neoplastic agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine and etoposide. Miscellaneous cytotoxic / anti- neoplastic agents include taxol and its derivatives, L-asparaginase, anti-tumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine. Anti-angiogenic agents are well known to those of skill in the art. Suitable anti- angiogenic agents for use in the methods and compositions of the present disclosure include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferons, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2. (TIMP-1 and -2). Small molecules, including topoisomerases such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, can also be used. Other anti-cancer agents that can be used in combination with the disclosed compounds include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone - 56 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; - 57 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; - 58 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; - 59 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. In some embodiments, the anti- cancer drug is 5-fluorouracil, taxol, or leucovorin. In some embodiments, the anti-cancer agent may be a prodrug form of an anti-cancer agent. As used herein, the term “prodrug form” and its derivatives is used to refer to a drug that has been chemically modified to add and / or remove one or more substituents in such a manner that, upon introduction of the prodrug form into a subject, such a modification may be reversed by naturally occurring processes, thus reproducing the drug. The use of a prodrug form of an anti-cancer agent in the compositions, among other things, may increase the concentration of the anti-cancer agent in the compositions of the present disclosure. In certain embodiments, an anti-cancer agent may be chemically modified with an alkyl or acyl group or some form of lipid. The selection of such a chemical modification, including the substituent(s) to add and / or remove to create the prodrug, may depend upon a number of factors including, but not limited to, the particular drug and the desired properties of the prodrug. One of ordinary skill in the art, with the benefit of this disclosure, will recognize suitable chemical modifications. Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle (LNP) of the present disclosure and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one adjuvant. In certain embodiments, the composition is a vaccine. Such a pharmaceutical composition may consist of at least one composition of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art. In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 1,000 mg / kg / day. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, - 60 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient. Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations. In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- - 61 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by - 62 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and / or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, “additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier. The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid. The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily - 63 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- - 64 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art. Administration / Dosing The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (i.e., composition) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range - 65 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) for a therapeutic composition of the disclosure is from about 0.01 mg / kg to 100 mg / kg of body weight / per day of active agent (i.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition without undue experimentation. The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic composition for the treatment of a disease or disorder in a patient. - 66 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account. The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 µg to about 7,500 mg, about 20 µg to about 7,000 mg, about 40 µg to about 6,500 mg, about 80 µ g to about 6,000 mg, about 100 µ g to about 5,500 mg, about 200 µ g to about 5,000 mg, about 400 µ g to about 4,000 mg, about 800 µ g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between. In some embodiments, the dose of active agent (i.e., nucleic acid) present in the composition of the disclosure is from about 0.5 µg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. - 67 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient. The term “container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient. Administration Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular - 68 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) formulations and compositions that are described herein. Parenteral Administration As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non- toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. - 69 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. EXAMPLES Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein. Materials and Methods Materials Amines, oleyl alcohol and linoleyl alcohol were purchased from Sigma Aldrich, Tokyo Chemical Industry (TCI) and Ambeed.2-Iminothiolane hydrochloride, acrylates, acryloyl chloride and triethylamine (TEA) were obtained from Sigma Aldrich. Mouse IL-6 uncoated ELISA, mouse TNF alpha uncoated ELISA, LysoTracker® Deep Red and 3,3’- Dioctadecyloxacarbocyanine perchlorate (DiO) were bought from Invitrogen.1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 1,2-di-O- octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) and cholesterol were obtained from Avanti Polar Lipids. DLin-MC3-DMA was purchased from MedChem Express. Cas9 mRNA (5moU, #L-7206) was bought from TriLink. GFP siRNA (#P-002048- 01-20) was obtained from Horizon Discovery. Highly modified sgRNA target mouse TTR (guide No. G211) was chemically synthesized by AxoLabs mRNA Synthesis Codon optimized SARS-CoV-2 di-proline modified Spike sequence, firefly Luciferase sequence, Cre recombinase sequence or human FGF21 sequence was cloned into an mRNA production plasmid (optimized 3’ and 5’ UTR and containing a 101 polyA tail), in vitro transcribed in the presence of 1-methyl pseudouridine modified nucleoside, co- transcriptionally capped using the CleanCapTMtechnology (TriLink) and cellulose purified to - 70 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) remove double-stranded RNAs. Purified mRNA was ethanol precipitated, washed, re- suspended in nuclease-free water, and subjected to quality control (electrophoresis, dot blot, endotoxin content and transfection into human DCs). All mRNAs were stored at -20 °C until use. Synthesis of oleyl acrylate (O18a) and linoleyl acrylate (O18b) Oleyl alcohol or linoleyl alcohol (1 mmol, 1 equiv.) in 10 mL CH2Cl2 was treated with TEA (3 mmol, 3 equiv.) and acryloyl chloride (1.5 mmol, 1.5 equiv.) at 0 °C for 30 and then at RT for overnight. The mixture was diluted with CH2Cl2 and washed with brine twice. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by a CombiFlash NextGen 300+ chromatography system (Teledyne ISCO, Lincoln, NE) with gradient elution from hexane to 80:20 hexane / ethyl acetate, and the desired fractions were collected as colorless oil (yield>90%). O18a was confirmed by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS-ESI: calculated for C21H38O2: 322.53, found [M+H]+= 323.51;1H NMR (400 MHz, CDCl3) δ: 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.6 Hz, 1H), 5.41 – 5.28 (m, 2H), 4.15 (t, J = 6.8 Hz, 2H), 2.01 (q, J = 6.5 Hz, 4H), 1.74 – 1.61 (m, 2H), 1.43 – 1.21 (m, 22H), 0.88 (t, J = 6.7 Hz, 3H). O18b was confirmed by MS and NMR. MS-ESI: calculated for C21H36O2: 320.52, found [M+H]+= 321.51;1H NMR (400 MHz, CDCl3) δ: 6.40 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.3, 10.4 Hz, 1H), 5.81 (dd, J = 10.4, 1.5 Hz, 1H), 5.44 – 5.27 (m, 4H), 4.15 (t, J = 6.7 Hz, 2H), 2.77 (t, J = 6.4 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.74 – 1.59 (m, 2H), 1.45 – 1.22 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H). High-throughput synthesis of AID-lipids Amine, 2-iminothiolane hydrochloride, acrylate and TEA were dissolved in ethanol in a 1.5 mL Eppendorf tube. All tubes were put on a floating rack and sonicated in an ultrasonic cleaner (Branson M2800) for 1 h at room temperature and at a frequency of 40 kHz. The yield was typically over 80%. Take 12T-O14 as an example: 8 mg of amine 12 (1 equiv.) and 7 mg of 2-iminothiolane hydrochloride (1.5 equiv.) were dissolved in 500 µL ethanol, followed by the addition of 12 mg of acrylate tail O14 (1.25 equiv.) and 7 mg of TEA (2 equiv.). All crude products were diluted with ethanol to prepare the stock solutions (40 mg / mL) for LNP preparation. Purification and characterization of 12T-O14 - 71 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) To purify the top-performing AID-lipid 12T-O14, its crude product was separated using a CombiFlash NextGen 300+ chromatography system with gradient elution from CH2Cl2 to 75:22:3 CH2Cl2 / MeOH / NH4OH (aq), and the desired fractions were collected as while solid (yield 84%).12T-O14 was characterized by MS and NMR. MS-ESI: calculated for C37H66N2O2S: 603.01, found [M+H]+= 603.73;1H NMR (400 MHz, MeOD) δ: 7.43 – 7.35 (m, 2H), 7.30 – 7.23 (m, 2H), 4.11 (t, J = 6.6 Hz, 2H), 2.86 (t, J = 7.0 Hz, 2H), 2.82 – 2.63 (m, 8H), 2.13 (p, J = 7.2 Hz, 2H), 1.66 (dtt, J = 13.0, 6.6, 3.8 Hz, 4H), 1.46 – 1.22 (m, 36H), 0.92 (t, J = 6.7 Hz, 6H);13C NMR (100 MHz, MeOD) δ: 173.17, 168.73, 144.88, 132.04, 130.72, 125.61, 65.25, 35.83, 35.04, 32.61, 32.39, 31.87, 31.17, 30.11, 30.08, 30.04, 29.98, 29.92, 29.79, 29.77, 29.67, 29.06, 27.49, 27.09, 26.35, 23.05, 13.75. LNP formulation and optimization For high-throughput in vitro and in vivo screening, AID-LNPs were prepared by pipette mixing the ethanolic phase containing AID-lipid, DOPE, cholesterol and DMG-PEG with an aqueous phase (10 mM citrate buffer, pH 3) containing mRNA at a volume ratio of 1:3 and diluted in culture medium or 1× PBS for cell or animal treatment, respectively. The weight ratio of AID-lipid, DOPE, cholesterol, DMG-PEG and mRNA was 16:10:10:3:1.6. To optimize the LNP formulation, pipette mixed LNPs with different weight ratios of 12T-O14, cholesterol, DOPE, DMG-PEG and mRNA were tested and screened in vitro and in vivo, and an optimized weight ratio of 16:15:10:3:1.6 was chosen for further studies. To formulate 12T-O14 LNP and DOTAP LNP by microfluidic mixing, the ethanol phase containing lipids was mixed with the aqueous phase containing mRNA at a flow rate of 1:3 in a microfluidic chip device. LNPs were dialyzed against 1× PBS in a 20 kDa MWCO cassette for 2 h, filtered through a 0.22 μM filter and stored at 4 °C. DiO-labeled LNPs were obtained by mixing DiO (1 mol% of total lipids) with LNPs before dialysis. SM-102 (or MC3) LNP was formulated by SM-102, DSPC, cholesterol and DMG- PEG at a molar ratio of 50:10:38.5:1.5 using microfluidic mixing. MC3 / 12T-O14 LNP was formulated by MC3, DSPC, cholesterol, DMG-PEG and 12T-O14 at a molar ratio of 25:5:19.25:0.75:50 at a total lipids:mRNA weight ratio of 40, 20, 10 or 5. Specifically, lung- targeted MC3 / 12T-O14 LNP and MC3 / DOTAP SORT LNP with a total lipids:mRNA weight ratio of 40 was formulated by pipette mixing. After dialysis, these LNPs were directly used without filtration. Characterization - 72 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 1H NMR and13C NMR spectra were recorded using a Bruker 400 MHz NMR spectrometer. LC-MS was performed on a Waters Acquity LCMS system equipped with UV- Vis detectors. The hydrodynamic size, polydispersity index (PDI) and zeta potential of LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The morphology of LNPs was characterized by a cryo-electron microscope (Titan Krios, Thermo Fisher). The mRNA encapsulation efficiency and the pKaof LNP were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen) and a 6-(p- toluidinyl)naphthalene-2-sulfonic acid (TNS) assay, respectively. Cell culture and animal studies HepG2 cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin. ACE-II expressing human embryonic kidney 293 (HEK-293 / ACE-II) cells were purchased from Takara and maintained in RPMI-1640 medium. Cells were cultured at 37 °C in a humidified incubator of 5% CO2, and routinely tested for mycoplasma contamination. C57BL / 6 and Ai6 female mice (6-8 weeks, 18-20 g) were purchased from Jackson Laboratory. High-throughput in vitro screening of AID-LNPs HepG2 were seeded into 96-well plates at a density of 5,000 per well overnight and mLuc-loaded AID-LNPs (15 ng / well) were used to treat cells for 24 h. Luciferase expression was evaluated by Luciferase Reporter 1000 Assay System (Promega, #E4550) and cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, #G7572) according to manufacturer’s protocols. In vivo screening of AID-LNPs Mice were anesthetized with 2.5% isoflurane, and mLuc-loaded AID-LNPs (2 μg mRNA in 40 μL PBS) were injected into the left or right gastrocnemius muscle using a 29- gauge needle.4 h later, mice were intraperitoneally (i.p.) injected with D-luciferin potassium salt (150 mg / kg), and bioluminescence imaging was performed on an IVIS imaging system (PerkinElmer). Molecular dynamics simulations Lipid dynamics simulations of 12T-O14, 4T-O14 and DOTAP were optimized at - 73 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) CHARMm force field. The lipid simulations were run up to 4000 steps to achieve the energy- minimized structures. All bonds containing intermolecular interactions were constrained using the Smart Minimizer algorithm (i.e., 1000 steps of Steepest Descent with Root-Mean- Squared (RMS) gradient of 3 and Conjugate Gradient minimization). The overall RMS gradient tolerance was set to 0.01. Momany-Rone method was assigned for partial charge estimation using Discovery Studio 2018 (Accelrys). The dimensionless packing parameter P of a lipid molecule was calculated as P = V / (AL) based on its Van der Waals molecule volume (V), cross section area of polar head (A) and average tail length (L). V, A and L were derived from the optimized conformation of the lipid as well as the estimated atomic Van der Waals radius. Cellular uptake HepG2 cells were seeded into 35-mm glass-bottom dishes for 24 h and treated with DiO-labelled LNPs (300 ng mRNA / mL) for 2 h. Afterwards, cells were stained with LysoTracker Deep Red (100 nM) for 45 min and Hoechst 33342 (10 μg / mL) for 5 min. Images were taken immediately using a confocal laser scanning microscope (LSM 710, Zeiss). Hemolysis assay Mouse red blood cells (RBCs) were isolated and washed twice with 1× PBS by centrifugation at 700 g for 5 min. Next, RBCs were diluted in PBS (pH 7.4 or 6.0) to a 4% vol / vol RBC solution and incubated with LNPs at a concentration of 3 μg mRNA / mL at 37 °C for 1 h. Afterwards, RBC solution was centrifuged at 700 g for 5 min and 100 μL of the supernatant was transferred into a 96-well plate. The absorption at 540 nm was determined with a plate reader. Positive and negative controls were carried out with 0.1% Triton-X and 1× PBS, respectively. Local mRNA delivery Mice were i.m. injected with SARS-CoV-2 Spike mRNA-loaded LNPs (2 μg mRNA in 40 μL PBS) twice using a prime-boost strategy at a three-week interval. Blood was collected into serum separator tubes (BD, #365967) through the retro-orbital route. Serum was acquired after centrifugation at 10,000 g for 5 min and stored at -20 °C until used. Systemic mRNA delivery - 74 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Mice were i.v. injected with 100 μL of mLuc-loaded LNPs (2 μg mRNA / mouse). In vivo bioluminescence imaging was performed at 4 h post-injection as described above. Mice were euthanized and major organs (heart, liver, spleen, lung and kidney) were acquired for ex vivo bioluminescence imaging immediately. For systemic FGF21 mRNA delivery, Mice were i.v. injected with 100 μL of FGF21 mRNA-loaded LNPs (5 μg mRNA / mouse). Serum was collected at 0, 6 and 24 h post- injection and stored at -20 °C until analysis of FGF21 by ELISA (R&D, #DF2100). Liver function was examined by measuring the activities of serum aspartate aminotransferase (Cayman, #701640) and serum alanine aminotransferase (Cayman, #700260).13 mouse cytokines were examined using a LEGENDplex™ multi-analyte flow assay kit (BioLegend, #740621) according to the manufacturer’s instruction. Major organs were collected at 24 h post-injection and processed for histological examination after stained with hematoxylin and eosin (H&E). Lung-targeted and spleen-targeted mRNA delivery mLuc delivery: To use 12T-O14 as a fifth component to navigate liver-tropic LNP to transfect the lung, MC3 / 12T-O14 LNP with a total lipids:mRNA weight ratio of 40 was i.v. injected at a dose of 2 μg mLuc per mouse. Lung-targeted MC3 / DOTAP LNP was used a positive control. To optimize MC3 / 12T-O14 LNP formulation for spleen targeting, MC3 / 12T-O14 LNPs with different total lipids:mRNA weight ratios (40, 20, 10 or 5) were i.v. injected at a dose of 2 μg mLuc per mouse. In vivo and ex vivo bioluminescence imaging were performed at 4 h post-injection. Cre mRNA delivery and flow cytometry: Lung-tropic MC3 / 12T-O14 LNP with a total lipids:mRNA weight ratio of 40 was i.v. injected into Ai6 mice at a dose of 5 μg Cre mRNA per mouse. Mice were euthanized on day 2, and lungs were harvested. Lungs were digested with 1.5 mg / mL Collagenase A (Sigma, #COLLA-RO) and 0.1 mg / mL DNase I (Sigma, #10104159001) for 1 h, filtered through a 70 µm cell strainers in complete DMEM, centrifuged, and red blood cells lysed in ACK lysis buffer to obtain a clear single cell suspension. Samples were stained with Live / Dead Aqua for 5 minutes, blocked using anti- mouse CD16 / 32 antibody for 20 min, and strained extracellularly for 30 min using antibodies. Cells were washed in FACs buffer and fixed with 300 µL (1% paraformaldehyde). Samples were analyzed on a BD LSR II equipped with 4 laser lines and 18 PMTs. The gating strategy for endothelial cells (PECAM+), macrophages (CD11b+), monocytes (CD64+), neutrophils (Ly6G+) and DCs (CD11c+), as well as the antibody list, is - 75 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) provided. Spleen-tropic MC3 / 12T-O14 LNP with a total lipids:mRNA weight ratio of 10:1 was i.v. injected into Ai6 mice at a dose of 5 μg Cre mRNA per mouse. Mice were euthanized at 2 d post-injection, and spleens were harvested. Spleens were processed, filtered through a 70 µm cell strainers in complete DMEM, centrifuged, and red blood cells lysed in ACK lysis buffer to obtain a clear single cell suspension. Samples were stained and analyzed as above described. The gating strategy for leukocytes (CD45+), macrophages (CD11b+), monocytes (CD64+), neutrophils (Ly6G+) and DCs (CD11c+), as well as the antibody list, is provided. Lung-targeted Cas9 mRNA / sgTTR co-delivery: Cas9 mRNA and sgTTR (4:1, wt:wt) were encapsulated into MC3 / 12T-O14 LNP at a total lipids:mRNA weight ratio of 40. C57BL / 6 mice were i.v. administrated with this lung-tropic MC3 / 12T-O14 LNP on Day 0, 1, 3 and 4 for four injections (totally 1 mg / kg). On day 7, lungs were harvested to determine the indels of TTR by Next-Generation Sequencing (NGS) or processed for histological examination after stained with H&E. MC3 LNP was used as a positive control for liver gene editing and as a negative control for lung gene editing. For TTR on-target DNA sequencing, DNA was extracted using the Qiagen Puregene Tissue Kit (#158063) and quantified using a Nanodrop 2000. PCR amplification of the TTR target site was carried out using Q5 High- Fidelity DNA Polymerase (New England Biolabs M0491) and the following primer sequences: mTTR-exon2-F, 5’-CGGTTTACTCTGACCCATTTC-3’ (SEQ ID NO.1) and mTTR-exon2-R, 5’-GGGCTTTCTACAAGCTTACC-3’ (SEQ ID NO.2). Deep sequencing of the TTR amplicons and determination of the on-target indel frequency was performed essentially as described except that 150 bp pair end reads were produced. Spleen-targeted mRNA vaccine delivery: SARS-CoV-2 Spike mRNA was encapsulated into MC3 / 12T-O14 LNP at a total lipids:mRNA weight ratio of 10. C57BL / 6 mice were i.v. administrated with this spleen-tropic MC3 / 12T-O14 LNP twice (2 μg mRNA each) using a prime-boost strategy at a three-week interval. Serum was collected for analysis. Determination of anti-Spike IgG titers using ELISA 96-well clear polystyrene high bind Stripwell™ microplates (Corning) were coated overnight with 1 μg / mL purified SARS-CoV-2 His tagged Spike protein (Sino Biological, #40589-V08H8). Plates were washed once with wash buffer (0.05% Tween-20 in PBS), and blocked for two hours at room temperature using a solution of heat-inactivated, IgG-depleted, protease-free bovine serum albumin (2% w / v BSA in PBS). After blocking, plates were washed three times, and mouse sera was serially diluted in the blocking solution and - 76 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) incubated for 2 h at RT. Plates were washed three times before the addition of HRP- conjugated anti-mouse secondary antibody specific to IgG (1:10,000, Abcam, #ab97040) in blocking buffer. Plates were incubated for 1.5 h, washed three times before the addition of 100 µL per well of KPL TMB substrate for 8 min. The reaction was stopped by adding 50 µL of 2N sulfuric acid, and the absorbance was measured at 450 nm using a SpectraMax™ 190 microplate reader. Spike-specific IgG end-point dilution titer was defined as the highest dilution of serum to give an OD greater than the cut-of OD value determined using the Frey method. SARS-CoV-2 pseudovirus neutralization assay Pseudovirus neutralization assay was performed using a lentivirus-based system expressing the D614G Spike protein. HEK-293 / ACE-II cells were seeded at a density of 20,000 cells / well in 96-well plates for overnight. Serum samples were serially diluted in RPMI-1640 medium and incubated with an equivalent volume (1:1, v / v) of lentivirus-based pseudovirus expressing Renilla Luciferase (50 foci forming units). The lentivirus-serum mixture was incubated at 37 °C for 1 h to allow interaction between neutralizing antibodies and pseudovirus particles. The mixture (100 µL) was then added to HEK-293 / ACE-II cells and incubated for 48 h. Luciferase expression was evaluated by the Renilla-Glo® Luciferase Assay System (Promega, #E2710) according to manufacturer’s protocols. The neutralization titer 50% (NT50) was determined as the greatest serum dilution at which the luminescence was reduced by 50% relative to control cells treated with the pseudovirus in the absence of serum. NT50 titers for each sample were measured in two technical replicates performed on separate days. Statistical analysis Data are presented as mean ± SD. Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey test was applied for comparison between two groups or among multiple groups, respectively. p < 0.05 was considered to be statistically significant. Example 1: Optimization of amine–thiol–acrylate conjugation for AID-lipid synthesis To confirm the feasibility of the amine-thiol-acrylate conjugation, a model reaction between amine 19, Traut’s reagent, and acrylate O12 was performed (FIG.2A). Several synthetic conditions were tested and the reaction progress was monitored by liquid chromatography-mass spectrometry (LC-MS), wherein the desired target was observed by - 77 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) LCMS (FIG.2B). The reaction proceeded very quickly at RT with the presence of the base triethylamine (TEA) in either dimethyl sulfoxide (DMSO) or ethanol (EtOH) with minimum byproducts, and was complete in 1 h with isolation yields over 80% for the target product 19T-O12. Since the conversion of the primary amine to the thiolated product by Traut’s reagent is typically complete within 1 h and the subsequent nucleophilic thiol-Michael addition proceeds instantly, the combination of these two reactions in a one-pot profile greatly simplifies and accelerates the synthetic process. Example 2: High-throughput synthesis and screening of AID-lipids Next, this T-MCR method was utilized to synthesize a series of AID-lipids. EtOH was selected as the solvent, as it is a green solvent and the resulting solution is ready for LNP formulation. Twenty-five amines, including alkylamines, anilines and hydrazides, were reacted in a combinatorial manner with four alkyl acrylates (O9, O12, O14 and O16) to form 100 AID-lipids containing amidine and ester linkages (FIG.1A) (Tables 1-3). To enable fast and high-throughput synthesis, individual reactions were performed in 1.5 mL Eppendorf tubes, where all tubes were sonicated simultaneously for 1 h (FIG.3A). Table 1. Exemplary amines No. Structure Name 1decan-1-amine2 dodecan-1-amine3 tetradecan-1-amine4 hexadecan-1-amine5 octadecan-1-amine6 (Z)-octadec-9-en-1-amineH 7 N NH2palmitohydrazide O 8 stearoylhydrazide 9 2-hexyldecan-1-amine 10 octyl glycinate 11 4-octylaniline - 78 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 12 4-decylaniline 13 4-dodecylaniline 14 4-(nonyloxy)aniline 15 ethane-1,2-diamine16 propane-1,3-diamine17 pentane-1,5-diamine18 N1-(2-aminoethyl)ethane- 1,2-diamine 19 N1-(2-aminoethyl)-N1- methylethane-1,2-diamine N1,N1-bis(2- 20 aminoethyl)ethane-1,2- diamine N1-(3-aminopropy121 l)- N - methylpropane-1,3-diamine N1,N1-bis(3- 22 aminopropyl)propane-1,3- diamine 23 3,3’-(piperazine-1,4- diyl)bis(propan-1-amine) N1-(2-(4-(2- 24 aminoethyl)piperazin-1- yl)ethyl)ethane-1,2-diamine N1,N1’-(butane-1,4- diyl)b125 is(N -(3- aminopropyl)propane-1,3- diamine) Table 2. Exemplary acrylates No. Structure Name O9 nonyl acrylate O12 dodecyl acrylate O14 tetradecyl acrylate O16 hexadecyl acrylate - 79 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Table 3. Exemplary substituted amidine lipids No. Compound 1T- O9nonyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate 1T- O12dodecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate 1T- O14tetradecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate 1T- O16hexadecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate 2T- O9nonyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate 2T- O12dodecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate 2T- O14tetradecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate 2T- O16hexadecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate 3T- O9nonyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate 3T- O12dodecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate - 80 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 3T- O14tetradecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate 3T- O16hexadecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate 4T- O9nonyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate 4T- O12dodecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate 4T- O14tetradecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate 4T- O16hexadecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate 5T- O9nonyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate 5T- O12dodecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate 5T- O14tetradecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate 5T- O16hexadecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate 6T- O9nonyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate - 81 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 6T- O12dodecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate 6T- O14tetradecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate 6T- O16hexadecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate 7T- O9nonyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate 7T- O12dodecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate 7T- O14tetradecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate 7T- O16hexadecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate 8T- O9nonyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate 8T- O12dodecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate 8T- O14tetradecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate 8T- O16hexadecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate - 82 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 9T- O9 nonyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate 9T- O12 dodecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate 9T- O14 tetradecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate 9T- O16 hexadecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate 10T -O9nonyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate 10T - O12dodecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate 10T - O14tetradecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate 10T - O16hexadecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate 11T -O9nonyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate 11T - O12 dodecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate - 83 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 11T - O14 tetradecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate 11T - O16 hexadecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate 12T -O9nonyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate 12T - O12 dodecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate 12T - O14 tetradecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate 12T - O16 hexadecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate 13T -O9nonyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate 13T - O12 dodecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate 13T - O14 tetradecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate 13T - O16 hexadecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate - 84 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 14T -O9 nonyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate 14T - O12dodecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate 14T - O14tetradecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate 14T - O16hexadecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate 15T -O9 dinonyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate 15T - O12didodecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate 15T - O14ditetradecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate 15T - O16dihexadecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate 16T -O9dinonyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate 16T - O12 didodecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate - 85 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 16T - O14 ditetradecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate 16T - O16 dihexadecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate 17T -O9dinonyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate 17T - O12 didodecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate 17T - O14 ditetradecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate 17T - O16 dihexadecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate 18T -O9dinonyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate 18T - O12 didodecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate 18T - O14 ditetradecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate 18T - O16 dihexadecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate - 86 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 19T -O9dinonyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate 19T - O12 didodecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate 19T - O14 ditetradecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate 19T - O16 dihexadecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate 20T -O9 dinonyl 8,16-diimino-12-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate 20T - O12 didodecyl 12-(2-(4-((3-(dodecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)- 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate 20T - O14 ditetradecyl 8,16-diimino-12-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15- triazatricosanedioate - 87 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 20T - O16 dihexadecyl 12-(2-(4-((3-(hexadecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)- 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate 21T -O9 dinonyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate 21T - O12didodecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate 21T - O14 ditetradecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate 21T - O16 dihexadecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate 22T -O9 dinonyl 8,18-diimino-13-(3-(3-((3-(nonyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17- triazapentacosanedioate 22T - O12 didodecyl 13-(3-(3-((3-(dodecyloxy)-3-oxopropyl)thio)propanimidamido)propyl)- - 88 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 8,18-diimino-4,22-dithia-9,13,17-triazapentacosanedioate 22T - O14 ditetradecyl 8,18-diimino-13-(3-(3-((3-oxo-3- (tetradecyloxy)propyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17- triazapentacosanedioate 22T - O16 dihexadecyl 13-(3-(3-((3-(hexadecyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-8,18-diimino-4,22-dithia-9,13,17- triazapentacosanedioate 23T -O9 dinonyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate 23T - O12 didodecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate - 89 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 23T - O14 ditetradecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate 23T - O16 dihexadecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate 24T -O9 nonyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate 24T - O12 dodecyl 3-((4-((2-((2-(4-(2-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate 24T - O14 tetradecyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-oxo-3- - 90 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) (tetradecyloxy)propyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate 24T - O16 hexadecyl 3-((4-((2-((2-(4-(2-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate 25T -O9 dinonyl 8,23-diimino-13,18-bis(3-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22- tetraazatriacontanedioate 25T - O12 didodecyl 13,18-bis(3-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate - 91 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) 25T - O14 ditetradecyl 8,23-diimino-13,18-bis(3-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22- tetraazatriacontanedioate 25T - O16 dihexadecyl 13,18-bis(3-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate The resulting 100 AID-lipids were formulated into 100 AID-LNPs along with excipients including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). To evaluate the mRNA delivery efficacy of this lipid library and study the SAR, 1- methylpseudouridine (m1ψ)-modified luciferase mRNA (mLuc) was encapsulated into these LNPs and luciferase protein expression in HepG2 cells was determined. Of the 100 AID-lipid members, none showed toxicity (i.e., cell viability < 80%, FIG.4). Moreover, 98 AID-lipids were able to deliver mRNA in vitro (FIGs.3B-3C), of which, 23 mediated luciferase expression up to 100,000 units, which is generally considered very high performance. Analysis of these 23 outstanding AID-lipids revealed that they were synthesized either by long-chain (C14-C18) alkylamines, ring-alkyl anilines, diamines or partial polyamines (FIG. 3D), whose relative hit rates (i.e., percentage in the group with luminescence > 100,000 units – percentage in library) are 10.1, 14.1, 9.7 and -5.9, respectively (FIG.3E). - 92 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Although monoamines overall afforded less effective AID-lipids (i.e., relative hit rate of about -3.8), two monoamine subclasses (i.e., long-chain alkylamine and ring-alkyl anilines) generally yielded outstanding AID-lipids (FIG.3E). Interestingly, ring-alkyl anilines tended to generate more potent AID-lipids (luminescence > 200,000 units) compared to long- chain alkylamines, despite having the same number of carbon atoms (e.g., 12T-O14 versus 4T-O14; 13T-O14 versus 5T-O14, FIG.3C). With respect to tail length, O14 generally outperformed others to produce efficacious AID-lipids with the highest relative hit rate of 9.8 (FIG.3F). Since ring-alkyl aniline has the highest hit rate and such structures (either as amino heads or as tails) have never been reported in previous combinatorial libraries, this family of AID-lipids was further examined. It is noteworthy that anilines 11-14 not only provide a primary amine for amidine formation, but may also provide one of the two tails in the corresponding asymmetric AID-lipids. Therefore, the length and hydrophobicity of the alkyl substituent on the benzene ring should greatly impact mRNA delivery. Indeed, the elongation of the alkyl substituent appeared to increase delivery efficiency (11-13; FIG.3C and FIG.5), but replacement of one carbon with the polar oxygen atom in the alkyl substituent dramatically decreased efficacy presumably due to its reduced hydrophobicity (12 versus 14; and FIG.6). The in vivo delivery efficacy of exemplary AID-lipids was next examined. The top 8 AID-lipid candidates (luminescence > 200,000 units) from the in vitro screening were tested in mice after intramuscular (i.m.) injection. Despite all AID-LNPs showing similar in vitro potency and achieving strong transfection at an mRNA dose as low as 3.7 ng per well (FIG. 3G), their in vivo performance was disparate (FIG.3H). Two AID-lipids, 12T-O14 and 13T- O14, mediated significantly higher mLuc transfection than others. Interestingly, both possess a ring-alkyl aniline and an O14 tail, correlating well with their high relative hit rates. A few exemplary AID-lipids were purified, and the consistency of their in vivo performance was confirmed with their corresponding crude counterparts, as well as the superiority of ring-alkyl aniline (i.e., 12 and 13) and O14 tail. Furthermore, two unsaturated acrylate tails (O18a and O18b) were prepared and then applied T-MCR to generate 12T-O18a and 12T-O18b. Despite their improved in vitro mRNA delivery efficiency compared to 12T-O14, neither of them achieved better in vivo performance. Here, the top-performing AID-lipid 12T-O14 was purified, characterized (FIGs 7A- 7E, FIGs.8-9, and Table 4), and used for subsequent studies. - 93 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Table 4. Exemplary 12T-O14-LNP formulation Component Weight ratio (relative) Molar ratio (%) 12T-O14 16 33 Cholesterol 15 48.5 DOPE 10 17 DMG-PEG 3 1.5 mRNA 1.6 - Example 3: Optimization and characterization of AID-LNP After acquiring the purified 12T-O14, the LNP formulation was next systematically optimized using an in vitro / vivo hybrid method (FIGs.10A-10C), which resulted in the molar ratio of 12T-O14, cholesterol, DOPE and DMG-PEG of about 33:48.5:17:1.5. Next, the physicochemical properties of the 12T-O14 LNP formulated by microfluidic mixing of lipids (in ethanol) and mRNA (in citrate buffer, pH 3.0) were characterized. The mRNA encapsulation efficiency (EE) of this LNP was 93.4%, suggesting its strong ability to condense and entrap mRNA molecules. The hydrodynamic diameter of 12T-O14 LNP was approximately 73.4 nm (FIGs.12A-12C) with a low polydispersity index (PDI = 0.075) and a neutral surface charge (ζ = 1.63 mV). The apparent pKa of 12T-O14 LNP was determined to be 8.15 (FIG.13), suggesting that 12T-O14 is protonated at pH 7.4 due to the basicity of the amidine. Owing to this feature, 12T-O14 LNP could also efficiently encapsulate mRNA through electrostatic interaction in neutral PBS buffer with an EE of 88.1%. The representative cryogenic electron microscopy (cryo-EM) image showed that 12T-O14 LNP was spherical with a multi-lamellar shell and an amorphous core (FIG.11A). Table 5. Selected 12T-O14 LNP characterization data LNP 12T-O14 LNP Ethanolic (lipid) phase 12T-O14:DOPE:chol:DMG-PEG (33:17:48.5:1.5) Aqueous (mRNA) phase Citrate buffer (pH 3.0) PBS buffer (pH 7.4) Diameter (nm) 73.4 ± 1.6 93.1 ± 1.2 PDI 0.075 0.061 Zeta potential (mV) 1.63 ± 2.8 2.33 ± 3.2 EE (%) 93.4 88.1 pKa8.48 - 94 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Table 6. Selected data for certain exemplary lipids Packing 12T-O14 4T-O14 DOTAP parameters V 1231.21 1173.27 1413.98 A 12.33 12.33 32.01 L 21.19 23.14 21.8 P value 4.71 4.11 2.03 Table 7. Selected characterization of certain exemplary LNPs LNP Diameter PDI Zeta Encapsulation (nm) potential Efficiency (%) (mv) DOTAP LNP 81.6 ± 2.7 0.102 2.9 ± 1.2 90.3 ± 1.8 SM-102 LNP 67.5 ± 3.1 0.082 -1.1 ± 0.3 93.4 ± 2.7 MC3 LNP 69.2 ± 1.7 0.068 -0.5 ± 0.9 93.1 ± 1.3 MC3 / DOTAP LNP (40:1)a165.7 ± 6.3 0.230 5.9 ± 3.2 95.8 ± 2.5 MC3 / 12T-O14 LNP (40:1)a159.2 ± 7.4 0.226 6.1 ± 2.8 96.5 ± 0.7 MC3 / 12T-O14 LNP (20:1) 83.5 ± 6.7 0.127 -1.2 ± 0.5 95.4 ± 1.2 MC3 / 12T-O14 LNP (10:1) 89.4 ± 5.3 0.115 -13.8 ± 1.4 92.6 ± 0.6 MC3 / 12T-O14 LNP (5:1) 83.7 ± 5.4 0.137 -18.9 ± 0.7 74.8 ± 3.4 C12-200 / 18PA LNP 111.8 ± 4.8 0.183 -8.5 ± 1.7 46.0 ± 2.8 Example 4: AID-lipid aids safe and effective intracellular mRNA delivery Previous studies have suggested that cationic lipids are prone to induce immunotoxicity and cytotoxicity. Therefore, the immunostimulatory effect of 12T-O14 LNP was compared with a series of benchmark LNPs in RAW264.7 macrophages (FIG.14). Two proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were examined by enzyme-linked immunosorbent assay (ELISA).12T-O14 LNP induced marginal levels of TNF-α and undetectable levels of IL-6, which were comparable with DOTMA, DOTAP, MC3 and SM-102 LNPs but were significantly lower than C12-200 LNP. These results suggest that 12T-O14 LNP had a low immunostimulatory effect. Next, the cytotoxicity of 12T-O14 LNP was investigated in HepG2 cells (FIG.11B). LNPs formulated - 95 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) by the traditional cationic lipid DOTAP and the state-of-the-art ionizable cationic lipid SM- 102 were chosen for comparison. The results showed that 12T-O14 LNP and SM-102 LNP were more biocompatible than DOTAP LNP, as DOTAP LNP significantly reduced cell viability at a high dose. After confirming the low toxicity profile of 12T-O14 LNP, the intracellular mRNA delivery efficiency of the compounds were compared.12T-O14 LNP remarkably outperformed DOTAP LNP in delivery of mLuc into HepG2 cells, though it was inferior to SM-102 LNP (FIG.11C). In terms of siRNA delivery, 12T-O14 LNP mediated greater gene knockdown than DOTAP LNP, which was comparable to the gold standard siRNA-delivering system MC3 LNP (FIG.15). These results suggest that 12T-O14 LNP is able to efficiently deliver different RNA constructs into cells. Example 5: AID-lipid uses benzene ring to aid membrane disruption Endosomal entrapment of LNPs is a critical barrier for intracellular mRNA delivery. Therefore, the superior performance of 12T-O14 LNP prompted the investigation of its endosomal escape properties. The confocal laser scanning microscope (CLSM) images revealed that, similar to SM-102 LNP, 12T-O14 LNP escaped from endosomes more efficiently than DOTAP LNP (FIG.11D), as more cytosolic distribution of LNPs (green) and less co-localization between LNPs and endosomes was observed in 12T-O14 LNP-treated cells. It was observed that a major structural difference between 12T-O14 and DOTAP (or many other AID-lipids) is that the former has a benzene ring. Thus, without wishing to be bound by any theory, it was hypothesized that this bulky group could act as a wedge that increases the distance between two tails to produce a cone shape (termed “Wedge effect”, FIG.11E), which can form a more cone-shaped ion pair with the anionic membrane phospholipid to enhance endosome disruption. To verify this hypothesis, the packing parameter (P) of the lipid molecule was calculated based on molecular dynamics simulations (FIG.11F), which is correlated to the cone angle from the polar head to terminal tails. For more rigid comparison, 4T-O14, which contains a C6 alkyl chain instead of a benzene ring, was included (FIG.11E). It was found that the P value of 12T-O14 (P=4.71) was larger than that of 4T-O14 (P=4.11), DOTAP (P=2.03) and SM-102 (P=3.60). These computational results suggest that 12T-O14 possesses a more cone-shaped structure and is more likely to induce the rupture of endosomal membrane. - 96 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Next, the membrane-disruptive activities of these lipids was evaluated using a hemolysis assay (FIG.11G).12T-O14 LNP exhibited significantly stronger hemolysis than 4T-O14 LNP, DOTAP LNP and SM-102 LNP at neutral pH. Notably, similar to SM-102 LNP, the hemolytic activity of 12T-O14 LNP was dramatically enhanced at acidic pH, suggesting that the bulky benzene ring (to produce a cone-shaped structure) and the cationic amidine (to bind with the anionic membrane phospholipid) cooperatively contribute to membrane disruption. It is worth mentioning that the strong hemolytic activity of 12T-O14 LNP at neutral pH was reduced in the presence of serum (FIG.16 and Table 8), indicating that improved hemocompatibility can be achieved after protein absorption. Together, these results strongly validate the importance of the benzene ring in aiding the endosomal escape of 12T-O14 LNP. Table 8. Antibody panel for lung cells Marker Fluorophore Aqua live / dead - PECAM-1 APC CD11c Pacific Blue Ly6G PE / Cy7 CD64 PE / CF594 CD11b PerCP / Cy5.5 Example 6: AID-lipid aids local mRNA vaccine delivery and systemic mRNA drug delivery Inspired by the synthetic simplicity, in vitro potency, marginal immunotoxicity and low cytotoxicity of 12T-O14, its potential for vaccine and protein replacement therapy development by delivering functional mRNAs in vivo was further explored. DOTAP and SM- 102 were chosen for comparison due to their prevalence in the delivery of mRNA vaccines / therapeutics. Local administration of LNP-formulated mRNA encoding antigen has been widely adopted for vaccination to trigger antigen-specific antibody in both preclinical and clinical settings. After confirming that 12T-O14 LNP mediated more protein expression than DOTAP LNP but less protein expression than SM-102 LNP following i.m. injection of mLuc-loaded LNPs (FIG.17A), the antibody responses of SARS-CoV-2 Spike mRNA-encapsulated LNP - 97 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) vaccines were then tested (FIG.17B). The anti-Spike specific binding immunoglobulin G (IgG) was measured in mice following a prime and boost immunization regimen. The results showed that, in five mice receiving 12T-O14 LNP vaccine, one mouse (20%) responded after priming and all mice (100%) responded after boosting, which was similar to SM-102 LNP vaccine and significantly outperformed DOTAP LNP vaccine with no responded mouse (0%) after priming and only two responded mice (40%) after boosting. Moreover, the average anti-Spike IgG titer in 12T-O14 LNP vaccinated mice reached 4.7×105, which was on bar with that in SM-102 LNP vaccinated mice and 1161-fold higher than that in DOTAP LNP vaccinated mice. Neutralization antibodies (NAbs) against SARS- CoV-2 pseudovirus were measured using a lentivirus-based system expressing Spike protein (FIG.18).12T-O14 LNP vaccine triggered a significantly higher titer of NAbs than DOTAP LNP vaccine, though it was inferior to SM-102 LNP vaccine. These results suggest that 12T- O14 LNP can be a promising vector for local mRNA vaccine delivery. Next, it was explored whether 12T-O14 LNP was able to deliver functional mRNA systemically, as systemic delivery of mRNA-encoding therapeutic proteins holds great potential to treat many diseases. First, the in vivo transfection profile was determined by examining luciferase expression following intravenous (i.v.) injection of mLuc-loaded LNPs. The whole body bioluminescence imaging results showed that 12T-O14 LNP enabled 28- fold stronger luciferase expression than DOTAP LNP (FIG.17C). Predominant transfection of liver, spleen and lung was observed in 12T-O14 LNP-treated mice (FIG.17D), whose total flux was 72-, 108- and 28-fold higher than that in DOTAP LNP-treated mice. Next, the systemic delivery of mRNA encoding human fibroblast growth factor 21 (FGF21), a metabolic hormone primarily secreted by the liver, was tested, which is a promising therapeutic protein to treat obesity, type 2 diabetes and non-alcoholic steatohepatitis. While a high level of circulating FGF21 was detected in 12T-O14 LNP- treated mice at 6 h post-treatment (FIG.17E), none was detected in DOTAP LNP-treated mice due to its faint liver transfection. The state-of-the-art SM-102 LNP enabled stronger liver transfection and higher FGF21 expression compared to 12T-O14 LNP (FIGs.17C-17E). The toxicities of LNPs at 24 h post treatment were further analyzed. None of the three LNPs caused damage to the liver or other organs, as no significant increases of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as no histological abnormalities were observed (FIG.19). Moreover, cytokine analysis revealed that all 13 cytokines tested were either undetectable or at low levels (< 5 pg / mL) for all three LNPs. These results indicate that 12T-O14 LNP can - 98 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) aid systemic mRNA delivery for potential protein replacement therapy with good tolerability. Table 9. Innate immune responses after LNP treatment (n=3) LNPs No. IFN-γCXC L1 TNF-αCCL2IL-IL-1β 12p70 CCL51 <0.86 0.59 <0.49 <2.56 <0.09 <0.39 0.27 Control 2 <0.86 0.61 <0.49 <2.56 0.09 <0.39 <0.27 3 <0.86 0.83 <0.49 <2.56 0.10 <0.39 <0.27 4 <0.86 0.56 <0.49 <2.56 <0.09 0.83 <0.27 12T-O14 LNP 5 <0.86 0.57 <0.49 <2.56 <0.09 0.55 <0.27 6 <0.86 0.66 <0.49 <2.56 <0.09 0.93 <0.27 7 <0.86 0.59 <0.49 <2.56 <0.09 <0.39 <0.27 DOTAP LNP 8 <0.86 0.53 <0.49 <2.56 <0.09 <0.39 <0.27 9 <0.86 0.57 <0.49 <2.56 <0.09 0.42 <0.27 10 <0.86 0.70 <0.49 <2.56 <0.09 0.66 <0.27 SM-102 LNP 11 <0.86 0.82 <0.49 <2.56 <0.09 0.42 0.30 12 <0.86 0.64 <0.49 <2.56 <0.09 0.53 <0.27 LNPs No. CXCL10 GM-CSF IL-10 IFN-β IFN-α IL-6 1 <1.84 <0.36 <0.44 <0.41 <0.38 <0.29 Control 2 <1.84 <0.36 0.67 <0.41 <0.38 <0.29 3 1.96 <0.36 0.48 <0.41 <0.38 <0.29 4 4.39 <0.36 0.53 <0.41 0.75 0.40 12T-O14 LNP 5 4.62 <0.36 0.45 <0.41 0.71 <0.29 6 4.17 <0.36 0.51 <0.41 0.54 <0.29 7 2.68 <0.36 <0.44 <0.41 <0.38 <0.29 DOTAP LNP 8 2.38 <0.36 0.57 <0.41 <0.38 <0.29 9 2.58 <0.36 0.66 <0.41 <0.38 <0.29 10 3.51 <0.36 <0.44 <0.41 0.63 <0.29 SM-102 LNP 11 3.39 <0.36 0.52 <0.41 0.73 <0.29 12 3.75 <0.36 <0.44 <0.41 0.67 <0.29 “<” indicates the cytokine concentration is lower than the minimum detectable concentration. FGF21 mRNA-loaded LNPs (5 μg mRNA / mouse) were i.v. injected into mice and serum was collected at 24 h post treatment for cytokine analysis. All cytokines were either undetectable or at low levels (< 5 pg / mL) for all three LNPs, which were comparable with the untreated control group. Example 7: AID-lipid aids liver-tropic LNP to selectively target lung or spleen While most LNPs tend to accumulate in the liver, there is a huge demand to develop LNP technologies for non-liver mRNA delivery, such as to the lung or spleen. Yet so far, very few studies have successfully engineered liver-tropic LNPs to selectively target the lung or spleen, let alone toggle between the two. Previous studies have indicated that manipulating the internal and / or external charge of LNP formulations can tune organ tropism, known as selective organ targeting (SORT) technology. It was hypothesized that 12T-O14 could also be used as a supplementary cationic - 99 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) lipid to develop lung-targeted LNPs similar to the previous reported SORT LNP. An FDA- approved liver-tropic MC3 LNP was chosen as the framework formulation and it was supplemented with 50% 12T-O14 to develop MC3 / 12T-O14 LNP (MC3:DSPC:Cholesterol:DMG-PEG:12T-O14 = 25:5:19.25:0.75:50). The standard lung-tropic 50% DOTAP-supplemented MC3 SORT LNP (termed MC3 / DOTAP LNP) was prepared accordingly and served as a positive control. The weight ratio of total lipids:mRNA was kept at 40:1 to ensure excess positive charge for both LNPs. The in vivo and ex vivo imaging results showed that while both mLuc-loaded LNPs predominantly transfected the lungs (FIG.20A), MC3 / 12T-O14 LNP was approximately 50- fold more potent than MC3 / DOTAP LNP. It is worth mentioning that very strong transfection was achieved in the lungs (total flux > 1×109p / s) by MC3 / 12T-O14 LNP at a low mRNA dose (0.1 mg / kg), highlighting its potential for lung-targeted mRNA deliver. Next, it was examined whether 12T-O14 supplementation could redirect liver-tropic MC3 LNP to the spleen. Since negatively charged LNPs have been shown to preferentially target the spleen, the weight ratio of total lipids:mRNA was gradually decreased from 40:1 to 10:1 in order to obtain excess negative charge in MC3 / 12T-O14 LNP while keeping the lipid molar ratio unchanged. The decrease of weight ratio shifted the surface charge of MC3 / 12T- O14 LNP from slightly positive (40:1, 6.1 mV) to near-neutral (20:1, -1.2 mV) and finally to negative (10:1, -13.8 mV) without significantly reducing mRNA EE. Correspondingly, the luciferase expression shifted from lung (40:1) to lung and spleen (20:1) and finally to spleen (10:1, FIG.20B). Interestingly, while MC3 LNP predominantly transfected the liver with a luminescence flux above 1×108p / s, 12T-O14 supplementation almost completely shifted the transfection to lung and / or spleen with designated fluxes maintained above 1×108p / s (FIGs.20C-20D). Notably, although lung and spleen both can be transfected by 12T-O14 LNP as previously shown (FIG.17D), this 12T-O14 supplementation strategy can enable much stronger and more precise lung and spleen transfection. Further decrease of the weight ratio to 5:1 resulted in a low EE of mRNA (~75%) and a declined splenic signal below 1×107p / s (FIG.21), and thus was not considered for subsequent studies. Altogether, these results suggest that 12T-O14 can serve as a supplementary cationic lipid in liver-tropic LNPs to develop potent lung-tropic or spleen- tropic LNPs via simple alterations of the LNP formulation. Table 10. Antibody panel for spleen cells - 100 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Marker Fluorophore Aqua live / dead - CD45 APC CD11c Pacific Blue Ly6G PE / Cy7 CD64 PE / CF594 CD11b PerCP / Cy5.5 Example 8: AID-lipid enables liver-tropic LNP to transfect non-liver cells Inspired by the superior ability of 12T-O14 to redirect liver-tropic LNP to lung or spleen selectively, the transfection of specific cell types in these organs was examined using an activatable Cre / loxP Ai6 reporter mouse model. Following LNP delivery of Cre recombinase-encoded mRNA (Cre mRNA), the translated Cre protein can cut the STOP cassette therefore turning on the expression of ZsGreen1 in transfected cells (FIG.22A). It was first confirmed that strong ZsGreen1 signal was detected in lung sections of lung-tropic MC3 / 12T-O14 LNP (40:1)-treated mice and in spleen sections of spleen-tropic MC3 / 12T-O14 LNP (10:1)-treated mice. Next, the transfected cell populations were analyzed using flow cytometry. Lung-tropic MC3 / 12T-O14 LNP was able to deliver mRNA to ~6% of total cells and primarily to endothelial cells (EC, ~60%) in the lung (FIG.22B), which was comparable to previously reported top-performing lung-targeted LNP systems. Recently, there has been a growing interest in genome editing through co-delivery of Cas9 mRNA and sgRNA as exemplified by Intellia’s LNP-mediated liver transthyretin (TTR) knockout, yet few studies have successfully used this combination to knock out an endogenous gene in the lung. Accordingly, the potential of lung-tropic MC3 / 12T-O14 LNP to co-deliver Cas9 mRNA and sgRNA for gene editing was next tested. TTR was selected as a model editing target and the generation of insertions and deletions (indels) was quantified by Next-Generation Sequencing (NGS). Lung-tropic MC3 / 12T-O14 LNP induced roughly 2% TTR indels in the lung at a total RNA dose of 1 mg / kg (FIG.22C) without causing noticeable pulmonary damage. As expected, liver-tropic MC3 LNP was unable to edit TTR in the lung (indels < 0.05%) despite it inducing roughly 6% TTR indels in the liver. These results suggest that 12T-O14 expands the potential utility of liver-tropic LNP for targeted mRNA delivery and gene editing in the lung. Furthermore, the transfected splenocytes were analyzed after systemic delivery of Cre - 101 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) mRNA using spleen-tropic MC3 / 12T-O14 LNP. This LNP formulation mainly transfected splenic antigen presenting cells (APCs), including ~15% of macrophages and ~10% of dendritic cells (DCs). Since there is a growing interest to deliver mRNA vaccines to splenic APCs as exemplified by BioNTech’s RNA-LPX system, this potential in spleen-tropic MC3 / 12T-O14 LNP was examined. SARS-CoV-2 Spike mRNA was chosen as a model antigen-encoded mRNA, which enables facile determination of the magnitude of adaptive immune responses based on serum anti-Spike IgG titer and SARS-CoV-2 pseudovirus NAb titer. After the prime and boost immunization of spleen-tropic MC3 / 12T-O14 LNP vaccine, the average anti-Spike IgG titer and NAb titer in mice reached 9.9×105and 1.0×104, respectively (FIG.22E), which were higher than those in mice vaccinated with 12T-O14 LNP vaccine or SM-102 LNP vaccine via i.m. injection (FIG.17B and FIG.18). These results demonstrate the great potential of spleen-tropic MC3 / 12T-O14 LNP for splenic APC-targeted mRNA vaccine delivery. Sequence Listing SEQ ID NO:1 (mTTR-exon2-F) 5’-CGGTTTACTCTGACCCATTTC-3’ SEQ ID NO:2 (mTTR-exon2-R) 5’-GGGCTTTCTACAAGCTTACC-3’ Enumerated Embodiments The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance: Embodiment 1 provides a compound of formula (I), or a salt or solvate thereof: (I), wherein: A is selected from the group consisting ; L comprises at least one divalent moiety of - (optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, - (optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2-C8heterocycloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, and -(optionally substituted - 102 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) C2-C10 heteroarylenyl)-, wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each occurrence of G is ; each occurrence of T1, T2, and T3is independently ; each occurrence of R1a, R1b, and R1cis ; R2is selected from the group consisting of optionally substituted C1-C20heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3alkylenyl)C(=O)OR8, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, and C2- C10heteroaryl in R2are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R7is selected from the group consisting of is selected from the group consisting of optionally substituted C1-C20alkyl, optionally substituted C1-C20heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20alkynyl, and optionally substituted C2-C20heteroalkynyl; R8is selected from the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, and optionally substituted C2-C20 heteroalkynyl; - 103 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) RAand RBare each independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C3-C8cycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C10 heteroaryl; X1is O; and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Embodiment 2 provides the compound of Embodiment 1, wherein the compound of formula (I) is selected from the group consisting of: R2N T1R1a, are group - (optionally substituted C2-C6 alkylenyl)-, -(optionally substituted C1-C6 heteroalkylenyl)-, - (optionally substituted C1-C6alkylenyl)(optionally substituted C2-C8heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-, and -(optionally substituted C1-C6heteroalkylenyl)(optionally substituted C2-C8heterocycloalkylenyl)(optionally substituted C1-C6 alkylenyl)-. Embodiment 3 provides the compound of Embodiment 1 or 2, wherein R2is selected from the group consisting of C1-C20 alkyl, C2-C20 alkenyl, -NHC(=O)(C1-C20 alkyl), -(C1-C3 alkylenyl)C(=O)O(C1-C20alkyl), and phenyl, wherein the phenyl is optionally substituted with at least one C1-C20 alkyl or C1-C20 alkoxy. Embodiment 4 provides the compound of any one of Embodiments 1-4, wherein R2is selected from the group consisting of -(CH2)9CH3, -(CH2)11CH3, -(CH2)13CH3, -(CH2)15CH3, - each independently selected from the group consisting of -(CH2)2-, -(CH2)3-, -(CH2)4-, - - 104 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) (CH2)5-, -(CH2)2NH(CH2)2-, -(CH2)2NCH3(CH2)2-, -(CH2)3NCH3(CH2)3-, 1-5, wherein at (a) each occurrence of R4aand R4bare H; and (b) m is 2 or 3. Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein at least one of the following applies: (a) at least one of R5a, R5b, R5c, R5d, R5e, and R5fis H; (b) at least two of R5a, R5b, R5c, R5d, R5e, and R5fare H; (c) at least three R5a, R5b, R5c, R5d, R5e, and R5fare H; (d) at least four of R5a, R5b, R5c, R5d, R5e, and R5fare H; (e) at least five of R5a, R5b, R5c, R5d, R5e, and R5fare H; and (f) each of R5a, R5b, R5c, R5d, R5e, and R5fare H. Embodiment 8 provides the compound of any one of Embodiments 1-7, wherein T1, T2, and T3are each independently . Embodiment 9 provides of any one of Embodiments 1-8, wherein at least one of the following applies: (a) at least one of R6a, R6b, R6c, and R6dis H; (b) at least two of R6a, R6b, R6c, and R6dare H; (c) at least three of R6a, R6b, R6c, and R6dare H; and (d) each of R6a, R6b, R6c, and R6dare H. Embodiment 10 provides the compound of any one of Embodiments 1-9, wherein R1a, R1b, and R1care each . Embodiment 11 provides any one of Embodiments 1-10, wherein R7is selected from the group consisting of nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. Embodiment 12 provides the compound of any one of Embodiments 1-11, wherein R1a, R1b, and R1care each independently selected from the group consisting of - (CH2)2C(=O)O(CH2)8CH3, -(CH2)2C(=O)O(CH2)11CH3, -(CH2)2C(=O)O(CH2)13CH3, and - - 105 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) (CH2)2C(=O)O(CH2)15CH3. Embodiment 13 provides the compound of any one of Embodiments 1-12, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, optionally substituted heterocycloalkylenyl, optionally substituted arylenyl, and optionally substituted heteroarylenyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6alkyl, C3-C8cycloalkyl, C1-C6haloalkyl, C1-C3haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O)2N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O)2R’’, C2-C8heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C1-C6alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl. Embodiment 14 provides the compound of any one of Embodiments 1-13, which is selected from the group consisting of: nonyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; nonyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; - 106 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) dodecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; nonyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; dodecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; tetradecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; hexadecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; nonyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; nonyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate hexadecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; - 107 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) hexadecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dinonyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; didodecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; ditetradecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dihexadecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dinonyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; didodecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; ditetradecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; dihexadecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 12-(2-(4-((3-(dodecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)-8,16- diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 12-(2-(4-((3-(hexadecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)- 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; - 108 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) didodecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dihexadecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dinonyl 8,18-diimino-13-(3-(3-((3-(nonyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17-triazapentacosanedioate; didodecyl 13-(3-(3-((3-(dodecyloxy)-3-oxopropyl)thio)propanimidamido)propyl)- 8,18-diimino-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-(3-(3-((3-oxo-3- (tetradecyloxy)propyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17- triazapentacosanedioate; dihexadecyl 13-(3-(3-((3-(hexadecyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-8,18-diimino-4,22-dithia-9,13,17- triazapentacosanedioate; dinonyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; didodecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; ditetradecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; dihexadecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; nonyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-((2-((2-(4-(2-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-((2-((2-(4-(2-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; dinonyl 8,23-diimino-13,18-bis(3-(4-((3-(nonyloxy)-3- - 109 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) oxopropyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22-tetraazatriacontanedioate; didodecyl 13,18-bis(3-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; ditetradecyl 8,23-diimino-13,18-bis(3-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; and dihexadecyl 13,18-bis(3-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate. Embodiment 15 provides a lipid nanoparticle (LNP) composition comprising: (a) at least one ionizable lipid compound of any one of Embodiments 1-14; (b) at least one helper lipid; (c) at least one cholesterol lipid; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof. Embodiment 16 provides the LNP of Embodiment 15, wherein the at least one helper lipid comprises about 1 mol% to about 30 mol% of the LNP. Embodiment 17 provides the LNP of any one of Embodiments 15 or 16, wherein the at least one helper lipid comprises about 5 mol% or about 17 mol% of the LNP. Embodiment 18 provides the LNP of any one of Embodiments 15-17, wherein the at least one helper lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) and diastereoylphosphatidylcholine (DSPC). Embodiment 19 provides the LNP of any one of Embodiments 15-18, wherein the cholesterol comprises about 10 mol% to about 60 mol% of the LNP. Embodiment 20 provides the LNP of any one of Embodiments 15-19, wherein the cholesterol comprises about 19.25 mol% or about 48.5 mol% of the LNP. Embodiment 21 provides the LNP of any one of Embodiments 15-20, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 0.1 mol% to about 10 mol% of the LNP. Embodiment 22 provides the LNP of any one of Embodiments 15-21, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 0.75 mol% or about 1.5 mol% of the LNP. Embodiment 23 provides the LNP of any one of Embodiments 15-22, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises 1,2- - 110 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000). Embodiment 24 provides the LNP of any one of Embodiments 15-23, wherein the at least one ionizable lipid comprises about 10 mol% to about 90 mol% of the LNP. Embodiment 25 provides the LNP of any one of Embodiments 15-24, wherein the at least one ionizable lipid consists essentially of at least one compound of formula (I) of any one of Embodiments 1-14. Embodiment 26 provides the LNP of Embodiment 25, wherein the at least one ionizable lipid comprises about 10 mol% to about 70 mol% of the LNP. Embodiment 27 provides the LNP of Embodiment 25 or 26, wherein the LNP has a molar ratio of (a):(b):(c):(d) of about 33:17:48.5:1.5. Embodiment 28 provides the LNP of any one of Embodiments 1-24, wherein the at least one ionizable lipid comprises at least one compound of formula (I) of any one of Embodiments 1-14 and at least one additional ionizable lipid. Embodiment 29 provides the LNP of Embodiment 28, wherein the at least one additional ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3). Embodiment 30 provides the LNP of Embodiment 28 or 29, wherein the at least one compound of formula (I) comprises about 40 mol% to about 60 mol% of the LNP. Embodiment 31 provides the LNP of any one of Embodiments 28-30, wherein the at least one compound of formula (I) comprises about 50 mol% of the LNP. Embodiment 32 provides the LNP of any one of Embodiments 28-31, wherein the at least one additional ionizable lipid comprises about 10 mol% to about 40 mol% of the LNP. Embodiment 33 provides the LNP of any one of Embodiments 28-32, wherein the at least one additional ionizable lipid comprises about 25 mol% of the LNP. Embodiment 34 provides the LNP of any one of Embodiments 28-33, wherein the LNP has a ratio of the compound of formula (I) to the at least one additional ionizable lipid of about 2:1. Embodiment 35 provides the LNP of any one of Embodiments 28-34, wherein the LNP has a molar ratio of (a):(b):(c):(d) of about 75:5:19.25:0.75. Embodiment 36 provides the LNP of any one of Embodiments 15-35, wherein the LNP further comprises at least one cargo molecule. Embodiment 37 provides the LNP of Embodiment 36, wherein the cargo molecule is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof. - 111 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) Embodiment 38 provides the LNP of Embodiment 36 or 37, wherein the cargo comprises a nucleic acid. Embodiment 39 provides the LNP of Embodiment 38, wherein the nucleic acid cargo is DNA or RNA. Embodiment 40 provides the LNP of Embodiment 38 or 39, wherein the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof. Embodiment 41 provides the LNP of any one of Embodiments 36-40, wherein the cargo is at least partially encapsulated in the LNP. Embodiment 42 provides the LNP of any one of Embodiments 36-41, wherein the LNP has a weight ratio of total lipid ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1 to about 1:1 (total lipid:cargo (w / w)), optionally wherein the weight ratio is about 40:1, 20:1, 10:1, or about 5:1. Embodiment 43 provides a method of preparing an ionizable lipid of formula (II): (II), wherein: A is selected from the group consisting of ; L comprises at least one divalent moiety from of - (optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, - (optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2-C8heterocycloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, and -(optionally substituted C2-C10heteroarylenyl)-, wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each occurrence of G is ; each occurrence of T1, T2, and T3is independently ; each occurrence of R9a, R9b, and R9cis selected from the group consisting of - 112 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) of optionally substituted C1-C20alkyl, substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, optionally substituted C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3alkylenyl)C(=O)OR8, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, and C2- C10heteroaryl are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R8is selected from the group consisting of optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, and optionally substituted C2-C20 heteroalkynyl; R10is selected from the group consisting of is selected from the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C10heteroaryl; X2is selected from the group consisting of a bond, O, S, and N(RA); X3is selected from the group consisting of O, S, and N(RA); and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; the method comprising: (a) contacting an amine compound of formula (A1) or (A2): - 113 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) , moiety selected from the group alkylenyl)-, -(optionally substituted C1-C12heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2-C8 heterocycloalkylenyl)-, - (optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C10heteroarylenyl)-, wherein each divalent moiety in Z is optionally substituted with at least one occurrence of Y; each occurrence of Y is ; and a compound of formula (B): ; to form a thiolate (b) contacting the thiolate intermediate and a compound of formula compound of formula (C1), (C2), or (C3): , Embodiment 44 provides the method of Embodiment 43, wherein at least one of the following applies: (a) the compound of formula (A1) or (A2) is selected from the group consisting of decan-1-amine, dodecan-1-amine, tetradecan-1-amine, hexadecan-1-amine, octadecan-1-amine, (Z)-octadec-9-en-1-amine, palmitoylhydrazide, stearoylhydrazide, 2-hexyldecan-1-amine, octyl glycinate, 4-octylaniline, 4- octylaniline, 4-decylaniline, 4-dodecylaniline, 4-(nonyloxy)aniline, ethane- 1,2-diamine, propane-1,3-diamine, pentane-1,5-diamine, N1-(2- aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2- - 114 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) diamine, N1,N1-bis(2-aminoethyl)ethane-1,2-diamine, N1-(3-aminopropyl)- N1- methylpropane-1,3-diamine, N1,N1-bis(3-aminopropyl)propane-1,3-diamine, 3,3’-(piperazine-1,4-diyl)bis(propan-1-amine), N1-(2-(4-(2- aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, and N1,N1’-(butane-1,4- diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine); (b) the compound of formula (B) is dihydrothiophen-2(3H)-imine; and (c) the compound of formula (C1), (C2), or (C3) is selected from the group consisting of nonyl acrylate, dodecyl acrylate, tetradecyl acrylate, and hexadecyl acrylate. Embodiment 45 provides a pharmaceutical composition comprising the lipid nanoparticle of any one of Embodiments 15-42 and at least one pharmaceutically acceptable carrier. Embodiment 46 provides the pharmaceutical composition of Embodiment 45, wherein the composition further comprises at least one adjuvant. Embodiment 47 provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of Embodiments 15-42. Embodiment 48 provides the method of Embodiment 47, wherein the LNP is formulated as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier. Embodiment 49 provides the method of Embodiment 47 or 48, wherein the disease is cancer. Embodiment 50 provides the method of Embodiment 49, wherein the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. Embodiment 51 provides the method of Embodiment 49 or 50, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and / or ameliorating cancer in the subject. Embodiment 52 provides a method of delivering at least one cargo molecule to the lung and / or spleen of a subject, the method comprising administering to the subject a lipid nanoparticle (LNP) comprising: - 115 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) (a) at least one ionizable lipid compound of any one of Embodiments 1-14; (b) at least one helper lipid; (c) at least one cholesterol lipid; (d) at least one polymer-conjugated lipid and / or a modified derivative thereof; and (e) at least one cargo molecule. Embodiment 53 provides the LNP of Embodiment 52, wherein the wherein the cargo comprises a nucleic acid. Embodiment 54 provides the LNP of Embodiment 53, wherein the nucleic acid cargo is DNA or RNA. Embodiment 55 provides the method of Embodiment 53 or 54, wherein the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof. Embodiment 56 provides the method of any one of Embodiments 52-55, wherein the cargo is at least partially encapsulated in the LNP. Embodiment 57 provides the method of any one of Embodiments 52-56, wherein the LNP has a weight ratio of total lipids ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1 to about 1:1 (total lipid:cargo (w / w)), optionally wherein the weight ratio is about 40:1, 20:1, 10:1, or about 5:1. Embodiment 58 provides the method of Embodiment 57, wherein the nucleic caid cargo is RNA, optionally wherein the RNA is mRNA. Embodiment 59 provides the method of Embodiment 57 or 58, wherein the LNP has a weight ratio of total lipids to nucleic acid cargo of about 40:1 (w / w). Embodiment 60 provides the method of Embodiment 59, wherein the nucleic acid cargo is selectively delivered to the lung. Embodiment 61 provides the method of Embodiment 57 or 58, wherein the LNP has a weight ratio of total lipids to nucleic acid cargo of about 10:1 (w / w). Embodiment 62 provides the method of Embodiment 61, wherein the nucleic acid cargo is selectively delivered to the spleen. Embodiment 63 provides the method of any one of Embodiments 47-62, wherein the subject is a mammal. Embodiment 64 provides the method of Embodiment 63, wherein the mammal is a human. The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding - 116 - 53096686.3 Attorney Docket No.046483-7437WO1(03698) any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application. - 117 - 53096686.3

Claims

Attorney Docket No.046483-7437WO1(03698) CLAIMS What is claimed is:

1. A compound of formula (I), or a salt or solvate thereof: (I), wherein: A is selected from the group consisting of ;L comprises at least one divalent moiety selected from the group consisting of - (optionally substituted C1-C12alkylenyl)-, -(optionally substituted C1-C12heteroalkylenyl)-, - (optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C10 heteroarylenyl)-, wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each occurrence of G is ;each occurrence of T1, T2, and T3is ;each occurrence of R1a, R1b, and R1cis ; 2R is selected from the group consisting of optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, optionally substituted C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3alkylenyl)C(=O)OR8, wherein the C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, and C2- C10heteroaryl in R2are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of - 118 - 53096686.3Attorney Docket No.046483-7437WO1(03698) H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R7is selected from the group consisting of is selected from the group consisting of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; R8is selected from the group consisting of optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, and optionally substituted C2-C20 heteroalkynyl; RAand RBare each independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; X1is O; and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

2. The compound of claim 1, wherein the compound of formula (I) is selected from the group consisting of: R2,L1, L2, and L3are each independently selected from the group consisting of - (optionally substituted C2-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, - (optionally substituted C1-C6 alkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-, and -(optionally substituted C1-C6 heteroalkylenyl)(optionally substituted C2-C8 heterocycloalkylenyl)(optionally substituted C1-C6alkylenyl)-. - 119 - 53096686.3Attorney Docket No.046483-7437WO1(03698) 3. The compound of claim 1 or 2, wherein R2is selected from the group consisting of C1-C20 alkyl, C2-C20 alkenyl, -NHC(=O)(C1-C20 alkyl), -(C1-C3 alkylenyl)C(=O)O(C1-C20 alkyl), and phenyl, wherein the phenyl is optionally substituted with at least one C1-C20alkyl or C1-C20 alkoxy.

4. The compound of any one of claims 1-4, wherein R2is selected from the group consisting of -(CH2)9CH3, -(CH2)11CH3, -(CH2)13CH3, -(CH2)15CH3, -(CH2)17CH3, -5. The compound of claim 2, wherein L1, L2, and L3are each independently selected from the group consisting of -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)2NH(CH2)2-, -6. The compound of any one of claims 1-5, wherein at least one of the following applies: (a) each occurrence of R4aand R4bare H; and (b) m is 2 or 3.

7. The compound of any one of claims 1-6, wherein at least one of the following applies: (a) at least one of R5a, R5b, R5c, R5d, R5e, and R5fis H; (b) at least two of R5a, R5b, R5c, R5d, R5e, and R5fare H; (c) at least three R5a, R5b, R5c, R5d, R5e, and R5fare H; (d) at least four of R5a, R5b, R5c, R5d, R5e, and R5fare H; (e) at least five of R5a, R5b, R5c, R5d, R5e, and R5fare H; and - 120 - 53096686.3Attorney Docket No.046483-7437WO1(03698) (f) each of R5a, R5b, R5c, R5d, R5e, and R5fare H.

8. The compound of any one of claims 1-7, wherein T1, T2, and T3are each independently .

9. The any one of claims 1-8, wherein at least one of the following applies: (a) at least one of R6a, R6b, R6c, and R6dis H; (b) at least two of R6a, R6b, R6c, and R6dare H; (c) at least three of R6a, R6b, R6c, and R6dare H; and (d) each of R6a, R6b, R6c, and R6dare H.

10. The compound of any one of claims 1-9, wherein R1a, R1b, and R1care each .

11. The compound of any one of claims 1-10, wherein R7is selected from the group consisting of nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

12. The compound of any one of claims 1-11, wherein R1a, R1b, and R1care each independently selected from the group consisting of -(CH2)2C(=O)O(CH2)8CH3, - (CH2)2C(=O)O(CH2)11CH3, -(CH2)2C(=O)O(CH2)13CH3, and -(CH2)2C(=O)O(CH2)15CH3.

13. The compound of any one of claims 1-12, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, optionally substituted heterocycloalkylenyl, optionally substituted arylenyl, and optionally substituted heteroarylenyl is independently optionally substituted with at least one substituent selected from the group consisting of C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C3 haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R’’), - 121 - 53096686.3Attorney Docket No.046483-7437WO1(03698) C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O)2N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O)2R’’, C2-C8heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C1-C6alkyl, C3-C8cycloalkyl, C1-C6haloalkyl, benzyl, and phenyl.

14. The compound of any one of claims 1-13, which is selected from the group consisting of: nonyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(decylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(dodecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(tetradecylamino)butyl)thio)propanoate; nonyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-(hexadecylamino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(octadecylamino)butyl)thio)propanoate; nonyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; dodecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; tetradecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; hexadecyl (Z)-3-((4-imino-4-(octadec-9-en-1-ylamino)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; - 122 - 53096686.3Attorney Docket No.046483-7437WO1(03698) hexadecyl 3-((4-imino-4-(2-palmitoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; dodecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-(2-stearoylhydrazineyl)butyl)thio)propanoate; nonyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-((2-hexyldecyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((2-(octyloxy)-2-oxoethyl)amino)butyl)thio)propanoate; nonyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-octylphenyl)amino)butyl)thio)propanoate; nonyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate hexadecyl 3-((4-((4-decylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; dodecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; tetradecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; hexadecyl 3-((4-((4-dodecylphenyl)amino)-4-iminobutyl)thio)propanoate; nonyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dodecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; tetradecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-imino-4-((4-(nonyloxy)phenyl)amino)butyl)thio)propanoate; dinonyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; didodecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; ditetradecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dihexadecyl 8,13-diimino-4,17-dithia-9,12-diazaicosanedioate; dinonyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; - 123 - 53096686.3Attorney Docket No.046483-7437WO1(03698) didodecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; ditetradecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; dihexadecyl 8,14-diimino-4,18-dithia-9,13-diazahenicosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,15-diazatricosanedioate; dinonyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 8,16-diimino-12-methyl-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,16-diimino-12-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; didodecyl 12-(2-(4-((3-(dodecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)-8,16- diimino-4,20-dithia-9,12,15-triazatricosanedioate; ditetradecyl 8,16-diimino-12-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)-4,20-dithia-9,12,15-triazatricosanedioate; dihexadecyl 12-(2-(4-((3-(hexadecyloxy)-3-oxopropyl)thio)butanimidamido)ethyl)- 8,16-diimino-4,20-dithia-9,12,15-triazatricosanedioate; dinonyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; didodecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dihexadecyl 8,18-diimino-13-methyl-4,22-dithia-9,13,17-triazapentacosanedioate; dinonyl 8,18-diimino-13-(3-(3-((3-(nonyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17-triazapentacosanedioate; didodecyl 13-(3-(3-((3-(dodecyloxy)-3-oxopropyl)thio)propanimidamido)propyl)- 8,18-diimino-4,22-dithia-9,13,17-triazapentacosanedioate; ditetradecyl 8,18-diimino-13-(3-(3-((3-oxo-3- (tetradecyloxy)propyl)thio)propanimidamido)propyl)-4,22-dithia-9,13,17- triazapentacosanedioate; - 124 - 53096686.3Attorney Docket No.046483-7437WO1(03698) dihexadecyl 13-(3-(3-((3-(hexadecyloxy)-3- oxopropyl)thio)propanimidamido)propyl)-8,18-diimino-4,22-dithia-9,13,17- triazapentacosanedioate; dinonyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; didodecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; ditetradecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; dihexadecyl 3,3’-((((piperazine-1,4-diylbis(propane-3,1-diyl))bis(azanediyl))bis(4- iminobutane-4,1-diyl))bis(sulfanediyl))dipropionate; nonyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; dodecyl 3-((4-((2-((2-(4-(2-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; tetradecyl 3-((4-imino-4-((2-((2-(4-(2-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)ethyl)piperazin-1- yl)ethyl)amino)ethyl)amino)butyl)thio)propanoate; hexadecyl 3-((4-((2-((2-(4-(2-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)ethyl)piperazin-1-yl)ethyl)amino)ethyl)amino)-4- iminobutyl)thio)propanoate; dinonyl 8,23-diimino-13,18-bis(3-(4-((3-(nonyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22-tetraazatriacontanedioate; didodecyl 13,18-bis(3-(4-((3-(dodecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; ditetradecyl 8,23-diimino-13,18-bis(3-(4-((3-oxo-3- (tetradecyloxy)propyl)thio)butanimidamido)propyl)-4,27-dithia-9,13,18,22- tetraazatriacontanedioate; and dihexadecyl 13,18-bis(3-(4-((3-(hexadecyloxy)-3- oxopropyl)thio)butanimidamido)propyl)-8,23-diimino-4,27-dithia-9,13,18,22- tetraazatriacontanedioate. - 125 - 53096686.3Attorney Docket No.046483-7437WO1(03698) 15. A lipid nanoparticle (LNP) composition comprising: (a) at least one ionizable lipid compound of any one of claims 1-14; (b) at least one helper lipid; (c) at least one cholesterol lipid; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof.

16. The LNP of claim 15, wherein the at least one helper lipid comprises about 1 mol% to about 30 mol% of the LNP.

17. The LNP of any one of claims 15 or 16, wherein the at least one helper lipid comprises about 5 mol% or about 17 mol% of the LNP.

18. The LNP of any one of claims 15-17, wherein the at least one helper lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) and diastereoylphosphatidylcholine (DSPC).

19. The LNP of any one of claims 15-18, wherein the cholesterol comprises about 10 mol% to about 60 mol% of the LNP.

20. The LNP of any one of claims 15-19, wherein the cholesterol comprises about 19.25 mol% or about 48.5 mol% of the LNP.

21. The LNP of any one of claims 15-20, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 0.1 mol% to about 10 mol% of the LNP.

22. The LNP of any one of claims 15-21, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises about 0.75 mol% or about 1.5 mol% of the LNP.

23. The LNP of any one of claims 15-22, wherein the at least one polymer-conjugated lipid and / or a modified derivative thereof comprises 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG-2000). - 126 - 53096686.3Attorney Docket No.046483-7437WO1(03698) 24. The LNP of any one of claims 15-23, wherein the at least one ionizable lipid comprises about 10 mol% to about 90 mol% of the LNP.

25. The LNP of any one of claims 15-24, wherein the at least one ionizable lipid consists essentially of at least one compound of formula (I) of any one of claims 1-14.

26. The LNP of claim 25, wherein the at least one ionizable lipid comprises about 10 mol% to about 70 mol% of the LNP.

27. The LNP of claim 25 or 26, wherein the LNP has a molar ratio of (a):(b):(c):(d) of about 33:17:48.5:1.

5.

28. The LNP of any one of claims 1-24, wherein the at least one ionizable lipid comprises at least one compound of formula (I) of any one of claims 1-14 and at least one additional ionizable lipid.

29. The LNP of claim 28, wherein the at least one additional ionizable lipid comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3).

30. The LNP of claim 28 or 29, wherein the at least one compound of formula (I) comprises about 40 mol% to about 60 mol% of the LNP.

31. The LNP of any one of claims 28-30, wherein the at least one compound of formula (I) comprises about 50 mol% of the LNP.

32. The LNP of any one of claims 28-31, wherein the at least one additional ionizable lipid comprises about 10 mol% to about 40 mol% of the LNP.

33. The LNP of any one of claims 28-32, wherein the at least one additional ionizable lipid comprises about 25 mol% of the LNP.

34. The LNP of any one of claims 28-33, wherein the LNP has a ratio of the compound of formula (I) to the at least one additional ionizable lipid of about 2:

1. - 127 - 53096686.3Attorney Docket No.046483-7437WO1(03698) 35. The LNP of any one of claims 28-34, wherein the LNP has a molar ratio of (a):(b):(c):(d) of about 75:5:19.25:0.

75.

36. The LNP of any one of claims 15-35, wherein the LNP further comprises at least one cargo molecule.

37. The LNP of claim 36, wherein the cargo molecule is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof.

38. The LNP of claim 36 or 37, wherein the cargo comprises a nucleic acid.

39. The LNP of claim 38, wherein the nucleic acid cargo is DNA or RNA.

40. The LNP of claim 38 or 39, wherein the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof.

41. The LNP of any one of claims 36-40, wherein the cargo is at least partially encapsulated in the LNP.

42. The LNP of any one of claims 36-41, wherein the LNP has a weight ratio of total lipid ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1 to about 1:1 (total lipid:cargo (w / w)), optionally wherein the weight ratio is about 40:1, 20:1, 10:1, or about 5:

1.

43. A method of preparing an ionizable lipid of formula (II): (II), wherein: A is selected from the group consisting ; L comprises at least one divalent moietyof - (optionally substituted C1-C12alkylenyl)-, -(optionally substituted C1-C12heteroalkylenyl)-, - - 128 - 53096686.3Attorney Docket No.046483-7437WO1(03698) (optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C10 heteroarylenyl)-, wherein each divalent moiety in L is optionally substituted with at least one occurrence of G; each occurrence of G is ;each occurrence of T1, T2, and T3is ; each occurrence of R9a, R9b, and R9cis selectedof , and R10;of optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C6-C10 aryl, C2-C10 heteroaryl, -N(RA)C(=O)(R8), and -(optionally substituted C1-C3 alkylenyl)C(=O)OR8, wherein the C3-C8cycloalkyl, C3-C8heterocycloalkyl, C6-C10aryl, and C2- C10 heteroaryl are optionally substituted with at least one substituent selected from the group consisting of R8, OR8, N(RA)(R8), S(R8), halogen, CN, and NO2; R3aand R3b, and R3care each independently H; each occurrence of R4aand R4bis independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R5a, R5b, R5c, R5d, R5e, and R5fare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R6a, R6b, R6c, and R6dare each independently selected from the group consisting of H, halogen, CN, NO2, and optionally substituted C1-C6 alkyl; R8is selected from the group consisting of optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20alkynyl, and optionally substituted C2-C20 heteroalkynyl; R10is selected from the group consisting of is selected from the group consisting of - 129 - 53096686.3Attorney Docket No.046483-7437WO1(03698) optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, and optionally substituted C2-C20 heteroalkynyl; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C10heteroaryl; X2is selected from the group consisting of a bond, O, S, and N(RA); X3is selected from the group consisting of O, S, and N(RA); and m is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; the method comprising: (a) contacting an amine compound of formula (A1) or (A2): , moiety selected from thegroup - alkylenyl)-, -(optionally substituted C1-C12heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2-C8 heterocycloalkylenyl)-, - (optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C10heteroarylenyl)-, wherein each divalent moiety in Z is optionally substituted with at least one occurrence of Y; each occurrence of Y is a compound of formula (B):; to form a thiolate(b) contacting the thiolate intermediate and a compound of formula compound of formula (C1), (C2), or (C3): - 130 - 53096686.3Attorney Docket No.046483-7437WO1(03698) ,44. The method of claim 43, wherein at least one of the following applies: (a) the compound of formula (A1) or (A2) is selected from the group consisting of decan-1-amine, dodecan-1-amine, tetradecan-1-amine, hexadecan-1-amine, octadecan-1-amine, (Z)-octadec-9-en-1-amine, palmitoylhydrazide, stearoylhydrazide, 2-hexyldecan-1-amine, octyl glycinate, 4-octylaniline, 4- octylaniline, 4-decylaniline, 4-dodecylaniline, 4-(nonyloxy)aniline, ethane- 1,2-diamine, propane-1,3-diamine, pentane-1,5-diamine, N1-(2- aminoethyl)ethane-1,2-diamine, N1-(2-aminoethyl)-N1-methylethane-1,2- diamine, N1,N1-bis(2-aminoethyl)ethane-1,2-diamine, N1-(3-aminopropyl)- N1- methylpropane-1,3-diamine, N1,N1-bis(3-aminopropyl)propane-1,3-diamine, 3,3’-(piperazine-1,4-diyl)bis(propan-1-amine), N1-(2-(4-(2- aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, and N1,N1’-(butane-1,4- diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine); (b) the compound of formula (B) is dihydrothiophen-2(3H)-imine; and (c) the compound of formula (C1), (C2), or (C3) is selected from the group consisting of nonyl acrylate, dodecyl acrylate, tetradecyl acrylate, and hexadecyl acrylate.

45. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 15-42 and at least one pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, wherein the composition further comprises at least one adjuvant.

47. A method of treating, preventing, and / or ameliorating a disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 15-42.

48. The method of claim 47, wherein the LNP is formulated as a pharmaceutical - 131 - 53096686.3Attorney Docket No.046483-7437WO1(03698) composition comprising at least one pharmaceutically acceptable carrier.

49. The method of claim 47 or 48, wherein the disease is cancer.

50. The method of claim 49, wherein the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer.

51. The method of claim 49 or 50, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and / or ameliorating cancer in the subject.

52. A method of delivering at least one cargo molecule to the lung and / or spleen of a subject, the method comprising administering to the subject a lipid nanoparticle (LNP) comprising: (a) at least one ionizable lipid compound of any one of claims 1-14; (b) at least one helper lipid; (c) at least one cholesterol lipid; (d) at least one polymer-conjugated lipid and / or a modified derivative thereof; and (e) at least one cargo molecule.

53. The method of claim 52, wherein the wherein the cargo comprises a nucleic acid.

54. The method of claim 53, wherein the nucleic acid cargo is DNA or RNA.

55. The method of claim 53 or 54, wherein the nucleic acid is selected from the group consisting of mRNA, cDNA, pDNA, microRNA, sgRNA, siRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof.

56. The method of any one of claims 52-55, wherein the cargo is at least partially encapsulated in the LNP. - 132 - 53096686.3Attorney Docket No.046483-7437WO1(03698) 57. The method of any one of claims 52-56, wherein the LNP has a weight ratio of total lipids ((a), (b), (c), and (d)) to nucleic acid cargo ranging from about 60:1 to about 1:1 (total lipid:cargo (w / w)), optionally wherein the weight ratio is about 40:1, 20:1, 10:1, or about 5:

1.

58. The method of claim 57, wherein the nucleic caid cargo is RNA, optionally wherein the RNA is mRNA.

59. The method of claim 57 or 58, wherein the LNP has a weight ratio of total lipids to nucleic acid cargo of about 40:1 (w / w).

60. The method of claim 59, wherein the nucleic acid cargo is selectively delivered to the lung.

61. The method of claim 57 or 58, wherein the LNP has a weight ratio of total lipids to nucleic acid cargo of about 10:1 (w / w).

62. The method of claim 61, wherein the nucleic acid cargo is selectively delivered to the spleen.

63. The method of any one of claims 47-62, wherein the subject is a mammal.

64. The method of claim 63, wherein the mammal is a human. - 133 - 53096686.3

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