Propargyl amino-ionizable lipid compounds, lipid nanoparticles (LNPS), and methods of making and using same for gene editing

Abstract

The present disclosure relates, in one aspect, to propargyl amino-ionizable lipid compounds of formula (I), and methods of preparing the same. In another aspect, the disclosure relates to lipid nanoparticles (LNPs) comprising at least one ionizable lipid of formula (I) or (III). In another aspect, the LNPs of the disclosure are useful for gene editing applications. In another aspect, the LNPs of the disclosure are useful for splenic delivery of therapeutic cargo.

Description

[0001] TITLE

[0002] Propargyl Amino-Ionizable Lipid Compounds, Lipid Nanoparticles (LNPs). and Methods of Making and Using Same for Gene Editing

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 710,254, filed October 22, 2024, and U. S. Provisional Patent Application No. 63 / 848,636, filed July 22, 2025, both of which are hereby incorporated herein by reference in their entireties.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under TR002776 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The XML file named “046483-7486WO1 - Sequence Listing.xml” created on October 20, 2025, comprising 4,722 bytes, is incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] Messenger RNA (mRNA)-based therapies have revolutionized modem medicine’s approach to treat or prevent diseases. The recent approval of three lipid nanoparticle (LNP)-based RNA drugs (Patisiran, mRNA-1273, and BNT162b2) and continuing advances in LNP technologies for mRNA therapeutics / vaccines and CRISPR gene editing have sparked great interest in this safe and effective nonviral vector. The classical LNP formulation comprises four components: ionizable lipid(s), neutral or helper phospholipid(s), cholesterol, and polyethylene glycol (PEG)-conjugated lipids. An ionizable lipid ty pically comprises an amino headgroup and two (or more) alkyl tails that are connected by linkers, which play a pivotal role in protecting and transporting RNA cargos. Under acidic LNP formulation conditions, ionizable lipids are protonated and switched to a positively charged mode, allowing for complexation with and encapsulation of anionic RNA molecules. At physiological pH, ionizable lipids remain neutral, thereby circumventing charge-related toxicity and improving the pharmacokinetic profile of LNPs. Following cellular uptake, the acidic environment of the endosome facilitates the protonation of ionizable lipids, leading to endosomal escape and release of mRNA.

[0010] Notably, ionizable lipids not only govern the potency but also the biocompatibility of LNPs. Therefore, the current research is focused on structural optimization of ionizable lipids with improved activity and biodegradability. Ionizable lipids are conventionally synthesized via medicinal chemistry or combinatorial chemistry. The medicinal chemistry approach involves rational design and multi-step organic synthesis of ionizable lipids, guided by the putative in vivo mechanism of action and empirical structural criteria. DLin-MC3-DMA (MC3) is a representative ionizable lipid developed based on this approach. Although this approach ensures a high success rate to generate desirable lipid candidates, it is limited by laborious, low-throughput synthesis, making it difficult to create structurally diverse lipids and systematically investigate structure-activity relationships (SARs). The combinatorial chemistry approach typically involves one-pot synthesis of ionizable lipids based on highly efficient two-component reactions or multi-component reactions (MCRs). It enables rapid, high-throughput synthesis of structurally diverse lipids, yet it suffers from a low hit rate for lipids with both high potency and biodegradability due to limitations regarding readily available starting materials.

[0011] Combining medicinal chemistry-based rational design of optimal building blocks with combinatorial chemistry-based high-throughput synthesis exploits the advantages of both approaches to generate desired ionizable lipid structures. However, this combined approach is still highly dependent on user’s empiricism and trial-and-error practices, motivating the development of a rationale-driven and stepwise method to structurally optimize ionizable lipids with desired properties.

[0012] Additionally, gene editing involves the precise manipulation of DNA sequences to modulate cellular phenotypes, offering a novel therapeutic strategy to cure monogenic diseases. CRISPR-Cas9, the most widely used genome editor, is an RNA-guided DNA-cutting enzyme complex that creates double stranded DNA breaks (DSBs) at target positions in the DNA of eukary otic cells. Repair of these breaks most commonly occurs via error-prone non-homologous end joining (NHEJ), which leads to small insertions and deletions (indels) at the break site that may interrupt gene function. Early clinical data suggest that NHEJ-mediated gene knockout can reduce the expression of disease-causing proteins or induce the expression of therapeutic proteins. Indeed, the FDA recently approved the first genome editing drugs that use CRISPR-Cas9 technology, CASGEVY™ and LYFGENIA™, for the treatment of sickle cell disease and transfusion-dependent beta-thalassemia. To fully realize the therapeutic potential of genome editing, CRISPR-Cas9 must be delivered safely and effectively to target cells in vivo.

[0013] There is thus a need in the art for novel ionizable lipids and rapid methods for preparing and identifying the same. Further, there is a need in the art for compositions suitable for the delivery of base editing machinery (e.g., Cas9 mRNA and targeted sgRNA) and methods of using same. The present disclosure addresses these unmet needs.

[0014] BRIEF SUMMARY OF THE INVENTION

[0015] In one aspect, the disclosure provides a compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:

[0016] R1a— -A — R1b(I),

[0017] wherein:

[0018] A is selected from the group consisting of

[0019]

[0020] each occurrence of L1, if present, is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Cs-Cio arylenyl)-X-, and -(optionally substituted C2-C8 heteroarylenyl)-X-;

[0021] each occurrence of X, if present, is independently selected from the group consisting of -N(Rle)-, -[N(CH2)i-3N(Rle)(Rle)]-, -N(RA)-, and -O-;

[0022] Rla. Rlb, Rlc, Rld, and each occurrence of R16, if present, are each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and -CH(R3a)(R3b), wherein

[0023] at least one of Rla, Rlb, and Rlc, if present, is -CH(R3a)(R3b),

[0024] one of Rlaand Rlhcan combine with R2to form an optionally substituted C2-C8 heterocycloalkyl, and

[0025] one of Rla, Rlb, Rlc, and Rldcan combine with one occurrence of L1to form an optionally substituted C2-C8 heterocycloalkyl; R2is selected from the group consisting of optionally substituted Ci-Ce alky l, optionally substituted C2-C6 alkenyl, optionally substituted Cs-Cs cycloalkyl. optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);

[0026] R3aand R3bare each independently selected from the group consisting of optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, optionally substituted C2-C24 alkynyl, optionally substituted C1-C24 heteroalkyl, optionally substituted C2-C24 heteroalkenyl, optionally substituted C2-C24 heteroalkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C8 heteroaryl;

[0027] m is 1, 2, 3. 4, 5, 6. 7, 8, 9, or 10; and

[0028] each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl.

[0029] In certain embodiments, the compound of Formula (I) is:

[0030]

[0031] formula (II), or a salt, stereoisomer, or isotopologue thereof:

[0032] Rx

[0033] R2— N

[0034] R5a(II).

[0035] the method comprising:

[0036] RY

[0037] R2— N

[0038] contacting a compound of formula (A): H (A),

[0039] a compound of formula (

[0040]

[0041] O

[0042] A

[0043] a compound of formula (C):H R<3(C),

[0044] in the presence of a copper catalyst;

[0045] wherein: R2is selected from the group consisting of optionally substituted Ci-Ce alky l, optionally substituted C2-C6 alkenyl, optionally substituted Cs-Cs cycloalkyl. optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);

[0046] Rxis selected from the group consisting of R5band optionally substituted Ci-Ce alkyl, or

[0047] Rxand R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyl;

[0048] RYis selected from the group consisting of H and optionally substituted Ci-Ce alkyl, or

[0049] RYand R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyl;

[0050] R5aand R5b, if present, are each independently

[0051]

[0052] R6is selected from the group consisting of R4and

[0053]

[0054] each occurrence of R4is C1-C16 alkyd or C2-C16 alkenyl;

[0055] each occurrence of L2is independently selected from the group consisting of -CH2-, -O-, -C(=O)-, and -(optionally substituted phenylenyl)-; and

[0056] each occurrence of n and o is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0057] In another aspect, the disclosure provides a lipid nanoparticle (LNP) comprising: (a) at least one ionizable lipid comprising the compound of the disclosure;

[0058] (b) at least one neutral lipid;

[0059] (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof. The disclosure provides pharmaceutical composition comprising a lipid nanoparticle (LNP) of the disclosure and at least one pharmaceutically acceptable carrier.

[0060] In another aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or infection in a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the disclosure.

[0061] In another aspect, the disclosure provies lipid nanoparticle (LNP) comprising:

[0062] (a) at least one ionizable lipid compound of Formula (III), or a salt, solvate, stereoisomer, or isotopologue thereof:

[0063]

[0064] wherein:

[0065] R3a

[0066]

[0067] Rlaand Rlbare each independently R3b;

[0068] R2a, R2b, R2C, R2d, R2e, R2f, R2g, and R2hare each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted Ce-Cio ar l, and optionally substituted C2-C10 heteroaiyl;

[0069] each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H, -(optionally substituted Ci-Ce alkylenyl)-C(=O)OR4, -(optionally substituted Ci-Ce alkylenyl)-C(=O)N(R4)(R3), -(optionally substituted Ci-Ce alkylenyl)-C(=O)R4, -(optionally substituted Ci-C6alkylenyl)-(R4), -C(=O)OR4, -C(=O)N(R4)(R5), -C(=O)R4, and R4,

[0070] wherein no more than one of each occurrence of R3a, R3b, and R3cis H;

[0071] R4is selected from the group consisting of optionally substituted C1-C28 alkyl, optionally substituted C2-C28 heteroalkyl, optionally substituted Cs-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C28 alkenyl, and optionally substituted C2-C28 alkynyl;

[0072] R5is selected from the group consisting of H and optionally substituted Ci-Ce alkyl; each occurrence of L is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-. -(optionally substituted C1-C12 heteroalky lenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-, -X-(optionally substituted C2-C12 alkenylenyl)-, -X-(optionally substituted C1-C12 alkynylenyl)-, -X-(optionally substituted C1-C12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocyloalkylenyl;

[0073] each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3c)-, and -O-; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4;

[0074] (b) at least one neutral lipid;

[0075] (c) at least one sterol;

[0076] (d) at least one polymer conjugated lipid; and

[0077] (e) nucleic acid cargo comprising at least one messenger RNA (mRNA), wherein the at least one mRNA has a size ranging from about 2 kilobases to about 10 kilobases.

[0078] In certain embodiments, the ionizable lipid of Formula (III) is:

[0079]

[0080] l,l'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2- hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-ol),

[0081] (C14-494).

[0082] In another aspect, method of treating, preventing, and / or ameliorating a genetic disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of the disclosure or a pharmaceutical composition thereof.

[0083] In another aspect, the disclosure provides a method of genome editing a mutated gene sequence associated with a disease or disorder in a subject.

[0084] In certain embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) of the disclosure or a pharmaceutical composition thereof.

[0085] BRIEF DESCRIPTION OF THE FIGURES

[0086] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0087] FIGs. 1A-1B: A3-coupling and activity- and degradability-driven directed chemical evolution of UPenn-lipids. FIG. 1A: The reaction scheme and mechanism of A3-coupling. An alkynyl-Cu intermediate is formed, which reacts with in situ produced iminium ion originated from aldehyde and amine to form a propargylamine with concomitant regeneration of the Cu+catalyst. FIG. IB: A workflow of activity- and degradability-driven directed chemical evolution of UPenn-lipids. The process starts with a pilot library of UPenn-lipids established by readily available starting materials. After screening and analysis, the selected UPenn-lipid is used as the starting point for subsequent rounds of rationale-driven, customized mutation and selection, gradually improving the desired activity and biodegradability over multiple iterations. This directed chemical evolution approach enables accelerated discovery of desired ionizable lipids and valuable design criteria. The desired UPenn-lipids comprise two ionizable nitrogens, an amino-diacid motif, two 2° esters and pseudo-three tails with an asymmetric tail structure.

[0088] FIGs. 2A-2C: Combinatorial screening of UPenn-lipids in Library 1. FIG. 2A:

[0089] Combinatorial synthesis of UPenn-lipids via A3-coupling of commercially available amines (1-30), formaldehyde (a) and alkynes (A-F). A representative synthesis of ll(aB)2 through A3-coupling twice is shown. FIGs. 2B: In vitro mLuc expression in a heat map (n=2, initial screening). HepG2 cells were treated with mLuc-loaded LNPs at an mRNA dose of 15 ng / well for 24 h. RLU, relative light unit. Untreated cells typically exhibited <100 RLU. FIG.

[0090] 2C: Distribution of UPenn-lipids with in vitro mLuc expression >2.000 RLU across alkyne structure.

[0091] FIGs. 3A-3F: Optimization of symmetric UPenn-lipids with biodegradable alkynes in Library' 2 and asymmetric UPenn-lipids with long-chain aldehydes in Library 3. FIG. 3 A: Biodegradability -driven directed evolution of UPenn-lipids starting from ll(aB)2. Cleavable ester linkers were introduced into 11 (aB)2 and six biodegradable symmetric UPenn-lipids with linear or branched tails were generated. FIG. 3B: In vitro mLuc expression. HepG2 cells were treated with mLuc-loaded LNPs at an mRNA dose of 15 ng / well for 24 h. FIG. 3C: In vivo mLuc expression. Mice were injected i.v. with mLuc-loaded LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h post-treatment and whole-body total flux was quantified. FIG. 3D: Correlation between in vitro and in vivo mLuc expression. FIG. 3E: Asymmetry -driven directed evolution of UPenn-lipids starting from 1 l(aL)2. The optimized ionizable head structure (highlighted in grey) and one alkynyl tail L were maintained, while a long-chain aldehyde was introduced as another tail to generate asymmetric UPenn-lipid. FIG. 3F: In vivo mLuc expression. Mice were injected i.v. with mLuc-loaded LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h post-treatment and whole-body total flux was quantified. Data are presented as mean ± SD (n=3).

[0092] FIGs. 4A-4B: Optimization of biodegradable tails and headgroups for asy mmetric UPenn-lipids in Libraries 4 and 5. FIG. 4A: Optimization of biodegradable tails in asymmetric UPenn-lipids in Library 4. 35 asymmetric UPenn-lipids were combinatorially synthesized via A3-coupling of amine 31, five biodegradable aldehydes (e-i) and seven biodegradable alkynes (M-R). A representative synthesis of 31hP through A3-coupling is shown. Mice were injected i.v. with mLuc-loaded LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h post-treatment and whole-body total flux was quantified. FIG. 4B: Optimization of ionizable headgroup in asymmetric UPenn-lipids in Library75. 26 additional asymmetric UPenn-lipids were combinatorially synthesized via A3-coupling of 26 secondary amines (32-57), biodegradable aldehyde h and biodegradable alkyne P. Mice were injected i.v. with mLuc-loaded LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h posttreatment and whole-body total flux was quantified. Data are presented as mean ± SD (n=3).

[0093] FIGs. 5A-5I: Characterization of UPenn-LNP and its application in delivering mRNA-based gene editors and vaccines. FIG. 5 A: Physicochemical parameters of 31hP LNP (n=3). FIG. 5B: A representative cryo-EM image of 31hP LNP. Scale bar = 50 nm. FIG. 5C: In vitro transfection of 3 IhP LNP in the presence of various endocytic inhibitors (n=3). Amiloride, an inhibitor of macropinocytosis; Chlorpromazine, an inhibitor of clathrin-mediated endocytosis; Genistein, an inhibitor of caveolae-mediated endocytosis; M -CD, an inhibitor of lipid raft-mediated endocytosis. FIG. 5D: A scheme of LNP-enabled co-dehvery of Cas9 and TTR sgRNA. Mice were i.v. injected with LNPs encapsulating Cas9 mRNA / TTR sgRNA (4:1, wt:wt) at a total RNA dose of 1 mg / kg. On day 7, DNA was extracted from the liver to determine on-target indel frequency by next-generation sequencing (NGS). and serum was collected to determine TTR concentration by ELISA. FIG. 5E: Indels at TTR locus (n=3). FIG. 5F: Serum TTR (n=3). FIG. 5G: A scheme of LNP-enabled delivery of SARS-CoV-2 Spike mRNA vaccine. Mice were vaccinated i.m. twice using a prime-boost strategy at a three-week interval. Serum was collected at the indicated time points to determine anti-Spike IgG titers and SARS-CoV-2 pseudovirus neutralization antibody titers. FIG. 5H: Anti-Spike IgG titers (n=5). FIG. 51: SARS-CoV-2 pseudovirus neutralization antibody titers (n=5). Data are presented as mean ± SD.

[0094] FIGs. 6A-6E: General methods for the synthesis of biodegradable aldehydes and alkynes.

[0095] FIG. 7: Cell viability of UPenn-LNPs in Library 1. HepG2 cells were treated with various LNPs at a dose of 15 ng mRNA / well for 24 h. No major cytotoxicity was observed for each LNP. The dashed line indicates 80% cell viability. Data are presented as mean ± SD (n=2, initial screening).

[0096] FIGs. 8A-8B: Representative purification traces of UPenn-lipids. FIG. 8A: The purification trace of H(aL)2 recorded by a CombiFlash NextGen 300+ chromatography system equipped with evaporative light scattering (ELS) detectors. The crude product was purified with gradient elution from 100% DCM to 60% DCM / Me0H / NH40H (75:22:3, aq). FIG. 8B: The purification trace of 31hP recorded by a CombiFlashNextGen 300+ chromatography system equipped with ELS detectors. The crude product was purified with gradient elution from 100% DCM to 80% DCM / Me0H / NH40H (75:22:3, aq).

[0097] FIG. 9: Cell viability of UPenn-LNPs in Library 2. HepG2 cells were treated with various LNPs at a dose of 15 ng mRNA / well for 24 h. No major cytotoxicity was observed for each LNP. The dashed line indicates 80% cell viability. Data are presented as mean ± SD (n=3).

[0098] FIG. 10: In vivo mLuc expression. Mice were injected i.v. with mLuc- loaded LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h post-treatment and total flux was quantified. The dash line indicates the MC3 LNP level. Data are presented as mean ± SD (n=3).

[0099] FIG. 11: Mass spectrum of 31hP UPenn-lipid.

[0100] FIG. 12: H-NMR spectrum of 31hP UPenn-lipid in CDCh.

[0101] FIG. 13: Representative in vivo bioluminescence imaging results of 31 hP LNP and MC3 LNP. Mice were i.v. injected with mLuc-loaded UPenn-LNPs at an mRNA dose of 0.1 mg / kg. Images were taken at 4 h post-treatment. The signal mainly localized in the upper abdomen with predominant liver transfection.

[0102] FIG. 14: TNS assay was used to determine the apparent pa of 31hP LNP. TNS fluorescence signal corresponds to ionization. pa is calculated as the pH corresponding to half of the maximum TNS fluorescence value.

[0103] FIG. 15: Hemolysis of 31hP LNP at pH 7.4 or 6.0. RBCs were incubated with 31hP LNP at an mRNA concentration of 3 pg / rnL at 37 °C for 1 h. Positive and negative controls were carried out with 0.1% Triton-X (100% hemolysis) and 1 x PBS (0% hemolysis), respectively. Data are presented as mean ± SD (n=3).

[0104] FIG. 16: ALT and AST analysis. Cas9 mRNA / TTR sgRNA-loaded LNPs (1 mg / kg) were i.v. injected into mice. Serum was collected at 24 h post-treatment for ALT and AST analysis. Data are presented as mean ± SD (n=3).

[0105] FIG. 17: Cytokine responses. Cas9 mRNA / TTR sgRNA-loaded LNPs (1 mg / kg) were i.v. injected into mice (n=3). Serum was collected at 24 h post-treatment for flow-based multiplex cytokine analysis. Cytokines were all at low levels (< 10 pg / mL) after LNP treatment. Data with cytokine level lower than the minimum detectable concentration are not shown on the chart.

[0106] FIG. 18: In vivo mLuc expression after i.m. injection. mLuc-loaded UPenn-LNPs (2 pg mRNA in 50 pL PBS) were injected into the gastrocnemius muscle. Images were taken at 4 h post-treatment and total flux at injection site or liver region was quantified. Data are presented as mean ± SD (n=3).

[0107] FIG. 19: Exemplary lipids of Library’ 2.

[0108] FIG. 20: Exemplary lipids of Library’ 3.

[0109] FIGs. 21A-21B: Exemplary lipids of Library' 4.

[0110] FIGs. 22A-22B: Exemplary' lipids of Library' 5.

[0111] FIGs. 23A-23H: Optimization of LNP conditions for co-delivery of gene editing cargo. FIG. 23 A: Schematic describing the overall study design, where microfluidic and lipid excipient parameters were optimized to produce an LNP formulation with enhanced gene editing efficacy. FIG. 23B: Schematic depicting the two nucleic cargos trialed and compared across microfluidic flow rates. FIGs. 23C-23D: Size of LNPs produced at different total flow rates (0.3 - 3.6 mL / min) encapsulating either GFP mRNA (FIG. 23C) or SpCas9 mRNA and GFP sgRNA (FIG. 23D). FIGs. 23E-23F: mRNA encapsulation efficiency of LNPs produced at different flow rates, encapsulating either GFP mRNA (FIG. 23E) or SpCas9 mRNA and GFP sgRNA (FIG. 23F). FIG. 23G: GFP transfection resulting from delivery' of GFP mRNA to HepG2 cells via LNPs produced at different flow rates. FIG. 23H: Gene editing resulting from delivery’ of Cas9 mRNA and GFP sgRNA to HepG2-GFP cells via LNPs produced at different flow rates. One-way ANOVA with post hoc Dunnett’s test was used for statistical comparison (ns = non-significant, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001); all data reported as mean ± SEM (minimum n = 3).

[0112] FIGs. 24A-24F: Optimization of phospholipid structure for co-delivery of gene editing cargo. FIG. 24A: Structures of phospholipids incorporated into LNPs. FIGs. 24B-24D: Size (FIG. 24B), RNA encapsulation efficiency (EE%) (FIG. 24C), and Zeta potential (FIG. 24D) of LNPs produced with different phospholipids to encapsulate SpCas9 mRNA and GFP sgRNA. FIG. 24E: Gene editing resulting from delivery of Cas9 mRNA and GFP sgRNA to HepG2-GFP cells via LNPs produced with different phospholipids. FIG. 24F: Viability of cells treated with LNPs produced with different phospholipids. One-way ANOVA with post hoc Dunnett’s test was used for statistical comparison (ns = nonsignificant, * = p < 0.05, ** = p < 0.01, **** = p < 0.0001); all data reported as mean ± SEM (minimum n = 5).

[0113] FIGs. 25A-25F: Optimization of cholesterol structure for co-delivery of gene editing cargo. FIG. 25A: Structures of cholesterol analogues and / or derivatives incorporated into LNPs. FIGs. 25B-25D: Size (FIG. 25B), RNA encapsulation efficiency (EE%) (FIG. 25C), and Zeta potential (FIG. 25D) of LNPs produced with different cholesterols to encapsulate SpCas9 mRNA and GFP sgRNA. FIG. 25E: Delivery of Cas9 mRNA and GFP sgRNA to HepG2-GFP cells via LNPs produced with different cholesterols. FIG. 25F: Viability of cells treated with LNPs produced with different cholesterols. One-way ANOVA with post hoc Dunnett’s test was used for statistical comparison (ns = non-significant, ** = p < 0.01 *** = p < 0.001, **** = p < 0.0001); all data reported as mean ± SEM (minimum n = 5).

[0114] FIGs. 26A-26F: Optimization of lipid-PEG structure for co-delivery of gene editing cargo. FIG. 26A: Structures oflipid-PEGs incorporated into LNPs. FIGs. 26B-26D: Size (FIG. 26B), RNA encapsulation efficiency (EE%) (FIG. 26C), and Zeta potential (FIG. 26D) of LNPs produced with different lipid-PEGs to encapsulate SpCas9 mRNA and GFP sgRNA. FIG. 26E: Delivery of Cas9 mRNA and GFP sgRNA to HepG2-GFP cells via LNPs produced with different lipid-PEGs. FIG. 26F: Viability of cells treated with LNPs produced with different lipid-PEGs. One-way ANOVA with post hoc Dunnett’s test was used for statistical comparison (ns = non-significant, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001); all data reported as mean ± SEM (minimum n = 5).

[0115] FIGs. 27A-27G: Delivery7of gene editing cargo in vitro and in vivo with excipient-optimized LNPs. FIG. 27A: Delivery of Cas9 mRNA and GFP sgRNA to HepG2-GFP cells via LNPs produced with different combinations of excipients. FIG. 27B: Viability of cells treated with LNPs produced with different combinations of excipients. FIG. 27C:

[0116] Experimental groups for in vivo assessment of LNP gene editing efficacy. PBS was used as a negative control. MC3 LNPs are an FDA-approved LNP formulation. Rl, R3, R4, and R8 LNPs were the top-performers from the in vitro screen and were compared against R0 LNPs. All LNPs were formulated to encapsulate SpCas9 mRNA and TTR sgRNA. FIG. 27D:

[0117] C57BL / 6 mice were treated with LNPs at a dose of 1 mg / kg and sacrificed after 5 days later. Serum was collected before and after LNP treatment and analyzed for TTR protein via ELISA. FIGs. 27E-27F: AST (FIG. 27E) and ALT (FIG. 27F) levels in the serum of mice treated with LNPs relative to PBS-injected controls. FIG. 27G: Next-generation sequencing analysis for insertions and deletions (indels) at the expected locus for TTR gene editing in liver genomic DNA of PBS-, R0 LNP-, or R4 LNP -treated animals. One-way ANOVA with post hoc Dunnett’s test was used for statistical comparison with those directly adjacent to bars representing comparison against R0 LNPs (ns = non-significant. * = p < 0.05, *** = p < 0.001, **** = p < 0.0001); all data reported as mean ± SEM (minimum n = 5). 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.

[0118] 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 notjust 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.

[0119] 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.

[0120] 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. Propargyl Amino-Ionizable Lipid Compounds, Lipid Nanoparticles (LNPs) Comprising the Same, and Methods of Making and Using Same

[0121] The ionizable lipid is a critical component of lipid nanoparticles (LNPs) that governs the activity and biocompatibility of LNPs in mRNA delivery. While extensive efforts have been put into the development of potent and biodegradable ionizable lipids through rational design and / or combinatorial synthesis, there is a lack of robust methodologies to guide the stepwise, rational optimization of ionizable lipid structure.

[0122] In one aspect, the disclosure describes a directed evolution approach for rationale-driven, iterative structural optimization of unsaturated propargylamine-linked ionizable (UPenn) lipids based on an innovative Al dehyde-Alkyne- Amine coupling reaction to improve activity and biodegradability over multiple iterations. Through five cycles of directed chemical evolution, dozens of biodegradable, asymmetric UPenn-lipids were identified with comparable or enhanced activities to an industry standard ionizable lipid, and the key structure-activity relationships related to the head group, ester linkage, and tail are summarized herein. The non-limiting lead UPenn-lipid achieved superior hepatic delivery of mRNA-based CRISPR gene editors and improved intramuscular delivery of SARS-CoV-2 Spike mRNA vaccines compared to two industry standard lipids, demonstrating great promise for both systemic therapeutic and intramuscular vaccine applications. It is anticipated that the adapted directed evolution methodology and discovered structural criteria can be extended to accelerate the development of ionizable lipids with desired properties for a wide array of nucleic acid delivery applications.

[0123] Lipid Nanoparticles (LNPs) and Methods of Using Same for Gene Editing Ionizable lipid nanoparticles (LNPs) are the most clinically advanced non-viral RNA delivery platform due to their excellent biocompatibility and efficacy in preclinical and clinical models. Conventionally, LNPs are composed of an RNA cargo that is microfluidically mixed with four organic components: (1) an ionizable lipid core that gains positive charge in acidic environments for RNA binding during nanoparticle formulation and subsequent RNA release during cellular endocytosis, (2) a phospholipid to facilitate nanoparticle cellular uptake (z.e., a “helper” or “neutral” lipid), (3) cholesterol for nanoparticle structural stability, and (4) a polymer conjugated lipid (<?.g., polyethylene glycol (PEG) conjugated lipid) coat to reduce opsonization and clearance of nanoparticles from the bloodstream. Many studies have investigated the optimal formulation parameters for LNP-mediated delivery of siRNA and mRNA. This has facilitated FDA approval of siRNA-LNP drugs (e.g., ONPATTRO®) and mRNA-LNP vaccines (e.g., Modema and Pfizer SARS-CoV2 vaccines). However, few studies have systematically optimized LNPs for the delivery of mRNA-based CRISPR-Cas9 platforms.

[0124] Given the inherent chemical and structural differences between siRNA, mRNA, and mRNA-based CRISPR-Cas9 platforms in terms of size, stability and charge density of the nucleic acids, it yvas hypothesized that the optimal LNP formulation parameters to facilitate CRISPR-Cas9 gene editing would vary from those developed for siRNA or reporter mRNA delivery.

[0125] The disclosure describes the study of both the microfluidic formulation parameters and organic excipient selection for LNPs co-encapsulating Cas9 mRNA and a single guide RNA (sgRNA) (FIG. 23A). The in vitro optimization process yielded an LNP formulation that facilitated 3-fold greater therapeutic genome editing in vivo in the liver in comparison to the LNP formulation previously used for mRNA delivery and an FDA- approved LNP formulation. Key features of the optimized LNP were the incorporation of new cholesterol and lipid-PEG analogs. The disclosure elucidates the importance of LNP formulation parameters for in vivo gene editing and presents an optimized delivery platform for the treatment of metabolic liver disease.

[0126] Definitions

[0127] The term “about’7as 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.

[0128] 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.

[0129] 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.

[0130] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl 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.

[0131] 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 alkyd 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-ocl l 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.

[0132] 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.

[0133] 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 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 sen e 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.

[0134] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)s 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, heterocyclyl amines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialky larylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0135] 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 -NR?. 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 20060083780 and 20060240554; U. S. Patent Nos. 5,208,036; 5,264,618; 5,279,833;

[0140] 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, Cis alkyl 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.

[0141] The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cy clobutyl, cyclopentyl, cyclohexyl, cy cloheptyl, 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, norbomyl, 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 norbomyl 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] In particular, in the case of amRNA, 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 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 (z.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.

[0147] 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 (z.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.

[0148] 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.

[0149] 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.

[0150] The term “haloalky I” group, as used herein, includes mono-halo alkyl groups, polyhalo 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 -di chloroethyl, 1,2-dichloroethyL l,3-dibromo-3,3- difluoropropyl, perfl uorobutyl, and the like.

[0151] The term “helper lipid’7as 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. Nonlimiting examples of helper lipids include 1.2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin (POPC) and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0152] 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 quatemized. 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.

[0153] 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 heteroary l 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.

[0154] 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, 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 ary l 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, benzhy dry 1, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,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-benzofb] 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|bJfuranyl), 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,

[0155] 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,1 l-dihydro-5H-dibenz[b,f] azepine (10,1 l-dihydro-5H-dibenz[b,f]azepine-l-yl, 10,ll-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,ll-dihydro-5H-dibenz[b,f|azepine-3-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,1 l-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 tetrahydrofuiyl.

[0156] The term “heterocyclyF’ 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, 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.

[0157] 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.

[0158] 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, (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Ci), ethyl (C2), propyl (C3), or butyl (C4), and (Co-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

[0159] 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.

[0160] 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. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7.

[0161] 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.

[0162] 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.

[0163] 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).

[0164] 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.

[0165] 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 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 enzy matic degradation.

[0166] 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.

[0167] 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.

[0168] The term “neutral lipid” or “helper 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.

[0169] The term “non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0170] 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, -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., Mai. Cell. Probes, 8:91-98 (1994)).

[0171] “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.

[0172] 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)).

[0173] The terms “patient / ’ “subject,” or “individual” are used interchangeably herein, and 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.

[0174] 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 w hich it is contained.

[0175] 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.

[0176] 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, P-hy dr oxy butyric, salicylic, galactaric and galacturonic acid.

[0177] 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.

[0178] 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, 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 com 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, com 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.

[0179] 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 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.

[0180] 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-hpid conjugates, such as PEG coupled to dialkydoxypropyls, 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.

[0181] 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.

[0182] 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 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 “does not substantially,” when used in conjunction with a process or event indicates that the process or event does not occur or occurs in a trivial amount, such that the process or event occurs about 0% or less than about 0.5, 1.0. 1.5, 2.0, 2.5, 3.0. 3.5, 4.0, 4.5. or about 5.0% of the maximal amount that the process or event could occur.

[0183] 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 hydroxygroups. 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.

[0184] C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(0)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(O)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)C0N(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(0)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, (Ci-C100) hydrocarbyl, alkyl, acyl, cycloalkyd, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroary lalkyl; 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.

[0185] A “therapeutic’" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0186] The term “therapeutic cargo” as used herein refers to any molecule (e.g, small molecules and macromolecules) or compound that provides a therapeutic or functional benefit to the targeted cell or tissue when delivered.

[0187] 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 severity7of 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-a, TGF-, TNF, HGF, IGF, and IL-1-8, inter alia) cytokines, paratopes, Fabs (fragments, antigen binding), and antibodies.

[0188] 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. Propargyl Amino-Ionizable Lipid Compounds

[0189] In one aspect the disclosure provides a compound of formula (I), or a salt stereoisomer, or isotopologue thereof:

[0190]

[0191] wherein:

[0192] A is selected from the group consisting of

[0193]

[0194] each occurrence of L1, if present, is independently selected from the group consisting of -(optionally substituted C1-C12 alkyleny l)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, and -(optionally substituted C2-C8 heteroarylenyl)-X-;

[0195] each occurrence of X, if present, is independently selected from the group consisting of -N(Rle)-, -[N(CH2)i-3N(R16)(Rle)]-, -N(RA)-, and -O-;

[0196] Rla, Rlb, Rlc, Rld, and each occurrence of Rle, if present, are each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and -CH(R3a)(R3b), wherein

[0197] at least one of Rla, Rlb, and Rlc, if present, is -CH(R3a)(R3b).

[0198] one of Rlaand Rlbcan combine with R2to form an optionally substituted C2-C8 heterocycloalkyl, and

[0199] one of Rla, Rlb, Rlc, and Rldcan combine with one occurrence of L1to form an optionally substituted C2-C8 heterocycloalkyl;

[0200] R2is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);

[0201] R3aand R3bare each independently selected from the group consisting of optionally substituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, optionally substituted C2-C24 alkynyl, optionally substituted C1-C24 heteroalkyl, optionally substituted C2-C24 heteroalkenyl, optionally substituted C2-C24 heteroalkynyl, optionally substituted C'3-C's cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C8 heteroaryl;

[0202] m is 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10; and

[0203] each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl.

[0204] In certain embodiments, R2is CHs. In certain embodiments, R2is (CH2)CH3. In certain embodiments, R2is (CH2)4ORA. In certain embodiments, R2is (CH2)C(=O)ORA. In certain embodiments, R2is (CH2)I-3N(RA)(RB). In certain embodiments, R2is (CH2)i- 3(optionally substituted pyrrolidinyl). In certain embodiments, R2is (CH2)i-3(optionally substituted imidazolyl). In certain embodiments, R2is (CH2)i-3(optionally substituted piperizinyl). In certain embodiments, R2is (CH2)i-3(optionally substituted piperidinyl). In certain embodiments, R2is (CH2)i-3(optionally substituted morpholinyl). In certain embodiments, R2is optionally substituted cyclohexyl. In certain embodiments, R2is optionally substituted phenyl.

[0205] R!1a

[0206] In certain embodiments, compound of formula (I) is

[0207]

[0208] . In certain embodiments,

[0209]

[0210] compound of formula (I) is R1aIn certain embodiments, compound of formula (I)

[0211]

[0212]

[0213] In certain embodiments, compound of formula (I) is ' R1a. In certain

[0214] embodiments, compound of formula (I) is

[0215]

[0216] . In certain embodiments,

[0217]

[0218] formula (I) is R'aIn certain embodiments, compound of formula (I) is

[0219]

[0220] N-YN-R13

[0221]

[0222] . In certain embodiments, compound of formula (I) is — / . In Y 1,~Y / YrCR13

[0223] certain embodiments, compound of formula (I) is ' — / . In certain embodiments,

[0224] compound of formula

[0225]

[0226] certain embodiments, compound of formula

[0227] O^Y"N-'R,a(I) is ' —. In certain embodiments, compound of formula (I) is

[0228]

[0229] . In certain embodiments, compound of formula (I) is

[0230]

[0231] . In certain

[0232] embodiments, compound of formula (I) is

[0233]

[0234] In certain embodiments,

[0235] compound of formula (

[0236]

[0237] certain embodiments, compound of

[0238] formula (I) is

[0239]

[0240] . In certain embodiments, compound of formula (I) is

[0241]

[0242] In certain embodiments, compound of formula (

[0243]

[0244] certain embodiments, compound of formula

[0245]

[0246] certain

[0247] embodiments, compound of formula certain embodiments.

[0248] \|- /

[0249]

[0250] compound of formula (I) is —. In certain embodiments, compound of

[0251] formula (I) is

[0252]

[0253] In certain embodiments, compound of formula (I) is

[0254]

[0255] certain embodiments, compound of formula (I) is R1a. In certain embodiments.

[0256] R i1a

[0257] N,1b

[0258] compound of formula (I) isR. In certain embodiments, compound of formula (I) is R1aO R3

[0259]

[0260] . In certain embodiments, compound of formula (1) is

[0261]

[0262] . In certain R i3

[0263] embodiments, compound of formula (I) is

[0264]

[0265] . In certain embodiments,

[0266] R r1a

[0267] \ / s,b

[0268] compound of formula (I) is O R1. in certain embodiments, compound of formula R I1a

[0269] ' N / - / N'R1b

[0270] (I) is I. In certain embodiments, compound of formula (I) is

[0271] R1a

[0272] R1a

[0273]

[0274] ., p

[0275] In certain embodiments, compound of formula (

[0276]

[0277] certain

[0278] embodiments, compound of formula (I) is

[0279]

[0280] . In certain embodiments,

[0281] compound of formula (I) is

[0282]

[0283] In certain embodiments, compound of

[0284]

[0285] , p . In certain embodiments, compound of formula (I) is

[0286]

[0287] . In certain

[0288] embodiments, compound of formula (I) is

[0289]

[0290] . In certain embodiments, compound R i1a

[0291]

[0292] of formula (I) is R1b. In certain embodiments, compound of formula (I) is R ilaR I1b

[0293] embodiments, compound of formula (I) is

[0294] . In certain embodiments, compound of formula (I) is

[0295] R1a

[0296]

[0297]

[0298] . in embodiments, compound of formula (I) is R1d

[0299]

[0300] embodiments, compound of formula (I) isr1c Rla. In certain embodiments,

[0301] compound of formula (I) is

[0302]

[0303] . In certain embodiments,

[0304] R1b

[0305]

[0306] compound of formula (I) is R1c. In certain embodiments,

[0307] compound of formula

[0308]

[0309] R3a

[0310] A,b

[0311] In certain embodiments, Rldis H. In certain embodiments, Rldis ' R. In certain R3a

[0312] embodiments, Rlais ''Rjb. In certain embodiments, Rlbis H. In certain embodiments, Rlb R3aR3a

[0313]

[0314]

[0315] . In certain embodiments, Rlbis ' R“b. In certain embodiments, Rlcis H. In R3aR3a

[0316] certain embodiments, Rlcis

[0317]

[0318] . In certain embodiments, Rlcis

[0319]

[0320] . In certain R3a

[0321] embodiments, Rldis H. In certain embodiments, Rldis

[0322]

[0323] . In certain embodiments, RldR3aR3a

[0324] A

[0325] is ' R3b. In certain embodiments, Rleis H. In certain embodiments, Rleis ' R3°. In

[0326] certain embodiments,

[0327]

[0328] In certain embodiments, each occurrence of R3aand R3bis independently selected4— ===— L2- R4- " X- (■ L2j-~ R4

[0329] from the group consisting of H, R,n, and "0, wherein:

[0330] each occurrence of R4is Ci-Cie alkyl or C2-C16 alkenyl;

[0331] each occurrence of L2is independently selected from the group consisting of -CH2-, - O-, -C(=O)-, and -(optionally substituted phenylenyl)-; and

[0332] each occurrence of n and o is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0333] In certain embodiments, at least one of R3aand R?bin each occurrence of

[0334] - EEE— (-L2)— R4

[0335] CH(R3a)(R3b) is

[0336] In certain embodiments, -(L2)„- is

[0337]

[0338] In certain embodiments, -(L2),,- is

[0339]

[0340] O. In certain embodiments, -(L2)„- is

[0341]

[0342] . In certain

[0343] embodiments, -

[0344]

[0345] In certain embodiments, -(L2)o- is -O-. In certain embodiments, -(L2)o- is -C(=O)O-. In certain embodiments, -(L2)o- is -OC(=O)-.

[0346] In certain embodiments, R4is

[0347]

[0348] [ncertain embodiments, R4is

[0349]

[0350] , certain embodiments, R4is

[0351]

[0352] In certain embodiments, R4is

[0353]

[0354] certain embodiments, R4is

[0355]

[0356]

[0357] In certain embodiments, R,ais

[0358]

[0359] In certain embodiments,

[0360]

[0361]

[0362]

[0363] n certain embodiments, R3ais

[0364] . In certain embodiments, R3ais

[0365] . In certain embodiments, ’ais

[0366]

[0367] n certain embodiments, R3ais

[0368]

[0369] ain embodiments, R3ais

[0370] odiments, R3ais

[0371] ain embodiments, R3bis

[0372]

[0373] . In certain embodiments,

[0374]

[0375] certain embodiments,

[0376]

[0377] ,

[0378]

[0379] ,

[0380]

[0381]

[0382] , R3bis

[0383]

[0384]

[0385] ''N"'

[0386] I

[0387] In certain embodiments, A is R2, Rlaand Rlbare each independently CH(R3a)(Rab), both occurrences of R3aare H, and neither occurrence of R3bis H, optionally wherein both occurrences of R3bcomprise an optionally substituted C2-C24 alkynyl. In certain ''N

[0388] lZ

[0389] embodiments, A isr2, Rlaand Rlbare each independently CH(R3a)(R3b), both occurrences of R3bare H, and neither occurrence of R3ais H, optionally wherein both occurrences of R3acomprise an optionally substituted C2-C24 alkynyl. In certain

[0390] ''N'"

[0391] I

[0392] embodiments, A is R2, Rlais H, methyl, or ethyl, and Rlbis CH(R3a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted C6-C10 aryl or optionally substituted C1-C24 alky l. In certain embodiments, A is R2, Rlbis H, methyl, or ethyl, and Rlais CH(R"a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl. and one of R3aand R3bis optionally substituted

[0393]

[0394] C6-C10 aryl or optionally substituted C1-C24 alkyl. In certain embodiments, A is R2. Rlacombines with R2to form an optionally substituted C2-C8 heterocycloalkyl and Rlbis CH(R3a)(R3b, wherein neither R3anor R?bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R?aand R3bis optionally substituted Ce-Cio

[0395]

[0396] aryl or optionally substituted C1-C24 alky l. In certain embodiments, A is R2, Rlbcombines with R2to form an optionally substituted C2-C8 heterocycloalkyl and Rlais CH(R3a)(R3b, wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Ce-Cio aryl or optionally substituted C1-C24 alky l.

[0397] Exemplary nomenclature used herein to describe ionizable lipids of the disclosure includes ”#(xY)n" and ”#xY.” wherein “#” refers to an integer ranging from 1 to 57 which indicates the identity7of the amine core starting material (Table 1), “x” refers to a lower case letter which indicates the identity7of the aldehyde starting material (Table 2), “Y” refers to an upper case letter which indicates the identity' of the alkyne starting material (Table 3), and

[0398]

[0399] is 1 or 2. In certain embodiments, when amine starting material (z.e., “#”) comprises a secondary amine (e.g., (CTE^NH), ‘77” is 1. In certain embodiments, when amine starting material (i.e.,

[0400]

[0401] comprises a primary7amine (e.g., (CH3)NH2), “n” is 2. The chemical structure of exemplary compound 31hP (“#’’ is 31, “x” is h. “Y” is P, and n is 1) is provided herein for illustrative purposes:

[0402]

[0403] The present disclosure contemplates all appropriate combinations of

[0404]

[0405] “x,"’ “Y,’" One of ordinary skill in the art. in view of the specification as filed, understands the chemical structures referenced using the nomenclature provided herein, which provides the identity of the starting materials, because one of ordinary skill in the art understands how the starting materials combine to arrive at the ionizable lipid products. Further, one of ordinary skill in the art understands which combinations of

[0406]

[0407] are appropriate and / or feasible.

[0408] In certain embodiments, the compound is selected from the group consisting of 31aA, 31aB, 31aC, 31aD. 31aE, 31aF, 31aG. 31aH, 31al, 31aJ, 31aK, 31aL, 31aM, 31aN, 31aO, 31aP, 31aQ, 31aR. 31bA, 31bB. 31bC, 31bD. 31bE, 31bF, 31bG. 31bH, 31bl, 31bJ, 31bK, 31bL, 31bM, 31bN, 31bO, 31bP, 31bQ, 31bR, 31cA, 31cB, 31cC, 31cD, 31cE, 31cF, 31cG, 31cH, 31cl, 31cJ, 31cK, 31cL, 31cM, 31cN, 31cO, 31cP, 31cQ, 31cR, 31dA, 31dB, 31dC, 31dD, 31dE, 31dF. 31dG, 31dH. 31dl. 31dJ, 31dK, 31dL. 31dM, 31dN, 31dO, 31dP. 31dQ, 31dR, 31eA, 31eB, 31eC, 31eD. 31eE, 31eF, 31eG. 31eH, 31el, 31eJ, 31eK, 31eL. 31eM, 31eN, 31eO, 31eP, 31eQ, 31eR, 31fA, 31fB, 31fC, 31fD, 31fE, 3 IfF, 31fG, 31fH, 31fl, 31fJ, 3IfK, 31fL, 31fM, 31fN, 31fO, 31fP, 31fQ, 31fR, 31gA, 31gB, 31gC, 31gD, 31gE, 31gF, 31gG, 31gH, 31gl, 31gJ, 31gK, 31gL, 31gM, 31gN, 31gO, 31gP, 31gQ, 31gR, 31hA, 31hB, 31hC, 31hD, 31hE, 31hF, 31hG. 31hH, 31hl, 31hJ, 31hK, 31hL, 31hM, 31hN, 31hO, 31hP, 31hQ, 31hR, 311A. 311B, 3 liC, 311D, 311E. 311F. 311G. 311H, 3111. 3 liJ, 311K. 3 IiL. 311M, 31iN, 31iO, 31iP, 311Q, 31iR, ll(aA)2, ll(aB)2, ll(aC)2, ll(aD)2, H(aE)2, l l(aF)2, ll(aG)2, ll(aH)2, ll(al)2, 1 l(aJ)2, ll(aK)2, ll(aL)2, ll(aM)2, ll(aN)2, ll(aO)2, ll(aP)2, ll(aQ)2, ll(aR)2, ll(bA)2, ll(bB)2, ll(bC)2, l l(bD)2, H(bE)2. ll(bF)2, H(bG)2, H(bH)2. U(bl)2, I l(bJ)2, l l(bK)2. 1 l(bL)2, H(bM)2, ll(bN)2, 1 l(bO)2. 1 l(bP)2, H(bQ)2, ll(bR)2. 11(CA)2, I I (CB)2, 11 (cC)2, 11 (CD)2, 11 (CE)2, 11 (CF)2, 11 (CG)2, 11 (cH)2, 11 (cl)2, 11 (cJ)2, 11 (cK)2, 11(CL)2, 11(CM)2, 11(CN)2, 11(CO)2, 11(CP)2, 11 (CQ)2, 11 (CR)2, H(dA)2, ll(dB)2, ll(dC)2, ll(dD)2, ll(dE)2, ll(dF)2, H(dG)2, ll(dH)2, 1 l(dl)2, 1 l(dJ)2, ll(dK)2, 1 l(dL)2, ll(dM)2, ll(dN)2, ll(dO)2, ll(dP)2. U(dQ)2, l l(dR)2, H(eA)2. ll(eB)2, ll(eC)2, H(eD)2. ll(eE)2, 1 l(eF)2, H(eG)2, l l(eH)2, ll(el)2, 1 l(eJ)2, H(eK)2, 1 l(eL)2, H(eM)2, ll(eN)2, 1 l(eO)2, 11(eP)2, H(eQ)2, ll(eR)2, ll(fA)2, ll(fB)2, ll(fC)2, ll(fD)2, ll(fE)2, 1 l(fF)2, ll(fG)2, ll(fH)2, 1 l(fl)2, ll(fj)2, ll(fK)2, 1 l(fL)2, ll(fM)2, ll(fN)2, H(fO)2, 1 l(fP)2, ll(fQ)2, 1 l(fR)2, ll(gA)2, H(gB)2, ll(gC)2. ll(gD)2, l l(gE)2, H(gF)2, ll(gG)2, H(gH)2, ll(gl)2, 1 l(gj)2. H(gK)2, l l(gL)2, l l(gM)2, l l(gN)2. l l(gO)2. l l(gP)2, H(gQ)2. H(gR)2, H(hA)2, l l(hB)2, I l(hC)2, H(hD)2, H(hE)2, l l(hF)2, H(hG)2, ll(hH)2, 11(hl)2, 1 l(hJ)2, H(hK)2, 1 l(hL)2, I I (hM)2, 11 (hN)2, 11 (hO)2, 11 (hP)2, 11 (hQ)2, 11 (hR)2, 11 (i A)2, 11 (1B)2, 11 (iC)2, 11 (iD)2, 11(IE)2, 1 l(iF)2, H(iG)2, U(iH)2, Il(il)2, Il(iJ)2, Il(iK)2, 11(IL)2, ll(iM)2, 1 l(iN)2, 1 l(iO)2, 1 l(iP)2, H(iQ)2, and l l(iR)2.

[0409] In certain embodiments, the compound is:

[0410]

[0411] In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, 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 Ci-Ce alkyl, Cs-Cs cycloalkyl, Ci-Ce haloalkyl, C1-C3 haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R”), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=0)N(R’)(R”), S(=O)2N(R’)(R”), N(R’)C(=0)R”, N(R’)S(=O)2R”, C2-Cs 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, Ci-Cs alkyl, C3-C8 cycloalkyl, Ci-Ce haloalkyl, benzyl, and phenyl, or wherein R and R” can combine with the nitrogen atom to which they are bound to form a C2-Cs heterocycloalkyl (e.g., cyclic tertiary amine).

[0412] Lipids Compounds of Formula (III)

[0413] In one aspect, the present disclosure provides an ionizable lipid of Formula (III), or a salt, solvate, stereoisomer, or isotopologue thereof:

[0414]

[0415] wherein:

[0416] Rlaand Rlbare each independently

[0417]

[0418] •

[0419] R2a, R2b, R2C, R2d, R2e, R2f, R2g, and R2hare each independently selected from the group consisting of H, optionally substituted Ci-Ci2alkyl, optionally substituted C2-Ci2heteroalkyl, optionally substituted Cti-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C10 heteroaryl;

[0420] each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H, -(optionally substituted C1-C6 alkylenyl)-C(=O)OR4, -(optionally substituted Ci-Ce alkylenyl)-C(=O)N(R4)(R5), -(optionally substituted Ci-Ce alkylenyl)-C(=O)R4, -(optionally substituted Ci-C6alkylenyl)-(R4), -C(=O)OR4, -C(=O)N(R4)(R5), -C(=O)R4, and R4,

[0421] wherein no more than one of each occurrence of R3a, R3b, and R3cis H; R4is selected from the group consisting of optionally substituted C1-C28 alkyl, optionally substituted C2-C28 heteroalkyl, optionally substituted Ch-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C28 alkenyl, and optionally substituted C2-C28 alkynyl;

[0422] R5is selected from the group consisting of H and optionally substituted Ci-Ce alkyl; each occurrence of L is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-, -X-(optionally substituted C2-C12 alkenylenyl)-, -X-(optionally substituted C1-C12 alkynylenyl)-, -X-(optionally substituted C1-C12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl;

[0423] each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3C)-, and -O-; and

[0424] each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4.

[0425] In certain embodiments, at least one selected from the group consisting of R2a, R2b, R2C, R2d, R2e, R2f, R2g, and R2his H. In certain embodiments, at least two selected from the group consisting of R2a, R2h, R2c, R2d, R2e, R2f. R2g, and R2bare H. In certain embodiments, at least three selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R211are H. In certain embodiments, at least four selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2hare H. In certain embodiments, at least five selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f. R2g, and R2bare H. In certain embodiments, at least six selected from the group consisting of R2a, R2b, R2c. R2d, R2e, R2f, R2g, and R2hare H. In certain embodiments, at least seven selected from the group consisting of R2a, R2b, R2c,

[0426]

[0427] embodiments, each of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2hare H.

[0428] In certain embodiments, L is -CH2-. In certain embodiments, L is -(CH2)2-. In certain embodiments, L is -(CH2)s-. In certain embodiments, L is -(CH2)IO-. In certain embodiments, L is -(CH2)2O-. In certain embodiments, L is -(CH2)3O-. In certain embodiments, L is -CH2CH(OR5)CH2-. In certain embodiments, L is -(CH2)2NR3c-. In --( / N— certain embodiments, L is. In certain embodiments, L is

[0429]

[0430] certain embodiments, L is

[0431]

[0432] For instances of L which are asymmetric (e g., -(CFh^O-) it is understood that the present disclosure encompasses both possible orientations (e g., -(CH2)3O- and -O(CH2)3-).

[0433] In certain embodiments, the ionizable lipid of Formula (III) is:

[0434]

[0435] (Illa). In certain embodiments, the ionizable

[0436]

[0437] lipid of Formula (III) is: R3bR3c(Illb). In certain embodiments, the ionizable lipid of Formula (III) is:

[0438]

[0439] certain embodiments, the ionizable lipid of

[0440] Formula (III) is:

[0441]

[0442] (Illd). In certain embodiments, the ionizable lipid of Formula (III) is:

[0443]

[0444] (Ille). In certain embodiments, the ionizable lipid of Formula (III) is:

[0445]

[0446] certain embodiments, the ionizable lipid of Formula (III) is:

[0447]

[0448] (Illg). In certain embodiments, the ionizable lipid of Formula (III) is:

[0449]

[0450] In certain embodiments, the ionizable

[0451] lipid of Formula (III) is:

[0452]

[0453] In certain embodiments, R3ais H. In certain embodiments, R'ais - CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, R3ais -CH2CH(OH)(optionally substituted C2-C28 alkenyl). In certain embodiments, R3ais -CH2CH2C(=O)O(optionally substituted C1-C28 alkyl). In certain embodiments, R3ais -CH2CH2C(=O)NH(optionally substituted C1-C28 alkyl). In certain embodiments, R3bis H. In certain embodiments, R3bis -CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, R3bis -CH2CH(OH)(optionally substituted C2-C28 alkenyl). In certain embodiments, R3bis -CH2CH2C(=O)O(optionally substituted C1-C28 alkyl). In certain embodiments, R3bis -CH2CH2C(=O)NH(optionally substituted C1-C28 alky l). In certain embodiments, R'cis H. In certain embodiments, R?cis -CH2CH(OH)(optionally substituted C1-C28 alkyl). In certain embodiments, R3cis -CH2CH(OH)(optionally substituted C2-C28 alkenyl). In certain embodiments, R3cis -CH2CH2C(=O)O(optionally substituted C1-C28 alkyl). In certain embodiments, R3cis -CH2CH2C(=O)NH(optionally substituted C1-C28 alkyl).

[0454] In certain embodiments, R3ais -CH2CH(OH)(CH2)9CH3. In certain embodiments, R3a is -CH2CH(OH)(CH2)HCH3. In certain embodiments, R3ais -CH2CH(OH)(CH2)i3CH3. In certain embodiments, R3bis -CH2CH(OH)(CH2)9CH3. In certain embodiments, R3bis - CH2CH(OH)(CH2)IICH. In certain embodiments, R3bis -CH2CH(OH)(CH2)i3CH3. In certain embodiments, R3cis -CH2CH(OH)(CH2)9CH3. In certain embodiments, R3cis - CH2CH(OH)(CH2)HCH3. In certain embodiments, R3cis -CH2CH(OH)(CH2)i3CH3.

[0455] In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroar l, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of Ci- Ce alkyl, Cs-Cs cycloalkyl, Ci-Ce haloalkyl, C1-C3 haloalkoxy, 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, Ci-Ce alkyl, Cs-C8cycloalkyl, Ci-Ce haloalkyl, benzyl, and phenyl. In certain embodiments, the ionizable lipid of Formula (III) is:

[0456]

[0457] l,l'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2- hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-ol),

[0458] (C14-494)

[0459] Ionizable Lipids and / or Cationic Lipids or Lipidoids

[0460] The scope of ionizable lipids contemplated for use in the present disclosure is not limited to ionizable lipidoids of formula (I) or (III). 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,l- 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]-l-piperazinyl]ethyl]imino]bis-2-dodecanol (Cl 2-200), l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3- 45 dimethylaminopropyl)- l,3]-di oxolane (D Lin-K-C3-D MA), 2, 2-dilinoleyl-4-(4-dimethylaminobutyl)-[l,3]-di oxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[ l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ), 2,2-dili-noleyl-4-dimethylaminomethyl-[1,3] -dioxolane (DLin-KDMA), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (D Lin-C-DAP). l,2-dilinoleyoxy-3-(dimethylaminoacetoxypropane (DLin-DAC), 1-2dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (D Lin-MPZ), 3-(N, N-dilinoleylamino)-l,2-propanediol (D LinAP), 3-(N, N-dioleylamino)-l,2-propanedio (DOAP), l,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-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(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 ammonium 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)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3-dimethyl-l-(cis,cis-9’,l-2’-octadecadienoxy) propane (CpLinDMA), N, N-dimethyl-3,4-dioleyloxy benzylamine (DMOBA), l,2-N, N’dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), l,2-N, N’-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA C’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.

[0461] 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, DLm-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.

[0462] 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0463] Non-Cationic Lipid

[0464] 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.

[0465] 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.

[0466] Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, 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), lysophosphatidylchohne, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0467] 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 ’-hydroxy ethyl ether, cholesteryl-4’-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof.

[0468] Polymer-Conjugated Lipid(s)

[0469] 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.

[0470] 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 gly colsuccinate (MePEGS), monomethoxypolyethylene glycolsuccinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycolamine (MePEG-NEb), 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.

[0471] 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 (Cis), or mixtures thereof.

[0472] 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.

[0473] 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.

[0474] 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, polymethacrylamide and poly dimethylacrylamide, polylactic acid, polygly colic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose.

[0475] 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.

[0476] 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.

[0477] 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-hpid 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.

[0478] Lipid Nanoparticles (LNPs) Comprising Propargyl Amino-Ionizable Lipids

[0479] In another aspect, the disclosure provides a lipid nanoparticle (LNP). In certain embodiments, the LNP comprises at least one ionizable lipid. In certain embodiments, the ionizable lipid is a compound of formula (I). In certain embodiments, the LNP comprises a neutral lipid. In certain embodiments, the LNP comprises at least one cholesterol lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises at least one polymer-conjugated lipid and / or a modified derivative thereof.

[0480] In certain embodiments, the at least one ionizable lipid compound 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, 70. 71. 72. 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound 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, 70, 71, 72, 73. 74. 75. 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86. 87. 88. 89. or 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises greater 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, 70, 71, 72. 73. 74. 75. 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 mol% of the LNP.

[0481] In certain embodiments, the at least one ionizable lipid compound comprises less than about 40 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises about 40 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises greater than about 40 mol% of the LNP.

[0482] In certain embodiments, the at least one ionizable lipid compound comprises or consists essentially of:

[0483]

[0484] In certain embodiments, the at least one neutral 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, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral 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, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater 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. or about 40 mol% of the LNP.

[0485] In certain embodiments, the at least one neutral lipid comprises less than about 10 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 10 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 10 mol% of the LNP.

[0486] In certain embodiments, the neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). In certain embodiments, the neutral lipid consists essentially of dioleoylphosphatidy lethanolamine (DOPE). In certain embodiments, the neutral lipid comprises distearoylphosphatidylcholine (DSPC). In certain embodiments, the neutral lipid consists essentially of distearoylphosphatidylcholine (DSPC). In certain embodiments, the neutral lipid comprises dioleoylphosphatidylcholine (DOPC). In certain embodiments, the neutral lipid consists essentially of dioleoylphosphatidylcholine (DOPC).

[0487] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises less than about 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, or about 75 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises about 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, or about 75 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises greater than about 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, or about 75 mol% of the LNP.

[0488] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises less than about 48.8 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises about 48.8 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises greater than about 48.8 mol% of the LNP.

[0489] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol. In certain embodiments, the at least one cholesterol lipid and / or modified denvative thereof consists essentially of cholesterol.

[0490] 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, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3. 11.4. 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or about 15 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, 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, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3. 11.4. 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or about 15 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises greater 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. 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2. 11.3. 11.4. 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or about 15 mol% of the LNP.

[0491] In certain embodiments, the at least one polymer-conjugated lipid comprises less than about 1.5 mol%. In certain embodiments, the at least one polymer-conjugated lipid comprises about 1.5 mol%. In certain embodiments, the at least one polymer-conjugated lipid comprises greater than about 1.5 mol%.

[0492] In certain embodiments, the at least one polymer-conjugated lipid comprises 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (C14PEG2K). In certain embodiments, the at least one polymer-conjugated lipid consists essentially of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (C14PEG2K).

[0493] In certain embodiments, the LNP has a molar ratio of (a): (b): (c): (d) of about 40: 10: 48.8: 1.5. In certain embodiments, the LNP further comprises at least one cargo molecule. In certain embodiments, the at least one cargo molecule comprises or consists of a therapeutic cargo molecule.

[0494] 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.

[0495] In certain embodiments, the cargo molecule comprises a nucleic acid.

[0496] In certain embodiments, the nucleic acid 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.

[0497] In certain embodiments, the mRNA encodes a receptor. In certain embodiments, the mRNA encodes an antigen binding domain. In certain embodiments, the receptor or antigen binding domain comprises a SARS-CoV-2 spike protein. In certain embodiments, the mRNA encodes an enzyme.

[0498] In certain embodiments, the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein. In certain embodiments, the CRISPR associated protein is Cas9. In certain embodiments, the nucleic acid cargo further comprises sgRNA.

[0499] Lipid Nanoparticle (LNP) Compositions

[0500] In one aspect, the disclosure provides a lipid nanoparticle (LNP). In certain embodiments, the LNP comprises at least one ionizable lipid. In certain embodiments, the LNP comprises at least one neutral lipid. In certain embodiments, the LNP comprises at least one sterol. In certain embodiments, the LNP comprises at least one polymer conjugated lipid. In certain embodiments, the LNP comprises nucleic acid cargo. In certain embodiments, the nucleic acid cargo comprises at least one messenger RNA (mRNA). In certain embodiments, the mRNA has a size of about 1, 2, 3, 4, 5, 6, 7, 8. 9, or about 10 kilobases. In certain embodiments, the LNP comprises nucleic acid cargo comprising a messenger RNA (mRNA) encoding a base editor and a single guide RNA (sgRNA).

[0501] In certain embodiments, the at least one ionizable lipid compound comprises a compound of Formula (III), or a salt, solvate, stereoisomer, or isotopologue thereof:

[0502]

[0503] wherein:

[0504] R1aand R1bare each independently

[0505]

[0506] ;

[0507] R2a, R2b, R2C, R2d, R2e, R2f, R2g, and R2hare each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl;

[0508] each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H, -(optionally substituted Ci-Ce alkylenyl)-C(=O)OR4, -(optionally substituted Ci-Ce alkylenyl)-C(=O)N(R4)(R5), -(optionally substituted Ci-Ce alkylenyl)-C(=O)R4, -(optionally substituted Ci-C6alkylenyl)-(R4), -C(=O)OR4, -C(=O)N(R4)(R5), -C(=O)R4, and R4,

[0509] wherein no more than one of each occurrence of R3a, R3b, and R3cis H; R4is selected from the group consisting of optionally substituted C1-C28 alkyl, optionally-substituted C2-C28 heteroalkyl, optionally substituted Cs-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C28 alkenyl, and optionally substituted C2-C28 alkynyl;

[0510] R5is selected from the group consisting of H and optionally substituted Ci-Cs alkyl; each occurrence of L is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-. -X-(optionally substituted C2-C12 alkenylenyl)-, -X-(optionally substituted C1-C12 alkynylenyl)-, -X-(optionally substituted C1-C12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl;

[0511] each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3e)-. and -O-; and

[0512] each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4.

[0513] In certain embodiments, the ionizable lipid of Formula (III) is:

[0514]

[0515] 1, 1 '-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2- hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-ol),

[0516] (C14-494)

[0517] In certain embodiments, the at least one ionizable lipid compound 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 at least one ionizable lipid compound 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 at least one ionizable lipid compound 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.

[0518] In certain embodiments, the at least one ionizable lipid compound comprises about 35 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises less than about 35 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises more than about 35 mol% of the LNP.

[0519] In certain embodiments, the neutral lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In certain embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In certain embodiments, the neutral lipid is l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). In certain embodiments, the neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0520] In certain embodiments, the at least one neutral lipid comprises about 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 neutral 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, or about 30 mol% of the LNP. In certain embodiments, the at least one neutral 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. or about 30 mol% of the LNP.

[0521] In certain embodiments, the at least one neutral lipid comprises about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises more than about 16 mol% of the LNP.

[0522] In certain embodiments, the sterol is cholesterol. In certain embodiments, the sterol is 24-a-methyl-cholesterol (campesterol). In certain embodiments, the sterol is 24-a-ethyl-cholestanol (stigmastanol). In certain embodiments, the sterol is 24-a-ethyl-cholesterol (B-sitosterol).

[0523] In certain embodiments, the polymer conjugated lipid is l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In certain embodiments, the polymer conjugated lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In certain embodiments, the polymer conjugated lipid is 1,2-dimyristoyl-rac-glycerol (DMG). In certain embodiments, the polymer conjugated lipid is N, N-ditetradecylacetamide (DTA).

[0524] In certain embodiments, the at least one lipid is covalently conjugated to a polyethylene glycol. In certain embodiments, the polyethylene glycol has a molecular weight selected from the group consisting of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500. 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400. 2500, 2600, 2700, 2800, 2900. 3000. 3100, 3200, 3300, 3400. 3500, 3600, 3700, 3800. 3900. 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000. 9100, 9200, 9300, 9400. 9500, 9600, 9700, 9800, 9900. or about 10000 kDa.

[0525] In certain embodiments, the polymer conjugated lipid is l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG). In certain embodiments, the polymer conjugated lipid is l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG). In certain embodiments, the polymer conjugated lipid is l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG). In certain embodiments, the polymer conjugated lipid is methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

[0526] 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, 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, 9.8, 9.9, or about 10 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, 9.8, 9.9. or about 10 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, 9.8, 9.9, or about 10 mol% of the LNP.

[0527] In certain embodiments, the at least one polymer conjugated lipid comprises about 2.5 mol% of the LNP. In certain embodiments, the at least one polymer conjugated lipid comprises less than about 2.5 mol% of the LNP. In certain embodiments, the at least one polymer conjugated lipid comprises more than about 2.5 mol% of the LNP.

[0528] In certain embodiments, the at least one sterol comprises cholesterol and the polymer conjugated lipid does not comprise a polyethylene glycol (PEG)-substituted 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In certain embodiments, the at least one sterol consists essentially of cholesterol and the polymer conjugated lipid does not comprise a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In certain embodiments, the at least one sterol comprises cholesterol and the polymer conjugated lipid does not consist essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). In certain embodiments, the at least one sterol consists essentially of cholesterol and the polymer conjugated lipid does not consist essentially of a polyethylene glycol (PEG)-substituted 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE).

[0529] In certain embodiments, the at least one polymer conjugated lipid comprises a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the at least one sterol does not comprise cholesterol. In certain embodiments, the at least one polymer conjugated lipid comprises a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the at least one sterol does not consist essentially of cholesterol. In certain embodiments, the at least one polymer conjugated lipid consists essentially of a polyethylene glycol (PEG)-substituted 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the at least one sterol does not comprise cholesterol. In certain embodiments, the at least one polymer conjugated lipid consists essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and the at least one sterol does not consist essentially of cholesterol.

[0530] In certain embodiments, the at least one neutral lipid comprises or consists essentially of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG).

[0531] In certain embodiments, the at least one neutral lipid comprises or consists essentially of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

[0532] In certain embodiments, the at least one neutral lipid comprises or consists essentially of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol). and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

[0533] In certain embodiments, the at least one neutral lipid comprises or consists essentially of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

[0534] In certain embodiments, the LNP has a molar ratio of (a): (b): (c): (d) of about 35: 15: 46.5: 2.5.

[0535] In certain embodiments, the LNP has a mass ratio of mRNA to sgRNA ranging from about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1. 0.9:1, 0.8:1, 0.7:1, 0.6: 1, 0.5:1, 0.4:1, 0.3:1. 0.2:1, or about 0.1:1. In certain embodiments, the LNP has a ratio of mRNA to sgRNA of about 4: 1.

[0536] In certain embodiments, the nucleic acid cargo (z.e., mRNA + sgRNA) has a concentration in the LNP ranging from about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130. 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250. 260, 270, 280, 290, 300, 310. 320, 330, 340. 350, 360. 370, 380, 390, 400, 410. 420, 430. 440, 450, 460. 470, 480, 490, or about 500 ng / pL. In certain embodiments, the nucleic acid cargo (i.e., mRNA + sgRNA) has a concentration in the LNP of about 75 ng / pL.

[0537] In certain embodiments, the ionizable lipid and mRNA in the LNP have a mass ratio selected from the group consisting of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1. 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and about 1:1 (ionizable lipid: mRNA).

[0538] In certain embodiments, the mRNA encodes a base editor. In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the base editor is an adenine base editor. In certain embodiments, the mRNA encodes a clustered regularly interspaced short palindromic repeat (CRISPR) associated protein. In certain embodiments, the CRISPR associated protein is CRISPR associated protein 9 (Cas9). In certain embodiments, the Cas9 is Streptococcus pyogenes Cas9 (SpCas9).

[0539] In certain embodiments, the sgRNA is engineered to target a DNA sequence in a eukaryotic cell. In certain embodiments, the DNA sequence in a eukaryotic cell comprises a gene implicated in a genetic disease or disorder. In certain embodiments, the genetic disease or disorder is a monogenic disease or disorder.

[0540] In certain embodiments, the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafiiess, Duchenne muscular dystrophy, familial hypercholesterolemia, Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease and Tay-Sachs disease.

[0541] In certain embodiments, the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

[0542] In certain embodiments, the LNP is prepared by microfluidic mixing using a total flow rate (TFR) ranging from 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, or about 4.0 mL / min. In certain embodiments, the LNP is prepared by microfluidic mixing using a total flow rate (TFR) of about 2.4 mL / min. Base-editing

[0543] In certain embodiments of the present disclosure, a subject is administered a baseeditor and / or base-editing complex for in utero gene editing. In certain embodiments, the base-editor and / or base-editing complex comprises an adenine base-editor (ABE). In certain embodiments, the base-editor and / or base-editing complex comprises a cytosine base-editor (CBE).

[0544] In certain embodiments, the ABE or CBE complex comprises a catalytically impaired Streptococcus pyogenes Cas9 (SpCas9) protein, unable to make DSBs, fused to either a cytosine deaminase domain from a nucleic acid-editing protein or a modified tRNA adenosine deaminase. The SpCas9 and sgRNA tether the base editor at the genome target site, and the cytosine deaminase converts a nearby cytosine into uracil and, ultimately, thymine (resulting in either C T or G A changes in the coding sequence of a gene, depending on which strand is targeted). The cytosine deaminase can introduce nonsense mutations in a site-specific fashion. Alternatively, the adenine deaminase converts a nearby adenine into inosine and, ultimately, guanine and can correct a disease-causing G A mutation. Unlike HDR, base editing does not require proliferating cells to efficiently introduce mutations. Non-limiting examples of ABE and / or CBE complexes are described in International Application No. PCT / US2021 / 071011, which is incorporated herein by reference in its entirety.

[0545] Methods for selecting and preparing sgRNA molecules suitable for binding to an appropriate mutated site in a genome are known to those of ordinary ■ skill in the art, and specific mutations underlying a number of genetic diseases, including neurological diseases, characterized by a single nucleotide mutation are known to those of ordinary skill in the art. Non-limiting examples single nucleotide mutations underlying genetic diseases and / or sgRNA sequences suitable for binding such sequences in the genome of a subject are described in International Application No. PCT / US2021 / 071011, which is incorporated herein by reference in its entirely.

[0546] In certain embodiments, the cargo comprises at least one selected from the group consisting of a nucleic acid molecule, small molecule, protein, therapeutic agent, antibody, and any combinations thereof.

[0547] Methods

[0548] Therapeutic Methods In one aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or infection in 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 an ionizable lipid compound of Formula (I). In certain embodiments, the LNP of the disclosure comprises an ionizable lipid compound of Formula (III).

[0549] In certain embodiments, the disease is at least one selected from the group consisting of cancer, cardiovascular disease, and a metabolic disease. In certain embodiments, the viral infection comprises COVID- 19.

[0550] In certain embodiments, the therapeutic 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.

[0551] In certain embodiments, the therapeutic cargo molecule comprises a nucleic acid. In certain embodiments, the nucleic acid is DNA or RNA.

[0552] 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.

[0553] In certain embodiments, the mRNA encodes an enz me. In certain embodiments, the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9. In certain embodiments, the nucleic acid cargo further comprises sgRNA.

[0554] In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

[0555] In another aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a genetic 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 or the pharmaceutical composition of the disclosure. In certain embodiments, the LNP of the disclosure comprises an ionizable lipid compound of Formula (I). In certain embodiments, the LNP of the disclosure comprises an ionizable lipid compound of Formula (III).

[0556] In certain embodiments, the LNP comprises nucleic acid cargo. In certain embodiments, the nucleic acid cargo comprises at least one messenger RNA (mRNA). In certain embodiments, the mRNA has a size of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 kilobases. In certain embodiments, the LNP comprises nucleic acid cargo comprising a messenger RNA (mRNA) encoding a base editor and a single guide RNA (sgRNA). In certain embodiments, the genetic disease or disorder is a monogenic disease or disorder. In certain embodiments, the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafness, Duchenne muscular dystrophy, familial hypercholesterolemia, Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease and Tay-Sachs disease.

[0557] In certain embodiments, the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

[0558] In another aspect, the disclosure provides a method of genome editing a mutated gene sequence associated with 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 or the pharmaceutical composition of the disclosure. In certain embodiments, the LNP comprises nucleic acid cargo. In certain embodiments, the nucleic acid cargo comprises at least one messenger RNA (mRNA). In certain embodiments, the mRNA has a size of about 1, 2, 3, 4. 5, 6, 7, 8, 9, or about 10 kilobases. In certain embodiments, the LNP comprises nucleic acid cargo comprising a messenger RNA (mRNA) encoding a base editor and a single guide RNA (sgRNA).

[0559] In certain embodiments, the sgRNA is targeted to a DNA sequence of the mutated gene sequence associated with the disease or disorder in the subject.

[0560] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0561] Synthetic Methods

[0562] In another aspect, the disclosure provides a method for preparing a compound of formula (II), or a salt, stereoisomer, or isotopologue thereof

[0563] Rx

[0564] R2-N

[0565] R5a(II),

[0566] the method comprising:

[0567] Y

[0568] R2“N

[0569] contacting a compound of formula (A): H (A),

[0570] a compound of formula (

[0571]

[0572] a compound of formula (

[0573]

[0574] in the presence of a copper catalyst;

[0575] wherein:

[0576] R2is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Cs-Cs cycloalkyl, optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);

[0577] Rxis selected from the group consisting of R5band optionally substituted Ci-Ce alkyl, or

[0578] Rxand R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyd;

[0579] RYis selected from the group consisting of H and optionally substituted Ci-Ce alkyl, or

[0580] R^ and R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyl;

[0581] R3aand R3b, if present, are each independently

[0582]

[0583] R6is selected from the group consisting of R4and

[0584]

[0585] each occurrence of R4is C1-C16 alkyl or C2-C16 alkenyl;

[0586] each occurrence of L2is independently selected from the group consisting of -CH2-, -O-, -C(=O)-, and -(optionally substituted phenylenyl)-; and

[0587] each occurrence of n and o is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0588] In certain embodiments, the copper catalyst comprises CuCl. In certain embodiments, the copper catalyst has a concentration of about 10 mol%. In certain embodiments, the contacting occurs at a temperature of about 50 °C. In certain embodiments, the contacting occurs for a period of about 48 h.

[0589] In certain embodiments, -(L2)„- is

[0590]

[0591] in certain embodiments, -(L2)„- is

[0592]

[0593] embodiments, -

[0594]

[0595] .

[0596] In certain embodiments, -(L2)o- is -O-. In certain embodiments, -(L2)o- is -C(=O)O-. In certain embodiments, -(L2)o- is -OC(=O)-.

[0597] In certain embodiments, R4is

[0598]

[0599] . in certain embodiments, R4is

[0600]

[0601] , certain embodiments, R4is

[0602]

[0603] In certain embodiments, R4is

[0604]

[0605] Pharmaceutical Compositions

[0606] 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.

[0607] 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.

[0608] 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.

[0609] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, 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.

[0610] 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.

[0611] 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.

[0612] 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 t pe and age of the veterinary or human patient being treated, and the like.

[0613] 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.

[0614] 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- 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.

[0615] 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.

[0616] 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.

[0617] 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).

[0618] 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 including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0619] 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 earner.

[0620] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total w eight 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 benzy l alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzy l alcohol and 0.05-0.5% sorbic acid.

[0621] The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol 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 w eight by total w eight 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 w eight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total w eight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelflife 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 w ould be known to those skilled in the art.

[0622] 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, w ater, and isotonic saline. Oily vehicles 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. 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, heptadecaethyleneoxy cetanol, 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 / 7-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0623] 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.

[0624] 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- 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.

[0625] 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 phy siologically 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.

[0626] Administration / Dosing

[0627] 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.

[0628] 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 (z.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 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.

[0629] 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.

[0630] 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.

[0631] 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.

[0632] 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. 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.

[0633] The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 p g to about 6,000 mg, about 100 p g to about 5,500 mg, about 200 p g to about 5,000 mg, about 400 p g to about 4.000 mg, about 800 p 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.

[0634] In some embodiments, the dose of active agent (z.e., nucleic acid) present in the composition of the disclosure is from about 0.5 pg 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. 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.

[0635] 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 know n 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 abi 1 i ty to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

[0636] Administration

[0637] 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, intrapulmonaiy. intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0638] 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 formulations and compositions that are described herein.

[0639] Parenteral Administration

[0640] 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, intrastemal injection, and kidney dialytic infusion techniques.

[0641] 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 (PC A) 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.

[0642] 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 anon-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. 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.

[0643] EXAMPLES

[0644] 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.

[0645] Materials and Methods (Examples 1-8)

[0646] Materials

[0647] Amines, aldehydes, alkynes, alcohols, carboxylic acids, N, N’- Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and copper(I) chloride were purchased from Sigma Aldrich, Tokyo Chemical Industry and Ambeed. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG 2000) and cholesterol were obtained from Avanti Polar Lipids. DLin-MC3-DMA and SM-102 were purchased from MedChem Express. Nucleoside-modified Cas9 mRNA (5moU) was bought from TriLink. Highly modified sgRNA target mouse TTR (guide No. G211) was chemically synthesized by a commercial supplier (AxoLabs) based on a previous publication.

[0648] mRNA Synthesis

[0649] Codon optimized firefly Luciferase sequence or SARS-CoV-2 Spike sequence was cloned into a proprietary mRNA production plasmid (optimized 3’ and 5’ UTR with a 101 poly A tail), in vitro transcribed in the presence of 1 -methyl pseudouridine modified nucleoside, co-transcriptionally capped using the CleanCap technology (TriLink) and cellulose purified to remove double-stranded RNAs. Purified mRNA was ethanol precipitated, washed, re-suspended in nuclease-free water, and subjected to quality control. All mRNAs were stored at -20 °C until use.

[0650] Combinatorial synthesis of UPenn-lipids in Library 1 and 2

[0651] These UPenn-lipids were synthesized via solvent-free, copper(I) chloride catalyzed A3-coupling of amine, formaldehyde and alkyne. Excess formaldehyde and alkyne (1.25 eq. of -NH-) were used to ensure repeated A3-coupling reactions. Taking amine 11 as an example, amine 11 (0.1 mmol, 1 eq.), formaldehyde (0.25 mmol, 37% in water, 2.5 eq.), alkyne (0.25 mmol, 2.5 eq.) and copper(I) chloride (0.01 mmol, 0.1 eq.) were combined in a glass vial and stirred at 50 °C for 48 h. Excess TEA was added to the reaction if the amine was in the salt form. The yield was -50% for aromatic alkynes and <10% for aliphatic alkynes. The crude products in Library 1 were dissolved in ethanol and directly used for in vitro screening. The crude products in Library 2 were purified using a CombiFlash NextGen 300+ chromatography system (Teledyne Isco) with gradient elution from 100% DCM to 60% DCM / MeOH / NH₄OH (75:22:3, aq) and target products were confirmed by MS.

[0652] Combinatorial synthesis ofUPenn-lipids in Library 3-5

[0653] These UPenn-lipids were synthesized via A3-coupling of amine, long-chain aldehyde and long-chain alky ne. Excess amine (1.25 eq. of aldehyde / alkyne) was used. Taking amine 31 as an example, amine 31 (0.125 mmol, 1.25 eq.), long-chain aldehyde (0.1 mmol, 1 eq.), long-chain alkyne (0.1 mmol, 1 eq.) and copper(I) chloride (0.01 mmol, 0.1 eq.) were combined in a glass vial and stirred at 50 °C for 48 h. The crude products in Library 3-5 were purified using a CombiFlash NextGen 300+ chromatography system with gradient elution from 100% DCM to 80% DCM / MeOH / NH4OH (75:22:3, aq) and target products were confirmed by MS. The yield was typically -50%. The lead UPenn-lipid 31hP was collected as brown oil (y ield 51%). MS-ESI: calculated for C49H86N2O4: 767.24, found [M+H]+= 768.03. 'H NMR (400 MHz. CDCL) 8 8.04 - 7.96 (m, 2H), 7.64 (d. J = 8.2 Hz. 2H), 5.06 (p. J = 6.1 Hz, 1H), 4.89 (p, J = 6.2 Hz, 1H), 4.79 (s, 1H), 2.68 - 2.33 (m, 10H), 2.17 (s, 3H), 1.91 (p, J = 7.2 Hz, 2H), 1.75 - 1.60 (m, 4H), 1.52 (d, J = 6.4 Hz, 4H), 1.26 (d, J = 6.9 Hz, 36H), 1.00 (t, J = 7.1 Hz, 6H), 0.94 (td, J = 7.4, 0.9 Hz, 3H), 0.87 (td, J = 6.9, 2.4 Hz, 9H).

[0654] LNP formulation and optimization

[0655] For initial in vitro and in vivo screening, UPenn-LNPs were prepared by pipette mixing of the ethanolic phase containing UPenn-lipid, DOPE, cholesterol and DMG-PEG with the aqueous phase (10 mM citrate buffer, pH 3) containing mRNA at a volume ratio of 1:3 and then diluted in culture medium or 1 x PBS for cell or animal treatment, respectively. The weight ratio of UPenn-lipid: DOPE: cholesterol: DMG-PEG: mRNA was fixed at 16:10:10:3:1.6. To formulate 31hP LNP by microfluidic mixing, the ethanolic phase containing lipids (31hP / DOPE / Chol / DMG-PEG = 40:10:48.8:1.5) was mixed with the aqueous phase containing mRNA at a flow rate ratio of 1:3 and at a 31hP / mRNA weight ratio of 10:1 in a microfluidic chip device. The standard MC3 LNP (or SM-102 LNP) was formulated with MC3 (or SM-102), DSPC, cholesterol and DMG-PEG at a molar ratio of 50:10:38.5:1.5 using microfluidic mixing at an ionizable lipid / mRNA weight ratio of 10:1. LNPs were dialyzed against 1 x PBS in a 20 kDa MWCO cassette for 2 h, filtered through a 0.22 µM filter and stored at 4 °C.

[0656] Characterization

[0657] ’H-NMR was recorded using a Bruker 400 MHz NMR spectrometer. MS was performed on a Waters Acquity LCMS system equipped with UV-Vis and MS detectors. The hydrodynamic size, poly dispersity index (PDI) and zeta potential of LNPs were measured using a Malvern Zetasizer Nano ZS90. The morphology of LNPs was characterized by a cryo-electron microscope (Titan Krios, Thermo Fisher) equipped with a K3 Bioquantum. The mRNA encapsulation efficiency and the pKa of LNP were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen) and a 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS) assay, respectively.

[0658] Cell culture and animal studies

[0659] The human hepatocellular carcinoma HepG2 cell line was purchased 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 pg / mL streptomycin. Cells were cultured at 37 °C in a humidified incubator of 5% CO2, and routinely tested for mycoplasma contamination. C57BL / 6 mice (6-8 weeks, 18-20 g) were purchased from Jackson Laboratory7.

[0660] In vitro mLuc delivery by UPenn-LNPs

[0661] HepG2 cells were seeded in 96-well plates at a density of 5,000 per well overnight and mLuc-loaded UPenn-LNPs (15 ng mRNA / well) were used to treat cells for 24 h.

[0662] 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 mLuc delivery by UPenn-LNPs

[0663] Mice were i.v. injected with mLuc-loaded UPenn-LNPs at an mRNA dose of 0.1 mg / kg. For i.m. delivery, mLuc-loaded UPenn-LNPs (2 pg mRNA in 50 pL PBS) were injected into the gastrocnemius muscle. 4 h later, mice were intraperitoneally (i.p.) injected with D-luciferin potassium salt (150 mg / kg), and bioluminescence imaging was performed using an in vivo imaging system (PerkinElmer).

[0664] Hemolysis assay

[0665] Mouse red blood cells (RBCs) were isolated and washed three times with 1 * PBS by centrifugation at 700 g for 5 min. Next, RBCs were diluted to a 4% vol / vol RBC solution either in neutral (pH 7.4) or acidic PBS (pH 6.0), and incubated with LNPs at a final mRNA concentration of 3 pg / mL at 37 °C for 1 h. Finally, the RBC solution was centrifuged at 700 g for 5 min and 100 pL 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 lx PBS, respectively.

[0666] Cellular internalization inhibition

[0667] HepG2 cells were seeded in a 96-well plate at a density of 5,000 per well overnight. Cells were pre-treated with 5 mM amiloride, 20 pM chlorpromazine, 0.2 mM genistein or 5 mM M[3-CD for 30 min. Control group was pre-treated with dimethylsulfoxide (DMSO). Then, cells were treated with mLuc-loaded LNPs (15 ng / well) for 24 h. Luciferase expression was determined as described above and normalized to control.

[0668] Systemic delivery of CRISPR gene editor

[0669] Mice were i.v. injected with Cas9 mRNA / TTR sgRNA (4:1, wt:wt)-loaded LNPs at a total RNA dose of 1 mg / kg. Serum was collected at on day 7 and analyzed by ELISA (Aviva Systems Biology, #OKIA00111). Mice were euthanized, and livers were collected to determine the on-target indel frequency by next-generation sequencing (NGS) as described previously.

[0670] Intramuscular delivery of SARS-CoV-2 mRNA vaccine

[0671] Mice were i.m. injected with SARS-CoV-2 Spike mRNA-loaded LNPs (2 pg mRNA in 50 pL PBS) twice using a prime-boost strategy at a three-week interval. Serum was acquired at 0, 3, and 5 weeks post prime vaccination. Anti-Spike IgG titers and pseudovirus neutralization antibody titers against D614G-mutated SARS-CoV-2 ancestor strain were determined as described previously.

[0672] Safety evaluations

[0673] Serum was collected at 24 h post-treatment of Cas9 mRNA / TTR sgRNA-loaded LNPs (1 mg / kg). ALT and AST activities were determined by alanine transaminase colorimetric activity assay kit (#700260. Cayman) and aspartate aminotransferase colorimetric activity assay kit (#701640, Cayman), respectively. 13 pro-inflmmatory cytokines were examined using a LEGENDplex™ multi-analyte flow assay kit (BioLegend, #740621) according to the manufacturer’s instruction.

[0674] Statistical analysis

[0675] Data are presented as mean ± SD. Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test was applied for comparison between two groups or among multiple groups, respectively, p < 0.05 was considered to be statistically significant.

[0676] Example 1: Chemical Synthesis of Intermediates

[0677]

[0678] G. Colorless oil, yield 91%. MS-ESI: calculated for C15H18O2: 230.13, found [M+H]+ = 231.12. 'H NMR (400 MHz. CDCh) 57.51 - 7.42 (m. 2H), 7.31 - 7.27 (m, 2H), 5.11 (s, 2H), 3.07 (s, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.63 (p, J = 7.3 Hz, 2H), 1.35 - 1.21 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H).

[0679]

[0680] H: Colorless oil, yield 90%. MS-ESI: calculated for C17H22O2: 258.16, found [M+H]+ = 259.14. 'H NMR (400 MHz, CDCh) 57.50 - 7.43 (m, 2H), 7.34 - 7.28 (m, 2H), 5.10 (s, 2H), 3.09 (s, 1H), 2.37 (tt, J = 8.9, 5.3 Hz, 1H), 1.58 (dq, J = 12.7, 6.6 Hz, 2H), 1.44 (ddd. J = 14.2, 8.1. 5.2 Hz. 2H), 1.25 (h. J = 6.4 Hz. 4H), 0.88 (td, J = 6.9, 4.2 Hz, 6H).

[0681]

[0682] I: Colorless oil, yield 92%. MS-ESI: calculated for C17H22O2: 258.16, found [M+H]+ = 259.15. 'H NMR (400 MHz, CDCh) 57.51 - 7.44 (m, 2H), 7.33 - 7.27 (m, 2H), 5.11 (s, 2H), 3.07 (s, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.65 (p, J = 7.3 Hz, 2H), 1.37 - 1.22 (m, 8H), 0.87 (t, J = 6.8 Hz, 3H).

[0683]

[0684] J: Colorless oil, yield 91%. MS-ESI: calculated for C21H30O2: 314.22, found [M+H]+ = 315.21.1H NMR (400 MHz, CDCh) 57.52 - 7.43 (m, 2H), 7.33 - 7.27 (m, 2H), 5.10 (s, 2H), 3.07 (s, 1H), 2.38 (tt, J = 8.9. 5.3 Hz. 1H), 1.60 (dq, J = 12.7, 6.6 Hz, 2H), 1.46 (ddd. J = 14.2. 8.1. 5.2 Hz. 2H). 1.24 (h. J = 6.4 Hz. 12H), 0.88 (td. J = 6.9. 4.2 Hz, 6H).

[0685]

[0686] K: Colorless oil, yield 92%. MS-ESI: calculated for C19H26O2: 286.19, found [M+H]+ = 287.17. 'H NMR (400 MHz. CDCh) 57.52 - 7.44 (m. 2H), 7.33 - 7.28 (m, 2H), 5.10 (s, 2H), 3.08 (s, 1H), 2.35 (t, J = 7.5 Hz, 2H), 1.64 (p, J = 7.3 Hz, 2H), 1.36 - 1.20 (m, 12H), 0.88 (t, J = 6.8 Hz, 3H).

[0687]

[0688] L: Colorless oil, yield 90%. MS-ESI: calculated for C25H38O2: 370.29, found [M+H]+ = 371.30. 'H NMR (400 MHz, CDCh) 57.51 - 7.44 (m, 2H), 7.34 - 7.27 (m, 2H), 5.11 (s, 2H), 3.08 (s, 1H), 2.38 (tt, J = 8.9, 5.3 Hz, 1H), 1.59 (dq, J = 12.7, 6.6 Hz, 2H), 1.45 (ddd. J = 14.2, 8.1. 5.2 Hz. 2H), 1.24 (h. J = 6.4 Hz. 20H), 0.87 (td, J = 6.9. 4.2 Hz, 6H).

[0689]

[0690] M: Colorless oil, yield 94%. MS-ESI: calculated for C17H30O2: 266.22, found [M+H]+ = 267.21. 'H NMR (400 MHz, CDCh) 54.10 (t, J = 6.5 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 2.25 (td, J = 7.0, 2.7 Hz, 2H), 1.97 (t, J = 2.7 Hz, 1H), 1.80 - 1.71 (m, 2H), 1.67 - 1.54 (m, 2H), 1.37 - 1.22 (m, 16H), 0.92 - 0.81 (m, 3H).

[0691]

[0692] N: Colorless oil, yield 91%. MS-ESI: calculated for C18H32O2: 280.24, found [M+H]+ = 281.25. 'H NMR (400 MHz, CDCh) 54.84 (tt, J = 6.9, 5.5 Hz, 1H), 2.43 (t, J = 7.4 Hz. 2H), 2.28 (td, J = 7.0, 2.6 Hz, 2H), 1.97 (t, J = 2.7 Hz, 1H), 1.87 (p, J = 7.2 Hz. 2H), 1.61 - 1.49 (m, 2H), 1.26 (m, 19H), 0.91 - 0.82 (m, 3H).

[0693]

[0694] O: Colorless oil, yield 91%. MS-ESI: calculated for C17H30O2: 266.22, found [M+H]+ = 267.23. 'H NMR (400 MHz, CDCh) 54.83 (tt, J = 6.9, 5.5 Hz, 1H), 2.44 (t, J = 7.4 Hz, 2H), 2.27 (td, J = 7.0, 2.6 Hz, 2H), 1.97 (t, J = 2.7 Hz, 1H), 1.86 (p. J = 7.2 Hz. 2H), 1.62 - 1.49 (m. 4H), 1.26 (m, 12H), 0.91 - 0.84 (m, 6H).

[0695]

[0696] P: Colorless oil, yield 90%. MS-ESI: calculated for C23H42O2: 350.32, found [M+H]+ = 351.30. 'H NMR (400 MHz, CDCh) 54.88 (p, J = 6.3 Hz, 1H), 2.43 (t, J = 7.4 Hz, 2H), 2.26 (td, J = 7.0, 2.7 Hz, 2H), 1.96 (t, J = 2.7 Hz, 1H), 1.85 (p, J = 7.2 Hz, 2H), 1.55 - 1.46 (m, 4H). 1.26 (s, 24H), 0.91 - 0.85 (m, 6H).

[0697]

[0698] Q: Colorless oil, yield 92%. MS-ESI: calculated for C16H28O2: 252.21, found [M+H]+ = 253.19. 'H NMR (400 MHz, CDCh) 54.09 (t. J = 6.5 Hz. 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.23 (td, J = 7.0, 2.7 Hz, 2H), 1.95 (t, J = 2.7 Hz, 1H), 1.80 - 1.70 (m, 2H), 1.66 - 1.54(m, 4H), 1.36 - 1.23 (m, 12H), 0.88 (td, J = 6.9, 1.8 Hz, 3H).

[0699]

[0700] R: Colorless oil, yield 90%. MS-ESI: calculated for C22H40O2: 336.30, found [M+H]+ = 337.30.1H NMR (400 MHz, CDCh) 54.10 (t, J = 6.4 Hz, 2H), 2.31 (tt, J = 8.9, 5.3 Hz. 1H), 2.23 (td, J = 7.0, 2.6 Hz, 2H), 1.95 (t, J = 2.6 Hz, 1H), 1.82 - 1.54 (m, 6H), 1.43 (dt. J = 7.9, 5.6 Hz, 2H). 1.26 (d, J = 4.2 Hz, 20H), 0.92 - 0.83 (m, 6H).

[0701]

[0702] e: Colorless oil, yield 90%. MS-ESI: calculated for C23H36O3: 360.27, found [M+H]+ = 361.25. 'H NMR (400 MHz, CDCh) 5 10.00 (s, 1H), 7.97 - 7.88 (m, 2H), 7.27 - 7.22 (m, 2H), 2.60 (tt, J = 8.9, 5.4 Hz, 1H), 1.83 - 1.69 (m, 2H), 1.66 - 1.53 (m, 2H), 1.46 - 1.22 (m, 20H), 0.89 (td, J = 6.9, 4.4 Hz, 6H).

[0703]

[0704] f: Colorless oil, yield 94%. MS-ESI: calculated for C19H28O3: 304.20, found [M+H]+ = 305.22.1H NMR (400 MHz, CDCh) 5 10.11 (s, 1H), 8.23 - 8.18 (m, 2H). 8.01 -7.92 (m, 2H), 4.10 (t, J = 6.5 Hz, 2H), 2.25 (td, J = 7.0, 2.7 Hz, 2H), 1.49 - 1.19 (m, 16H), 0.92 - 0.82 (m, 3H).

[0705]

[0706] g: Colorless oil, yield 91%. MS-ESI: calculated for C20H3003: 318.22, found [M+H]+ = 319.23. 'H NMR (400 MHz, CDCh) 5 10.12 (s, 1H), 8.23 - 8.18 (m, 2H), 8.01 -7.92 (m, 2H), 5.12 (m, 1H), 1.81 - 1.60 (m, 2H), 1.48 - 1.18 (m, 19H), 0.91 - 0.83 (m, 3H).

[0707]

[0708] h: Colorless oil, yield 91%. MS-ESI: calculated for C19H28O3: 304.20, found [M+H]+ = 305.18.1H NMR(400 MHz, CDCh) 5 10.11 (s, 1H), 8.24 - 8.18 (m, 2H), 8.00 -7.91 (m, 2H), 5.10 (ddd, J = 12.6, 6.9, 5.6 Hz, 1H), 1.79 - 1.58 (m, 4H), 1.45 - 1.17 (m, 12H), 0.96 (t. J = 7.4 Hz. 3H), 0.92 - 0.82 (m, 3H).

[0709]

[0710] i: Colorless oil, yield 89%. MS-ESI: calculated for C25H40O3: 388.30, found [M+H]+ = 389.31.1H NMR (400 MHz, CDCh) 5 10.11 (s, 1H), 8.23 - 8.16 (m, 2H), 7.99 -7.91 (m, 2H), 5.15 (tt, J = 7.1, 5.3 Hz, 1H), 1.77 - 1.58 (m, 4H), 1.41 - 1.16 (m, 24H), 0.91 -0.83 (m, 6H).

[0711] Example 2: Generating UPenn-lipids via commercially available starting materials The three-component coupling of an amine, an aldehyde and an alkyne, commonly known as A3-coupling, has been established as a one-pot, convenient and general approach towards unsaturated propargylamine derivatives. This reaction can be performed under ambient, solvent-free conditions with good tolerance of many functional groups (e.g., alcohols, carboxylic acids, and esters). A3-coupling is atom-economical and eco-friendly with water as the only by-product. Moreover, depending on the amine and aldehyde used, symmetric dipropargylamines or asymmetric monopropargylamines can be synthesized, which increases the structural diversity of products. Therefore, A3-coupling is an ideal combinatorial chemistry reaction for ionizable lipid synthesis.

[0712] Since A3-coupling has never been adapted for synthesizing ionizable lipids, a pilot library of 180 UPenn-lipids were first established to evaluate the potential of this reaction using commercially available amines (1-30), formaldehyde (a) and alkynes (A-F) (FIG. 2A). Notably, excess aldehyde and alkyne were used to enable repeated A3-coupling in order to produce tertiary (3°) amine-based UPenn-lipids. It was noticed that aromatic alkynes (A-C) typically gave moderate yields (-50%) while aliphatic alkynes (D-F) typically gave poor yields (<10%). This observation is consistent with previous studies, where aliphatic alkynes were found to be less efficient to form dipropargylamines compared to aromatic alkynes. Next, 90 aromatic alkyne-derived UPenn-lipids were tested for their in vitro mRNA delivery efficiency due to their decent yields (FIG. 2B). Crude UPenn-lipids were formulated into UPenn-LNPs along with other standard lipid excipients and firefly luciferase mRNA (mLuc). HepG2 cells (a human hepatocellular carcinoma cell line) were treated with UPenn-LNPs at a low mRNA dose (15 ng / well) to avoid potential cytotoxicity (FIG. 7). Among this library of UPenn-lipids, amine 11 -derived symmetric 1 l(aB)2 demonstrated the highest transfection and its analogs (i.e., 1 l(aA)2 and ll(aC)2) also showed some transfection (>100 RLU, FIG. 2B). Moreover, the alkyl substituent on the aromatic alkyne greatly affected mRNA transfection and the elongation of the alkyl substituent appeared to increase the delivery efficiency (FIG. 2C). Through this pilot study, the feasibility of A3-coupling to produce ionizable lipids and the potential of amine 11 -based UPenn-lipids for mRNA delivery was verified. However, the lead UPenn-lipid 11 (aB)2 is not optimal for in vivo mRNA delivery applications, since it is not biodegradable and has a short tail length relative to the structure of FDA-approved ones.

[0713] Exemplary amine, alkyne, and aldehyde components used to prepare certain exemplary ionizable lipid compounds of the disclosure are provided herein (Tables 1-3).

[0714] Table 1. Exemplary amine components

[0715]

[0716]

[0717]

[0718] Table 2. Exemplary aldehyde components

[0719]

[0720]

[0721] Table 3. Exemplary alkyne components

[0722]

[0723]

[0724] Example 3: Mutating symmetric UPenn-lipids via biodegradable alkynes

[0725] Based on the strong in vitro transfection efficacy, ll(aB)2 was chosen as the starting point for iterative directed evolution. In this cycle, both efficacy and biodegradability were considered, since biodegradable ionizable lipids are highly preferred for in vivo mRNA delivery. Therefore, cleavable ester linkers were introduced into the tail region of UPenn-lipids by customizing 6 aromatic alkynes (G-L) via a one-step esterification reaction (FIG.

[0726] 3 A). G, I and K were aromatic alkynes with elongated linear al k l substituents, while H, J and L were their branched counterparts. Branched alkynyl tails were included since branching allows the ionizable lipid to adopt a more cone-shaped structure that is beneficial for endosomal escape and mRNA delivery.

[0727] The six customized biodegradable UPenn-lipids (H(aG)2-ll(aL)2, FIG. 19) were purified (FIGs. 8A-8B) and tested for in vitro and in vivo mUuc deliver}7. With increasing tail length and branching, UPenn-lipids generally showed increased in vitro transfection efficacy (FIG. 3B and FIG. 9), but their in vivo activities were disparate (FIG. 3C). Linear 1 l(aG)2 and 1 l(aK)2 showed limited in vivo transfection efficacy presumably due to their low and high lipophilicity7, respectively. Branched UPenn-lipids generally outperformed their linear counterparts both in vitro and in vivo, apart from 1 l(aJ)2 which showed slightly lower in vivo activity than 1 l(al)2. 1 l(aL)2 with a symmetric structure and two biodegradable branched tails was identified as the lead UPenn-lipid in this library with the highest in vivo mRNA delivery efficiency. After analyzing the relationship between in vitro and in vivo results, it was found that there was a poor correlation (FIG. 3D), which has also been observed by others. Therefore, in vivo mRNA delivery efficiency of UPenn-lipids was only tested in subsequent directed evolution cycles. Example 4: Mutating asymmetric UPenn-lipids via long-chain aldehydes

[0728] While the lead ll(aL)2 achieved a whole-body total flux of ~5xl08p / s, it did not reach the transfection level of the industry standard lipid MC3, which has been predetermined to be ~1 x 109p / s. Previous studies have suggested that asymmetric ionizable lipids with two different alkyl tails are prone to show better in vivo delivery efficacy than their symmetric analogs. It is hypothesized that asymmetric tails lower the packing density, decrease the crystallization tendency, and increase the fluidity of the lipid bilayer. Therefore, for the next round of directed evolution, the optimized ionizable head structure and alkynyl tail L (tail one) was maintained, while mutating the aldehyde (tail two).

[0729] Notably, owing to the flexibility of A3-coupling, asymmetric UPenn-lipids can be easily synthesized from an amine, a long-chain aldehyde, and a long-chain alkyne via A3-coupling once. To maintain the optimized ionizable head structure, a secondary' (2°) amine 31 (a methyl derivative of amine 11) was used to ensure that only 3° amines exist in the UPenn-lipid structure (FIG. 3E). Three commercially available long-chain aldehydes (b-d) were used to establish a pilot library of asymmetric UPenn-lipids to verily the hypothesis (FIG. 20). Excitingly, all three UPenn-lipids (31bL-31dL) surpassed the in vivo performance of MC3 (FIG. 3F). These data strongly demonstrate the merit of an asymmetric UPenn-lipid structure. Moreover, asymmetric UPenn-lipids seem to have a well tolerability of long-chain aldehydes as both aliphatic and aromatic ones can afford potent ones.

[0730] An asymmetric, 2° amine-containing UPenn-lipid 1 IbL was also synthesized via A3-coupling once of amine 11, aldehyde b, and alkyne L (FIG. 20). However, 1 IbL did not outperform the MC3 lipid or its 3° amine-containing analog 3 IbL (FIG. 3F). Due to the scope of this study, subsequent rounds of directed evolution were focused on the asymmetric, 3° amine-based UPenn-lipids.

[0731] Example 5: Mutating asymmetric UPenn-lipids via biodegradable aldehydes and alkynes

[0732] Inspired by findings on asymmetric UPenn-lipid performance, it was further optimized amine 31-based asymmetric UPenn-lipids by introducing biodegradability into the aldehyde structures and diversifying the tail structures. In this cycle of directed evolution, five biodegradable aldehydes (e-i) with linear or branched structures were synthesized via a one-step esterification reaction (FIG. 4A). Similarly, six additional biodegradable alkynes (M-R) with linear or branched structures were synthesized. Through combinatorial synthesis. 35 asymmetric UPenn-lipids were created in this library (FIG. 4A and FIGs. 21 A-21B). In contrast to library 1, it was found that aliphatic alkynes (M-R) gave moderate yields (-50%) similar to the aromatic alkyne L, likely due to the fact that only a single A3-coupling reaction was required here to generate the monopropargylamine product.

[0733] After testing the in vivo mLuc delivery of Library 4, eight asymmetric UPenn-lipids were identified wi th greater transfection efficacy as compared to MC3 lipid (FIG. 4A and FIG. 10). Specifically, four asymmetric UPenn-lipids (3IiN, 31 iO, 31gP and 31hP) facilitated more than 5-fold greater luciferase expression relative to MC3 with 31hP identified as the top-performer (8-fold higher). Interestingly, all these four UPenn-lipids comprise an aminodiacid motif, two 2° esters and two major tails with one long branch tail (pseudo-three tails). Deviating from these structural criteria generally led to the reduced in vivo activity. The potency of the lead UPenn-lipids is supported by previous studies that have shown that (1) negatively charged carboxylates attached to the ionizable headgroup can neutralize the positive charge, facilitating accelerated dissociation of the mRNA-LNP complex and release of mRNA; (2) 2° esters show slower degradation than primary ones, prolonging the availability of the mRNA-LNP complex; (3) branched tail structures allow the ionizable lipid to adopt a more cone-shaped structure for enhanced endosomal disruption.

[0734] Example 6: Mutating headgroup of asymmetric UPenn-lipids

[0735] With the optimal linker and tail structure identified, the ionizable headgroup was reevaluated in the final cycle of direct evolution. The headgroup was mutated while maintaining the optimal tail region as hP and screened 26 more 2° amines with only one site (-NH-) for the A3-coupling reaction (FIG. 4B and FIGs. 22A-22B). Notably, these amines have not been previously tested.

[0736] In this library, 11 more asymmetric UPenn-lipids were identified that reached the transfection efficacy of MC3 lipid. However, none of these UPenn-lipids surpassed the potency of 31hP. Interestingly, two ionizable nitrogens were observed in all potent UPenn-lipids (31-42 hP), but this feature alone was not adequate to afford potent ones (e.g., 43hP, 45-49 hP). UPenn-lipids with one ionizable nitrogen (51hP, 54-57hP) or three ionizable nitrogens (44hP, 50hP and 52hP) were inferior for in vivo mRNA delivery, which may be a detrimental consequence of dramatically changed polarity7.

[0737] Example 7: Characterization of UPenn-LNPs

[0738] After five iterations of activity- and biodegradability-driven directed chemical evolution of UPenn-lipids, 31hP was identified as the top-performing ionizable lipid candidate (characterization data in FIGs. 8A-8B and FIGs. 11-12). Notably, throughout this process, it was found that 31hP LNP and other potent UPenn-LNPs predominantly transfected the liver upon i.v. administration, which is consistent with MC3 LNP and the majority7of LNPs reported in the literature (FIG. 13).

[0739] The physicochemical properties of the 31hP LNP formulated by microfluidic mixing were next characterized (FIG. 5A). The hydrody namic size of 31hP LNP was approximately 80 nm with a poly dispersity index (PDI) of 0.093 and a neutral surface charge ( = -0.27 mV). The mRNA encapsulation efficiency (EE) was determined to be approximately 93%. The apparent pVa of LNP was determined to be 6.25 (FIG. 14). Owning to its ionization property. 31hP LNP showed good hemocompatibility at pH 7.4 and increased hemolysis at pH 6.0 (FIG. 15). Cryogenic electron microscopy (Cryo-EM) imaging results showed that 3 IhP LNP had a dense spherical structure with a lamellar shell and an amorphous core (FIG.

[0740] 5B). To investigate the key pathways governing the cellular uptake of 31hP LNP, an internalization inhibition study was performed in vitro (FIG. 5C). The significantly reduced transfection efficacy in the presence of amiloride or methyl-P-cyclodextrin (M -CD) suggests that the cellular uptake of 31hP LNP is mainly facilitated through macropinocytosis and lipid raft-mediated endocytosis.

[0741] Example 8: Delivering mRNA-based gene editors and vaccines using UPenn-lipids Following characterization, the utility of the UPenn-lipids was demonstrated by delivering therapeutic mRNAs. The 31hP LNP was first chosen for the hepatic delivery of mRNA-based CRISPR gene editors, which are much larger than mLuc and as such are challenging to deliver in vivo. Liver-tropic MC3 LNP was selected as the industry standard control LNP (Table 4). In a proof-of-principle study, LNPs co-encapsulating Cas9 mRNA and an sgRNA specific for the transthyretin (TTR) gene, which is implicated in hereditary amyloidosis, were formulated. A single injection of 31hP LNP co-delivering Cas9 mRNA and TTR sgRNA at 1 mg total RNA / kg dose achieved -18% on-target editing at the TTR locus in the liver and -30% reduction (p=0.0024) of TTR in the serum (FIGs. 5D-5F). In contrast, MC3 LNP only achieved -6% on-target editing at the TTR locus and -14% reduction ( / >=().0746) of TTR. Importantly, 31hP LNP was well-tolerated in mice with no abnormal increases in alanine transaminase (ALT), aspartate aminotransferase (AST) or pro-inflammatory cytokines at 24 h post-treatment (FIGs. 16-17).

[0742]

[0743] scatering (DLS) measurement in PBS (pH 7.4). The mRNA encapsulation efficiency (EE) was determined using a modified Quant-iT RiboGreen RNA assay. Data are presented as mean ± SD (n=3).

[0744] Finally, the potential of 31hP LNP for mRNA vaccine applications was explored. Recently, the FDA approved Modema’s SARS-CoV-2 mRNA-LNP vaccine, which uses an asymmetric ionizable lipid (SM-102) with similar ester orientation and pseudo-three tails to the 31hP (Table 4). Therefore,, the immunogenicity of 31hP LNP and SM-102 LNP encapsulating SARS-CoV-2 Spike mRNA after intramuscular vaccination were directly compared. In a standard prime-boost rodent vaccine model, 31hP LNP vaccine elicited a significantly higher anti-Spike antibody titer than SM-102 LNP vaccine (FIGs. 5-5H).

[0745] Specifically, all five mice (100%) generated strong antibody responses after a single vaccination of 31hP LNP vaccine, while only two mice (40%) in the SM-102 LNP vaccine group demonstrated moderate antibody responses after the prime vaccination. Using a lentivirus-based SARS-CoV-2 pseudovirus neutralization assay, it was further confirmed that 3 IhP LNP vaccine triggered a significantly higher neutralization antibody titer than SM-102 LNP vaccine after prime and boost vaccination (FIG. 51). Interestingly, it was found that 31hP LNP enabled comparable mRNA expression in muscle to SM-102 LNP with less off-target expression in liver (FIG. 18), suggesting that other factors (e.g., adjuvant effects) might play a role in the significantly higher immunogenicity7for the 31hP LNP vaccine.

[0746] Taken together, these results strongly support that 31hP is a superior ionizable lipid relative to industry standard ionizable lipids and hold the promise in the systemic delivery of mRNA therapeutics and intramuscular delivery of mRNA vaccines.

[0747] Materials and Methods (Examples 9-13)

[0748] Ionizable lipid synthesis

[0749] Cl 4-494 ionizable lipids were synthesized as previously described (see International Patent Application Nos. PCT / US2020 / 056255 and PCT / US2020 / 056252). Briefly, 2-{2-[4-(2- { [2-(2-aminoethoxy)ethy 1] amino} ethy l)piperazin- 1 -y 1] ethoxy } ethan- 1 -amine (referred to herein as “494,” Enamine, Kyiv, Ukraine) was reacted with excess 1,2-epoxy tetradecane (referred to herein as “Cl 4,” MilliporeSigma, Burlington. MA) in a 4 mL glass scintillation vial with a magnetic stir bar for 2 d at 80 °C. The product was transferred to a Rotovapor R-300 for solvent evaporation. For purification, lipid fractions were separated via a CombiFlash Nextgen 300+ chromatography system (Teledyn ISCO, Lincoln, NE). The fraction containing C 14-494 ionizable lipid was identified via liquid chromatography-mass spectrometry (LC-MS). Cl 4-494 ionizable lipid was suspended in ethanol prior to experimentation.

[0750] mRNA production

[0751] GFP and SpCas9 mRNA were sourced from TriLink Biotechnologies (San Diego, C A) with CleanCap® modifications. GFP CRISPRevolutionTM sgRNA was sourced from Synthego (Redwood City; CA) with a sequence of 5 -GGGCGAGGAGCUGUUCACCG - 3’ (SEQ ID NO:1). TTR sgRNA was sourced from Synthego (Redwood City, CA) with a sequence of 5’-UUACAGCCACGUCUACAGCA-3’ (SEQ ID NO:2).

[0752] LNP formulation and characterization

[0753] Cl 4-494 ionizable lipid was combined in ethanol with cholesterol (MilliporeSigma), 1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti Polar Lipids, Alabaster, AL), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG, Avanti Polar Lipids) to a total volume of 112.5 pL at a molar ratio of 35:46.5: 16:2.5. A separate aqueous phase was prepared from 25 pg of GFP mRNA or 25 pg of a combination of SpCas9 mRNA and sgRNA (4: 1 mass ratio) in 10 mM citrate bulfer to a total volume of 337.5 pL. The ethanol and aqueous phases were chaotically mixed at flow rates specified in the main text via a herringbone microfluidic device to produce LNPs. LNPs were dialyzed against IX PBS in Slide- A-Lyzer G220 kDa dialysis cassettes (Thermo Fisher Scientific) for 2 h, sterilized using 0.22 or 0.45 pm filters, and stored at 4 °C.

[0754] To produce phospholipid-substituted LNPs, DOPE was replaced during LNP formulation with either l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids), l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC, Avanti Polar Lipids) or 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids). To produce cholesterol-substituted LNPs, cholesterol was replaced during LNP formulation with either 24-a-methyl-cholesterol (campesterol, Cayman Chemical, Ann Arbor, Michigan), 24-a-ethyl-cholestanol (stigmastanol, Avanti Polar Lipids), or 24-a-ethyl-cholesterol (B-sitosterol, Avanti Polar Lipids). To produce PEG-substituted LNPs, DMPE-PEG was replaced during LNP formulation with either l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG, Avanti Polar Lipids), 1,2-dimyristoyl-rac- glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG, Avanti Polar Lipids), or methoxypoly ethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG, Avanti Polar Lipids). To produce LNPs with multiple organic excipient substitutions, the appropriate changes were made, as described elsewhere herein, during LNP formulation.

[0755] DynaPro® Plate Reader III (Wyatt Technology', Santa Barbara, CA) was used to measure the z average diameter, poly dispersity’ index (PDI), and zeta potential of LNPs. Encapsulation efficiency was measured using a Quant-iT-RiboGreen (Thermo Fisher Scientific) assay via manufacturer specifications. All LNP characterization data is reported as the mean of triplicate measurements. All materials were prepared and handled nuclease-free throughout synthesis, formulation, and characterization steps.

[0756] In vitro assessment of gene editing LNPs

[0757] To test the in vitro efficacy of gene editing LNPs, HepG2-GFP cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine (Gibco, Dublin, Ireland) supplemented with 10% volume / volume of fetal bovine serum (Gibco) and 1% volume / volume penicillin-streptomycin (Gibco). HepG2-GFP cells were seeded at a density of 20,000 cells / 100 pL. LNPs containing a total of 150 ng total RNA were used to treat cells, and cells were grown for a total of 5 days. At harvest, cells were isolated and resuspended in flow cytometry buffer (Ca2+ / Mg2+Free PBS, 0.5% BSA, 0.5 mM EDTA). Samples were analyzed for fluorescence (GFP1) via flow cytometry (BD FACSAriaTM Cell Sorter.

[0758] Haryana, India). Viability was assessed in a duplicate plate of treated cells via Live / DeadTM Cytotoxicity7Kit (Thermo Fisher Scientific). To test the in vitro efficacy of reporter mRNA LNPs, the same protocols were followed with HepG2-GFP cells replaced with HepG2 cells.

[0759] In vivo assessment of gene editing LNPs

[0760] C57BL / 6J mice (#000664) were purchased from The Jackson Laboratory7(Bar Harbor, ME). For in vivo gene editing experiments, adult female C57BL / 6J mice were injected with LNPs encapsulating SpCas9 mRNA and TTR sgRNA at a dose of 1 mg / kg of total RNA via standard access of the lateral tail vein. After five days, tissues were harvested and genomic DNA was extracted using a DNEasy7Blood and Tissue Kit according to manufacturer’s instructions (Qiagen, Hilden, Germany). PCR amplification of the target amplicon w as carried out using SuperFi II Hi-Fidelity DNA Polymerase (Thermo Fisher Scientific) with a universal annealing temperature of 60 °C and the following primer sequences: mTTR-exon2-F, 5 -CGGTTTACTCTGACCCATTTC-3’ (SEQ ID NO:3) and mTTR-exon2-R, 5 -GGGCTTTCTACAAGCTTACC-3’ (SEQ ID N0:4). Full-length Illumina sequencing adapters were then added to PCR products using a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA). Pooled samples were sequenced using an Illumina MiSeq system. Alignment of fastq files to the target amplicon and quantification of editing frequency at the TTR locus was performed using CRISPResso2.

[0761] Mouse serum from experimental animals prior to LNP treatment and five days after LNP treatment was harvested and analyzed via ELISA for quantification of TTR protein (#OKIAOOH1, Aviva Systems Biology, San Diego, CA) per manufacturer specifications. Mouse serum was analyzed for AST and ALT activity via a Roche Cobas Chemistry Analyzer (Roche, Basel, Switzerland).

[0762] Statistical analysis

[0763] All statistical analyses were carried out using GraphPad Prism 9 software. Unpaired Student’s t tests or one-way ANOVA with post hoc Dunnetf s test were used to determine significance between experimental groups.

[0764] Example 9: Optimization of microfluidic total flow rate for LNP co-delivery of SpCas9 mR A and sgRNA

[0765] Microfluidic methods based on stepwise ethanol dilution can generate LNPs with precisely defined physiochemical properties. Microfluidic approaches to generate LNPs encapsulating diverse nucleic acid cargos, including DNA, siRNA, reporter mRNA, and protein have been performed. In these studies, an ethanol-based solution of ionizable lipid and other organic excipients (i.e., phospholipid, cholesterol, lipid-PEG) and a buffered solution of nucleic acids were prepared, loaded into a glass syringes, and rapidly mixed. The mixing step was performed in a microfluidic device fabricated with poly dimethylsiloxane (PDMS) and etched via soft lithography with channels designed to promote chaotic mixing. Syringe pumps were used to control the inlet flow rates, while the outlet of the microfluidic device was connected to a dialysis cassette for buffer exchange.

[0766] Using this microfluidic system, the effect of the total flow rate (TFR) of mixing on the physiochemical properties and in vitro efficacy of LNPs encapsulating either a GFP reporter mRNA (996 nucleotides) or both SpCas9 mRNA (4521 nucleotides) and GFP sgRNA (-100 nucleotides) was investigated (FIG. 23B). It was hypothesized that encapsulation of larger and more charge dense mRNA-based cargos would require more precise microfluidic control of LNP formulation. To isolate the effect of microfluidic TFR, the organic phase was prepared identically for both nucleic acid conditions, using a multi-tail piperazine-based ionizable lipid previously shown to facilitate mRNA delivery to the liver (i.e., C14-494) and organic excipients (z.e., DOPE, cholesterol, DMPE-PEG) known to enhance intracellular mRNA delivery. The molar ratio of organic components (35:16:46.5:2.5) and N: P ratio between the ionizable lipid and mRNA (10:1) was also held constant for these experiments.

[0767] The TFR in the microfluidic system was varied from 0.3 mL / min to 3.6 mL / min, while the flow rate ratio (FRR) between the aqueous and organic phases were maintained at 3:1. For both LNPs encapsulating GFP mRNA and LNPs encapsulating SpCas9 mRNA and GFP sgRNA, an increase in TFR led to a corresponding decrease in average LNP diameter (FIGs. 23C-23D) and an increase in RNA encapsulation efficiency (EE%) (FIGs. 23E-23F).

[0768] To investigate the in vitro transfection efficacy of LNPs prepared at different TFR, either HepG2 cells (immortalized human hepatoma cell line) or HepG2-GFP cells (HepG2 cell line constitutively expressing GFP) were treated with LNPs at a dose of 150 ng total RNA per 20,000 cells. Transfection for LNPs encapsulating GFP mRNA was quantified via flow cytometry by resultant GFP expression in HepG2 cells, while transfection for LNPs encapsulating SpCas9 mRNA and GFP sgRNA was quantified by knockout of GFP expression in HepG2-GFP cells.

[0769] Variation of TFR to produce LNPs encapsulating GFP mRNA did not alter in vitro transfection efficacy in HepG2 cells (FIG. 23G). In contrast, an increase in TFR from 0.3 mL / min to 2.4 mL / min resulted in gene editing LNPs that mediated nearly 2-fold greater editing at the GFP locus in HepG2-GFP cells (FIG. 23H). For gene editing LNPs, low TFR may lead to early precipitation of lipid components within the microfluidic channel, resulting in sub-optimal self-assembly into LNPs. Indeed, gene editing LNPs produced at a TFR of 0.3 mL / min were large (300 nm) and poorly encapsulated their RNA cargo (< 80%). Notably, at the highest TFR that was tested (3.6 mL / min), LNPs had significantly decreased in vitro gene editing efficacy. While LNPs produced at a TFR of 3.6 mL / min had macro-scale

[0770] phy siochemical properties (e.g, size, EE%) akin to those produced at 2.4 mL / min, higher TFR may lead to increased shear stress during LNP formulation, leading to modulation of internal lipid bilayer structure and RNA packing and ultimately negatively impacting delivery of gene editing cargos. Based on these data, an optimal TFR of 2.4 mL / min was maintained for downstream formulation of LNPs with varying organic excipient identities.

[0771] Example 10: Impact of phospholipid structure on LNP-mediated gene editing

[0772] The structural and biological properties of LNPs are not solely ascribed to a single lipid excipient component. Rather, each lipid excipient component plays a fundamental role in LNP bioactivity. The ionizable lipid typically possesses a tertiary amine that is protonated in pH conditions below the acid dissociation constant (pKa) of the lipid and deprotonated in neutral conditions. Ionizable lipid pH sensitivity enables nucleic acid encapsulation during formulation and nucleic acid release via endosomal disruption. In this study, ionizable lipid C 14-494 was chosen for all LNP formulations given its strong in vitro and in vivo evidence of mRNA delivery efficacy to the liver. Phospholipids play several critical roles in LNP delivery, including enhancing endosomal escape, aiding in solubilization of RNAs inside aqueous pockets, and even driving organ tropism in vivo. Computational and experimental studies have suggested that phospholipids with phosphatidylcholine (PC) head groups provide stability to the bilayer membrane, while those with phosphatidylethanolamine (PE) head groups introduce membrane curvature, increase tension, and, in turn, endosomal disruption. The relative saturation of phospholipid tails can also modulate the fluidity' of the lipid bilayer and promote either the formation of a less stable hexagonal phase (unsaturated tails) or a more stable lamellar phase (saturated tails). However, the selection of phospholipids in LNPs co-encapsulating SpCas9 mRNA and sgRNA has not been well-studied.

[0773] To probe the effect of phospholipid structure on the gene editing efficacy of LNPs, C 14-494 LNPs were formulated to encapsulate SpCas9 mRNA and GFP sgRNA with DSPC, DOPC. or SOPC substituted for DOPE (FIG. 24A). In comparison to DOPE, a PE phospholipid with unsaturated lipid tails, DSPC is a PC phospholipid with saturated lipid tails, while DOPC and SOPC are PC phospholipids with unsaturated lipid tails. DOPE, DSPC, DOPC, and SOPC LNPs were characterized for size (FIG. 24B), EE% (FIG. 24C), and zeta potential (FIG. 24D). DOPE LNPs had an average diameter of 70 nm. which was similar to the size of DSPC LNPs (68 nm) but significantly smaller than DOPC LNPs (182 nm) and SOPC LNPs (155 nm) (FIG. 24B). Although DOPE LNPs boasted the highest EE% (89%), all LNP formulations had greater than 80% encapsulation efficiency (FIG. 24C). DOPE LNPs had a nearly neutral surface zeta potential (+0.33 mV), while DSPC, DOPC, and SOPC LNPs all had positive zeta potential values (FIG. 24D). In HepG2-GFP cells, DOPE LNPs and DSPC LNPs both facilitated gene editing at similar rates, resulting in GFP knockout in 15% of cells (FIG. 24E). In contrast, DOPC and SOPC LNPs mediated gene editing in only ~8% of cells. All LNP formulations resulted in greater than 80% cell viability (FIG. 24F).

[0774] The data convey that inclusion of DOPE and DSPC in the organic phase produces LNPs with similar physiochemical properties and gene editing efficacy in vitro. The smaller size of DOPE LNPs and DSPC LNPs likely allows for enhanced cellular uptake, leading to greater intracellular delivery of gene editing cargos. LNPs produced with PC containing phospholipids mediated higher levels of gene editing as saturation of lipid tails increased (DOPC SOPC DSPC) (FIG. 24E). With more saturated hydrocarbon chains, the phospholipid assumes a more cylindrical shape, tending toward the formation of a more stable bilayer phase, which may be necessary to better encapsulate large mRNA cargos. In contrast, when comparing the impact of saturated phospholipids with distinct head groups on LNP gene editing efficacy, LNPs formulated with PE head group phospholipid (DOPE) outperformed those formulated with PC head group phospholipid (DOPC) by two-fold (FIG.

[0775] 24E). possibly due to promotion of a non-bilayer inverted hexagonal phase, which facilitates improved endosomal membrane fusion of phospholipids. Thus, there appears to be a balance, and likely a complex interplay, between lipid bilayer stabilizing and destabilizing properties conveyed by phospholipids for maximal delivery7of gene editing cargo. More broadly, these data imply that either partially or fully saturated phospholipids with a PE head group (e.g, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l,2-distearoyl-sn-glycero-3-phosphoethanolamine) may further optimize LNPs for gene editing applications.

[0776] Example 11: Impact of cholesterol structure on LNP-mediated gene editing Cholesterol plays several important roles in LNPs. The tetracyclic core of cholesterol confers a planar structure that allows for intercalation into bilayer structures, which decreases membrane permeability and affords the bilayer a greater resistance to destabilization by serum components. The inclusion of cholesterol in LNPs has also been shown to be fundamental in nucleic acid delivery, potentially by promoting endosomal membrane fusion and interacting with subcellular lipid transport pathways. Recently, structure-activity analysis of cholesterol analogs revealed that incorporation of C-24 alkyl phytosterols into LNPs in the place of cholesterol enhanced mRNA transfection. Here, the effect of cholesterol substitution with three analogs on LNP gene editing efficacy was assessed.

[0777] To this end, Cl 4-494 LNPs were formulated to encapsulate SpCas9 mRNA and GFP sgRNA with campesterol, P-sitosterol, and stigmastanol (FIG. 25A) substituted for cholesterol. In comparison to cholesterol, campesterol and P-sitosterol possess alkyl substitutions in the tail region, while stigmastanol has both an alkyl-substituted tail and reduction of a double bond within a sterol ring in the body region. Cholesterol, campesterol, P-sitosterol, and stigmastanol LNPs were characterized for size (FIG. 25B), EE% (FIG. 25C), and zeta potential (FIG. 25D). All three cholesterol analogs doubled the size of LNPs (-140 nm) from the original cholesterol formulation (70 nm) (FIG. 25B) with a corresponding reduction in encapsulation efficiency (FIG. 25C). Substitution with cholesterol analogs had no effect on the surface zeta potential of LNPs, as all formulations remained nearly neutral (FIG. 25D). In HepG2-GFP cells, P-sitosterol LNPs and stigmastanol LNPs enhanced gene editing efficacy by two-fold relative to the original cholesterol formulation (FIG. 25E). In contrast, campesterol LNPs mediated similar levels of knockout to cholesterol LNPs. There was no impact of cholesterol substitution on cell viability (FIG. 25F).

[0778] Previous studies have shown that the C-24 alkyl group in cholesterol imparts crystal defects in the ordering of the lipid bilayer and that the frequency of defects is directly proportional to the length of the alkyl side chain. LNPs produced with C-24 alkyl analogs may have a more faceted surface geometry than the relatively uniform curvature of cholesterol LNPs, facilitating cellular uptake and potentially biasing subcellular trafficking toward pathways that promote endosomal escape. The results described herein demonstrate that gene editing was enhanced with the introduction of the longer C-24 ethyl group ( -sitosterol, stigmastanol) relative to a C-24 alkyl group (campesterol) or the native, unsubstituted tail (cholesterol) (FIG. 25E). As has been shown previously for reporter mRNA delivery7, the gene editing efficacy of C-24 ethyl cholesterol analogs did not vary with the presence of a A-5 double bond (P-sitosterol vs. stigmastanol), demonstrating that the flexibility endowed by saturation of this double bond does not impact LNP gene editing efficacy. Together, these results corroborate that cholesterol analogs can enhance mRNA transfection and, more specifically, improve the delivery7of mRNA-based gene editing cargos.

[0779] Example 12: Impact of lipid-PEG structure on LNP-mediated gene editing

[0780] The incorporation of a hydrophilic lipid-PEG into LNPs provides a steric barrier that drives lipid bilayer self-assembly during formulation. Lipid-PEGs are widely used to enhance in vivo pharmacokinetic properties of LNPs. including serum stability, circulation half-life, and evasion of the mononuclear phagocyte system. However, PEGylation has also been show n to negatively impact LNP nucleic acid delivery, since the addition of lipid-PEG introduces significant steric hindrance and subsequently hinders cellular uptake and endosomal escape. Given these competing effects, it was hypothesized that the choice of lipid-PEG would impact the gene editing efficacy of LNPs.

[0781] To test this, Cl 4-494 LNPs were formulated to encapsulate SpCas9 mRNA and GFP sgRNA with DSPE-PEG, DMG-PEG, or DTA-PEG substituted for DMPE-PEG (FIG. 26 A). In comparison to DMPE-PEG (C14), DSPE-PEG (C18) possesses an additional four methylene groups in its acyl tails. DMG-PEG and DTA-PEG utilize a glycerol linker or an amide linker, respectively, instead of the glycero-3-phosphoethanolamine linkage used in DMPE-PEG between the lipophilic tails and the PEG chain. Notably, DMG-PEG is the lipid-PEG used in the formulation of the Pfizer SARS-CoV-2 vaccine, while DTA-PEG is the lipid-PEG used in the formulation of the Modema SARS-CoV-2 vaccine. All lipid-PEGs had an average polymer molecular weight of 2000 (PEG-2000). DMPE-PEG, DSPE-PEG, DMG-PEG, and DTA-PEG LNPs were characterized for size (FIG. 26B), EE% (FIG. 26C), and zeta potential (FIG. 26D). Substitution of DMPE-PEG with DSPE-PEG, DMG-PEG, or DTA-PEG PEG resulted in an increase in LNP size from 70 nm to 90-100 nm (FIG. 26B), while encapsulation efficiency remained above 80% for all LNP formulations (FIG. 26C). Like DMPE-PEG LNPs, DSPE-PEG LNPs had a neutral surface zeta potential, while DMG-PEG and DTA-PEG PEG LNPs had a positive surface zeta potential between 5-7 mV (FIG. 26D). In HepG2-GFP cells, DSPE-PEG LNPs mediated 3-fold less GFP knockout relative to DMPE-PEG LNPs, while DMG-PEG LNPs and DTA-PEG LNPs significantly enhanced gene editing efficacy (FIG. 26E). None of the lipid-PEG analogs tested were cytotoxic (FIG.

[0782] 26F).

[0783] Given their linker chemistries, DMPE-PEG and DPSE-PEG can be classified as anionic lipid-PEGs, while DMG-PEG and DTA-PEG can be classified as neutral lipid-PEGs. In line with their charge properties, LNPs coated with DMG-PEG and DTA-PEG had more positive surface zeta potentials than DMPE-PEG and DSPE-PEG LNPs (FIG. 26D).

[0784] Interestingly, LNPs produced with neutral lipid-PEGs also facilitated higher levels of gene editing (FIG. 26E). This result may be attributed to less favorable charge interactions between anionic lipid-PEGs and large, highly anionic Cas9 mRNA and sgRNA cargo. Relative to DMPE-PEG LNPs, DSPE-PEG LNPs demonstrated poor gene editing efficacy (FIG. 26E). Although DSPE-PEG is less easily shed in vivo, allowing for extension of circulation time, longer lipid-PEG tails have been shown to decrease cellular uptake, supporting the observed decrease in LNP transfection efficacy. Thus, for hepatic gene editing applications, the selection of a shorter lipid-PEG (DMPE-PEG, DMG-PEG, DTA-PEG) in LNP formulations maximizes both cellular uptake and liver targeting due to rapid dissociation and replacement with an ApoE rich protein corona to facilitate effective liver targeting. However, for extrahepatic gene editing applications, selection of a lipid-PEG may need to be more nuanced, considering the competing contributions of this excipient to both in vivo biodistribution and transfection.

[0785] Example 13: Effect of excipient combinations on LNP-mediated gene editing in vitro and in vivo

[0786] Next, the effect of combining the top-performing lipid excipients in distinct LNP formulations was evaluated. All possible combinations of Cl 4-494 LNPs encapsulating SpCas9 mRNA and GFP sgRNA were formulated using the best two phospholipids (DOPE and DSPC), cholesterol analogs (stigmastanol and P-sitosterol), and lipid-PEGs (DMG-PEG and DTA-PEG) (Table 5).

[0787] Table 5. Exemplary LNPs of the disclosure

[0788]

[0789] phospholipids (DOPE, DSPC), cholesterols (stigmastanol, P-sitosterol), and lipid-PEGs (DMG-PEG, DTA-PEG). Formulations are labeled R1-R8. Base formulation is represented as RO.

[0790] The eight formulations (R1-R8) were screened against the base Cl 4-494 LNP formulation (RO) in HepG2-GFP cells. Six out of eight LNPs (Rl, R2, R3, R4, R6. R8) enhanced GFP knockout in HepG2-GFP cells relative to RO LNPs (FIG. 27 A). The top four LNPs (Rl, R3, R4, R8) resulted in greater than 2-fold gene editing at the GFP locus without cellular toxicity (FIG. 27B). Notably, Rl, R3, R4, and R8 LNPs all achieved higher levels of gene editing than any LNP formulation with a single lipid excipient changed (FIG. 24E, FIG.

[0791] 25E, and FIG. 26E).

[0792] To test the in vivo efficacy of the lead LNP formulations, the TTR gene was chosen as a representative target. The TTR gene is currently being investigated in clinical trials for patients with hereditary transthyretin amyloidosis (AFTR). Knockout of the TTR gene leads to decreased levels of misfolded TTR protein and thereby limits the buildup of pathogenic amyloid plaques. Rl, R3, R4, and R8 LNPs were reformulated to encapsulate SpCas9 mRNA and TTR sgRNA, and compared to the base LNP formulation (RO) and an FDA-approved LNP formulation (MC3) as controls. All LNPs were administered via tail vein injection in 8-week-old C57BL / 6 mice at a dose of 1 mg / kg total RNA (FIG. 27C). TTR protein levels were assessed before and after treatment via serum ELISA. Three out of four excipient-optimized LNPs (i.e., R3, R4, and R8) resulted in significantly greater reduction in serum TTR protein in comparison to the base RO LNP formulation (FIG. 27D). Treatment with R4 LNPs also resulted in greater TTR knockdown than the clinically-approved MC3 LNP formulation (FIG.

[0793] 27D). None of the LNPs that were tested in vivo led to elevations in hepatic enzymes, demonstrating safety of these delivery carriers (FIGs. 27E-27F). The livers of mice treated with PBS, RO LNPs, or R4 LNPs were subsequently harvested and digested to isolate genomic DNA. Next-generation sequencing analysis of these samples at the expected locus of genome editing within the TTR gene revealed that optimized R4 LNPs outperformed unoptimized RO LNPs by nearly 4-fold (FIG. 27G). Thus, the top-performing LNP formulation, which was optimized systematically for lipid excipients to enhance delivery of gene editing cargo, demonstrated strong potential for mRNA-based gene editing therapies in the liver.

[0794] Sequence Listing

[0795] SEQ ID NO: 1

[0796] 5' -GGGCGAGGAGCUGUUCACCG-3 '

[0797] SEQ ID NO: 2

[0798] 5' -UUACAGCCACGUCUACAGCA-3 '

[0799] SEQ ID NO: 3

[0800] 5' -CGGTTTACTCTGACCCATTTC-3 '

[0801] SEQ ID NO: 4

[0802] 5' -GGGCTTTCTACAAGCTTACC-3 '

[0803] Enumerated Embodiments

[0804] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0805] Embodiment 1: A compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:

[0806]

[0807] A is selected from the group consisting of ^ R2 and

[0808]

[0809] each occurrence of L1, if present, is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, and -(optionally substituted C2-C8 heteroarylenyl)-X-:

[0810] each occurrence of X, if present, is independently selected from the group consisting of -N(Rle)-, -[N(CH2)i-3N(Rle)(Rle)]-, -N(RA)-, and -O-;

[0811] Rla, Rlb, Rlc, Rld, and each occurrence of Rle, if present, are each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and -CH(R3a)(R3b), wherein

[0812] at least one of Rla, Rlb, and Rlc, if present, is -CH(R3a)(R3b),

[0813] one of Rlaand Rlbcan combine with R2to form an optionally substituted C2-C8 heterocycloalkyl, and

[0814] one of Rla, Rlb, Rlc, and Rldcan combine with one occurrence of L1to form an optionally substituted C2-C8 heterocycloalkyl;

[0815] R2is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C8 cycloalkyl. optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, andN(RA)(RB);

[0816] R3aand R3bare each independently selected from the group consisting of optionally substituted C1-C24 alky l, optionally substituted C2-C24 alkenyl, optionally substituted C2-C24 alkynyl, optionally substituted C1-C24 heteroalkyl, optionally substituted C2-C24 heteroalkenyl, optionally substituted C2-C24 heteroalkynyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C8 heteroaryl:

[0817] m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl.

[0818] Embodiment 2: The compound of Embodiment 1, wherein R2is selected from the group consisting of CH3, (CH2)CH3, (CH2)4ORA, (CH2)C(=O)ORA, (CH2)I-3N(RA)(RB), (CH2)i-3(optionally substituted pyrrolidinyl), (CH2)i-3(optionally substituted imidazolyl), (CH2)i-3(optionally substituted piperizinyl), (CH2)i-3(optionally substituted piperidinyl), (CH2)i-3(optionally substituted morpholinyl), optionally substituted cyclohexyl, and optionally substituted phenyl.

[0819] Embodiment 3: The compound of Embodiment 1 or 2, wherein the compound of formula (I) is selected from the group consisting of:

[0820]

[0821]

[0822] Embodiment 4: The compound of any one of Embodiments 1-3, wherein Rla, Rlb, Rlc, Rld, and each occurrence of Rle, if present, are each independently selected from the

[0823] group consisting

[0824]

[0825] Embodiment 5: The compound of any one of Embodiments 1-4, wherein each occurrence of R3aand R3bis independently selected from the group consisting of H, R4,

[0826]

[0827] , wherein:

[0828] each occurrence of R4is C1-C16 alkyl or C2-C16 alkenyl;

[0829] each occurrence of L2is independently selected from the group consisting of -CH2-, -O-, -C(=O)-, and -(optionally substituted phenylenyl)-;

[0830] each occurrence of n and o is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0831] Embodiment 6: The compound of Embodiment 5, wherein at least one of R3aand R?b

[0832] in each occurrence

[0833]

[0834] Embodiment 7: The compound of Embodiment 5 or 6, wherein at least one of the following applies:

[0835] (a) -(L2)n- is selected from the group consisting of

[0836]

[0837]

[0838] (b) -(L2)o- is selected from the group consisting of -O-, -C(=O)O-, and -OC(=O)-.

[0839] Embodiment 8: The compound of any one of Embodiments 5-7, wherein R4is selected from the group consisting of

[0840]

[0841]

[0842] occurrence of R3aand R’bis independently selected from the group consisting of:

[0843]

[0844]

[0845] Embodiment 10: The compound of any one of Embodiments 1-9, wherein A is ''N''

[0846] I

[0847] R2and one of the following applies:

[0848] (a) Rlaand Rlbare each independently CH(R?a)(R3b), both occurrences of R?aare H, and neither occurrence of R3bis H, optionally wherein both occurrences of R3bcomprise an optionally substituted C2-C24 alkynyl;

[0849] (b) Rlaand Rlbare each independently CH(R3a)(R3b), both occurrences of R3bare El, and neither occurrence of R3ais H, optionally wherein both occurrences of R?acomprise an optionally substituted C2-C24 alkynyl:

[0850] (c) Rlais H, methyl, or ethyl, and Rlbis CH(R3a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Ce-Cio aryl or optionally substituted Ci- C24 alkyl:

[0851] (d) Rlbis H, methyl, or ethyl, and Rlais CH(R3a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Ce-Cio aryl or optionally substituted Ci- C24 alkyl;

[0852] (e) Rlacombines with R2to form an optionally substituted C2-C8 heterocycloalkyl and Rlbis CH(R3a)(R3b, wherein neither R-anor R 'bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl. and one of R3aand R3bis optionally substituted Cs-Cio aryl or optionally substituted C1-C24 alkyl; and

[0853] (f) Rlbcombines with R2to form an optionally substituted C2-C8 heterocycloalkyl and Rlais CH(R3a)(R3b, wherein neither R?anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Cs-Cio aryl or optionally substituted C1-C24 alkyl.

[0854] Embodiment 11: The compound of any one of Embodiments 1-10, wherein the compound is selected from the group consisting of 31aA, 31aB, 31aC, 31aD, 31aE, 31aF, 31aG, 31aH, 31al, 31aJ, 31aK, 31aL, 31aM, 31aN, 31aO, 31aP, 31aQ, 31aR, 31bA, 31bB, 31bC, 31bD. 31bE, 31bF, 31bG. 31bH, 3 Ibl, 31bJ, 31bK, 31bL, 31bM, 31bN, 31bO, 31bP, 31bQ, 31bR, 31cA, 31cB, 31cC, 31cD, 31cE, 31cF, 31cG, 31cH, 31cl, 31cJ, 31cK, 31cL, 31cM, 31cN, 31cO, 31cP, 31cQ, 31cR, 31dA, 31dB, 31dC, 31dD, 31dE, 31dF, 31dG, 31dH, 31dl, 31dJ, 31dK, 31dL, 31dM, 31dN, 31dO, 31dP, 31dQ, 31dR, 31eA, 31eB, 31eC, 31eD, 31eE, 31eF, 31eG, 31eH, 31el, 31eJ, 31eK, 31eL. 31eM, 31eN. 31eO, 31eP, 31eQ, 31eR, 31fA, 31fB, 31fC, 31fD, 31fE, 3 HF, 31fG, 31fH, 3 Iff, 31fJ, 31fK. 31fL. 31fM, 31fN, 31fO.

[0855] 3 IfP, 31fQ, 31fR, 31gA, 31gB, 31gC, 31gD, 31gE, 31gF, 31gG, 31gH, 31gl, 31gJ, 31gK, 31gL, 31gM, 31gN, 31gO, 31gP, 31gQ, 31gR, 31hA, 31hB, 31hC, 31hD, 31hE, 31hF, 31hG, 31hH, 31hl, 31hJ, 31hK. 31hL, 31hM, 31hN, 31110, 31hP, 31hQ, 31hR, 31iA, 31iB, 31iC, 3HD, 3 HE, 3HF, 31iG, 31iH, 3 HI, 3HJ, 3HK, 3HL, 3HM. 3HN, 31iO, 3 liP, 3HQ, 3HR, 11 (aA)2, 11 (aB)2, 11 (aC)2, 11 (aD)2, 11 (aE)2, 1 1 (aF)2, 11 (aG)2, 11 (aH)2, 1 1 (al)2, 11 (aJ)2, 11 (aK)2, 11 (aL)2, 11 (aM)2, 11 (aN)2, 11 (aO)2, 11 (aP)2, 11 (aQ)2, 11 (aR)2, 11 (b A)2, 11 (bB)2, 1 l(bC)2, H(bD)2, ll(bE)2, 1 l(bF)2, ll(bG)2, ll(bH)2, ll(bl)2, 1 l(bJ)2, ll(bK)2, ll(bL)2, ll(bM)2, ll(bN)2, ll(bO)2, ll(bP)2. U(bQ)2, ll(bR)2, 11(CA)2. 11(CB)2, 1 l(cC)2, H(cD)2.

[0856] 11(CE)2, 11(CF)2, 1 1(CG)2, 11(CH)2, ll(cl)2, ll(cJ)2, H(cK)2, H(cL)2, 11(CM)2, 11(CN)2, l l(cO)2, ll(cP)2, 11(CQ)2, 11(CR)2, H(dA)2, I l(dB)2, ll(dC)2, ll(dD)2, ll(dE)2, ll(dF)2, ll(dG)2, ll(dH)2, ll(dl)2, ll(dJ)2, ll(dK)2, ll(dL)2, ll(dM)2, ll(dN)2, ll(dO)2, ll(dP)2, U(dQ)2, H(dR)2, H(eA)2. ll(eB)2, ll(eC)2, H(eD)2, ll(eE)2, H(eF)2, ll(eG)2, ll(eH)2, 1 l(el)2. 1 l(eJ)2, l l(eK)2. l l(eL)2, l l(eM)2, l l(eN)2, l l(eO)2. 1 l(eP)2, H(eQ)2. l l(eR)2, 1 l(fA)2, 1 l(fB)2, l l(fC)2, 1 l(fD)2, l l(fE)2, H(fF)2, H(fG)2, ll(fH)2, 1 l(fl)2, 1 l(fj)2, 1 l(fk)2, 1 l(fL)2, ll(fM)2, ll(fN)2, 1 l(fO)2, 1 l(fP)2, H(fQ)2, H(fR)2, H(gA)2, ll(gB)2, ll(gC)2, ll(gD)2, ll(gE)2, 1 l(gF)2, ll(gG)2, H(gH)2, 1 l(gl)2, 1 l(gj)2, ll(gK)2, ll(gL)2, ll(gM)2, ll(gN)2, ll(gO)2, l l(gP)2. H(gQ)2, H(gR)2, H(hA)2, ll(hB)2, ll(hC)2, H(hD)2, 1 l(hE)2.

[0857] 1 l(hF)2, ll(hG)2, H(hH)2, l l(hl)2, ll(hJ)2, H(hK)2, H(hL)2, l l(hM)2, l l(hN)2, ll(hO)2, ll(hP)2, H(hQ)2, ll(hR)2, 11(IA)2, ll(iB)2, 1 l(iC)2, ll(iD)2, H(iE)2, ll(iF)2, ll(iG)2, 1 l(iH)2, ll(il)2, 1 l(iJ)2, H(iK)2, H(iL)2, ll(iM)2, 1 l(iN)2, 1 l(iO)2, 1 l(iP)2. U(iQ)2, and 11(IR)2.

[0858] Embodiment 12: The compound of any one of Embodiments 1-11, wherein the compound is:

[0859]

[0860] Rx

[0861] R2— N

[0862] stereoisomer, or isotopologue thereof:Roa(II), the method comprising: contacting a RY

[0863] R2“"NH_=_.L2^_R4 compound of formula (A): H (A), a compound of formula (B): ” (B),

[0864] O

[0865] 6

[0866] and a compound of formula (C):H R(C), in the presence of a copper catalyst; wherein:

[0867] R2is selected from the group consisting of optionally substituted Ci-Ce alkyl. optionally substituted C2-C6 alkenyl, optionally substituted -Cs cycloalkyl, optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);

[0868] Rxis selected from the group consisting of R5band optionally substituted Ci-Ce alkyl, or

[0869] Rxand R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyd;

[0870] RYis selected from the group consisting of H and optionally substituted Ci-Ce alkyl, or

[0871] R^ and R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyd;

[0872] R6

[0873] R3aand R5b, if present, are each independently

[0874]

[0875] ; R6is selected from the group consisting of R4and

[0876]

[0877] ;

[0878] each occurrence of R4is Ci-Cie alkyl or C2-C16 alkenyl;

[0879] each occurrence of L2is independently selected from the group consisting of -CH2-, -O-, -C(=O)-, and -(optionally substituted phenylenyl)-; and

[0880] each occurrence of n and o is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0881] Embodiment 14: The method of Embodiment 13, wherein the copper catalyst comprises CuCl, optionally wherein the copper catalyst has a concentration of about 10 mol%.

[0882] Embodiment 15: The method of Embodiment 13 or 14, wherein the contacting occurs at a temperature of about 50 °C.

[0883] Embodiment 16: The method of any one of Embodiments 13-15, wherein the contacting occurs for a period of about 48 h.

[0884] Embodiment 17: The method of any one of Embodiments 13-16, wherein at least one of the following applies:

[0885]

[0886] (b) -(L2)o- is selected from the group consisting of -O-, -C(=O)O-, and -OC(=O)-. Embodiment 18: The compound of any one of Embodiments 13-17, wherein R4is selected from the group consisting of:

[0887]

[0888] Embodiment 19: A lipid nanoparticle (LNP) comprising:

[0889] (a) at least one ionizable lipid comprising the compound of any one of Embodiments 1-12;

[0890] (b) at least one neutral lipid; (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof. Embodiment 20: The LNP of Embodiment 19, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 90 mol% of the LNP, optionally wherein the at least one ionizable lipid compound comprises about 40 mol% of the LNP.

[0891] Embodiment 21: The LNP of Embodiment 19 or 20, wherein the at least ionizable lipid compound comprises or consists essentially of:

[0892]

[0893] Embodiment 22: The LNP of any one of Embodiments 19-21, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 10 mol% of the LNP.

[0894] Embodiment 23: The LNP of any one of Embodiments 19-22, wherein the neutral lipid comprises or consists essentially of at least one neutral lipid selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC), optionally wherein the neutral lipid comprises or consists essentially of dioleoylphosphatidylethanolamine (DOPE).

[0895] Embodiment 24; The LNP of any one of Embodiments 19-23, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 75 mol% of the LNP, optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 48.8 mol% of the LNP.

[0896] Embodiment 25: The LNP of any one of Embodiments 19-24, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol.

[0897] Embodiment 26: The LNP of any one of Embodiments 19-25, wherein the at least one polymer-conjugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer-conjugated lipid comprises about 1.5 mol%.

[0898] Embodiment 27: The LNP of any one of Embodiments 19-26, wherein the at least one polymer-conjugated lipid comprises or consists essentially of l,2-dimyristoyl-.w-glycero-3 -phosphoethanolamine-jV- [methoxy (poly ethyleneglycol)-2000] (C14PEG2K). Embodiment 28: The LNP of any one of Embodiments 19-27, wherein the LNP has a molar ratio of (a): (b): (c): (d) of about 40: 10: 48.8: 1.5.

[0899] Embodiment 29: The LNP of any one of Embodiments 19-28, wherein the LNP further comprises at least one cargo molecule, optionally wherein the at least one cargo molecule comprises or consists of a therapeutic cargo molecule.

[0900] Embodiment 30: The LNP of Embodiment 29, 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.

[0901] Embodiment 31: The LNP of Embodiment 29 or 30, wherein the cargo molecule comprises a nucleic acid.

[0902] Embodiment 32: The LNP of Embodiment 31, wherein the nucleic acid is DNA or RNA.

[0903] Embodiment 33: The LNP of Embodiment 31 or 32, 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.

[0904] Embodiment 34: The LNP of Embodiment 33, wherein the mRNA comprises a receptor or antigen binding domain, optionally wherein the receptor or antigen binding domain comprises a SARS-CoV-2 spike protein.

[0905] Embodiment 35: The LNP of Embodiment 33, wherein the mRNA encodes an enzyme.

[0906] Embodiment 36: The LNP of Embodiment 33, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9.

[0907] Embodiment 37: The LNP of Embodiment 36, wherein the nucleic acid cargo further comprises sgRNA.

[0908] Embodiment 38: A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of Embodiments 19-37 and at least one pharmaceutically acceptable carrier.

[0909] Embodiment 39: A method of treating, preventing, and / or ameliorating a disease or infection in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of Embodiments 29-37.

[0910] Embodiment 40: The method of Embodiment 39, wherein the disease is at least one selected from the group consisting of cancer, cardiovascular disease, metabolic disease, or viral infection, optionally wherein the viral infection comprises COVID- 19. Embodiment 41: The method of Embodiment 39 or 40, wherein the LNP comprises at least one therapeutic cargo molecule.

[0911] Embodiment 42: The method of Embodiment 41, wherein the therapeutic 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.

[0912] Embodiment 43: The method of Embodiment 41 or 42, wherein the therapeutic cargo molecule comprises a nucleic acid.

[0913] Embodiment 44: The method of Embodiment 43, wherein the nucleic acid is DNA or RNA.

[0914] Embodiment 45: The method of Embodiment 43 or 44, 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.

[0915] Embodiment 46: The method of Embodiment 45, wherein the mRNA comprises a receptor or antigen binding domain, optionally wherein the receptor or antigen binding domain comprises a SARS-CoV-2 spike protein.

[0916] Embodiment 47: The method of Embodiment 45, wherein the mRNA encodes an enzyme.

[0917] Embodiment 48: The method of Embodiment 45, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9.

[0918] Embodiment 49: The method of Embodiment 48, wherein the nucleic acid cargo further comprises sgRNA.

[0919] Embodiment 50: The method of any one of Embodiments 39-49, wherein the subject is a mammal.

[0920] Embodiment 51: The method of Embodiment 50, wherein the mammal is a human. Embodiment 52: A lipid nanoparticle (LNP) comprising:

[0921] (a) at least one ionizable lipid compound of Formula (III), or a salt, solvate, stereoisomer, or isotopologue thereof:

[0922]

[0923] wherein: , R3a

[0924] — ('L^N

[0925] R1aand R1bare each independently R30;

[0926] R2a. R2b, R2C, R2d, R2e, R2f, R2g. and R2hare each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C10 heteroaryl;

[0927] each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H, -(optionally substituted Ci-Ce alkylenyl)-C(=O)OR4, -(optionally substituted C1-C6 alkylenyl)-C(=O)N(R4)(R5), -(optionally substituted C1-C6 alkylenyl)-C(=O)R4, -(optionally substituted Ci-Ce alkylenyl)-(R4), -C(=O)OR4, -C(=O)N(R4)(R5). -C(=O)R4, and R4,

[0928] wherein no more than one of each occurrence of R3a, R3b, and R3cis H;

[0929] R4is selected from the group consisting of optionally substituted C1-C28 alkyl, optionally substituted C2-C28 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C28 alkenyl, and optionally substituted C2-C28 alky nyl;

[0930] R3is selected from the group consisting of H and optionally substituted Ci-Ce alkyl; each occurrence of L is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalky lenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-, -X-(optionally substituted C2-C12 alkenylenyl)-, -X-(optionally substituted C1-C12 alkynylenyl)-, -X-(optionally substituted C1-C12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocyloalkylenyl;

[0931] each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3c)-, and -O-; and

[0932] each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4;

[0933] (b) at least one neutral lipid;

[0934] (c) at least one sterol;

[0935] (d) at least one polymer conjugated lipid; and (e) nucleic acid cargo comprising at least one messenger RNA (mRNA), wherein the at least one mRNA has a size ranging from about 2 kilobases to about 10 kilobases.

[0936] Embodiment 53: The LNP of Embodiment 52, wherein one of the following applies: (a) the at least one sterol comprises or consists essentially of cholesterol and the polymer conjugated lipid does not comprise or consist essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE); and

[0937] (b) the at least one polymer conjugated lipid comprises or consists essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE) and the at least one sterol does not comprise or consist essentially of cholesterol.

[0938] Embodiment 54: The LNP of Embodiment 52 or 53, wherein the ionizable lipid of Formula (III) is:

[0939]

[0940] l,l'-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2- hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-ol),

[0941] (C14-494)

[0942] Embodiment 55: The LNP of any one of Embodiments 52-54, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 60 mol% of the LNP. optionally wherein the at least one ionizable lipid compound comprises about 35 mol% of the LNP.

[0943] Embodiment 56: The LNP of any one of Embodiments 52-55, wherein the neutral lipid is at least one selected from the group consisting of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), optionally wherein the at least one neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0944] Embodiment 57: The LNP of any one of Embodiments 52-56, wherein the at least one neutral lipid comprises about 10 mol% to about 30 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 16 mol% of the LNP. Embodiment 58: The LNP of any one of Embodiments 52-57, wherein the sterol is at least one selected from the group consisting of cholesterol, 24-a-methyl-cholesterol (campesterol), 24-a-ethyl-cholestanol (stigmastanol), and 24-a-ethyl-cholesterol (B-sitosterol).

[0945] Embodiment 59: The LNP of any one of Embodiments 52-58, wherein the polymer conjugated lipid comprises at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-gly cero-3-phosphoethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-rac-glycerol (DMG), and N, N-ditetradecylacetamide (DTA), wherein the at least one lipid is covalently conjugated to a polyethylene glycol, optionally wherein the polyethylene glycol has a molecular weight ranging from about 100 kDa to about 10,000 kDa.

[0946] Embodiment 60: The LNP of any one of Embodiments 52-59, wherein the polymer conjugated lipid is at least one selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (poly ethylene glycol)-2000] (DSPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxy poly ethylene gly col-2000 (DMG-PEG), and methoxypoly ethyl eneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

[0947] Embodiment 61: The LNP of any one of Embodiments 52-60, wherein the at least one polymer conjugated lipid comprises about 0.1 mol% to about 10 mol% of the LNP, optionally wherein the at least one polymer conjugated lipid comprises about 2.5 mol% of the LNP.

[0948] Embodiment 62: The LNP of any one of Embodiments 52-61, wherein one of the following applies:

[0949] (a) the at least one neutral lipid comprises or consists essentially of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of 1,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG); (b) the at least one neutral lipid comprises or consists essentially of 1.2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG);

[0950] (c) the at least one neutral lipid comprises or consists essentially of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG); and

[0951] (d) the at least one neutral lipid comprises or consists essentially of 1.2-distearoyl- sn-glycero-3-phosphocholine (DSPC), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG);

[0952] Embodiment 63: The LNP of any one of Embodiments 52-62, wherein the LNP has a molar ratio of (a): (b): (c): (d) of about 35: 15: 46.5: 2.5.

[0953] Embodiment 64: The LNP of any one of Embodiments 52-63, wherein the LNP has a mass ratio of mRNA to sgRNA ranging from about 20: 1 to about 0.1:1, optionally wherein the LNP has a ratio of mRNA to sgRNA of about 4:1.

[0954] Embodiment 65: The LNP of any one of Embodiments 52-64, wherein the nucleic acid cargo (z.e., mRNA + sgRNA) has a concentration in the LNP ranging from about 1 ng / pL to about 500 ng / pL, optionally wherein the nucleic acid cargo (z.e., mRNA + sgRNA) has a concentration in the LNP of about 75 ng / pL.

[0955] Embodiment 66: The LNP of any one of Embodiments 52-65, wherein the nucleic acid cargo comprises mRNA encoding a base editor and a single guide RNA (sgRNA). Embodiment 67: The LNP of Embodiment 66, wherein the base editor is selected from the group consisting of a cytosine base editor and an adenine base editor.

[0956] Embodiment 68: The LNP of any one of Embodiments 52-67, wherein the mRNA encodes a clustered regularly interspaced short palindromic repeat (CRISPR) associated protein, optionally wherein the CRISPR associated protein is CRISPR associated protein 9 (Cas9), and optionally wherein the Cas9 is Streptococcus pyogenes Cas9 (SpCas9).

[0957] Embodiment 69: The LNP of any one of Embodiments 66-68, wherein the sgRNA is engineered to target a DNA sequence in a eukaryotic cell.

[0958] Embodiment 70: The LNP of Embodiment 69, wherein the DNA sequence in a eukaryotic cell comprises a gene implicated in a genetic disease or disorder, optionally wherein the genetic disease or disorder is a monogenic disease or disorder.

[0959] Embodiment 71: The LNP of Embodiment 70, wherein the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafness, Duchenne muscular dystrophy, familial hypercholesterolemia, Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease, and Tay-Sachs disease.

[0960] Embodiment 72: The LNP of any one of Embodiments 66-71, wherein the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

[0961] Embodiment 73: The LNP of any one of Embodiments 52-72, wherein the LNP is prepared by microfluidic mixing using a total flow rate (TFR) ranging from about 0.1 mL / min to about 4.0 mL / min, optionally wherein the LNP is prepared by microfluidic mixing using a total flow rate (TFR) of about 2.4 mL / min.

[0962] Embodiment 74: A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of Embodiments 52-73 and at least one pharmaceutically acceptable excipient.

[0963] Embodiment 75: A method of treating, preventing, and / or ameliorating a genetic disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of Embodiments 52-73 and / or the pharmaceutical composition of Embodiment 74.

[0964] Embodiment 76: The method of Embodiment 75, wherein the nucleic acid cargo comprises a mRNA encoding a base editor and a single guide RNA (sgRNA).

[0965] Embodiment 77: The method of Embodiment 75 or 76, wherein the genetic disease or disorder is a monogenic disease or disorder.

[0966] Embodiment 78: The method of Embodiment 77, wherein the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafness, Duchenne muscular dystrophy, familial hypercholesterolemia. Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease and Tay-Sachs disease.

[0967] Embodiment 79 provides the method of any one of Embodiments 76-78, wherein the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

[0968] Embodiment 80: A method of genome editing a mutated gene sequence associated with 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 52-73 and / or the pharmaceutical composition of Embodiment 74.

[0969] Embodiment 81: The method of Embodiment 80, wherein the nucleic acid cargo comprises a mRNA encoding a base editor and a single guide RNA (sgRNA). Embodiment 82: The method of Embodiment 81, wherein the sgRNA is targeted to a DNA sequence of the mutated gene sequence associated with the disease or disorder in the subject.

[0970] Embodiment 83: The method of any one of Embodiments 75-82, wherein the subject is a mammal.

[0971] Embodiment 84: The method of any one of Embodiments 75-83, wherein the subject is a human.

[0972] 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 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.

Claims

CLAIMSWhat is claimed is:

1. A compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:R1a— A — R1b(I),wherein:Ijl A is selected from the group consisting of R2andeach occurrence of L1, if present, is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroaiylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-. -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, and -(optionally substituted C2-C8 heteroarylenyl)-X-;each occurrence of X, if present, is independently selected from the group consisting of -N(Rle)-, -LN(CH2)i-3N(Rle)(Rle)J-, -N(RA)-, and -O-;Rla, Rlh, Rlc, Rld, and each occurrence of Rle, if present, are each independently selected from the group consisting of H, optionally substituted Ci-Ce alkyd, and -CH(R3a)(R3b), whereinat least one of Rla, Rlb, and Rlc, if present, is -CH(R3a)(R3b).one of Rlaand Rlbcan combine with R2to form an optionally substituted C2-C8 heterocycloalkyl, andone of Rla, Rlb, Rlc, and Rldcan combine with one occurrence of L1to form an optionally substituted C2-C8 heterocycloalkyl;R2is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C6 heteroalky l, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);R3aand R3bare each independently selected from the group consisting of optionallysubstituted C1-C24 alkyl, optionally substituted C2-C24 alkenyl, optionally substituted C2-C24 alkynyl, optionally substituted C1-C24 heteroalkyl, optionally substituted C2-C24 heteroalkenyl, optionally substituted C2-C24 heteroalkynyl, optionally substituted C'3-Cs cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted Ce-Cio aryl, and optionally substituted C2-C8 heteroaryl;m is 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10; andeach occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl.

2. The compound of claim 1, wherein R2is selected from the group consisting of CH3, (CH2)CH3, (CH2)4ORA, (CH2)C(=O)ORA. (CH2)I-3N(RA)(RB), (CH2)i-3(optionally substituted pyrrolidinyl), (CH2)i- (optionally substituted imidazolyl), (CH2)i- (optionally substituted piperizinyl), (CH2)i-3(optionally substituted piperidinyl), (CH2)i-s(optionally substituted morpholinyl), optionally substituted cyclohexyl, and optionally substituted phenyl.

3. The compound of claim 1 or 2, wherein the compound of formula (I) is selected from the group consisting of:

4. The compound of any one of claims 1-3. wherein Rla, Rlb, Rlc, Rld, and each occurrence of Rle, if present, are each independently selected from the group consisting of H,5. The compound of any one of claims 1-4, wherein each occurrence of Raaand R3bis.....=„.L2:R4 independently selected from the group consisting of H, R4, 'n, and, wherein:each occurrence of R4is C1-C16 alkyl or C2-C16 alkenyl;each occurrence of L2is independently selected from the group consisting of -CH2-, - O-, -C(=O)-, and -(optionally substituted phenylenyl)-;each occurrence of n and o is independently 0. 1, 2, 3. 4, 5, 6.

7. or 8.

6. The compound of claim 5, wherein at least one of R3aand R3bin each occurrence of7. The compound of claim 5 or 6, wherein at least one of the following applies:(a) -(L2)„- is selected from the group consisting of(b) -(L2)o- is selected from the group consisting of -O-, -C(=O)O-, and -OC(=O)-.

8. The compound of any one of claims 5-7, wherein R4is selected from the group9. The compound of any one of claims 5-8, wherein each occurrence of R3aand R3bis independently selected from the group consisting of:''NI.Z10. The compound of any one of claims 1-9. wherein A isr2and one of the following applies:(a) Rlaand Rlbare each independently CH(R3a)(R3b), both occurrences of R?aare H, and neither occurrence of R3bis H. optionally wherein both occurrences of R3bcomprise an optionally substituted C2-C24 alkynyl;(b) Rlaand Rlbare each independently CH(R3a)(R3b), both occurrences of R3bare H, and neither occurrence of R3ais H, optionally wherein both occurrences of R?acomprise an optionally substituted C2-C24 alkynyl:(c) Rlais H, methyl, or ethyl, and Rlbis CH(R3a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted C6-C10 aryl or optionally substituted Ci- C24 alkyl:(d) Rlbis H, methyl, or ethyl, and Rlais CH(R3a)(R3b), wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted C6-C10 aryl or optionally substituted Ci- C24 alkyl;(e) Rlacombines with R2to form an optionally substituted C2-C8 helerocycloalk I and Rlbis CH(R3a)(R3b, wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Ce-Cio aryl or optionally substituted C1-C24 alkyl; and(f) Rlbcombines with R2to form an optionally substituted C2-C8 heterocycloalkyl and Rlais CH(R3a)(R3b. wherein neither R3anor R3bis H, optionally wherein one of R3aand R3bis optionally substituted C2-C24 alkynyl, and one of R3aand R3bis optionally substituted Ce-Cio aryl or optionally substituted C1-C24 alkyl.

11. The compound of any one of claims 1-10, wherein the compound is selected from the group consisting of 31 aA, 31aB, 31aC, 31aD, 31aE, 31aF, 31aG, 31aH, 31al, 31aJ, 31aK, 31aL, 31aM, 31aN, 31aO, 31aP, 31aQ, 31aR, 31bA, 31bB, 31bC, 31bD, 31bE, 31bF, 31bG, 31bH, 31bl, 31bJ, 31bK, 31bL, 31bM, 31bN, 31bO, 31bP, 31bQ, 31bR, 31cA, 31cB, 31cC, 31cD, 31cE, 31cF, 31cG, 31cH, 31cl, 31cJ, 31cK, 31cL, 31cM. 31cN, 31cO. 31cP, 31cQ, 31cR, 31dA, 31dB, 31dC, 31dD, 31dE, 31dF. 31dG, 31dH. 3 Idl. 3 IdJ. 31dK, 31dL. 31dM, 31dN, 31dO, 31dP, 31dQ, 31dR, 31eA, 31eB, 31eC, 31eD, 31eE, 31eF, 31eG, 31eH, 31el, 31eJ, 31eK, 31eL, 31eM, 31eN, 31eO, 31eP, 31eQ, 31eR, 31fA, 31fB, 31fC, 31fD, 31fE, 31fF, 31fG, 31fH, 31fl, 31fJ, 31fK, 31fL, 31fM, 31fN, 31fO, 31fP, 31fQ, 31fR, 31gA, 31gB, 31gC, 31gD, 31gE, 31gF, 31gG. 31gH, 3 Igl, 31gJ, 31gK, 31gL, 31gM, 31gN, 31gO, 31gP, 31gQ, 31gR, 31hA, 31hB, 31hC, 31hD, 31hE, 31hF, 31hG, 31hH, 31hl, 31hJ, 31hK, 31hL,31hM, 31hN, 31hO, 31hP, 31hQ, 31hR, 31iA, 311B, 311C, 31iD, 31iE, 3 liF, 31iG, 31iH, 3 lil, 3 liJ, 31iK, 31iL, 31iM, 31iN, 31iO, 3 liP, 3 liQ, 31iR. ll(aA)2, ll(aB)2, ll(aC)2. ll(aD)2, H(aE)2, 1 l(aF)2, H(aG)2, H(aH)2, 1 l(al)2, 1 l(aJ)2, ll(aK)2, 1 l(aL)2, H(aM)2, ll(aN)2, ll(aO)2, ll(aP)2, H(aQ)2, ll(aR)2, ll(bA)2, ll(bB)2, ll(bC)2, 11 (bD)2, 11 (bE)2, 1 l(bF)2, ll(bG)2, ll(bH)2, ll(bl)2, ll(bJ)2, H(bK)2, ll(bL)2, ll(bM)2, ll(bN)2, ll(bO)2, ll(bP)2, H(bQ)2, ll(bR)2, 11(CA)2, 11(CB)2, 11(CC)2, 11(CD)2, 11(CE)2. 11(CF)2, 11(CG)2, 11(CH)2, l l(cl)2. 1 l(cJ)2. 11(CK)2, 11(CL)2, 11(CM)2, 11(CN)2. 11(CO)2, 11(CP)2, 11(CQ)2, 11(CR)2, H(dA)2, ll(dB)2, ll(dC)2, H(dD)2, 1 l(dE)2, 1 l(dF)2, H(dG)2, ll(dH)2, ll(dl)2, 1 l(dJ)2, ll(dK)2, ll(dL)2, ll(dM)2, ll(dN)2, ll(dO)2, 1 l(dP)2, 1 l(dQ)2, ll(dR)2, ll(eA)2, U(eB)2, ll(eC)2, ll(eD)2, ll(eE)2, H(eF)2, ll(eG)2, H(eH)2, 1 l(el)2, ll(eJ)2, H(eK)2, ll(eL)2, H(eM)2, H(eN)2. ll(eO)2, 1 l(eP)2, ll(eQ)2. H(eR)2, H(fA)2. ll(fB)2, 1 l(fC)2. 1 l(fD)2, ll(ffi)2, ll(fF)2, H(fG)2, 11(1H)2, ll(fl)2, ll(fJ)2, 11(1K)2, ll(fL)2, ll(fM)2, H(fN)2, ll(fO)2, ll(fP)2, ll(fQ)2, H(fR)2, H(gA)2, H(gB)2, ll(gC)2, H(gD)2, ll(gE)2, H(gF)2, ll(gG)2, ll(gH)2, 1 l(gl)2, ll(gj)2, ll(gK)2, ll(gL)2, ll(gM)2, ll(gN)2, ll(gO)2, ll(gP)2, U(gQ)2, H(gR)2, H(hA)2. ll(hB)2, 1 l(hC)2, H(hD)2, ll(hE)2, ll(hF)2, H(hG)2, ll(hH)2, 1 l(hl)2, 1 l(hJ)2, ll(hK)2, 1 l(hL)2. ll(hM)2. ll(hN)2, ll(hO)2, 1 l(hP)2. ll(hQ)2, H(hR)2, 1 l(iA)2, H(iB)2, 11(1C)2, H(iD)2, 11(IE)2, 11(IF)2, H(iG)2, ll(iH)2, 11 (il)2, 1 l(iJ)2, H(iK)2, 1 l(iL)2, ll(iM)2, 1 l(iN)2, 1 l(iO)2, 1 l(iP)2, ll(iQ)2, and 11(IR)2.

12. The compound of any one of claims 1-11, wherein the compound is:

13. A method for preparing a compound of formula (II), or a salt, stereoisomer, or isotopologue thereof:RxR2— NR5a(II).the method comprising:RYR2—Ncontacting a compound of formula (A): H (A),a compound of formulao-A6a compound of formula (C): H R (C),in the presence of a copper catalyst;wherein:R2is selected from the group consisting of optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Cs-Cs cycloalkyl, optionally substituted C2-C6 heteroalkyl, optionally substituted C3-C6 heteroalkenyl, optionally substituted C2-C8 heterocycloalkyl, and N(RA)(RB);Rxis selected from the group consisting of R5band optionally substituted Ci-Ce alkyl, orRxand R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 hctcrocycloalk l;RYis selected from the group consisting of H and optionally substituted Ci-Cs alkyl, orR^ and R2combine with the nitrogen atom to which they are bound to form an optionally substituted C2-C8 heterocycloalkyd;R3aand R5b, if present, are each independently;R6is selected from the group consisting of R4and;each occurrence of R4is C1-C16 alkyl or C2-C16 alkenyl;each occurrence of L2is independently selected from the group consisting of -CH2-, -O-, -C(=O)-, and -(optionally substituted phenylenyl)-; andeach occurrence of n and o is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

14. The method of claim 13, wherein the copper catalyst comprises CuCl, optionally wherein the copper catalyst has a concentration of about 10 mol%.

15. The method of claim 13 or 14, wherein the contacting occurs at a temperature of about 50 °C.

16. The method of any one of claims 13-15, wherein the contacting occurs for a period of about 48 h.

17. The method of any one of claims 13-16, wherein at least one of the following applies:(a) -(L2)„- is selected from the group consisting of(b) -(L2)o- is selected from the group consisting of -O-, -C(=O)O-, and -OC(=O)-.

18. The compound of any one of claims 13-17, wherein R4is selected from the group consisting of:

19. A lipid nanoparticle (LNP) comprising:(a) at least one ionizable lipid comprising the compound of any one of claims 1- 12;(b) at least one neutral lipid;(c) at least one cholesterol lipid and / or a modified derivative thereof; and(d) at least one polymer-conjugated lipid and / or a modified derivative thereof.

20. The LNP of claim 19, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 90 mol% of the LNP, optionally wherein the at least one ionizable lipid compound comprises about 40 mol% of the LNP.

21. The LNP of claim 19 or 20, wherein the at least ionizable lipid compound comprises or consists essentially of:

22. The LNP of any one of claims 19-21, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 10 mol% of the LNP.

23. The LNP of any one of claims 19-22, wherein the neutral lipid comprises or consists essentially of at least one neutral lipid selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholme (DOPC), optionally wherein the neutral lipid comprises or consists essentially of dioleoylphosphatidylethanolamine (DOPE).

24. The LNP of any one of claims 19-23, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 75 mol% of the LNP. optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 48.8 mol% of the LNP.

25. The LNP of any one of claims 19-24, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol.

26. The LNP of any one of claims 19-25, wherein the at least one polymer-conj ugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer-conj ugated lipid comprises about 1.5 mol%.

27. The LNP of any one of claims 19-26, wherein the at least one polymer-conj ugated lipid comprises or consists essentially of l,2-dimyristoyl-577-glycero-3-phosphoethanolamine- / V-[methoxy(polyethyleneglycol)-2000] (C 14PEG2K).

28. The LNP of any one of claims 19-27, wherein the LNP has a molar ratio of (a): (b):(c): (d) of about 40: 10: 48.8: 1.5.

29. The LNP of any one of claims 19-28, wherein the LNP further comprises at least one cargo molecule, optionally wherein the at least one cargo molecule comprises or consists of a therapeutic cargo molecule.

30. The LNP of claim 29, 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.

31. The LNP of claim 29 or 30, wherein the cargo molecule comprises a nucleic acid.

32. The LNP of claim 31, wherein the nucleic acid is DNA or RNA.

33. The LNP of claim 31 or 32, 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.

34. The LNP of claim 33, wherein the mRNA comprises a receptor or antigen binding domain, optionally wherein the receptor or antigen binding domain comprises a SARS-CoV-2 spike protein.

35. The LNP of claim 33, wherein the mRNA encodes an enzyme.

36. The LNP of claim 33, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9.

37. The LNP of claim 36, wherein the nucleic acid cargo further comprises sgRNA.

38. A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of claims 19-37 and at least one pharmaceutically acceptable carrier.

39. A method of treating, preventing, and / or ameliorating a disease or infection in asubject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 29-37.

40. The method of claim 39, wherein the disease is at least one selected from the group consisting of cancer, cardiovascular disease, metabolic disease, or viral infection, optionally wherein the viral infection comprises COVID- 19.

41. The method of claim 39 or 40, wherein the LNP comprises at least one therapeutic cargo molecule.

42. The method of claim 41, wherein the therapeutic 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.

43. The method of claim 41 or 42, wherein the therapeutic cargo molecule comprises a nucleic acid.

44. The method of claim 43, wherein the nucleic acid is DNA or RNA.

45. The method of claim 43 or 44. 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.

46. The method of claim 45, wherein the mRNA comprises a receptor or antigen binding domain, optionally wherein the receptor or antigen binding domain comprises a SARS-CoV-2 spike protein.

47. The method of claim 45, wherein the mRNA encodes an enzyme.

48. The method of claim 45, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9.

49. The method of claim 48, wherein the nucleic acid cargo further comprises sgRNA.

50. The method of any one of claims 39-49, wherein the subject is a mammal.

51. The method of claim 50, wherein the mammal is a human.

52. A lipid nanoparticle (LNP) comprising:(a) at least one ionizable lipid compound of Formula (III), or a salt, solvate, stereoisomer, or isotopologue thereof:wherein:Rlaand Rlbare each independently;R2a, R2b, R2C, R2d, R2e, R2f, R2g. and R2hare each independently selected from the group consisting of H, optionally substituted C1-C12 alky l, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C7-C13 aralkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl;each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H, -(optionally substituted Ci-Ce alkylenyl)-C(=O)OR4. -(optionally substituted C1-C6 alkylenyl)-C(=O)N(R4)(R3), -(optionally substituted Ci-Ce alkylenyl)-C(=O)R4, -(optionally substituted Ci-C6alkylenyl)-(R4), -C(=O)OR4, -C(=O)N(R4)(R5), -C(=O)R4, and R4,wherein no more than one of each occurrence of R3a, R3b, and R3cis H;R4is selected from the group consisting of optionally substituted C1-C28 alkyl, optionally substituted C2-C28 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C28 alkenyl, and optionally-substituted C2-C28 alkynyl;R5is selected from the group consisting of H and optionally substituted Ci-Ce alkyl; each occurrence of L is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-. -(optionally substituted C1-C12 heteroalkylenyl)-X-, -X-(optionally substituted C1-C12 alkylenyl)-, -X-(optionally substituted C2-C 12 alkenylenyl)-, -X-(optionally substituted C1-C12 alkynylenyl)-, -X-(optionally substituted C1-C12 heteroalkylenyl)-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocyloalkylenyl;each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3c)-, and -O-; andeach occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4;(b) at least one neutral lipid;(c) at least one sterol;(d) at least one polymer conjugated lipid; and(e) nucleic acid cargo comprising at least one messenger RNA (mRNA), wherein the at least one mRNA has a size ranging from about 2 kilobases to about 10 kilobases.

53. The LNP of claim 52, wherein one of the following applies:(a) the at least one sterol comprises or consists essentially of cholesterol and the polymer conjugated lipid does not comprise or consist essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE); and(b) the at least one polymer conjugated lipid comprises or consists essentially of a polyethylene glycol (PEG)-substituted l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE) and the at least one sterol does not comprise or consist essentially of cholesterol.

54. The LNP of claim 52 or 53, wherein the ionizable lipid of Formula (III) is:l, T-((2-(2-(4-(2-((2-(2-(bis(2-hydroxytetradecyl)amino)ethoxy)ethyl)(2-hydroxytetradecyl)amino)ethyl)piperazin-l-yl)ethoxy)ethyl)azanediyl)bis(tetradecan-2-ol),(C14-494)55. The LNP of any one of claims 52-54, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 60 mol% of the LNP, optionally wherein the at least one ionizable lipid compound comprises about 35 mol% of the LNP.

56. The LNP of any one of claims 52-55, wherein the neutral lipid is at least one selected from the group consisting of l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 -stearoy l-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), optionally wherein the at least one neutral lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

57. The LNP of any one of claims 52-56, wherein the at least one neutral lipid comprises about 10 mol% to about 30 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 16 mol% of the LNP.

58. The LNP of any one of claims 52-57, wherein the sterol is at least one selected from the group consisting of cholesterol, 24-a-methyl-cholesterol (campesterol). 24-a-ethyl-cholestanol (stigmastanol), and 24-a-ethyl-cholesterol (B-sitosterol).

59. The LNP of any one of claims 52-58, wherein the polymer conjugated lipid comprises at least one lipid selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-rac-glycerol (DMG), and N, N-ditetradecylacetamide (DTA), wherein the at least one lipid is covalently conjugated to a polyethylene glycol, optionally wherein the polyethylene glycol has a molecular weight ranging from about 100 kDa to about 10,000 kDa.

60. The LNP of any one of claims 52-59, wherein the polymer conjugated lipid is at least one selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG), 1.2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG),l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG), and methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA-PEG).

61. The LNP of any one of claims 52-60, wherein the at least one polymer conjugated lipid comprises about 0.1 mol% to about 10 mol% of the LNP, optionally wherein the at least one polymer conjugated lipid comprises about 2.5 mol% of the LNP.

62. The LNP of any one of claims 52-61, wherein one of the following applies:(a) the at least one neutral lipid comprises or consists essentially of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of 1,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG); (b) the at least one neutral lipid comprises or consists essentially of 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholestanol (stigmastanol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG);(c) the at least one neutral lipid comprises or consists essentially of 1.2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG); and(d) the at least one neutral lipid comprises or consists essentially of 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), the at least one sterol comprises or consists essentially of 24-a-ethyl-cholesterol (B-sitosterol), and the at least one polymer conjugated lipid comprises or consists essentially of methoxypolyethyleneglycoloxy(2000)-N, N-ditetradecylacetamide (DTA- PEG);63. The LNP of any one of claims 52-62, wherein the LNP has a molar ratio of (a): (b): (c): (d) of about 35: 15: 46.5: 2.5.

64. The LNP of any one of claims 52-63, wherein the LNP has a mass ratio of mRNA to sgRNA ranging from about 20: 1 to about 0.1:1, optionally wherein the LNP has a ratio of mRNA to sgRNA of about 4: 1.

65. The LNP of any one of claims 52-64, wherein the nucleic acid cargo (i.e., mRNA + sgRNA) has a concentration in the LNP ranging from about 1 ng / pL to about 500 ng / pL. optionally wherein the nucleic acid cargo (i.e., mRNA + sgRNA) has a concentration in the LNP of about 75 ng / pL.

66. The LNP of any one of claims 52-65, wherein the nucleic acid cargo comprises mRNA encoding a base editor and a single guide RNA (sgRNA).

67. The LNP of claim 66, wherein the base editor is selected from the group consisting of a cytosine base editor and an adenine base editor.

68. The LNP of any one of claims 52-67, wherein the mRNA encodes a clustered regularly interspaced short palindromic repeat (CRISPR) associated protein, optionally wherein the CRISPR associated protein is CRISPR associated protein 9 (Cas9), and optionally wherein the Cas9 is Streptococcus pyogenes Cas9 (SpCas9).

69. The LNP of any one of claims 66-68, wherein the sgRNA is engineered to target a DNA sequence in a eukary otic cell.

70. The LNP of claim 69, wherein the DNA sequence in a eukaryotic cell comprises a gene implicated in a genetic disease or disorder, optionally wherein the genetic disease or disorder is a monogenic disease or disorder.

71. The LNP of claim 70, wherein the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafness, Duchenne muscular dystrophy, familial hypercholesterolemia, Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease, and Tay-Sachs disease.

72. The LNP of any one of claims 66-71, wherein the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

73. The LNP of any one of claims 52-72, wherein the LNP is prepared by microfluidic mixing using a total flow rate (TFR) ranging from about 0.1 mL / min to about 4.0 mL / min, optionally wherein the LNP is prepared by microfluidic mixing using a total flow rate (TFR) of about 2.4 mL / min.

74. A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of claims 52-73 and at least one pharmaceutically acceptable excipient.

75. A method of treating, preventing, and / or ameliorating a genetic disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 52-73 and / or the pharmaceutical composition of claim 74.

76. The method of claim 75, wherein the nucleic acid cargo comprises a mRNA encoding a base editor and a single guide RNA (sgRNA).

77. The method of claim 75 or 76, wherein the genetic disease or disorder is a monogenic disease or disorder.

78. The method of claim 77, wherein the monogenic disease or disorder is selected from the group consisting of transthyretin amyloidosis (ATTR), muscular dystrophy, cystic fibrosis, congenital deafness, Duchenne muscular dystrophy, familial hypercholesterolemia, Hemochromatosis, Neurofibromatosis type 1 (NF1), Sickle cell disease and Tay-Sachs disease.

79. The method of any one of claims 76-78, wherein the sgRNA is engineered to target a DNA sequence encoding transthyretin (TTR).

80. A method of genome editing a mutated gene sequence associated with 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 52-73 and / or the pharmaceutical composition of claim 74.

81. The method of claim 80, wherein the nucleic acid cargo comprises a mRNA encoding a base editor and a single guide RNA (sgRNA).

82. The method of claim 81, wherein the sgRNA is targeted to a DNA sequence of the mutated gene sequence associated with the disease or disorder in the subject.

83. The method of any one of claims 75-82, wherein the subject is a mammal.

84. The method of any one of claims 75-83, wherein the subject is a human.

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