Branched lipid compositions, lipid nanoparticles (LNPs) containing same, and methods of using same

Abstract

The present disclosure provides modified immune cells or their precursor cells comprising a chimeric antigen receptor and a biologically inert molecule bound to its surface. Also included are methods for preparing the modified immune cells and methods for treating toxicity associated with CAR treatment in a subject in need thereof, the methods comprising the modified immune cells. TIFF2025529217000215.tif67128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 373,793, filed August 29, 2022, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under TR002776 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing The XML file entitled "046483-7399WO1 - Sequence Listing.xml", created on August 24, 2023, and comprising 3.8 kilobytes, is incorporated herein by reference in its entirety. [Background technology]

[0004] background In recent years, messenger RNA (mRNA), a transient intermediate between genes and proteins, has emerged as a new and promising approach for therapeutic applications, including protein replacement therapy, vaccines, and gene editing.However, mRNA is rapidly degraded, and due to its large size and negative charge, it cannot easily pass through cell membranes.Therefore, the use of cargo (e.g., DNA, RNA, small molecules, and / or polypeptides), especially mRNA medicines, requires a safe, effective, and stable delivery system to prevent degradation and achieve cellular uptake and cellular functionality.

[0005] Thus, there is a need in the art for lipid nanoparticles (LNPs) suitable for targeted delivery of cargo, ionizable lipids for preparing the same, and methods for using the same. The present disclosure addresses such a need. Summary of the Invention

[0006] overview In one aspect, the present disclosure provides several branched, ionizable lipid (IL) compounds, methods for preparing them, lipid nanoparticles (LNPs) containing them, and methods for using them to deliver cargo (e.g., mRNA).

[0007] In one aspect, the present disclosure provides a compound of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: TIFF2025529217000002.tif22128 is provided, where R 1a , R 1b , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is defined elsewhere herein.

[0008] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising at least one ionizable lipid compound of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof. In some embodiments, the LNP comprises at least one neutral lipid. In some embodiments, the LNP comprises cholesterol. In some embodiments, the LNP comprises at least one complex lipid. In some embodiments, the LNP further comprises at least one cargo molecule.

[0009] In another aspect, the present disclosure provides a pharmaceutical composition comprising at least one LNP of the present disclosure and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present disclosure provides a method for treating, preventing, and / or ameliorating a disease in a subject. In some embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure.

[0011] In another aspect, the present disclosure provides a method for delivering a nucleic acid or therapeutic agent to the liver of a subject. In some embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure.

[0012] In another aspect, the present disclosure provides a method for preparing modified immune cells or progenitor cells thereof. In some embodiments, the method includes contacting immune cells or progenitor cells thereof with at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure. [Brief explanation of the drawings]

[0013] The drawings illustrate generally, by way of example, and not by way of limitation, various aspects of the present application. [Figure 1] 1 shows a non-limiting embodiment of the synthesis of an epoxide intermediate used in preparing the ionizable lipid compositions of the present disclosure. [Figure 2A] 2A-2B show non-limiting embodiments of the synthesis of ionizable lipid compositions of the present disclosure by SN2 addition of an exemplary polyamine core to an exemplary epoxide substrate. [Figure 2B] See legend to Figure 2A. [Figure 3A]Figures 3A-3H: In vitro and in vivo fluc mRNA delivery by LNPs, including exemplary linear and branched LNPs prepared from polyamine core 494. Figures 3A-3B: LNPs containing core 494 IL and encapsulating fluc mRNA were first incubated with HeLa cells at 20 ng of mRNA per 20,000 cells. After 24 hours, luminescence (Figure 3A) and cell viability (Figure 3B) were assessed. Normalized luciferase expression was averaged over n = 4 technical replicates, and is expressed as the mean ± SEM of n = 3 biological replicates. Percent cell viability was normalized to untreated cells, and is expressed as the mean ± SEM of n = 3 biological replicates, and is expressed as the mean ± SEM of n = 3 biological replicates. Figures 3C-3F: C57BL / 6J mice were intravenously injected with LNPs at 0.1 mg / kg mRNA. After 12 hours, whole-body luminescence (Figure 3C) and images (Figure 3D) were acquired by IVIS. Mice were then sacrificed, and organ luminescence (Figure 3E) and images (Figure 3F) were collected by IVIS. Total luminescence is expressed as the mean ± SEM of n = 3. Two-way ANOVA with Student's post-hoc t-test with Holm-Sidak correction for multiple comparisons was used to compare normalized luciferase expression between branching groups and linker lengths (Figure 3A). One-way ANOVA with Student's post-hoc t-test with Holm-Sidak correction for multiple comparisons was used to compare either cell viability between treatment groups relative to the untreated group (Figure 3C) or total luminescence between treatment groups relative to C12-200 (Figures 3C and 3E). *p ≦ 0.05, **p ≦ 0.01, ***p ≦ 0.001, ****p ≦ 0.0001. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F]See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 4A] Figures 4A-4E: In vitro and in vivo fluc mRNA delivery by LNPs containing exemplary linear and branched LNPs prepared from a polyamine core 200. Figures 4A-4B: Exemplary linear and branched LNPs containing core 200 IL and encapsulating fluc mRNA were first incubated with HeLa cells at 20 ng of mRNA per 20,000 cells. After 24 hours, luminescence (Figure 4A) and cell viability (Figure 4B) were assessed. Normalized luciferase expression was averaged over n = 4 technical replicates, and is expressed as the mean ± SEM of n = 3 biological replicates. Percent cell viability was normalized to untreated cells, and is expressed as the mean ± SEM of n = 3 biological replicates, and is expressed as the mean ± SEM of n = 3 technical replicates. Figures 4C-4E: C57BL / J mice were intravenously injected with LNPs at 0.1 mg / kg mRNA. After 12 hours, whole-body luminescence was acquired by IVIS (Figure 4C). Mice were then sacrificed, and organ luminescence (Figure 4D) and images (Figure 4E) were collected by IVIS. Total luminescence is expressed as the mean ± SEM of n = 3. Two-way ANOVA with Student's post-hoc t-test with Holm-Sidak correction for multiple comparisons was used to compare normalized luciferase expression between branching groups and linker lengths (Figure 4A). One-way ANOVA with Student's post-hoc t-test with Holm-Sidak correction for multiple comparisons was used to compare either cell viability between treatment groups relative to the untreated group (Figure 4B) or total luminescence between treatment groups relative to C12-200 (Figure 4C-D). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. [Figure 4B] See legend to Figure 4A. [Figure 4C]See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 5A] 5A-5D provide bar graphs depicting luminescence data for combined organs (i.e., liver, spleen, lung, kidney, and heart) of Black 6 mice administered exemplary LNPs of the present disclosure, including LNPs containing TriLink luciferase mRNA encapsulated therein, including LNPs containing ionizable lipids prepared with polyamine core 494 (FIG. 5A) and LNPs containing ionizable lipids prepared with polyamine core 200 (FIG. 5B), compared to selected controls (e.g., LNPs containing ionizable lipids C8, C12, and / or MC3). FIGS. 5C-5D provide enlarged images of the data shown in FIGS. 5A-5B, respectively, thereby showing data for organs other than the liver (i.e., heart, kidney, lung, and spleen). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A] Figures 6A-6C: Eight exemplary LNPs containing ionizable lipids prepared from a polyamine core 200 were analyzed for TTR knockdown in the liver. LNPs were reconstituted with Cas9 mRNA and TTR single-stranded guide RNA (sgRNA). Mice were injected with the combined RNA at a dose of 1.0 mg per kg body weight (Figure 6A). Seven days later, blood was collected, and serum TTR levels were measured by ELISA compared to pre-injection serum (Figure 6B). In addition, livers were harvested, and indels were measured by next-generation sequencing. Overall, branched LNPs appear to significantly enhance Cas9-mediated gene editing (Figure 6C). [Figure 6B]See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7A] Figures 7A-7N: Analysis of the physicochemical properties, shape, and stability of several exemplary LNPs, including unbranched and branched IL-containing LNPs. The in vivo liver luminescence of eight exemplary LNPs, including linear and branched IL-containing LNPs prepared from polyamine core 200, was plotted and fitted using third-order least-squares regression to the hydrodynamic diameter (Figure 7A), PDI (Figure 7B), zeta potential (Figure 7C), pKa (Figure 7D), encapsulation efficiency (Figure 7E), and HeLa cell luminescence (Figure 7F). Figures 7G-7J: Cryo-TEM images of C8-200 (Figure 7G), E4i-200 (Figure 7H), E4t-200 (Figure 7I), and E4s-200 (Figure 7J). Figures 7K-7N: The stability of eight exemplary LNPs containing ILs prepared from polyamine core 200 was assessed by measuring the hydrodynamic diameter and PDI every hour for 24 hours immediately after incubation at 37°C in 1X PBS (Figures 7K-7L) or DMEM supplemented with 10% FBS (Figures 7M-7N). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 7G] See legend to Figure 7A. [Figure 7H] See legend to Figure 7A. [Figure 7I] See legend to Figure 7A. [Figure 7J] See legend to Figure 7A. [Figure 7K] See legend to Figure 7A. [Figure 7L] See legend to Figure 7A. [Figure 7M] See legend to Figure 7A. [Figure 7N] See legend to Figure 7A. [Figure 8A] Figures 8A-8G: Exemplary data for branched IL-containing LNPs and linear IL-containing LNPs in the liver and in artificial endosomes. C57BL / 6J mice were intravenously injected with 1.0 mg of clodronate. 24 hours later, the mice were intravenously injected again with eight exemplary LNPs containing IL prepared from polyamine core 200, which further contained fluc mRNA at a dose of 0.1 mg / kg. Figure 8A: After a total of 36 hours, the mice were sacrificed and dissected, and the luminescence of the liver was measured using IVIS. Total luminous flux is expressed as the mean ± SEM of n = 3. Apolipoprotein E (APOE) knockout mice were intravenously injected with four exemplary LNPs containing fluc mRNA at a dose of 0.1 mg / kg (Figure 8B). After 12 hours, the mice were sacrificed and dissected, and the luminescence of the liver was measured using IVIS. Total luminous flux is expressed as the mean ± SEM of n = 3. Figures 8C-8D: C57BL / 6J mice were injected with eight types of Core 200 LNPs containing 1 mol% DiR. 12 hours later, the mice were sacrificed, and organ fluorescence (Figure 8C) and images (Figure 8D) were collected. Total radiant efficiency is expressed as the mean ± SEM of n = 3. Figures 8F-8G: Eight exemplary representative LNPs were mixed with artificial endosomes containing FRET pairs. The percentage of endosome disruption upon mixing was measured by examining the increase in donor fluorescence, and the measurements were performed at several time points. Compared to linear LNPs, branched LNPs significantly increased endosome disruption (molar ratio of endosomes: DOPS (25%), DOPC (25%), DOPE (48%), NBD-PE (1%), and Rho-PE (1%)). [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 9A] 9A-9B: Exemplary toxicity data (FIG. 9A) and exemplary luciferase expression data (FIG. 9B) in CAL-27 cells treated with several exemplary LNPs of the present disclosure, including luciferase mRNA. LNPs were incubated with luciferase mRNA and applied to cells at a dose of 20 ng of mRNA per 20,000 cells. After 24 hours, luciferase assays and cell viability assays were performed to analyze luciferase expression and toxicity, respectively. [Figure 9B] See legend to Figure 9A. [Figure 10A] 10A-10B: Exemplary tumor luminescence data using a CAL-27 tumor-induced mouse model administered several exemplary LNPs of the present disclosure, including luciferase-containing LNPs. One million CAL-27 cells were inoculated into the right flank of Nu / J mice, and after tumor growth for two weeks, five types of LNPs encapsulating luciferase mRNA were injected intratumorally at a dose of 0.1 mg / kg, including two types of branched LNPs and three types of linear LNPs, including C12-200. As a control, one group was injected with PBS. Six hours later, the mice were sacrificed, and major organs and tumors were imaged for luminescence. [Figure 10B] See legend to Figure 10A. [Figure 11-1]Figures 11A-11D: Exemplary viability data for several squamous cell carcinoma cell lines treated with exemplary LNPs of the present disclosure, including human p53 mRNA and / or luciferase mRNA. CAL-27 cells (Figures 11A-11B) and OECM-1 cells (Figures 11C-11D) were treated with LNPs E10i-494, which contained either human p53 mRNA or luciferase mRNA. Cells were treated for different time intervals, and overall viability was measured after 24 hours (Figures 11A and 11C) and 48 hours (Figures 11B and 11D) using a cytotoxicity assay. [Figure 11-2] See description of Figure 11-1. [Figure 12] 12A-12B show an initial assessment of the performance of exemplary LNPs of the present disclosure by measuring relative light production (FIG. 12A) and viability (FIG. 12B) in induced pluripotent stem cells (iPSC-SV20) after 24 hours, where the exemplary LNPs contain 20 ng of luciferase mRNA per 15,000 cells. [Figure 13] Figures 13A-13B show a dose-escalation study of exemplary LNPs of the present disclosure, comprising luciferase mRNA, in iPSC-SV20 cells, where luminescence (Figure 13A) and viability (Figure 13B) were measured against mRNA doses (e.g., 20 ng, 50 ng, 100 ng, 200 ng, 400 ng, and 600 ng per well). [Figure 14-1]Figures 14A-14H: Exemplary data demonstrating successful transfection of mCherry mRNA into induced pluripotent stem cells (iPSC-SV20) by applying exemplary LNPs of the present disclosure, which contain an mCherry mRNA cargo. A total of 90,000 iPSC-SV20 cells were plated in a 24-well plate and allowed to reach 50% confluence overnight. The cells were treated with various concentrations of exemplary LNPs of the present disclosure and harvested 24 hours later. Flow cytometry was performed to determine the percentage of successfully transfected cells (Figures 14A-14F). The cells were further imaged by fluorescence microscopy to visualize the mCherry signal (Figures 14G-14H). [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 15-1] Figures 15A-15H provide exemplary flow cytometry graphs of fluorescence-activated cell sorting (FACS) of induced pluripotent stem cells (iPSCs), which were transfected with mCherry mRNA by application of a non-limiting exemplary LNP (E4i-200) of the present disclosure, containing an mCherry mRNA cargo. A total of 50,000 iPSCs were plated in a 24-well plate overnight, after which 366 ng of mCherry mRNA encapsulated in E4i-200 LNP (Figures 15E-15H) or control LNP (Figures 15A-15D) was applied. Cells were harvested after 24 hours (Figures 15A and 15E), 48 hours (Figures 15B and 15F), 72 hours (Figures 15C and 15G), and 96 hours (Figures 15D and 15H). [Figure 15-2] See description of Figure 15-1. [Figure 15-3]See description of Figure 15-1. [Figure 15-4] See description of Figure 15-1. [Figure 16A] 16A-16B provide bar graphs depicting relative light output data (FIG. 16A) and viability data (FIG. 16B) of activated primary T cells (CD4+:CD8+ 1:1) from healthy human donors, compared to selected controls (e.g., LNPs containing ionizable lipids C8, C12, and / or MC3), where the cells were incubated for 24 hours with exemplary LNPs of the present disclosure, including LNPs containing IL prepared with polyamine core 494 and LNPs containing IL prepared with polyamine core 200, and the LNPs contain luciferase mRNA encapsulated therein (200 ng luciferase mRNA per 60,000 cells). [Figure 16B] See legend to Figure 16A. [Figure 17] 17A-17B provide bar graphs depicting relative light output data (FIG. 17A) and viability data (FIG. 17B) of activated primary T cells (1:1 CD4+:CD8+) from healthy human donors, which were incubated for 24 hours with exemplary LNPs of the present disclosure containing encapsulated luciferase mRNA (200 ng luciferase mRNA per 60,000 cells), and all LNPs formulated with ionizable lipid:DOPE:cholesterol:C14PEG 2000 in a ratio of 40:30:25:2.5. [Figure 18] 1 provides a bar graph showing relative luminescence data for NK-92MI cells (i.e., immortalized human NK cells that autologously express IL-2) incubated with some exemplary LNPs of the present disclosure over a 24-hour period compared to a control, wherein the LNPs comprise luciferase mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention Reference will now be made in detail to several aspects of the disclosed subject matter, some examples of which are illustrated in the accompanying drawings. While the disclosed subject matter is set forth in conjunction with numbered claims, it will be understood that the exemplary subject matter is not intended to limit the scope of the claims to the disclosed subject matter.

[0015] Throughout this specification, values ​​expressed in range format should be interpreted in a flexible manner, including not only the numerical values ​​explicitly set forth as the range endpoints, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly set forth. For example, a range of "about 0.1% to about 5%" or a range of "about 0.1% to 5%" should be interpreted not only to include only about 0.1% to about 5%, but also to include individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%, etc.) within the specified range. The term "about X to Y" has the same meaning as "about X to about Y" unless otherwise specified. Similarly, the term "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise specified.

[0016] As used herein, the terms "a," "an," or "the" are used to include one or more unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise specified. The phrases "at least one of A and B" or "at least one of A or B" have the same meaning as "either A or B, or A and B." Additionally, it is understood that any phrases or terms used herein that are not otherwise defined are for descriptive purposes only and not for limiting purposes. Any section headings used are intended to aid in the reading of this specification and should not be construed as limiting; information associated with a section heading may be found within or outside of that particular section. All publications, patents, and patent documents referenced herein are incorporated by reference in their entirety, just as if individually incorporated by reference.

[0017] In the methods described herein, operations may be performed in any order unless a temporal or operational order is explicitly specified. Furthermore, certain operations may be performed simultaneously unless the explicit claim language specifies that the operations be performed separately. For example, if a claimed operation of performing X and a claimed operation of performing Y can be performed simultaneously in a single operation, the resulting process is within the literal scope of the claimed process.

[0018] explanation Lipid nanoparticles (LNPs) have emerged as superior drug delivery vehicles for mRNA therapy. One of the key factors that led to the success of LNPs over previous lipid-based particles was the transition from permanently cationic lipids, which can induce severe toxicity, to ionizable lipids (ILs), which are cationic only under low pH conditions. Extensive efforts have been expended to optimize the structure of ILs, which contain an amine core complexed to a long lipid tail, because small molecule modulation can dramatically change the overall efficacy of the resulting LNPs.

[0019] In one aspect, the present disclosure relates to the design and evaluation of LNPs that contain ionizable lipids that contain terminally branched lipid tails.For the exemplary lipids described herein, a simplified and modular synthesis scheme has been developed, which allows for easy adjustment of length and terminal branching.Furthermore, the present disclosure describes the application of this synthesis scheme to prepare non-limiting ILs, utilizing several exemplary polyamine cores.

[0020] In one aspect, the present disclosure relates to the observation that compared with non-branched lipid, branched lipid significantly increases the transfection into liver, including in gene editing model.Furthermore, the present disclosure utilizes physicochemical evaluation and a number of experiments related to hepatocyte targeting to identify that branched lipid can more induce mRNA endosomal escape.

[0021] The present disclosure further provides non-limiting exemplary uses of the LNPs described herein that comprise branched ILs, including mRNA delivery for mRNA therapy, gene editing in the liver, stem cell reprogramming, and CAR T-cell therapy and / or CAR NK-cell therapy.

[0022] definition The term "about," as used herein, allows for a degree of variability in values ​​or ranges, such as within 10%, within 5%, or within 1% of a specified value or of the ends of a specified range, and includes the specified value or range itself.

[0023] The term "acyl," as used herein, refers to a group containing a carbonyl moiety, wherein the group is attached via the carbonyl carbon atom. The carbon atom of the carbonyl is bonded to a hydrogen forming a "formyl" group or to another carbon atom, which may be part of an alkyl group, an aryl group, an aralkyl group, a cycloalkyl group, a cycloalkylalkyl group, a heterocyclyl group, a heterocyclylalkyl group, a heteroaryl group, a heteroarylalkyl group, etc. The acyl group may contain 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. The acyl group may contain double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. The acyl group may also contain heteroatoms within the meaning herein. The nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups. When the group containing the carbon atom bonded to the carbonyl carbon atom contains a halogen, the group is called a "haloacyl" group. One example is a trifluoroacetyl group.

[0024] The term "adjuvant," as used herein, is defined as any molecule that enhances the antigen-specific adaptive immune response.

[0025] The term "alkenyl," as used herein, refers to straight- and branched-chain alkyl groups, as well as cyclic alkyl groups, as defined herein, that differ by the presence of at least one double bond between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or from 2 to about 20 carbon atoms, or from 2 to 12 carbon atoms, or in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to, vinyl, -CH=C=CCH2, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, among others.

[0026] The term "alkoxy," as used herein, refers to an alkyl group bonded to an oxygen atom and includes cycloalkyl groups, as 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 groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can contain from about 1 to about 12, from about 1 to about 20, or from about 1 to about 40 carbon atoms bonded to the oxygen atom, and may further contain double or triple bonds and may also contain heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group, provided that two adjacent atoms in the structure are so substituted.

[0027] The term "alkyl," as used herein, refers to straight-chain and branched alkyl groups, as well as cycloalkyl groups, having 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbon atoms, or, in some embodiments, 1 to 8 carbon atoms. Examples of straight-chain alkyl groups include those having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups, as well as other branched forms of alkyl. Representative substituted alkyl groups may be substituted one or more times with any of the groups enumerated herein, such as amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0028] The term "alkynyl," as used herein, refers to straight- and branched-chain alkyl groups that differ by the presence of at least one triple bond between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, from 2 to about 20 carbon atoms, or from 2 to 12 carbon atoms, or in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to, -C≡CH, -C≡C(CH), -C≡C(CHCH), -CHC≡CH, -CHC≡C(CHCH), and -CHC≡C(CHCH), among others.

[0029] The terms "alkylene" or "alkylenyl," as used herein, refer to a divalent saturated aliphatic radical (e.g., -CH-, -CHCH-, and -CHCHCH-, among others). In certain embodiments, the terms can refer to a moiety derived from an alkene by cleavage of a double bond, or to a moiety derived from an alkane by removal of two hydrogen atoms, either from the same carbon atom (e.g., -CH-) or from separate carbon atoms (e.g., -CHCH-). Similarly, the terms "heteroalkylenyl," "cycloalkylenyl," "heterocycloalkylenyl," and the like, as used herein, refer to a divalent radical of a moiety corresponding to a parent group (e.g., heteroalkyl, cycloalkyl, and / or heterocycloalkyl). A divalent radical has two vacant valencies at any position of the group. Thus, a divalent radical can form single bonds with two different atoms or two different groups, or it can form a double bond with one atom.

[0030] The term "amine," as used herein, refers to primary amines, secondary amines, and tertiary amines, e.g., having the formula N(group), where each group can independently be H or other than H, e.g., alkyl, aryl, etc. Amines include, but are not limited to, R-NH (e.g., alkylamines, arylamines, alkylarylamines); RNH where each R is independently selected (e.g., dialkylamines, diarylamines, aralkylamines, heterocyclylamines, etc.); and RN where each R is independently selected (e.g., trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, etc.). The term "amine," as used herein, also encompasses ammonium ions.

[0031] The term "amino group" as used herein includes -NH2, -NHR, -NR2, -NR3 +Substituents of the type -NR3, where each R is independently selected and cannot be protonated. + The terms "amino" and "alkylamino" refer to the respective protonated forms, except for "amino" and "alkylamino" which refer to the respective protonated forms. Thus, any compound substituted with an amino group can be considered an amine. Within the meaning herein, an "amino group" can be a primary amino group, a secondary amino group, a tertiary amino group, or a quaternary amino group. An "alkylamino" group includes monoalkylamino groups, dialkylamino groups, and trialkylamino groups.

[0032] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH.Such lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG) and other anionic modified groups that are attached to neutral lipids.

[0033] The term "aralkyl," as used herein, refers to an alkyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group, as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups, and also fused (cycloalkylaryl)alkyl groups, such as 4-ethyl-indanyl. An aralkenyl group is an alkenyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group, as defined herein.

[0034] The term "aryl," as used herein, refers to a cyclic aromatic hydrocarbon group that does 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 from about 6 to about 14 carbon atoms in the ring portion of the group. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono- or multiply substituted, such as, but not limited to, phenyl substituted at one or more of the 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or naphthyl substituted at one or more of the 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring.

[0035] The term "atm" as used herein refers to atmospheric pressure under standard conditions. Thus, 1 atm is an atmosphere of 101 kPa, 2 atm is an atmosphere of 202 kPa, and so on.

[0036] The term "cationic lipid" refers to any of a number of lipid species that retain a net positive charge at a selected pH, such as physiological pH (e.g., a pH of about 7.0). Cationic lipids that contain alkyl chains with multiple unsaturated sites, such as at least two or three unsaturated sites, have been found to be particularly useful for creating lipid particles with increased membrane fluidity. Many cationic lipids and related analogues that are also useful in the present disclosure are described in US Patent Application Publication No. 20060083780 and US Patent Application Publication No. 20060240554; US Patent No. 5,208,036; US Patent No. 5,264,618; US Patent No. 5,279,833; US Patent No. 5,283,185; US Patent No. 5,753,613; and US Patent No. 5,785,992; and PCT Publication WO 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes.Non-limiting examples of cationic lipids are described in detail herein.In some examples, cationic lipids have a head group of protonatable tertiary amine (for example, pH-titrating one), C 18 These lipids contain an alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Examples of such lipids include DSDMA, DLinDMA, DLenDMA, and DODMA.

[0037] The term "cycloalkyl," as used herein, refers to cyclic alkyl groups, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, cycloalkyl groups can have from 3 to about 8-12 ring members, while in other embodiments, they can have a number ranging from 3 to 4, 5, 6, or 7 ring carbon atoms. Cycloalkyl groups further include polycyclic cycloalkyl groups, such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and further include fused rings, such as, but not limited to, decalinyl. Cycloalkyl groups also include rings substituted with straight-chain or branched-chain alkyl groups, as defined herein. Representative substituted cycloalkyl groups may be mono- or multiply substituted, such as, but not limited to, 2,2-disubstituted, 2,3-disubstituted, 2,4-disubstituted, 2,5-disubstituted, or 2,6-disubstituted cyclohexyl groups, or mono-, di-, or tri-substituted norbornyl or cycloheptyl groups, which may be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl," alone or in combination, refers to a cyclic alkenyl group.

[0038] The terms "epoxy-functional" or "epoxy-substituted" as used herein refer to a functional group in which the oxygen atoms of the epoxy substituents are directly bonded to two adjacent carbon atoms in a carbon chain or ring system. Examples of epoxy-substituted functional groups include, but are not limited to, 2,3-epoxypropyl, 3,4-epoxybutyl, 4,5-epoxypentyl, 2,3-epoxypropoxy, epoxypropoxypropyl, 2-glycidoxyethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2-(glycidoxycarbonyl)propyl, 3-(3,4-epoxycyclohexyl)propyl, 2-(3,4-epoxycyclohexyl)ethyl, 2-(2,3-epoxycyclopentyl)ethyl, 2-(4-methyl-3,4-epoxycyclohexyl)propyl, 2-(3,4-epoxy-3-methylcyclohexyl)-2-methylethyl, and 5,6-epoxyhexyl.

[0039] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which, if the disease is not ameliorated, the animal's well-being continues to deteriorate.

[0040] In contrast, a "disorder" in an animal is a state of health in an animal that allows the animal to maintain homeostasis, but in which the animal's state of health is less favorable than would occur in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health.

[0041] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

[0042] As used herein, the terms "effective amount," "pharmacologically effective amount," and "therapeutically effective amount" refer to a non-toxic amount of an agent sufficient to produce a desired biological result. The result may be a reduction and / or alleviation of signs, symptoms, or causes of a disease, or any other desired change in a biological system. Those skilled in the art will be able to determine the appropriate therapeutic amount in any individual case using routine experimentation.

[0043] In particular, in the case of mRNA, an "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid with respect to the mRNA is an amount sufficient to produce a desired effect, such as mRNA-directed expression of an amount of protein that causes a desired biological effect in the organism in which the protein is expressed. For example, in some embodiments, the expressed protein is an active form of a protein normally expressed in a certain cell type in the body, and a therapeutically effective amount of mRNA is an amount that produces an amount of the protein it encodes 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 protein normally expressed in that cell type in a healthy individual. For example, in some embodiments, the expressed protein is a protein normally expressed in a certain cell type in the body, and a therapeutically effective amount of mRNA is an amount that produces an expression level in an individual with abnormal expression of the protein (i.e., an individual deficient in the protein) similar to that observed in a healthy individual. Suitable assays for measuring mRNA or protein expression include, but are not limited to, dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays known to those skilled in the art.

[0044] The term "encode," as used herein, refers to the specification of a product (e.g., protein and RNA) by a given nucleotide sequence in a nucleic acid (i.e., DNA and / or RNA) upon transcription from DNA or translation from RNA. In certain embodiments, the term "encode" refers to an RNA sequence being specified by transcription of a DNA sequence. In certain embodiments, the term "encode" refers to an amino acid sequence (e.g., a polypeptide or protein) being specified by translation of an mRNA. In certain embodiments, the term "encode" refers to an amino acid sequence being specified by transcription of DNA into mRNA followed by translation of the mRNA encoded by the DNA sequence. In certain embodiments, an encoded product can include a direct transcript or a translation. In certain embodiments, an encoded product can include post-translational modifications that are understood or reasonably expected by one of skill in the art.

[0045] The term "fully encapsulated" means that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or nuclease or protease assay (which can significantly degrade free DNA, RNA, or protein). In a fully encapsulated system, in a procedure that normally degrades 100% of the free active agent or therapeutic agent, preferably, less than about 25% of the active agent or therapeutic agent in the particle is degraded, more preferably, less than about 10% of the active agent or therapeutic agent in the particle is degraded, 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, complete encapsulation can be determined by the OLIGREEN® assay. OLIGREEN® is an extremely sensitive nucleic acid fluorescent dye for quantifying oligonucleotides and single-stranded DNA or RNA in solution (commercially available from Invitrogen Corporation; Carlsbad, Calif.). "Fully encapsulated" also indicates that the lipid particle is stable in serum, i.e., when administered in vivo, the lipid particle does not break down into its constituent parts within a short period of time.

[0046] 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.

[0047] The term "haloalkyl" group, as used herein, includes monohaloalkyl and polyhaloalkyl groups, where all halo atoms may be the same or different, and also includes perhaloalkyl groups, where all hydrogen atoms have been replaced with halogen atoms, such as fluorine atoms. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.

[0048] The term "heteroaryl," as used herein, refers to an aromatic ring compound containing five or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S; for example, heteroaryl rings can have from five to about 8-12 ring members. Heteroaryl groups are a variety of heterocyclyl groups that have an aromatic electronic structure. A heteroaryl group referred to as a C2-heteroaryl can be a five-membered ring with two carbon atoms and three heteroatoms, a six-membered ring with two carbon atoms and four heteroatoms, and others. Similarly, a C4-heteroaryl can be a five-membered ring with one heteroatom, a six-membered ring with two heteroatoms, and others. The sum of the number of carbon atoms and the number of heteroatoms equals the total number of atoms in the ring. 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. Heteroaryl groups can be unsubstituted or substituted with groups as discussed herein. Representative substituted heteroaryl groups may be substituted one or more times, eg, with groups such as those enumerated herein.

[0049] Further examples of aryl and heteroaryl groups include 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, benzyl, benzo ... hydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl) , pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), ), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-Dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl) nyl), 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 zolyl, 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-dibenzo[b,f]azepine (5H-dibenzo[b,f]azepin-1-yl, 5H-dibenzo[b,f]azepin-2-yl, 5 H-dibenzo[b,f]azepin-3-yl, 5H-dibenzo[b,f]azepin-4-yl, 5H-dibenzo[b,f]azepin-5-yl), 10,11-dihydro-5H-dibenzo[b,f]azepine (10,11-dihydro-5H-dibenzo[b,f]azepin-1-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-2-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-4-yl, 10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl), etc.,

[0050] The term "heteroarylalkyl," as used herein, refers to an alkyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a heteroaryl group, as defined herein.

[0051] The term "heterocyclylalkyl," as used herein, refers to an alkyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group, as defined herein, is replaced with a bond to a heterocyclyl group, as defined herein. Representative heterocyclylalkyl groups include, but are not limited to, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, tetrahydrofuran-2-ylethyl, and indol-2-ylpropyl.

[0052] The term "heterocyclyl," as used herein, refers to aromatic and non-aromatic ring compounds containing three or more ring members, where one or more of the ring members is a heteroatom, such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl or heteroaryl, or, if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups contain from 3 to about 20 ring members, while other such groups have from 3 to about 15 ring members. A heterocyclyl group referred to as a C2-heterocyclyl can be a five-membered ring having two carbon atoms and three heteroatoms, a six-membered ring having two carbon atoms and four heteroatoms, and so forth. Similarly, a C4-heterocyclyl can be a five-membered ring having one heteroatom, a six-membered ring having 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 may also contain one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The term "heterocyclyl group" includes species that are fused rings, including those containing fused aromatic and non-aromatic groups. For example, both dioxolanyl and benzodioxolanyl ring systems (methylenedioxyphenyl ring systems) are heterocyclyl groups within the meaning herein. The term also includes polycyclic ring systems containing heteroatoms, such as, but not limited to, quinuclidyl. A heterocyclyl group may be unsubstituted or 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 may be mono- or multiply substituted, such as, but not limited to, piperidinyl or quinolinyl groups substituted at the 2-, 3-, 4-, 5-, or 6-position or disubstituted with groups such as those listed herein by way of example.

[0053] The terms "hydrocarbon" or "hydrocarbyl," as used herein, refer to molecules or functional groups that contain carbon and hydrogen atoms. These terms can also refer to molecules or functional groups that normally contain both carbon and hydrogen atoms, but in which all hydrogen atoms have been replaced with other functional groups.

[0054] As used herein, the term "hydrocarbyl" refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbyl group is defined as any of the following: (C a -C b)hydrocarbyl, where a and b are integers, meaning any number of carbon atoms from a to b. For example, (C1-C4)hydrocarbyl means a hydrocarbyl group that can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and can be any number of carbon atoms from (C0-C b ) Hydrocarbyl means that in some embodiments there are no hydrocarbyl groups present.

[0055] The term "independently selected from," as used herein, means that the referenced groups are the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, "X 1 , X 2 , and X 3 are independently selected from the noble gases, for example, X 1 , X 2 , and X 3 are all the same, X 1 , X 2 , and X 3 In the situation where all are different, X 1 and X 2 is the same but X 3 may include different situations, as well as other similar permutations.

[0056] The term "ionizable lipid," as used herein, refers to a lipid (e.g., a cationic lipid) that has at least one protonatable or deprotonatable group such that the lipid is positively charged at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH. Those skilled in the art will understand that, because the addition or removal of protons as a function of pH is an equilibrium process, reference to a charged or neutral lipid refers to the nature of the predominant species, and not all of the lipid must be present in a charged or neutral form. Generally, ionizable lipids have a pK of the protonatable group in the range of about 4 to about 7.a It has.

[0057] The term "local delivery," as used herein, refers to the direct delivery of an active agent or therapeutic agent, such as messenger RNA, to a target site in an organism. For example, an agent can be delivered locally by direct injection into a disease site, such as a tumor, or by direct injection into another target site, such as a site of inflammation, or by direct injection into a target organ, such as the liver, heart, pancreas, kidney, etc.

[0058] The term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, and characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0059] The term "lipid complex" refers to a complex lipid that inhibits lipid particle aggregation. Such lipid complexes include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid complexes), PEG-lipid complexes, such as PEG-coupled dialkyloxypropyl, PEG-coupled diacylglycerol, PEG-coupled cholesterol, PEG-coupled phosphatidylethanolamine, PEG-conjugated ceramide, etc. (e.g., U.S. Patent No. 5,885,613, the disclosure of which is incorporated herein by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG may be directly conjugated to lipids or may be linked to lipids via a linker moiety. Any linker moiety suitable for coupling PEG to lipids may be used, including, for example, linker moieties other than esters and linker moieties containing esters. In some preferred embodiments, linker moieties other than esters are used.

[0060] As used herein, "lipid-encapsulated" can refer to a lipid particle that provides complete encapsulation, partial encapsulation, or both, of an active agent or therapeutic agent, such as a nucleic acid (e.g., a protein cargo). In one preferred embodiment, the nucleic acid is completely encapsulated in the lipid particle (e.g., forming an SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle).

[0061] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., 1-1,000 nm), which comprises one or more lipids and / or additional agents.

[0062] The term " lipid particle " is used herein to refer to the lipid formulation that can be used to deliver active agent or therapeutic agent, such as nucleic acid (for example, mRNA, etc.) to target site of interest.The lipid particle of the present disclosure is typically formed by cationic lipid, non-cationic lipid and the complex lipid that inhibits particle aggregation, and in this lipid particle, active agent or therapeutic agent can be encapsulated in lipid, thereby protecting the active agent from enzymatic degradation.

[0063] The term "monovalent," as used herein, refers to a substituent being attached to the substituted molecule via a single bond. When substituents are monovalent, such as F or Cl, they are attached to the atom they replace by a single bond.

[0064] The term "neutral lipid" refers to any of a number of lipid species that exist in either an uncharged form or a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0065] The term "non-cationic lipid" refers to amphipathic lipids, as well as any other neutral or anionic lipid.

[0066] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides and either single-stranded or double-stranded, and includes DNA and RNA. DNA can be in the form of, for example, an antisense molecule, a plasmid DNA, a precondensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, a chromosomal DNA, or derivatives and combinations thereof. RNA can be in the form of siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), or combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or modified linkages, which are synthetic, natural, and non-natural, and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs).Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides and have similar binding properties to the reference nucleic acid.Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its explicitly specified sequence, as well as its conservatively modified variants (e.g., variants with degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences.Specifically, degenerate codon substitution can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mal. Cell. Probes, 8:91-98 (1994)).

[0067] As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides and longer fragments referred to as polynucleotides. In certain embodiments, oligonucleotides of the present disclosure are about 15 to about 60 nucleotides in length. Nucleic acids may be administered alone in lipid particles of the present disclosure, or may be administered in combination (e.g., co-administered) with lipid particles of the present disclosure that contain peptides, polypeptides, or small molecules, such as conventional drugs. In other embodiments, nucleic acids may be administered in viral vectors.

[0068] A "nucleotide" comprises a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked to each other via the phosphate group. "Base" includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs, and also synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications in which new reactive groups are placed, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0069] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., variants with degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, along with the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by creating 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)).

[0070] The term "organic group," as used herein, refers to any functional group containing carbon. Examples include oxygen-containing groups (e.g., alkoxy, aryloxy, aralkyloxy, and oxo(carbonyl) groups); carboxyl groups (including carboxylic acids, carboxylates, and carboxylic acid esters); sulfur-containing groups (e.g., alkylsulfide and arylsulfide groups); and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R), CN, CF, OCF, R, C(O), methylenedioxy, ethylenedioxy, N(R), SR, SOR, SO, SO, N(R), SO, R, C(O)R, C(O)C(O)R, C(O)CHC(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R), OC(O)N(R), C(S)N(R), (CH). 0-2 N(R)C(O)R, (CH2) 0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)S02R, N(R)S02N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1-C 100 ) hydrocarbyl, where R can be hydrogen (in instances containing other carbon atoms) or a carbon-based moiety, which can be substituted or unsubstituted.

[0071] The terms "patient," "subject," and "individual" are used interchangeably herein and refer to any animal or cell thereof, whether in vitro or in situ, that is amenable to the methods described herein. In non-limiting embodiments, the patient, subject, or individual is a human.

[0072] As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not interfere with the biological activity or properties of the compound and that is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0073] As used herein, the expression "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from a pharmaceutically acceptable non-toxic acid or base, including an inorganic acid or base, an organic acid or base, a solvate thereof, a hydrate thereof, or a clathrate thereof.

[0074] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic acids or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid (including sulfate and hydrogen sulfate ions), and phosphoric acid (including hydrogen phosphate and dihydrogen phosphate ions). Suitable organic acids can be selected from the aliphatic acid class, alicyclic acid class, aromatic acid class, araliphatic acid class, heterocyclic acid class, carboxylic acid class, and sulfonic acid class, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, malonic acid, saccharinic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, lactic ... These include benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, beta-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0075] Suitable pharmaceutically acceptable base addition salts of the compounds described herein include, for example, ammonium salts, and also include metal salts, including alkali metal salts, alkaline earth metal salts, and transition metal salts, such as calcium salts, magnesium salts, potassium salts, sodium salts, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts formed from basic amines, such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding compound, for example, by reacting the compound with an appropriate acid or base.

[0076] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, which is involved in carrying or transporting a compound described herein within or to a patient so that the compound can perform its intended function. Typically, such a compound is carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compounds described herein, and not harmful to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include sugars (such as lactose, glucose, and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (such as cocoa butter and suppository wax); oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); glycols (such as propylene glycol); polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol); esters (such as ethyl oleate and ethyl laurate); agar; buffers (such as magnesium hydroxide and aluminum hydroxide); surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, compatible substances used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any coatings, antibacterial and antifungal agents, absorption delaying agents, and the like that are compatible with the activity of the compounds described herein and are physiologically acceptable to the patient.Active auxiliary compounds may also be incorporated into the composition. "Pharmaceutically acceptable carriers" may further include pharmaceutically acceptable salts of the compounds described herein. Other additional ingredients that may be included in the pharmaceutical compositions used in conjunction with the methods or compounds described herein are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences" (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0077] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short and long chains, where short chains are commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0078] The term "polymer-conjugated lipid" refers to a molecule that contains both a lipid portion and a polymer portion. One example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that contains both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), DSPE-PEG-DBCO, DOPE-PEG-azide, DSPE-PEG-azide, DPPE-PEG-azide, DSPE-PEG-carboxy-NHS, DOPE-PEG-carboxylic acid, DSPE-PEG-carboxylic acid, etc.

[0079] The term "room temperature" as used herein refers to a temperature of about 15 to 28 degrees.

[0080] The term "solvent" as used herein refers to a liquid capable of dissolving a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0081] The term "substantially" as used herein refers to a majority or majority, such as 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," as used herein, can mean that a composition has no or only such an insignificant amount of a substance that the amount of the substance present does not affect important properties of the composition in which it is included, such as about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or an amount that is less than, equal to, or greater than about 4.5 wt%, 4 wt%, 3.5 wt%, 3 wt%, 2.5 wt%, 2 wt%, 1.5 wt%, 1 wt%, 0.9 wt%, 0.8 wt%, 0.7 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, 0.01 wt%, or about 0.001 wt%, or less. The term "substantially free" can mean that the composition has only an insignificant amount of the substance, such as about 0 wt% to about 5 wt%, or about 0 wt% to about 1 wt%, or less than or equal to, or greater than about 4.5 wt%, 4 wt%, 3.5 wt%, 3 wt%, 2.5 wt%, 2 wt%, 1.5 wt%, 1 wt%, 0.9 wt%, 0.8 wt%, 0.7 wt%, 0.6 wt%, 0.5 wt%, 0.4 wt%, 0.3 wt%, 0.2 wt%, 0.1 wt%, 0.01 wt%, or about 0.001 wt%, or less, or about 0 wt%.

[0082] The term "substituted," as used herein in conjunction with a molecule or organic group as defined herein, refers to a state in which one or more hydrogen atoms therein are replaced with one or more atoms other than hydrogen. The term "functional group" or "substituent," as used herein, refers to a group that can be substituted with a molecule or organic group, or a group that is substituted with a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogens (e.g., F, Cl, Br, and I); oxygen atoms in groups (e.g., hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups (including carboxylic acids, carboxylates, and carboxylic acid esters)); sulfur atoms in groups (e.g., thiol groups, alkylsulfide groups and arylsulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups); nitrogen atoms in groups (e.g., amines, hydroxylamine, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines); and other heteroatoms in various other groups. Non-limiting examples of substituents that can be attached to a substituted carbon atom (or other atom) include F, Cl, Br, I, OR, OC(O)N(R), CN, NO, NO, ONO, azido, CF, OCF, R, O(oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R), SR, SOR, SO, SO, N(R), SO, R, C(O)R, C(O)C(O)R, C(O)CHC(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R), OC(O)N(R), C(S)N(R), (CH) 0-2 N(R)C(O)R, (CH2) 0-2N(R)N(R), N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R), N(R)SOR, N(R)SON(R), N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R), N(R)C(S)N(R), N(COR)COR, N(OR)R, C(=NH)N(R), C(O)N(OR)R, and C(=NOR)R, where R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C-C 100 ) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or two R groups attached to or adjacent to a nitrogen atom may together with one or more of the nitrogen atoms form a heterocyclyl.

[0083] A "therapeutic" treatment is a treatment administered to a subject who exhibits symptoms of a pathological condition with the intent of reducing or eliminating those symptoms.

[0084] The term "therapeutic protein," as used herein, refers to a protein or peptide that, when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's abnormality or disease state. In certain embodiments, a therapeutic protein or peptide has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein or peptide can have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes the entire protein or peptide, and can also refer to therapeutically active fragments thereof. The term can also include therapeutically active variants of proteins. Exemplary therapeutic proteins include, but are not limited to, analgesic proteins, anti-inflammatory proteins, antiproliferative proteins, pro-apoptotic proteins, anti-angiogenic proteins, cytotoxic proteins, cytostatic proteins, cytokines, chemokines, growth factors, wound healing proteins, pharmaceutical proteins, or prodrug-activating proteins. Therapeutic proteins may include growth factors (e.g., EGF, TGF-α, TGF-β, TNF, HGF, IGF, and IL-1 through IL-8, among others), cytokines, paratopes, Fabs (antigen-binding fragments), and antibodies.

[0085] The terms "treat," "treating," and "treatment," as used herein, refer to a reduction in the frequency or severity of symptoms of a disease or disorder experienced by a subject resulting from the administration of an agent or compound to the subject.

[0086] lipid compounds In one aspect, the present disclosure provides an ionizable lipid compound of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: TIFF2025529217000003.tif22128, wherein: R 1a and R 1b are each independently TIFF2025529217000004.tif11128; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is H, optionally substituted C1-C 12 Alkyl, optionally substituted C-C 12 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 12 Alkenyl, optionally substituted C-C 12 Alkynyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, TIFF2025529217000005.tif18138; R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4d Each occurrence of, if present, is optionally substituted C1-C 12 independently selected from the group consisting of alkyl, halogen, CN, and NO2; R5 Each occurrence of is an optionally substituted C1-C3 alkyl, optionally substituted C3-C 12 Cycloalkyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C 12 Cycloalkyl, C2-C 12 Heterocycloalkyl, optionally substituted C-C 12 Aralkyl, optionally substituted C-C 12 Aryl, optionally substituted C-C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ) independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C-C 12 alkylenyl)-X-, -(optionally substituted C-C 12 alkenylenyl)-X-, -(optionally substituted C-C 12 alkynylenyl)-X-, -(optionally substituted C-C 12 independently selected from the group consisting of (heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )-, and -O-; Each occurrence of Y, if present, is a bond, -N(R a )—, and —O—; Each occurrence of Z is C1-C 24 is alkylenyl, C1-C at each occurrence of Z 24Alkylenyl is C1-C 12 Alkyl and C1-C 12 independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C1-C at each occurrence of Z 24 The alkylenyl is optionally further independently substituted; R a and R b Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 Aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ) independently selected from the group consisting of: R c and R d Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 independently selected from the group consisting of aralkyl, optionally substituted phenyl, and optionally substituted C-C heteroaryl; and Each occurrence of m is independently 1, 2, 3, or 4.

[0087] In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least one selected from the group consisting of is H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f, R 2g , and R 2h At least two selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least three selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least four selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least five selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least six selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least seven selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h Each of is H.

[0088] In some embodiments, L is —(CH) 1-10 In some embodiments, L is -(CH) 2-10 NR 3c In some embodiments, L is -(CH) 2-10 In some embodiments, L is -(CH2). 1-3 -CH(OR a )-(CH2) 1-3 In some embodiments, L is piperazinylenyl. In some embodiments, L is cyclohexylenyl.

[0089] In some embodiments, L is -CH2-. In some embodiments, L is -(CH2)2-. In some embodiments, L is -(CH2)3-. In some embodiments, L is -(CH2) 10 In some embodiments, L is —(CH2)2O—. In some embodiments, L is —(CH2)3O—. In some embodiments, L is —CH2CH(OR a )CH2-. In some embodiments, L is -(CH2)2NR 3c In some embodiments, L is TIFF2025529217000006.tif9128. In some embodiments, L is TIFF2025529217000007.tif9128. In some embodiments, L is TIFF2025529217000008.tif12128.

[0090] In some embodiments, the compound of formula (I) TIFF2025529217000009.tif18128.

[0091] In some embodiments, the compound of formula (I) The file is TIFF2025529217000010.tif23128.

[0092] In some embodiments, the compound of formula (I) The file is TIFF2025529217000011.tif20128.

[0093] In some embodiments, the compound of formula (I) TIFF2025529217000012.tif23128.

[0094] In some embodiments, the compound of formula (I) The file is TIFF2025529217000013.tif23132.

[0095] In some embodiments, the compound of formula (I) The file is TIFF2025529217000014.tif24141.

[0096] In some embodiments, the compound of formula (I) TIFF2025529217000015.tif20128.

[0097] In some embodiments, the compound of formula (I) TIFF2025529217000016.tif23128.

[0098] In some embodiments, the compound of formula (I) The file is TIFF2025529217000017.tif31128.

[0099] In some embodiments, R 4a is H. In some embodiments, R 4b is H. In some embodiments, R 4c is H. In some embodiments, R 4d is H.

[0100] In some embodiments, R 5 is methyl.

[0101] In some embodiments, R 6 is H.

[0102] In some embodiments, each occurrence of Z is independently TIFF2025529217000018.tif14128, wherein: R 7a , R 7b , R 7c , and R 7d each occurrence of is independently selected from the group consisting of H, C-C alkyl, and C-C haloalkyl; R 7a , R 7b , R 7c , and R 7d at least one of is not H; and Each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0103] In some embodiments, R 7a is H. In some embodiments, R 7b is H. In some embodiments, R 7c is H. In some embodiments, R 7d is H. In some embodiments, R 7a is methyl. In some embodiments, R 7b is methyl. In some embodiments, R 7c is methyl. In some embodiments, R 7d is methyl.

[0104] In some embodiments, Z is —(CH) 4-10 In some embodiments, Z is -(CH) 4-10 In some embodiments, Z is -(CH) 4-10 -CH(CH3)-CH2-*.

[0105] In some embodiments, R 3a teeth TIFF2025529217000019.tif13128. In one embodiment, R 3a teeth TIFF2025529217000020.tif13128. In one embodiment, R 3a teeth TIFF2025529217000021.tif13128. In one embodiment, R 3a teeth TIFF2025529217000022.tif13128. In one embodiment, R 3a teeth TIFF2025529217000023.tif13128. In one embodiment, R 3a teeth TIFF2025529217000024.tif13128. In one embodiment, R 3a teeth TIFF2025529217000025.tif13128. In one embodiment, R 3a teeth TIFF2025529217000026.tif13128. In one embodiment, R 3a teeth TIFF2025529217000027.tif13128. In one embodiment, R 3a teeth TIFF2025529217000028.tif13128. In one embodiment, R 3a teeth TIFF2025529217000029.tif13128. In one embodiment, R 3a teeth The file is TIFF2025529217000030.tif13128.

[0106] In some embodiments, R 3b teeth TIFF2025529217000031.tif13128. In one embodiment, R 3b teeth TIFF2025529217000032.tif13128. In one embodiment, R 3b teeth TIFF2025529217000033.tif13128. In one embodiment, R3b teeth TIFF2025529217000034.tif13128. In one embodiment, R 3b teeth TIFF2025529217000035.tif13128. In one embodiment, R 3b teeth TIFF2025529217000036.tif13128. In one embodiment, R 3b teeth TIFF2025529217000037.tif13128. In one embodiment, R 3b teeth TIFF2025529217000038.tif13128. In one embodiment, R 3b teeth TIFF2025529217000039.tif13128. In one embodiment, R 3b teeth TIFF2025529217000040.tif13128. In one embodiment, R 3b teeth TIFF2025529217000041.tif13128. In one embodiment, R 3b teeth The file is TIFF2025529217000042.tif13128.

[0107] In some embodiments, R 3c teeth TIFF2025529217000043.tif13128. In one embodiment, R 3c teeth TIFF2025529217000044.tif13128. In one embodiment, R 3c teeth TIFF2025529217000045.tif13128. In one embodiment, R 3c teeth TIFF2025529217000046.tif13128. In one embodiment, R 3c teeth TIFF2025529217000047.tif13128. In one embodiment, R 3c teeth TIFF2025529217000048.tif13128. In one embodiment, R 3c teeth TIFF2025529217000049.tif13128. In one embodiment, R 3c teeth TIFF2025529217000050.tif13128. In one embodiment, R 3c teeth TIFF2025529217000051.tif13128. In one embodiment, R 3c teeth TIFF2025529217000052.tif13128. In one embodiment, R 3c teeth TIFF2025529217000053.tif13128. In one embodiment, R 3c teeth The file is TIFF2025529217000054.tif13128.

[0108] 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is optionally substituted with at least one substituent, and said at least and at least one substituent is selected from the group consisting of C-C alkyl, C-C cycloalkyl, C-C haloalkyl, C-C haloalkoxy, phenoxy, halogen, CN, NO, OH, N(R')(R''), C(=O)R', C(=O)OR', OC(=O)OR', C(=O)N(R')(R''), S(=O)N(R')(R''), N(R')C(=O)R'', N(R')S(=O)R'', C-C 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, C-C alkyl, C-C cycloalkyl, C-C haloalkyl, benzyl, and phenyl.

[0109] In some embodiments, the compound of formula (I) TIFF2025529217000055.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000056.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000057.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000058.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000059.tif33128. In some embodiments, the compound of formula (I) TIFF2025529217000060.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000061.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000062.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000063.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000064.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000065.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000066.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000067.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000068.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000069.tif36128. In some embodiments, the compound of formula (I) TIFF2025529217000070.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000071.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000072.tif36128. In some embodiments, the compound of formula (I) TIFF2025529217000073.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000074.tif35128. In some embodiments, the compound of formula (I) The file is TIFF2025529217000075.tif36128.

[0110] Ionizable and / or cationic lipids The range of ionizable lipids intended for use in the present disclosure is not limited to the ionizable lipids of formula (I).In the lipid nanoparticles of the present disclosure, cationic lipids or ionizable lipids include, for example, the following: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLinMC3DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{ (2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-1,3]-dioxolane (DLin-K-C3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane Lan (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-KDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-dimethylaminoacetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyloxy-3-dimethylaminopropane (DLin-DAP ...2-Dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (D Lin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (D LinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (D Lin-EG-D MA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSD MA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxymethyl)] Amidoethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,The cationic lipid may include one or more of N'dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or a mixture thereof. In some embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA ("XTC2"), or a mixture thereof. Ionizable lipids are not limited to those listed herein, and may further include ionizable lipids known to those skilled in the art or described in the following disclosures, the entire contents of which are incorporated herein by reference: PCT Application No. PCT / US2020 / 056255 and / or PCT Application No. PCT / US2020 / 056252.

[0111] For example, 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 the synthesis of additional cationic lipids, is described in U.S. Patent Application Publication No. 2011 / 0256175, the disclosure of which is incorporated herein by reference in its entirety for all purposes. For example, the synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as the synthesis of additional cationic lipids, is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes. For example, the synthesis of cationic lipids such as CLinDMA, as well as the synthesis of additional cationic lipids, is described in U.S. Patent Application Publication No. 20060240554, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0112] Non-cationic lipids In the nucleic acid-lipid particles of the present disclosure, the non-cationic lipid may comprise, for example, 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) phospholipid; or (3) a mixture of phospholipid and cholesterol or a derivative thereof.

[0113] 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 those skilled in the art and is 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 incorporated herein in their entirety for all purposes.

[0114] Non-limiting examples of non-cationic lipids include phospholipids (e.g., lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyl ...DPPG), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyloleoylphosphatidylcholine (DPPG), palmitoyloleoylphosphatid phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0115] Other phospholipids that are diacylphosphatidylcholines and other phospholipids that are diacylphosphatidylethanolamines may also be used. The acyl groups in these lipids may be, for example, C 10 -C 24The acyl group may be derived from a fatty acid having a carbon chain of, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Further examples of non-cationic lipids include sterols, such as cholesterol and its derivatives, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. In some embodiments, the phospholipid is DPPC, DSPC, or a mixture thereof.

[0116] complex lipid In the nucleic acid-lipid particles of the present disclosure, the conjugated lipid that inhibits particle aggregation can include, for example, one or more of the following: polyethylene glycol (PEG) lipid conjugates, polyamide (ATTA)-lipid conjugates, cationic polymer-lipid conjugates (CPL), or mixtures thereof. In some embodiments, the nucleic acid-lipid particles include either PEG-lipid conjugates or ATTA-lipid conjugates.

[0117] PEG is a linear, water-soluble polymer consisting of repeating ethylene PEG units with two terminal hydroxyl groups. PEGs are classified by their molecular weight; 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, monomethoxypolyethylene glycol (MePEGOH), monomethoxypolyethylene glycol succinate (MePEGS), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol tresylate (MePEG-TRES), and monomethoxypolyethylene glycol imidazolylcarbonyl (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 incorporated herein by reference in their entirety for all purposes.In addition, monomethoxypolyethylene glycol acetic acid (MePEG-CH2COOH) is particularly useful for preparing PEG-lipid conjugates, including, for example, PEG-DAA conjugates.

[0118] In some embodiments, PEG-lipid conjugates or ATTA-lipid conjugates are used with CPL. The conjugated lipids that inhibit particle aggregation can include, for example, PEG-lipids, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEGDAA conjugates can be used with PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), PEG-distearyloxypropyl (C 18), or a mixture thereof.

[0119] Other PEG-lipid conjugates suitable for use in the present disclosure include, but are not limited to, mPEG2000-1,2-diO-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT application number PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.More PEG-lipid conjugates suitable for use in the present disclosure include, but are not limited to, 1-[8'-(1,2-dimyristoyl-3-propaneoxy)-carboxamide-3',6'-dioxaoctanyl]carbamoyl-methyl-poly(ethylene glycol) (2KPEG-DMG).The synthesis of 2KPEG-DMG is described in US Patent No. 7,404,969, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0120] The PEG moiety of the PEG-lipid conjugates described herein can have an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In some examples, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, 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.

[0121] In addition, it will be readily apparent to those skilled 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, polyhydroxypropylmethacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0122] In addition to the above components, particles (e.g., LNPs) of the present disclosure can further include cationic poly(ethylene glycol) (PEG) lipids, or CPLs (e.g., Chen et al., Bioconj. Chem., 11:433-437 (2000)). SPLPs and SPLP-CPLs suitable for use in the present disclosure, as well as methods of making and using SPLPs and SPLP-CPLs, are disclosed, for example, in U.S. Pat. No. 6,852,334 and PCT Publication WO 00 / 62813, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0123] In some examples, the conjugated lipid (e.g., PEG-lipid conjugate) that inhibits particle aggregation comprises about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 1 mol% to about 1.8 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, about 1.4 mol% to about 1.5 mol%, or about 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol% or about 1.5 mol% of the total lipid present in the particle. mol%, 1.8 mol%, 1.9 mol%, or 2 mol% (or any fraction or range therein).

[0124] In the lipid nanoparticles of the present disclosure, active agent or therapeutic agent can be completely encapsulated in the lipid portion of particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation.In some embodiments, the nucleic acid-lipid particle that comprises nucleic acid, such as messenger RNA (i.e., mRNA), completely encapsulates nucleic acid in the lipid portion of particle, thereby protecting the nucleic acid from nuclease degradation.In some examples, the nucleic acid in nucleic acid-lipid particle is not substantially decomposed even after the particle is exposed to nuclease at 37 degrees for at least about 20 minutes, 30 minutes, 45 minutes or 60 minutes. In some other examples, the nucleic acid in nucleic acid-lipid particles is not substantially decomposed after the particles are incubated in serum at 37 degrees for at least about 30 minutes, 45 minutes or 60 minutes, or at least about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours or 36 hours.In other embodiments, active agent or therapeutic agent (for example, nucleic acid, such as siRNA) is complexed with the lipid part of particle.One of the advantages of the formulation of the present disclosure is that lipid particle composition is substantially non-toxic to mammals, for example, humans.

[0125] synthesis The present disclosure further provides methods for preparing the compounds of the present disclosure. The compounds of the present teachings can be prepared according to the procedures described herein from commercially available starting materials, from compounds known in the literature, or from easily prepared intermediates, utilizing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and for functional group transformations and manipulations can be readily obtained from the relevant scientific literature or standard textbooks in the art.

[0126] Where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are described, it is understood that other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented can be varied in order to optimize the production of the compounds described herein.

[0127] The processes described herein can be monitored according to any suitable technique known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), mass spectrometry, or product formation can also be monitored by chromatography, such as high pressure liquid chromatography (HPLC), gas chromatography (GC), gel permeation chromatography (GPC), or thin layer chromatography (TLC).

[0128] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., "Protective Groups in Organic Synthesis", 2d. Ed. (Wiley & Sons, 1991), the entire disclosure of which is incorporated herein by reference for all purposes.

[0129] The reactions or processes described herein may be carried out in a suitable solvent, which can be easily selected by those skilled in the art of organic synthesis.Suitable solvents are typically substantially non-reactive with the reactants, intermediates, and / or products at the temperature at which the reaction is carried out, i.e., a temperature that may range from the solvent's freezing temperature to the solvent's boiling temperature.A given reaction may be carried out in one solvent or in a mixture of multiple solvents.A suitable solvent for a particular reaction step may be selected depending on the particular reaction step.

[0130] In some embodiments, compounds synthesized using the methods described herein may contain one or more chiral carbon atoms, resulting in two or more isomers. Absolute stereochemistry may be represented using a wedge bond (bold or parallel). In some embodiments, the product formed in any of the reactions described may be racemic. If a racemate is formed, the isomers that make up the racemate may be separated using any suitable method for chiral resolution known to those skilled in the art. Suitable methods for chiral resolution include, but are not limited to, supercritical fluid chromatography (SFC), chiral HPLC, crystallization, derivatization, or any combination thereof.

[0131] In some embodiments, separation of isomers formed in one or more separate reactions may require the formation of derivatives prior to chiral resolution. A non-limiting example of derivatization is to protect one or more functional groups present in the compound using known protecting groups (e.g., esters, amides, carbamates, ethers, etc.), followed by separation of the isomers by an appropriate method. The desired compound is finally obtained by removing the protecting group.

[0132] The present disclosure provides ionizable lipid compounds of formula (I) and also provides non-limiting exemplary methods for preparing the same. Those skilled in the art will recognize that other techniques and / or methods may also be suitable for synthesizing compounds of formula (I).

[0133] In certain embodiments, compounds of the present disclosure may be prepared as provided in Schemes 1-5, wherein X 1 , X 2 , and X 3 are each independently a halogen; s is an integer ranging from 1 to 24; R' is an optionally substituted alkyl containing at least one branched or tertiary carbon (i.e., a carbon atom covalently bonded to at least three carbon atoms); and L, m, and Y are as defined within the scope of this disclosure.

[0134] In some embodiments, homologated haloalkenes 1-2 can be prepared by selectively removing one halogen from dihaloalkane 1-1 in a suitable solvent, including but not limited to, tetrahydrofuran (THF), under suitable conditions, including but not limited to, a temperature of about 70° C., for a period of time, including but not limited to, about 16 hours. In some embodiments, alkenes 1-3 ... using a Grignard reagent (i.e., R'-MgX 3) in the presence of a suitable catalyst, including but not limited to, LiCuCl, in the presence of a polar solvent and / or polar additive, a non-limiting example of which is N-methylpyrrolidone (NMP), and in the presence of a suitable solvent, including but not limited to, THF, under suitable reaction conditions, including a reaction temperature of room temperature, for a period of time, including but not limited to, 1 hour. In certain embodiments, epoxide 1-4 can be prepared from alkene 1-3 by epoxidation with a suitable epoxidizing reagent, including but not limited to, m-chloroperbenzoic acid (mCPBA), under suitable reaction conditions, including a reaction temperature of room temperature, for a period of time, including but not limited to, 16 hours. An exemplary embodiment of the synthetic route shown in Scheme 1 is provided in FIG. 1.

[0135] TIFF2025529217000077.tif32144 In some embodiments, polyamine core 2-1 can be alkylated with four or more equivalents of epoxide 1-4 to provide ionizable lipid compound 2-2, in the presence of a suitable solvent, including but not limited to ethanol (EtOH), under suitable reaction conditions, including but not limited to a temperature of about 80°C, for a period of time, including but not limited to 48 hours. In some embodiments, L comprises a primary or secondary amine. In such embodiments, more than four equivalents of 1-4 are required for complete alkylation in embodiments where L comprises a primary or secondary amine. In some embodiments, a greater than stoichiometric amount of 1-4 can be used to facilitate alkylation. An exemplary embodiment of the synthetic route shown in Scheme 2 is provided in Figure 2.

[0136] TIFF2025529217000078.tif35151 In some embodiments, polyamine core 2-1 may be complexed with four or more equivalents of an α,β-unsaturated carbonyl compound 3-1 (i.e., an α,β-unsaturated ketone, an α,β-unsaturated ester, and / or an α,β-unsaturated amide) via [1,4]-conjugate addition (i.e., a Michael addition reaction) in the presence of a suitable solvent and under suitable reaction conditions to provide an ionizable lipid compound 3-2.

[0137] TIFF2025529217000079.tif34150 In some embodiments, polyamine core 2-1 may be complexed with four or more equivalents of α,β-epoxy-substituted carbonyl compound 4-1 by nucleophilic addition to provide ionizable lipid compound 4-2, in the presence of a suitable solvent under suitable reaction conditions.

[0138] TIFF2025529217000080.tif32148 In some embodiments, polyamine core 2-1 may be complexed with four or more equivalents of aldehyde 5-1 by reductive amination and / or reductive alkylation to provide ionizable lipid compound 5-2 in the presence of a suitable hydride reducing agent, in the presence of a suitable solvent, and under suitable reaction conditions.

[0139] Further, the present disclosure exemplifies the synthesis of ionizable lipids of formula (I) (e.g., Compound 2-2, Compound 3-2, Compound 4-2, and Compound 5-2 in Schemes 2-5), where polyamine 2-1 comprises polyamine core 494 (i.e., 2-(2-aminoethoxy)-N-(2-(4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)ethyl)ethan-1-amine), and polyamine core 200 (i.e., N 1The disclosure is not limited to the embodiments specifically exemplified herein, and other polyamine cores are contemplated for use in the disclosure, including but not limited to 3-(4-(3-aminopropyl)piperazin-1-yl)-N-(2-(4-(3-(4-(3-aminopropyl)piperazin-1-yl)propyl)piperazin-1-yl)ethyl)propan-1-amine, 2-(2-(2-aminoethoxy)ethoxy)-N-(2-(4-(2-(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl)ethyl)ethan-1-amine, N-(4-(2-(2-aminoethoxy)ethoxy)ethyl)piperazin-1-yl)ethyl)ethane-1-amine, N-(4-(2-(2-amino ... 1 -(2-(4-(10-aminodecyl)piperazin-1-yl)ethyl)decane-1,10-diamine, 3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)-N-(2-(4-(3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)piperazin-1-yl)ethyl)propan-1-amine, N 1 -(2-(4-(3-amino-2-ethoxypropyl)piperazin-1-yl)ethyl)-2-ethoxypropane-1,3-diamine, N-((1-(aminomethyl)cyclohexyl)methyl)-2-(4-((1-(aminomethyl)cyclohexyl)methyl)piperazin-1-yl)ethan-1-amine, and N 1 -(2-(4-(4-aminohexyl)piperazin-1-yl)ethyl)cyclohexane-1,4-diamine.

[0140] Table 1: Exemplary ionizable lipids of the present disclosure TIFF2025529217000081.tif180146TIFF2025529217000082.tif210146TIFF2025529217000083.tif188146TIFF2025529217000084.tif193146

[0141] Lipid nanoparticles (LNPs) In another aspect, the present disclosure provides lipid nanoparticle (LNP) compositions.

[0142] In certain embodiments, the LNP composition comprises at least one ionizable lipid compound having the structure of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: TIFF2025529217000085.tif22128, wherein: R 1a and R 1b are each independently TIFF2025529217000086.tif11128; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is H, optionally substituted C1-C 12 Alkyl, optionally substituted C-C 12 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 12 Alkenyl, optionally substituted C-C 12 Alkynyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, TIFF2025529217000087.tif18138; R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4d Each occurrence of, if present, is optionally substituted C1-C12 independently selected from the group consisting of alkyl, halogen, CN, and NO2; R 5 Each occurrence of is an optionally substituted C1-C3 alkyl, optionally substituted C3-C 12 Cycloalkyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C 12 Cycloalkyl, C2-C 12 Heterocycloalkyl, optionally substituted C-C 12 Aralkyl, optionally substituted C-C 12 Aryl, optionally substituted C-C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ) independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C-C 12 alkylenyl)-X-, -(optionally substituted C-C 12 alkenylenyl)-X-, -(optionally substituted C-C 12 alkynylenyl)-X-, -(optionally substituted C-C 12 independently selected from the group consisting of (heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )—, and —O—; Each occurrence of Y, if present, is a bond, -N(R a )—, and —O—; Each occurrence of Z is C1-C 24is alkylenyl, C1-C at each occurrence of Z 24 Alkylenyl is C1-C 12 Alkyl and C1-C 12 independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C1-C at each occurrence of Z 24 The alkylenyl is optionally further independently substituted; R a and R b Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 Aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ) independently selected from the group consisting of: R c and R d Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 independently selected from the group consisting of aralkyl, optionally substituted phenyl, and optionally substituted C-C heteroaryl; and Each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4.

[0143] In certain embodiments, the LNP composition comprises at least one neutral lipid.

[0144] In certain embodiments, the LNP composition comprises cholesterol.

[0145] In certain embodiments, the LNP composition comprises at least one complex lipid.

[0146] In certain embodiments, the LNP composition comprises at least one nucleic acid cargo and / or therapeutic drug cargo that is at least partially encapsulated in the LNP composition.

[0147] In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least one selected from the group consisting of is H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least two selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least three selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least four selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least five selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e, R 2f , R 2g , and R 2h At least six selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least seven selected from the group consisting of are H. In some embodiments, R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h Each of is H.

[0148] In some embodiments, L is —(CH) 1-10 In some embodiments, L is -(CH) 2-10 NR 3c In some embodiments, L is -(CH) 2-10 In some embodiments, L is -(CH2). 1-3 -CH(OR a )-(CH2) 1-3 In some embodiments, L is piperazinylenyl. In some embodiments, L is cyclohexylenyl.

[0149] In some embodiments, L is -CH2-. In some embodiments, L is -(CH2)2-. In some embodiments, L is -(CH2)3-. In some embodiments, L is -(CH2) 10 In some embodiments, L is —(CH2)2O—. In some embodiments, L is —(CH2)3O—. In some embodiments, L is —CH2CH(OR a )CH2-. In some embodiments, L is -(CH2)2NR 3c In some embodiments, L is TIFF2025529217000088.tif9128. In some embodiments, L is TIFF2025529217000089.tif9128. In some embodiments, L is The file is TIFF2025529217000090.tif12128.

[0150] In some embodiments, the compound of formula (I) The file is TIFF2025529217000091.tif19128.

[0151] In some embodiments, the compound of formula (I) The file is TIFF2025529217000092.tif23128.

[0152] In some embodiments, the compound of formula (I) TIFF2025529217000093.tif20128.

[0153] In some embodiments, the compound of formula (I) The file is TIFF2025529217000094.tif23128.

[0154] In some embodiments, the compound of formula (I) The file is TIFF2025529217000095.tif23132.

[0155] In some embodiments, the compound of formula (I) The file is TIFF2025529217000096.tif24141.

[0156] In some embodiments, the compound of formula (I) TIFF2025529217000097.tif20128.

[0157] In some embodiments, the compound of formula (I) TIFF2025529217000098.tif23128.

[0158] In some embodiments, the compound of formula (I) The file is TIFF2025529217000099.tif31128.

[0159] In some embodiments, R 4a is H. In some embodiments, R 4b is H. In some embodiments, R 4c is H. In some embodiments, R 4d is H.

[0160] In some embodiments, R 5 is methyl.

[0161] In some embodiments, R 6 is H.

[0162] In some embodiments, each occurrence of Z is independently TIFF2025529217000100.tif13128, wherein: R 7a , R 7b , R 7c , and R 7d each occurrence of is independently selected from the group consisting of H, C-C alkyl, and C-C haloalkyl; R 7a , R 7b , R 7c , and R 7d at least one of is not H; and Each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0163] In some embodiments, R 7a is H. In some embodiments, R 7b is H. In some embodiments, R 7c is H. In some embodiments, R 7d is H. In some embodiments, R 7a is methyl. In some embodiments, R 7b is methyl. In some embodiments, R 7c is methyl. In some embodiments, R7d is methyl.

[0164] In some embodiments, Z is —(CH) 4-10 In some embodiments, Z is -(CH) 4-10 In some embodiments, Z is -(CH) 4-10 -CH(CH3)-CH2-*.

[0165] In some embodiments, R 3a teeth TIFF2025529217000101.tif13128. In one embodiment, R 3a teeth TIFF2025529217000102.tif13128. In one embodiment, R 3a teeth TIFF2025529217000103.tif13128. In one embodiment, R 3a teeth TIFF2025529217000104.tif13128. In one embodiment, R 3a teeth TIFF2025529217000105.tif13128. In one embodiment, R 3a teeth TIFF2025529217000106.tif13128. In one embodiment, R 3a teeth TIFF2025529217000107.tif13128. In one embodiment, R 3a teeth TIFF2025529217000108.tif13128. In one embodiment, R 3a teeth TIFF2025529217000109.tif13128. In one embodiment, R 3a teeth TIFF2025529217000110.tif13128. In one embodiment, R 3a teeth TIFF2025529217000111.tif13128. In one embodiment, R 3a teeth The file is TIFF2025529217000112.tif13128.

[0166] In some embodiments, R 3b teeth TIFF2025529217000113.tif13128. In one embodiment, R 3b teeth TIFF2025529217000114.tif13128. In one embodiment, R 3b teeth TIFF2025529217000115.tif13128. In one embodiment, R 3b teeth TIFF2025529217000116.tif13128. In one embodiment, R 3b teeth TIFF2025529217000117.tif13128. In one embodiment, R 3b teeth TIFF2025529217000118.tif13128. In one embodiment, R 3b teeth TIFF2025529217000119.tif13128. In one embodiment, R 3b teeth TIFF2025529217000120.tif13128. In one embodiment, R 3b teeth TIFF2025529217000121.tif13128. In one embodiment, R 3b teeth TIFF2025529217000122.tif13128. In one embodiment, R 3b teeth TIFF2025529217000123.tif13128. In one embodiment, R 3b teeth The file is TIFF2025529217000124.tif13128.

[0167] In some embodiments, R 3c teeth TIFF2025529217000125.tif13128. In one embodiment, R 3c teeth TIFF2025529217000126.tif13128. In one embodiment, R 3c teeth TIFF2025529217000127.tif13128. In one embodiment, R 3c teeth TIFF2025529217000128.tif13128. In one embodiment, R 3c teeth TIFF2025529217000129.tif13128. In one embodiment, R 3c teeth TIFF2025529217000130.tif13128. In one embodiment, R 3c teeth TIFF2025529217000131.tif13128. In one embodiment, R 3c teeth TIFF2025529217000132.tif13128. In one embodiment, R 3c teeth TIFF2025529217000133.tif13128. In one embodiment, R 3c teeth TIFF2025529217000134.tif13128. In one embodiment, R 3c teeth TIFF2025529217000135.tif13128. In one embodiment, R 3c teeth The file is TIFF2025529217000136.tif13128.

[0168] 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is optionally substituted with at least one substituent, and said at least and at least one substituent is selected from the group consisting of C-C alkyl, C-C cycloalkyl, C-C haloalkyl, C-C haloalkoxy, phenoxy, halogen, CN, NO, OH, N(R')(R''), C(=O)R', C(=O)OR', OC(=O)OR', C(=O)N(R')(R''), S(=O)N(R')(R''), N(R')C(=O)R'', N(R')S(=O)R'', C-C 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, C-C alkyl, C-C cycloalkyl, C-C haloalkyl, benzyl, and phenyl.

[0169] In some embodiments, the compound of formula (I) TIFF2025529217000137.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000138.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000139.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000140.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000141.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000142.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000143.tif32128. In some embodiments, the compound of formula (I) TIFF2025529217000144.tif33128. In some embodiments, the compound of formula (I) TIFF2025529217000145.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000146.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000147.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000148.tif31128. In some embodiments, the compound of formula (I) TIFF2025529217000149.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000150.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000151.tif36128. In some embodiments, the compound of formula (I) TIFF2025529217000152.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000153.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000154.tif36128. In some embodiments, the compound of formula (I) TIFF2025529217000155.tif35128. In some embodiments, the compound of formula (I) TIFF2025529217000156.tif35128. In some embodiments, the compound of formula (I) The file is TIFF2025529217000157.tif36128.

[0170] In some embodiments, the at least one ionizable lipid of Formula (I) comprises about 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 mol%, 83 mol%, 84 mol%, 85 mol%, 86 mol%, 87 mol%, 88 mol%, 89 mol%, 90 mol%, 91 mol%, mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 mol%, 83 mol%, 84 mol%, 85 mol%, 86 mol%, 87 mol%, 88 mol%, 89 mol%, or approximately 90 mol%.

[0171] In some embodiments, the at least one ionizable lipid of formula (I) comprises less than about 10 mol%, less than 11 mol%, less than 12 mol%, less than 13 mol%, less than 14 mol%, less than 15 mol%, less than 16 mol%, less than 17 mol%, less than 18 mol%, less than 19 mol%, less than 20 mol%, less than 21 mol%, less than 22 mol%, less than 23 mol%, less than 24 mol%, less than 25 mol%, less than 26 mol%, less than 27 mol%, less than 28 mol%, less than 29 mol%, less than 30 mol%, less than 31 mol%, less than 32 mol%, less than 33 mol%, less than 34 mol%, less than 35 mol%, less than 36 mol%, less than 37 mol%, less than 38 mol%, less than 39 mol%, less than 40 mol%, less than 41 mol% of the LNP. Less than 42 mol%, Less than 43 mol%, Less than 44 mol%, Less than 45 mol%, Less than 46 mol%, Less than 47 mol%, Less than 48 mol%, Less than 49 mol%, Less than 50 mol%, Less than 51 mol%, Less than 52 mol%, Less than 53 mol%, Less than 54 mol%, Less than 55 mol%, Less than 56 mol%, Less than 57 mol%, Less than 58 mol%, Less than 59 mol%, Less than 60 mol%, Less than 61 mol%, Less than 62 mol%, Less than 63 mol%, Less than 64 mol%, Less than 65 mol%, Less than 66 mol%, Less than 67 mol%, Less than 68 mol%, Less than 69 mol%, Less than 70 mol%, Less than 71 mol%, Less than 72 mol%, Less than 73 mol%, Less than 74 mol%, Less than 75 mol%, 76 mol%, less than 77 mol%, less than 78 mol%, less than 79 mol%, less than 80 mol%, less than 81 mol%, less than 82 mol%, less than 83 mol%, less than 84 mol%, less than 85 mol%, less than 86 mol%, less than 87 mol%, less than 88 mol%, less than 89 mol%, or less than about 90 mol%.

[0172] In certain embodiments, the at least one ionizable lipid of formula (I) comprises more than about 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, or ...% of the LNP. More than mol%, more than 42 mol%, more than 43 mol%, more than 44 mol%, more than 45 mol%, more than 46 mol%, more than 47 mol%, more than 48 mol%, more than 49 mol%, more than 50 mol%, more than 51 mol%, more than 52 mol%, more than 53 mol%, more than 54 mol%, more than 55 mol%, more than 56 mol%, 57 More than mol%, more than 58 mol%, more than 59 mol%, more than 60 mol%, more than 61 mol%, more than 62 mol%, more than 63 mol%, more than 64 mol%, more than 65 mol%, more than 66 mol%, more than 67 mol%, more than 68 mol%, more than 69 mol%, more than 70 mol%, more than 71 mol%, more than 72 mol%, 73 More than mol%, more than 74 mol%, more than 75 mol%, 76 mol%, greater than 77 mol%, greater than 78 mol%, greater than 79 mol%, greater than 80 mol%, greater than 81 mol%, greater than 82 mol%, greater than 83 mol%, greater than 84 mol%, greater than 85 mol%, greater than 86 mol%, greater than 87 mol%, greater than 88 mol%, greater than 89 mol%, or greater than about 90 mol%.

[0173] In some embodiments, the at least one ionizable lipid of Formula (I) comprises about 35 mol% of the LNP. In some embodiments, the at least one ionizable lipid of Formula (I) comprises less than about 35 mol% of the LNP. In some embodiments, the at least one ionizable lipid of Formula (I) comprises more than about 35 mol% of the LNP.

[0174] In some embodiments, the at least one ionizable lipid of Formula (I) comprises about 40 mol% of the LNP. In some embodiments, the at least one ionizable lipid of Formula (I) comprises less than about 40 mol% of the LNP. In some embodiments, the at least one ionizable lipid of Formula (I) comprises more than about 40 mol% of the LNP.

[0175] In certain embodiments, the at least one neutral lipid comprises about 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, or about 40 mol% of the LNP. Constitutes mol%.

[0176] In certain embodiments, the at least one neutral lipid comprises less than about 1 mol%, less than 2 mol%, less than 3 mol%, less than 4 mol%, less than 5 mol%, less than 6 mol%, less than 7 mol%, less than 8 mol%, less than 9 mol%, less than 10 mol%, less than 11 mol%, less than 12 mol%, less than 13 mol%, less than 14 mol%, less than 15 mol%, less than 16 mol%, less than 17 mol%, less than 18 mol%, less than 19 mol%, less than 20 mol%, less than 21 mol%, less than 22 mol%, less than 23 mol%, less than 24 mol%, less than 25 mol%, less than 26 mol%, less than 27 mol%, less than 28 mol%, less than 29 mol%, less than 30 mol%, less than 31 mol%, less than 32 mol%, less than 33 mol%, less than 34 mol%, less than 35 mol% of the LNP. mol%, less than 36 mol%, less than 37 mol%, less than 38 mol%, less than 39 mol%, or less than about 40 mol%.

[0177] In certain embodiments, the at least one neutral lipid comprises more than about 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, or more than about 1 mol% of the LNP. mol%, greater than 36 mol%, greater than 37 mol%, greater than 38 mol%, greater than 39 mol%, or greater than about 40 mol%.

[0178] In some embodiments, the at least one neutral lipid comprises about 16 mol% of the LNP. In some embodiments, the at least one neutral lipid comprises less than about 16 mol% of the LNP. In some embodiments, the at least one neutral lipid comprises more than about 16 mol% of the LNP.

[0179] In some embodiments, the at least one neutral lipid comprises about 30 mol% of the LNP. In some embodiments, the at least one neutral lipid comprises less than about 30 mol% of the LNP. In some embodiments, the at least one neutral lipid comprises more than about 30 mol% of the LNP.

[0180] In some embodiments, at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). In some embodiments, at least one neutral lipid comprises distearoylphosphatidylcholine (DSPC). In some embodiments, at least one neutral lipid comprises dioleoylphosphatidylcholine (DOPC).

[0181] In some embodiments, cholesterol comprises from about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, or about 75 mol% of the LNP.

[0182] In some embodiments, cholesterol is less than about 20 mol%, less than 21 mol%, less than 22 mol%, less than 23 mol%, less than 24 mol%, less than 25 mol%, less than 26 mol%, less than 27 mol%, less than 28 mol%, less than 29 mol%, less than 30 mol%, less than 31 mol%, less than 32 mol%, less than 33 mol%, less than 34 mol%, less than 35 mol%, less than 36 mol%, less than 37 mol%, less than 38 mol%, less than 39 mol%, less than 40 mol%, less than 41 mol%, less than 42 mol%, less than 43 mol%, less than 44 mol%, less than 45 mol%, less than 46 mol%, less than 47 mol%, less than 48 mol%, less than 49 mol%, less than 50 mol%, less than 51 mol%, less than 52 mol%, less than 53 mol%, less than 54 mol%, less than 55 mol%, less than 56 mol%, less than 57 mol%, less than 58 mol%, less than 59 mol%, less than 60 mol%, less than 61 mol%, less than 62 mol%, less than 63 mol%, less than 64 mol%, less than 65 mol%, less than 66 mol%, less than 67 mol%, less than 68 mol%, less than 69 mol%, less than 70 mol%, less than 71 mol%, less than 72 mol%, less than 73 mol%, less than 74 mol%, or less than about 75 mol%.

[0183] In some embodiments, cholesterol is greater than about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, or ... mol%, greater than 54 mol%, greater than 55 mol%, greater than 56 mol%, greater than 57 mol%, greater than 58 mol%, greater than 59 mol%, greater than 60 mol%, greater than 61 mol%, greater than 62 mol%, greater than 63 mol%, greater than 64 mol%, greater than 65 mol%, greater than 66 mol%, greater than 67 mol%, greater than 68 mol%, greater than 69 mol%, greater than 70 mol%, greater than 71 mol%, greater than 72 mol%, greater than 73 mol%, greater than 74 mol%, or greater than about 75 mol%.

[0184] In some embodiments, cholesterol comprises about 25 mol% of the LNP. In some embodiments, cholesterol comprises less than about 25 mol% of the LNP. In some embodiments, cholesterol comprises more than about 25 mol% of the LNP.

[0185] In some embodiments, cholesterol comprises about 46.5 mol% of the LNP. In some embodiments, cholesterol comprises less than about 46.5 mol% of the LNP. In some embodiments, cholesterol comprises more than about 46.5 mol% of the LNP.

[0186] In certain embodiments, the at least one complex lipid comprises about 0.1 mol%, 0.2 mol%, 0.3 mol%, 0.4 mol%, 0.5 mol%, 0.6 mol%, 0.7 mol%, 0.8 mol%, 0.9 mol%, 1.0 mol%, 2.0 mol%, 3.0 mol%, 4.0 mol%, 5.0 mol%, 6.0 mol%, 7.0 mol%, 8.0 mol%, 9.0 mol%, 10.0 mol%, 11.0 mol%, 12.0 mol%, 13.0 mol%, 14.0 mol%, or about 15.0 mol% of the LNP.

[0187] In certain embodiments, the at least one complex lipid comprises less than about 0.1 mol%, less than 0.2 mol%, less than 0.3 mol%, less than 0.4 mol%, less than 0.5 mol%, less than 0.6 mol%, less than 0.7 mol%, less than 0.8 mol%, less than 0.9 mol%, less than 1.0 mol%, less than 2.0 mol%, less than 3.0 mol%, less than 4.0 mol%, less than 5.0 mol%, less than 6.0 mol%, less than 7.0 mol%, less than 8.0 mol%, less than 9.0 mol%, less than 10.0 mol%, less than 11.0 mol%, less than 12.0 mol%, less than 13.0 mol%, less than 14.0 mol%, or less than about 15.0 mol% of the LNP.

[0188] In certain embodiments, the at least one complex lipid comprises more than about 0.1 mol%, more than 0.2 mol%, more than 0.3 mol%, more than 0.4 mol%, more than 0.5 mol%, more than 0.6 mol%, more than 0.7 mol%, more than 0.8 mol%, more than 0.9 mol%, more than 1.0 mol%, more than 2.0 mol%, more than 3.0 mol%, more than 4.0 mol%, more than 5.0 mol%, more than 6.0 mol%, more than 7.0 mol%, more than 8.0 mol%, more than 9.0 mol%, more than 10.0 mol%, more than 11.0 mol%, more than 12.0 mol%, more than 13.0 mol%, more than 14.0 mol%, or more than about 15.0 mol% of the LNP.

[0189] In some embodiments, at least one conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000 ) is included.

[0190] In some embodiments, the LNPs have a molar ratio of (a):(b):(c):(d) of about 35:16:46.5:2.5, hi some embodiments, the LNPs have a molar ratio of (a):(b):(c):(d) of about 40:30:25:2.5.

[0191] In some embodiments, the nucleic acid molecule is a therapeutic agent. In some embodiments, the nucleic acid molecule comprises RNA. In some embodiments, the nucleic acid molecule comprises DNA. In some embodiments, the nucleic acid molecule comprises mRNA. In some embodiments, the nucleic acid molecule comprises cDNA. In some embodiments, the nucleic acid molecule comprises miRNA. In some embodiments, the nucleic acid molecule comprises siRNA. In some embodiments, the nucleic acid molecule comprises modified RNA.

[0192] In certain embodiments, the nucleic acid molecule is mRNA.

[0193] In some embodiments, the LNPs have a mass ratio of (a):mRNA 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, or about 5:1 (w / w). In some embodiments, the LNPs have a mass ratio of (a):mRNA of about 10:1.

[0194] In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is specific for binding to a surface antigen of a pathogenic cell or tumor cell. In some embodiments, the surface antigen is selected from the group consisting of CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169 ... D161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-1 2, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE A1, HAL-A2 NY-ESO-1, PSC1, folate receptor alpha, 8H9, NCAM, VEGF, 5T4, fetal AchR, NKG2D ligand, TEM1, and TEM8.

[0195] In some embodiments, the mRNA encodes an enzyme. In some embodiments, the mRNA encodes a CRISPR (clustered regularly interspaced short palindrome repeats)-associated protein, optionally the CRISPR-associated protein is Cas9. In some embodiments, the mRNA further encodes a small-guiding RNA (sgRNA).

[0196] method In another aspect, the present disclosure provides a method for treating, preventing, and / or ameliorating a disease in a subject. In some embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure.

[0197] In some embodiments, the LNPs of the present disclosure are useful for the treatment, prevention, and / or amelioration of any of a number of diseases and / or disorders. In some embodiments, the LNPs of the present disclosure are suitable for the treatment, prevention, and / or amelioration of diseases and / or disorders for which mRNA delivery and / or gene therapy (e.g., cancer immunotherapy) is useful. In some embodiments, the LNPs of the present disclosure are suitable for the treatment, prevention, and / or amelioration of diseases and / or disorders, including, inter alia, autoimmune disorders, cardiovascular diseases, and neurological disorders. In some embodiments, the LNPs of the present disclosure are suitable for gene editing applications, including gene editing of the liver, brain, lungs, and / or hematopoietic cells for monogenic diseases.

[0198] In some embodiments, the disease is cancer.In some embodiments, the cancer is at least one selected from the group consisting of pancreatic cancer, colon cancer, bladder cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small cell lung cancer or colon cancer.In some embodiments, the subject is further administered at least one additional agent or therapy that is useful for treating, preventing and / or ameliorating cancer in the subject.

[0199] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

[0200] In another aspect, the present disclosure provides a method for delivering a nucleic acid or therapeutic agent to the liver of a subject. In some embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure.

[0201] In another aspect, the present disclosure provides a method for preparing modified immune cells or progenitor cells thereof. In some embodiments, the method includes contacting immune cells or progenitor cells thereof with at least one lipid nanoparticle (LNP) of the present disclosure and / or at least one pharmaceutical composition of the present disclosure.

[0202] In some embodiments, the engineered immune cells or precursors thereof are αβ T cells, γδ T cells, CD8+ T cells, CD4+ helper T cells, CD4+ regulatory T cells, NK T cells, NK cells, and any combination thereof.

[0203] In some embodiments, the modified immune cells or precursors thereof are T cells, and optionally the T cells are CD4+ T cells. In some embodiments, the modified immune cells or precursors thereof are NK cells.

[0204] In another aspect, the present disclosure provides a compound of formula (2-2), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotope thereof: A method for preparing TIFF2025529217000158.tif26128 is provided.

[0205] In some embodiments, the method comprises reacting a compound of formula (1-4): TIFF2025529217000159.tif11128 and a compound of formula (2-1): TIFF2025529217000160.tif10128, wherein Each occurrence of L is a bond, -(optionally substituted C-C 12 alkylenyl)-X-, -(optionally substituted C-C 12alkenylenyl)-X-, -(optionally substituted C-C 12 alkynylenyl)-X-, -(optionally substituted C-C 12 independently selected from the group consisting of (heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl; Each occurrence of R' is independently an optionally substituted C-C 12 alkyl, where each occurrence of R' contains at least one tertiary carbon; each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; and Each occurrence of s is independently an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0206] In some embodiments, the contacting step is performed in the presence of a solvent, where optionally, the solvent comprises ethanol (EtOH). In some embodiments, the contacting step is performed at a temperature of about 70° C. to about 90° C., where optionally, the contacting step is performed at a temperature of about 80° C. In some embodiments, the contacting step is performed for a period of about 1 hour to about 72 hours, where optionally, the contacting step is performed for a period of about 48 hours.

[0207] In some embodiments, the compound of formula (1-4) is a compound of formula (1-3): TIFF2025529217000161.tif9128 and the formula R'-MgX 3 with an organomagnesium compound of In the formula, X 3 is selected from the group consisting of Cl, Br, and I.

[0208] In some embodiments, the contacting step is carried out in the presence of a suitable epoxidizing reagent, where optionally the epoxidizing reagent is m-chloroperbenzoic acid. In some embodiments, the contacting step is carried out in the presence of a solvent, where optionally the solvent comprises dichloromethane.

[0209] In some embodiments, the compound of formula (1-3) is a compound of formula (1-2): TIFF2025529217000162.tif10128 and the formula R'-MgX 3 and an organomagnesium compound of formula X 1 and X 3 are each independently selected from the group consisting of Cl, Br, and I.

[0210] In certain embodiments, the contacting step is carried out in the presence of a suitable catalyst, where optionally the catalyst comprises LiCuCl. In certain embodiments, the contacting step is carried out in the presence of a solvent, where optionally the solvent comprises at least one selected from the group consisting of tetrahydrofuran (THF) and N-methylpyrrolidone (NMP).

[0211] In some embodiments, the compound of formula (1-2) is a compound of formula (1-1): TIFF2025529217000163.tif10128 with an alkoxide base, wherein X 1 , X 2 , and X 3 are each independently selected from the group consisting of Cl, Br, and I.

[0212] In some embodiments, the contacting step is performed in the presence of a solvent, wherein the solvent optionally comprises THF. In some embodiments, the alkoxide base comprises a tert-butoxide base, wherein the tert-butoxide base is optionally potassium tert-butoxide. In some embodiments, the contacting step is performed at a temperature of about 60°C to about 80°C.

[0213] Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticles (LNPs) of the present disclosure and at least one pharmaceutically acceptable carrier. In some embodiments, the composition further comprises at least one adjuvant.

[0214] Such pharmaceutical compositions may consist of at least one composition of the present invention in a form suitable for administration to a subject, or may comprise at least one composition of the present invention and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combination thereof. At least one composition of the present 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.

[0215] In some embodiments, pharmaceutical compositions useful for practicing the methods of the present invention can be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day, hi other embodiments, pharmaceutical compositions useful for practicing the present invention can be administered to deliver a dose of 1 ng / kg / day to 1,000 mg / kg / day.

[0216] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention may vary depending on their identities, their sizes, and the condition of the subject being treated, as well as the route by which the composition is administered. As an example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0217] Pharmaceutical compositions useful in the methods of the present invention may be suitably formulated for nasal, inhalation, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, epidural, intrathecal, intravenous, or other route of administration. Compositions useful in the methods of the present invention may be administered directly to the brain, brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include protruding nanoparticles, microspheres, liposome preparations, coated particles, polymer complexes, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

[0218] In some embodiments, the compositions of the present invention are part of a pharmaceutical matrix, which allows for the manipulation of insoluble substances and the improvement of their bioavailability, the development of controlled-release or sustained-release products, and the creation of uniform compositions. For example, pharmaceutical matrices can be prepared using hot-melt extrusion, solid solutions, solid dispersions, size reduction techniques, molecular complexes (e.g., cyclodextrins and others), microparticulation, and particle and formulation coating processes. In such processes, amorphous or crystalline phases can be used.

[0219] The route of administration will be readily apparent to one of skill in the art, and will vary depending on any number of factors, including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, etc.

[0220] The formulation of the pharmaceutical composition described herein can be prepared by any method known in the field of pharmacology and pharmacy or any method that will be developed in the future.In general, such preparation methods include combining the active ingredient 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.

[0221] As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of 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 one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose, or may be for one of multiple daily doses (e.g., about 1 to 4 times per day or more). When multiple daily doses are used, the unit dosage form may be for each dose being the same or each dose being different.

[0222] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans as prescription drugs, those skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and veterinary pharmacologists of ordinary skill can design and implement such modifications with no more than routine experimentation, if any. It is intended that the pharmaceutical compositions of the present invention be administered to humans and other primates, as well as mammals, including commercially relevant mammals, including, but not limited to, cows, pigs, horses, sheep, cats, and dogs.

[0223] In some embodiments, compositions of the present invention are formulated with one or more pharmaceutically acceptable excipients or carriers. In some embodiments, pharmaceutical compositions of the present invention comprise a therapeutically effective amount of at least one compound of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatin (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions, such as phosphate solutions and organic acid salt solutions. Examples of these and other pharmaceutically acceptable carriers are described in "Remington's Pharmaceutical Sciences" (1991, Mack Publication Co., New Jersey).

[0224] The carrier can be a solvent or dispersion medium, and these include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), recombinant human albumin, solubilized gelatin, suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Often, the composition includes an isotonic agent, such as sugar, sodium chloride, or a polyalcohol, such as mannitol and sorbitol. Prolonged absorption of injectable compositions can be achieved by including in the composition a substance that delays absorption, such as aluminum monostearate or gelatin.

[0225] The formulation may be used in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalation, intravenous, subcutaneous, transdermal, enteral, or any other suitable administration mode, and known in the art. The pharmaceutical preparation may be sterilized, and if desired, the pharmaceutical preparation may be mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic buffering, coloring substances, flavoring substances, and / or aroma substances. If desired, the above may also be combined with other active ingredients, such as other analgesics, anxiolytics, hypnotics, etc. As used herein, "additional ingredients" includes, but is not limited to, one or more ingredients that can be used as pharmaceutical carriers.

[0226] The compositions of the present invention may contain a preservative in an amount of about 0.005% to 2.0% by weight of the total composition. Preservatives are used to prevent deterioration when exposed to environmental contaminants. Examples of preservatives useful in the present invention include, but are not limited to, those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea, and any combination thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05 to 0.5% sorbic acid.

[0227] The compositions may include antioxidants and chelating agents, which inhibit the degradation of the compounds. Antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid in an exemplary range of about 0.01% to 0.3% by weight, or BHT in an exemplary range of 0.03% to 0.1% by weight, based on the total weight of the composition. The chelating agent may be present in an amount of 0.01% to 0.5% by weight, based on the total weight of the composition. Exemplary chelating agents include edetate (e.g., edetate disodium) and citric acid in an exemplary range of about 0.01% to 0.20% by weight, or 0.02% to 0.10% by weight, based on the total weight of the composition. Chelating agents are useful for chelating metal ions in the compositions, which may impair the shelf life of the formulation. BHT and edetate disodium are exemplary antioxidants and exemplary chelating agents, respectively, for some compounds, but these may be substituted with other antioxidants and chelating agents that are suitable and equivalent and would therefore be known to those skilled in the art.

[0228] Liquid suspensions can be prepared by conventional methods to suspend active ingredients in aqueous or oily vehicles.Aqueous vehicles include, for example, water and isotonic saline.Oil vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.Liquid suspensions can further contain one or more additional ingredients, including but not limited to suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners.Oil suspensions can also contain thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats and oils, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include naturally occurring phosphatides such as lecithin, as well as condensation products of alkylene oxides with fatty acids, long-chain aliphatic alcohols, partial esters derived from fatty acids and hexitols, or partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, gum arabic, and ionic or nonionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoate, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0229] Liquid solutions in which the active ingredient is in an aqueous or oily solvent can be prepared in substantially the same manner as liquid suspensions, with the primary difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, "oily" liquids are those that contain carbon-containing liquid molecules and exhibit lower polarity than water. Liquid solutions that are pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, although it is understood that the suspending agent may not necessarily support the 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 peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0230] Pharmaceutical compositions of the present invention may also be prepared, packaged, or sold in the form of oil-in-water or water-in-oil emulsions. The oil phase may be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or a combination thereof. Such compositions may further comprise one or more emulsifiers, such as naturally occurring gums, such as gum arabic or gum tragacanth; naturally occurring phosphatides, such as soy phosphatides or lecithin phosphatides; esters or partial esters derived from the combination 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, sweeteners or flavoring agents.

[0231] Methods for impregnating or coating a substance with a chemical composition are known in the art, including methods of depositing or bonding a chemical composition to a surface, methods of incorporating a chemical composition into the structure of a substance (i.e., a physiologically degradable substance, etc.) during its synthesis, and methods of absorbing an aqueous or oily solution or suspension into an absorbent substance, which may or may not be followed by a drying step. Methods of mixing the ingredients include physical grinding, the use of pellets in solid and suspension formulations, and mixing into transdermal patches, as known to those skilled in the art.

[0232] Administration / Dosage Dosage regimen may affect what constitutes an effective amount.The therapeutic preparation can be administered to patients either before or after the onset of disease or disorder.Furthermore, the dosage can be divided into several portions and administered daily or sequentially, and the dosage can be staggered and administered daily or sequentially, or the dosage can be continuously infused or bolus injected.Furthermore, when indicated by the urgency of treatment or prevention, the dosage of the therapeutic preparation can be increased or decreased proportionately.

[0233] Administration of the compositions of the present disclosure to patients, e.g., mammals, e.g., humans, can be carried out using known procedures at dosages and for periods of time effective to treat the diseases or disorders contemplated herein. The effective amount of therapeutic agent (i.e., composition) required to achieve a therapeutic effect can vary depending on factors such as: the activity of the particular therapeutic agent utilized; the time of administration; the rate at which the composition is excreted; the duration of treatment; other drugs, compounds, or other substances used in combination with the composition; the state of the disease or disorder, age, sex, weight, health status, general health, and medical history of the patient being treated, and similar factors well known in the medical field. Dosage regimens can be adjusted to provide the best therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced if indicated by the exigencies of the treatment situation. For the therapeutic compositions of the present disclosure, a non-limiting example of an effective dosage range is from about 0.01 mg / kg body weight to 100 mg / kg body weight of the active agent (i.e., nucleic acid) per day. One of ordinary skill in the art would be able to examine the factors related to the effective amount of a therapeutic composition and make the determination regarding the effective amount without undue experimentation.

[0234] The composition may be administered to an animal several times a day, or the composition 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 few months or once a year, or even less frequently. It is understood that the amount of the composition divided into daily doses can be administered, for example, every day, every other day, every two days, every three days, every four days, or every five days, as a non-limiting example. For example, in every other day administration, a dose of 5 mg per day can be administered starting on Monday, followed by a dose of 5 mg per day on Wednesday, followed by a dose of 5 mg per day on Friday, and so on. The frequency of administration is readily apparent to those skilled in the art and varies depending on several factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the animal.

[0235] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, a particular composition, and a particular mode of administration, without causing toxicity to the patient.

[0236] A medical doctor, such as a physician or veterinarian, having ordinary skill in the art could 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 present disclosure utilized in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0237] In certain embodiments, it is particularly advantageous to formulate the compound into a unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for a patient to be treated; each unit contains a predetermined amount of a therapeutic composition that, in association with the required pharmaceutical vehicle, produces a desired therapeutic effect. The unit dosage forms of the present disclosure are defined by and directly vary according to (a) the characteristics specific to the therapeutic composition and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art in compounding / formulating such therapeutic compositions to treat diseases or disorders in patients.

[0238] In some embodiments, the compositions of the present disclosure are administered to patients at a dosage frequency ranging from 1 to 5 times per day or more. In other embodiments, the compositions of the present disclosure are administered to patients at dosage frequencies ranging from, but not limited to, once per day, once every two days, once every three days to once per week, and once every two weeks. Those skilled in the art will readily appreciate that the administration frequency of the various combination compositions of the present disclosure will vary from subject to subject depending on many factors, including, but not limited to, age, the disease or disorder being treated, gender, general health, and other factors. Therefore, the present disclosure should not be construed as being limited to any particular dosing regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0239] The amount of active agent in the compositions of the present disclosure for administration may range from about 1 μg to about 7,500 mg, from about 20 μg to about 7,000 mg, from about 40 μg to about 6,500 mg, from about 80 μg to about 6,000 mg, from about 100 μg to about 5,500 mg, from about 200 μg to about 5,000 mg, from about 400 μg to about 4,000 mg, from about 800 μg to about 3,000 mg, from about 1 mg to about 2,500 mg, from about 2 mg to about 2,000 mg, from about 5 mg to about 1,000 mg, from about 10 mg to about 750 mg, from about 20 mg to about 600 mg, from about 30 mg to about 500 mg, from about 40 mg to about 400 mg, from about 50 mg to about 300 mg, or from about 60 The dosage may be in the range of about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any whole or partial increment therebetween.

[0240] In some embodiments, the amount of active agent (i.e., nucleic acid) present in the compositions of the present disclosure is from about 0.5 μg to about 5,000 mg. In some embodiments, the amount of active agent present in the compositions of the present disclosure used in the 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, the dose of the second compound 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 whole or partial increments thereof.

[0241] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a composition of the present 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.

[0242] The term "container" includes any receptacle for holding a pharmaceutical composition or for managing stability or moisture absorption. For example, in some embodiments, the container is a package containing the pharmaceutical composition, such as a liquid (solution and suspension) formulation, a semisolid formulation, a lyophilized solid formulation, a solution and powder formulation, or a lyophilized formulation present in a dual chamber. In other embodiments, the container is not a package containing the pharmaceutical composition, i.e., the container is a container, such as a box or vial, containing a packaged or unpackaged pharmaceutical composition and instructions for using the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. It should be understood that a package containing a pharmaceutical composition may include instructions for using the pharmaceutical composition, thereby increasing the functional relevance of the packaged product. However, it should be understood that the instructions may include information related to the ability of the compound to perform its intended function, such as treating, preventing, or reducing a disease or disorder in a patient.

[0243] Administration Routes of administration of any of the compositions of the present disclosure include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivaginal), nasal, and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, epidural, intrathoracic, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0244] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gelcaps, 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, etc. It should be understood that the formulations and compositions that may be useful in the present disclosure are not limited to the particular formulations and compositions described herein.

[0245] Parenteral administration As used herein, "parenteral administration" of pharmaceutical compositions includes any administration route characterized by creating a physical opening in the target tissue and administering the pharmaceutical composition through the opening in the tissue.Therefore, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the pharmaceutical composition, applying the pharmaceutical composition through a surgical incision, applying the pharmaceutical composition through a non-surgical wound that reaches the tissue, etc.In particular, parenteral administration is intended to include, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and kidney dialysis infusion techniques.

[0246] Pharmaceutical compositions suitable for parenteral administration include 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 continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold as devices, such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in aqueous or oily vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry form (i.e., powder or granular form) for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) and subsequent parenteral administration of the reconstituted composition.

[0247] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Such suspensions or solutions may be formulated according to known techniques and may contain additional ingredients in addition to the active ingredient, such as dispersing agents, wetting agents, or suspending agents described herein. For example, such sterile injectable formulations may be prepared using a non-toxic, parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol. 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 diglycerides. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in recombinant human albumin, in fluid gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained release or implantable compositions may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. [Example]

[0248] Various aspects of the present application may be better understood with reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the present application to the examples provided herein.

[0249] material and method material All LNP lipid excipients except IL and lipids for artificial endosome production were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cas9 and firefly luciferase mRNA with 5-methoxyuridine substitutions were purchased from TriLink Biotechnologies (San Diego, CA, USA). The TTR sgRNA had the following sequence: It was synthesized by Axolabs (Kulmbach, Germany) using TIFF2025529217000164.tif11150, where N refers to an RNA residue, n is a 2'-O-methyl residue, and s is a phosphorothioate backbone modification.

[0250] The chemicals 1,2-epoxyoctane, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, and 8-bromo-1-octene were purchased from TCI (Montgomeryville, PA, USA); Triton X-100 was purchased from Alfa Aesar (Haverhill, MA, USA); 1,12-dibromododecane and N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine were purchased from AmBeed (Arlington Heights, IL, USA); anhydrous 1-methyl-2-pyrrolidinone and chloroform-d were purchased from Acros Organics (Geel, Belgium); all non-anhydrous solvents, anhydrous magnesium sulfate, and 1 N hydrochloric acid were purchased from Fisher Scientific (Waltham, MA, USA). 10-Bromo-1-decene was purchased from Oakwood Chemicals (Estill, SC, USA); 6-Bromo-1-hexene was purchased from Asta Tech (Bristol, PA, USA); and 2-{2-[4-(2-{[2-(2-aminoethoxy)ethyl]amino}ethyl)piperazin-1-yl]ethoxy}ethan-1-amine was purchased from Enamine (Kiev, Ukraine). All other chemical reagents were purchased from MilliporeSigma (St. Louis, MO, USA).

[0251] synthesis All flash chromatography was performed using RediSep Gold® silica gel disposable flash columns on a Teledyne Isco (Lincoln, NE, USA) CombiFlash NextGen 300+ equipped with an evaporative light scattering detector. Evaporation of solvents was performed using a Buchi (New Castle, DE, USA) Rotavapor® R-300 System Professional. 1 H and 13 C NMR spectra were obtained in d-chloroform using an Avance Neo 400 MHz spectrometer (Bruker, Billerica, MA, USA). LC-MS spectra were obtained in ethanol using an Acquity UPLC (Milford, MA, USA) equipped with an SQD, using a C8 column with a 2-minute wash followed by a mobile phase gradient of 50% water (1% trifluoroacetic acid) and 50% acetonitrile (1% trifluoroacetic acid) to 100% acetonitrile (1% trifluoroacetic acid).

[0252] 12-Bromododec-1-ene To a 250 mL round-bottom flask were added 1,12-dibromododecane (8.00 g, 24.4 mmol, 2.0 equiv.) and anhydrous tetrahydrofuran (20 mL). Next, potassium tert-butoxide (2.74 g, 24.4 mmol, 1.0 equiv.) in anhydrous THF (50 mL) was added dropwise. The reaction was stirred at 70 °C for 16 h. The reaction was then quenched with deionized water (40 mL) and extracted with hexane (3 x 35 mL). The organic fractions were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by CombiFlash-assisted flash chromatography by injecting the liquid onto an 80 g column. The mobile phase was isocratic hexane for 10 min at a flow rate of 20 mL / min. The product was isolated as a clear oil in 25.8% yield. TIFF2025529217000165.tif31141

[0253] General Procedure "A": Synthesis of Branched Alkenes A 100 mL Schlenk flask was purged with nitrogen. Anhydrous tetrahydrofuran (5 mL or 10 mL), N-methylpyrrolidinone (48.0 mmol, 4.0 equiv.), dilithium tetrachlorocuprate (0.1 M tetrahydrofuran; 0.36 mmol, 0.03 equiv.), and the corresponding bromoalkene (12.0 mmol, 1.0 equiv.) were added to the flask. The solution was stirred at room temperature under nitrogen for 5 minutes. The flask was then placed in a room-temperature water bath. The corresponding Grignard reagent (13.2 mmol, 1.1 equiv.) was then added dropwise. After 5 minutes, the flask was removed from the water bath and then stirred at room temperature for 1 hour. The flask was then cooled to 0 °C and then slowly quenched with hydrochloric acid (1 M; 40 mL). The aqueous phase was extracted with hexane (3 x 20 mL), and the organic layers were combined, washed with hydrochloric acid (1 M; 1 x 40 mL), brine (2 x 40 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by CombiFlash-assisted flash chromatography by injecting the liquid onto a 40 g column. The mobile phase was isocratic hexane, and a flow rate of 7 mL / min was used. The product was isolated as a clear oil. Specific conditions for each reaction are described in the Supporting Information.

[0254] General Procedure "B": Synthesis of Branched Epoxides To a 100 mL round-bottom flask were added the corresponding branched alkene (1.0 equiv.) and dichloromethane (5 mL). The flask was mixed for 1 minute and then cooled to 0 °C. Next, half of a solution of meta-chloroperbenzoic acid (70% purity; 2.0 equiv.) dissolved in dichloromethane (30 mL) was added dropwise. The mixture was stirred for 1 hour, after which the second half of the meta-chloroperbenzoic acid solution in dichloromethane was added dropwise. After 1 hour, the reaction flask was removed from the 0 °C water bath and stirred at room temperature for 14 hours. The reaction was quenched by adding 20 mL of a 1:1 solution of saturated sodium bicarbonate and saturated sodium thiosulfate. The layers were separated, and the organic layer was washed with brine (1 x 30 mL). The aqueous layers were then combined and extracted with DCM (3 x 15 mL). The organic layers were combined, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified by CombiFlash-assisted flash chromatography by loading the liquid onto a 24 g column. The mobile phase had a gradient from 100% hexane to 90% hexane and 10% ethyl acetate over 15 min, using a flow rate of 35 mL / min. The product was isolated as a clear oil. Specific conditions for each reaction are described in the Supporting Information.

[0255] General Procedure "C": Synthesis of Ionizable Lipids To a 1-dram vial were added the corresponding polyamine core (1.0 equiv.), the corresponding epoxide (6.0 equiv. or 7.0 equiv.), and ethanol (0.3 mL). The reaction was stirred at 80°C for 48 hours. The solution was then diluted with dichloromethane (0.7 mL). The solution was purified by CombiFlash-assisted flash chromatography by injecting the liquid onto a 12 g column. The mobile phase had a gradient from 95% dichloromethane and 5% Ultrasolution (75% dichloromethane, 22% methanol, and 3% aqueous ammonium hydroxide) to 80% dichloromethane and 20% Ultrasolution over 35 minutes, using a flow rate of 7 mL / min. The product was isolated as a viscous, yellow to clear oil. Specific conditions for each reaction are described in the Supporting Information.

[0256] LNP formulation An ethanol phase containing each lipid (i.e., ionizable lipid, neutral lipid, and lipid complex) used in the formulation, as well as cholesterol, and an aqueous phase containing mRNA were mixed using a microfluidic device to prepare LNPs. The ethanol phase consisted of the corresponding ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000), at fixed molar ratios of 35%, 16%, 46.5%, and 2.5%, respectively. The aqueous phase consisted of 10 mM citrate buffer in which the mRNA had been dissolved. The aqueous and ethanol phases were mixed at flow rates of 1.8 mL / min and 0.6 mL / min, respectively, using a Pump33DS syringe pump. The LNPs were dialyzed in 1× phosphate-buffered saline for 2 hours using a microdialysis cassette (20,000 MWCO, Thermo Fisher Scientific, Waltham, MA) and then filtered through a 0.22 μm filter.

[0257] Encapsulation efficiency of LNPs The mRNA encapsulation efficiency of each LNP formulation was calculated using the Quant-iT-RiboGreen (Thermo Fisher Scientific, Waltham, MA) assay as previously described (Heyes et al., 2005, J. Controlled Release. 107:276-287). Each LNP sample was diluted to approximately 2 ng / μL in two microcentrifuge tubes containing 1X TE buffer or 0.1% (v / v) Triton X-100 (Sigma-Aldrich). After 20 minutes, LNPs in TE buffer and Triton X-100, as well as mRNA standards, were plated in triplicate in a black 96-well plate, and fluorescent RiboGreen reagent was added according to the manufacturer's instructions. Fluorescence intensity was read at an excitation wavelength of 480 nm and an emission wavelength of 520 nm on an Infinite 200 Pro plate reader (Tecan). RNA content was estimated by comparison with a standard curve estimated using least-squares linear regression (LSLR). Encapsulation efficiency was calculated as (B A) / B 100, where A is the RNA content in TE buffer and B is the RNA content in Triton X-100. Encapsulation efficiency is expressed as the mean ± standard deviation (n = 3).

[0258] Dynamic Light Scattering and Zeta Potential To measure baseline dynamic light scattering (DLS), 10 μL of each LNP solution was diluted 100-fold with 1X PBS in a 4 mL disposable cuvette. To measure baseline zeta potential, 20 μL of each LNP solution was diluted 50-fold with deionized water in a zeta potential cuvette, DTS1070 (Malvern Panalytical, Malvern, UK). Four measurements, each with at least 10 runs, were recorded for each sample using a Zetasizer Nano (Malvern Instruments, Malvern, UK). Data are presented as mean ± standard deviation (n = 3–4 measurements).

[0259] Determination of pKa of LNPs Surface ionization measurements to calculate the pKa of each LNP formulation were performed as previously described (Hajj et al., 2019, Small, 15:1805097). Buffer solutions containing 150 mM sodium chloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 mM ammonium citrate were adjusted to pH 2–12 in 0.5 increments. 125 μL of each pH-adjusted solution and 5 μL of each LNP formulation were added in triplicate to a black 96-well plate. 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS) was then added to each well to a final TNS concentration of 6 μM. Fluorescence intensity was read at an excitation wavelength of 322 nm and an emission wavelength of 431 nm on an Infinite 200 Pro plate reader (Tecan, Morrisville, NC). The pKa was obtained using least squares regression as the pH corresponding to half-maximal fluorescence intensity, i.e., 50% protonation.

[0260] LNP-mediated in vitro delivery of luciferase mRNA into HeLa cells HeLa cells (ATCC No. CCL-2) were cultured in L-glutamine-containing DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were plated at 20,000 cells per well in 100 μL of medium in tissue culture-treated 96-well plates and allowed to adhere overnight. The LNP formulation was used to treat cells at a dose of 20 ng of mRNA (i.e., TriLink luciferase) per 20,000 cells. One group of cells, designated "untreated," was treated with medium alone. After 24 hours of treatment with LNP, the medium was removed. 50 μL of 1X lysis buffer (Promega, Madison, WI) was added to each well, followed by 100 μL of luciferase assay substrate (Promega). After a 10-minute incubation, luminescence was quantified using an Infinite 200 Pro plate reader (Tecan). The luminescence signal for each condition was normalized by dividing it by the luminescence signal of the LNP (i.e., C14-494) referred to as the control.

[0261] To assess cytotoxicity, additional plates were prepared as described elsewhere herein. After 24 hours, 100 μL of CellTiter-Glo (Promega) was added to each well, and after 10 minutes of incubation, luminescence corresponding to ATP production was quantified using a plate reader. Luminescence for each group was normalized by dividing by the luminescence signal of untreated control cells.

[0262] Luciferase expression and percent viability are expressed as mean ± standard deviation (n = 3–4 biological replicates and at least 4 technical replicates per plate). For luciferase expression, a two-way ANOVA with Dunnett's multiple comparison test was used to compare means among formulations and treatment conditions.

[0263] Luciferase imaging and quantification Luciferase signals were assessed in Black 6 mice after intravenous tail vein injection of luciferase mRNA. Specifically, mice were imaged 12 hours after IV injection of LNP or PBS. Luciferase imaging was performed using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). Mice were intraperitoneally injected with 150 mg / kg D-luciferin and potassium salt (Biotium, Fremont, CA) 10 minutes before sacrifice and imaging. Subsequently, the mouse liver, spleen, lungs, kidneys, and heart were excised and imaged using IVIS. Image analysis was performed using Living Image software (PerkinElmer). Whole-body and organ bioluminescence values ​​shown represent the mean ± standard deviation (SD) (n ≥ 3). IVIS images of representative organs are shown, representing the maximum luminescence values ​​for each treatment condition.

[0264] In vivo biodistribution studies Female C57BL / 6J mice, 6–8 weeks old and weighing an average of 20 g, were purchased from Jackson Laboratory (Bar Harbor, ME). Fluc mRNA-encapsulated LNPs were injected into the lateral tail vein at a dose of 0.1 mg of mRNA per kg of body weight (mg / kg). After 12 hours, the ventral hair of the mice was removed using Veet Gel Cream Hair Remover (Reckitt Benckiser, Slough, UK). Then, the mice received an intraperitoneal injection of D-luciferin (0.2 mL, 15 mg / mL; Biotium, Fremont, CA). Five minutes later, whole-body luminescence images were acquired using an in vivo imaging system (IVIS; PerkinElmer, Waltham, MA). The mice were then euthanized, and the heart, lungs, liver, kidneys, and spleen were removed and imaged for luminescence using the IVIS. Luminescent flux was quantified with Living Image software (PerkinElmer) by setting a rectangular region of interest (ROI) surrounding the whole-body or organ image and maintaining the same ROI size between each whole body or organ. Total flux is expressed as the mean ± SEM of n = 3 biological replicates.

[0265] TTR gene editing The day before injection, blood was collected from mice via retroorbital bleeding. Serum was isolated by centrifuging the blood at 3,500 rpm for 15 minutes in Microtainer blood collection tubes (BD, Franklin Lakes, NJ, USA) containing serum separator gel. Mice were injected via the lateral tail vein with LNPs containing a 1:3 molar ratio of Cas9 mRNA and TTR sgRNA at a dose of 1.0 mg / kg. Seven days later, serum was isolated as described above, mice were euthanized, and livers were removed. Serum TTR levels were measured using a mouse prealbumin ELISA kit (Aviva Systems Biology, San Diego, CA, USA) according to the manufacturer's instructions.

[0266] For indel analysis, DNA was extracted from the liver using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and quantified using the nanodrop plate attachment on an Infinite 200 Pro plate reader (Tecan). PCR amplification of the TTR target site was performed using Q5 high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and the following primer sequences: mTTR-exon2-F, mTTR-exon3-F, mTTR-exon4-F, mTTR-exon5-F, mTTR-exon6-F, mTTR-exon7-F, mTTR-exon8-F, mTTR-exon9-F, mTTR-exon10-F, mTTR-exon11-F, mTTR-exon2-F, mTTR-exon3-F, mTTR-exon4-F, mTTR-exon5-F, mTTR-exon12-F, mTTR-exon2-F, mTTR-exon2-F, mTTR-exon3-F, mTTR-exon4-F, mTTR-exon5-F, mTTR-exon1 ...2-F, mTTR-exon3-F, mTTR-exon4-F, mTTR-exon4-F, mTTR-exon5-F, mTTR-exon1 TIFF2025529217000166.tif4128 and mTTR-exon2-R, Deep sequencing of TTR amplicons and determination of the frequency of on-target indels was performed essentially as described, except that 150-bp paired-end reads were performed.

[0267] Primary T cell luminescence In certain embodiments, the present disclosure provides primary T cell luminescence data for evaluating the LNPs of the present disclosure using T cells from healthy human donors. Cells were plated at 60,000 cells per well with 200 ng of mRNA (i.e., TriLink luciferase) per well and treated with the LNPs of the present disclosure.

[0268] One group of cells, designated "untreated," was treated with medium alone. After 24 hours of treatment with LNP, the cells were centrifuged at 700 g for 5 minutes, and the medium was removed. 50 μL of 1X lysis buffer (Promega, Madison, WI) was added to each well, followed by 100 μL of luciferase assay substrate (Promega). After a 10-minute incubation, luminescence was quantified using an Infinite 200 Pro plate reader (Tecan). The luminescence signal for each condition was normalized by dividing it by the luminescence signal of the LNP (i.e., C14-494), designated as the control. For luciferase expression, a one-way ANOVA with Sidak's multiple comparisons correction was performed using the B10 formulation as the control. Additionally, a two-way ANOVA with Dunnett's multiple comparisons test was used to compare means between formulations and treatment conditions.

[0269] To assess cytotoxicity, additional plates were prepared as described elsewhere herein. After 24 hours, cells were centrifuged at 700 g for 5 minutes, and 100 μL of CellTiter-Glo (Promega) was added to each well. After 10 minutes of incubation, luminescence corresponding to ATP production was quantified using a plate reader. Luminescence for each group was normalized by dividing by the luminescence signal of untreated control cells.

[0270] Cryo-transmission electron microscopy Shape and size were analyzed by cryo-TEM by applying 3 μL of LNP at a concentration of 50 ng / μL mRNA to glow-discharged Quantifoil porous carbon grids. Grids were blotted and flash-frozen in liquid ethane using a Vitrobot Mark IV. Imaging was performed on a Titan Krios equipped with a K3 Bioquantum at the Beckman Center for Cryo-EM.

[0271] Stability analysis of LNPs LNPs were diluted 10-fold with either 1X PBS or additive-containing DMEM (Gibco). The hydrodynamic diameter and PDI of LNPs were measured every hour at 37°C using a DynaPro plate reader III (Wyatt Technology) as described above. All samples were run in duplicate. Additive-containing DMEM was used as a control.

[0272] Knockdown of Kupffer cells Mice were depleted of macrophage cells by injecting 0.2 mL of clodronate liposomes (Liposoma, Amsterdam, Netherlands) at a dose of 5 mg / mL via the lateral tail vein. 24 hours later, mice were reinjected via the lateral tail vein with fluc-encapsulated LNPs at a dose of 0.1 mg / kg. After 12 hours, liver luminescence was quantified as described above.

[0273] To confirm Kupffer cell depletion, a subset of mice was injected with clodronate liposomes as described above. After 24 hours, the liver was perfused with 10 mL of 1X PBS, followed by 10 mL of supplemented DMEM containing 5 mg / mL collagenase IV (STEMCELL Technologies, Vancouver, Canada). The liver was then removed and placed in 5 mL of RPMI containing 5 mg / mL collagenase IV for 1 hour at room temperature. The liver lobes were then separated, crushed using a syringe slider, and passed through a 70 μm filter. The suspension was then centrifuged at 100 rpm for 3 minutes to pellet the hepatocytes. The supernatant was transferred to a new tube, and up to 30 mL of supplemented DMEM was added. The centrifugation process was repeated two more times. The three separate supernatants were then centrifuged at 200 rpm for 10 minutes, and the upper portion of the supernatant was discarded, leaving approximately 5 mL in each tube. The fractions were combined into one tube and centrifuged at 25 g for 5 minutes. The cell pellet was washed with 6 mL of DMEM containing additives, centrifuged again, and the upper half of the supernatant was collected. Cells were counted as described above and then analyzed by flow cytometry.

[0274] LNP accumulation LNPs were reconstituted with fluc mRNA as described above. They were then mixed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR; 5 μM in DMSO; Thermo Fisher Scientific) at a volume ratio of 50:1. The solution was placed on a plate shaker at 200 rpm for 25 minutes at room temperature. Mice were then administered the LNPs and dissected as described above. Organ fluorescence was captured using the specific "DiR" settings in Living Image. ROIs were captured as described above. Total radiant efficiency is expressed as the mean ± SEM of n = 3 biological replicates.

[0275] Artificial endosome disruption assay Artificial endosomes were prepared by the lipid thin film hydration method. DOPS, DOPC, DOPE, NBD-PE, and Rho-PE were added to a 3 mL vial in a molar ratio of 25:25:48:1:1. To prevent photobleaching, the rotary evaporator was wrapped in aluminum foil and the lipids were concentrated under vacuum. After 2 h of concentration, the samples were rehydrated with 1X PBS (pH 7.4) to a final concentration of 1 mM for 20 min at room temperature using a Branson 3800 ultrasonic cleaner (Brookfield, CT, USA).

[0276] The assay was performed in a black-bottom 96-well plate. 0.1 mL PBS (pH 5.5, 0.1 M), 1 μL of artificial endosomes, and an amount of LNP equivalent to 400 ng were added to each well.

[0277] As a negative control, LNP was replaced with additional PBS, and as a positive control, LNP was replaced with 2% Triton-X100. Plates were wrapped in aluminum foil and incubated at 37°C. Fluorescence was measured at various time points over a 24-hour period.

[0278] Example 1: Synthesis of certain exemplary ionizable lipids, preparation of lipid nanoparticle (LNP) formulations, and their selected properties Ionizable lipids (ILs) can be prepared in one step by reacting monoamines or polyamines with lipids containing electrophilic functional groups (e.g., epoxides). This allows for the rapid generation of combinatorial libraries of ILs by simply mixing different amine cores with epoxides of different lengths. Thus, with the goal of generating ILs with greater structural diversity, a synthetic approach was developed to synthesize epoxides of any desired length and with any desired terminal branching, which can then be further reacted with a number of polyamine or monoamine reagents.

[0279] To enable flexible synthesis of epoxides of diverse structures, primary bromoalkenes were coupled to branched halomagnesium alkyls via copper-catalyzed Grignard C-C coupling to generate branched alkenes (Figure 1). Primary bromoalkenes are common reagents and are therefore used herein to generate epoxides of various lengths. Longer bromoalkenes can be generated by monoselective elimination of dibromoalkanes with tert-butoxide. Many Grignard-functionalized alkyls are also commercially available due to their widespread use in other synthetic procedures and are utilized herein to generate terminally branched groups. Terminally branched alkenes were converted to the corresponding branched epoxides by mCBPA-mediated epoxidation. The C-C coupling and epoxidation steps can be completed and purified in less than 24 hours, demonstrating the simplicity of the method.

[0280] In this study, 12 different branched epoxides were synthesized, including non-limiting examples of branched groups of four different lipid lengths: isopropyl, tert-butyl, and sec-butyl. To generate the ILs, the epoxides were first S N The polyamine cores were reacted with 2-(2-aminoethoxy)-N-(2-(4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)ethyl)ethan-1-amine (494) or N-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine (200) via a 2 reaction (Figures 2A-2B). Polyamine cores 494 and 200 have previously been used to generate ILs and corresponding LNPs for several applications, including CAR T cell therapy, intrauterine delivery, and placental delivery, and therefore represent interesting structural motifs for these preliminary studies. As controls, four linear ILs with the same relative lipid lengths as the branched library were also prepared using the same S NA total of 16 exemplary ILs were synthesized using 494.

[0281] The nomenclature used herein to describe the branched lipids of the present disclosure uses the formula "AXb-C", where A is the reactive moiety of the lipid tail, b is the type of branching, C is the lipid core number, and X is the linker length or number of methylene units between A and B. The abbreviations for branching are "i" for isopropyl, "t" for tert-butyl, and "s" for sec-butyl. For unbranched ILs, their traditional names are used.

[0282] In one aspect, the present disclosure relates to lipid nanoparticles, comprising one or more ionizable lipid compounds of formula (I), one or more neutral lipids, cholesterol, and one or more complex lipids.In some embodiments, one or more complex lipids inhibit the aggregation of two or more lipid nanoparticles.In some embodiments, lipid nanoparticles further comprise one or more nucleic acid cargoes.The preparation and properties of such lipid nanoparticles are provided in more detail herein.

[0283] In some embodiments, LNPs of the present disclosure were prepared using a microfluidic device to prepare LNPs having the following molar ratios of components: (a) an ionizable lipid of Formula (I) (35 mol%); (b) DOPE (16 mol%); (c) cholesterol (46.5 mol%), and DMG-PEG. 2000(2.5 mol%). In some embodiments, the ionizable lipid of formula (I) is selected from the group consisting of E4i-494, E6i-494, E8i-494, E10i-494, E4t-494, E6t-494, E8t-494, E10t-494, E4s-494, E6s-494, E8s-494, E10s-494, E4i-200, E8i-200, E4t-200, E8t-200, E4s-200, and E8s-200 (Table 1). LNPs comprising each of the above-described ionizable lipids of formula (I) and having the above-described molar ratios are exemplified herein, and such LNP formulations are disclosed herein by reference to identifiers that refer to each of the ionizable lipids used to prepare the LNPs (e.g., the ionizable lipid E4i-494 was used to prepare an LNP designated E4i-494).

[0284] Because the exemplary LNPs of the present application and used in some of the exemplary uses and / or studies described herein are formulated using the same non-limiting molar ratios of excipients and using the same phospholipids and PEGylated lipids, the difference between each LNP formulation is the structure of the IL.

[0285] For consistency, as described herein, each LNP is named after its corresponding IL. LNPs were initially loaded with firefly luciferase (fluc) mRNA at a weight ratio of IL to mRNA of 10:1, and LNPs were characterized for their encapsulation efficiency, hydrodynamic diameter, zeta potential, and pK a The LNPs were characterized for their ion exchange potential (PDI) and ion exchange potential (PDI) (Table 2). Multiple parameters were evaluated, and no notable differences existed between LNPs using linear, isopropyl, tert-butyl, and sec-butyl ILs. All exemplary LNPs had encapsulation efficiencies >80% and ranged in size from 70 to 160 nm, with an average PDI of approximately 0.2. The zeta potential of the LNPs also ranged from 5.57 to -28.1, indicating that the majority had a neutral or weakly negative charge. Finally, the majority of LNPs had a pK near 6.a It has.

[0286] Table 2. Selected properties of exemplary LNPs and controls of the present disclosure. TIFF2025529217000168.tif172149 LNPs C8-494, C10-494, C12-494, and C12-200 correspond to LNPs having the same formulation as those disclosed herein, with differences in the ionizable lipid component, where the ionizable lipid was prepared using either polyamine core 494 or polyamine core 200 with an epoxide having a linear alkyl group with a linker length indicated by C and an integer (i.e., C10-494 corresponds to an ionizable lipid prepared using polyamine core 494 with 2-undecyloxirane).

[0287] Example 2: LNPs containing branched ionizable lipids enhance delivery of mRNA in vivo, ex vivo, and in vitro The present disclosure provides data regarding in vivo, ex vivo, and in vitro delivery of exemplary LNPs of the present disclosure.

[0288] The efficacy of exemplary LNPs of the present disclosure was evaluated in both in vitro and in vitro models. Each LNP was evaluated for transfection efficacy in HeLa cells by incubating the LNPs at a dose of 20 ng of mRNA per 20,000 cells for 24 hours. The results of luminescence showed that branching significantly enhanced LNP transfection by 10-fold at the shortest and longest lipid lengths, while branching did not significantly change or significantly reduce transfection at intermediate lengths (Figure 3A). Toxicity was investigated using the CellTiter-Glo assay, and statistically significant toxicity was demonstrated for seven LNPs; however, none of the LNPs had a viability below 80% (Figure 3B).

[0289] To verify the consistency of this trend, LNPs were intravenously administered to C57BL / 6J mice at a dose of 0.1 mg / kg. DLin-MC3-DMA (MC3), an FDA-approved LNP formulation for siRNA delivery, and C12-200, a LNP with a polyamine core similar to 494 but more potent for mRNA delivery, were also administered to serve as positive controls. After 12 hours, whole-body and organ luminescence measurements were acquired (Figures 3C-3D). Unlike the in vitro results, all LNPs with branched ILs (branched LNPs) performed similarly to or better than their linear counterparts (linear LNPs) for systemic and liver delivery of RNA. With regard to systemic delivery, six branched LNPs and only one linear LNP induced luciferase expression to the same extent as C12-200, whereas E4t-494 and E6t-494 outperformed C12-200, and linear LNP did not outperform C12-200.

[0290] All LNPs transfected preferentially into the liver over other organs, and the six LNPs with branched ILs performed comparable to or better than C12-200 in terms of liver luminescence, with E4i-494 producing 1.5-fold higher luminescence (Figures 3E–3G). Notably, none of the linear LNPs achieved a signal statistically equal to or greater than C12-200. When compared with MC3 but not C12-200, the four branched LNPs had significantly increased luminescence compared with MC3, whereas the linear LNPs did not have significantly increased luminescence compared with MC3.

[0291] To further explore the branched lipid paradigm, we investigated whether lipid branching could enhance the delivery of liver-trophic amine cores, particularly core 200 exemplified herein. To this end, six branched epoxides were coupled to core 200 to generate six new branched ILs, including core 200 (Figure 2B). The successful synthesis of core 200 branched ILs highlights the powerful nature of the original branched epoxide design, given its applicability to many different amine cores. While the three branched groups were evaluated at the corresponding lipid lengths of 8 and 12 carbons, respectively, the present disclosure is not limited to such chain lengths, and the corresponding linear versions, C8-200 and C12-200, were utilized as controls. While C12-200 is considered a standard liver-trophic IL, C8-200 performed significantly worse, providing an important test of whether branching could also transform this IL into a potent liver-targeting LNP.

[0292] These eight ILs were then formulated as LNPs containing fluc mRNA and characterized using the same methodology as described above (Figure 4A). Similar to the studies with 494 LNPs, studies in HeLa cells revealed contradictory results: C8-200 performed better or the same as its branched counterpart, while the two longer branched LNPs outperformed C12-200 (Figure 4B). None of the LNPs demonstrated in vitro toxicity at the concentrations tested in the luciferase assay. Core 200 LNPs were then intravenously injected into C57BL / 6J mice with 0.1 mg / kg fluc mRNA. All branched LNPs, including the C8-200 branched lipid, performed similarly to or better than C12-200 in terms of systemic luminescence and liver delivery (Figures 4C–4E), with E4s-200 inducing fivefold greater luminescence in the liver. These results demonstrate that branching at the lipid terminus enhances delivery to the liver regardless of the lipid core, providing essential design criteria for future ILs.

[0293] In some embodiments, the present disclosure provides delivery data, including luminescence data (e.g., whole-body, liver, and / or combined organ luminescence data) obtained by administering exemplary LNPs containing TriLink luciferase to Black 6 mice. In some embodiments, the mice were also administered one or more controls (e.g., LNPs containing MC3 and / or LNPs containing unbranched ionizable lipids). In some embodiments, a subset of LNPs containing ionizable lipids prepared from a polyamine core 494 was evaluated in this manner, and the data demonstrate that some LNPs of the present disclosure enhanced luminescence compared to LNPs containing unbranched ionizable lipids in terms of organ luminescence (FIG. 5A). In some embodiments, a subset of LNPs containing ionizable lipids prepared from a polyamine core 200 was evaluated in this manner, and the data demonstrate that some LNPs of the present disclosure enhanced luminescence compared to LNPs containing unbranched ionizable lipids in terms of combined organ luminescence (FIG. 5B).

[0294] Furthermore, the experiments provided herein demonstrate that the LNPs of the present disclosure were selective for distribution to the liver compared to the spleen, lung, kidney, and / or heart, with little or no luminescence observed in organs other than the liver (Figures 5A-5D).

[0295] Example 3: Branched LNPs induce potent gene editing LNP delivery was then evaluated in a clinically relevant mouse model of transthyretin (TTR)-mediated amyloidosis. TTR is a protein that, when overexpressed in the liver, generates amyloid fibrils that can induce restrictive cardiomyopathy and heart failure. LNP-based siRNA therapy, such as the MC3-based ONPATTRO®, has been used to silence TTR translation in the liver; however, due to the limited therapeutic window of siRNA, the therapy may require multiple injections. Alternatively, delivery of gene editing machinery may provide a single-dose therapy that can permanently reduce TTR to safe levels. Therefore, eight core 200 LNPs were reconstituted to encapsulate Cas9 mRNA and a single-stranded guide RNA (sgRNA) targeting TTR. C57BL / 6J mice were then injected with either 0.3 mg / kg or 1.0 mg / kg of the Cas9 and TTR sgRNA combination. Seven days later, blood was collected, serum was isolated, and TTR protein levels were assessed compared with serum isolated from the same mice 24 hours prior to testing (FIG. 6A).

[0296] After blood collection, mice were sacrificed, and livers were harvested, DNA isolated, and indels were analyzed using next-generation sequencing (NGS). While LNP C12-200 reduced TTR levels by 70%, several LNPs with branched ILs reduced TTR levels by 80–90% (Figure 6B). Additionally, these latter LNPs induced 50–60% indels, whereas LNP C12-200 induced only 40% indels (Figure 6C). These results demonstrate that lipid branching can enhance delivery independent of mRNA and demonstrate the versatility of this design.

[0297] To assess toxicity, mice were reinjected with the same formulation. Blood was collected 12 hours later, and major organs were removed, H&E stained, and analyzed for toxicity markers by independent experts 24 hours later. Blood analysis for liver damage markers revealed that E4i-200, E4s-200, and E8t-200 significantly elevated AST levels compared with the control group, whereas C12-200 did not. However, E4s-200, C12-200, E8i-200, and E8t-200 all had higher ALT levels than PBS.

[0298] Example 4: Branched ILs are more effective at promoting endosomal escape To identify the mechanism for the increased efficacy of branched LNPs, several potential explanations were evaluated. First, it was assessed whether lipid branching results in LNPs with optimal physicochemical parameters, which were determined by comparing the liver luminescence obtained for each of the LNPs from this study that contained fluc mRNA with the particle hydrodynamic diameter, PDI, zeta potential, and pK a This was achieved by correlating the lipid branching with the globular structure of the LNPs (Figures 7A-7E). Successful liver delivery was further correlated with transfection of HeLa cells (Figure 7F). Interestingly, after fitting the data using cubic least-squares regression, no parameters predicted successful liver delivery. Furthermore, HeLa cell transfection was also a very poor predictor of in vivo delivery. Following this study, we analyzed whether the shape of the branched LNPs was significantly different from that of linear LNPs; however, when their structures were analyzed by cryo-TEM, all LNPs had a similar ovoid shape (Figures 7G-7J). This suggests that lipid branching in this shape may have minimal impact on the spherical configuration of the LNPs, although it does not exclude the possibility of more subtle changes in bilayer and monolayer configurations.

[0299] Next, LNP stability was assessed by incubating exemplary LNPs at 37°C in PBS and in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The size and PDI of the LNPs were measured hourly by dynamic light scattering immediately after incubation for 24 hours (Figures 7K-7N). Consistent with the characterization results, no significant destabilization was observed in either PBS or DMEM, and in the latter, all LNPs formed a protein corona of similar size.

[0300] Because the physicochemical properties, shape, and stability of the particles appear unaffected by lipid branching, their role in hepatic transport and uptake was investigated. Several studies have demonstrated that IL structure can influence the specific hepatocytes that take up LNPs. For example, Kupffer cells, which are hepatic macrophages, may preferentially capture some types of LNPs, potentially resulting in differential intrahepatic distribution. Therefore, C57BL / 6J mice were injected with clodronate liposomes to deplete macrophages. 24 hours later, the mice were reinjected with eight core 200 LNPs encapsulating fluc mRNA (Figure 8E). After a total of 36 hours, liver luminescence imaging revealed a decrease in luminescence for all LNPs except C8-200 and E4i-200 (Figure 8A).

[0301] These data suggest that branching is unlikely to be a factor in macrophage uptake. Furthermore, the two LNPs with the shortest lipid length, C8-200 and E4i-200, transfected less frequently into macrophages than nonmacrophage cells; however, screening a larger library would be necessary to verify this trend.

[0302] Next, the role of protein corona interaction with the liver was investigated by injecting the fluc-containing LNPs E4i-200, E4t-200, E4s-200, and C12-200 at 0.1 mg / kg into apolipoprotein E (APOE) knockout mice. Liver luminescence was measured 12 hours later, and all four LNPs showed a dramatic decrease in luminescence, ranging from 100- to 1000-fold, compared to wild-type mice (Figure 8B). This suggests that both branched and unbranched LNPs target the liver via an APOE-mediated mechanism, as has been demonstrated with many other liver-targeting LNPs.

[0303] Next, particle accumulation in the liver was analyzed. LNPs containing fluc were reconstituted with 1 mol% DiR, a lipophilic carbocyanine near-infrared fluorescent dye. This fluorescent LNP was injected into C57BL / 6J mice at 0.1 mg / kg fluc mRNA. 12 hours later, major organs were harvested and fluorescently imaged. Here, fluorescence is a marker of LNP accumulation. Although mRNA transfection and translation occurred exclusively in the liver, LNP fluorescence was observed in both the liver and spleen (Figures 8C-8D). More interestingly, all LNPs showed similar fluorescence in both organs.

[0304] Without wishing to be bound by any theory, it was hypothesized that if all eight LNPs accumulated in the liver in relatively similar amounts, but branched LNPs promoted mRNA translation more, this result may be due to the branched ILs enabling endosomal escape and cytosolic translocation of a larger number of mRNA molecules. Although other reports exist that some structural moieties of ILs affect endosomal escape, the specific role of terminal branching has not yet been elucidated in the art.

[0305] To verify these results, eight types of LNPs were tested for their interaction with artificial endosomes, a useful strategy for examining their role in endosomal escape. Exemplary endosomes were prepared by thin-film hydration using DOPE, 18:1 Δ9-cis phosphocholine (DOPC), 18:1 phospho-L-serine (DOPS), 18:1 phosphoethanolamine complexed with NBD (NBD-PE), and 18:1 phosphoethanolamine complexed with Lissamine-Rhodamine B (Liss-Rhod-PE) in a molar ratio of 48:25:25:1:1, respectively. The latter two lipids are a FRET pair, which was used to monitor endosomal integrity. Dissociation of the FRET pair upon endosome disruption was accompanied by a corresponding increase in the fluorescence intensity of the donor fluorophore. For this test, each LNP was diluted 6-fold in pH 5.5 buffer to mimic endosomal conditions and then mixed with the artificial endosomes. The fluorescence intensity of the donor fluorophore was measured at various time points. Branched LNPs induced 2- to 3-fold higher fluorescence intensity than linear LNPs for both the C8-200 (Figure 8F) and C12-200 (Figure 8G) groups, supporting the idea that lipid branching promotes more pronounced endosomal escape, and the linear versions did not.

[0306] Example 5: Oral Cancer Tumor Suppressor Therapy The present disclosure further provides exemplary data related to the use of the LNPs described herein to treat and / or ameliorate oral cancer tumors. Exemplary LNPs of the present disclosure utilized LNPs containing ionizable lipids prepared from a polyamine core 494, including C8-494 to C14-494, E4i-494 to E10i-494, E4t-494 to E10t-494, and E4s-494 to E10s-494, while C12-200 was utilized as a positive control.

[0307] In this study, CAL-27 cells were used as a model to evaluate the usefulness of the LNPs of the present disclosure in delivering certain mRNA cargoes. CAL-27 cells are an HPV-negative human tongue squamous cell carcinoma, which is considered the gold standard cell line for studying HPV-negative oral cancer.

[0308] Exemplary LNPs were incubated with luciferase mRNA, and the resulting LNPs were then administered to cells at a dose of 20 ng of mRNA per 20,000 cells. After 24 hours, luciferase assays (FIG. 9A) and cell viability assays (FIG. 9B) were performed to analyze luciferase expression and toxicity, respectively. Luciferase assay data were normalized to C12-200. Some of the evaluated LNPs (e.g., E10i-494 and E10s-494) performed better than the C12-200 LNP, with two LNPs containing branched, ionizable lipids performing significantly better than the C12-200 LNP.

[0309] The present disclosure also describes the use of a mouse tumor model in which CAL-27 cells were inoculated into the right flank of Nu / J mice and tumors were allowed to grow for two weeks. Five exemplary LNPs containing luciferase mRNA, including two containing branched ionizable lipids and three containing linear ionizable lipids, were then injected intratumorally at a dose of 0.1 mg / kg. Some mice also received PBS as a control. The mice were sacrificed, and major organs and tumors were harvested and fluorescently imaged (Figures 10A-10B). The results presented herein demonstrate that the top-performing LNP (i.e., E10i-494), which contains branched ionizable lipids, exhibited a signal greater than 10-fold higher than that observed with LNPs containing C12-200.

[0310] The present disclosure further describes the use of the LNPs of the present disclosure in a viability assay (i.e., killing assay) using CAL-27 and OECM-1 cell lines, which are also HPV-negative squamous cell carcinoma cell lines. The exemplary LNPs described herein and utilized in this study contain p53 cargo. In one aspect, p53 is an ideal target for LNP-mediated inhibitor therapy, as over 70% of oral squamous cell carcinomas have mutated or downregulated p53. A range of mRNA doses (i.e., 5 ng / μL, 20 ng / μL, 50 ng / μL, 100 ng / μL, and 250 ng / μL) were utilized at different time intervals (i.e., 24 or 48 hours), and overall viability was measured (Figures 11A-11D). Low viability (i.e., cell death) was observed after both 24 and 48 hours when exemplary LNPs containing p53 mRNA at least partially encapsulated therein were applied.

[0311] Example 6: Stem Cell Reprogramming The present disclosure further provides exemplary data regarding the use of the LNPs described herein to reprogram stem cells (e.g., induced pluripotent stem cells (iPSCs)). iPSCs are derived from skin or blood cells that have been reprogrammed back to an embryonic pluripotent state, which can generate a source of any type of human cell needed for therapeutic purposes. For example, iPSCs can be differentiated into blood cells to create new blood that is cancer-free for leukemia patients.

[0312] Transfection of iPSCs with mRNA cargo allows for the expression of several biomolecules of interest, and may also allow differentiation into cell lines of interest (e.g., neurons and / or muscle cells) if desired. In the experiments described herein, iPSCs were transfected with mRNA at least partially encapsulated in exemplary LNPs of the present disclosure, which contained ionizable lipids prepared from polyamine core 494 and ionizable lipids prepared from polyamine core 200, while LNPs containing C12-200 served as a positive control.

[0313] An initial screening of exemplary LNP performance was performed using LNPs of the present disclosure containing luciferase mRNA (20 ng / 15,000 cells), and luciferase expression (FIG. 12A) and viability (FIG. 12B) were measured. Similar viability was observed for each of the exemplary LNPs, while some LNPs (i.e., E4i-200, E4t-200, and E4s-200) exhibited high relative fluorescence.

[0314] Dose-response experiments of luciferase luminescence (Figure 13A) and viability (Figure 13B) were performed on the top-performing LNPs identified in the screening study. LNP E4i-200 was identified as the top performer in terms of relative luminescence. LNP E4i-200 was reconstituted with mCherry. iPSC-SV20 cells were plated in 24-well plates overnight and then treated with mCherry-containing E4i-LNPs. Flow cytometry was performed to determine the percentage of successfully transfected cells, and the cells were imaged by fluorescence microscopy to visualize the mCherry signal (Figures 14A-14H).

[0315] Timepoint studies were performed in which mCherry (or control) transfected iPSCs were harvested after 24 hours (Figures 15A and 15E), 48 hours (Figures 15B and 15F), 72 hours (Figures 15C and 15G), and 96 hours (Figures 15D and 15H). The results provided herein demonstrate that the mCherry signal remained strong over 48 hours, as determined by FACS analysis.

[0316] Example 7: CAR-T cell therapy and CAR-natural killer (NK) cell therapy In another aspect, the present disclosure relates to the use of the LNPs of the present disclosure for use in CAR-T and CAR-NK therapy.

[0317] The experiments described herein utilize LNPs containing ionizable lipids prepared from polyamine core 494 and LNPs containing ionizable lipids prepared from polyamine core 200. Initial screening of LNPs was performed using exemplary LNPs containing luciferase mRNA, which were incubated with activated primary T cells (CD4+:CD8+ 1:1) from healthy human donors, where luciferase was present at a concentration of 200 ng mRNA / 60,000 cells. Luciferase expression (FIG. 16A) and cell viability (FIG. 16B) were measured after 24 hours, and LNPs E8i-200 and E10s-200 were found to perform better than LNP formulation B10 and exhibit minimal toxicity.

[0318] The formulation of LNP components in the top-performing LNPs identified in the screens described herein was modified to match the formulation used for LNP B10 (i.e., ionizable lipid:DOPE:cholesterol:C14PEG2000, 40:30:25:2.5), and the relative light output (FIG. 17A) and viability (FIG. 17B) of the exemplary LNPs in primary T cells were measured. LNP E8i-200 was identified as a top performer in this experiment, demonstrating nearly six-fold increased light output compared to B10, with minimal change in viability.

[0319] The present disclosure further describes the evaluation of exemplary LNPs for transfection of NK-92MI cells (i.e., immortalized human natural killer cells that autologously express IL-2). In these experiments, LNPs containing luciferase mRNA (200 ng mRNA / 60,000 cells) were incubated with NK-92MI cells, and luciferase expression was measured 24 hours later. Some exemplary LNPs containing branched ionizable lipids (e.g., E6i-200 and E8i-200) performed significantly better than LNPs containing C12-200, thus demonstrating the utility of the disclosed LNPs for transfection of immune cells (e.g., T cells and NK cells) (Figure 18).

[0320] Array List TIFF2025529217000169.tif56144

[0321] Numbered Aspects Exemplary aspects are provided below, the numbering of which should not be construed as indicating any level of importance.

[0322] Aspect 1 provides: An ionizable lipid compound of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: TIFF2025529217000170.tif22128In formula, R 1a and R 1b But each independently TIFF2025529217000171.tif11128; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h H, optionally substituted C1-C 12 Alkyl, optionally substituted C-C 12 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 12 Alkenyl, optionally substituted C-C 12 Alkynyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, TIFF2025529217000172.tif18138; R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4d Each occurrence of, if present, is optionally substituted C-C 12 independently selected from the group consisting of alkyl, halogen, CN, and NO2; R 5 each occurrence of is optionally substituted C1-C3 alkyl, optionally substituted C3-C 12Cycloalkyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C 12 Cycloalkyl, C2-C 12 Heterocycloalkyl, optionally substituted C-C 12 Aralkyl, optionally substituted C-C 12 Aryl, optionally substituted C-C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ) independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C-C 12 alkylenyl)-X-, -(optionally substituted C-C 12 alkenylenyl)-X-, -(optionally substituted C-C 12 alkynylenyl)-X-, -(optionally substituted C-C 12 independently selected from the group consisting of (heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )—, and —O—; Each occurrence of Y, if present, is a bond, -N(R a )—, and —O—; Each occurrence of Z is C1-C 24 is alkylenyl, C1-C at each occurrence of Z 24 Alkylenyl is C1-C 12 Alkyl and C1-C 12independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C1-C at each occurrence of Z 24 The alkylenyl is optionally and independently further substituted; R a and R b each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 Aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ) independently selected from the group consisting of: R c and R d each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 independently selected from the group consisting of aralkyl, optionally substituted phenyl, and optionally substituted C-C heteroaryl; and Each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4. Aspect 2 provides: below: (a) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least one selected from the group consisting of is H; (b) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R2g , and R 2h at least two selected from the group consisting of are H; (c) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least three selected from the group consisting of are H; (d) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least four selected from the group consisting of are H; (e) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least five selected from the group consisting of are H; (f) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least six selected from the group consisting of are H; (g) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least seven selected from the group consisting of are H; and (h) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R2g , and R 2h Each of these is H The compound of embodiment 1, wherein at least one of Aspect 3 provides: Each occurrence of L is -(CH2) 1-10 -, -(CH2) 2-10 NR 3c -, -(CH2) 2-10 O-, -(CH2) 1-3 -CH(OR a )-(CH2) 1-3 The compound of embodiment 1 or 2, wherein L is independently selected from the group consisting of -, piperazinylenyl, and cyclohexylenyl. Aspect 4 provides: Each occurrence of L is -CH2-, -(CH2)2-, -(CH2)3-, -(CH2) 10 -, -(CH2)2O-, -(CH2)3O-, -CH2CH(OR a )CH2-, -(CH2)2NR 3c -, TIFF2025529217000173.tif13128. Aspect 5 provides: wherein said compound of formula (I) TIFF2025529217000174.tif236141. Aspect 6 provides: R 4a , R 4b , R 4c , and R 4d The compound of any one of embodiments 1-5, wherein each occurrence of is independently H. Aspect 7 provides: R 5 The compound of any one of embodiments 1-6, wherein each occurrence of is independently methyl. Aspect 8 provides: R 6 The compound of any one of embodiments 1-7, wherein each occurrence of is independently H. Aspect 9 provides: Each occurrence of Z, independently, TIFF2025529217000175.tif14128, wherein: R 7a , R 7b , R 7c , and R 7d each occurrence of is independently selected from the group consisting of H, C-C alkyl, and C-C haloalkyl; R 7a , R 7b , R 7c , and R 7d at least one of is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; The compound of any one of embodiments 1 to 8. Aspect 10 provides: R 7a , R 7b , R 7c , and R 7d The compound of embodiment 9, wherein each occurrence of is selected from the group consisting of H and methyl. Aspect 11 provides: Each occurrence of Z is -(CH2) 4-10 -CH(CH3)-*, -(CH2) 4-10 -C(CH3)2-*, and -(CH2) 4-10 The compound of any one of embodiments 1-10, independently selected from the group consisting of: —CH(CH 3 )—CH 2 —*. Aspect 12 provides: R 3a , R 3b , and R 3c Each occurrence of 12. The compound of any one of embodiments 1 to 11, independently selected from the group consisting of: TIFF2025529217000176.tif80141. Aspect 13 provides: 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent, wherein said at least one substituent is C1- 13. The compound of any one of embodiments 1-12, wherein each occurrence of R' and R'' is independently selected from the group consisting of H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, 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-C8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R' and R'' is independently selected from the group consisting of H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl. Aspect 14 provides: 14. The compound of any one of embodiments 1-13, wherein the compound is selected from the group consisting of: TIFF2025529217000177.tif210102 TIFF2025529217000178.tif210111 TIFF2025529217000179.tif191128 TIFF2025529217000180.tif152128 Aspect 15 provides: A lipid nanoparticle (LNP) composition comprising: (a) at least one ionizable lipid compound having the structure of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: TIFF2025529217000181.tif22128In formula, R 1a and R 1b But each independently TIFF2025529217000182.tif11128; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h H, optionally substituted C1-C 12 Alkyl, optionally substituted C-C 12 Heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C 12 Alkenyl, optionally substituted C-C 12 Alkynyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, TIFF2025529217000183.tif18138; R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4dEach occurrence of, if present, is optionally substituted C-C 12 independently selected from the group consisting of alkyl, halogen, CN, and NO2; R 5 each occurrence of is optionally substituted C1-C3 alkyl, optionally substituted C3-C 12 Cycloalkyl, optionally substituted C-C 10 Aryl and optionally substituted C-C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C 12 Cycloalkyl, C2-C 12 Heterocycloalkyl, optionally substituted C-C 12 Aralkyl, optionally substituted C-C 12 Aryl, optionally substituted C-C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ) independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C-C 12 alkylenyl)-X-, -(optionally substituted C-C 12 alkenylenyl)-X-, -(optionally substituted C-C 12 alkynylenyl)-X-, -(optionally substituted C-C 12 independently selected from the group consisting of (heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )—, and —O—; Each occurrence of Y, if present, is a bond, -N(R a )—, and —O—; Each occurrence of Z is C1-C 24 is alkylenyl, C1-C at each occurrence of Z 24 Alkylenyl is C1-C 12 Alkyl and C1-C 12 independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C1-C at each occurrence of Z 24 The alkylenyl is optionally and independently further substituted; R a and R b each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 Aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ) independently selected from the group consisting of: R c and R d each occurrence of is selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C 12 independently selected from the group consisting of aralkyl, optionally substituted phenyl, and optionally substituted C-C heteroaryl; and each 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) cholesterol; and (d) at least one complex lipid; Aspect 16 provides: (e) at least one nucleic acid cargo and / or therapeutic drug cargo, at least partially encapsulated in said LNP; 16. The LNP of embodiment 15, further comprising: Aspect 17 provides: below: (a) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least one selected from the group consisting of is H; (b) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least two selected from the group consisting of are H; (c) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least three selected from the group consisting of are H; (d) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least four selected from the group consisting of are H; (e) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least five selected from the group consisting of are H; (f) R 2a , R2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least six selected from the group consisting of are H; (g) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least seven selected from the group consisting of are H; and (h) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h Each of these is H 17. The LNP of embodiment 15 or 16, wherein at least one of: Aspect 18 provides: Each occurrence of L is -(CH2) 1-10 -, -(CH2) 2-10 NR 3c -, -(CH2) 2-10 O-, -(CH2) 1-3 -CH(OR a )-(CH2) 1-3 18. The LNP of any one of embodiments 15-17, wherein L is independently selected from the group consisting of -, piperazinyl, and cyclohexylenyl. Aspect 19 provides: Each occurrence of L is -CH2-, -(CH2)2-, -(CH2)3-, -(CH2) 10 -, -(CH2)2O-, -(CH2)3O-, -CH2CH(OR a )CH2-, -(CH2)2NR 3c -, 19. The LNP of any one of embodiments 15 to 18, independently selected from the group consisting of: TIFF2025529217000184.tif13128. Aspect 20 provides: wherein said compound of formula (I) 20. The LNP of any one of embodiments 15 to 19, wherein the LNP is selected from the group consisting of: TIFF2025529217000185.tif235141. Aspect 21 provides: R 4a , R 4b , R 4c , and R 4d 21. The LNP of any one of embodiments 15-20, wherein each occurrence of is independently H. Aspect 22 provides: R 5 22. The LNP of any one of embodiments 15-21, wherein each occurrence of is independently methyl. Aspect 23 provides: R 6 23. The LNP of any one of embodiments 15-22, wherein each occurrence of is independently H. Aspect 24 provides: Each occurrence of Z, independently, TIFF2025529217000186.tif14128, wherein: R 7a , R 7b , R 7c , and R 7d each occurrence of is independently selected from the group consisting of H, C-C alkyl, and C-C haloalkyl; R 7a , R 7b , R 7c , and R 7d at least one of is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; The LNP of any one of embodiments 15 to 23. Aspect 25 provides: R 7a , R 7b , R 7c , and R 7d 25. The LNP of embodiment 24, wherein each occurrence of is selected from the group consisting of H and methyl. Aspect 26 provides: Each occurrence of Z is -(CH2) 4-10 -CH(CH3)-*, -(CH2) 4-10 -C(CH3)2-*, and -(CH2) 4-10 26. The LNP of any one of embodiments 15-25, wherein each of the LNPs is independently selected from the group consisting of: —CH(CH3)—CH2—*. Aspect 27 provides: R 3a , R 3b , and R 3c Each occurrence of 27. The LNP of any one of embodiments 15 to 26, independently selected from the group consisting of: TIFF2025529217000187.tif80141. Embodiment 28 provides: 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent, wherein said at least one substituent is selected from the group consisting of C1-C6 28. The LNP of any one of embodiments 15-27, wherein R′ and R″ are independently selected from the group consisting of C-C alkyl, C-C cycloalkyl, C-C haloalkyl, C-C haloalkoxy, phenoxy, halogen, CN, NO, OH, N(R′)(R″), C(═O)R′, C(═O)OR′, OC(═O)OR′, C(═O)N(R′)(R″), S(═O)N(R′)(R″), N(R′)C(═O)R″, N(R′)S(═O)R″, C-C 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, C-C alkyl, C-C cycloalkyl, C-C haloalkyl, benzyl, and phenyl. Aspect 29 provides: TIFF2025529217000188.tif174128TIFF2025529217000189.tif211111TIFF2025529217000190.tif188128TIFF2025529217000191.tif191128. Embodiment 30 provides: The LNP of any one of embodiments 15-29, wherein said at least one ionizable lipid of Formula (I) comprises between about 10 mol% and about 90 mol% of said LNP. Aspect 31 provides: The LNP of any one of embodiments 15-30, wherein the at least one ionizable lipid according to Formula (I) constitutes about 35 mol % or about 40 mol % of the LNP. Aspect 32 provides: The LNP of any one of embodiments 15-31, wherein the at least one neutral lipid comprises between about 1 mol % and about 40 mol % of the LNP. Aspect 33 provides: The LNP of any one of embodiments 15-32, wherein the at least one neutral lipid comprises about 16 mol% or about 30 mol% of the LNP. Aspect 34 provides:

[0039] 34. The LNP of any one of embodiments 15-33, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC). Embodiment 35 provides: The LNP of any one of embodiments 15-34, wherein the at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). Embodiment 36 provides the following: The LNP of any one of embodiments 15-35, wherein cholesterol comprises between about 20 mol% and about 75 mol% of the LNP. Aspect 37 provides: The LNP of any one of embodiments 15-36, wherein cholesterol comprises about 25 mol% or about 46.5 mol% of the LNP. Embodiment 38 provides the following: The LNP of any one of embodiments 15-37, wherein the at least one complex lipid comprises between about 0.1 mol % and about 15 mol % of the LNP. Aspect 39 provides: The LNP of any one of embodiments 15-38, wherein the at least one complex lipid comprises about 2.5 mol % of the LNP. Embodiment 40 provides: At least one conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000 40. The LNP of any one of embodiments 15-39, comprising: Aspect 41 provides the following: 41. The LNP of any one of embodiments 15-40, wherein (a):(b):(c):(d) has a molar ratio of about 35:16:46.5:2.5 or about 40:30:25:2.5. Aspect 42 provides: The LNP of any one of aspects 16-41, wherein the nucleic acid molecule is a therapeutic agent. Aspect 43 provides: The LNP of any one of aspects 16 to 42, wherein the nucleic acid molecule is at least one selected from the group consisting of RNA and DNA. Aspect 44 provides the following: The LNP of any one of aspects 16 to 43, wherein the nucleic acid molecule is at least one selected from the group consisting of mRNA, cDNA, miRNA, siRNA, and modified RNA. Embodiment 45 provides: The LNP of any one of aspects 42 to 44, wherein the nucleic acid is mRNA. Embodiment 46 provides the following: 46. ​​The LNP of embodiment 45, having a mass ratio of (a):mRNA of about 20:1 to about 5:1 (w / w), optionally having a mass ratio of (a):mRNA of about 10:1. Aspect 47 provides: 47. The LNP of embodiment 45 or 46, wherein the mRNA encodes a chimeric antigen receptor (CAR). Embodiment 48 provides the following: The LNP of embodiment 47, wherein the CAR is specific for binding to a surface antigen of a pathogenic or tumor cell. Aspect 49 provides: The surface antigens are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD16 1, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12 , TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE Al, HAL-A2 NY-ESO-1, PSC1, folate receptor alpha, 8H9, NCAM, VEGF, 5T4, fetal AchR, NKG2D ligand, TEM1, and TEM8. Embodiment 50 provides: 47. The LNP of embodiment 45 or 46, wherein the mRNA encodes an enzyme. Aspect 51 provides: 50. The LNP of any one of embodiments 45-46 and 49, wherein the mRNA encodes a CRISPR (clustered regularly interspaced short palindrome repeats) associated protein, and optionally the CRISPR associated protein is Cas9. Embodiment 52 provides: A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of embodiments 15 to 51 and at least one pharmaceutically acceptable carrier. Embodiment 53 provides: 53. The pharmaceutical composition of embodiment 52, further comprising at least one adjuvant. Embodiment 54 provides: 52. A method for treating, preventing, and / or ameliorating a disease in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of embodiments 16 to 51 and / or at least one pharmaceutical composition of embodiment 52 or 53. Embodiment 55 provides: 55. The method of embodiment 54, wherein the disease is selected from the group consisting of cancer, an autoimmune disorder, a cardiovascular disease, and a neurological disease. Embodiment 56 provides: 56. The method of embodiment 55, wherein the cancer is at least one selected from the group consisting of oral cancer, pancreatic cancer, colon cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small cell lung cancer, or colon cancer. Embodiment 57 provides: The method of embodiment 54 or 56, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and / or ameliorating cancer in the subject. Embodiment 58 provides: The method of any one of embodiments 54 to 57, wherein the subject is a mammal. Embodiment 59 provides: The method of embodiment 58, wherein the mammal is a human. Embodiment 60 provides: 52. A method for delivering a nucleic acid or therapeutic agent to the liver of a subject, comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of embodiments 16 to 51 and / or at least one pharmaceutical composition of embodiment 52 or 53. Embodiment 61 provides the following: 52. A method for preparing a modified immune cell or a progenitor thereof, the method comprising contacting an immune cell or a progenitor thereof with a lipid nanoparticle (LNP) of any one of embodiments 16 to 51 and / or at least one pharmaceutical composition of embodiment 52 or 53. Embodiment 62 provides: 62. The method of embodiment 61, wherein the modified immune cell or precursor thereof is an αβ T cell, a γδ T cell, a CD8+ T cell, a CD4+ helper T cell, a CD4+ regulatory T cell, an NK T cell, an NK cell, and any combination thereof. Embodiment 63 provides: 63. The method of embodiment 62, wherein the modified immune cell or precursor thereof is a T cell, and optionally the T cell is a CD4+ T cell. Embodiment 64 provides the following: The method of embodiment 62, wherein the modified immune cell or a precursor thereof is an NK cell.

[0323] The terms and expressions used herein are used as terms of description and not of limitation, and the use of such terms and expressions is without any intention to exclude the features shown and described or any equivalents of portions thereof, with the understanding that various modifications are possible within the scope of the aspects of this application. Thus, while this application describes specific embodiments and optional features, it should be understood that those skilled in the art may utilize modifications and variations of the compositions, methods, and concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the aspects of this application.

Claims

1. An ionizable lipid compound of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: During the ceremony, R 1a and R 1b But each independently and R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h H, optionally substituted C 1 -C 12 Alkyl, optionally substituted C 2 -C 12 Heteroalkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 2 -C 8 Heterocycloalkyl, optionally substituted C 2 -C 12 Alkenyl, optionally substituted C 2 -C 12 Alkynyl, optionally substituted C 6 -C 10 Aryl and optionally substituted C 2 -C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, are independently selected from the group consisting of R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4d Each occurrence of, if present, may be substituted C 1 -C 12 Alkyl, halogen, CN, and NO 2 are independently selected from the group consisting of: R 5 Each occurrence of may be substituted C 1 -C 3 Alkyl, optionally substituted C 3 -C 12 Cycloalkyl, optionally substituted C 6 -C 10 Aryl and optionally substituted C 2 -C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 12 Cycloalkyl, C 2 -C 12 Heterocycloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted C 6 -C 12 Aryl, optionally substituted C 2 -C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 Cycloalkylenyl, and optionally substituted C 2 -C 8 independently selected from the group consisting of heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )-, and -O-; Each occurrence of Y, if present, is a bond, -N(R a )-, and -O-; Each occurrence of Z is C 1 -C 24 is alkylenyl, C in each occurrence of Z 1 -C 24 Alkylenyl is C 1 -C 12 Alkyl and C 1 -C 12 independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C in each occurrence of Z 1 -C 24 The alkylenyl is optionally and independently further substituted; R a and R b Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 1 -C 6 Haloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted phenyl, optionally substituted C 2 -C 8 Heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d independently selected from the group consisting of: R c and R d Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 1 -C 6 Haloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted phenyl, and optionally substituted C 2 -C 8 independently selected from the group consisting of heteroaryl; and Each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4.

2. below: (a) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least one selected from the group consisting of is H; (b) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least two selected from the group consisting of are H; (c) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least three selected from the group consisting of are H; (d) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least four selected from the group consisting of are H; (e) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least five selected from the group consisting of are H; (f) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least six selected from the group consisting of are H; (g) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least seven selected from the group consisting of are H; and (h) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h Each of these is H The compound according to claim 1, wherein at least one of the following applies:

3. Each occurrence of L is -(CH 2 ) 1-10 -, -(CH 2 ) 2-10 NR 3c -, -(CH 2 ) 2-10 O-, -(CH 2 ) 1-3 -CH(OR a )-(CH 2 ) 1-3 3. The compound of claim 1 or 2, wherein L is independently selected from the group consisting of -, piperazinylenyl, and cyclohexylenyl.

4. Each occurrence of L is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 10 -, -(CH 2 ) 2 O-, -(CH 2 ) 3 O-, -CH 2 CH(OR a )CH 2 -, -(CH 2 ) 2 NR 3c -, The compound of any one of claims 1 to 3, independently selected from the group consisting of:

5. wherein said compound of formula (I) The compound according to any one of claims 1 to 4, selected from the group consisting of:

6. R 4a , R 4b , R 4c , and R 4d 6. The compound of any one of claims 1 to 5, wherein each occurrence of is independently H.

7. R 5 7. The compound of any one of claims 1 to 6, wherein each occurrence of is independently methyl.

8. R 6 8. The compound of any one of claims 1 to 7, wherein each occurrence of is independently H.

9. Each occurrence of Z, independently, wherein: R 7a , R 7b , R 7c , and R 7d Each occurrence of H, C 1 -C 6 Alkyl, and C 1 -C 6 haloalkyl; R 7a , R 7b , R 7c , and R 7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; A compound according to any one of claims 1 to 8.

10. R 7a , R 7b , R 7c , and R 7d 10. The compound of claim 9, wherein each occurrence of is selected from the group consisting of H and methyl.

11. Each occurrence of Z is -(CH 2 ) 4-10 -CH(CH 3 )-*, -(CH 2 ) 4-10 -C(CH 3 ) 2 -* and -(CH 2 ) 4-10 -CH(CH 3 )-CH 2 11. The compound of any one of claims 1 to 10, independently selected from the group consisting of:

12. R 3a , R 3b , and R 3c Each occurrence of 12. The compound of any one of claims 1 to 11, independently selected from the group consisting of:

13. 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently substituted with at least one substituent, wherein said at least one substituent is selected from the group consisting of: C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 Haloalkyl, C 1 -C 3 Haloalkoxy, phenoxy, halogen, CN, NO 2 , OH, N(R')(R''), C(=O)R', C(=O)OR', OC(=O)OR', C(=O)N(R')(R''), S(=O) 2 N(R')(R''), N(R')C(=O)R'', N(R')S(=O) 2 R'', C 2 -C 8 and phenyl optionally substituted with at least one halogen, wherein each occurrence of R′ and R″ is selected from the group consisting of H, C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 13. The compound of any one of claims 1 to 12, independently selected from the group consisting of haloalkyl, benzyl, and phenyl.

14. A compound according to any one of claims 1 to 13, selected from the group consisting of:

15. A lipid nanoparticle (LNP) composition comprising: (f) at least one ionizable lipid compound having the structure of formula (I), or a salt thereof, a solvate thereof, a stereoisomer thereof, or an isotopologue thereof: During the ceremony, R 1a and R 1b But each independently and R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h H, optionally substituted C 1 -C 12 Alkyl, optionally substituted C 2 -C 12 Heteroalkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 2 -C 8 Heterocycloalkyl, optionally substituted C 2 -C 12 Alkenyl, optionally substituted C 2 -C 12 Alkynyl, optionally substituted C 6 -C 10 Aryl and optionally substituted C 2 -C 10 heteroaryl; R 3a , R 3b , and R 3c Each occurrence of H, are independently selected from the group consisting of R 3a , R 3b , and R 3c at most one of is H; R 4a , R 4b , R 4c , and R 4d Each occurrence of, if present, may be substituted C 1 -C 12 Alkyl, halogen, CN, and NO 2 are independently selected from the group consisting of: R 5 Each occurrence of may be substituted C 1 -C 3 Alkyl, optionally substituted C 3 -C 12 Cycloalkyl, optionally substituted C 6 -C 10 Aryl and optionally substituted C 2 -C 10 independently selected from the group consisting of heteroaryl; R 6 Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 12 Cycloalkyl, C 2 -C 12 Heterocycloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted C 6 -C 12 Aryl, optionally substituted C 2 -C 12 Heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b independently selected from the group consisting of: Each occurrence of L is a bond, -(optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 Cycloalkylenyl, and optionally substituted C 2 -C 8 independently selected from the group consisting of heterocycloalkylenyl; Each occurrence of X, if present, is a bond, -N(R 3c )-, and -O-; Each occurrence of Y, if present, is a bond, -N(R a )-, and -O-; Each occurrence of Z is C 1 -C 24 is alkylenyl, C in each occurrence of Z 1 -C 24 Alkylenyl is C 1 -C 12 Alkyl and C 1 -C 12 independently substituted with at least one substituent selected from the group consisting of haloalkyl; and C in each occurrence of Z 1 -C 24 The alkylenyl is optionally and independently further substituted; R a and R b Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 1 -C 6 Haloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted phenyl, optionally substituted C 2 -C 8 Heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d independently selected from the group consisting of: R c and R d Each occurrence of 1 -C 6 Alkyl, optionally substituted C 3 -C 8 Cycloalkyl, optionally substituted C 1 -C 6 Haloalkyl, optionally substituted C 6 -C 12 Aralkyl, optionally substituted phenyl, and optionally substituted C 2 -C 8 independently selected from the group consisting of heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; (g) at least one neutral lipid; (h) cholesterol; and (i) At least one complex lipid.

16. (j) at least one nucleic acid cargo and / or therapeutic drug cargo, at least partially encapsulated in said LNP; 16. The LNP of claim 15, further comprising:

17. below: (a) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h At least one selected from the group consisting of is H; (b) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least two selected from the group consisting of are H; (c) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least three selected from the group consisting of are H; (d) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least four selected from the group consisting of are H; (e) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least five selected from the group consisting of are H; (f) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least six selected from the group consisting of are H; (g) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h at least seven selected from the group consisting of are H; and (h) R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h Each of these is H 17. The LNP of claim 15 or 16, wherein at least one of the following applies:

18. Each occurrence of L is -(CH 2 ) 1-10 -, -(CH 2 ) 2-10 NR 3c -, -(CH 2 ) 2-10 O-, -(CH 2 ) 1-3 -CH(OR a )-(CH 2 ) 1-3 18. The LNP of any one of claims 15-17, wherein L is independently selected from the group consisting of -, piperazinylenyl, and cyclohexylenyl.

19. Each occurrence of L is -CH 2 -, -(CH 2 ) 2 -, -(CH 2 ) 3 -, -(CH 2 ) 10 -, -(CH 2 ) 2 O-, -(CH 2 ) 3 O-, -CH 2 CH(OR a )CH 2 -, -(CH 2 ) 2 NR 3c -, 19. The LNP of any one of claims 15 to 18, independently selected from the group consisting of:

20. wherein said compound of formula (I) 20. The LNP of any one of claims 15 to 19, selected from the group consisting of:

21. R 4a , R 4b , R 4c , and R 4d 21. The LNP of any one of claims 15-20, wherein each occurrence of is independently H.

22. R 5 22. The LNP of any one of claims 15-21, wherein each occurrence of is independently methyl.

23. R 6 23. The LNP of any one of claims 15-22, wherein each occurrence of is independently H.

24. Each occurrence of Z, independently, wherein: R 7a , R 7b , R 7c , and R 7d Each occurrence of H, C 1 -C 6 Alkyl, and C 1 -C 6 haloalkyl; R 7a , R 7b , R 7c , and R 7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; 24. The LNP of any one of claims 15 to 23.

25. R 7a , R 7b , R 7c , and R 7d 25. The LNP of claim 24, wherein each occurrence of is selected from the group consisting of H and methyl.

26. Each occurrence of Z is -(CH 2 ) 4-10 -CH(CH 3 )-*, -(CH 2 ) 4-10 -C(CH 3 ) 2 -* and -(CH 2 ) 4-10 -CH(CH 3 )-CH 2 26. The LNP of any one of claims 15 to 25, independently selected from the group consisting of: -*.

27. R 3a , R 3b , and R 3c Each occurrence of 27. The LNP of any one of claims 15 to 26, independently selected from the group consisting of:

28. 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 heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently substituted with at least one substituent, wherein said at least one substituent is selected from the group consisting of: C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 Haloalkyl, C 1 -C 3 Haloalkoxy, phenoxy, halogen, CN, NO 2 , OH, N(R')(R''), C(=O)R', C(=O)OR', OC(=O)OR', C(=O)N(R')(R''), S(=O) 2 N(R')(R''), N(R')C(=O)R'', N(R')S(=O) 2 R'', C 2 -C 8 and phenyl optionally substituted with at least one halogen, wherein each occurrence of R′ and R″ is selected from the group consisting of H, C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl, C 1 -C 6 28. The LNP of any one of claims 15-27, wherein the LNP is independently selected from the group consisting of haloalkyl, benzyl, and phenyl.

29. An LNP described in any one of claims 15 to 28, selected from the group consisting of:

30. 30. The LNP of any one of claims 15-29, wherein the at least one ionizable lipid of formula (I) comprises from about 10 mol% to about 90 mol% of the LNP.

31. 31. The LNP of any one of claims 15 to 30, wherein the at least one ionizable lipid of formula (I) comprises about 35 mol% or about 40 mol% of the LNP.

32. 32. The LNP of any one of claims 15-31, wherein the at least one neutral lipid comprises between about 1 mol% and about 40 mol% of the LNP.

33. 33. The LNP of any one of claims 15-32, wherein at least one neutral lipid comprises about 16 mol% or about 30 mol% of the LNP.

34. 34. The LNP of any one of claims 15-33, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC).

35. 35. The LNP of any one of claims 15-34, wherein at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE).

36. 36. The LNP of any one of claims 15-35, wherein cholesterol comprises between about 20 mol% and about 75 mol% of the LNP.

37. 37. The LNP of any one of claims 15-36, wherein cholesterol comprises about 25 mol% or about 46.5 mol% of the LNP.

38. 38. The LNP of any one of claims 15 to 37, wherein at least one complex lipid comprises from about 0.1 mol% to about 15 mol% of the LNP.

39. 39. The LNP of any one of claims 15 to 38, wherein at least one complex lipid comprises about 2.5 mol% of the LNP.

40. At least one conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000 40. The LNP of any one of claims 15 to 39, comprising:

41. 41. The LNP of any one of claims 15-40, wherein (a):(b):(c):(d) has a molar ratio of about 35:16:46.5:2.5 or about 40:30:25:2.

5.

42. The LNP of any one of claims 16 to 41, wherein the nucleic acid molecule is a therapeutic agent.

43. The LNP of any one of claims 16 to 42, wherein the nucleic acid molecule is at least one selected from the group consisting of RNA and DNA.

44. The LNP of any one of claims 16 to 43, wherein the nucleic acid molecule is at least one selected from the group consisting of mRNA, cDNA, miRNA, siRNA, and modified RNA.

45. The LNP of any one of claims 42 to 44, wherein the nucleic acid is mRNA.

46. 46. ​​The LNP of claim 45, having a mass ratio of (a):mRNA of about 20:1 to about 5:1 (w / w), optionally having a mass ratio of (a):mRNA of about 10:

1.

47. 47. The LNP of claim 45 or 46, wherein the mRNA encodes a chimeric antigen receptor (CAR).

48. 48. The LNP of claim 47, wherein the CAR is specific for binding to a surface antigen of a pathogenic cell or tumor cell.

49. The surface antigens are CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD16 1, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12 , TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR IVIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE A1, HAL-A2 NY-ESO-1, PSC1, folate receptor alpha, 8H9, NCAM, VEGF, 5T4, fetal AchR, NKG2D ligand, TEM1, and TEM8.

50. 47. The LNP of claim 45 or 46, wherein the mRNA encodes an enzyme.

51. 50. The LNP of any one of Claims 45-46 and 49, wherein the mRNA encodes a CRISPR (clustered regularly interspaced short palindrome repeats) associated protein, and optionally the CRISPR associated protein is Cas9.

52. A pharmaceutical composition comprising a lipid nanoparticle (LNP) according to any one of claims 15 to 51 and at least one pharmaceutically acceptable carrier.

53. 53. The pharmaceutical composition of claim 52, further comprising at least one adjuvant.

54. 52. A method for treating, preventing, and / or ameliorating a disease in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) according to any one of claims 16 to 51 and / or at least one pharmaceutical composition according to claim 52 or 53.

55. 55. The method of claim 54, wherein the disease is selected from the group consisting of cancer, an autoimmune disorder, a cardiovascular disease, and a neurological disease.

56. 56. The method of claim 55, wherein the cancer is at least one selected from the group consisting of oral cancer, pancreatic cancer, colon cancer, bladder cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small cell lung cancer, or colon cancer.

57. 57. The method of claim 54 or 56, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and / or ameliorating cancer in the subject.

58. 58. The method of any one of claims 54 to 57, wherein the subject is a mammal.

59. 59. The method of claim 58, wherein the mammal is a human.

60. 52. A method for delivering a nucleic acid or therapeutic agent to the liver of a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 16 to 51 and / or at least one pharmaceutical composition of claim 52 or 53.

61. 54. A method for preparing modified immune cells or precursor cells thereof, the method comprising contacting immune cells or precursor cells thereof with a lipid nanoparticle (LNP) according to any one of claims 16 to 51 and / or at least one pharmaceutical composition according to claim 52 or 53.

62. 62. The method of claim 61, wherein the modified immune cell or precursor thereof is an αβ T cell, a γδ T cell, a CD8+ T cell, a CD4+ helper T cell, a CD4+ regulatory T cell, a NK T cell, a NK cell, and any combination thereof.

63. 63. The method of claim 62, wherein the modified immune cell or precursor thereof is a T cell, and optionally the T cell is a CD4+ T cell.

64. 63. The method of claim 62, wherein the modified immune cell or a precursor thereof is a NK cell.

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