Cationic degradable lipid compositions, lipid nanoparticles (LNPS) comprising same, and methods of use thereof

Abstract

The disclosure relates, in one aspect, to certain cationic degradable lipid compounds. In another aspect, the disclosure relates to lipid nanoparticle (LNP) compositions comprising the cationic degradable lipid compounds of the disclosure. In another aspect, the disclosure relates to methods for delivering cargo a subject's lung and / or treating, preventing, and / or ameliorating lung-associated diseases and / or disorders in a subject with administration of LNP compositions of the disclosure.

Description

[0001] TITLE OF THE INVENTION

[0002] Cationic Degradable Lipid Compositions, Lipid Nanoparticles (LNPs) Comprising Same, and Methods of Use Thereof

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under TR002776 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 559,424, filed February 29, 2024, which is incorporated herein by reference in its entirety.

[0007] BACKGROUND

[0008] Lipid nanoparticles (LNPs) are clinically relevant delivery agents capable of delivering messenger RNA (mRNA)-based therapeutics, holding great promise for use in vaccination, protein replacement therapy, cancer immunotherapy, and CRISPR-Cas-based gene editing. Recently, the US Food and Drug Administration (FDA) fully approved two mRNA vaccines against COVID- 19 enabled by LNPs; clinical trials have also demonstrated robust in vivo CRISPR gene editing to treat hereditary transthyretin amyloidosis in patients, which represents a significant milestone for mRNA therapeutics.

[0009] Unfortunately, there have also been clinical failures driven in part due to inefficient delivery. These advancements and failures underscore the need to develop potent ionizable lipids to facilitate efficient mRNA delivery for disease treatments; however, extrahepatic mRNA delivery has remained challenging, owing to the slow blood flow and the discontinuous vasculature of hepatic sinusoids that enhance liver delivery. The lungs represent a compelling target for mRNA delivery due to the diverse range of pathological targets affecting endothelial, epithelial, and immune cells in lung-associated diseases.

[0010] Various approaches have been utilized to target the lungs and their respective cellular populations, including pre-treating animals to overwhelm the liver or reduce drug activity in specific cell types to shift tropism, conjugating receptor ligands onto LNPs surface for active targeting, and interacting with serum proteins for endogenous targeting. Although some of these approaches have resulted in advanced phase 1 / 2 clinical studies, the multistep strategy and safety concerns have limited their applicability to evaluate large lipid libraries.

[0011] To identify the lead-performers of ionizable lipids for mRNA delivery in each large, chemically distinct new lipid library, scientists must explore the transfection efficacy of each LNP formulation in delivering its payload into target tissues and cells in vivo. Because injecting and sacrificing thousands of mice per lipid library is challenging, typically only a fraction of the LNP candidates can be evaluated in vivo, limiting the amount of data available for the remainder of the initial large lipid library. It has also been reported that in vitro delivery profiles are usually a poor predictor of in vivo nanoparticle delivery; thus, development of high-throughput methods for in vivo screening can accelerate the discovery of LNPs with new chemical structure and properties that can overcome delivery barriers for gene therapy applications in the lungs.

[0012] Thus, in one aspect, there is a need in the art for LNPs comprising cationic degradable lipids and methods of use thereof for selective delivery of therapeutic cargo to the lungs. The present disclosure addresses these needs.

[0013] BRIEF SUMMARY

[0014] In one aspect, the disclosure provides a compound of formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof, wherein R1, L1, R2a, R?a, and m are defined elsewhere herein:

[0015] In certain embodiments, the compound of formula (I) is:

[0016] In certain embodiments, the compound of formula (I) is:

[0017]

[0018] 6b)

[0019] In certain embodiments, the compound of formula (I) is:

[0020] In certain embodiments, the compound of formula (I) is:

[0021] In another aspect, the disclosure provides a lipid nanoparticle (LNP) composition. In certain embodiments, the LNP comprises at least one compound of formula (I). In certain embodiments, the LNP comprises at least one neutral lipid. In certain embodiments, the LNP comprises at least one cholesterol lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises at least one polymer conjugated lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises at least one cargo molecule.

[0022] In another aspect, the disclosure provides a pharmaceutical composition comprising the LNP of the disclosure and a pharmaceutically acceptable carrier. In another aspect, the disclosure provides a method for delivering a cargo to a subject’s lungs. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle of the disclosure or the pharmaceutical composition of the disclosure. In another aspect, the disclosure provides a method for treating, preventing, and / or ameliorating a lung-associated disease or disorder in a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle of the disclosure or the pharmaceutical composition of the disclosure.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0025] FIGs. 1A-1C: High-throughput LNP screening facilitates the discovery of cationic degradable (CAD) lipid-like materials for mRNA delivery' to the lungs. FIG. 1 A: CAD LNPs were first formulated using a microfluidic mixing device by mixing nucleic acid with CAD lipids, helper lipid, cholesterol, and PEG-lipid. Following in vitro high-throughput screening, a series of CAD LNPs were selected and formulated to co-encapsulate b-DNA and mRNA, pooled, and systemically administered to mice, allowing for quantification of accumulation in each organ (e.g., heart, liver, spleen, lungs, and kidneys) using deep-sequencing to identify CAD lipid candidates for lung-targeted mRNA delivery. FIG. IB: combinatorial library of CAD lipids was chemically synthesized following a Schiff base reduction scheme, using amine heads and aldehyde degradable tails. FIG. 1C: overview of 12 amines cores and 15 aldehyde degradable tails used to synthesize 180 CAD lipids.

[0026] FIG. 2: Model reaction between amine head 5 and nd their respective1H NMR spectra.

[0027] FIGs. 3A-3H: Investigation of structure-activity relationship of CAD LNPs for firefly luciferase (FLuc) mRNA delivery in vitro. FIG. 3A: CAD LNP formulation parameters. CAD LNPs were formulated with one of 180 distinct CAD lipids. DOPE, cholesterol, and C14PEG2K at a molar ratio of 35: 16:46.5:2.5, for a total of 180 distinct LNP formulations. FIG. 3B: representative cryogenic transmission electron microscopy (cryo-TEM) image of CAD LNP morphology. Scale bar: 100 nm. FIG. 3C: hydrodynamic size distribution of representative CAD LNP revealed by DLS. FIG. 3D: a heatmap of luciferase expression following treatment of HeLa cells with CAD LNPs (10 ng luciferase mRNA. n > 3 replicates). Relative luminescence unit (RLU) values of > 100 were classified as hits for hit rate calculation. FIG. 3E: relative hit rate of CAD LNPs with different secondary' amine numbers. FIG. 3F: relative hit rate of CAD LNPs with different tail architectures. FIG. 3G: relative hit rate of CAD LNPs with different tail substitution numbers on each aldehyde. FIG. 3H: relative hit rate of CAD LNPs with different tail lengths. FIG. 4: mRNA encapsulation efficiency of 180 CAD-LNPs used for in vitro screening on HeLa cell (n = 3 replicates). Data are represented as ± s.e.m.

[0028] FIG. 5: Cell viability after 24 h transfection of 180 CAD LNPs in HeLa cells. 5,000 cells were plated per well and treated by 10 ng mRNA (n = 3 replicates). Data are represented as mean ± s.e.m.

[0029] FIGs. 6A-6E: In vivo structure-activity relationship analysis of 96 chemically distinct CAD lipids and organ tropism. FIG. 6A: schematic illustration of barcoding approach to probe biodistribution profile of newly designed CAD LNPs. LNPs were formulated by pipette mixing to encapsulate barcoded DNA (b-DNA) and FLuc mRNA. Lipid phases consisted of one of 96 CAD lipids, DOPE, cholesterol, and C14PEG2K at a molar ratio of 35: 16:46.5:2.5. LNP formulations were pooled together and administered systemically to C57BL / 6J mice (n = 5). Tissues were isolated 6 h post-administration, DNA was extracted, and accumulation of b-DNAs was quantified by deep sequencing. FIG. 6B: hydrodynamic diameter of all administered CAD LNPs. The diameter of the LNP pool control (red triangle symbol) falls within the range of the CAD LNPs composing the pool. FIG. 6C: zeta potential of all administered CAD LNPs. The zeta potential of the pooled LNP (red triangle symbol) falls within the range of the CAD LNPs composing the pool. FIG. 6D: heatmap visualizing accumulation of CAD LNPs in different organs as measured by deep sequencing. Dark clusters represent higher relative accumulation of b-DNA in a specific tissue sample. Structural details of CAD lipids used in each LNP formulation are described above the heatmap. FIG. 6E: volcano plots summarizing enrichment analysis of barcodes in the lungs, liver, and spleen. All sequencing data were normalized to the uninjected pool of LNPs.

[0030] FIG. 7 : Heatmap demonstrates the accumulation of CAD LNPs in the heart and kidneys. Dark clusters represent higher accumulation of a b-DNA in a specific tissue sample. Structural details of CAD lipids in each LNP formulation is provided, n = 5 mice.

[0031] FIGs. 8A-8I: Validation of lead LNP formulations for mRNA delivery to the lungs. FIG. 8 A: four lead LNP formulations were discovered from a 180-CAD lipid library after high-throughput in vitro and in vivo screening. FIG. 8B: whole body and ex vivo imaging of luciferase expression mediated by LNP-CAD3, 4. 9, and 10 at 6 h post-injection (0.1 mg / kg FLuc mRNA). H: heart; Li: liver; S: spleen; Lu: lung; K: kidney. Quantification of luciferase expression in the lungs (FIG. 8C), liver (FIG. 8D), and spleen (FIG. 8E) using region-of- interest (ROI) analysis. FIG. 8F: relative luciferase expression in each measured organ. FIG. 8G: Ail 4 mice were treated with LNP-CAD9 or MC3 / DOTAP encapsulating Cre mRNA 3 days prior to analysis (0.3 mg / kg). Lungs were digested and stained for quantifying cell populations for tdTomato+expression. PBS was injected as negative control. FIG. 8H: proportion of tdTomato cells in the lung assessed by flow cytometry. FIG. 81: representative immunostaining demonstrating signal overlap between tdTomato+cells and the EC marker platelet endothelial cell adhesion molecule 1 (PECAM1). DAPI was used for nuclear staining. Statistical significance in FIG. 8H was calculated using Student’s t test with unpaired design. **P < 0.01. Data are presented as mean ± s.e.m. (n = 3-4 mice)

[0032] FIGs. 9A-9C: Ex vivo luminescence intensity of organs in mice after administration of LNP-CAD20 delivering FLuc-mRNA at a dosage of 0.25 mg / kg. FIG. 9A: luminescence imaging of the body after LNP treatment. FIG. 9B: luminescence imaging of organs from FIG. 9A. FIG. 9C: luminescence quantification of the organs from FIG. 9B. H: heart; Li: liver; S: spleen; Lu: lung; K: kidney. Data are represented as mean ± s.e.m. (n = 3 mice).

[0033] FIG. 10: Representative gating strategy of diverse tdTomato+cells in the lungs. Draq7 was used for distinguish live and dead cells. CD45+antibody was used to stain immune cells, then CD457CD31+was used for lung endothelial cells, CD457CD317EPCAM+was used for lung epithelial cells, and the rest CD457CD3T / EPCAM- was stained as others. Ail4 mice was administrated with PBS or Cre mRNA LNP (MC3 / DOTAP LNP or LNP-CAD9) at a total mRNA dosage of 0.3 mg / kg.

[0034] FIG. 11: Tissue section histology of PBS and LNP-CAD9 treated samples. Mice were administered intravenously at mRNA dosage of 1.0 mg / kg, and PBS treated group was used as the negative control. Tissue sections of heart, liver, spleen, lung, and kidney were acquired 12 h post-injection and prepared for H&E staining (n = 3 mice). Scale bar = 100 μm.

[0035] FIGs. 12A-12H: antiangiogenic therapy through knocking out VEGFR2 by LNPs in orthotropic lung cancer model. FIG. 12A: schematic illustration of lung tumor implantation through i.v. injection of Lewis Lung Carcinoma cell lines expressing GFP (LLC-GFP) and treatment protocol of C57BL / 6J female mice. On day 20 after tumor cell inoculation, mice were randomly assigned to four groups: PBS treated control (Gl), LNP-CAD9 encapsulating Cas9 mRNA / scrambled sgRNA treatment (G2), LNP-CAD9 encapsulating Cas9 mRNA / VEGFR2 sgRNA treatment (G3), and MC3 / DOTAP LNP encapsulating Cas9 mRNA / VEGFR2 sgRNA treatment (G4). Mice were treated two days later for a total of 2 doses (2.0 mg kg-1of RNA per injection). Seven days after the last administration, 6 mice in each group were euthanized and their lungs were isolated for antiangiogenic analysis. The remaining mice were used for survival evaluation. FIG. 12B: antiangiogenic mechanism through LNP-mediated VEGFR2 knockout. LNPs encapsulating Cas9 mRNA / VEGFR2 sgRNA demonstrated the ability to reduce the expression of VEGFR2 in lung endothelial cells, which further inhibited the VEGF-VEGFR2 pathway. KO: knockout. FIG. 12C: RT- qPCR measurement of VEGFR2 level in different treatment groups (n = 3 mice for G1 and G2 groups; n = 4 mice for G3 and G4 groups). FIG. 12D: quantification of tumor areas per lung of different treatment groups (n = 6 mice). FIG. 12E: representative H&E staining of lung tissue after sacrifice (n = 6 mice). Arrows indicate tumor areas in the lungs. Scale bar: 1 mm. FIG. 12F: percent survival of mice under different treatments (n = 6 mice). FIG. 12G: representative immunostaining of tumor areas in the lungs. Endothelial cells were stained by CD31 antibody. DAPI was used for nuclear staining. Scale bar: 100 μm. FIG. 12F: microvascular density (MVD) in the tumor area under different treatments (n = 6 mice). Statistical significance in FIGs. 12C-12D, FIG. 12F, and FIG. 12H was calculated using one- way analysis of variance (ANOVA). followed by Dunnetfs multiple comparison test. 0.001; **p < 0.01; *p < 0.05; p > 0.05, not significant. Data are presented as mean ± s.e.m.

[0036] FIG. 13 A: structural morphology' of 3-A2-7b LNP encapsulating b-DNA / FLuc (with a weight ratio of 10: 1) as visualized by Cryo-TEM. Scale bar: 100 nm. FIG. 13B: distribution of hydrodynamic diameter of 3-A2-7b LNP encapsulating b-DNA / FLuc mRNA (with a weight ratio of 10: 1) acquired by DLS. FIG. 13C: luciferase expression following treatment of HeLa cells with 3-A2-7b LNP encapsulating FLuc mRNA and b-DNA / FLuc mRNA (10 ng luciferase mRNA, n = 5 replicates). Statistical significance in (FIG. 13C) was calculated using Student’s t test with unpaired design. P > 0.05, not significant (ns).

[0037] FIG. 14: correlation of LNPs size and their respective lung delivery barcoding rank number.

[0038] FIG. 15 A: representative gating strategy for tdTomato+ endothelial cells in the liver. 7AAD was used to distinguish live and dead cells. CD45+ antibody was used to stain immune cells, then CD45- / CD31+ was used to identify liver endothelial cells. Ail4 mice was administered with PBS or LNP-CAD9 delivering Cre mRNA at a total dosage of 0.3 mg kg- 1. The mice were necropsied 3 days post-injection for flow cytometry' studies. FIG. 15B: proportion of tdTomato+ endothelial cells in the liver assessed by flow cytometry'. Data are presented as ± s.e.m. (n = 4 mice).

[0039] FIG. 16A: representative gating strategy for tdTomato+ endothelial cells in the heart. 7AAD was used to distinguish live and dead cells. CD45+ antibody was used to stain immune cells, then CD45- / CD31+ w'as used to identify heart endothelial cells. Ail4 mice was administered with PBS or LNP-CAD9 delivering Cre mRNA at a total dosage of 0.3 mg kg- 1. The mice were necropsied 3 days post-injection for flow cytometry’ studies. FIG. 16B: proportion of tdTomato+ endothelial cells in the heart assessed by flow' cytometry. Data are presented as ± s.e.m. (n = 4 mice).

[0040] FIGs. 17A-17F: representative TNS assay curves for determining the apparent pKa of LNP-CAD20 (FIG. 17 A), LNP-CAD95 (FIG. 17B), LNP-CAD10 (FIG. 17C), LNP-CAD3 (FIG. 17D), LNP-CAD14 (FIG. 17E) and LNPCAD73 (FIG. 17F). Apparent pKa was defined as the point at which 50% of maximum TNS fluorescence was achieved.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0043] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g, 0.1% to 0.5%, 1. 1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

[0044] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0045] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0046] Description

[0047] Although mRNA therapeutics have made significant progress through systemic and local administration in preclinical / clinical trials, extrahepatic mRNA delivery still poses a huge challenge. The development of novel ionizable lipids has been identified as means to facilitate efficient mRNA delivery for hepatic and extrahepatic applications. However, identification of top performers in a chemically distinct lipid library’ for mRNA delivery is tedious, typically requiring multiple screening steps both in vitro and in vivo. Moreover, in vitro delivery profiles are poor predictors of in vivo nanoparticle delivery , and false negatives abound, especially for extrahepatic organs. Thus, the expeditious deployment of high- throughput screening technology to enhance screening efficacy for extrahepatic mRNA delivery is of paramount importance.

[0048] Due to the importance of chemical characteristics of LNPs in determining delivery behavior and the tenuous relationship between in vitro and in vivo performance, there is a need for simple and rapid high-throughput screening techniques. High-throughput barcoding technology has been used for quantifying how hundreds of different LNPs deliver mRNA in vivo. Historically, this barcoding approach is usually based on exploring formulation parameters orthogonally with a small chemically distinct lipid library, leaving the inherent structure-activity relationship of ionizable lipids somewhat overlooked for extrahepatic mRNA delivery. This barcoded high-throughput screening system was employed to identify a CAD LNP capable of delivering mRNA at a clinically relevant dose to the lungs from a chemically^ diverse library of 180 CAD lipids.

[0049] Additionally, the apparent pKa of LNPs can exert influence over tissue-specific mRNA delivery activity. To investigate this, representative liver-enriched (LNP-CAD20 and LNP-CAD95). lung-enriched (LNP-CAD10 and LNP-CAD3), and spleen-enriched (LNP- CAD14 and LNP-CAD73) LNPs were selected for examination. In this investigation, LNP- CAD20 and LNP-CAD95 exhibited apparent pKa comfortably residing within the well- established range of 6 to 7 for liver-targeted LNPs, whereas LNP-CAD14 and LNP-CAD73 displayed lower pKa between ranging from 4 to 5 for spleen-targeted LNPs (FIGs. 17A- 17F). In the case of lung-enriched LNPs (LNP-CAD10 and LNP-CAD3), the observed measurements revealed pKa of 4.905 and 5.806 (FIGs. 17A-17F), respectively. Notably, these values do not exceed the pivotal threshold of 9, as stipulated for effective lung-targeting LNP deliver}’ in previous reports. Cumulatively, these results provide compelling evidence that the pKa of LNPs represents only one facet of the complicated landscape governing tissue-specific mRNA delivery activity'.

[0050] In summary’, a class of CAD lipids were designed using a facile Schiff base reduction methodology, leading to synthesis of a combinatonal library of 180 chemically distinct CAD lipids in hours. These CAD lipids were formulated into CAD LNPs carrying FLuc mRNA to evaluate their transfection potential in HeLa cells in vitro. From these preliminary- results, 96 promising CAD lipids were identified and used to generate LNPs co-encapsulating b-DNA and FLuc mRNA, injecting these LNPs as a pool into C57BL / 6J female mice to enable quantification of cargo accumulation via deep sequencing. Enrichment analysis of the large resultant dataset identified 21 CAD LNPs yvith lung affinity’, among yvhich 4 LNPs demonstrated highly enriched pulmonary accumulation without enrichment the liver and spleen, suggestive of strongly preferential pulmonary delivery’. Low-throughput counters creenmg was employed to verify functional delivery by delivering mRNA encoding FLuc, identifying LNP-CAD9 as the lead candidate for pulmonary mRNA deliver}’, with -90% of total luciferase expression observed in the lungs. Further investigation revealed that LNP-CAD9 delivering Cre mRNA can preferentially edit lung endothelial cells at the clinically relevant dose of 0.3 mg / kg, substantially outperforming a gold standard lung-tropic MC3 / DOTAP LNP system. These findings demonstrate that high-throughput barcoding technology’ can be utilized as an efficient and effective screening tool for identifying structurally distinct nanoparticles for extrahepatic deliver}' to the lungs. It is envisaged that the present disclosure provides the potential to unlock mRNA delivery of neyvly designed lipid libraries for extrahepatic protein replacement, vaccine, and genetic disease treatments.

[0051] Definitions

[0052] The term "about" as used herein can allow for a degree of variability in a value or range, for example, yvithin 10%. within 5%. or yvithin 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0053] The term "acyl" as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a "formyl" group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralky l cycloalkyl, cycloalkylalkyl, heterocyclyl. heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A 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 and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a "haloacyl" group. An example is a trifluoroacetyl group.

[0054] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen- specific adaptive immune response.

[0055] The term "alkenyl" as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

[0056] The term "alkoxy" as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxy ethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0057] The term "alkyl" as used herein refers to straight chain and branched alkyd groups and cycloalky l groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-penty 1, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-buty l, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term "alkyd" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0058] The term "alkynyl" as used herein refers to straight and branched chain alkyd groups, except that at least one triple bond exists between two carbon atoms, Thus. alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - CACH. -C =C(CH3). -C=C(CH2CH3). -CH2OCH, -CH2OC(CH3), and -CH2OCXCH2CH3) among others.

[0059] The term “alkylene” or “alkydenyl” as used herein refers to a bivalent saturated aliphatic radical (e.g., -CH2-, -CH2CH2-, and -CH2CH2CH2-, inter aha). In certain embodiments, the term may be regarded as a moiety derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from the same (e.g. -CH2-) different (e.g, -CH2CH2-) carbon atoms. Similarly, the terms “heteroalkyl enyl”, “cycloalkylenyl”, “heterocycloalkydenyl”, and the like, as used herein refer to a divalent radical of the moiety corresponding to the base group (e.g, heteroalkyl, cycloalkyd, and / or heterocycloalkyl). A divalent radical possesses two open valencies at any position(s) of the group. Thus, the divalent radical may form a single bond to two distinct atoms or groups, or may form a double bond with one atom.

[0060] The term "amine" as used herein refers to primary, secondary, and tertiary' amines having, e.g. , the formula N(group)s wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkydamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term "amine" also includes ammonium ions as used herein.

[0061] The term "amino group" as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an ammo group can be viewed as an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary' amino group. An "alkylamino" group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0062] The term '‘anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0063] The term "aralkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

[0064] The term "aryl" as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

[0065] The term '‘cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). It has been found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390. the disclosures of which are herein incorporated by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cat-ionic lipids comprise a protonatable tertiary amine (e.g.. pH titratable) head group, Cis alky l chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e g., DSDMA, DLinDMA, DLenDMA, and DODMA.

[0066] The term "cycloalkyl" as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cycloocty l groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4. 5. 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination denotes a cyclic alkenyl group.

[0067] The terms "epoxy -functional" or "epoxy-substituted" as used herein refers to a functional group in which an oxygen atom, the epoxy substituent, is directly attached to two adjacent carbon atoms of 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, epoxy propoxypropyl, 2-glycidoxy ethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2-(glycidoxycarbonyl)propyl, 3-(3,4-epoxycylohexyl)propyl, 2-(3,4- epoxycyclohexyljethyl, 2-(2,3-epoxycylopentyl)ethyl, 2-(4-methyl-3,4- epoxycyclohexyljpropyl, 2-(3.4-epoxy-3-methylcylohexyl)-2-methylethyl, and 5,6- epoxyhexyl.

[0068] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0069] In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0070] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0071] As used herein, the terms "effective amount." "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0072] In particular, in the case of a mRNA, and "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid as relating to a mRNA is an amount sufficient to produce the desired effect, e.g, mRNA-directed expression of an amount of a protein that causes a desirable biological effect in the organism within which the protein is expressed. For example, in some embodiments, the expressed protein is an active form of a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g. , at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the protein that is normally expressed in the cell type of a healthy individual. For example, in some embodiments, the expressed protein is a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces a similar level of expression as observed in a healthy individual in an individual with aberrant expression of the protein (i.e.. protein deficient individual). Suitable assays for measuring the expression of an mRNA or protein include, but are not limited to dot blots. Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0073] The term "encode" as used herein refers to the product specified (e.g., protein and RNA) by a given sequence of nucleotides in a nucleic acid (i.e., DNA and / or RNA), upon transcription or translation of the DNA or RNA, respectively. In certain embodiments, the term "encode" refers to the RNA sequence specified by transcription of a DNA sequence. In certain embodiments, the term "encode" refers to the amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In certain embodiments, the term "encode" refers to the amino acid sequence specified by transcription of DNA to mRNA and subsequent translation of the mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may comprise a direct transcription or translation product. In certain embodiments, the encoded product may comprise post-translational modifications understood or reasonably expected by one skilled in the art. The term "fully encapsulated" indicates that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particle is degraded in a treatment that would normally degrade 100% of free active agent or therapeutic agent, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particle is degraded. In the context of nucleic acid therapeutic agents, full encapsulation may be determined by an Oligreen® assay. Oligreen® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif). "Fully encapsulated" also indicates that the lipid particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0074] 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.

[0075] The term "haloalkyl" group, as used herein, includes mono-halo alkyl groups, poly- halo alky l groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl. 1,1 -di chloroethyl, 1.2-di chloroethyl, l,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.

[0076] The term "heteroaryl" as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and St for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroary l group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with tw o carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl. azaindolyl. indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl. isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

[0077] Additional examples of ary l and heteroary l groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl. N- hydroxytriazolyl, N-hydroxyimidazolyl. anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2 -thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazoly 1, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1 -imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl ( 1,2, 3 -triazol- 1-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl. l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2 -pyridyl, 3 -pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3 -isoquinolyl, 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b] furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7- 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),

[0078] 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo [b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1 -indolyl, 2-indolyl,

[0079] 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl,

[0080] 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl. 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl,

[0081] 7 -benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b.f| azepine (5H-dibenz[b,f] azepin- 1 -yl, 5H-dibenz[b,f] azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,1 l-dihydro-5H-dibenz[b,f| azepine (10,1 l-dihydro-5H-dibenz[b,f|azepine-l-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-3-yl. 10,l l-dihydro-5H-dibenz[b,f|azepine-4-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-5-yl), and the like.

[0082] The term "heteroarylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

[0083] The term "heterocyclylalkyl" as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alky l group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methy l, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

[0084] The term "heterocyclyl" as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O. and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase "heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl. isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.

[0085] The term "hydrocarbon" or "hydrocarbyl" as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.

[0086] As used herein, the term "hydrocarbyl" refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alky l, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Ci), ethyl (C2), propyl (C3), or butyl (C4), and (Co-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

[0087] The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X1, X2, and X3are independently selected from noble gases" would include the scenario where, for example, X1, X2, and X3are all the same, where X1. X2. and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0088] The term "ionizable lipid" as used herein refers to a lipid (e.g., a cationic lipid) having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g.. pH 7.4), and neutral at a second pH. preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKaof the protonatable group in the range of about 4 to about 7.

[0089] The term "local delivery," as used herein, refers to delivery of an active agent or therapeutic agent such as a messenger RNA directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like. The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0090] The term "lipid conjugate" refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to. polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (e.g., U.S. Pat. No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moi eties and ester- containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

[0091] As used herein, "lipid encapsulated" can refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a protein cargo), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an SPLP. pSPLP, SNALP. or other nucleic acid-lipid particle).

[0092] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids and / or additional agents.

[0093] The term "lipid particle" is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest. In the lipid particle of the disclosure, which is ty pically formed from a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle, the active agent or therapeutic agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.

[0094] The term "monovalent" as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

[0095] The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0096] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0097] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'- O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary' sequences as well as the sequence explicitly indicated. Specifically , degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem, 260:2605-2608 (1985); Rossolini et al, Mai. Cell. Probes, 8:91-98 (1994)).

[0098] As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides. In particular embodiments, oligonucleotides of the disclosure are from about 15 to about 60 nucleotides in length. Nucleic acid may be administered alone in the lipid particles of the disclosure, or in combination (e.g., co-administered) with lipid particles of the disclosure comprising peptides, polypeptides, or small molecules such as conventional drugs. In other embodiments, the nucleic acid may be administered in a viral vector.

[0099] "Nucleotides" contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to. amines, alcohols, thiols, carboxylates, and alkyl halides.

[0100] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs. and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al. , J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0101] The term "organic group" as used herein refers to any carbon-containing functional group. Examples can include an oxy gen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester: a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R. C(O)C(O)R, C(O)CH2C(O)R. C(S)R, C(O)OR. OC(O)R. C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2. (CH2)O-2N(R)C(O)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl. wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0102] The terms "patient," "subject," or "individual" are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0103] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0104] As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

[0105] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic. sulfanilic, cyclohexylaminosulfonic, stearic, alginic. P-hydroxybutyric, salicylic, galactaric and galacturonic acid.

[0106] Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

[0107] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc: excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0108] The terms "peptide," "polypeptide." and "protein" are used interchangeably herein, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many ty pes. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0109] The term "polymer conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term "pegy lated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1 -(monomethoxy -poly ethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG), DSPE-PEG- DBCO, DOPE-PEG-Azide, DSPE-PEG-Azide, DPPE-PEG-Azide, DSPE-PEG-Carboxy- NHS, DOPE-PEG-Carboxylic Acid, DSPE-PEG-Carboxylic acid and the like.

[0110] The term "room temperature" as used herein refers to a temperature of about 15 to 28 °C.

[0111] The term "solvent" as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0112] The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of' can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0113] The term "substituted" as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g, F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, 0C(0)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo). S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy. N(R)2, SR. SOR. SO2R, SO2N(R)2, SO3R. C(O)R. C(O)C(O)R.

[0114] C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(0)N(R)2, 0C(0)N(R)2, C(S)N(R)2, (CH2)O- 2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)C0N(R)2, N(R)SO2R. N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(0)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C 1- C100) hydrocarbyl, alkyd, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroary lalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0115] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0116] The term “therapeutic protein” as used herein refers to a protein or peptide which has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In certain embodiments, a therapeutic protein or peptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein or peptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “therapeutic protein” includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Exemplary' therapeutic proteins include, but are not limited to, an analgesic protein, an anti-inflammatory protein, an anti-proliferative protein, an proapoptotic protein, an anti-angiogenic protein, a cytotoxic protein, a cytostatic protein, a cytokine, a chemokine, a growth factor, a wound healing protein, a pharmaceutical protein, or a pro-drug activating protein. Therapeutic proteins may include growth factors (EGF, TGF-a, TGF- P, TNF, HGF, IGF, and IL-1-8, inter alia) cytokines, paratopes, Fabs (fragments, antigen binding), and antibodies.

[0117] The terms "treat," "treating" and "treatment," as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

[0118] Ionizable Lipids and / or Cationic Degradable Lipids

[0119] In one aspect, the present disclosure provides a cationic degradable lipid compound of formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein:

[0120] R1is selected from the group consisting of N(RA)(RB) and N(R2b)(R3b); each occurrence of L1is independently selected from the group consisting of -N(R2d)-, -N(RA)-, -(optionally substituted C1-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2- Cs heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C8heteroarylenyl)-. wherein one occurrence of L1is optionally substituted with -(optionally substituted C1-C6alkylenyl)-N(R2c)(R3c); each occurrence of R2a, R2b, R2c, and R2dis independently "■L2— A(R4)„ . each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H and optionally substituted C1-C6alky l; each occurrence of A is independently optionally substituted C6-C10aryl and optionally substituted C2-C8heteroaryl; each occurrence of R4is -OC(=O)(optionally substituted C1-C24 alkyl); each occurrence of L2is independently -(optionally substituted C1-C6alkydenyl)-; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted cycloalkyl, optionally substituted C2-C8heterocycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C8heteroaryl, or RAand RBcan combine with the N atom to which they are bound to form a C2-C8heterocycloalkyl; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each occurrence of n is independently 1, 2, or 3.

[0121] In certain embodiments, L1is -(CH2)2-. In certain embodiments, L1is -(CILjs-. In certain embodiments, L1is -N(CH3)-. In certain embodiments, L1is -N[(optionally substituted C1-C6alkylenyl)-N(R2c)(R3c)]-. In certain embodiments, L1is

[0122] In certain embodiments, RAand RBcombine with the N atom to which they are bound to form in certain embodiments, RAis methyl. In certain embodiments, RAis ethyl. In certain embodiments, RBis methyl. In certain embodiments, RBis ethyl.

[0123] In certain embodiments, L1and m are selected such that -(L')rn- is -( In certain embodiments, L1and m are selected such that - is In certain embodiments. L1and m are selected such that - is In certain embodiments, L 1and m are selected such that - i In certain embodiments, L1 and m are selected such that is . in certain embodiments, L1and m are selected such that is . In certain embodiments, L1 and m are selected such that

[0124] In certain embodiments, R1is NMe2. In certain embodiments, R1is In certain embodiments, R1is In certain embodiments,

[0125] In certain embodiments, the compound of formula (I) is . In certain embodiments, the compound of formula (I) is . In certain embodiments. the compound of formula (I) is certain embodiments, the compound of formula (1) i In certain embodiments, the compound of formula (1) is

[0126] wherein: each occurrence of R5a, R5b, and R3c. if present, is independently each occurrence of R6a, R6b, R7a, R7b, R8a, R8b, and R9is independently H or C1-C6 alkyl. wherein R6a, R6b, R7a. R7b, R8a, R8b. and R9are selected such that each occurrence of R5a, R3b, and R5ccomprise a Ci-C24 alkyl.

[0127] In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8his methyl, and R9is ethyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8bis methy l, and R9is propyl. In certain embodiments, one of R6a, R6b. R7a, R7b, R8a. and R8bis methyl, and R9is butyl. In certain embodiments, one of R6a. R6b, R7a, R7b. R8a, and R8bis methyl, and R9is pentyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8bis methyl, and R9is hexyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is ethyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R81’ is ethyl, and R9is propyl. In certain embodiments, one of R6a, R6b, R7a. R7b, R8a, and R8bis ethyl, and R9is butyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is pentyl. In certain embodiments, one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is hexyl.

[0128] In certain embodiments, R5ais 1 -methylpenty l. In certain embodiments, R5ais 1- methylhexyl. In certain embodiments, R5ais 1 -methylheptyl. In certain embodiments, R5ais

[0129] 1 -methyloctyl. In certain embodiments, R5ais 1 -methylnonyl. In certain embodiments, R5ais

[0130] 1 -ethylpentyl. In certain embodiments, R5ais 1-ethylhexyl. In certain embodiments, R5ais 1- ethylheptyl. In certain embodiments, R5ais 1 -ethyloctyl. In certain embodiments, R 'ais 2- methylpentyl. In certain embodiments, R5ais 2-methylhexyl. In certain embodiments, R5ais

[0131] 2 -methylheptyl. In certain embodiments, R5ais 2-methyloctyl. In certain embodiments, R5ais 2-methylnonyl. In certain embodiments, R5ais 2-ethylpentyl. In certain embodiments, R5ais 2-ethylhexyl. In certain embodiments, R5ais 2-ethylheptyl. In certain embodiments, R5ais

[0132] 2-ethyloctyl. In certain embodiments, R5ais 3-methylpentyl. In certain embodiments, R5ais

[0133] 3-methylhexyl. In certain embodiments, R5ais 3-methylheptyl. In certain embodiments, R5ais 3-methyloctyl. In certain embodiments, R5ais 3-methylnonyl. In certain embodiments, R’ais 3-ethylpentyl. In certain embodiments, R5ais 3-ethylhexyl. In certain embodiments, R5ais

[0134] 3-ethylheptyl. In certain embodiments, R5ais 3-ethylocty l. In certain embodiments, R5ais 3- ethylnonyl.

[0135] In certain embodiments, R5bis 1 -methylpentyl. In certain embodiments, R5bis 1- methylhexyl. In certain embodiments, R5bis 1 -methylheptyl. In certain embodiments, R5bis 1 -methyloctyl. In certain embodiments, R5bis 1 -methylnonyl. In certain embodiments, R5bis 1 -ethylpentyl. In certain embodiments, R5bis 1-ethylhexyl. In certain embodiments, R5bis 1 -ethylheptyl. In certain embodiments, R5bis 1 -ethyloctyl. In certain embodiments, R5bis 2- methylpentyl. In certain embodiments, R5bis 2-methylhexyl. In certain embodiments, R5bis 2 -methylheptyl. In certain embodiments, R5bis 2-methyloctyl. In certain embodiments, R5bis 2-methylnonyl. In certain embodiments, R5bis 2-ethylpentyl. In certain embodiments. R5bis 2-ethylhexyl. In certain embodiments, R5bis 2-ethylheptyl. In certain embodiments, R3bis

[0136] 2-ethyloctyl. In certain embodiments, R5bis 3-methylpentyl. In certain embodiments, R5bis

[0137] 3-methylhexyl. In certain embodiments, R5bis 3 -methylheptyd. In certain embodiments, R5bis 3-methyloctyl. In certain embodiments, R5bis 3-methylnonyl. In certain embodiments, R5bis 3-ethylpentyl. In certain embodiments, R5bis 3-ethylhexyl. In certain embodiments. R5bis 3-ethylheptyl. In certain embodiments, R5bis 3-ethylocty 1. In certain embodiments, R5bis 3-ethylnonyl.

[0138] In certain embodiments, R5cis 1 -methylpentyl. In certain embodiments, R5cis 1 - methylhexyl. In certain embodiments, R5cis 1 -methylheptyl. In certain embodiments, R5cis

[0139] 1 -methyloctyd. In certain embodiments, R5cis 1 -methylnonyl. In certain embodiments, R5cis

[0140] 1 -ethylpentyl. In certain embodiments, R5cis 1 -ethylhexyd. In certain embodiments, R5cis 1- ethylheptyl. In certain embodiments, R5cis 1 -ethylocty l. In certain embodiments, R3Cis 2- methylpentyl. In certain embodiments, R5cis 2-methylhexyl. In certain embodiments, R5cis

[0141] 2 -methylheptyl. In certain embodiments, R5cis 2-methyloctyl. In certain embodiments, R5cis 2-methylnonyl. In certain embodiments, R5cis 2-ethylpentyl. In certain embodiments, R5cis 2-ethylhexyl. In certain embodiments, R5cis 2-ethydheptyl. In certain embodiments, R5cis

[0142] 2-ethyloctyl. In certain embodiments, R5cis 3-methylpentyl. In certain embodiments, R5cis

[0143] 3-methylhexyl. In certain embodiments. R5cis 3 -methylheptyd. In certain embodiments, R5cis 3-methylocty 1. In certain embodiments, R5cis 3-methylnonyl. In certain embodiments, R’cis 3-ethylpenty 1. In certain embodiments, R5cis 3-ethylhexyl. In certain embodiments, R5cis

[0144] 3-ethylheptyl. In certain embodiments, R5cis 3-ethyloctyl. In certain embodiments, R5cis 3- ethylnonyl.

[0145]

[0146] In certain embodiments, certain embodiments. R2ais certain embodiments, R2ais , certain embodiments, R2ais

[0147]

[0148] Ionizable Lipids and / or Cationic Lipids The scope of ionizable lipids contemplated for use in the present disclosure is not limited to ionizable lipids of Formula (I). In the lipid nanoparticles of the disclosure, the cationic lipid or ionizable lipid may comprise, e.g., one or more of the following: (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLinMC3DMA), [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315). heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-

[0149] (undecyloxy)hexyl] amino} octanoate (SM-102), 1 ,1 '-[[2-[4-[2-[[2-[bis(2- hy droxy dodecy l)amino] ethyl] (2-hy droxy dodecyl)amino] ethyl] - 1 - piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200), l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3- 45 dimethylaminopropyl)- 1,3] -dioxolane (D Lin-K-C3-D MA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), 2,2- dilinoleyl-5-dimethylaminomethyl-[l ,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N- methylpepiazino-[ 1 ,3]-dioxolane (DLin-K-MPZ), 2.2-dili-noleyl-4-dimethylaminomethyl- [l,3]-dioxolane (DLin-KDMA). 1.2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (D Lin-C-DAP), l,2-dilinoleyoxy-3-(dimethylaminoacetoxypropane (DLin-DAC), 1- 2dilinoley oxy-3-morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy- 3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (D Lin-MPZ), 3-(N,N-dilinoleylamino)-l,2-propanediol (D LinAP), 3-(N,N-dioleylamino)-l,2-propanedio (DOAP), l,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (D Lin-EG-D MA), N,N-dioleyl-N,N-dimethylanrmonium chloride (DODAC), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2- distearyloxy-N,N-dimethylaminopropane (DSD MA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB). N-(l-(2,3-dioleoyloxy)propyl)-N,N. N-trimethylammonium chloride (DOTAP), 3- (N-(N'.N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol). N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl anrmonium bromide (DMRIE), 2,3- dioleyloxy-N-[2 (spermine-carboxamidoethyl]-N,N-dimethy 1-1- propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3- dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy) propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA ("XTC2"), or mixtures thereof. The ionizable lipids are not limited to those recited herein, and can further include ionizable lipids known to those skilled in the art, or described in PCT Application No. PCT / US2020 / 056255 and / or PCT Application No. PCT / US2020 / 056252, the disclosures of which are herein incorporated by reference in its entirety.

[0150] The synthesis of cationic lipids such as DLin-K-C2-DMA ("XTC2"), DLin-K-C3- DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U.S. Application Publication No. US 2011 / 0256175, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DUin-DAC, DLin-MA, DLinDAP, DUin-S-DMA, DUm-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DUinAP, DOAP, and DLin-EG-DMA. as well as additional cationic lipids, is described in PCT Application No. PCT / US08 / 88676. filed December 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0151] Non-ccitionic Lipid

[0152] In the nucleic acid-lipid particles of the present disclosure, the non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. In some embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0153] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is known to one skilled in the art and described in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378, all of which are hereby incorporated herein in their entireties for all purposes.

[0154] Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylchohne (DOPC). dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), ioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0155] Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids can be. for example, acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl- 2'-hydroxy ethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof.

[0156] Conjugated Lipid

[0157] In the nucleic acid-lipid particles of the present disclosure, the conjugated lipid that inhibits aggregation of particles may comprise, e.g.. one or more of the following: a polyethyleneglycol (PEG) lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic- poly mer-lipid conjugates (CPLs), or mixtures thereof. In some embodiments, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.

[0158] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with tw o terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEGOH), monomethoxy poly ethylene glycolsuccinate (MePEGS), monomethoxypolyethylene glycolsuccinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycolamine (MePEG-NEE), monomethoxypolyethylene glycoltresylate (MePEG-TRES), and monomethoxypolyethylene glycolimidazolylcarbonyl (MePEG-IM). Other PEGs such as those described in U.S. Patent Nos. 6.774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CEECOOH) is particularly useful for preparing PEG-lipid conjugates including, e.g, PEG-DAA conjugates.

[0159] In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. The conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEGDAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C 14), a PEG- dipalmityloxypropyl (Cie), a PEG-distearyloxypropyl (Cis), or mixtures thereof.

[0160] Additional PEG-lipid conjugates suitable for use in the 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 No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8'-(l,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'- dioxaoctanyl] carbamoyl-methyl-poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S. Patent No. 7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0161] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g, from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2.000 daltons, etc.). In some embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0162] In addition to the foregoing, it will be readily apparent to those of skill in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and poly dimethylacrylamide, polylactic acid, polygly colic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0163] In addition to the foregoing components, the particles (e.g, LNP) of the present disclosure can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (e.g, Chen et al., Bioconj. Chem., 1 1:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in the present disclosure, and methods of making and using SPLPs and SPLP-CPLs, are disclosed, e.g, in U.S. Patent No. 6,852,334 and PCT Publication No. WO 00 / 62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0164] In certain instances, the conjugated lipid that inhibits aggregation of particles (e.g, PEG-lipid conjugate) may comprise from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%. from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 1 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, from about 1.4 mol% to about 1.5 mol%. or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, or 2 mol% (or any fraction thereof or range therein) of the total lipid present in the particle.

[0165] In the lipid nanoparticles of the present disclosure, the active agent or therapeutic agent may be fully encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation. In some embodiments, a nucleic acid-lipid particle comprising a nucleic acid such as a messenger RNA (z.e., mRNA) is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37° C. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agent or therapeutic agent (e.g., nucleic acid such as siRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulations of the present disclosure is that the lipid particle compositions are substantially non-toxic to mammals such as humans.

[0166] Lipid Nanoparticle (LNP) Compositions

[0167] In one aspect, the present disclosure provides a lipid nanoparticle (LNP) composition. In certain embodiments, at least one neutral lipid. In certain embodiments, at least one cholesterol lipid and / or a modified derivative thereof. In certain embodiments, at least one polymer conjugated lipid and / or modified derivative thereof. In certain embodiments, the LNP further comprises at least one nucleic acid and / or therapeutic cargo, wherein the cargo is at least partially encapsulated therein. In certain embodiments, the LNP further comprise an ionizable lipid. In certain embodiments, at least one ionizable lipid is a cationic degradable lipid. In certain embodiments, the LNP comprises at least one cationic degradable lipid compound of formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein:

[0168] R1is selected from the group consisting of N(RA)(RB) and N(R2b)(R3b); each occurrence of L1is independently selected from the group consisting of -N(R2d)-, -N(RA)-, -(optionally substituted C1-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2- Cs heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C8heteroarylenyl)-, wherein one occurrence of L1is optionally substituted with -(optionally substituted C1-C6alkylenyl)-N(R2c)(R3c); each occurrence of R2a, R2b, R2c, and R2dis independently L2— A(R4)n. each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H and optionally substituted C1-C6alkyl; each occurrence of A is independently optionally substituted C6-C10ary l and optionally substituted C2-C8heteroaryl; each occurrence of R4is -OC(=O)(optionally substituted C1-C24alkyl); each occurrence of L2is independently -(optionally substituted C1-C6alkylenyl)-; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C3-C8cycloalkyl, optionally substituted C2-C8heterocycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C8heteroaryl. or RAand RBcan combine with the N atom to which they are bound to form a C2-C8heterocycloalkyl; m is 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10; and each occurrence of / n is independently 1. 2, or 3.

[0169] In certain embodiments, the compound of formula (I) is:

[0170]

[0171] In certain embodiments, the at least one cationic degradable lipid comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

[0172] 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP.

[0173] In certain embodiments, the at least one cationic degradable lipid comprises more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

[0174] 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44. 45. 46. 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,

[0175] 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP.

[0176] In certain embodiments, the at least one cationic degradable lipid comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

[0177] 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

[0178] 61. 62. 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the at least one cationic degradable lipid comprises less than about 35 mol% of the LNP. In certain embodiments, the at least one cationic degradable lipid comprises more than about 35 mol% of the LNP. In certain embodiments, the at least one cationic degradable lipid comprises about 35 mol% of the LNP.

[0179] In certain embodiments, the at least one neutral lipid comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,

[0180] 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 16 mol% of the LNP.

[0181] In certain embodiments, the at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). In certain embodiments, the at least one neutral lipid comprises distearoylphosphatidylcholine (DSPC).

[0182] In certain embodiments, the cholesterol and / or modified derivative thereof comprises less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the cholesterol and / or modified derivative thereof comprises more than about 20, 21, 22, 23, 24, 25, 26, 27. 28. 29. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

[0183] 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the cholesterol and / or modified derivative thereof comprises about 20,

[0184] 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33. 34. 35. 36. 37, 38, 39, 40, 41, 42, 43, 44, 45,

[0185] 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mol% of the LNP. In certain embodiments, the cholesterol and / or modified derivative thereof comprises about 46.5 mol% of the LNP. In certain embodiments, the cholesterol and / or modified derivative thereof is cholesterol.

[0186] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5. 10.0, 10.5, 11.0, 11.5, 12.0. or about 12.5 mol% of the LNP. In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, or about 12.5 mol% of the LNP. In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, or about 12.5 mol% of the LNP. In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises less than about 2.5 of the LNP. In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises more than about 2.5 of the LNP. In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 2.5 of the LNP.

[0187] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col- 2000:

[0188] In certain embodiments, the at least one polymer conjugated lipid and / or modified derivative thereof comprises l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000] (ammonium salt):

[0189] In certain embodiments, the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35: 16:46.5:2.5. In certain embodiments, the LNP comprises at least one cargo molecule. In certain embodiments, the cargo is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof. In certain embodiments, the cargo is a nucleic acid.

[0190] In certain embodiments, the nucleic acid is DNA or RNA. In certain embodiments, the nucleic acid is selected from the group consisting of siRNA, mRNA, cDNA, pDNA, microRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof.

[0191] In certain embodiments, the cargo is at least partially encapsulated in the LNP. In certain embodiments, the cargo is siRNA, optionally wherein the siRNA is a TAK1 targeting siRNA.

[0192] In certain embodiments, the LNP comprises a nucleic acid molecule. In certain embodiments, the nucleic acid molecule is at least one selected from the group consisting of DNA and RNA. In certain embodiments, the nucleic acid molecule is selected from the group consisting of cDNA, mRNA, miRNA, siRNA, modified RNA, antagomir, antisense molecule, and a targeted nucleic acid, or any combination thereof. In certain embodiments, the nucleic acid molecule encodes a chimeric antigen receptor (CAR). In certain embodiments, the CAR is specific for binding to a surface antigen of a pathogenic cell or a tumor cell. In certain embodiments, the surface antigen is selected from the group consisting of CD4, CD8, CD1, CD2, CD3, CD5, CD7, CD16, CD19, CD20. CD22, CD25, CD26, CD27, CD28, CD30. CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD 103, CD1 19, CD123, CD 126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, 0X40, 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 ligands, B7-H6, IL13R-a2, 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-a, 8H9, NCAM, VEGF, 5T4, Fetal AchR. NKG2D ligands, TEML and TEM8.

[0193] In certain embodiments, the nucleic acid molecule encodes at least one selected from the group consisting of mRNA and sgRNA. In certain embodiments, the mRNA encodes a therapeutic protein. In certain embodiments, the therapeutic protein is a CRISPR-associated protein. In certain embodiments, the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9). In certain embodiments, the sgRNA is VEGFR2. In certain embodiments. the sgRNA is a TTR guide RNA. In certain embodiments, the therapeutic protein encodes FGF21. In certain embodiments, the therapeutic proteins encodes SARS-CoV-2 spike protein. In certain embodiments, the therapeutic agent is a CRISPR-associated protein. In certain embodiments, the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9). In certain embodiments, the therapeutic protein is a CRISPR-associated protein. In certain embodiments, the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9) and the sgRNA is VEGFR2.

[0194] Methods

[0195] In one aspect, the present disclosure provides a method for delivering a cargo to a subject's lungs, the method comprising administering to the subject at least one lipid nanoparticle of the disclosure and / or the pharmaceutical composition of the disclosure. In certain embodiments, the cargo is delivered to a lung epithelial cell.

[0196] In certain embodiments, the cargo is selectively delivered to the lung over at least one selected from the group consisting of the liver, heart, kidney, and spleen.

[0197] In another aspect, the present disclosure provides a method for treating, preventing, and / or ameliorating a lung-associated disease or disorder, the method comprising administering to the subject at least one lipid nanoparticle of the disclosure and / or the pharmaceutical composition of the disclosure.

[0198] In certain embodiments, the lung-associated disease or disorder is at least one selected from the group consisting of acute respiratory distress syndrome (ARDS), asthma, bronchiectasis, bronchopulmonary' dysplasia (BPD), chronic obstructive pulmonary disorder (COPD), cystic fibrosis, hypersensitivity' pneumonitis, interstitial lung disease (ILD), lung abscess, lung cancer, occupational lung diseases, pleural effusion, pneumonia, pulmonary edema, pulmonary embolism, pulmonary fibrosis, pulmonary hypertension, sarcoidosis, and tuberculosis.

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

[0200] Pharmaceutical Compositions

[0201] In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle (LNP) of the present disclosure and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one adjuvant. Such a pharmaceutical composition may consist of at least one composition of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0202] In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 1,000 mg / kg / day.

[0203] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will van', depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0204] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.

[0205] In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavail ability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0206] The formulations of the pharmaceutical compositions described herein may be prepared by any method know n or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessors' ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

[0207] As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0208] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0209] In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®). solubilized gelatins (e.g, GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991 , Mack Publication Co., New Jersey).

[0210] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0211] Formulations may be employed in admixtures with conventional excipients, z.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and / or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

[0212] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total w eight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzy l alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzy l alcohol and 0.05-0.5% sorbic acid.

[0213] The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%. or BHT in the range of 0.03% to 0. 1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0214] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxy cetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to. methyl, ethyl, or / 7-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0215] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil. fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0216] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0217] Methods for impregnating or coating a material with a chemical composition are known in the art. and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (z.<?., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdennal patch, as known to those skilled in the art.

[0218] Administration / Dosing

[0219] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0220] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (i.e., composition) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic composition of the disclosure is from about 0.01 mg / kg to 100 mg / kg of body weight / per day of active agent (i.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition without undue experimentation.

[0221] The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day. once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.

[0222] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0223] A medical doctor, e.g.. physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0224] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic composition for the treatment of a disease or disorder in a patient.

[0225] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every7two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to. age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

[0226] The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 p g to about 6,000 mg, about 100 p g to about 5,500 mg, about 200 p g to about 5,000 mg, about 400 p g to about 4.000 mg, about 800 p g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg. about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

[0227] In some embodiments, the dose of active agent (z.e., nucleic acid) present in the composition of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg. or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0228] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0229] The term "container" includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i. e. , the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g. treating, preventing, or reducing a disease or disorder in a patient.

[0230] Administration Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal, and (trans )rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0231] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0232] Parenteral Administration

[0233] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to. administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0234] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to. suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to. suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0235] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non- toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0236] EXAMPLES

[0237] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

[0238] Materials and Methods

[0239] Chemicals and reagents for synthesis

[0240] 2.2'-Diamino-N-methyldi ethylamine (98%, TCI America), 3,3'-diamino-N- methyldipropylamine (98%. TCI America), 2.2’- (piperazine- 1.4-diyl)di ethanamine (ArnBeed), l,4-bis(3-aminopropyl)piperazine (98%, TCI America), N,N- dimethylethylenediamine (98%, TCI America), N,N-dimethyl-l,3- propanediamine (99%, TCI America), N,N-diethylethylenediamine (98%, TCI America), N,N-diethyl-l,3- diaminopropane (99%. TCI America). l-(2-aminoethyl)-4- methylpiperazine (97%, Alfa Aesar), l-(3-aminopropyl)-4-methylpiperazine (98%, Alfa Aesar), 1,3-diaminopropane (99%, Sigma-Aldrich), tris(2-aminoethyl)amine (98%, TCI America), 3,5-dihydroxybenzaldehyde (AinBeed). 2,4,6-trihydroxybenzaldehyde (AinBeed). heptanoic acid (98%, TCI America), 2- methylhexanoic acid (98%, TCI America), n-octanoic acid (98%, TCI America), 2- methylheptanoic acid (98%, TCI America), 2-ethylhexanoic acid (99%, TCI America), nonanoic acid (98%, TCI America), 4-methyl-n-octanoic acid (98%, TCI America), decanoic acid (98%, TCI America), 4-methylnonanoic acid (98%, TCI America), 4-ethyloctanoic acid (98%, TCI America), 4-dimethylaminopyridine (DMAP, 98%, TCI America), l-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1, 98%, Thermo Scientific), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, AvantiPolarLipids), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC, AvantiPolarLipids), 1.2-dioleoyl-3- trimethylammonium-propane (DOTAP, AvantiPolarLipids), D-Lin-MC3-DMA (MC3, MedChemExpress), cholesterol (Sigma- Aldrich) and l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethyleneglycol)-2000] (C 14PEG2K, AvantiPolarLipids) were used as received. Organic solvents were purchased from Fisher Scientific. Chloroform-d (CDCh) were purchased from Acros Organics.

[0241] Nucleic acids and other reagents for biological assays

[0242] Firefly luciferase (FLuc) mRNA (5moU)-(L-7202) and Cre mRNA (5moU)-(L-7211) were purchased from TriLink BioTechmologies. DNA barcode (b-DNA) design parameters followed previous reports. Scrambled negative control sgRNA (Cat#A35526, Thermo Fisher Scientific) and VEGFR2 sgRNA (Synthego) were used as received. All oligonucleotides were purchased from Integrated DNA Technologies and were purified through stand desalting procedures. Luciferase 1000 Assay System (Ref. E4550) and CellTiter-Glo Luminescent Cell Viability (Ref. G7572) were purchased from Promega Corporation. Alanine Transaminase (ALT) Colorimetric Activity Assay Kit (Item. 700260) and Aspartate Aminotransferase (AST) Colorimetric Activity Assay Kit (Item.701640) were purchased from Cayman Chemical. Anti-mouse CD31 antibody (AF488, Cat#102514), CD45 antibody (Brilliant Violet 421. Cat#103134), EpCAM (AF647, Cat#l 18212) were purchased from BioLegend. Live / Dead staining Draq7 was purchased from BioLegend.

[0243] Cell culture

[0244] Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco containing high glucose. L-glutamine. phenol red, and without sodium pyruvate and HEPES. Trypsin- EDTA (0.25%), penicillin streptomycin (P / S) were purchased from Gibco. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich. HeLa cells (Cat#CCL-2, ATCC. Manassas, Virginia, USA) were cultured in DMEM supplemented with 10% FBS and 1% P / S. GFP expressing Lewis Lung Carcinoma (LLC-GFP) cells were cultured in DMEM supplemented with 10% FBS and 1% P / S. LLC cells were obtained from ATCC (Cat#CRL-1642) and were transduced with GFP according to methods previously described in the literature.

[0245] Animal studies

[0246] C57BL / 6J (female, 6-8 weeks, 18-20 g) and B6.Cg-Gt(ROSA)26Sortml4(CAG- tdTomato)Hze / J (Ail4, female, 6-8 weeks, 18-20 g) mice were purchased from Jackson Laboratory. All mice were housed in a specific-pathogen-free animal facility at ambient temperature, air humidity (40-70%), and 12h dark / 12h light cycle and had free access to water and chow (Cat$5053, LabDiet).

[0247] Instruments

[0248] !H NMR spectrum were performed on a NEO 400 MHz spectrometer. LC-MS was performed on an Agilent LCMS system equipped with UV-Vis and evaporative light scattering detectors (ELSD). Flash chromatography was conducted on a Teledyne Isco CombiFlash Rf-200i chromatography system equipped with UV-Vis and evaporative light scattering detectors (ELSD). LNPs were formulated by a Pump33DS syringe pump (Harvard Apparatus, Holliston, MA). Particle size and zeta potentials were measured by Dynamic Light Scattering (DLS) with Malvern Zetasizer Nano ZS. Particle morphology was measured by Cryo-TEM. Flow cytometry was performed using a Canto Hill machine. In vitro luminescent intensity, cell viability , ALT qualification and AST qualification were quantified using an Infinite M Plex plate reader (Tecan, Morrisville, NC).

[0249] Lipid nanoparticles (LNPs) formulations

[0250] All LNPs encapsulating mRNA used in this study were prepared as follows. An ethanol phase containing all hpids and an aqueous phase containing mRNA (FLuc mRNA. Cre mRNA) were mixed using a microfluidic device to formulate LNPs. Ethanol phase contained CAD lipid, DOPE, cholesterol and C14-PEG2K, with a molar ratio of 35%, 16%, 46.5% and 2.5%. Aqueous phase was composed of mRNA dissolved in 10 mM citrate buffer. The ethanol and aqueous phases were mixed at a flow rate of 1.8 ml / min and 0.6 ml / min (3: 1) using Pump33DS syringe pumps. LNPs were then dialyzed in lx PBS using a microdialysis cassette (20,000 MWCO, Thermo Fisher Scientific, Waltham, MA) for 2 h and filtered through a 0.45 μm filter. Zetasizer Nano was used to measure the Z-average diameters, poly dispersity index (PDI) and Zeta potential. mRNA concentration and encapsulation efficiency of LNP formulation were measured using a modified Quant-iT RiboGreen (ThermoFisher) assay on a plate reader.

[0251] LNPs encapsulating DNA barcodes (b-DNA) and FLuc mRNA were prepared as follows. Ethanol phase contained CAD lipid, DOPE, cholesterol and C14-PEG2K. with a molar ratio of 35%, 16%, 46.5% and 2.5%. Aqueous phase was composed of b- DNAs and FLuc mRNA (w / w, 10: 1) dissolved in 10 mM citrate buffer. LNPs were formulated by pipette mixing the lipid solution into the nucleic acid-containing citrate buffer at a volume ratio of 1:3 (v / v). The resulting LNPs were dialyzed against lx PBS in a 96-well microdialysis plate (10, 000 MWCO, Thermo Fisher Scientific) at room temperature for 2 h. 50 pL of each LNP formulation was pooled together to make LNP pool for dosing. DNA concentration in LNP formulations was determined by a NanoDrop Spectrophotometer. Zetasizer Nano was used to measure the Z-average diameters, poly dispersity index (PDI) and Zeta potential.

[0252] Gold-standard lung-tropic MC3 / DOTAP LNP was used as positive control followed a similar formulation process, where ethanol phase contains MC3 lipid (25%), 1,2- dioleoyl-3- trimethylammonium-propane (DOTAP, 50%), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC, 5%). cholesterol (19.25%), and C14-PEG2K (0.75%).

[0253] In vitro FLuc mRNA LNP library screening

[0254] In a white transparent 96-well plate, HeLa cells were seeded at a density of 5 x 103cells per well in 100 pL growth medium (DMEM. 10% FBS, 1% P / S), and were incubated at 37 °C in 5% CO2. The medium was exchanged for fresh growth medium, and then LNPs were treated at a dose of 10 ng FLuc mRNA per well. Luciferase expression was measured 24 h after LNP transfection using a Luciferase Assay System (Promega) according to the manufacturer’s protocol. The luminescent signal was normalized to PBS treated cells. Cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega), in which the luminescence was normalized to PBS treated cells according to the manufacturer’s protocol. In vivo barcoded LNP delivery

[0255] All animal procedures were performed on female C57BL / 6J mice aged 6-8 weeks. Mice were administered with a pool of different b-DNAs LNPs. along with a naked b-DNAs (served as a negative control), at a dosage of 1.0 mg / kg via tail vein injection. Tissues samples were harvested 6 h post-administration, snap-frozen in liquid nitrogen, disrupted into power using a Geno / Grinder and stored in -80 °C until further analyzed.

[0256] NGS library pool preparation

[0257] To prepare samples, approximately 30 pg of dry homogenized sample was suspended in a DNA-stabilizing lysis buffer containing 100 rnM tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 0,2% sodium dodecyl sulfate (SDS), and 200 mM NaCl. To remove protein and RNA contaminants, 20 pg of RNase A (New England Biolabs) and 100 pg of proteinase K (New England Biolabs) was added to each sample. Barcoded DNA (b-DNA) was subsequently extracted using a Zymo Oligo Clean and Concentrator kit (Zymo Research) following the manufacturer’s instructions. Extracted b-DNA was amplified by PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs) with 16 denaturation-annealing-extension cycles using overhanging primers to add adapter (P5 / P7) and index (i7) sequences for Illumina sequencing. The polymerase chain reaction (PCR) cleanup was performed using AMPure XP solid-phase reversible immobilization (SPRI) beads (Beckman Coulter Life Sciences) at a 1.8: 1 bead:reaction volume ratio. Resultant library concentration was quantified using a Qubit IX dsDNA High Sensitivity assay on a Qubit Flex fluorometer (Thermo Fisher Scientific). Libraries were combined in equimolar amounts to produce a library pool for next-generation sequencing (NGS), which was stored at -20 °C until sequencing. NGS was performed using an Illumina MiSeq series sequencer (RRID:SCR_022382) with a 5% phiX sequencing control (Illumina) spike-in.

[0258] NGS data analysis and visualization

[0259] NGS data were demultiplexed to produce FASTQ files using a standard Illumina sequencing workflow (bc!2fastq2). MD5 checksums were employed to ensure successful data transfer and data integrity. FASTQ files were processed until the UMI- tools Python package to extract unique molecular identifier (UMI) and barcode sequences. All analysis downstream of sequence extraction used a combination of shell scripting and R scripts. GNU sed and awk were used to extract barcode and UMI pairs to tabular data files. To collapse UMIs, an R script was employed using the Rncc, dplyr, multidplyr, stringi, and vroom packages. Further data processing was performed using the dplyr, forcats, readxl, stringi, and tidyr packages. Visualization were created using the ggplot2 package, with ggrepel used for labeling of enriched barcodes for hit identification. The Nix package manager was used for dependency management for all analyses to reproducibility, and GNU Make was used to orchestrate processing steps.

[0260] Counter-screening of FLuc mRNA delivery in vivo

[0261] After analyzing in vivo b-DNA delivery, LNPs-CAD3, CAD4, CAD9, CAD10, and CAD20 were selected for counter-screening by delivering FLuc mRNA. Mice were administered FLuc mRNA-LNP via tail vein injection. The luciferase expression was evaluated using an IVIS Spectrum imaging system (Caliper Life Sciences) 12 h post- injection. Mice were then injected D-luciferin (PerkinElmer) at a dose of 150 mg / kg by intraperitoneal injection (i.p.). Bioluminescence was quantified by measuring total flux in the region of interest where signal emanated using Living IMAGE Software provided by Caliper. Ex vivo imaging was performed on heart, liver, spleen, lung, and kidney after resection.

[0262] Flow cytometry of tdTomato+cell types in the lung

[0263] Ail4 Mice were administered a single intravenous Cre mRNA LNP-CAD9 at a dosage of 0.3 mg / kg via tail vein injection. MC3 / DOTAP LNPs formulating with Cre mRNA was used as positive control. After 3 days post-injection, mice were firstly anesthetized by isoflurane, then perfused with lx PBS. Afterwards the lung was collected, cut into small pieces, and digested by DMEM medium containing collagen IV (0.5 mg / mL). The above cell suspension was then filtered, centrifuged (5 min. 600 g) and lysed by ACK lysis buffer (1 mL) for 5 min. Single-cell suspensions were collected by centrifugation (5 min, 600 g) and resuspended in lx PBS (500 pL), which were further stained by anti -mouse Alexa Fluor 488 CD31 antibody (1:200, Biolegend, Cat#102514). Brilliant Violet 421 CD45 antibody (1:200. Biolegend. Cat#103134), AF647 CD326 antibody (1:200. EpCAM. Biolegend, Cat#l 18212) at 4 °C for 30 min. At last, the above suspensions were centrifuged and resuspended in Draq7 dyed lx PBS (0.5 mL, 0,2%) for flow cytometry analysis.

[0264] Immunofluorescence of the lungs

[0265] Mouse lung was obtained, transported to laboratory on ice and fixed with 3.2% PFA as previously reported. Lung sections were then blocked in PBS + 1% BSA. 5% donkey serum. 0.1% Triton X-100, and 0.02% sodium azide for 1 h at room temperature, followed by incubated with primary antibody (CD31, BioLegend, Cat# 102514) overnight at 4 °C. Afterwards, slides were washed and incubated with fluorophore- conjugated secondary antibody (Alexa Fluor™ 488-conjugated donkey anti-rat, 1 : 1000, Thermo Fisher Scientific. Cat#A-21208) for 2 h. which were further washed and incubated with DAPI for 5 min and mounted using ProLong Gold (Life Sciences, Cat#P36930). Imaging was conducted with a Leica DMi8 microscope and analyzed with LAS X software (Leica).

[0266] In vivo CRISPR-Cas9 VEGFR2 editing for antitumor therapy

[0267] LLC-GFP lung tumor model was established through tail veil injection of 1.0 x 106LLC-GFP cell into C57BL / 6J female mice. On day 20 after tumor cell inoculation, mice were randomly assigned to four groups: PBS treated group (n = 12, Gl), LNP-CAD9 encapsulating Cas9 mRNA / scramble sgRNA treated group (n = 12, G2), LNP-CAD9 encapsulating Cas9 mRNA / VEGFR2 sgRNA treated group (n = 12, G3), and MC3 / DOTAP encapsulating Cas9 mRNA / VEGFR2 sgRNA treated group (n = 12, G4). Mice were treated every other day for a total of 2 times (2.0 mg kg’1of RNA per injection). 6 of the mice in each group were euthanized 7 days post- administration of LNPs, and their lungs were collected for analyses. The rest mice were subjected for survival evaluation. For survival analysis, mice were euthanized upon reaching a body weight loss exceeding 20% via carbon dioxide asphyxiation. The sequence of VEGFR2 sgRNA (5'-GTCCCGGTACGAGCACTTGT-3') were used according to a previous study. Tumor-bearing lung tissue were fixed with 3.2% PFA and then subjected for paraffin wax section. The obtained lung section slides were dewaxed, then blocked in PBS+3% BSA, and 0.1% Triton X-10 for 30 mins at room temperature, follow ed by incubated with primary antibody CD31 (Cat#AF3628, 1 :200, R&D Systems) and GFP (Cat#ab 183734. 1 :200, Abeam) overnight at 4 °C. Afterwards, slides were washed and incubated with secondary antibody (AF488-conjugated donkey anti-rabbit, 1 : 1000, Thermo Fisher Scientific. Cat#A-21206; AF555 -conjugated donkey anti-goat. 1: 1000, Thermo Fisher Scientific. Cat#A32816) for 2 h, which were further washed and incubated with DAPI for 5 min and mounted. Imaging was conducted with a Leica DMi8 microscope and analyzed with LAS X software (Leica).

[0268] Statistics and Reproducibility

[0269] Two-sided Wilcoxon rank-sum test was used for the analysis of normalized accumulation of LNP formulation. One-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test was utilized for statistical analysis, a value were applied to annotate statistical significance. All in vitro experiment were performed independently for at three times. In vivo barcoded experiment was performed with animal size of 5 female mice. IVIS imaging was performed with animal size of 3 female mice. Cre mRNA delivery' was performed with animal size of 4 female mice. Tumor inoculation was performed using a cohort of 12 female mice. All data were presented as mean ± s.e.m.

[0270] Example 1: Chemical synthesis

[0271] (a) Synthesis of aldehyde di-degradable tails

[0272] Taking 5 -formyl- 1,3 -phenylene diheptanoate (A2-6) as an example, briefly, 3,5- dihydroxybenzaldehyde (1.38 g, 10 mmol, 1.0 equiv). heptanoic acid (3.91 g, 30 mmol, 3.0 equiv), l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HC1, 5.75 g, 30 mmol, 3.0 equiv) and 4-dimethylaminopyridine (DMAP, 366 mg, 3 mmol, 0.3 equiv) were dissolved in anhydrous dichloromethane (DCM; 50 mL) and the mixture was cooled to 0°C on an ice bath, the reaction was then allowed to warm to room temperature overnight. The supernatant was washed with HC1 (1%; 2 x 50 mL), brine (2 x 50 mL), saturated sodium bicarbonate (2 x 50 mL) and brine (2 x 50 mL). The organic layer was collected, dried over Na2SO4, and concentrated in vacuo. The final pure monomer was further purified by flash chromatographic (silica gel, hexane to DCM / hexane = 2 / 1) as light-yellow oil.JH NMR (400 MHz, 9.98 (s, 1H), 7.52 (s, 2H), 7.21 (s, 1H). 2.63-2.56 (m, 4H). 1.82-1.71 (m, 4H), 1.49-1.30 (m, 12H), 0.98-0.89 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 363.4, Found:

[0273] 363.4.

[0274] A2-6b. ‘H NMR (400 MHz, CDCl3) δ: 9.98 (s, 1H), 7.51 (s, 2H), 7.18 (s, 1H), 2.63- 2.56 (m, 2H), 1.91-1.53 (m, 4H), 1.51-1.27 (m, 14H), 0.98-0.88 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 363.4, Found: 363.4.

[0275] Az-7. ‘H NMR (400 MHz, CDCl3) δ: 9.97 (s, 1H), 7.52 (s, 2H), 7.21 (s, 1H), 2.76- 2.67 (m, 2H), 1.81-1.71 (m, 4H), 1.47-1.25 (m, 16H), 0.98-0.87 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 391.5, Found: 391.5.

[0276] A2-7b. ‘H NMR (400 MHz, CDCl3) δ: 9.98 (s, 1H), 7.51 (s, 2H), 7.17 (s, 1H), 2.76- 2.66 (m, 2H), 1.88-1.53 (m, 4H), 1.50-1.26 (m, 18H), 0.97-0.87 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 391.5, Found: 391.5.

[0277] A2-7b2. ‘H NMR (400 MHz, CDCl3) δ: 9.99 (s, 1H), 7.52 (s, 2H), 7.16 (s, 1H), 2.59- 2.52 (m, 2H), 1.86-1.74 (m, 4H), 1.70-1.58 (m, 4H), 1.44-1.28 (m, 8H), 1.09-1.01 (m, 6H), 0.97-0.89 (m, 6H). LC-MS (m / z): Calcd for [M+H]~: 391.5, Found: 391.5.

[0278] Az-8. ‘H NMR (400 MHz, CDCl3) δ: 9.97 (s, 1H), 7.51 (s, 2H), 7.19 (s, 1H), 2.64-

[0279] 2.54 (m, 4H), 1.84-1.70 (m, 4H), 1.50-1.24 (m, 20H), 0.98-0.87 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 419.5, Found: 419.5.

[0280] A2-8b. ‘H NMR (400 MHz, CDCl3) δ: 9.97 (s, 1H), 7.51 (s. 2H), 7.17 (s, 1H), 2.68-

[0281] 2.53 (m, 4H), 1.90-1.77 (m, 2H), 1.66-1.53 (m, 4H), 1.41 -1.20 (m, 12H), 1 .00-0.89 (m, 12H).

[0282] LC-MS (m / z): Calcd for [M+H] ": 419.5, Found: 419.5.

[0283] Az-9. ‘H NMR (400 MHz, CDCl3) δ: 9.97 (s, 1H), 7.51 (s, 2H), 7.22 (s, 1H), 2.63-

[0284] 2.55 (m, 4H), 1.82-1.73 (m, 4H), 1.50-1.22 (m, 24H), 0.97-0.87 (m, 6H). LC-MS (m / z): Calcd for [M+H]+: 447.6, Found: 447.6.

[0285] A2-9b. ‘H NMR (400 MHz, CDCl3) δ: 9.97 (s, 1H), 7.51 (s. 2H), 7.19 (s, 1H), 2.68-

[0286] 2.55 (m, 4H), 1.89-1.78 (m, 2H), 1.66-1.52 (m, 4H), 1.41-1.22 (m, 16H), 0.99-0.89 (m, 12H). LC-MS (m / z): Calcd for [M+H]’: 447.6, Found: 447.6.

[0287] A2-9b2. 'H NMR (400 MHz, CDCL) d. 9.99 (s, 1H), 7.52 (s, 2H), 7.22 (s, 1H). 2.63- 2.55 (m, 4H), 1.80-1.71 (m, 4H), 1.44-1.22 (m, 18H), 0.98-0.89 (m, 12H). LC-MS (m / z): Calcd for [M+H]+: 447.6, Found: 447.6.

[0288] Synthetic route of aldehyde tri-degradable tails used in the disclosure. Taking 2- formylbenzene-l,3,5-triyl tris(2-methylhexanoate) (Aj-6b) as an example, briefly, 2,4,6- trihydroxybenzaldehyde (1.54 g. 10 mmol, 1.0 equiv), heptanoic acid (5.86 g, 45 mmol, 4.5 equiv), l-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC HC1, 8.62 g, 45 mmol, 4.5 equiv) and 4-dimethylaminopyridine (DMAP, 550 mg, 4.5 mmol, 0.45 equiv) were dissolved in anhydrous dichloromethane (DCM; 80 mL) and the mixture was cooled to 0 °C on an ice bath, then the reaction was then allowed to warm to room temperature overnight. The supernatant was washed with HC1 (1%; 2 x 50 mL), brine (2 x 50 mL), saturated sodium bicarbonate (2 x 50 mL) and brine (2 x 50 mL). The organic layer was collected, dried over concentrated Na2SO4, and concentrated in vacuo. The final pure monomer was further purified by flash chromatographic (silica gel, hexane to ethyl acetate / hexane = 1 / 6) as orange oil.!H NMR (400 MHz, CDCl3) δ. 10.15 (s, 1H), 6.93 (s, 2H), 2.84-2.69 (m, 3H), 1.96-1.76 (m, 6H), 1.61-1.19 (m, 21H), 0.98-0.83 (m, 9H). LC-MS (m / z): Calcd for [M+H]+: 491.6, Found: 491.6. A3-7b. ‘H NMR (400 MHz, CDCl3) δ: 10.15 (s, 1H), 6.90 (s, 2H), 2.83-2.69 (m, 3H), 1.91-1.71 (m, 6H), 1.47-1.22 (m, 27H), 0.98-0.88 (m, 9H). LC-MS (m / z): Calcd for [M+H]+: 533.7, Found: 533.7.

[0289] A3-7b2. 'H NMR (400 MHz, CDCl3) δ: 10.15 (s, 1H). 6.91 (s, 2H). 2.66-2.56 (m, 3H), 1.91-1.77 (m, 6H), 1.73-1.59 (m, 6H), 1.46-1.36 (m, 12H), 1.10-0.89 (m, 18H). LC-MS (m / z): Calcd for [M+H]+: 533.7, Found: 533.7.

[0290] A3-8b. 'H NMR (400 MHz, CDCl3) δ: 10.06 (s, 1H), 6.95 (s, 2H), 2.73-2.53 (m, 3H), 1.92-1.74 (m, 6H), 1.66-1.47 (m, 6H), 1.44-1.15 (m, 21H), 1.02-0.85 (m, 18H). LC-MS (m / z): Calcd for [M+H]+: 575.8, Found: 575.8.

[0291] A3-9b2. 'HNMR (400 MHz, CDCl3) δ: 10.15 (s, 1H), 6.66-6.61 (s, 2H), 2.69-2.34 (m. 6H), 1.82-1.69 (m. 6H), 1.68-1.60 (m. 3H), 1.43-1.21 (m. 24H), 0.99-0.86 (m, 18H). LC- MS (m / z): Calcd for [M+H]+: 617.8, Found: 617.8.

[0292] (c) Synthesis of cationic degradable lipid (CAD) libraries

[0293] A cationic degradable lipid library (180 lipids) was prepared by one-pot, two steps “Schiff-base” reduction reaction between twelve different amine heads and fifteen different aldehyde degradable tails. Taking synthesis of 5-A2-7b2 as an example, amine head 5 (17.63 mg, 0.2 mmol, 1 equiv) and A2-7b2 (93.73 mg, 0.24 mmol, 1.2 equiv) were added in a glass vial equipped with a stir bar dissolved in ethanol. Then acetic acid (30 mg, 0.48 mmol, 2,4 equiv) was added into the above solution and the reaction was stirred at 80 °C for 3 hours. Sodium borohydride (NaBH-i. 75 mg, 2 mmol, 10 equiv) was further added to react for 1 hour at room temperature. Then dichloromethane was added into the above solution, which was further washed by saturated sodium chloride (NaCl x 3) and dried by sodium sulfate (NaSOi). The crude product was afforded by removing the solvents and could be used to screen the library for FLuc mRNA delivery without further purification.

[0294] In another trial, the crude product can be further purified by flash chromatography (DCM to DCM / MeOH=10 / l). 5-A2-7b2. NMR (400 MHz, CDCl3) δ: 6.96 (s, 2H), 6.75 (s, 1H), 3.83 (s, 2H), 2.77-2.68 (m, 2H), 2.55-2.44 (m, 4H), 2.27-2.22 (s, 6H), 1.85-1.54 (m, 8H), 1.46-1.34 (m. 8H), 1.08-0.98 (m. 6H), 0.97-0.90 (m. 6H). LC-MS (m / z): Calcd for [M+H]+: 464.7, Found: 464.7.

[0295] Example 2: Combinatorial design of CAD lipids

[0296] It has been reported that incorporating atypical chemical motifs can alter protein corona composition on LNPs and shift organ tropism. Notably, positively charged molecules enable the binding of distinct proteins which can interact with specific cellular receptors highly expressed within the lungs for extrahepatic nucleic acid delivery. These molecules are typically utilized as a fifth constituent incorporated into the LNP formulation for tissue- specific mRNA delivery; however, identifying the interplay between the structure of the lipids themselves and lung tropism remains challenging.

[0297] Ionizable lipids were rationally designed according to a Schiff base reduction scheme, which links amine heads and aldehyde degradable alkyl tails (FIG. IB). In brief, combinatorial reactions between 12 amine heads and 15 aldehyde degradable tails were conducted for 3 h to yield Schiff base intermediates under acetic acid (AcOH) (FIG. 1C and FIG. 2) A subsequent reduction under sodium borohydride was conducted for 1 h and led to the final cationic degradable (CAD) lipids. This library was broadened by varying amine core structures, tail architecture, tail substitution numbers, and tail lengths, giving the resulting 180 CAD lipids the nomenclature X-Ay-Z, where “X” indicates the order of amine cores in this study, “Ay-Z” represents aldehyde degradable tails (“y” represents the tail number; “Z” represents the carbon number on each tail). This “two-step, one-pof ’ reaction is simple and robust, yielding CAD lipids in several hours, which is significantly faster than the widely used “Michael addition” reaction. Moreover, the final product can be used without further purification (FIG. 2). It was envisioned that this combinatorial CAD ionizable lipid library could extend the chemical diversity of ionizable lipid formulations for nucleic acid delivery applications.

[0298] Example 3: In vitro high-throughput screening to identify novel lipid-like materials for potent mRNA transfection

[0299] The structure-activity relationship (SAR) of novel CAD lipid-like materials for mRNA delivery was first investigated in vitro. CAD LNPs encapsulating firefly luciferase (FLuc) mRNA were used to transfect HeLa cells. CAD LNPs were formulated using CAD lipids, the phospholipid DOPE, cholesterol, and lipid-anchored poly(ethylene glycol) (C14PEG2K) (35: 16:46.5:2.5 molar ratio) and were mixed with FLuc mRNA via perfusion through a microfluidic mixing device designed with staggered herringbone features (FIG. 3A). The resulting CAD LNPs showed mRNA encapsulation efficiencies ranging from 74% to 95% (FIG. 4). CAD LNPs showed uniform solid core morphology when investigated using cryo-transmission electron microscopy (cryo-TEM) (FIGs. 3B-3C). Additionally, all CAD LNPs exhibited low cytotoxicity (cell viability >85%) (FIG. 5).

[0300] From in vitro screening in HeLa cells, a heat map of mRNA delivery by CAD lipids was generated by calculating the relative hit rate (relative luminescence units, RLU > 100) of different CAD lipid parameters to evaluate which structural parameters are most important for mRNA delivery in vitro (FIG. 3D). It was first investigated if the cationic amine number of each CAD lipid influenced mRNA delivery efficacy, observing that CAD lipids with two secondary7amines per lipid exhibited the highest mRNA delivery efficacy, with a hit rate of -13% over the whole library (FIG. 3E). It was postulated that CAD lipids with a greater number of secondary amine groups (>2) had a relatively higher binding efficacy with mRNA, making it difficult to release hydrophilic mRNA compounds into the cytoplasm for efficient deliver^'. Additionally, it was observed that CAD lipids with branched architecture exhibited substantially higher mRNA delivery than linear structured ones (FIG. 3H). which corroborates a previous report that branched tails may increase endosomal escape for mRNA delivery. Importantly, tail number and tail length on each aldehyde were very influential for mRNA deliver)', where a tail number of 2 and a tail length of 7 on each aldehyde resulted in the highest hit rates (FIGs. 3G-3H). These observations are in accordance with previously reported LNP systems, where efficacy generally correlated with tail substitution sites and diversity. Within this library, 96 CAD LNP formulations which showed effective transfection in HeLa cells were then selected for subsequent in vivo studies.

[0301] Example 4: Understanding CAD lipid structure and organ tropism relationships in vivo

[0302] To better understand the relationship between CAD lipid structure and their organ tropism, 96 CAD LNPs were evaluated in vivo through a barcode DNA-based assay, which can quantify how hundreds of different LNPs deliver mRNA in vivo. LNP1, with chemical composition 1, was formulated to carry b-DNA 1 and FLuc mRNA, and LNP N, with chemical composition N, to carry b-DNA N and FLuc mRNA, at a weight ratio of 10: 1, using pipette mixing (FIG. 6A). To investigate the potential influence of b-DNA on LNP structure and the efficacy of mRNA deliver)' in vitro, 3-A2-7b LNP platform was used as a representative example to encapsulate b-DNA / FLuc mRNA (at a weight ratio of 10: 1) and FLuc mRNA, respectively. These two LNPs exhibit similar structural morphology (FIG. 3B and FIG. 13A), however, LNP carrying b-DNA / FLuc mRNA displayed a reduction in average particle size (FIG. 3C and FIG. 13B), which is attributed to the smaller size of b- DNA compared to mRNA. Nonetheless, despite these differences in particle size, no appreciable distinction in transfection efficacy was observed between these two LNP formulations in vitro (FIG. 13C)

[0303] By integrating unique DNA barcodes in each LNP, the organ tropism of each tested LNP was assessed through deep sequencing. The hydrodynamic diameter of these LNPs was evaluated as a qualify control measure, showing that the size of these LNPs ranged from 100 to 250 nm (FIGs. 6B and Table 1), consistent with previous reports that pipette-mixed LNPs are generally larger in size. Because CAD lipids were incorporated into the formulation, over 65% of the resulting CAD LNPs exhibited a positive charge (FIG. 6C and Table 1). Furthermore, the diameter and zeta potential of the pool of CAD LNPs were tested and it was found that they were within the range of the diameter and zeta potential of the 96 individual CAD LNPs, respectively, indicating that mixing the CAD LNPs did not adversely affect solubility (FIGs. 6B-6C).

[0304] Table 1. Particle size, PDI, zeta potential, and encapsulation efficiency (EE) of CAD 1-96 based barcode LNPs and its respective CAD lipid information.

[0305] After characterizing the pool of 96 CAD LNPs, the LNPs were then intravenously (i.v.) administered to C57BL / 6J mice at a total nucleic acid dose of 1.0 mg / kg (averaging a 0.01 mg total nucleic acid / kg / particle, for all 96 CAD LNPs), and tissues (e.g., heart, liver, spleen, lung, and kidney) were isolated 6 h post-injection and DNA extracted from these tissues (FIG. 6A) Extracted DNA samples were amplified by polymerase chain reaction (PCR) and deep sequenced to compare the relative accumulation of CAD LNPs in different tissues through comparison to the uninjected LNP pool. This approach allowed us to identify CAD LNPs with preferential accumulation in specific organs.

[0306] This large dataset was used to analyze a comprehensive in vivo structure-activity relationship. First, a heat map was generated based on the normalized accumulation of each barcoded oligomer in different organs (FIG. 6D and FIG. 7). Within the heat map, darker red represents higher relative accumulation in a tissue of interest. The secondary amine number of each CAD lipid, tail architecture, tail length, and tail number on each aldehyde group appeared to significantly affect their structure-activity relationship. Specifically, more secondary amine-based CAD lipids (secondary amine number >2) preferentially delivered cargoes to the lungs compared to monoamine CAD lipids (FIG. 6D). It was hypothesized that more strongly cationic CAD lipids may result in a relatively positive charge in the LNP formulation, which is supported by the fact that -60% of these LNPs display a positive charge. This conclusion is also in accordance with previous studies reporting that cationic lipids facilitate LNP formulations deliver genetic cargos into the lungs. The correlation between LNP size and lung-tropic activity was further investigated, and only a weak relationship was observed (FIG. 14). To visualize the in vivo activity of tested CAD LNPs in more detail, volcano plots were generated to show the results of enrichment analysis (FIG. 6E). Through this enrichment analysis, it was found that 21 of the tested CAD LNPs can efficiently deliver genetic cargo into the lungs. Notably, both LNP-CAD24 and LNP-CAD56 displayed the greatest enrichment within the lungs. However, both LNP-CAD24 and LNP-56 displayed at least sone enrichment in the liver and spleen, whereas LNP-CAD3, LNP-CAD4, LNP-CAD9, and LNP-CAD10 demonstrated highly enriched accumulation in the lungs with depleted or not notably enriched delivery in the liver and spleen (FIG. 6E), bringing these LNP formulations to the fore in the search for LNPs mediating mRNA delivery to the lungs.

[0307] Example 5: Validation of top performing LNPs for mRNA delivery to the lungs

[0308] Through high-throughput screening both in vitro and in vivo, 180 chemically distinct CAD LNPs were screened to discover that 21 of them show lung-targeting delivery in vivo. By further enrichment analysis, 4 lead LNP formulations were identified which efficiently deliver genetic cargoes to the lungs (FIG. 8A). To verify that measures of b-DNA accumulation can accurately identify’ CAD LNPs for mRNA delivery, the lead identified liver formulation, LNP-CAD20, was used (FIGs. 6D-6E), to deliver FLuc mRNA in vivo. The specific luciferase expression in the liver supported the high-throughput barcoded screening results (FIGs. 9A-9C). To further validate mRNA delivery efficacy to the lungs by the lead lung-tropic LNP formulations, LNP-CAD3, 4, 9, and 10 were formulated with FLuc mRNA and systemically injected into C57BL / 6J mice at a dose of 0.1 mg / kg (FIG. 9B). Bioluminescence imaging confirmed that the selected 4 CAD LNPs can functionally deliver mRNA the lungs as expected from high-throughput screening results. By analyzing luminescence of the lungs, liver, and spleen, LNP-CAD9 was identified as the top performing LNP candidate for pulmonary mRNA delivers’ (FIGs. 8C-8F), which exhibited luciferase expression predominantly in the lungs (-90% of total luminescence flux).

[0309] Next, it was explored whether LNP-CAD9 delivered mRNA at a clinically relevant dose. For this, an Ail4 (constitutive loxP-STOP-loxP-tdTomato) mouse model was employed, in which the translated Cre protein excises the stop cassette to produce tdTomato fluorescence only in transfected cells upon intracellular delivery of Cre recombinase mRNA (FIG. 8H). Following a single administration of 0.3 mg / kg Cre mRNA using LNP-CAD9, efficient lung gene editing was observed 3 days post-administration (FIG. 8H and FIG. 10), resulting in -60% tdTomato+ endothelial cells. To benchmark the lung delivery potency of the identified LNP-CAD9. a gold standard lung-tropic MC3 / DOTAP system, in which DOTAP was reported to be incorporated as a cationic lipid component for facilitating FDA- approved MC3 LNP delivers mRNA to the lungs, was produced and tested. LNP-CAD9 induced a significantly higher percentage of tdTomato+endothelial cells (FIG. 8H and FIG. 10), with a 2.7-fold increase compared to the MC3 / DOTAP LNP, which was further validated by increased tdTomato area from immunostaining (FIG. 81). LNP-CAD9 carrying Cre mRNA mainly reached the capillary endothelial cells of the vasculature in the lungs (FIG. 81). Moreover, LNP-CAD9 delivering Cre mRNA did not demonstrate discernable editing of endothelial cells within the liver and heart, highlighting the specific and targeted editing of lung endothelium achieved by LNP-CAD9 (FIGs. 15A-15B and FIGs. 16A-16B). Additionally, LNP-CAD9 displayed minimal in vivo toxicity from tissue section histology (FIG. 11), demonstrating the promising translational potential of CAD LNPs. These results led us to conclude that LNP-CAD9, discovered by high-throughput screening technology, preferentially delivered mRNA into lung endothelial cells at a clinically relevant dose, substantially outperforming a gold-standard lung-tropic MC3 / D0TAP formulation.

[0310] Example 6: Exploring therapeutic potential of top performing LNPs for in vivo gene editing for antitumor therapy

[0311] The therapeutic potential of this identified platform was assessed in vascular-relevant disease models, at least in part because LNP-CAD9 demonstrated promising editing efficacy of lung endothelial cells in vivo. Angiogenesis is a complex and vital physiological process, playing crucial roles in embryo development, wound healing, and collateral vessel formation. However, angiogenesis becomes aberrantly upregulated in tumorigenesis, supporting tumor progression by supplying essential oxygen and nutrients. Consequently, antiangiogenic therapy strategies aim to starve tumors by disrupting the delivery of these vital resources, thereby suppressing tumor growth, invasion, and metastasis. Among the numerous pro- angiogenic factors that have been discovered, vascular endothelial growth factor (VEGF) ranks among the most important. VEGF binds to its receptor VEGFR2, on tumor vascular endothelial cells, which subsequently promotes endothelial cell proliferation, migration and survival, leading to increased tumor vascularization through the growth of new blood vessels. Thus, the targeted disruption of VEGFR2 expression holds promise as an approach to inhibit the VEGF-VEGFR2 signaling pathway for antiangiogenic cancer therapy.

[0312] A representative orthotropic lung cancer model was established in female mice by intravenous administration of Lewis Lung Carcinoma cell lines expressing GFP (LLC-GFP). and antiangiogenic cancer therapy efficacy of LNP-CAD9 co-encapsulating Cas9 mRN A / VEGFR2 single guide RNA (sgRNA) was evaluated in vivo (FIG. 12A). MC3 / D0TAP LNPs served as a benchmark lung-tropic LNP control. After tumor inoculation for 20 days, the mice w ere randomly allocated into four groups and received i.v. administration of PBS (Gl), LNP-CAD9 co-delivering Cas9 mRNA / scrambled sgRNA (G2), LNP-CAD9 co-delivering Cas9 mRNA / VEGFR2 sgRNA (G3), or MC3 / DOTAP co- delivering Cas9 mRN A / VEGFR2 sgRNA (G4), with a total RNA dosage of 4.0 mg kg'1(administered in two days of 2.0 mg kg1each injection). Cas9 / VEGFR2 sgRNA complex would induce double-strand breaks and insertions / deletions within the VEGFR2 locus, and thereby inhibiting the VEGF-VEGFR2 signaling pathway (FIG. 12B). Seven days after the last administration, mice were euthanized, and their lungs were isolated to assess in vivo antitumor efficacy under the various treatment conditions. The mice that received LNPs co- delivering Cas9 mRNA / VEGFR2 sgRNA (G3 and G4) exhibited decreased VEGFR2 expression as quantified by RT-qPCR compared to PBS or scrambled sgRNA treated groups (G1 and G2) (FIG. 12C). Furthermore, evaluations of the tumor area per lung and histological examinations via hematoxylin and eosin (H&E) staining revealed that the administration of LNP-CAD9 co-delivering Cas9 mRNA / VEGFR2 sgRNA led to a significant reduction in lung tumor burden, outperforming MC3 / DOTAP treated groups (FIGs. 12D-12E). The survival analysis also demonstrated that administration of LNP-CAD9 co-delivering Cas9 mRNA / VEGFR2 sgRNA presented the highest tumor-inhibitory potential among all the treatments. This treatment regimen extended the median survival period from 32 days (Gl) to 52 days (G3) (FIG. 12F). Additionally, angiogenesis of tumor tissues was assessed by immunostaining vascular endothelial cells using CD31 antibody (FIGs. 12G- 12H). The results revealed a marked reduction in newly formed tumor blood vessels within the tumor site after treatment with LNP-CAD9 co-delivering Cas9 mRNA / VEGFR2 sgRNA (FIG. 12G). Quantification of microvascular density (MVD) in the tumor tissues showed that the administration of Cas9 mRNA / VEGFR2 sgRNA encapsulated by LNPs (G3 and G4) significantly reduced MVD compared to PBS or scrambled sgRNA treated groups (Gl and G2), indicating a pronounced antiangiogenic effect (FIG. 12H). Importantly, mice treated with LNP-CAD9 co-delivering Cas9 mRNA / VEGFR2 sgRNA demonstrated superior antitumor efficacy compared to MC3 / DOTAP LNPs. Collectively, these findings underscore the therapeutic potential of LNP-CAD9 platform for inhibiting tumor angiogenesis in the lung, resulting in effective suppression of tumor growth and far surpassing the lung-tropic gold-standard MC3 / DOTAP formulations.

[0313] Enumerated Embodiments

[0314] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0315] Embodiment 1 provides the compound of formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein:

[0316] R1is selected from the group consisting of N(RA)(RB) and N(R2b)(R3b); each occurrence of L1is independently selected from the group consisting of -N(R2d)-, -N(RA)-, -(optionally substituted C1-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2- Cs heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C8heteroarylenyl)-, wherein one occurrence of L1is optionally substituted with -(optionally substituted C1-C6alkylenyl)-N(R2c)(R3c); each occurrence of R2a, R2b, R2c, and R2dis independently L2— A(R4)n. each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H and optionally substituted C1-C6alkyl; each occurrence of A is independently optionally substituted C6-C10ary l and optionally substituted C2-C8heteroaryl; each occurrence of R4is -OC(=O)(optionally substituted C1-C24 alkyl); each occurrence of L2is independently -(optionally substituted C1-C6alkylenyl)-; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C3-C8cycloalkyl, optionally substituted C2-C8heterocycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C8heteroaryl. or RAand RBcan combine with the N atom to w hich they are bound to form a C2-C8heterocycloalkyl; m is 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10; and each occurrence of / 7 is independently 1. 2, or 3.

[0317] Embodiment 2 provides the compound of Embodiment 1, w herein at least one of the following applies:

[0318] (a) each occurrence of L1is independently selected from the group consisting of - (optionally substituted C1-C6alkylenyl)-

[0319] (b) RAand RBcombine with the N atom to which they are bound to form

[0320] (c) RAand RBare each independently methyl or ethyl.

[0321] Embodiment 3 provides the compound of Embodiment 1 or 2, wherein L1and m are selected such that (L ) / ??- is selected from the group consisting of -(CH2)2-, -(CH2)3-, -

[0322] Embodiment 4 provides compound of any one of Embodiments 1-3, wherein R1is selected from the group consisting of NMe2, NE an

[0323] Embodiment 5 provides the compound of any one of Embodiments 1-4, wherein the compound of formula (I) is selected from the group consisting of: Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein A,

[0324] R4, and n are selected such that -A(R4)nis: wherein: each occurrence of R5a, R5b, and R’c, if present, is independently each occurrence of R6a, R6b, R7a, R711, R8a, R8b, and R9is independently H or C1-C6alkyl. wherein R6a, R6b, R7a. R7b, R8a, R8b. and R9are selected such that each occurrence of R5a, R3b, and R5ccomprise a Ci-C24 alkyl.

[0325] Embodiment 7 provides the compound of Embodiment 6, wherein at least one of the following applies:

[0326] (a) one of R6a, R6b, R7a, R711, R8a, and R8bis methyl, and R9is ethyl;

[0327] (b) one of R6a, R6b. R7a, R711, R8a. and R8bis methyl, and R9is propyl;

[0328] (c) one of R6a, R6b, R7a, R7b, R8a, and R8bis methyl, and R9is butyl;

[0329] (d) one of R6a, R6b, R7a, R7b, R8a, and R8bis methyl, and R9is pentyl;

[0330] (e) one of R6a, R6b, R7a, R7b. R8a, and R8bis methyl, and R9is hexyl;

[0331] (a) one of R6a, R6b. R7a, R™, R8a. and R8bis ethyl, and R9is ethyl;

[0332] (b) one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is propyl;

[0333] (c) one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is butyl;

[0334] (d) one of R6a, R6b, R7a, R71’. R8a, and R8bis ethyl, and R9is pentyl; and

[0335] (e) one of R6a, R6b, R7a, R711, R8a, and R8bis ethyl, and R9is hexyl.

[0336] Embodiment 8 provides the compound of Embodiment 6 or 7, wherein R5a. R5b, and R5c, if present, are each independently selected form the group consisting of 1 -methylpentyl, 1 -methylhexyl, 1 -methylheptyl, 1 -methyloctyl, 1 -methylnonyl, 1 -ethylpentyl, 1 -ethylhexyl,

[0337] 1 -ethylheptyl, 1 -ethyloctyl, 2-methylpentyl, 2-methylhexyl, 2-methylheptyl, 2-methyloctyl,

[0338] 2 -methylnonyl, 2-ethylpentyl, 2-ethylhexyl, 2-ethylheptyl, 2-ethyloctyl, 3-methylpentyl, 3- methylhexyl, 3-methylheptyl, 3-methyloctyl, 3 -methylnonyl, 3 -ethylpentyl, 3-ethylhexyL 3- ethylheptyl, 3-ethyloctyl, and 3-ethylnonyl.

[0339] Embodiment 9 provides the compound of any one of Embodiments 1-8, wherein each occurrence of R2a, R2b. R2c, and R2d, if present, is independently selected from the group consisting of:

[0340] Embodiment 10 provides the compound of any one of Embodiments 1 -9, which is selected from the group consisting of:

[0341]

[0342] Embodiment 11 provides a lipid nanoparticle (LNP) composition comprising:

[0343] (a) at least one compound of any one of Embodiments 1 -10;

[0344] (b) at least one neutral lipid;

[0345] (c) at least one cholesterol lipid and / or a modified derivative thereof;

[0346] (d) at least one polymer conjugated lipid and / or a modified derivative thereof.

[0347] Embodiment 12 provides the LNP of Embodiment 11, further comprising at least one cargo molecule.

[0348] Embodiment 13 provides the LNP composition of Embodiment 12, wherein the cargo is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof.

[0349] Embodiment 14 provides the LNP composition of Embodiment 12 or 13, wherein the cargo is a nucleic acid.

[0350] Embodiment 15 provides the LNP composition of Embodiment 13 or 14, wherein the nucleic acid is DNA or RNA.

[0351] Embodiment 16 provides the LNP composition of any one of Embodiments 13-15, wherein the nucleic acid is selected from the group consisting of siRNA, sgRNA, mRNA, cDNA, pDNA, microRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof.

[0352] Embodiment 17 provides the LNP composition of Embodiment 16, wherein the mRNA encodes a therapeutic protein, optionally wherein the therapeutic protein is a CRISPR-associated protein, and optionally wherein the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9). Embodiment 18 provides the LNP of any one of Embodiments 12-17, wherein the cargo is at least partially encapsulated in the LNP.

[0353] Embodiment 19 provides the LNP of any one of Embodiments 11-18, wherein the compound of formula (I) is selected from the group consisting of: and

[0354] Embodiment 20 provides the LNP of any one of Embodiments 11-19. wherein the at least one cationic degradable lipid comprises about 10 mol% to about 70 mol% of the LNP, optionally wherein the at least one cationic degradable lipid compound comprises about 35 mol% of the LNP.

[0355] Embodiment 21 provides the LNP of any one of Embodiments 11-20, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylchohne (DSPC).

[0356] Embodiment 22 provides the LNP of any one of Embodiments 11-21, wherein the at least one neutral lipid comprises about 1 to about 30 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 16 mol% of the LNP.

[0357] Embodiment 23 provides the LNP of any one of Embodiments 11-22, wherein the at least one cholesterol lipid and / or modified derivative thereof is cholesterol.

[0358] Embodiment 24 provides the LNP of any one of Embodiments 11-23, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 70 mol% of the LNP, optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 46.5 mol% of the LNP.

[0359] Embodiment 25 provides the LNP of any one of Embodiments 11-24, wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises at least one selected from the group consisting of:

[0360] 1 ,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000, and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt).

[0361] Embodiment 26 provides the LNP of any one of Embodiments 11-25. wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 2.5 mol% of the LNP.

[0362] Embodiment 27 provides the LNP of any one of Embodiments 11-26, wherein the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35: 16:46.5:2.5

[0363] Embodiment 28 provides a pharmaceutical composition comprising the LNP of anyone of Embodiments 11-27 and a pharmaceutically acceptable carrier.

[0364] Embodiment 29 provides a method for delivering a cargo to a subject’s lungs, the method comprising administering to the subject at least one lipid nanoparticle of any one of Embodiments 12-27 and / or the pharmaceutical composition of Embodiment 28.

[0365] Embodiment 30 provides the method of Embodiment 29, wherein the cargo is delivered to a lung epithelial cell.

[0366] Embodiment 31 provides the method of Embodiment 29 or 30, wherein the cargo is selectively delivered to the lung over at least one selected from the group consisting of the liver, heart, kidney, and spleen.

[0367] Embodiment 32 provides a method for treating, preventing, and / or ameliorating a lung-associated disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle of any one of Embodiments 12-27 and / or the pharmaceutical composition of Embodiment 28.

[0368] Embodiment 33 provides the method of Embodiment 32, wherein the lung-associated disease or disorder is at least one selected from the group consisting of acute respiratory distress syndrome (ARDS), asthma, bronchiectasis, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disorder (COPD), cystic fibrosis, hypersensitivitypneumonitis, interstitial lung disease (ILD), lung abscess, lung cancer, occupational lung diseases, pleural effusion, pneumonia, pulmonary edema, pulmonary embolism, pulmonary fibrosis, pulmonary hypertension, sarcoidosis, and tuberculosis.

[0369] Embodiment 34 provides the method of any one of Embodiments 29-33, wherein the subject is a mammal.

[0370] Embodiment 35 provides the method of Embodiment 34, wherein the mammal is a human.

[0371] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art. and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Claims

CLAIMSWhat is claimed is:

1. A compound of formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein:R1is selected from the group consisting of N(RA)(RB) and N(R2b)(R3b); each occurrence of L1is independently selected from the group consisting of -N(R2d)-, -N(RA)-, -(optionally substituted C1-C6alkylenyl)-, -(optionally substituted C1-C6heteroalkylenyl)-, -(optionally substituted C3-C8cycloalkylenyl)-, -(optionally substituted C2- Cs heterocycloalkylenyl)-, -(optionally substituted C6-C10arylenyl)-, and -(optionally substituted C2-C8heteroarylenyl)-, wherein one occurrence of L1is optionally substituted with -(optionally substituted C1-C6alkylenyl)-N(R2c)(R3c); each occurrence of R2a, R2b, R2c, and R2dis independently L2— A(R4)n. each occurrence of R3a, R3b, and R3cis independently selected from the group consisting of H and optionally substituted C1-C6alkyl; each occurrence of A is independently optionally substituted C6-C10aryl and optionally substituted C2-C8heteroaryl; each occurrence of R4is -OC(=O)(optionally substituted C1-C24alkyl); each occurrence of L2is independently -(optionally substituted C1-C6alkylenyl)-; each occurrence of RAand RBis independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C3-C8cycloalkyl, optionally substituted C2-C8heterocycloalkyl, optionally substituted C6-C10aryl, and optionally substituted C2-C8heteroaryl. or RAand RBcan combine with the N atom to which they are bound to form a C2-C8heterocycloalkyl; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each occurrence ofis independently 1, 2, or 3.

2. The compound of claim 1, wherein at least one of the following applies:(a) each occurrence of L1is independently selected from the group consisting of -(CH2)2-, -(CH2)3-, -N(CH3)-, -N[(optionally substituted C1-C6alkylenyl)-N(R2C)(R3C)]-. and(b) RAand RBcombine with the N atom to which they are bound to formand(c) RAand RBare each independently methyl or ethyl.

3. The compound of claim 1 or 2. wherein L1and m are selected such thais selected from the group consisting of -(CH2)2-, -(CH2)3-, -(CH2)2N(CH3)(CH2)2-, -(CH2)3N(CH3)(CH2)3-4. The compound of any one of claims 1-3. wherein R1is selected from the group consisting of NMe2, NEt2, , and N(R2b)(R2b).

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

6. The compound of any one of claims 1-5. wherein A, R4. and n are selected such that -A(R4)nis:wherein: each occurrence of R5a, R5b, and R3c, if present, is independentlyeach occurrence of R6a, R6b, R7a, R71’. R8a, R8b. and R9is independently H or C1-C6alkyl. wherein R6a, R6b, R7a, R7b, R8a, R8b, and R9are selected such that each occurrence of R5a, R5b, and R5ccomprise a C1-C24alkyl.

7. The compound of claim 6, wherein at least one of the following applies:(a) one of R6a, R6b, R7a, R7b, R8a, and R8bis methyl, and R9is ethyl;(b) one of R6a, R6b, R7a, R71’. R8a, and R8bis methyl, and R9is propyl;(c) one of R6a, R6b, R7a, R711, R8a, and R8bis methyl, and R9is butyl;(d) one of R6a, R6b. R7a, R711, R8a. and R8bis methyl, and R9is pentyl;(e) one of R6a, R6b, R7a, R7b, R8a, and R8bis methyl, and R9is hexyl;(a) one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is ethyl;(b) one of R6a, R6b, R7a, R71’, R8a, and R8bis ethyl, and R9is propyl;(c) one of R6a, R6b. R7a, R711, R8a. and R8bis ethyl, and R9is butyl;(d) one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is pentyl; and(e) one of R6a, R6b, R7a, R7b, R8a, and R8bis ethyl, and R9is hexyl.

8. The compound of claim 6 or 7. wherein R5a, R5b, and R5c, if present, are each independently selected form the group consisting of 1 -methylpentyl, 1 -methylhexyl. 1-methylheptyl, 1 -methyloctyl, 1 -methylnonyl, 1 -ethylpentyl, 1 -ethylhexyl, 1 -ethylheptyl, 1- ethyloctyl, 2-methylpentyl, 2-methylhexyl, 2-methylheplyl. 2-methyloctyl, 2-methylnonyl. 2- ethylpentyl, 2-ethylhexyl, 2-ethylheptyl, 2-ethyloctyl. 3-methylpentyl, 3-methylhexyl, 3- methylheptyl, 3-methyloctyl, 3 -methylnonyl, 3-ethylpentyl, 3-ethylhexyl, 3-ethylheptyl. 3- ethyloctyl, and 3-ethylnonyl.

9. The compound of any one of claims 1-8. wherein each occurrence of R2a. R2b, R2c, and R2d, if present, is independently selected from the group consisting of:

10. The compound of any one of claims 1-9, which is selected from the group consisting of:l l. A lipid nanoparticle (LNP) composition comprising:(a) at least one compound of any one of claims 1-10;(b) at least one neutral lipid;(c) at least one cholesterol lipid and / or a modified derivative thereof;(d) at least one polymer conjugated lipid and / or a modified derivative thereof.

12. The LNP of claim 11, further comprising at least one cargo molecule.

13. The LNP composition of claim 12, wherein the cargo is at least one selected from the group consisting of a nucleic acid, small molecule, protein, therapeutic agent, antibody, and any combinations thereof.

14. The LNP composition of claim 12 or 13. wherein the cargo is a nucleic acid.

15. The LNP composition of claim 13 or 14, wherein the nucleic acid is DNA or RNA.

16. The LNP composition of any one of claims 13-15. wherein the nucleic acid is selected from the group consisting of siRNA, sgRNA, mRNA, cDNA, pDNA, microRNA, modified RNA, antagomir, antisense molecule, and any combinations thereof.

17. The LNP composition of claim 16. wherein the mRNA encodes a therapeutic protein, optionally wherein the therapeutic protein is a CRISPR-associated protein, and optionally wherein the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9).

18. The LNP of any one of claims 12-17, wherein the cargo is at least partially encapsulated in the LNP.

19. The LNP of any one of claims 11-18, wherein the compound of formula (I) is selected from the group consisting of:

20. The LNP of any one of claims 11 -19, wherein the at least one cationic degradable lipid comprises about 10 mol% to about 70 mol% of the LNP, optionally wherein the at least one cationic degradable lipid compound comprises about 35 mol% of the LNP.

21. The LNP of any one of claims 11-20, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC).

22. The LNP of any one of claims 11-21, wherein the at least one neutral lipid comprises about 1 to about 30 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 16 mol% of the LNP.

23. The LNP of any one of claims 11-22, wherein the at least one cholesterol lipid and / or modified derivative thereof is cholesterol.

24. The LNP of any one of claims 11-23, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 70 mol% of the LNP, optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 46.5 mol% of the LNP.

25. The LNP of any one of claims 11-24, wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises at least one selected from the group consisting of:l,2-dimyristoyl-rac-glycero-3 -methoxy poly ethylene gly col-2000, andl,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt).

26. The LNP of any one of claims 11-25, wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer conjugated lipid and / or modified derivative thereof comprises about 2.5 mol% of the LNP.

27. The LNP of any one of claims 11-26, wherein the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35: 16:46.5:2.

528. A pharmaceutical composition comprising the LNP of any one of claims 11-27 and a pharmaceutically acceptable earner.

29. A method for delivering a cargo to a subject’s lungs, the method comprising administering to the subject at least one lipid nanoparticle of any one of claims 12-27 and / or the pharmaceutical composition of claim 28.

30. The method of claim 29, wherein the cargo is delivered to a lung epithelial cell.

31. The method of claim 29 or 30. wherein the cargo is selectively delivered to the lung over at least one selected from the group consisting of the liver, heart, kidney, and spleen.

32. A method for treating, preventing, and / or ameliorating a lung-associated disease or disorder in a subject, the method comprising administering to the subject at least one lipid nanoparticle of any one of claims 12-27 and / or the pharmaceutical composition of claim 28.

33. The method of claim 32, wherein the lung-associated disease or disorder is at least one selected from the group consisting of acute respiratory distress syndrome (ARDS), asthma, bronchiectasis, bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disorder (COPD), cystic fibrosis, hypersensitivity pneumonitis, interstitial lung disease (ILD), lung abscess, lung cancer, occupational lung diseases, pleural effusion, pneumonia, pulmonary edema, pulmonary embolism, pulmonary fibrosis, pulmonary hypertension, sarcoidosis, and tuberculosis.

34. The method of any one of claims 29-33, wherein the subject is a mammal.

35. The method of claim 34, wherein the mammal is a human.

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