Lipid nanoparticles for targeted delivery of mRNA

Abstract

A composition is disclosed that includes a pharmaceutical agent assembled with a lipid composition, the lipid composition comprising a lipidoid having structural formula (I), or a pharma- ceutically acceptable salt thereof, wherein R a , R b1 , R b2 , R b3 , R b4 , n1 and n2 are as described herein. [Formula 1] JPEG2024504636000046.jpg27164

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 138,047, filed January 15, 2021, No. 63 / 256,811, filed October 18, 2021, and No. 63 / 251,791, filed October 4, 2021, the contents of all of which are incorporated by reference in their entireties. GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No. UG3 TR002636-01 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Genome editing technology allows for the permanent repair of disease-causing genetic mutations, however, application of this technology has been limited by the technical challenge of achieving safe, effective, and specific in vivo delivery of CRISPR-Cas9 genome editing components. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein is the development of a newly identified lipid nanoparticle (LNP) for specifically delivering CRISPR-Cas9 mRNA to the lung or liver. LNPs have recently been approved by the FDA for the delivery of siRNA to the liver, but described herein is their use for genome editing. In a direct comparison, the disclosed delivery platform significantly outperforms the FDA-approved LNP in efficiently delivering Cas9 mRNA for knocking down Angptl3 gene and subsequently regulating hypercholesterolemia, and matches the safety and specificity of the approved platform. [Brief description of the drawings]

[0005] [Figure 1]Schematic diagram of non-viral lipid nanoparticle (LNP)-mediated in vivo CRISPR-Cas9-based genome editing to induce loss-of-function mutations in Angptl3 to reduce blood lipid levels. Cas9 mRNA and Angptl3-specific single guide RNA (sgAngptl3) are encapsulated in LNPs and delivered to liver hepatocytes, where they cleave the Angptl3 target locus, resulting in a reduction in ANGPTL3 protein. The reduction in ANGPTL3 levels leads to the inhibition of lipoprotein lipase (LPL), which allows LPL to regulate the levels of therapeutically relevant circulating lipids such as low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG). [Figure 2A] 1 shows the synthesis of exemplary lipidoid nanoparticles. 2 shows the synthesis of lipidoid nanoparticles having a tail-branched bioreducible lipidoid chemical structure. [Figure 2B] 1 shows the synthesis of an exemplary lipidoid nanoparticle. Shows the systemic luciferase bioluminescence intensity of bioreducible LNPs vs. MC3 LNPs measured in Balb / c mice (n=3) 6 hours after administration at a dose of 0.5 mg / kg firefly luciferase (fLuc) mRNA. Formulation: lipid / cholesterol / DSPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), lipid / mRNA=12.5 / 1 (weight ratio). [Figure 2C] 1 shows the synthesis of an exemplary lipidoid nanoparticle. Size and distribution of 306-O12B LNP formulated with fLuc mRNA. [Figure 3A] Optimization of fLuc mRNA 306-O12B LNP formulation. Chemical structures of three different phospholipids are shown. [Figure 3B] Figure 3A shows the optimization of fLuc mRNA 306-O12B LNP formulation. Figure 3B shows the biodistribution of three different phospholipids. [Figure 3C]Figure 1 shows the optimization of fLuc mRNA 306-O12B LNP formulation. Biodistribution of fLuc mRNA LNPs formulated with cholesterol, DMG-PEG, and different phospholipids (DSPC or DOPE or DOPC). From top to bottom, luminescence signals are shown from the heart, liver, spleen, lung, and kidney of a representative mouse. *P<0.05, **P<0.01, ***P<0.001. [Figure 3D] Figure 1 shows optimization of fLuc mRNA 306-O12B LNP formulation. Formulation parameters of LNPs formulated with cholesterol, DOPC, and DMG-PEG at seven different molar ratios are shown. [Figure 3E] Optimization of fLuc mRNA 306-O12B LNP formulation. Systemic luciferase bioluminescence intensity 6 hours after injection of 0.5 mg / kg fLuc mRNA for LNPs formulated with cholesterol, DOPC, and DMG-PEG at different 306-O12B / mRNA weight ratios and at molar ratios of 50 / 38.5 / 10 / 1.5 (n=3). [Figure 3F] Figure 2 shows the optimization of fLuc mRNA 306-O12B LNP formulations. Figure 2 shows the whole body luciferase bioluminescence intensity of different formulations in Balb / c mice 6 hours after injection at a fLuc mRNA dose of 0.5 mg / kg. [Figure 4A] 306-O12B LNP mediated significant levels of in vivo genome editing in wild-type C57BL / 6 mice. Percentage of 306-O12B LNP-mediated indels of in vivo genome editing of Angptl3 and serum ANGPTL3 levels in wild-type C57BL / 6 mice. [Figure 4B]Figure 1 shows that 306-O12B LNPs mediated significant levels of in vivo genome editing in wild-type C57BL / 6 mice. The percentage of 306-O12B LNP-mediated indels following injection of 306-O12B LNPs formulated with Cas9 mRNA and sgAngptl3 at mass ratios of 2:1, 1:1.2, and 1:2 (n=5). 306-O12B LNPs were formulated with a molar ratio of [306-O12B:cholesterol:DSPC:DMG-PEG] of [50:38.5:10:1.5] and a 7.5 / 1 weight ratio of 306-O12B / total RNA. [Figure 5A] We show that 306-O12B LNPs are more efficient than MC-3LNPs in inducing loss-of-function mutations in Angptl3 via CRISPR-Cas9-based genome editing. Next generation sequencing analysis of indels in ANGPTL3 protein, triglyceride, and LDL-C levels in mice 7 days after administration of 306-O12B LNPs co-loaded with Cas9 mRNA and sgAngptl3 at a total RNA dose of 3.0 mg / kg. MC-3LNPs were used as positive controls. (n=5 or 6). *P<0.05, **P<0.01, ***P<0.001. [Figure 5B] Figure 2 shows that 306-O12B LNP is more efficient than MC-3 LNP in inducing loss-of-function mutations in Angptl3 via CRISPR-Cas9-based genome editing.The editing frequency of specific edited alleles in each treatment group is shown. [Figure 5C] Figure 1 shows that 306-O12B LNP is more efficient than MC-3 LNP in inducing loss-of-function mutations in Angptl3 via CRISPR-Cas9-based genome editing. Editing frequencies at the nine top predicted off-target sites are shown. [Figure 6A]Figure 1 shows that 306-O12B LNP-mediated CRISPR editing remains durable after 100 days. Next generation sequencing analysis of indels in ANGPTL3 protein, triglyceride, and LDL-C levels in mice 100 days after administration of 306-O12B LNPs co-loaded with Cas9 mRNA and sgAngptl3. [Figure 6B] Figure 1 shows that 306-O12B LNP-mediated CRISPR editing remains durable after 100 days. Serum levels of AST, ALT, and TNF-alpha measured 100 days after injection are shown. Female C57BL / 6 mice were systemically injected with 306-O12B LNPs co-formulated with Cas9 mRNA and sgAngptl3 at a single dose of 1.0, 2.0, and 3.0 mg / kg total RNA. Mouse serum was collected 100 days after injection. (n=5) *P<0.05, **P<0.01, ***P<0.001. [Figure 7A] Representative TEM image of 306-O12B LNPs before formulation with fLuc mRNA (Scale bar: 500 nm). [Figure 7B] Representative TEM image of 306-O12B LNPs after formulation with fLuc mRNA (Scale bar: 500 nm). [Figure 8] 1 shows in vivo imaging of mice injected with 306-O12B, 113-O12B, 306-O10B LNPs and MC-3 LNPs loaded with fLuc mRNA. Images were taken by IVIS imaging system 6 hours after injection. [Figure 9] The encapsulation efficiency of 306-O12B LNPs for fLuc mRNA is shown. [Figure 10A] Figure 3 shows that 306-O12B LNPs enabled sgLoxP-mediated genome editing in Ai14 / Cas9-bred mice. Ex vivo images of organs harvested from Ai14 / Cas9 mice administered LNPs encapsulating sgLoxP at a dose of 0.75 mg / kg, and tdTomato fluorescence was detected by the IVIS imaging system. [Figure 10B]Figure 2 shows that 306-O12B LNPs enabled sgLoxP-mediated genome editing in Ai14 / Cas9-crossed mice. Representative immunofluorescence images of liver sections showing that tdTomato signals were mainly expressed in liver hepatocytes. (Scale bar: 20 μm). [Figure 11A] 306-O12B LNPs enabled Cas9 / sgLoxP-mediated genome editing in Ai14 mice. Schematic diagram of simultaneous delivery of Cas9 mRNA and LoxP-targeted single guide RNA (sgLoxP) into genetically engineered tdTomato reporter Ai14 mice. [Figure 11B] Figure 3 shows that 306-O12B LNPs enabled Cas9 / sgLoxP-mediated genome editing in Ai14 mice. Ex vivo images and quantification of ROIs of organs harvested from Ai14 mice administered LNPs encapsulating Cas9 mRNA / sgLoxP (1 / 1.2 by weight) at a total RNA dose of 1.65 mg / kg are shown, and tdTomato fluorescence was detected by an IVIS imaging system. [Figure 11C] Figure 3 shows that 306-O12B LNPs enabled Cas9 / sgLoxP-mediated genome editing in Ai14 mice. Confocal fluorescence microscopy of liver sections showed that tdTomato signals were mainly expressed in liver hepatocytes. (Scale bar: 20 μm). [Figure 12] Shows the size distribution of Cas9 mRNA / sgAngptl3 co-loaded 306-O12B LNPs. [Figure 13] 1 shows a T7E1 assay performed with genomic DNA from liver samples from mice taken 7 days after receiving Cas9 mRNA / sgAngptl3 co-loaded 306-O12B LNP and MC-3 LNP. Arrows indicate cleavage products resulting from the T7E1 assay. [Figure 14] Serum levels of AST, ALT, and TNF-alpha measured 2 days after injection are shown (n=5). [Figure 15]IFN-α, IL-6, and IP-10 serum levels in mice were treated with 306-O12B LNPs co-formulated with Cas9 mRNA and sgAngptl3 at 3.0 mg / kg total RNA (n=5). [Figure 16] 306-O12B LNPs induced long-term editing of Angptl3 after a single dose. C57BL / 6 mice were administered 306-O12B LNPs co-formulated with 3 mg / kg Cas9 mRNA and sgAngptl3 (n=5 for day 7 and day 100 time points, n=3 for day 150 time point). [Figure 17] Representative liver-targeting lipids are shown. [Figure 18A] The effects of injection of various lipids are shown ex vivo-8 hours post-injection. [Figure 18B] The effects of injection of various lipids are shown ex vivo-8 hours post-injection. [Figure 18C] The effects of injection of various lipids are shown ex vivo-8 hours post-injection. [Figure 18D] The effects of injection of various lipids are shown ex vivo-8 hours post-injection. [Figure 19A] Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. Formulation: Lipid / Chol / DOPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), Lipid / mRNA=12.5 / 1 (weight ratio), Dose: 0.5mg / kg (10ug / mouse) (unmodified fLuc-mRNA). [Figure 19B] Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. Formulation: Lipid / Chol / DOPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), Lipid / mRNA=12.5 / 1 (weight ratio), Dose: 0.5mg / kg (10ug / mouse) (unmodified fLuc-mRNA). [Figure 19C]Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. Formulation: Lipid / Chol / DOPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), Lipid / mRNA=12.5 / 1 (weight ratio), Dose: 0.5mg / kg (10ug / mouse) (unmodified fLuc-mRNA). [Figure 19D] Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. Formulation: Lipid / Chol / DOPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), Lipid / mRNA=12.5 / 1 (weight ratio), Dose: 0.5mg / kg (10ug / mouse) (unmodified fLuc-mRNA). Luciferase protein / total protein in lungs 6 hours post-injection. [Figure 19E] Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. Formulation: lipid / Chol / DOPC / DMG-PEG=50 / 38.5 / 10 / 1.5 (molar ratio), lipid / mRNA=12.5 / 1 (weight ratio), dosage: 0.5mg / kg (10ug / mouse) (unmodified fLuc-mRNA). Analysis of lung cell types transfected using Ai14Cre / lox reporter mice. [Figure 20] Schematic diagram of in vivo Cre-mRNA delivery-306-N16B, with Cre-mRNA dosage: 7.5 μg / mouse / injection, for a total of two injections. [Figure 21A] Figure 1 shows that the in vivo selectivity of mRNA-encapsulated LNPs is governed by the chemical structure of the active lipidoid. Schematic diagram of the fine-tuning of the in vivo organ targeting behavior of mRNA-loaded LNPs by simply altering the linker group of the lipidoid tail. [Figure 21B] Figure 1 shows that the in vivo selectivity of mRNA-encapsulated LNPs is governed by the chemical structure of the active lipidoid. The synthetic pathway and representative chemical structures of lipidoids are shown. [Figure 21C]We show that the in vivo selectivity of mRNA-encapsulated LNPs is governed by the chemical structure of the active lipidoid. Representative whole-body bioluminescence images of mice taken by IVIS imaging system are shown. Mice were injected with a single dose of 0.5 mg / kg of either 306-O12B LNPs or 306-N12B LNPs loaded with Luc mRNA. Pictures were taken 6 hours after injection. (n=3). [Figure 22A] A second generation library of N-series LNPs confirms linker-driven lung selectivity. Second generation N-series LNPs were generated by reacting the N16B tail with the amine shown in Figure 22A. [Figure 22B] Figure 22 shows that the second generation library of N-series LNPs confirmed the linker-driven lung selectivity. Second generation N-series LNPs were generated by reacting the N16B tail with the amine shown in Figure 22A. IVIS images showing that N-series LNPs mediated specific delivery of fLuc mRNA to the lung. [Figure 23A] Figure 1 shows that 306-N16B LNPs enable specific delivery of Cre mRNA to lung endothelial cells.Figure 2 shows a schematic diagram of Cre mRNA delivery to the lung to activate tdTomato expression via Cre-mediated gene deletion of the stop cassette in tdTomato transgenic Ai14 mice. [Figure 23B] Figure 1. 306-N16B LNPs enable specific delivery of Cre mRNA to lung endothelial cells. Representative ex vivo images of tdTomato fluorescence in edited Ai14 mouse organs captured using an IVIS imaging system. Mice were intravenously injected with 306-N16B LNPs loaded with Cre mRNA at a single dose of 0.75 mg mRNA equivalent / kg. [Figure 23C]Figure 2 shows that 306-N16B LNPs enable specific delivery of Cre mRNA to pulmonary endothelial cells. Representative immunofluorescence images of lung tissue obtained by confocal microscopy are shown. Endothelial cells were stained with FITC-CD31 antibody, epithelial cells were stained with CD326-APC antibody, and macrophages were stained with F4 / 80-eFluor660 antibody. Bar: 20 μm. [Figure 23D] Figure 1 shows that 306-N16B LNPs enable specific delivery of Cre mRNA to pulmonary endothelial cells. Quantification of the percentage of tdTomato+ cells within defined cell types in the lung by FACS. (n=3). [Figure 24A] Bioinformatic classification of corona components. Quantification of the % of total protein of the top three protein components in the protein corona of 306-O12B LNP (n=3). [Figure 24B] Bioinformatic classification of corona components. Quantification of the % of total protein of the top three protein components in the protein corona of 306-N16B LNP (n=3). [Figure 24C] FIG. 1 shows a bioinformatic classification of corona components. The top 20 most abundant corona proteins are shown, classified based on their calculated molecular weight. [Figure 24D] Bioinformatic classification of corona components. The top 20 most abundant corona proteins are shown, classified based on their isoelectric points. [Figure 24E] Bioinformatic classification of corona components. The top 20 most abundant corona proteins are classified based on their biological functions. [Figure 25A] 1 shows the distribution of luciferase protein in mice treated with 306-O12B and 306-N16 LNPs. Ex vivo images of mouse organs and quantification of luciferase protein in different organs 6 hours after administration of 306-O12B LNPs encapsulating mRNA. [Figure 25B]1 shows the distribution of luciferase protein in mice treated with 306-N16B LNPs. Ex vivo images of mouse organs and quantification of luciferase protein in different organs 6 hours after administration of 306-O12B LNPs encapsulating mRNA. [Figure 26] Western blot analysis of Cas9 protein expression in different organs in Babl / c mice after injection of 306-N16B LNPs co-formulated with Cas9 mRNA. Dose: 0.5 mg equivalent Cas9 mRNA / kg. Organs were harvested 6 hours after injection. [Figure 27] FIG. 11 is a Venn diagram showing common and unique proteins absorbed by 306-O12B LNPs (light green) and 306-N16B LNPs (light blue). [Figure 28A] 1 shows the synthesis and in vivo screening of N-series LNPs. 2 shows the synthetic pathway and representative chemical structures of lipidoids. 3 shows representative whole-body bioluminescence images of mice. [Figure 28B] Figure 1 shows the synthesis and in vivo screening of N-series LNPs. Figure 2 shows the in vivo mRNA delivery efficacy of N-series LNPs measured by IVIS imaging system. Mice were injected with a single dose of 0.5mg / kg of either N-series LNPs loaded with Luc mRNA. Images were taken 6 hours after injection. (n=3). [Figure 28C] Figure 1 shows the synthesis and in vivo screening of N-series LNPs. Figure 2 shows the in vivo mRNA delivery efficacy of N-series LNPs measured by IVIS imaging system. Mice were injected with a single dose of 0.5mg / kg of either N-series LNPs loaded with Luc mRNA. Images were taken 6 hours after injection. (n=3). [Figure 29A] We show that different lung cell types can be targeted by tailoring the head structure of N-series LNPs. We show a schematic of the delivery of Cre mRNA to the lung to activate tdTomato expression via Cre-mediated gene deletion of the stop cassette in tdTomato transgenic Ai14 mice. [Figure 29B] We show that different lung cell types can be targeted by tailoring the head structure of N-series LNPs. We show chemical structures, representative ex vivo images of tdTomato fluorescence in edited Ai14 mouse organs captured using an IVIS imaging system, representative immunofluorescence images of lung tissue captured by confocal microscopy, and quantification of the percentage of tdTomato+ cells within defined cell types in the lung by FACS of 306-N16B LNPs. Mice were intravenously injected with Cre mRNA-loaded LNPs at a single dose of 0.75 mg mRNA equivalent / kg. Endothelial cells were stained with FITC-CD31 antibody, epithelial cells were stained with CD326-PE-Cy7 antibody, and macrophages were stained with F4 / 80-eFluor660 antibody. Bar: 20 μm. We show quantification of the percentage of tdTomato+ cells within defined cell types in the lung by FACS. (n=3). [Figure 29C] We show that different lung cell types can be targeted by adjusting the head structure of N-series LNPs. We show chemical structures, representative ex vivo images of tdTomato fluorescence in edited Ai14 mouse organs captured using an IVIS imaging system, representative immunofluorescence images of lung tissue taken by confocal microscopy, and quantification of the percentage of tdTomato+ cells within defined cell types in the lung with 113-N16B LNPs. Mice were intravenously injected with Cre mRNA-loaded LNPs at a single dose of 0.75 mg mRNA equivalents / kg. Endothelial cells were stained with FITC-CD31 antibody, epithelial cells were stained with CD326-PE-Cy7 antibody, and macrophages were stained with F4 / 80-eFluor660 antibody. Bar: 20 μm. We show quantification of the percentage of tdTomato+ cells within defined cell types in the lung by FACS. (n=3). [Figure 30A] 1 shows a proteomic study of the protein corona formed on LNPs. FIG. 2 is a schematic diagram of the different organ targeting of O-series LNPs and N-series LNPs. [Figure 30B] Figure 1 shows a proteomic study of the protein corona formed on LNPs. Figure 2 shows a schematic of the interaction of LNPs with proteins in blood vessels. [Figure 31A] FIG. 1 is a schematic diagram of co-delivery of Cas9 mRNA and sgLoxP to activate tdTomato expression in Ai14 mice. [Figure 31B] Representative ex vivo images of organs harvested from 306-N16B LNP-treated transgenic Ai14 mice co-loaded with Ca9 mRNA and sgLoxP were injected via the tail vein with a dose of 1.67 mg / kg total RNA. [Figure 31C] Representative microscopic images of lungs and livers dissected from Ai14 mice treated with 306-N16B LNPs co-encapsulated with Cas9 mRNA and sgLoxP are shown. Bar: 100 μm [Figure 32A]

[0023] Figure 1 shows that hybrid LNPs (hLNPs) enable specific delivery of mRNA to TTJ tumor cells in vivo. Schematic diagram of the preparation of hybrid LNPs. hLNPs were formulated with a molar ratio of 25:25:38.5:10:1.5 306-N16B:306-O12B:cholesterol:DOPC:DMG-PEG2000. [Figure 32B] We show that hybrid LNPs (hLNPs) enable specific delivery of mRNA to TTJ tumor cells in vivo. Representative images of immunohistochemistry (IHC) analysis of EGFP in mouse LAM lungs are shown. Syngeneic C57BL / 6J mice were injected with TSC2-deficient TTJ cells to form tumor nodules in the lungs. Mice were injected with hLNPs loaded with GFP mRNA into the tail vein. Lungs were harvested 6 hours after injection. [Figure 32C] Figure 1 shows that hybrid LNPs (hLNPs) enable specific delivery of mRNA to TTJ tumor cells in vivo. Multiplex immunofluorescence staining showing colocalization of pS6 (a tumor cell marker) with EGFP. (Left) pS6 staining; (Right) EGFP staining. [Figure 33A]The therapeutic effect of hLNPs loaded with TSC2 mRNA on anti-tumor growth is shown. Therapeutic design is shown. Syngeneic C57BL / 6J mice were injected with 2x106 TSC2-deficient TTJ cells via the tail vein to form tumor nodules in the lungs. 24 days after tumor cell inoculation, mice were randomized into three groups: untreated control, treatment with empty LNPs, and treatment with LNP / mRNA-TSC2 (0.75mg / kg mRNA per injection). Mice were treated every other day for a total of five times. (n=2). [Figure 33B] Figure 1 shows the therapeutic effect of TSC2 mRNA-loaded hLNPs on anti-tumor growth.H&E images of lungs dissected from mice receiving different treatments are shown. [Figure 33C] 1 shows the therapeutic effect of hLNPs loaded with TSC2 mRNA in anti-tumor growth. The percentage of tumor nodules per lung in the three treatment groups is shown. [Figure 33D] 1 shows the therapeutic effect of hLNPs loaded with TSC2 mRNA on anti-tumor growth. Representative images of IHC evaluation of ki67, cleaved caspase 3, macrophages, and CD3 in empty hLNPs and hLNPs loaded with TSC2 mRNA are shown. [Figure 33E] The therapeutic effect of hLNPs loaded with TSC2 mRNA in anti-tumor growth. Quantitative analysis of Figure 33D. 10 images were analyzed for each group. T cells: CD3 IHC, apoptosis: cleaved caspase 3 IHC, proliferation: Ki67 IHC: macrophages: F4 / 80 IHC. [Figure 34A] Figure 1 shows the in vitro transfection efficiency of LNPs in TTJ cells. The percentage of EGFP-positive TTJ cells measured by flow cytometry. [Figure 34B] Representative fluorescent EGFP images of transfected cells are shown. [Figure 35A] 1 shows in vitro transcription of full-length mouse Tsc2 mRNA assessed by bioanalyzer, demonstrating the integrity and full-length of the mRNA. [Figure 35B]Immunoblots show suppression of phospho-S6 by wild-type Tsc2 mRNA re-expression in TSC2 null TTJ cells from mice. [Figure 35C] FIG. 1 shows in vitro transcription of full-length human TSC2 mRNA as assessed by bioanalyzer. [Figure 35D] Immunoblots show suppression of phospho-S6 by wild-type TSC2 mRNA re-expression in patient-derived TSC2-deficient cells. 621-101: patient-derived TSC2-deficient cells; 621-103: TSC2 re-expressed in 621-101 cells; last two columns: two replicates of TSC2 mRNA treatment for 24 hours in 621-101 cells. [Diagram 36] Representative images of lungs dissected from mice receiving various treatments (n=2 mice in each group) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] In one embodiment, a highly potent new non-viral LNP-mediated CRISPR-Cas9 delivery system for hepatic delivery of Cas9 mRNA is disclosed and its efficacy is demonstrated by targeting the Angptl3 gene. The system is composed of a leading tail-branched bioreproducible lipidoid (306-O12B) co-formulated with an optimized mixture of excipient lipid molecules, and successfully co-delivers SpCas9 mRNA and a single guide RNA (sgAngptl3) targeting Angptl3 via at least one administration (Figure 1).

[0007] Using a library screening approach, it was confirmed that N-series LNPs (containing amide bonds in the tail) could selectively deliver mRNA to the lungs of mice, in contrast to O-series LNPs (containing ester bonds in the tail). Protein corona was analyzed on liver- and lung-targeted LNPs using LC / MS, identifying a group of unique plasma proteins specifically absorbed to the surface that may contribute to the targeting of these LNPs. The lipidoid tail structure significantly influences the interaction of LNPs with serum proteins, essentially governing the organ selectivity of LNPs. By simply tuning the headgroup structure of N-series LNPs, different lung cell types can be targeted. The first success of LNP-based RNA therapy is demonstrated herein in a preclinical model of lymphangioleiomyomatosis (LAM), a devastating lung disease caused by loss-of-function mutations in the Tsc2 gene. Lung-targeted LNPs showed highly efficient delivery of murine tuberous sclerosis complex 2 (Tsc2) mRNA for restoration of the TSC2 tumor suppressor in tumors, achieving remarkable therapeutic efficacy in reducing tumor burden. Overall, this study provides new insights into the rational and predictive design and engineering of next-generation organ- and cell-targeted LNPs for mRNA-based therapies.

[0008] Safe and effective systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo is a major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) co-formulated with mRNA is primarily limited to the liver and spleen. Current LNP development strategies rely primarily on labor- and cost-intensive in vitro and in vivo screening processes, and it is currently difficult to design tissue-specific targeted LNPs in a predictable manner. Using a library screening approach, it was confirmed that N-series LNPs (containing amide bonds in the tail), in contrast to O-series LNPs (containing ester bonds in the tail), can selectively deliver mRNA to the lungs of mice. Protein coronas were analyzed on liver- and lung-targeted LNPs using LC / MS to identify a group of unique plasma proteins specifically adsorbed to the surface that may contribute to the targeting of these LNPs. The lipidoid tail structure has a significant impact on the interaction of LNPs with serum proteins, essentially governing the organ selectivity of LNPs. By simply tuning the headgroup structure of N-series LNPs, different lung cell types can be targeted. Importantly, the first success of LNP-based RNA therapy is demonstrated herein in a preclinical model of lymphangioleiomyomatosis (LAM), a devastating lung disease caused by loss-of-function mutations in the Tsc2 gene. The disclosed lung-targeted LNPs showed highly efficient delivery of murine tuberous sclerosis complex 2 (Tsc2) mRNA for restoration of TSC2 tumor suppressor in tumors and achieved remarkable therapeutic efficacy in reducing tumor burden. Overall, this study provides new insights into the rational and predictive design and engineering of next-generation organ- and cell-targeted LNPs for mRNA-based therapy.

[0009] A major obstacle in the development of targeted LNPs is the limited understanding of nanobio interactions between nanoparticles (NPs) and biological components, making it difficult to predict the in vivo targeting behavior of newly designed LNPs. The outer surface of NPs can be quickly covered with a layer of serum proteins, termed the "protein corona," which remodels the surface properties of NPs and substantially influences the interaction of NPs with organs and cells. The lipidoid amine head structure can affect the delivery efficacy and even the in vivo targeting of mRNA-loaded LNPs. Imidazole-based synthetic lipids preferentially target mRNA to the spleen (X. Zhao et al., Imidazole-Based Synthetic Lipidoids for In Vivo mRNA Delivery into Primary T Lymphocytes. Angew Chem Int Ed Engl 59, 20083-20089 (2020). Regarding lipidoid tail chemistry, considerable progress has been made in understanding the effect of lipidoid tail length, degree of unsaturation, and branching on mRNA delivery potency, but the impact of lipidoid tail structure on the in vivo selectivity of LNPs remains poorly understood.

[0010] To address this important knowledge gap, we synthesized a library of amide bond-containing lipidoids (N-series LNPs) via a Michael addition reaction between the amine head and the acrylamide tail (Figure 28A). Surprisingly, in vivo screening revealed that N-series LNPs delivered mRNA almost exclusively to the lung after systemic administration (Figures 28B and 28C). Previous studies demonstrated that O-series lipidoids containing ester bonds in the tail tended to deliver mRNA to the liver (M. Qiu et al., Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc. Natl. Acad. Sci. USA118, e2020401118 (2021)). To better understand why such a small change induces such remarkable organ specificity, we further explored the underlying mechanisms of these delivery differences. Once injected into the bloodstream, LNPs can selectively control the adsorption of specific plasma proteins to serve as targeting ligands to direct LNPs to selected organs. Indeed, using proteomics, a group of unique plasma proteins was identified that specifically adsorb to the surface of two representative LNP candidates, 306-O12B and 306-N16B, and may affect the targeting of these LNPs. More importantly, distinct lung sub-cell populations can be targeted by modifying the lipidoid head structure of N-series LNPs. Furthermore, lung-targeted LNPs were evaluated for in vivo targeted delivery of Tsc2 mRNA to TSC2-deficient cells to restore expression of the TSC2 tumor suppressor for the treatment of pulmonary lymphangioleiomyomatosis (LAM), a rare genetic disorder caused by biallelic mutations and loss of function of TSC complex genes. This study provides a proof-of-concept that tuning the in vivo organ-targeting behavior of LNPs can be achieved by adjusting the composition of the protein corona via simple chemistry.This study provides a new strategy for the rational design of highly specific organ- and cell-selective LNPs for mRNA-based therapy. In one aspect, the following is disclosed.

[0011] Pharmaceutical Compositions The compositions and methods of the present invention may be utilized to treat individuals in need thereof. The pharmaceutical compositions described herein may include therapeutic or prophylactic compositions, or any combination thereof. In certain embodiments, the lipidoid composition may be assembled with an antigen, an immunomodulator, or any combination thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or lipidoid composition is preferably administered as a pharmaceutical composition, for example, comprising the lipidoid composition of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. The excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilisates for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0012] The pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a lipidoid composition, such as the lipidoid composition of the present invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may be a liposome or other polymer matrix, which may, for example, incorporate the lipidoid composition of the present invention. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to make and administer.

[0013] As used herein, the phrase "pharmacologically acceptable" is used to refer to those lipidoid compositions, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0014] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of materials that can function as pharma-ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; (10) glycerides, such as glycerides, ... (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer solution, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0015] Pharmaceutical compositions (preparations) can be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., as drenches such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue), absorption through the oral mucosa (e.g., sublingually), subcutaneously, transdermally (e.g., as a patch applied to the skin), and topically (e.g., as a cream, ointment, or spray applied to the skin). Lipidoid compositions may also be formulated for inhalation. In certain embodiments, the lipidoid composition may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0016] The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of lipidoid composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0017] Methods of preparing these formulations or compositions include the step of bringing into association an active composition, such as the lipidoid (e.g., nanoparticle) compositions described herein, with the carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the lipidoid (e.g., nanoparticle) compositions described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0018] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavor base, usually sucrose and acacia or tragacanth), lyophilisates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or mouthwash, etc., each containing as an active ingredient a predetermined amount of the lipid (e.g., nanoparticle) composition of the present invention containing a predetermined amount of the lipid (e.g., nanoparticle) composition described herein. The lipidoid composition may also be administered as a bolus, electuary, or paste.

[0019] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient may be mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar; Calcium carbonate, potato or tapioca starch, alginic acid, certain silicas, and sodium carbonate, (5) dissolution retarders such as paraffin, (6) absorption enhancers such as quaternary ammonium lipidoid compositions, (7) wetting agents such as cetyl alcohol and glycerol monostearate, (8) adsorbents such as kaolin and bentonite clay, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof, (10) complexing agents such as modified and unmodified cyclodex agents, and (11) colorants. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain buffering agents. Similar types of solid compositions can also be used as fillers for soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, and the like.

[0020] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered lipidoid composition moistened with an inert liquid diluent.

[0021] Tablets, and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain an opacifying agent, and can be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0022] Liquid dosage forms useful for oral administration include pharma- ceutically acceptable emulsions, lyophilisates for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0023] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0024] Suspensions may contain, in addition to the active lipidoid composition, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0025] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active lipidoid compositions may be mixed under sterile conditions with a pharma- ceutical acceptable carrier, and any preservatives, buffers, or propellants which may be required.

[0026] The ointments, pastes, creams and gels may contain, in addition to the active lipidoid composition, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0027] Powders and sprays can contain, in addition to the active lipidoid composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0028] Transdermal patches have the added advantage of providing controlled delivery of the lipidoid composition of the present invention to the body.Such dosage forms can be made by dissolving or dispensing the active lipidoid composition in a suitable medium.Absorption enhancers can also be used to increase the flux of the lipidoid composition through the skin.The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the lipid composition in a polymer matrix or gel.

[0029] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active lipidoid compositions in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0030] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0031] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0032] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be accomplished by using a liquid suspension of crystalline or amorphous substances that are poorly water-soluble. In this case, the rate of absorption of the drug depends on the rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0033] Injectable depot forms are made by forming microencapsulated matrices of the subject lipidoid compositions in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0034] For use in the methods of the invention, the active lipidoid composition may be provided per se or as a pharmaceutical composition containing, for example, 0.1-99.5% (more preferably 0.5-90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0035] The introduction method may be provided by a rechargeable or biodegradable device. For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained release polymeric devices have been developed and tested in vivo in recent years. Various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), can be used to form implants for sustained release of lipidoid compositions at specific target sites.

[0036] Actual dosage levels of the active ingredients in pharmaceutical compositions may be varied 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.

[0037] The selected dosage level will depend on a variety of factors, including the activity of the particular lipidoid composition or combination of lipidoid compositions, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the excretion rate of the particular lipidoid composition(s) being used, the duration of treatment, other drugs, lipidoid compositions and / or materials used in combination with the particular lipidoid composition(s) being used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0038] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the pharmaceutical composition or lipidoid composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. By "therapeutically effective amount" is meant a concentration of lipidoid composition sufficient to elicit the desired therapeutic effect. In general, it is understood that the effective amount of lipidoid composition varies according to the weight, sex, age, and medical history of the subject. Other factors that affect the effective amount include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the lipidoid composition, and another type of therapeutic agent that is administered together with the lipidoid composition of the present invention, if necessary. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0039] In general, a suitable daily dose of an active lipidoid composition used in the compositions and methods of the present invention will be that amount of the lipidoid composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0040] If desired, the effective daily dose of the active lipidoid composition may be administered as one, two, three, four, five, six or more separate doses administered at appropriate intervals throughout the day, optionally in unit dosage form. In certain embodiments of the present invention, the active lipidoid composition may be administered two or three times a day. In a preferred embodiment, the active lipidoid composition is administered once a day.

[0041] The patients receiving this treatment are animals in need of treatment, including primates, particularly humans, and other mammals such as horses, cows, pigs, sheep, cats, dogs, poultry, and pets in general.

[0042] In certain embodiments, the lipidoid compositions of the present invention may be administered alone or in combination with another type of therapeutic agent.

[0043] The present disclosure includes the use of pharma- ceutically acceptable salts of the lipidoid compositions of the present invention in the compositions and methods of the present invention.In certain embodiments, the contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts.In certain embodiments, the contemplated salts of the present invention include, but are not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.In certain embodiments, the contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present invention include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptan ... Acids that may be used include, but are not limited to, protonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenate.

[0044] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in the solvent of crystallization, the solvent of preparation or crystallization, or adventitious to such solvent.

[0045] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0046] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0047] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those skilled in the art. In general, the terminology and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0048] The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0049] As used herein, unless otherwise defined herein, chemical terms are used according to conventional usage in the art as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0050] All of the above, as well as any other publications, patents, and published patent applications referenced in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0051] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes examples when the event or circumstance occurs as well as examples when it does not occur. For example, "optionally substituted alkyl" refers to the fact that the alkyl may be substituted as well as the fact that the alkyl is not substituted.

[0052] It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by those skilled in the art to provide chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art, as well as those methods described below. When a substituent is itself substituted with more than one group, it is understood that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0053] As used herein, the term "optionally substituted" refers to the replacement of 1-6 hydrogen radicals in a given structure with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl). Preferably, "optionally substituted" refers to the replacement of 1-4 hydrogen radicals in a given structure with the above substituents. More preferably, 1-3 hydrogen radicals are replaced with the above substituents. It is understood that the substituents may be further substituted.

[0054] Articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or clear from the context. A claim or description containing "or" between one or more members of a group is considered satisfied when one, more than one, or all of the group members are present, employed, or otherwise relevant in a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of the group is present, employed, or otherwise relevant in a given product or process. The invention includes embodiments in which two or more, or all of the group members are present, employed, or otherwise relevant in a given product or process.

[0055] As used herein, the term "alkyl" includes, but is not limited to, C1 to C 10 Straight chain alkyl group or C1-C 10 It refers to saturated aliphatic groups, including branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. The "alkyl" group may be optionally substituted.

[0056] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0057] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0058] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0059] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0060] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0061] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight-chain or branched-chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C for straight chain). 1~30 , C for branched chain 3~30 ), more preferably 20 or less.

[0062] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0063] "C x~y " or "C x ~C yThe term "alkyl" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. CO alkyl indicates that the group is a hydrogen in a terminal position, while if internal, it is a bond. 1~6 An alkyl group contains, for example, 1 to 6 carbon atoms in the chain.

[0064] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0065] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0066] As used herein, the term "amide" refers to the group: [ka] In the formula, R 9 and R 10 each independently represents a hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure. When an amide is drawn without showing the groups required by the valence on the constituent nitrogen atoms (e.g., -C(O)-NR 9 and the like), it is implied and understood that a hydrogen atom is present on the nitrogen atom to satisfy the foregoing valence requirements (e.g., -C(O)-NHR 9 and so on).

[0067] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, e.g., [ka] In the formula, R 9 , R 10、 and R10 Each ' independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0068] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0069] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0070] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is 5-7 membered, more preferably 6 membered. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0071] The term "carbamate" is art-recognized and refers to the group: [ka] In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0072] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0073] The term "carbocycle" includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or more than two atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle, as long as valences permit. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions capable of carrying a hydrogen atom.

[0074] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0075] The term "carbonate" is art-recognized and refers to a -OCO2- group.

[0076] The term "carboxy," as used herein, refers to a group represented by the formula -CO2H.

[0077] The term "ester" as used herein means an ester group consisting of -C(O)OR 9 R 9 represents a hydrocarbyl group.

[0078] The term "ether" as used herein refers to a hydrocarbyl group linked to another hydrocarbyl group via oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0079] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo, and iodo.

[0080] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0081] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, in which the ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings, in which two or more carbons are common to two adjacent rings, and in which at least one of the rings is heteroaromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0082] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0083] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0084] The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, which ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0085] The term "hydrocarbyl" as used herein refers to a group that is bonded through a carbon atom that does not have =O or =S substituents, and that usually has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for the purposes of this application, while substituents such as acetyl (which has a =O substituent on the bonded carbon) and ethoxy (which is bonded through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0086] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0087] The term "lower" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups having 10 or fewer atoms in the substituent, preferably 6 or fewer atoms. For example, "lower alkyl" refers to an alkyl group having 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, an acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituent as defined herein is a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether appearing alone or in combination with other substituents (e.g., atoms in an aryl group are not counted when counting the carbon atoms of an alkyl substituent), such as in the descriptions hydroxyalkyl and aralkyl.

[0088] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings," and each ring of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms, preferably from 5 to 7 atoms, within the ring.

[0089] The term "sulfate" is art recognized and refers to the group -OSO3H, or a pharma- ceutically acceptable salt thereof.

[0090] The term "sulfonamide" is art-recognized and can be represented by the general formula: [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0091] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0092] The term "sulfonate" is art-recognized and refers to the group SO3H, or a pharma- ceutically acceptable salt thereof.

[0093] The term "sulfone" is art-recognized and refers to the group -S(O)2-.

[0094] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution results in a stable compound that does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like, subject to the permissible valences of the substituted atom and substituent. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain may themselves be substituted, where appropriate.

[0095] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0096] As used herein, the term "thioester" refers to a -C(O)SR 9 Or -SC(O)R 9 Refers to the base,

[0097] In the formula, R 9 represents hydrocarbyl.

[0098] The term "thioether" as used herein is the equivalent of an ether where the oxygen is replaced with a sulfur.

[0099] The term "urea" is art-recognized and may be represented by the general formula: [ka] In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0100] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0101] The phrase "pharmacologically acceptable" is art-recognized. In certain embodiments, this term is used to refer to those compositions, excipients, adjuvants, polymers and other materials and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0102] "Salt" is used herein to refer to an acid addition salt or a base addition salt.

[0103] Many of the lipidoid compositions (e.g., nanoparticles) useful in the methods and compositions of the present disclosure have at least one stereogenic center in their structure. This stereogenic center may be in R or S configuration, and R and S notation are used according to the rules described in Pure Appl.Chem.(1976),45,11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomers and diastereomers, of the compounds, salts, prodrugs, or mixtures thereof, including all possible mixtures of stereoisomers. See, for example, WO01 / 062726.

[0104] Additionally, certain compounds containing alkenyl groups may exist as Z (zusammen (together)) or E (entgegen (inverse)) isomers. In each case, the present disclosure includes both mixtures and separate individual isomers. Some lipidoid compositions (e.g., nanoparticles) may include chemical compounds that exist in tautomeric forms. Such forms, although not explicitly indicated in the formulas set forth herein, are intended to be included within the scope of the present disclosure.

[0105] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal government or a state government or its counterpart in a country other than the United States, or listed in the United States Pharmacopeia or other pharmacopoeia generally recognized for use in animals, or more specifically in humans.

[0106] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4 or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or salts formed when coordinated with an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc.

[0107] As used herein, the term "pharmaceutically acceptable basic addition salt" refers to any non-toxic organic or inorganic base addition salt of any acid compound disclosed herein. Exemplary inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of appropriate salts will be known to those skilled in the art.

[0108] The term "pharmacologically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, et al., J. Pharm. Sci. 66(1):1-79 (January 77)).

[0109] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered.

[0110] "Pharmaceutically acceptable metabolically cleavable group" refers to a group that is cleaved in vivo to provide a parent molecule of the structural formula shown herein. Examples of metabolically cleavable groups include -COR, -COOR, -CONRR, and -CH2OR radicals, where R is independently selected at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl, or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy, or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl, and trimethylsilyl groups.

[0111] As used herein, the phrase "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filter, diluent, excipient, solvent, or encapsulating material useful in formulating a drug for pharmaceutical or therapeutic use.

[0112] "Prodrug" refers to a compound that contains a derivative of the compound of the present invention that has a cleavable group and becomes a compound of the present invention that is pharmacologic in vivo by solvent decomposition or under physiological conditions. Such examples include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, and the like. Other derivatives of the compounds of the present invention are active in both their acid and acid derivative forms, but the acid-sensitive forms often offer advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs, pp.7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to those skilled in the art, such as esters prepared by reacting the parent acid with a suitable alcohol, or amides prepared by reacting the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acid groups pendant on the compounds of the present invention are specific prodrugs. In some cases, it may be desirable to prepare double ester type prodrugs, such as (acyloxy)alkyl esters or (alkoxycarbonyl)oxy)alkyl esters. In particular, the C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C 12 Substituted aryl and C7-C 12 It is an aryl alkyl ester.

[0113] "Solvate" refers to a form of a compound that is associated with a solvent or water (also referred to as a "hydrate"), usually by a solvent decomposition reaction. This physical association involves hydrogen bonding. Conventional solvents include water, ethanol, acetic acid, and the like. The compounds of the present invention may be prepared, for example, in crystalline form, and may be solvated or hydrated. Suitable solvates include pharma- ceutically acceptable solvates, such as hydrates, and further include both stoichiometric and non-stoichiometric solvates. In certain instances, a solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0114] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult) and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0115] "Effective amount" means the amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to effect such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. "Therapeutically effective amount" refers to an amount effective for therapeutic treatment. "Prophylactically effective amount" refers to an amount effective for prophylactic treatment.

[0116] "Preventing" or "prevention" or "prophylactic treatment" refers to reducing the risk of acquiring or developing a disease or disorder (i.e., preventing at least one of the clinical symptoms of the disease from developing in a subject who has not yet been exposed to a disease-causing agent or who is predisposed to the disease prior to the onset of the disease).

[0117] The term "prophylaxis" is related to "prevention" and refers to a strategy or approach whose purpose is to prevent disease rather than to treat or cure it. Non-limiting examples of preventive measures may include administration of vaccines, administration of low molecular weight heparin to hospitalized patients who are at risk of thrombosis due to immobility, and administration of antimalarial drugs such as chloroquine prior to visiting geographical areas where malaria is prevalent or where there is an increased risk of contracting malaria.

[0118] "Treating" or "treatment" or "therapeutic treatment" of any disease or disorder, in one embodiment, refers to alleviating the disease or disorder (i.e., arresting the disease or reducing the symptom, extent or severity of at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to improving at least one physical parameter, which cannot be discerned by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In a further embodiment, "treating" or "treatment" relates to slowing the progression of the disease.

[0119] "Administering" or "administration" of a substance, compound or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (e.g., by absorption through the skin tract). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations, which provide an extended, gradual or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. The appropriate method of administering a substance, compound or agent to a subject also depends, for example, on the age and / or physical condition of the subject, as well as the chemical and biological properties (e.g., solubility, digestibility, bioavailability, stability, and toxicity) of the compound or agent. In some embodiments, the compound or agent is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a slow or sustained release formulation or is administered using a device for such slow or sustained release.

[0120] As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic agents, such that the previously administered therapeutic agent is still effective in the body while the second agent is administered (e.g., the two agents are effective in the patient at the same time, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered either simultaneously or sequentially, either in the same formulation or in separate formulations. Thus, an individual undergoing such treatment may benefit from the combined effect of the different therapeutic agents.

[0121] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that has the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The exact effective amount required for a subject will depend, for example, on the subject's size, health and age, and the nature and extent of the condition being treated, such as cancer or MDS. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation.

[0122] As used herein, the term "isotopic variant" refers to a compound that contains unnatural proportions of an atomic isotope at one or more of the atoms that constitute such compound. For example, an "isotopic variant" of a compound can be, for example, an isotope of deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15( 15 In compounds where such isotopic substitutions are made, the following atoms, if present, may be changed so that, for example, any hydrogen is replaced with " 2 H / D" and any carbon 13 C or any nitrogen 15 It will be understood that the presence and location of such atoms may be determined within the skill of the art. Similarly, the present invention may include the preparation of isotopic variants with radioactive isotopes, for example, where the resulting compounds may be used in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C are particularly useful for this purpose given their ease of incorporation and ready means of detection. 11 C. 18 F, 15 O and 13Compounds substituted with positron emitting isotopes, such as N, may be prepared and may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. All isotopic variations of the compounds provided herein, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0123] It is also understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

[0124] Stereoisomers that are not mirror images of each other are called "diastereomers" and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers". For example, if a compound has an asymmetric center, it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers may be characterized by the absolute configuration of their asymmetric center and described by the R- and S-sequencing rules of Cahn and Prelog, or by the way in which the molecule rotates the plane of polarized light and is designated as right- or left-handed (i.e., as (+)- or (-)-isomers, respectively). Chiral compounds can exist as either individual enantiomers, or mixtures thereof. A mixture containing equal amounts of enantiomers is called a "racemic mixture".

[0125] "Tautomers" refer to compounds that are interchangeable forms of a particular compound structure, altered by the displacement of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the movement of electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro-forms of phenylnitromethane, which are similarly formed by treatment with acid or base. Tautomeric forms can be relevant to achieving optimal chemical reactivity and biological activity of a compound of interest.

[0126] As used herein, a pure enantiomer compound is substantially free of the other enantiomer or stereoisomer of the compound (i.e., enantiomeric excess). In other words, the "S" form of the compound is substantially free of the "R" form of the compound and is therefore in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" indicates that the compound contains more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight of the enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0127] As used herein, unless otherwise specified, the term "enantiomerically pure R-compounds" refers to at least about 95% by weight R-compounds and up to about 5% by weight S-compounds, at least about 99% by weight R-compounds and up to about 1% by weight S-compounds, or at least about 99.9% by weight R-compounds and up to about 0.1% by weight S-compounds. In certain embodiments, the weights are based on the total weight of the compounds.

[0128] As used herein, unless otherwise specified, the term "enantiomerically pure S-compound" or "S-compound" refers to at least about 95% by weight S-compound and up to about 5% by weight R-compound, at least about 99% by weight S-compound and up to about 1% by weight R-compound, or at least about 99.9% by weight S-compound and up to about 0.1% by weight R-compound. In certain embodiments, the weights are based on the total weight of the compound.

[0129] In the compositions provided herein, the enantiomerically pure compound, or its pharma- ceutically acceptable salt, solvate, hydrate, or prodrug, may be present together with other active or inactive ingredients. For example, a pharmaceutical composition comprising an enantiomerically pure R-compound may, for example, comprise about 90% of an excipient and about 10% of an enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such a composition may, for example, comprise at least about 95% by weight of the R-compound and up to about 5% by weight of the S-compound, based on the total weight of the compound. For example, a pharmaceutical composition comprising an enantiomerically pure S-compound may, for example, comprise about 90% of an excipient and about 10% of the enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such a composition may, for example, comprise at least about 95% by weight of the S-compound and up to about 5% by weight of the R-compound, based on the total weight of the compound. In certain embodiments, the active ingredients can be formulated with little or no excipients or carriers.

[0130] The compounds of the present invention may possess one or more asymmetric centers and therefore such compounds can be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof.

[0131] Unless otherwise indicated, the description or naming of a particular compound in the specification and claims is intended to include both the individual enantiomers and mixtures thereof, racemic or otherwise. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

[0132] Those skilled in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocycle, whether aromatic or non-aromatic, is determined by the size of the ring, the degree of unsaturation, and the valence of the heteroatoms. In general, a heterocycle may have from 1 to 4 heteroatoms, so long as the heteroaromatic ring is chemically feasible and stable.

[0133] List of embodiments The following listing of embodiments of the invention should be considered as disclosing various features of the invention, which features may be considered specific to the particular embodiment for which they are discussed or which may be combined with various other features as listed in other embodiments. Thus, merely because a feature is described under one particular embodiment does not necessarily limit the use of that feature to that embodiment.

[0134] Embodiment 1. A composition comprising a pharmaceutical agent assembled with a lipid composition, the lipid composition comprising a lipidoid having structural formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: R a is alkyl, n1 and n2 are each independently 1, 2, 3, or 4; R b1 , R b2 , R b3 , and R b4 are each independently H, [ka] and During the ceremony, * indicates the point of attachment to N each m is independently 1, 2, 3, 4, or 5; each q is independently 1, 2, 3, 4, or 5; Each R c are independently alkyl (e.g., C4 to C 20 ) or alkenyl (e.g., C4-C 20 ) and R b1 , R b2 , R b3 , and R b4 wherein at least one of is not H.

[0135] Embodiment 2. The composition of embodiment 1, wherein the composition is for preferential delivery to a target organ or target cell.

[0136] Embodiment 3. The composition of embodiment 1, wherein the composition is for modifying the expression profile of a target gene or its gene product in the target organ or the target cell.

[0137] Embodiment 4. The composition of embodiment 3, wherein the target gene or the gene product is a target protein or a functional variant thereof, or a target transcript.

[0138] Embodiment 5. A composition according to any one of embodiments 1 to 4, wherein the pharmaceutical agent is a therapeutic agent, a gene regulator, or a vaccine.

[0139] Embodiment 6.R a is C1-C4 alkyl.

[0140] Embodiment 7.R a The composition of embodiments 1-6, wherein is C1-C3 alkyl.

[0141] Embodiment 8.R a The composition of any one of embodiments 1-7, wherein is C1 alkyl.

[0142] Embodiment 9. The composition of any one of embodiments 1 to 8, wherein n1 is 1, 2, or 3.

[0143] Embodiment 10. The composition of embodiment 9, wherein n1 is 2 or 3.

[0144] Embodiment 11. The composition of any one of embodiments 1 to 10, wherein n2 is 1, 2, or 3.

[0145] Embodiment 12. The composition of embodiment 11, wherein n2 is 2 or 3.

[0146] Embodiment 13. A composition according to any one of embodiments 1 to 12, wherein n1 and n2 are the same.

[0147] Embodiment 14.R b1 , R b2 , R b3 , and R b4 The composition of any one of embodiments 1-13, wherein at least two of are not H.

[0148] Embodiment 15.R b1 , R b2 , R b3 and Rb4 are not H.

[0149] Embodiment 16.R b1 , R b2 , R b3 , and R b4 The composition of any one of embodiments 1-13, wherein none of

[0150] Embodiment 17.R b1 , R b2 , R b3 , and R b4 However, each independently, [ka] 14. The composition of any one of embodiments 1 to 13, wherein

[0151] Embodiment 18.R b1 , R b2 , R b3 , and R b4 However, each independently [ka] 14. The composition of any one of embodiments 1 to 13, wherein

[0152] Embodiment 19.R b1 , R b2 , Rb3 , and R b4 However, each independently [ka] 14. The composition of any one of embodiments 1 to 13, wherein

[0153] Embodiment 20. Each R c But independently, C4~C 16 Alkyl or C 4~16 The composition of any one of embodiments 1-19, wherein the alkyl group is alkenyl.

[0154] Embodiment 21. Each Rc is independently C6 to C 12 Alkyl or C6~C 12 The composition of any one of embodiments 1-20, wherein the alkyl group is alkenyl.

[0155] Embodiment 22. Each R c But independently, C4~C 20 Alkyl (e.g., C4-C 12 Alkyl, etc. C4-C 16 20. The composition of any one of embodiments 1-19, wherein R is 1 or 2.

[0156] Embodiment 23. Each R c But independently, C4~C 16 The composition of any one of embodiments 1-22, wherein is alkyl.

[0157] Embodiment 24. Each R C But independently, C4~C 12 The composition of any one of embodiments 1-23, wherein is alkyl.

[0158] Embodiment 25. The composition of any one of embodiments 1 to 22, wherein each m is 1, 2, or 3.

[0159] Embodiment 26. The composition of embodiment 25, wherein each m is 1 or 2.

[0160] Embodiment 27 The composition of embodiment 25, wherein each m is 1.

[0161] Embodiment 28. The composition of any one of embodiments 1 to 27, wherein each q is independently 1, 2, 3, or 4.

[0162] Embodiment 29. The composition of embodiment 28, wherein each q is independently 1, 2, or 3.

[0163] Embodiment 30 The composition of embodiment 28, wherein each q is 2.

[0164] Embodiment 31. The composition of any one of embodiments 1 to 30, wherein the lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is present in the lipid composition in a molar percentage of about 60% or less.

[0165] Embodiment 32. The composition of any one of embodiments 1 to 31, wherein the lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is present in the lipid composition in a molar percentage of about 50% or less.

[0166] Embodiment 33. The composition of any one of embodiments 1 to 32, wherein the lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is present in the lipid composition in a molar percentage of about 40% or less.

[0167] Embodiment 34. The composition of any one of embodiments 1 to 33, wherein the lipid composition further comprises a steroid or a steroid derivative.

[0168] Embodiment 35. The composition of any one of embodiments 1 to 34, wherein the lipid composition further comprises cholesterol or a cholesterol derivative.

[0169] Embodiment 36 The composition of embodiment 35, wherein the steroid or steroid derivative is present in the lipid composition in a molar percentage of about 50% or less.

[0170] Embodiment 37. The composition of any one of embodiments 1 to 36, wherein the lipid composition further comprises a polymer-conjugated lipid.

[0171] Embodiment 38. The composition of any one of embodiments 1 to 37, wherein the lipid composition further comprises a poly(ethylene glycol) (PEG)-conjugated lipid.

[0172] Embodiment 39. The composition of embodiment 37 or 38, wherein the polymer-conjugated lipid is present in the lipid composition in a molar percentage of about 10% or less.

[0173] Embodiment 40. The composition of any one of embodiments 1 to 39, wherein the lipid composition further comprises a phospholipid.

[0174] Embodiment 41. The composition of any one of embodiments 1 to 40, wherein the lipid composition further comprises a phosphoethanolamine lipid or a phosphocholine lipid.

[0175] Embodiment 42. The composition of embodiment 40, wherein the phospholipid is present in the lipid composition in a molar percentage of about 30% or less.

[0176] Embodiment 43. A composition according to any one of embodiments 1 to 42, wherein the lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is present in the composition in a mass or weight ratio of about 1:1 to about 200:1 relative to the pharmaceutical agent.

[0177] Embodiment 44. A composition according to any one of embodiments 1 to 43, wherein the lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is present in the composition in a mass or weight ratio of about 1:1 to about 100:1 relative to the pharmaceutical agent.

[0178] Embodiment 45. The composition of any one of embodiments 1 to 44, wherein the pharmaceutical agent comprises a polynucleotide.

[0179] Embodiment 46. The composition of any one of embodiments 1 to 44, wherein the pharmaceutical agent is a messenger ribonucleic acid (mRNA), an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof.

[0180] Embodiment 47. The composition of embodiment 46, wherein the polypeptide is a protein.

[0181] Embodiment 48. The composition of embodiment 45, wherein the polynucleotide is configured to upregulate or downregulate a target gene or its gene product.

[0182] Embodiment 49. The composition of embodiment 48, wherein the target gene or its gene product is a target protein or a functional variant thereof, or a target transcript.

[0183] Embodiment 50. The composition of any one of embodiments 1 to 49, wherein the pharmaceutical agent comprises a polynucleotide.

[0184] Embodiment 51. The composition of embodiment 50, wherein the polynucleotide is a messenger ribonucleic acid (mRNA) that encodes or is configured to upregulate or downregulate a target gene or its gene product.

[0185] Embodiment 52. The composition of embodiment 51, wherein the target gene or its gene product is a target protein or a functional variant thereof, or a target transcript.

[0186] Embodiment 53. The composition of any one of embodiments 1 to 52, wherein the target transcript is in a target organ or target cell.

[0187] 54. The pharmaceutical composition comprises: (a) a gene regulatory moiety configured to specifically bind to at least a portion of a target gene or its gene product; (b) A composition described in any one of embodiments 1 to 53, comprising a polynucleotide encoding the gene regulatory portion of (a).

[0188] Embodiment 55. The composition of embodiment 54, wherein the gene regulatory portion comprises a guide nucleic acid configured to complex with the target gene or at least a portion of its gene product, or a polynucleotide sequence encoding the guide nucleic acid.

[0189] Embodiment 56. The composition of embodiment 54 or 55, wherein the target gene or the gene product is a target protein or a functional variant thereof, or a target transcript.

[0190] Embodiment 57. The composition described in any one of embodiments 54 to 56, wherein the polynucleotide is a messenger ribonucleic acid (mRNA) encoding the gene regulatory portion of (a).

[0191] Embodiment 58. The composition according to any one of embodiments 54 to 57, wherein the gene regulatory portion comprises a heterologous endonuclease or a polynucleotide comprising a sequence encoding the heterologous endonuclease.

[0192] Embodiment 59. The composition of any one of embodiments 54 to 58, wherein the heterologous endonuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRSIPR)-associated (Cas) nuclease.

[0193] Embodiment 60. The composition of any one of embodiments 54 to 69, wherein the polynucleotide is a messenger ribonucleic acid (mRNA).

[0194] Embodiment 61. The composition of embodiment 60, wherein the heterologous endonuclease is present in the gene regulatory portion in a mass or weight ratio of about 1:20 to about 20:1 relative to the guide nucleic acid.

[0195] Embodiment 62. The composition of embodiment 61, wherein the heterologous endonuclease is present in the gene regulatory portion in a mass or weight ratio of about 1:10 to about 10:1 relative to the guide nucleic acid.

[0196] Embodiment 63. A composition described in any one of embodiments 1 to 62, wherein the target gene or its gene product is specific to or primarily found in a target organ or target cell of a subject.

[0197] Embodiment 64. The composition of embodiment 63, wherein the target gene or its gene product is a target protein or a functional variant thereof, or a target transcript.

[0198] Embodiment 65. The composition of embodiment 63 or 64, wherein the target gene or the gene product thereof is associated with a disease or disorder of the target organ or the target cell.

[0199] Embodiment 66. A composition described in any one of embodiments 54 to 65, wherein the gene regulatory portion is configured to provide an altered expression profile of the target gene or its gene product in a target organ or target cell of a subject.

[0200] Embodiment 67. A composition described in any one of embodiments 1 to 66, wherein the target organ is the lung or liver.

[0201] Embodiment 68. A composition described in any one of embodiments 1 to 67, wherein the target cell is a lung cell or a liver cell.

[0202] Embodiment 69. The composition of any one of embodiments 1 to 68, wherein the composition is formulated for systemic administration.

[0203] Embodiment 70. The composition of any one of embodiments 1 to 68, wherein the composition is formulated for topical administration.

[0204] Embodiment 71. The composition according to any one of embodiments 1 to 70, wherein the lipidoid of formula (I) or the pharma- ceutically acceptable salt thereof is R b1 , R b2 , R b3 , and R b4 are each independently H or [ka] and R b1 , R b2 , R b3 , and R b4 At least one of the [ka] The composition,

[0205] Embodiment 72.R b1 , R b2 , R b3 , and R b4 At least two (e.g., at least three, or all four) of [ka] 72. The composition of embodiment 71, wherein

[0206] Embodiment 73.R b1 , R b2 , R b3 , and R b4 At least three of the [ka] 73. The composition of embodiment 72, wherein

[0207] Embodiment 74.R b1 , R b2 , R b3 , and R b4 All four of these are independent of each other. [ka] 74. The composition of embodiment 73, wherein

[0208] Embodiment 75. The composition of any one of embodiments 71 to 74, wherein the composition is for preferential delivery of the pharmaceutical agent to the lungs.

[0209] Embodiment 76. The lipidoid of formula (I) or the pharma- ceutically acceptable salt thereof is represented by the structural formula (II A ), (II B ), (II C ), (II D ), or (II E ), [ka] or a pharma- ceutically acceptable salt thereof, m1, m2, m3, and m4, when present, are each independently 1, 2, or 3; q1, q2, q3, and q4, if present, are each independently 1, 2, 3, or 4; R c1 , R c2 , R c3 , and R c4 each independently, if present, C4 to C 20 Alkyl or C4-C 20 The composition of any one of embodiments 71-75, wherein the alkyl group is alkenyl.

[0210] Embodiment 77. The lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is a lipidoid of formula (II E ) or a pharma- ceutically acceptable salt thereof.

[0211] Embodiment 78.R c1 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 1278. The composition of embodiment 76 or 77, wherein R is 1 or 2; and R is 3 or 4.

[0212] Embodiment 79.R c1 But C6~C 12 Alkyl or C6~C 12 The composition of embodiment 78, wherein the alkyl group is alkenyl.

[0213] Embodiment 80. Formula (II B ), (II D ), or (II E ) in R c2 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 80. The composition of any one of embodiments 76-79, wherein R is 1 or 2.) alkenyl.

[0214] Embodiment 81. Formula (II B ), (II D ), or (II E ) in R c2 But C6~C 12 Alkyl or C6~C 12 The composition of any one of embodiments 76-80, wherein the alkyl group is alkenyl.

[0215] Embodiment 81. Formula (II C ), (II D ), or (II E ) in R c3 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 ) alkenyl.

[0216] Embodiment 83. Formula (II C ), (II D ), or (II E ) in R c3 But C6~C 12 Alkyl or C6~C12 The composition of any one of embodiments 76-82, wherein the alkyl group is alkenyl.

[0217] Embodiment 84. Formula (II E ) in R c4 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 ) alkenyl.

[0218] Embodiment 85. Formula (II E ) in R c4 But C6~C 12 Alkyl or C6~C 12 The composition of any one of embodiments 76-84, wherein the alkyl group is alkenyl.

[0219] Embodiment 86. A composition described in any one of embodiments 76 to 85, wherein m1 is 1 or 2.

[0220] Embodiment 87. Formula (II B ), (II D ), or (II E 87. The composition of any one of embodiments 76 to 86, wherein m2 is 1 or 2.

[0221] Embodiment 88. Formula (II C ), (II D ), or (II E 87. The composition of any one of embodiments 76 to 86, wherein m3 is 1 or 2.

[0222] Embodiment 89. Formula (II E 87. The composition of any one of embodiments 76 to 86, wherein m4 is 1 or 2.

[0223] The composition of any one of embodiments 76 to 89, wherein embodiment 90.q1 is 1, 2, or 3.

[0224] Embodiment 91. Formula (II B ), (II D ), or (II E ) wherein q2 is 1, 2, or 3.

[0225] Embodiment 92. Formula (II C ), (II D ), or (II E ) wherein q3 is 1, 2, or 3.

[0226] Embodiment 93. Formula (II E ) wherein q4 is 1, 2, or 3.

[0227] Embodiment 94. The lipidoid is: [ka] and any pharma- ceutically acceptable salt of any of the foregoing.

[0228] Embodiment 95. The compound of formula (I), (II A ), (II B ), (II C ), (II D ), or (II E ) or a pharma- ceutically acceptable salt thereof is present in the lipid composition in a molar percentage of about 20% to about 50%.

[0229] Embodiment 96. The composition of any one of embodiments 71 to 95, wherein the lipid composition comprises a steroid or steroid derivative in a molar percentage of about 10% to about 50%.

[0230] Embodiment 97. The compound of formula (I), (II A ), (II B ), (II C), (II D ), or (II E ) or said pharma- ceutical agent is present in said composition in a mass or weight ratio to said pharmaceutical agent of about 5:1 to about 100:1.

[0231] Embodiment 98. A composition described in any one of embodiments 71 to 97, wherein the composition is for preferential delivery of the pharmaceutical agent to the lung or lung cells compared to delivery to non-pulmonary organs or non-pulmonary cells.

[0232] Embodiment 99. The composition of embodiment 98, wherein the pharmaceutical agent is delivered to the lungs or lung cells of a subject.

[0233] Embodiment 100. The composition of embodiment 98, wherein the non-lung organ is a liver, heart, spleen, or kidney, and the non-lung cells are liver cells, heart cells, spleen cells, or kidney cells.

[0234] Embodiment 101. The composition of embodiment 98, wherein the pharmaceutical agent is configured to encode or upregulate a target gene or its gene product that is specific to or primarily found in the lung or lung cells.

[0235] Embodiment 102. The composition of embodiment 98, wherein the pharmaceutical agent is configured to encode or downregulate a target gene or its gene product that is specific to or primarily found in the lung or lung cells.

[0236] Embodiment 103. The composition of any one of embodiments 71 to 103, wherein the target gene or its gene product is a target protein or a functional variant thereof, or a target transcript.

[0237] Embodiment 104. The composition of embodiment 101, wherein the pharmaceutical agent is configured to provide an altered expression profile of the target gene or its gene product in the lung or lung cells.

[0238] Embodiment 105. The composition of any one of embodiments 98 to 104, wherein the pharmaceutical agent is associated with a pulmonary disease or disorder.

[0239] Embodiment 106. A method for preferentially delivering a pharmaceutical agent to the lungs or lung cells of a subject in need thereof, comprising administering a composition described in any one of embodiments 71 to 105, thereby providing a greater amount of expression or activity of the pharmaceutical agent (e.g., two-fold or more) in the lungs or lung cells of the subject compared to that achieved in a non-lung organ or non-lung cells of the subject.

[0240] Embodiment 107. The method of embodiment 106, wherein the method comprises administering a composition described in any one of embodiments 71 to 105, thereby providing at least a 5-fold greater amount of expression or activity of the pharmaceutical agent in the lung or lung cells of the subject compared to that achieved in a non-lung organ or non-lung cells.

[0241] Embodiment 108. The method of embodiment 106, wherein the method comprises administering a composition described in any one of embodiments 71 to 105, thereby providing at least a 10-fold greater amount of expression or activity of the pharmaceutical agent in the lung or lung cells of the subject compared to that achieved in a non-lung organ or non-lung cells.

[0242] Embodiment 109. The composition of any one of embodiments 71 to 108, wherein the non-lung organ is a liver, heart, spleen, or kidney, and the non-lung cells are liver cells, heart cells, spleen cells, or kidney cells.

[0243] Embodiment 110. A method for preferentially delivering a pharmaceutical agent to the lungs or lung cells of a subject in need thereof, comprising administering a composition described in any one of embodiments 71 to 109, thereby providing a greater amount of expression or activity (e.g., at least about two-fold greater amount) of the pharmaceutical agent in the lungs or lung cells of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0244] Embodiment 111. The method of embodiment 110, wherein the corresponding reference lipidoid has an ester-containing tail.

[0245] Embodiment 112. The method of embodiment 106 or 110, wherein the method modulates a greater amount (e.g., at least about 2-fold more) of the activity of a target gene or its gene product in the lung or lung cells of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0246] Embodiment 113. The method of embodiment 112, wherein the corresponding reference lipidoid has an ester-containing tail.

[0247] Embodiment 114 The method of embodiment 112, wherein the method provides an altered expression profile of the target gene or its gene product in the lung or lung cells of the subject.

[0248] Embodiment 115. In the lipidoid of formula (I) or the pharma- ceutically acceptable salt thereof, R b1 , R b2 , R b3 , and R b4 are each independently H or [ka] and R b1 , R b2 , R b3 , and R b4At least one of the [ka] The composition of any one of embodiments 1-114, wherein

[0249] Embodiment 116.R b1 , R b2 , R b3 , and R b4 At least two (e.g., at least three, or all four) of [ka] The composition of embodiment 115, wherein

[0250] Embodiment 117.R b1 , R b2 , R b3 , and R b4 At least three of the [ka] The composition of embodiment 116, wherein

[0251] Embodiment 118.R b1 , R b2 , R b3 , and R b4 All four of these are independent [ka] The composition of embodiment 117, wherein

[0252] Embodiment 119. The lipidoid of formula (I) or the pharma- ceutically acceptable salt thereof is represented by the structural formula (III A ), (III B ), (III C ), (III D ), or (III E ), [ka] or a pharma- ceutically acceptable salt thereof, m5, m6, m7, and m8 are each independently 1, 2, or 3; q5, q6, q7, and q8 are each independently 1, 2, 3, or 4; R c5 , R c6 , R c7 , and R c8 each independently, if present, C4 to C 20 Alkyl or C4-C 20 The composition of any one of embodiments 115-118, wherein the alkyl group is alkenyl.

[0253] Embodiment 120. The lipidoid of formula (I), or the pharma- ceutically acceptable salt thereof, is a lipidoid of formula (III) E ) or a pharma- ceutically acceptable salt thereof.

[0254] Embodiment 121.R c5 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 121. The composition of embodiment 119 or 120, wherein R is 1 or 2; and R is 3 or 4.

[0255] Embodiment 122. Formula (III B ), (III D ), or (III E ) in R c6 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 ) alkenyl.

[0256] Embodiment 123. Formula (III B ), (III D ), or (III E ) in Rc6 But C6~C 12 Alkyl or C6~C 12 The composition of any one of embodiments 119-122, wherein the alkyl group is alkenyl.

[0257] Embodiment 124. Formula (III C ), (III D ), or (III E ) in R c7 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 ) alkenyl.

[0258] Embodiment 125. Formula (III C ), (III D ), or (III E ) in R c7 But C6~C 12 Alkyl or C6~C 12 The composition of any one of embodiments 119-124, wherein the alkenyl.

[0259] Embodiment 126. Formula (III E ) in R c8 But C4~C 16 (For example, C6~C 12 ) Alkyl or C4-C 16 (For example, C6~C 12 ) alkenyl.

[0260] Embodiment 127. Formula (III E ) in R c8 But C6~C 12 Alkyl or C6~C 12 The composition of any one of embodiments 119-126, wherein the alkyl group is alkenyl.

[0261] Embodiment 128. A composition described in any one of embodiments 119 to 127, wherein m5 is 1 or 2.

[0262] Embodiment 129. Formula (III B ), (III D ), or (III E 129. The composition of any one of embodiments 119 to 128, wherein m6 is 1 or 2.

[0263] Embodiment 130. Formula (III C ), (III D ), or (III E 130. The composition of any one of embodiments 119 to 129, wherein m7 is 1 or 2.

[0264] Embodiment 131. Formula (III E 131. The composition of any one of embodiments 119 to 130, wherein m8 is 1 or 2.

[0265] A composition described in any one of embodiments 119 to 131, wherein embodiment 132.q5 is 1, 2, or 3.

[0266] Embodiment 133. Formula (III B ), (III D ), or (III E ) wherein q6 is 1, 2, or 3.

[0267] Embodiment 134. Formula (III C ), (III D ), or (III E ) wherein q7 is 1, 2, or 3.

[0268] Embodiment 135. Formula (III E ) wherein q8 is 1, 2, or 3.

[0269] Embodiment 136. The lipidoid is: [ka] [ka] and any pharma- ceutically acceptable salt of any of the foregoing.

[0270] Embodiment 137. The compound of formula (I), (III A ), (III B ), (III C ), (III D ), or (III E ) or a pharma- ceutically acceptable salt thereof is present in the lipid composition in a molar percentage of about 20% to about 50%.

[0271] Embodiment 138. The composition of any one of embodiments 115 to 137, wherein the lipid composition comprises a steroid or steroid derivative in a molar percentage of about 10% to about 50%.

[0272] Embodiment 139. The compound of formula (I), (III A ), (III B ), (III C ), (III D ), or (III E ) or said pharma- ceutical agent is present in said composition in a mass or weight ratio to said pharmaceutical agent of about 5:1 to about 100:1.

[0273] Embodiment 140. A composition described in any one of embodiments 115 to 139, wherein the composition is for preferential delivery of the pharmaceutical agent to the liver or hepatic cells compared to delivery to non-liver organs or non-hepatic cells.

[0274] Embodiment 141. The composition of embodiment 140, wherein the non-liver organ is lung and the non-liver cells are lung cells.

[0275] Embodiment 142. The composition of embodiment 140 or 141, wherein the pharmaceutical agent is configured to encode or upregulate a target gene or its gene product that is specific to the liver or hepatocytes or is found primarily in the liver or hepatocytes.

[0276] Embodiment 143. The composition of embodiment 140 or 141, wherein the pharmaceutical agent is configured to encode or downregulate a target gene or its gene product that is specific to the liver or hepatocytes or is found primarily in the liver or hepatocytes.

[0277] Embodiment 144. The composition of any one of embodiments 140 to 143, wherein the target gene is the angiopoietin-like 3 (ANGPTL3) gene.

[0278] Embodiment 145. The composition of embodiment 142 or 143, wherein the pharmaceutical agent is configured to provide an altered expression profile of the target gene or its gene product (e.g., the target protein or its functional variant, or the target transcript) in the liver or hepatic cells.

[0279] Embodiment 146. A composition described in any one of embodiments 140 to 145, wherein the pharmaceutical agent is associated with a liver disease or disorder.

[0280] Embodiment 147. A method for preferentially delivering a pharmaceutical agent to the liver or liver cells of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 146, thereby providing a greater amount of expression or activity of the pharmaceutical agent (e.g., at least about two-fold greater amount) in the lung or lung cells of the subject compared to that achieved in a non-liver organ or non-hepatic cells of the subject.

[0281] Embodiment 148. A method for preferentially delivering a pharmaceutical agent to the liver or hepatic cells of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 146, thereby providing at least about 5-fold greater expression or activity of the pharmaceutical agent in the liver or hepatic cells of the subject compared to that achieved in a non-liver organ or non-hepatic cells of the subject.

[0282] Embodiment 149. A method for preferentially delivering a pharmaceutical agent to the liver or hepatic cells of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 146, thereby providing at least about 10-fold greater expression or activity of the pharmaceutical agent in the liver or hepatic cells of the subject compared to that achieved in a non-liver organ or non-hepatic cells of the subject.

[0283] Embodiment 150. The method of any one of embodiments 147 to 149, wherein the non-liver organ is a lung and the non-liver cells are lung cells.

[0284] Embodiment 151. A method for preferentially delivering a pharmaceutical agent to the liver or hepatocytes of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 150, thereby providing a greater amount of expression or activity (e.g., at least about two-fold greater amount) of the pharmaceutical agent in the liver or hepatocytes of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0285] Embodiment 152. A method for preferentially delivering a pharmaceutical agent to the liver or hepatocytes of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 150, thereby providing at least about 5-fold greater expression or activity of the pharmaceutical agent in the liver or hepatocytes of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0286] Embodiment 153. A method for preferentially delivering a pharmaceutical agent to the liver or hepatocytes of a subject in need thereof, comprising administering a composition described in any one of embodiments 115 to 150, thereby providing at least about 10-fold greater expression or activity of the pharmaceutical agent in the liver or hepatocytes of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0287] Embodiment 154. The method of any one of embodiments 151 to 153, wherein the corresponding reference lipid has an amide-containing tail.

[0288] Embodiment 155. The amide-containing tail has structural formula (II A ), (II B ), (II C ), (II D ), or (II E 155. The method of embodiment 154, wherein the lipidoid has a corresponding structure having the formula:

[0289] Embodiment 156. The method of embodiment 147 or 155, wherein the method modulates a greater amount or activity of a target gene or its gene product in the liver or hepatic cells of the subject compared to that achieved with a corresponding reference lipid composition comprising a corresponding reference lipidoid.

[0290] Embodiment 157. The composition of embodiment 156, wherein the target gene is the angiopoietin-like 3 (ANGPTL3) gene.

[0291] Embodiment 158. The method of embodiment 156 or 157, wherein the corresponding reference lipidoid has an amide-containing tail.

[0292] Embodiment 159. The amide-containing tail has structural formula (II A ), (II B ), (II C ), (II D ), or (II E159. The method of embodiment 158, wherein the lipidoid has a corresponding structure having the formula:

[0293] Embodiment 160. The method of any one of embodiments 156 to 159, wherein the method provides an altered expression profile of the target gene or its gene product in the liver or hepatic cells of the subject.

[0294] Embodiment 161 The method according to any one of embodiments 1 to 160, wherein the gene product is the target protein or a functional variant thereof. EXAMPLES

[0295] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the compounds, compositions, materials, devices, and methods provided herein, and should not be construed in any way as limiting the scope thereof.

[0296] Materials and Methods Research design: The overall objective of this study is to explore a non-viral lipid nanoparticle (LNP)-mediated CRISPR-Cas9 mRNA-based strategy for in vivo organ-specific genome editing of Angptl3 for blood lipid regulation. This study includes: (i) identifying a liver-targeted bioreducible lipid nanoparticle platform that can effectively deliver mRNA to hepatocytes; (ii) optimizing the LNP formulation to maximize the efficacy of mRNA delivery in vivo; (iii) demonstrating the in vivo genome editing effect of 306-O12B LNP-mediated Cas9 mRNA delivery by targeting the therapeutically relevant Angptl3 gene, resulting in a significant reduction in serum ANGPTL3, low-density lipoprotein cholesterol, and triglyceride levels; and (iv) revealing that the therapeutic effect of LNP-mediated CRISPR-Cas9 mRNA-based genome editing of Angptl3 is stable for at least 100 days after a single dose. All animal studies were performed according to protocols approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) and in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. Genome editing efficacy, off-target side effects, safety profile, and changes in lipid levels were thoroughly evaluated.

[0297] LNP formulation: Lipidoids were synthesized according to previous reports (S. Tenzer et al., Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8, 772-781 (2013), KA Hajj et al., Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH. Small 15, e1805097 (2019)). LNPs were prepared using a NanoAssemblr microfluidic system (Precision Nanosystems). Briefly, lipidoids, cholesterol (Sigma), phospholipids (DSPC, DOPE, and DOPC, Avanti Polar Lipids), and methoxypolyethylene glycol (DMG-PEG) were mixed in a 100-mL PBS solution. 2000 ) (Avanti Polar Lipids) were dissolved in 100% ethanol in a molar ratio of 50 / 38.5 / 10 / 1.5 for a final lipidoid concentration of 10 mg / mL. The MC-3 LNP formulation was prepared according to the procedure previously described by Sedic et al., with minor modifications. In summary, the lipid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yi4-(dimethylamino)butanoate (MC-3), DSPC, cholesterol, and DMG-PEG were added to the MC-3 LNP formulation. 2000 was mixed with 50% MC-3, 38.5% cholesterol, 10% DSPC, and 1.5% DMG-PEG at a final MC-3 concentration of 10 mg / mL. 2000The lipid solution was then mixed with an acidic sodium acetate buffer containing mRNA (0.45 mg / mL, pH 4.0) using a NanoAssemblr microfluidic system. The resulting LNPs were dialyzed overnight at 4°C against PBS (pH 7.4, 10 mM). Cas9 mRNA and gRNA (either sgANGPTL3 or sgLoxP) were mixed in an appropriate weight ratio in sodium acetate buffer (25 mM, pH 5.2). The mRNA solution and lipid solution were injected into the NanoAssemblr microfluidic device in a ratio of 3:1, respectively, and the device resulted in rapid mixing of the two components and thus self-assembly of the LNPs. The formulation was further dialyzed against PBS (10 mM, pH 7.4) in a dialysis cassette at 4°C. The particle size of the formulation was measured by dynamic light scattering (DLS) using a ZetaPALS DLS machine (Brookhaven Instruments). RNA encapsulation efficiency was characterized by Ribogreen assay.

[0298] In vivo LNP delivery All procedures for all animal experiments were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals. All animals were ordered from Charles River. Female Balb / c mice (6-8 weeks) were used for in vivo firefly luciferase mRNA (fLuc mRNA, TriLink Biotechnologies) encapsulated LNP screening and formulation optimization. Briefly, fLuc mRNA LNPs were injected intravenously into mice at a dose of 0.5 mg / kg of mRNA. At the designated time points, mice were injected with 100 μL of D-luciferin potassium salt (Goldbio) solution (15 mg / mL in PBS), anesthetized under isoflurane anesthesia, and measured by an IVIS imaging system (Caliper Life Sciences).

[0299] Commercially available nucleic acids Cas9 mRNA was sourced from Tri-Link Biotechnologies. All sgRNAs used in this document were sourced from Synthego using their "end-modified" synthesis. Briefly, the first and last three bases of these gRNAs are synthesized using 2'-O-methyl nucleosides linked via 3' phosphorothioate linkages. All other bases throughout the remainder of the molecule are traditional ribonucleosides and phosphate linkages typical of natural RNA.

[0300] In vivo Cas9 mRNA / sgLoxP delivery Cas9 mRNA (TriLink Biotechnologies) and LoxP-targeted single guide RNA (sgLoxP, sequence: 5'-AAGTAAAACCTCTACAAATG, end-modified, Synthego) were co-loaded into 306-O12B LNPs and injected intravenously into female Ai14 mice at a dose of 1.65 mg / kg total RNA. Seven days after injection, mouse organs were collected and imaged by IVIS to detect the expression of tdTomato. Liver tissues were further sectioned.

[0301] immunostaining Tissue samples were embedded in OCT, completely frozen in liquid nitrogen, and stored at -80 °C until ready for sectioning. Frozen tissue blocks were sectioned to the desired thickness (10 μm) using a cryotome and placed on glass slides suitable for immunofluorescence staining. Tissue sections were fixed in pre-cooled acetone (-20 °C) for 10 min and then washed twice in PBS for 5 min each. Fixed tissue sections were incubated in 10% BSA blocking buffer for 1 h at room temperature (rt) and then washed with PBS. Hepatocyte-specific primary antibody (1:100 diluted in 1% BSA buffer, anti-hepatocyte-specific antigen (HepPar1), Novus) (43) was applied to the sections on the slides and incubated overnight in a humidified chamber at 4 °C. Slices were rinsed twice with PBS for 5 min each, then stained with eFlour660-conjugated F(ab')2-Goat anti-mouse secondary antibody (1:50, Invitrogen), incubated for 1 h in a light-tight humidified chamber at room temperature, and then washed three times with PBS. Slides were coverslipped using fluorescent mounting medium containing DAPI (Sigma). Sections were analyzed using a Leica SP8 confocal microscope.

[0302] In vivo genome editing of Angptl3: Guide RNA sequences targeting Angptl3 were designed using Benchling software. Female wild-type C57BL / 6 mice aged 6–8 weeks were intravenously administered 306-O12B LNPs co-loaded with Cas9 mRNA and ANGPTL3-targeting single guide RNA (sgAngptl3, sequence: 5'-AGCCCTTCAACACAAGGTCA, terminally modified, Synthego) at doses of 1.0, 2.0, and 3.0 mg / kg in total RNA. PBS-treated mice were treated as negative controls. On day 7 post-injection, mice were euthanized (without fasting), blood was collected for circulating ANGPTL3 protein and blood lipid quantification by ELISA, and liver tissue was harvested from the median and left lateral lobes for DNA extraction and next-generation sequencing (NGS) analysis. To control for the effect of circadian rhythm on serum lipid levels, serum was collected at approximately the same time (early afternoon) for all experiments. To further evaluate in vivo toxicity and immune responses, blood was collected and transferred to serum from mice 2 days after injection. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as well as tumor necrosis factor alpha (TNF-alpha) were measured using assay kits for AST (G-Biosciences), ALT (G-Biosciences), and TNF-alpha (R&D Systems) according to the manufacturer's protocols. Serum levels of IFN-α, IL-6, and IP-10 were measured using a mouse IFN-α ELISA kit (PBL Assay Science), a mouse IL-6 Quantikine ELISA kit (R&D Systems), and an Abcam mouse IP-10 ELISA kit, respectively.

[0303] NGS Sequencing Analysis: DNA was extracted from the median and left lateral lobes of the liver using a commercially available extraction kit (Qiagen DNEasy Blood & Tissue). PCR primers were designed to amplify regions surrounding the target site or predicted off-target sites in the Angptl3 gene (Table 1). Off-target sites were predicted using Cas-Off-Finder software (rgenome.net / cas-offinder / ). PCR amplicons were prepared for sequencing on an Illumina MiSeq (Tufts Genomics Core Facility). Sequencing data were analyzed using OutKnocker 2 software (outknocker.org / outknocker2.htm). [Table 1]

[0304] Serum ANGPTL3 protein, low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) analysis Mouse blood was collected without anticoagulant, allowed to clot for 2 h at room temperature, and mouse serum was collected by centrifugation at 2000 × g for 15–20 min at room temperature. Serum levels of ANGPTL3 protein, LDL-C, and TG were determined using a Mouse Angiopoietin-Like 3 Quantikine ELISA kit (R&D systems), a Mouse LDL-Cholesterol kit (Crystal Chem), and a Triglyceride Colorimetric Assay Kit (Cayman Chemical), respectively, according to the manufacturer's protocols.

[0305] T7E1 cleavage assay: Genomic regions flanking the target site were amplified using extracted genomic DNA template, Platinum SuperFi Green DNA polymerase (Invitrogen), and specific primers (see Table 1). The following cycles were performed: 98°C for 30 s, followed by 33 cycles of 98°C for 10 s, 65°C for 15 s, and 72°C for 30 s, followed by 72°C for 10 min. PCR products were purified using a GeneJET PCR purification kit (Thermo Scientific). 400 ng of purified PCR products were hybridized in NEBuffer2 (New England Biolabs) by heating to 95°C for 5 min, followed by a 2°C / s ramp to 85°C and a 0.1°C / s ramp to 25°C on an AppliedBiosystems PCR system (Thermo Fisher Scientific). The annealed samples were digested with T7 endonuclease I (New England Biolabs) for 15 min at 37° C., followed by incubation for 5 min at 65° C. to stop the reaction. The products were further purified and run on 4-20% Novex TBE gels (Invitrogen).

[0306] Statistical analysis: Data were expressed as mean ± SD. All data were analyzed using Graphpad Prism software. Statistical differences were analyzed by One-Way ANOVA. *p<0.05 was considered significant, **p<0.01, ***p<0.001 were considered highly significant.

[0307] Synthesis of lipidoid nanoparticles. Lipidoids were synthesized by a Michael addition reaction between an amine head and an alkyl-acrylate tail, as previously reported. Lipidoid nanoparticles were prepared by mixing the active lipidoid, cholesterol (Sigma Aldrich), DOPC (Avanti Polar Lipids), and mPEG using the NanoAssemblr microfluidic system. 2000-DMG (Avanti Polar Lipids) was formulated in ethanol solution at a molar ratio of 50:38.5:10:1.5 by rapid mixing with sodium acetate buffer (pH 5.2, 25 mM) containing mRNA. The final weight ratio of active lipidoid to mRNA was set at 10 / 1. The resulting LNPs were further dialyzed overnight at 4°C against PBS (10 mM, pH 7.4) in 3,500 MWCO cassettes (Thermo Scientific). The size and surface zeta potential of the LNPs were measured using a ZetaPALS DLS machine (Brookhaven Instruments).

[0308] In vivo mRNA delivery All animal experiments were performed in accordance with approved animal protocols. For in vivo firefly luciferase mRNA (fLuc mRNA) transfection, 6- to 8-week-old female Balb / c mice purchased from Charles River were administered fLuc mRNA-loaded LNPs (0.5 mg / kg of mRNA) intravenously (iv). Six hours after injection, mice were injected intraperitoneally with D-luciferin (Goldbio, 15 mg / mL in PBS) and imaged using an IVIS imaging system (Perkin Elmer). To further quantify luciferase protein expression in different organs, tissues were harvested, homogenized, centrifuged and the supernatants were collected. The supernatants were then assayed for luciferase expression using the Firefly Luciferase Assay Kit 2.0 (Biotium). Results were presented as nanograms luciferase protein per gram of tissue.

[0309] For transgenic Cre mRNA delivery, LNPs co-formulated with Cre mRNA were injected intravenously into Ai14Cre reporter mice (Jackson Laboratory). After 7 days, mice were euthanized and major organs were harvested and imaged using an IVIS imaging system. To further identify the specific cell populations transfected, tissues were sectioned to a depth of 10 μm and further imaged using an SP8 confocal microscope (Leica). For flow cytometry studies, lungs were minced and then processed with a mouse lung dissociation kit (Milteny Biotec). Next, the lung solution was filtered through a 70 μm strainer, proceeded to a single cell suspension, centrifuged, and then treated with red blood cell lysis buffer (eBioscience) for 5 min. The solution was then centrifuged again to collect the cell pellet. Cells were resuspended in flow cytometry staining buffer (eBioscience), and then antibodies were added and incubated on ice in the dark for 30 min. Stained cells were washed twice with cold PBS, resuspended in PBS, and measured using an LSR-II flow cytometer (BD Bioscience). The antibodies used in this study were as follows: CD326-PE-Cy7 (eBioscience), CD31-FITC (Biolegend), and F4 / 80-eFluor660 (eBioscience). All antibody dilutions were used according to the manufacturer's instructions.

[0310] For genome editing Cas9 mRNA delivery, Cas9 mRNA-loaded LNPs were injected intravenously into Balb / c mice (0.5 mg / kg). Six hours after injection, the mice were euthanized, organs were harvested, and Western blots were performed to detect Cas9 expression in different organs. Briefly, organs were homogenized in lysis buffer and centrifuged at 13,000 g for 10 min at 4 °C. The supernatant was collected and protein concentration was measured using a BCA assay kit (Thermo Fisher). Then, 20 μg of total protein was loaded onto a 4–20% polyacrylamide gel (Thermo Fisher), and the gel was then run under a steady voltage of 120 V for 90 min. The gel was cut, transferred to a PVDF membrane, blocked with 5% nonfat milk at room temperature for 1 h, and incubated with anti-CRISPR-Cas9 primary antibody (Abcam, ab189380) overnight at 4 °C. The membrane was washed five times with TBST and incubated with HRP rabbit anti-mouse secondary antibody (Abcam) for 1 h at room temperature and then imaged with ECL substrate using a gel imaging system.

[0311] Isolation of protein corona Mouse plasma was centrifuged at 13,000g at 4°C to remove protein aggregates before use. LNPs were mixed with an equal volume of C57BL / 6 mouse plasma to mimic in vivo protein concentration and incubated at 37°C under shaking for 1 h. Protein corona-coated LNPs were isolated by centrifugation at 13,000g for 30 min, followed by washing three times with cold PBS to remove unbound proteins. The same procedure was performed on plasma aliquots without added LNPs to verify the absence of protein precipitation. All experiments were performed in triplicate. The amount of protein in the protein corona-coated LNPs was determined using a BCA assay kit (Thermo Fisher). The resulting protein samples were further lyophilized and stored at -20°C for further experiments.

[0312] Protein reduction, alkylation, and digestion Lyophilized protein samples were reconstituted to 1 mg / mL in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific). For each sample, 100 μg of protein was reduced with 2.1 μL of 500 mM dithioerythritol (DTT), 99+% (Acros Organics) for 45 min at 50°C. Samples were then alkylated with 11.5 ul of iodoacetamide (Acros Organics) for 30 min at room temperature in the dark. Samples were then digested overnight with Trypsin / Lys-C Mix (Mass Spectrometry Grade, Promega) at 37°C with an enzyme-to-protein ratio of 1 to 50. Samples were then stored at -20°C until LC-MS analysis.

[0313] mass spectrometry The resulting samples were further analyzed by electrospray liquid chromatography-mass spectrometry (LC-MS / MS) using an Orbitrap Fusion Lumos mass spectrometer coupled online to an EASY-nLC 1200 (Thermo Fisher). Samples were run at a flow rate of 300 nL / min using the following gradient: 0–5% B for 0–4 min, 0–30% B for 4–74 min, 30–90% B for 74–79 min, 90% B for 79–89 min, 90–0% B for 89–94 min, and 0% B for 94–105 min (column re-equilibration). Mobile phase A was 0.1% FA in water / 0.1% FA in ACN at 96.1:3.9. Mobile phase B was 0.1% FA in water / 0.1% FA in ACN at 80.0:20.0. Samples were first desalted on a Thermo Fisher Scientific Acclaim PepMap 100C18 HPLC column (3 μm particle size, 75 μm × 2 cm, 100 Å) and then separated on a Thermo Fisher Scientific PepMap RSLC C18 EASY-Spray column (3 μm particle size, 75 μm × 15 cm, 100 Å). Data were analyzed using Proteome Discoverer 2.1.0.81 software (Thermo Fisher). The data was run against the Mus musculus Uniprot FASTA file (revised 26 August 2020) to identify mouse proteins within the corona.

[0314] In vitro mRNA delivery TSC2-deficient TTJ cells were cultured at 1 × 10 per well. 4Cells were seeded in 48-well plates with 100 μg / well of EGFP mRNA. After 24 h of incubation, cells were transfected with EGFP mRNA using LNP (100 ng of mRNA per well) for 24 h. Lipofectamine 2k was used as a positive control. To measure transfection efficiency, cells were digested with trypsin, collected, and GFP expression was further evaluated using flow cytometry (Attune NxT Cytometer, ThermoFisher). The percentage of EGFP-positive cells was calculated by Flowjo.

[0315] In vivo treatment of TTJ tumors with TSC2 mRNA C57BL / 6 mice were cultured with 2 × 10 TSC2-deficient TTJ cells. 6 The mice were injected into the tail vein to form tumor nodules in the lungs. 24 days after tumor cell inoculation, the mice were randomly assigned to three groups: an untreated control group, a group treated with empty LNPs, and a group treated with Tsc2 mRNA-loaded LNPs (dosage: 0.75 mg / kg mRNA per injection). The mice were treated every other day for a total of five times. After treatment, the mice were euthanized and the lungs were collected for further morphological and immunohistochemical analysis.

[0316] A highly potent novel non-viral LNP-mediated CRISPR-Cas9 delivery system for liver delivery of Cas9 mRNA is disclosed herein and its efficacy is demonstrated by targeting the Angptl3 gene. The system is composed of a leading tail-branched bioreproducible lipidoid (306-O12B) co-formulated with an optimized mixture of excipient lipid molecules, and successfully co-delivers SpCas9 mRNA and a single guide RNA (sgAngptl3) targeting Angptl3 via a single administration (Figure 1). 306-O12B LNPs specifically delivered Cas9 mRNA and sgAngptl3 to liver hepatocytes in wild-type C57BL / 6 mice, resulting in a median editing rate of 38.5% and a corresponding reduction in serum ANGPTL3 protein of 65.2%. Delivery using 306-O12B LNPs was more efficient than delivery using MC-3 LNPs, the gold standard LNP recently approved by the FDA for liver-targeted delivery of nucleic acids. Furthermore, liver-specific knockdown of Angptl3 resulted in a significant reduction in LDL-C and TG levels. Importantly, no evidence of off-target mutagenesis at the nine top predicted sites was observed, nor was any overt liver toxicity observed. CRISPR-mediated genome editing was maintained at therapeutically relevant levels for at least 100 days after injection of a single dose. This system provides a clinically viable approach for liver-specific delivery of CRISPR-Cas9-based genome editing tools.

[0317] Example 1: In vivo screening of lipid nanoparticles for mRNA delivery Tail-branched bioreducible lipidoids were prepared by a solvent-free combined Michael addition reaction between disulfide-bond-incorporated acrylate lipid tails and amine-containing heads, according to previous reports (Figure 2A). The in vivo mRNA delivery efficacy of these lipids was evaluated by encapsulating firefly luciferase mRNA (fLuc mRNA) within LNPs and delivering these LNPs intravenously to female wild-type Balb / c mice. These LNPs were formulated with the disclosed synthetic ionizable lipids along with the excipient compounds cholesterol, DSPC, and DMG-PEG. Representative transmission electron microscopy images of blank (unloaded) and fLuc mRNA-loaded LNPs are shown in Figures 7A and 7B. Gold standard MC-3 LNPs formulated with cholesterol, DSPC, and DMG-PEG were included as a positive control. Six hours after mRNA delivery, mice were intraperitoneally injected with luciferin substrate, and systemic fLuc activity was measured using an IVIS in vivo imaging system (PerkinElmer). As shown in Figure 2B, mRNA delivery by 306-O12B, 113-O12B, and 306-O10B LNPs resulted in comparable luciferase bioluminescence intensity compared to MC-3 LNP delivery. In vivo imaging of mice showed that luciferase protein was mainly expressed in the liver (Figure 8). Based on these preliminary results, 306-O12B LNPs were selected for further development due to their relatively high luciferase expression and high specificity. fLuc mRNA could be efficiently encapsulated in 306-O12B LNPs with an encapsulation efficiency of approximately 96% (Figure 9). After encapsulation of fLuc mRNA, 306-O12B LNPs had an average diameter of 112 nm (Figure 2C).

[0318] Example 2: Formulation optimization of 306-O12B LNPs To further increase luciferase expression in vivo, we optimized various formulation parameters used in the assembly of these LNPs, including the excipient phospholipid identity, the molar composition ratio of the four components of the LNP formulation, and the lipid / mRNA weight ratio of fLuc mRNA encapsulated 306-O12B LNPs.

[0319] First, two additional phospholipids, DOPE and DOPC (Figure 3A), with similar structures to the original DSPC phospholipid but different head group and tail saturation, were selected to evaluate the effect of phospholipid excipients on the efficacy of luciferase expression in vivo. DOPC and DOPE each contain one degree of unsaturation in the carbon tail, while DSPC is fully saturated. Furthermore, DSPC and DOPC each contain a quaternary amine head group, while DOPE contains a primary amine head group. Each of these features has been reported in the literature to affect LNP delivery efficiency. Quaternized amine head groups have been reported to exhibit a stronger proton sponge effect than primary amine head groups, which can facilitate endosomal escape of cargo mRNA into the cytoplasm, thus increasing mRNA translation. Furthermore, the saturation degree of the lipid tail has been shown to affect membrane fluidity, which may also affect endosomal escape. Unsaturated lipid tails may result in higher membrane fluidity, which may also help improve endosomal escape via destabilization of the endosomal membrane upon fusion of LNPs with the membrane. LNPs formed with DOPC containing quaternary amines and unsaturated tails would show the most efficient fLuc delivery. As shown in Figures 3B and 3C, fLuc mRNA LNPs formulated with DOPC actually resulted in significantly higher luciferase expression in the liver than those with DOPE and original DSPC phospholipids. fLuc mRNA delivered with DOPC-containing LNPs resulted in approximately 4-fold higher luminescence signal compared to original DSPC-containing LNPs.

[0320] In the initial screening, the active lipid and excipient components were formulated in a molar ratio of 50:38.5:10:1.5 lipid:cholesterol:DSPC:DMG-PEG. This excipient ratio has been reported in the literature to be effective. After identifying the optimal phospholipid as DOPC and adjusting the formulation accordingly, LNPs formulated in various molar ratios were tested to identify the optimal parameters (Figure 3D). As shown in Figure 3E, the original formulation O (306-O12B: Cholesterol:DOPC:DMG-PEG in a molar ratio of 50:38.5:10:1.5) showed the highest luciferase bioluminescence intensity, and none of the new formulation parameters surpassed the original parameters.

[0321] To further increase the in vivo efficacy, LNPs with this optimal ratio of the four components were formulated with different weight ratios of active lipid 306-O12B:mRNA (ranging from 5:1 to 25:1) ranging from 5:12. Interestingly, the highest efficacy was achieved when the weight ratio was 7.5:1. Increasing the amount of lipid beyond this point did not seem to improve the in vivo delivery efficacy (Figure 3F). Taken together, these results showed that the optimized formulation of 306-O12B LNPs had a molar composition of 50% 306-O12B, 38.5% cholesterol, 10% DOPC, and 1.5% DMG-PEG with a 7.5 / 1 weight ratio of 306-O12B / mRNA.

[0322] Example 3: In vivo hepatocyte-specific co-delivery of Cas9 mRNA and sgRNA with mRNA-optimized 306-O12B LNPs Identification of specific cell types preferentially edited by Cas9 mRNA / sgRNA LNPs is important for evaluating potential applications of CRISPR delivery systems. We used the Ai14 reporter mouse strain, genetically engineered with a LoxP-flanked STOP cassette that controls tdTomato expression. This mouse strain is frequently used with Cre recombinase, but successful CRISPR-mediated excision of the LoxP-flanked STOP codon also induces expression of tdTomato. When combined with Cas9 and a guide RNA targeting this site, examination of cells with tdTomato expression will indicate which cell types the LNP delivery system will target.

[0323] To validate this approach, 306-O12B LNPs were used to deliver LoxP-targeted sgRNA (sgLoxP) to mice engineered to express both Ai14 and constitutively expressed Cas9 constructs (Ai14+ / Cas9+ mouse model). Figures 10A and 10B show that 306-O12B LNPs enabled sgLoxP-mediated genome editing in Ai14 / Cas9-bred mice. As shown in Figure 10A, delivery of sgLoxP with the optimized 306-O12B LNP system resulted in red fluorescence that was specifically detected in the liver. Interestingly, further histological analysis, shown in Figure 10B, revealed that tdTomato signals were observed primarily in liver hepatocytes, suggesting that 306-O12B LNPs can also specifically deliver sgRNA to this therapeutically relevant cell type.

[0324] Next, Cas9 mRNA and sgLoxP were co-formulated into one single LNP and injected into Ai14 mice via the tail vein at a total RNA dose of 1.65 mg / kg (Figure 11A). Seven days after delivery, organs were harvested and imaged ex vivo using the IVIS system. Imaging of mouse organs showed that the system was indeed able to induce red fluorescence, indicating successful functional co-delivery of both mRNA and sgRNA components, and tdTomato signals were detected primarily in the liver (Figure 11B). Immunofluorescence staining of hepatocyte-specific antigens was also performed, and confocal images demonstrated that the majority of tdTomato protein was expressed in hepatocytes (Figure 11C). These findings indicate that 306-O12B LNPs can specifically transport Cas9 mRNA and gRNA to hepatocytes.

[0325] Example 4: In vivo genome editing of Angptl3 Encouraged by the positive results achieved above, we tested the ability of 306^O12B LNPs to deliver Cas9 mRNA to manipulate the expression of a functional endogenous gene, Angptl3. Angptl3 encodes angiopoietin-like 3 (ANGPTL3), a central regulator of lipoprotein metabolism that inhibits both lipoprotein lipase and endothelial lipase activity. We explored the ratio of Cas9 and guide RNA that may affect in vivo genome editing efficacy by injecting C57BL / 6 mice with 306-O12B LNPs co-formulated with different Cas9 mRNA and sgAngptl3 mass ratios of 2:1, 1:1.2, and 1:2 at a total RNA dose of 3.0 mg / kg. Nanoparticles were formulated at a 306-O12B:total RNA weight ratio of 7.5:1 using the optimized formulation parameters determined above, i.e., helper molecules at a molar ratio of 50:37.5:10:1.5 (306-O12B:cholesterol:DOPC:PEG). Seven days after injection, serum was collected for ELISA analysis of serum ANGPTL3 protein levels, and liver tissue samples were collected for DNA extraction and next-generation sequencing (NGS) to determine targeted Cas9-mediated genome editing. Genome editing in liver tissue is shown in Figure 4A, showing a reduction in serum ANGPTL3 protein levels at all Cas9 mRNA / sgAngptl3 ratios (Figure 4B), with no notable differences observed between the groups. A Cas9 mRNA / sgAngptl3 ratio of 1:1.2 was used for the following experiments. Cas9 mRNA / sgAngptl3 encapsulated 306-O12B LNPs had similar characteristics to fLuc mRNA LNPs with an average size of 110 nm (Figure 12).

[0326] Given the significant level of editing, more detailed in vivo editing experiments were performed. As a gold standard, 306-O12B LNP was compared with the FDA-approved liver-targeting lipid MC-3 (referred to as MC3 LNP), which encapsulates the same RNA component. Mice were administered 306-O12B LNP or MC-3 LNP at a total RNA dose of 3.0 mg / kg. Seven days after administration, editing was observed at the desired site in the liver using the T7E1 assay (Figure 13). NGS of Angptl3 target sites in liver samples further demonstrated that 306-O12B LNP-mediated delivery resulted in a median editing rate of 38.5%, which is significantly higher than the median editing rate of MC-3 LNP-mediated delivery (14.6%) (Figure 5A). More importantly, serum analysis revealed that serum ANGPTL3 protein, LDL-C, and TG levels in the 306-O12B LNP-treated group (65.2%, 56.8%, and 29.4% reductions, respectively) were significantly lower than those in MC-3 LNP-treated mice (25%, 15.7%, and 16.3% reductions, respectively) (Figure 5A). Detailed analysis of the NGS sequencing results revealed that the most frequent editing events were precise 1-nt deletions at the predicted Cas9 cleavage sites, followed by 1-nt insertions at the same positions (Figure 5B). As expected, the same editing events were observed in both MC3 LNP- and 306-O12B LNP-treated livers, with the main difference being the frequency of these events. This indicates that the observed reduction in serum components is likely a result of Cas9-mediated genome editing and implies that the observed differences in outcomes between the two lipids are simply a result of delivery efficiency rather than changes in the innate activity of Cas9 mRNA.

[0327] With any CRISPR delivery system, care must be taken to avoid off-target editing events or delivery-induced toxicity. The top nine most likely off-target genomic mutagenesis sites were computationally predicted and these loci were investigated via NGS of DNA extracted from liver. No evidence of editing was observed at any of the top nine predicted off-target mutagenesis sites (Figure 5C). To assess in vivo toxicity and potential immune inflammatory responses, serum levels of liver function markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as well as the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) were measured 48 hours post-administration. No significant changes in these markers were detected after treatment with Cas9 LNPs (Figure 14). In addition, serum cytokine levels, i.e., IFN-α, IL-6, and IP-10, were observed to be upregulated 6 hours post-administration. However, by 48 hours post-dose, levels of all cytokines returned to baseline, indicating that delivery did not induce a long-term systemic inflammatory response (Figure 15).

[0328] One of the major advantages of CRISPR-Cas9 over other therapies is its potential to produce long-term therapeutic efficacy with only a single injection. To assess the long-term tolerability of editing and knockdown in the liver, 306-O12B LNPs were injected into C57BL / 6 mice at total RNA doses of 1.0, 2.0, or 3.0 mg / kg. Importantly, it was found that the therapeutic effect was stable for at least 100 days after a single dose. Dose-dependent genome editing of Angptl3 in the liver and knockdown of serum ANGPTL3 were still observed 100 days after a single dose, with a significant reduction in serum ANGPTL3, LDL-C, and TG levels at the highest dose (60%, 48.1%, and 28.6% reduction, respectively) (Figure 6A). This indicates both the potential ability to titrate the knockdown effect by varying the total RNA dose, and the ability of a single dose to produce therapeutically relevant knockdown effects over longer timescales. No evidence of long-term systemic toxicity was observed (Figure 6B). Furthermore, continued observations showed that genome editing could still be detected at least 150 days after a single initial dose of 306-O12B LNP (Figure 16). Although further studies are needed to establish the maximum duration of genome editing effects after a single dose, as well as to ensure that serum lipid profiles continue to track with genome editing profiles over a longer period of time, this is a promising indicator of the long-term effectiveness of the delivery platform.

[0329] Taken together, these results demonstrated that 306-O12B LNPs were more efficient than the gold standard MC-3 LNPs in inducing loss-of-function mutations in Angptl3 by CRISPR-Cas9-based genome editing. The non-viral LNPs mediated in vivo genome editing of Angptl3 in the liver, resulting in therapeutically relevant reductions in blood lipid levels with minimal off-target editing events, systemic toxicity, or inflammation in mice.

[0330] Consideration Disclosed herein are highly potent lipidoid nanoparticles composed of synthetic bioreducible lipidoid 306-O12B capable of liver-specific delivery of Cas9 mRNA and gRNA for in vivo Angptl3 editing. In recent years, the CRISPR-Cas9 system has emerged as a powerful therapeutic platform to correct genetic disorders for disease treatment. However, the application of this technology has been significantly hindered for many reasons related to the lack of a safe and efficient delivery vehicle. First, the large size of the Cas9-gRNA complex makes it difficult to encode the entire complex within a single viral genome or to deliver the 160 kDa protein directly into cells. Second, the host immune response against the CRISPR mechanism or the delivery vector itself, as well as pre-existing anti-Cas9 or anti-AAV antibodies in humans, can dramatically reduce CRISPR efficacy and potential immunotoxicity. Finally, potential undesired host genome insertional mutagenesis of CRISPR constructs, or off-target genome editing events caused by insufficient specificity of the CRISPR mechanism, can induce lethal genotoxicity. Some of these issues can be ameliorated by delivering Cas9 as an mRNA complexed with a gRNA. For example, unlike plasmid DNA or viruses, the mRNA does not need to be transported into the cell nucleus, and translation of the mRNA into protein occurs in the cytoplasm, thus simplifying the delivery route and avoiding the potential for insertional mutagenesis of the CRISPR machinery. Furthermore, delivery of mRNA compounds results in transient expression of the CRISPR components, unlike viral delivery, which can result in prolonged expression. It has been suggested that transient expression may reduce off-target genome editing simply by decreasing the opportunity for such deleterious editing events to occur.

[0331] To facilitate clinical translation of mRNA-mediated CRISPR technology, a delivery system with excellent safety, high specificity and efficacy is required. Non-viral nanoparticles prepared from synthetic biomaterials are expected to improve the cytoplasmic delivery of nucleic acids in vivo. Lipid nanoparticles (LNPs) are one of the most developed formulations for RNA delivery. In August 2018, the US Food and Drug Administration (FDA) approved the first LNP-based siRNA formulation. Encouraged by the success of LNPs, there have been significant efforts to use LNPs for in vivo mRNA transfection.

[0332] However, the in vitro potency of an LNP formulation rarely reflects its in vivo performance, and therefore, a direct in vivo screening of a library of tail-branched bioreducible lipidoids was performed. The amine head and hydrophobic chain length in the lipidoid were found to play important roles in determining in vivo mRNA delivery efficacy. Lipid nanoparticle formulations are typically composed of an ionizable active lipid, a fluorescent helper lipid, cholesterol, and lipid-anchored polyethylene glycol (PEG). The relative ratios of these components can have a significant effect on in vivo mRNA delivery efficacy. The published formulation conditions of MC-3 LNP were used as the starting formulation. Although the structure of the lipidoid used is different from MC-3, the published LNP uses excipient lipids similar to those used in MC-3 LNP. The excipient lipid components may play similar roles in both types of LNPs, and therefore, using similar excipient ratios would provide a valid starting point. To further maximize protein expression in vivo, these formulation parameters were optimized for the LNPs used herein. The optimal formulation for LNPs contained 50% 306-O12B, 38.5% cholesterol, 10% DOPC, and 1.5% DMG-PEG (molar ratio) with a 7.5 / 1 weight ratio of 306-O12B / mRNA.

[0333] Previous reports have shown that inhibition of ANGPTL3 activity by ANGPTL3 antibodies or ASOs results in therapeutically beneficial reductions in triglyceride and LDL cholesterol levels in humans. Furthermore, complete loss of function of Angptl3, which naturally occurs in certain families, does not appear to cause adverse health effects in humans. Angptl3 is primarily expressed in the liver, an ideal target organ for in vivo genome editing due to the accessibility of that tissue by intravenous injection. Taken together, these characteristics make Angptl3 an attractive therapeutic target for genome editing. Genome editing enabled by delivery of Cas9 mRNA and gRNA by 306-O12B LNP occurs primarily in hepatocytes, as determined by the biodistribution of fluorescent signals in the Ai14 model mouse strain. Furthermore, serum triglyceride and LDL-C levels are effectively lowered as a result of hepatocyte-specific disruption of Angptl3.

[0334] 306-O12B LNPs produced more efficient genome editing than MC-3 LNPs at the same dose of mRNA. Notably, a recent preclinical study of ASO-mediated silencing of Angptl3 in a similar mouse model achieved a 50% reduction in serum ANGPTL3 protein levels, a 7% reduction in LDL-C, and a 35% reduction in TG levels. A single dose of CRISPR-mediated knockdown using 306-O12B LNPs matched or exceeded these knockdown benchmarks (65.2%, 56.8%, and 29.4% reduction, respectively), whereas MC3 LNPs did not (25%, 15.7%, and 16.3% reduction, respectively). That particular ASO mouse study was followed up in a Phase I human clinical trial, suggesting that the knockdown levels achieved were likely therapeutically relevant. However, direct comparison of mouse studies to human clinical trials for ANGPTL3 antibodies or ASO therapies is difficult due to differences in species and dosing regimens. Clinical trials of both ASO and antibody therapy have demonstrated the ability to titer the percentage of knockdown of these factors based on dosage and frequency, notably finding the strongest effects for multiple repeated doses of drug per patient at the highest experimental dose level. A dose-dependent reduction in serum ANGPTL3 levels was observed upon a single injection of 306-O12B LNPs. This indicates that in a clinical setting, the LNP system can achieve similar dose- and frequency-dependent knockdown, and that the correct dosing scheme can achieve knockdown of similar magnitude as reported in these clinical trials. The knockdown effect of a single dose, detectable at the genomic and protein levels, as well as in downstream therapeutic metrics, was stable for at least 100 days. Human clinical trials showed maximum knockdown effects within one week of administering the drug, with serum levels gradually returning to baseline over the following months. While this outcome is expected, this difference is particularly striking and could have tremendous therapeutic consequences, as CRISPR produces permanent changes to the genome of target cells while ASOs and antibodies are transient treatments.

[0335] Delivery of CRISPR mRNA in vivo can result in genome knockdown that is fully sustained for at least one year after administration. Although thorough analysis of serum lipid levels was only performed up to 100 days after administration, sequencing data showed durable genome editing up to at least 150 days. The disclosed data showed a very slight decrease in genome editing efficiency over time, which may reflect a slow turnover of hepatocytes in the liver with a predicted life span of about 300 days. The genome editing efficiency of the 3 mg / kg dose (31%) detected 150 days after injection is still higher than the lower editing efficiency of the 2 mg / kg dose (22%) detected only 100 days after injection. The genome editing rate of 22% was sufficient to generate a statistically significant reduction in ANGPTL3 protein, as well as serum cholesterol and LDL-C. Thus, it is very likely that a full therapeutic index will show significant knockout up to 5 months or more. This point requires further investigation.

[0336] In addition to genome editing efficacy, off-target effects are a major concern for clinical translation of CRISPR-Cas9 technology. Off-target effects can be discussed in two contexts: off-target delivery effects (i.e., liver drug delivery to the kidney) and biological off-target editing (i.e., editing events at unintended genomic loci). In this study, we used luciferase and tdTomato model systems to demonstrate specific delivery to the liver, specifically to hepatocytes, greatly minimizing off-target delivery effects. To address the issue of biological off-target editing, we designed a highly specific sgRNA target against Angptl3 by mitigating off-target editing events using publicly available computational-based design tools. Targeted NGS data confirmed that no editing was detected in the liver at the top nine off-target sites predicted by Cas-Off-Finder. Although further studies may be necessary to further validate the safety of the disclosed platform for general liver delivery applications using techniques such as whole genome sequencing and direct comparison with viral-mediated CRISPR delivery or other guide sequences, the data presented here suggests that under the conditions tested, 306-O12B LNP was found to be highly specific with respect to both off-target delivery and off-target editing. From a systemic perspective, the LNP system did not cause significant changes in ALT or AST levels, which are markers of liver health, and the disclosed data indicate that LNP is not detectably toxic to the liver. Similarly, the lack of significant changes in TNF-alpha indicates that LNP delivery does not induce a significant immune response. Together, this indicates that the disclosed treatment is safe in mice.

[0337] The results of this study may facilitate systemic delivery of CRISPR genome editing machinery in the clinic. More detailed preclinical studies of chronic tolerability, off-target effects, and efficacy in large animals should be performed in the future to realize eventual clinical application. Overall, 306-O12B LNP is a highly potent and safe non-viral delivery platform for clinically relevant liver-specific CRISPR-Cas9-based genome editing.

[0338] Example 5: Synthesis of lipidoids and in vivo screening of mRNA-loaded LNPs A set of bioreducible lipidoids was generated based on a specific amine head 3,3'-diamino-N-methyldipropylamine, designated 306 amine head, which has been previously demonstrated as a promising amine candidate for in vivo siRNA, ASO, and mRNA delivery. These lipidoids were made by reacting 306 amine head with three ester-linked alkyl-acrylate tails (O series) and three alkyl-acrylate tails (N series) containing amide bonds using a solvent-free Michael addition reaction (Figure 21B). LNPs were then formulated in the pre-optimized formulation conditions of 306-O12B LNPs for mRNA delivery, containing 50% synthetic active lipidoid, 38.5% cholesterol, 10% DOPC, and 1.5% DMG-PEG2000 in molar ratios. To investigate the in vivo systemic mRNA delivery profile of these LNPs, we encapsulated firefly luciferase mRNA (fLuc mRNA) encoding a reporter protein, firefly luciferase, which can be easily visualized in vivo by using an IVIS imaging system (Perkin Elmer). Bioluminescence signals in O-series LNPs (306-O12B, 306-O14B, and 306-O16B) treated mice were mainly located in the liver (Figures 21A, 21C, and 25A). Surprisingly, fLuc mRNA was delivered almost exclusively to the lungs by N-series LNPs (306-N12B, 306-N14B, and 306-N16B) (Figures 21A, 21C, and 25B). Quantification of luciferase protein in different tissue homogenates further demonstrated that the translation of luciferase-encoding mRNA with 306-O12B LNPs and 306-N16B LNPs was mainly located in the liver and lung, respectively (Figure 25A and Figure 25B). In addition to the different organ targeting profiles, tail length showed opposite effects on the mRNA delivery efficacy of these two series LNPs. For O series LNPs, 306-O12B showed the highest efficacy, while for N series LNPs, the efficacy trend was observed as 306-N16B>306-N14B>306-N12B.

[0339] Example 6: Second-generation library of N-series LNPs confirmed tail structure-dependent lung selectivity. Considering the fact that the amine head structure can affect the physicochemical properties of lipidoids as well as the resulting LNPs, it subsequently affects the in vivo fate of LNPs. Liver-targeted mRNA delivery permitted by O-series LNPs containing different amine and alkyl tail structures has been extensively explored and demonstrated. Thus, we confirmed the tail structure-dependent lung-targeted delivery of mRNA enabled by N-series LNPs using amines other than 306. Thus, a secondary library of N-series lipidoids was synthesized by reacting the N16 tail with 80, 113, 304, 400, and 401 amines (Figure 22A). fLuc mRNA-encapsulated LNPs with these lipidoids were prepared, and the in vivo organ selectivity of the resulting LNPs was investigated. As shown in Figure 22B, luciferase bioluminescence signals were observed primarily in the lung for all these N-series LNPs, confirming that the lung selectivity of these LNPs is determined by the lipidoid linker tail structure, i.e., the amide bond.

[0340] Example 7: 306-N16B LNPs enable delivery of mRNA to pulmonary endothelial cells. Clinical translation of mRNA-based therapeutics requires specific mRNA delivery to the organs and cell types of interest. Considering the ability of N-series LNPs to specifically deliver mRNA to the lungs, the major organs of the human respiratory system. The lungs can be affected by many respiratory diseases caused by dysfunction of endothelial, epithelial and immune cells. Therefore, it is important to identify which lung cell subpopulations were specifically transfected for further evaluation of the potential application of the disclosed lung-targeted LNPs in the treatment of lung diseases. We utilized the genetically engineered Cre / LoxP Ai9 reporter mouse strain, which is widely used to achieve cell type-specific expression of tdTomato protein. Ai9 mice were injected with 306-N16B LNPs carrying Cre mRNA encoding the Cre recombinase that cleaves the stop cassette to activate the expression of tdTomato fluorescent signal in edited cells (Figure 23A). As shown in the ex vivo images of organs harvested from treated mice, the red tdTomato signal was specifically detected in the lungs (Figure 23B). Tissue sections were immunostained with fluorochrome-labeled antibodies against endothelial, epithelial, and macrophage cells to gain initial insight into which specific sub-cell populations were edited in the lung. Interestingly, the red tdTomato signal was mainly localized to pulmonary endothelial cells, whereas little colocalization with epithelial or macrophage cells was observed (Figure 23C). To further quantify the specific cell types transfected in the lung, lung tissue was advanced to single cell suspension and analyzed using flow cytometry. Approximately 33.6% of pulmonary endothelial cells were transfected, whereas only a small fraction of epithelial (~1.66%) and macrophage cells (~1.92%) were transfected (Figure 23D). These findings suggest that 306-N16 LNPs can selectively deliver mRNA to pulmonary endothelial cells, which is important for further development of potential therapeutic applications for lung diseases associated with endothelial cells.

[0341] Example 8: 306-N16B LNPs enable lung-specific delivery of genome editing Cas9 mRNA. As mentioned above, 306-N16B LNPs can specifically deliver fLuc mRNA and Cre mRNA to lungs, and the length of these two mRNAs is less than 2000 nucleotides (1960 nucleotides for fLuc mRNA and 1350 nucleotides for Cre mRNA). Some specific diseases may require the delivery of even larger mRNA molecules. We tested whether 306-N16B LNPs have the potential to deliver longer mRNAs to lungs as well. 306-N16B LNPs were formulated with Cas9 mRNA, a genome editing mRNA with 4521 nucleotides, which is more than three times that of fLuc mRNA, and then LNPs were injected into Babl / c mice. After 6 hours, the mice were euthanized and organs were collected for further Western blot analysis to detect Cas9 protein expression in different organs. Notably, Cas9 protein can only be detected in the lungs (Figure 26), indicating that 306-N16B LNPs mediate the specific delivery of Cas9 mRNA to lungs. The 306-N16B LNP has great potential for application in the treatment of inherited lung diseases via specific genome editing.

[0342] Example 9: Characterization of proteins composing the protein corona in 306-O12B LNPs and 306-N16B LNPs The outer surface of nanoparticles (NPs) is believed to be immediately masked with a layer of biomolecular corona immediately after intravenous administration, which can dramatically change the surface properties of NPs and determine their in vivo fate. The controlled adsorption of specific plasma proteins on the NP surface would serve as targeting molecules that can selectively direct NPs to specific organs. The dramatically different in vivo organ targeting of 306-O12B and 306-N16B LNPs could be attributed to serum proteins formed on their surfaces. To identify and quantify proteins on 306-O12B and 306-N16B LNPs, these two LNPs were incubated with mouse plasma for 1 h at 37 °C, and the protein-coated LNPs were isolated and collected using centrifugation, followed by extensive washing with PBS. Proteomics was then performed to analyze the composition of proteins on the LNPs. 1838 and 1088 proteins were identified and quantified on 306-O12B LNPs and 306-N16B LNPs, respectively. The Venn diagram showed the common and unique proteins between 306-O12B LNPs and 306-N16B LNPs (Figure 27). For each LNP, the top 20 most abundant corona proteins that may play a dominant role in the protein corona are listed in Table 1 above. 306-O12B LNPs and 306-N16B LNPs shared only six common proteins among the top 20 proteins, demonstrating that proteins absorbed on the LNP surface play an important role in determining the targeting of LNPs. Previous studies reported apolipoprotein E (ApoE)-mediated delivery of LNPs to the liver. ApoE was identified as the second dominant protein composition in the corona of liver-targeted 306-O12B LNPs (Figure 24A), which was consistent with literature reports. Conversely, the top three protein compositions in the corona of lung-targeted 306-N16B LNPs are serum albumin, fibrinogen beta chain, and fibrinogen gamma chain (Figure 24B). It has been reported that fibrinogen coating can improve endothelial cell adhesion as well as endothelialization.However, whether 306-N16B LNP-mediated delivery of mRNA to the pulmonary endothelium was via a fibrinogen-dependent mechanism needs to be further demonstrated.

[0343] The top 20 proteins are classified according to their molecular weight (Mw) (Figure 24C). Minor differences between these two LNPs were observed, with the protein corona of 306-O12B LNPs being enriched in low molecular weight proteins, with 80% of the proteins having Mw<60 kDa. However, 55% of the proteins in the protein corona of 306-N16B LNPs are greater than 60 kDa. Proteins were classified based on their isoelectric point (pI). As shown in Figure 24D, 50% and 80% of the proteins exhibiting a negative charge (pI<7) at physiological pH 7.4 constitute the protein corona of 306-O12B LNPs and 306-N16B LNPs, respectively. It was suggested that positively charged NPs prefer to attract negatively charged proteins (MPMonopoli et al., Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc. 133, 2525-2534 (2011)). However, no significant difference in the zeta potential of the two LNPs was observed, and both LNPs showed slightly negative surface charges (Table 2), indicating that surface charge may not be the only factor affecting the interaction between nanoparticles and proteins in biological fluids. The proteins regarding their biological functions were further screened as shown in Figure 24E. From the functional point of view, the proteins abundant in 306-O12B LNPs and 306-N16B LNPs performed very similar functions, and they were all found to be related to the functions of lipid metabolism, complement activation, immune response, acute phase response, and coagulation. However, it is noted that apart from other plasma proteins, the highest enriched proteins in the corona of 306-O12B LNPs are apolipoproteins involved in lipid and cholesterol metabolism (Figure 24E). Coagulation-related corona proteins represent the largest proportion in the corona of 306-N16B LNPs (Figure 24E). [Table 2]

[0344] Taken together, these findings indicated that the protein coronas formed on these two 306-O12B and 306-N16B LNPs differed in protein composition, fractionation, and biological functions, which play a dominant role in determining the in vivo fate of these two LNPs.

[0345] Consideration In recent years, there has been growing interest in using LNPs to deliver messenger RNA (mRNA) therapeutics. In vitro synthesized mRNA has the potential to produce therapeutically relevant proteins "in vivo" to control and treat a wide range of diseases, including AIDS, Zika, rare diseases, cancer, and coronaviruses. To exploit this transformative technology to benefit human health, mRNA needs to be delivered to diseased organs and cells to enable specific expression of the therapeutic protein and subsequently produce the desired therapeutic effect. Lipid nanoparticles (LNPs) represent one of the major non-viral delivery systems for nucleic acid delivery, and considerable efforts have been made to use LNPs for mRNA delivery. However, the majority of developed LNPs target the liver after systemic administration, and new strategies are required to direct LNPs to other organs, such as the lung, kidney, heart, and brain. Some LNPs containing specific amine heads, e.g., imidazole, adamantyl, and neurotransmitter, are known to deliver mRNA, siRNA, and ASO to the spleen and brain. These LNPs were identified through labor-intensive screening approaches, and currently it remains impossible to generate common rules to guide the rational design of LNPs with predictable organ selectivity. More recently, the Siegwart laboratory discovered and developed the selective organ targeting (SORT) strategy to engineer LNPs to target extrahepatic tissues, such as the lung and spleen, by adding additional excipients. Although this advance is exciting, it split the original four-component system into five components, making the formulation somewhat complicated.

[0346] It is disclosed herein that the in vivo organ selectivity of LNPs can be precisely tuned by simply modifying the linker structure of the lipidoid tail without complicating the formulation of LNPs. By changing the linker from an ester bond (referred to as O-series) to an amide bond (referred to as N-series), the mRNA delivery specificity enabled by LNPs was switched from the liver to the lung. This finding was first observed from 306-amine head-based O-series and N-series LNPs. By examining the mRNA delivery targeting of a secondary library of N-series LNPs, it was confirmed that the lung targeting ability enabled by the linkage of amide bonds is generally applicable to other classes of amine heads. Targeted RNA delivery to the lung is an attractive concept, which offers a tremendous opportunity to target what are currently defined as "hard-to-drug" targets for the treatment of various lung diseases. In this study, it is demonstrated that 306-N16B LNPs can specifically transport mRNA to pulmonary endothelial cells, which are important therapeutic targets involved in acute respiratory distress syndrome, inflammation, and thrombosis.

[0347] A proteomic study was performed to try to explain why such small structural differences between O-series and N-series LNPs result in dramatically different organ selectivity. These two tail structures may have specific affinities for some different serum proteins that somehow regulate the protein composition of the corona that forms on the surface of LNPs during circulation. It was reported that the protein corona plays an important role in determining the in vivo fate of nanoparticles. The disclosed results revealed that the number and type of absorbed plasma proteins in the protein corona of 306-O12B and 306-N16 LNPs were different. Only six unique proteins were found among the top 20 most abundant proteins of these two LNPs. Even if the same set of proteins is found in both protein coronas, they may have completely different biological functions due to their random orientation in the protein corona layer. One example is ApoE; on the one hand, it has been widely reported that endogenous ApoE can be absorbed onto the LNP surface after iv injection to facilitate the transport of encapsulated RNA to hepatocytes by lipoprotein receptor-mediated endocytosis, while on the other hand, ApoE can be deployed to target nanoparticles to the brain.

[0348] In summary, this study demonstrated a strong correlation of structure-activity relationships between lipidoid tail chemistry and organ selectivity for mRNA delivery. Mechanistic studies using proteomics showed that the protein composition in the protein corona of these two LNPs differed, suggesting that the unique tail structures may affect the interactions of LNPs with biological fluids that may affect the targeting of LNPs. However, further studies should be performed to fully elucidate the effects of these unique proteins on organ targeting. Findings from this study provide a fundamental understanding of how lipid structure affects the in vivo fate of LNPs by manipulating protein corona function.

[0349] Example 10: Synthesis of N-series lipidoids and screening of mRNA-loaded LNPs in vivo Nine bioreducible N-series lipidoids were generated by reacting acrylamide tails (N16B, N14B, and N12B) with different commercially available amine heads using a solvent-free Michael addition reaction (Figure 28A). LNPs were then formulated with pre-optimized formulation conditions of LNPs for mRNA delivery, including 50% synthetic active lipidoid, 38.5% cholesterol, 10% DOPC, and 1.5% DMG-PEG2000 in molar ratios. To investigate the in vivo systemic mRNA delivery profile of these LNPs, firefly luciferase mRNA (fLuc mRNA) was encapsulated, which encodes firefly luciferase, a reporter protein that can be easily visualized in vivo by using an IVIS imaging system (Perkin Elmer). Interestingly, the bioluminescence signal in N-series LNP-treated mice was mainly located in the lungs (Figure 28B). In addition, it was found that the chemical structure of lipidoid plays an important role in determining the in vivo mRNA delivery efficacy of formulated LNPs. As shown in Figure 28C, the quantified luciferase bioluminescence intensity in the lungs 6 hours after injection shows that LNPs incorporating 306 amine heads showed the highest delivery efficacy compared to LNPs with 304 and 113. Delivery efficacy also increased with increasing lipidoid tail length, and lipidoids with N16B tails showed higher potency than lipidoids with N14B and N12B tails.

[0350] Example 11: Targeting distinct lung sub-cell populations by altering the lipidoid head structure of N-series LNPs Clinical translation of mRNA-based therapeutics requires specific mRNA delivery to the organ and cell type of interest. The lung can be affected by many diseases caused by dysfunction of multiple cellular compartments, including endothelial, epithelial and immune cells. To identify the specifically transfected lung cell subpopulations, we achieved cell type-specific expression of tdTomato protein using a genetically engineered Cre / LoxP Ai14 reporter mouse line. Ai14 mice were injected with the two most potent lung-targeted LNPs (306-N16B and 113-N16B). In these experiments, Cre mRNA encoding Cre recombinase was used as the mRNA cargo. Once delivered to the cells, Cre recombinase removes the stop cassette and activates the expression of tdTomato fluorescent signal in edited cells (Figure 29A). As shown in ex vivo images of organs harvested from treated mice (Figures 29B and 29C), red tdTomato signal was specifically detected in the lungs of both 306 and 113-N16B LNP-treated mice, and tdTomato-positive cells were observed using confocal imaging of lung sections. To further identify and quantify the specific cell types transfected in the lungs, lung tissue was processed into single cell suspensions and analyzed using flow cytometry. For 306-N16B LNPs, 33.6% of the lung endothelium was transfected, compared to 1.5% of the epithelium and 1.9% of the macrophages, indicating that 306-N16 LNPs can selectively deliver mRNA to lung endothelial cells. In contrast, 113-N16B LNPs preferentially delivered Cre mRNA to endothelial cells (69.6% of total endothelial cells were transfected), but also to macrophages (18.9%) and epithelia (7.3%) (Figure 29C). These findings set the stage for potential therapeutic applications of lung diseases associated with specific lung cells and demonstrate that different lung cell populations can be targeted simply by tailoring the head structure of N-series LNPs.

[0351] Example 12: Characterization of proteins composing the protein corona in 306-O12B LNPs and 306-N16B LNPs The outer surface of nanoparticles (NPs) is thought to be immediately masked with a layer of biomolecular corona immediately after intravenous administration, which can dramatically change the surface properties of NPs and determine their in vivo fate. Controlled adsorption of specific plasma proteins on the NP surface can selectively target NPs to specific organs. It was previously reported that O-series LNPs can specifically deliver mRNA to the liver (M. Qiu et al., Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc. Natl. Acad. Sci. USA118, e2020401118 (2021)). The dramatic difference in in vivo organ targeting of O-series and N-series LNPs (Figure 30A) may be due to serum proteins formed on their surfaces (Figure 30B). To test this, proteins on two representative LNPs, 306-O12B and 306-N16B, were identified and quantified. These two LNPs were incubated with mouse plasma for 1 h at 37°C, and the protein-coated LNPs were isolated and collected using centrifugation, followed by extensive washing with PBS. Proteomics was then performed to analyze the composition of proteins on the LNPs. 1838 and 1088 proteins were identified and quantified on 306-O12B LNPs and 306-N16B LNPs, respectively.

[0352] For each LNP, the top 20 most abundant corona proteins that may play a dominant role in the protein corona are listed in Table 3. 306-O12B LNPs and 306-N16B LNPs share only six common proteins among the top 20, supporting that proteins absorbed to the LNP surface may be involved in in vivo targeting. ApoE was identified as the second dominant protein composition in the corona of liver-targeted 306-O12B LNPs (Figure 24A), consistent with previous studies demonstrating apolipoprotein E (ApoE)-mediated delivery of LNPs to the liver. In contrast, the top three proteins in the corona of lung-targeted 306-N16B LNPs are serum albumin, fibrinogen beta chain, and fibrinogen gamma chain (Figure 24B). It has been reported that fibrinogen coating can improve endothelial cell adhesion and endothelialization. [Table 3]

[0353] The top 20 proteins were classified according to their molecular weight (Mw) (Figure 24C). The protein corona of 306-O12B LNPs was enriched in low molecular weight proteins with 80% of proteins having Mw<60 kDa, whereas 55% of proteins in the protein corona of 306-N16B LNPs are greater than 60 kDa. Proteins were also classified based on their isoelectric point (pI). As shown in Figure 24D, 50% and 80% of proteins exhibiting a negative charge (pI<7) at physiological pH 7.4 constitute the protein corona of 306-O12B LNPs and 306-N16B LNPs, respectively.

[0354] Finally, proteins were sorted according to their biological functions (Figure 24E). Proteins abundant in 306-O12B LNPs and 306-N16B LNPs are associated with lipid metabolism, complement activation, immune response, acute phase response, and coagulation. However, it is noted that apart from other plasma proteins, the highest enriched proteins in the corona of 306-O12B LNPs are apolipoproteins involved in lipid and cholesterol metabolism (Figure 24E), whereas coagulation-related corona proteins make up the largest proportion in the corona of 306-N16B LNPs (Figure 24E). Taken together, these findings indicated that the protein coronas formed on 306-O12B LNPs and 306-N16B LNPs differ in protein composition, fractions, and biological functions that play important roles in determining their tissue targeting in vivo.

[0355] Example 13: 306-N16B LNPs enable lung-specific delivery of genome editing Cas9 mRNA. As shown herein, 306-N16B LNP can specifically deliver fLuc mRNA and Cre mRNA to lung. The length of these two mRNAs is less than 2000 nucleotides (1960 nucleotides for fLuc RNA and 1350 nucleotides for Cre mRNA). Some specific diseases may require the delivery of larger mRNA molecules. We tested whether 306-N16B LNP has the potential to deliver longer mRNA to lung as well. 306-N16B LNP was formulated with Cas9 mRNA, a genome editing mRNA with 4521 nucleotides, which is more than three times that of fLuc mRNA, and then LNP was injected into Babl / c mice. After 6 hours, the mice were euthanized and the organs were collected for further Western blot analysis to detect Cas9 protein expression in different organs. Notably, Cas9 protein could be detected only in the lung (Figure 35), indicating that 306-N16B LNPs mediate specific delivery of Cas9 mRNA to the lung.

[0356] After confirming that 306-N16B LNPs can specifically deliver Cas9 mRNA to the lung, we tested whether Cas9 mRNA and single guide RNA in 306-N16B LNPs can be co-delivered to the lung to enable lung-specific CRISPR-Cas9 genome editing. To test this, Ai14 mice containing Cas9 mRNA and LoxP-targeting sgRNA (sgLoxP) were loaded with 306-N16B LNPs at a dose of 1.67 mg / kg total RNA (Cas9 mRNA / sgLoxP=1 / 1.2, wt / wt) and analyzed gene editing specificity 7 days after injection (Figure 31A). As shown in Figure 31B, the fluorescent signal was mainly seen in the lung, and a weak fluorescent signal was also observed in the liver. tdTomato-positive cells were seen from the microscopic images of tissue sections (Figure 31C). Results show that 306-N16B LNPs can simultaneously deliver Cas9 mRNA and sgRNA to both lung and liver in mice to enable genome editing applications. However, 306-N16B LNPs were found to show less tissue selectivity in simultaneously delivering Cas9 mRNA and sgRNA compared to single mRNA delivery, which can almost exclusively deliver mRNA to the lung. This indicates that the incorporation of shorter sgRNAs can change the physical properties of the formed LNPs and alter the organ tropism of the LNPs. The inclusion of additional components in the LNP formulation can alter delivery efficiency and tropism.

[0357] Example 14: LNPs for lung-targeted TSC2 mRNA delivery for the treatment of LAM in vivo Pulmonary lymphangioleiomyomatosis (LAM) is a rare inherited lung disease characterized by abnormal mTORC1 hyperactivity caused by inactivating mutations in the tuberous sclerosis 1 or 2 (TSC1 or TSC2) genes. Although the mTORC1 inhibitor rapamycin (sirolimus) has been approved by the FDA for the treatment of LAM, continued loss of lung function and the need for lung transplantation can still occur. Thus, there is an urgency to develop new therapies for the treatment of LAM. Therefore, we evaluated whether lung-targeted LNPs can deliver mRNA encoding the TSC2 gene for the treatment of LAM.

[0358] The feasibility of LNPs was tested to deliver EGFP mRNA to mouse TSC2-null TTJ cells, which is used in an established model of LAM. In vitro, lung-targeted 306-N16B LNPs showed low delivery efficacy in transporting mRNA to TTJ cells, whereas liver-targeted 306-O12B LNPs were found to exhibit relatively high transfection efficiency (Figure 34A). Therefore, hybrid LNPs (referred to as hLNPs) were designed that combined 50% of 306-O12B and 50% of 306-N16B in a molar ratio (Figure 32A). Such hybrid LNPs can efficiently deliver cargo to TTJ cells and also achieve lung-targeted delivery in vivo. Interestingly, such hLNPs showed much higher transfection efficiency to TTJ cells compared to 306-O12B and 306-N16B LNPs, and uniform expression of EGFP was observed (Figure 34B).

[0359] To determine whether hLNPs can deliver mRNA to TSC2-deficient cells in vivo, TTJ cells were intravenously injected into syngeneic 6-week-old C57BL / 6J mice to form LAM-like nodules in the lungs as previously reported. hLNPs encapsulating EGFP mRNA were injected via the tail vein. Mice were euthanized and lungs were harvested 6 hours after injection. EGFP expression in the lungs was analyzed by immunohistochemistry (IHC). Notably, as shown in Figure 32B, EGFP expression was detected primarily in tumor nodules derived from the TTJ, but not in adjacent normal lung tissues. Immunostaining for phospho-S6 (a marker for TSC2-deficient cells) revealed strong colocalization of phospho-S6 and EGFP (Figure 32C), confirming that hLNPs enable the specific delivery of EGFP mRNA to TTJ cells.

[0360] We next evaluated the therapeutic efficacy of Tsc2 mRNA-loaded hLNPs in this preclinical model. Full-length (5445 nucleotides) mouse wild-type Tsc2 mRNA was transcribed in vitro. In vitro, this mRNA suppressed the expression of phospho-S6 in TSC2-null cells (Figures 26A-26B), indicating successful suppression of the mTORC1 pathway. Similarly, re-expression of wild-type Tsc2 mRNA in patient-derived TSC2-deficient cells significantly inhibited mTORC1 signaling (Figures 26C-26D). TTJ tumor-bearing mice were treated with Tsc2 mRNA-encapsulated hLNPs (0.75 mg / kg) or empty hLNPs for a total of five doses every other day, starting on day 24 after TTJ cell inoculation, with two mice in each group. Untreated mice were also included as controls (Figure 33A). Tsc2 mRNA-loaded hLNPs significantly suppressed tumor growth compared to empty LNPs or compared to no treatment (Figure 36). The overall size of tumor nodules in mice treated with Tsc2 mRNA LNPs was reduced by 60% or 44% compared to untreated or empty LNP-treated groups, respectively (Figures 33B and 33C). No significant difference was observed between the empty LNP-treated and untreated groups. Furthermore, tumor growth and apoptosis were assessed using IHC for Ki67 and cleaved caspase 3, respectively (Figures 33D and 33E). Cell proliferation within the tumor module was reduced by Tsc2 mRNA LNP treatment compared to that of empty LNPs. No significant difference in apoptosis was observed between the two groups, suggesting that tumor apoptosis is not the primary mechanism of tumor reduction in this model. Thus, restoration of wild-type TSC2 by mRNA delivery may induce an immune-mediated mechanism that reduces tumor burden in TTJ cells. To test this possibility, we assessed macrophage and T cell tumor infiltration by IHC for F4 / 80 and CD3. While no differences in macrophage infiltration were observed between groups, the Tsc2 mRNA-loaded hLNP-treated group showed enhanced tumor T cell infiltration (Figure 33E), demonstrating that restoration of TSC2 protein expression induces an immune-mediated therapeutic response in this preclinical model of LAM.

[0361] Consideration In recent years, there has been growing interest in using LNPs to deliver messenger RNA (mRNA) therapeutics. In vitro synthesized mRNA has the potential to produce therapeutically relevant proteins "in vivo" to control and treat a wide range of diseases, including AIDS, Zika, rare diseases, cancer, and coronaviruses. To exploit this transformative technology to benefit human health, mRNA needs to be delivered to diseased organs and cells to enable specific expression of the therapeutic protein and subsequently produce the desired therapeutic effect. Lipid nanoparticles (LNPs) represent one of the main non-viral delivery systems for nucleic acid delivery, and considerable efforts have been made to use LNPs for mRNA delivery. However, the majority of developed LNPs target the liver after systemic administration, and new strategies are required to direct LNPs to other organs, such as the lung, kidney, heart, and brain. These LNPs were identified through labor-intensive screening approaches, and currently, it remains impossible to generate common rules to guide the rational design of LNPs with predictable organ selectivity. More recently, the Siegwart laboratory discovered and developed the selective organ targeting (SORT) strategy to engineer LNPs to target extrahepatic tissues, such as the lung and spleen, by adding additional excipients. Although this advance is exciting, it split the original four-component system into five components, making the formulation somewhat more complicated.

[0362] It is disclosed herein that the in vivo organ selectivity of LNPs can be precisely tuned by simply modifying the linker structure of the lipidoid tail without complicating the formulation of LNPs. By changing the linker from an ester bond (referred to as O-series) to an amide bond (referred to as N-series), the mRNA delivery specificity enabled by LNPs was switched from liver to lung. A proteomics study was performed to try to explain why such a small structural difference between O-series and N-series LNPs results in dramatically different organ selectivity. These two tail structures may have specific affinities for some different serum proteins that somehow regulate the protein composition of the corona that forms on the surface of LNPs during circulation. It has been reported that the protein corona plays an important role in determining the in vivo fate of nanoparticles. The disclosed results reveal that the number and type of absorbed plasma proteins in the protein corona of 306-O12B and 306-N16 LNPs are different. 14 unique proteins were found among the top 20 most abundant proteins of these two LNPs. Even if the same set of proteins is found in both protein coronas, they may have different biological functions because they are randomly oriented in the protein corona layer. One example is ApoE, where it has been widely reported that, on the one hand, endogenous ApoE can be absorbed to the LNP surface after iv injection to facilitate the transport of encapsulated RNA to hepatocytes by lipoprotein receptor-mediated endocytosis, while, on the other hand, ApoE can be deployed to target nanoparticles to the brain. The binding models of 306-O12B and 306-N16B to albumin, the most abundant protein identified in the protein corona of these two LNPs, were then demonstrated to be different, which may affect the orientation and biological function of albumin on the surface of the LNPs.

[0363] Targeted RNA delivery to the lung is an attractive concept, which offers enormous opportunities to target what are currently defined as "hard-to-druggable" targets for the treatment of a wide range of lung diseases. Here, it is demonstrated that different lung cells can be easily targeted by tailoring the head structure of the LNPs. Herein, it is disclosed that 306-N16B LNPs can specifically transport mRNA to pulmonary endothelial cells, which are important therapeutic targets involved in acute respiratory distress syndrome, inflammation, and thrombosis. However, mRNA containing 113-N16B LNPs could transfect not only pulmonary endothelial cells but also macrophages and even epithelial cells after systemic administration. Furthermore, it is also demonstrated that co-delivery of Cas9 mRNA and sgRNA into one single 306-N16B LNPs could achieve genome editing in the lungs of mice, which may enable further development of genome editing-based therapies for the treatment of lung genetic diseases. More importantly, the disclosed developed hybrid LNPs are shown to be able to specifically deliver Tsc2 mRNA to TSC2-deficient TTJ tumor cells in a preclinical LAM model and significantly inhibit tumor cell proliferation, providing an important proof-of-concept for the use of LNPs for the treatment of lung diseases.

[0364] In summary, this study demonstrated a strong correlation of structure-activity relationships between lipidoid tail chemistry and organ and cell selectivity for mRNA delivery. Mechanistic studies using proteomics showed that the protein composition in the protein corona of these two LNPs differed, suggesting that the unique tail linker structures may profoundly affect the interaction of LNPs with biological fluids, which substantially impacts the targeting of LNPs. However, further studies should be performed to fully elucidate the effects of these unique proteins on organ targeting. Findings from this study provide a fundamental understanding of how lipid structure can affect the in vivo fate of LNPs by manipulating protein corona function.

[0365] Incorporation by Reference All U.S. and PCT patent publications and U.S. patents referred to herein are hereby incorporated by reference in their entirety as if each individual patent publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0366] Other embodiments Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the invention as defined in the following claims.

Claims

1. 1. A composition comprising a pharmaceutical agent assembled with a lipid composition, the lipid composition comprising a compound represented by structural formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, During the ceremony, R ａ is alkyl, n1 and n2 are each independently 1, 2, 3, or 4; R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ are each independently H, 【Chemistry 2】 and Where: * indicates the point of attachment to N; each m is independently 1, 2, 3, 4, or 5; each q is independently 1, 2, 3, 4, or 5; Each R ｃ is independently alkyl or alkenyl; However, R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ wherein at least one of is not H.

2. R ａ C １ ~C ３ The composition of claim 1 wherein the alkyl group is an alkyl group.

3. n1 is 2 or 3, and / or n2 is 2 or 3, and / or n1 and n2 are the same; The composition of claim 2.

4. R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ The composition of claim 1 , wherein at least two of are not H.

5. R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ However, each independently, 【Chemistry 3】 2. The composition of claim 1 ,

6. Each R ｃ But independently, C ４ ~C １６ Alkyl or C ４～１６ The composition of claim 1 , wherein the alkyl group is alkenyl.

7. Each R ｃ But independently, C ４ ~C ２０ The composition of claim 1 wherein the alkyl group is an alkyl group.

8. 2. The composition of claim 1, wherein the lipidoid of formula (I) or a pharma- ceutically acceptable salt thereof is present in the lipid composition in a molar percentage of about 60% or less.

9. The composition of claim 1 , wherein the lipid composition further comprises a steroid or steroid derivative, a polymer-conjugated lipid, and / or a phospholipid.

10. A steroid or steroid derivative is present in the lipid composition in a molar percentage of about 50% or less; and / or the polymer-conjugated lipid is present in said lipid composition in a molar percentage of about 10% or less; and / or The phospholipid is present in the lipid composition in a molar percentage of about 30% or less. The composition of claim 9.

11. 2. The composition of claim 1, wherein the lipidoid of formula (I) or a pharma- ceutically acceptable salt thereof is present in the composition in a mass or weight ratio of about 1:1 to about 200:1 relative to the pharmaceutical agent.

12. (a) the pharmaceutical comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof, that modulates a target gene or its gene product; or (b) the pharmaceutical comprises a polynucleotide configured to encode or regulate a target gene or its gene product; The composition of claim 1.

13. The pharmaceutical product is (a) a gene regulatory moiety configured to specifically bind to at least a portion of a target gene or its gene product; (b) a polynucleotide encoding the gene regulatory portion of (a) The composition of claim 1 comprising:

14. (a) The pharmaceutical product comprises a guide nucleic acid configured to complex with at least a portion of a target gene or its gene product, or a polynucleotide sequence encoding said guide nucleic acid; or (b) the pharmaceutical comprises a heterologous endonuclease or a polynucleotide comprising a sequence encoding the heterologous endonuclease; The composition of claim 13.

15. The composition of claim 13, wherein the gene regulatory portion comprises a guide nucleic acid configured to complex with at least a portion of the target gene or its gene product, and a heterologous endonuclease, wherein the heterologous endonuclease is present in the gene regulatory portion in a mass or weight ratio of about 1:20 to about 20:1 relative to the guide nucleic acid.

16. The composition according to any one of claims 12 to 15, wherein the target gene or its gene product is specific to or is found predominantly in a target organ or target cell of a subject.

17. The composition of claim 16 , wherein the target gene or its gene product is associated with a disease or disorder of the target organ or target cell.

18. 18. The composition of claim 16 or 17, wherein the target organ is the lung or liver, or the target cell is a lung cell or a liver cell.

19. In the lipidoid of the formula (I) or a pharma- ceutically acceptable salt thereof, R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ are each independently H or 【Chemistry 4】 and R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ At least one of 【Chemistry 5】 2. The composition of claim 1 ,

20. R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ At least two of 【Chemistry 6】 20. The composition of claim 19, wherein

21. The lipidoid of formula (I) or a pharma- ceutically acceptable salt thereof is represented by the structural formula (II Ａ ), (II Ｂ ), (II Ｃ ), (II Ｄ ), or (II Ｅ ) a lipidoid having 【Chemistry 7】 or a pharma- ceutically acceptable salt thereof, m1, m2, m3, and m4, when present, are each independently 1, 2, or 3; q1, q2, q3, and q4, if present, are each independently 1, 2, 3, or 4; R ｃ１ , R ｃ２ , R ｃ３ , and R ｃ４ each independently, if present, ４ ~C ２０ Alkyl or C ４ ~C ２０ The composition of claim 19 which is alkenyl.

22. The lipidoid of the formula (I) or a pharma- ceutical acceptable salt thereof is Ｅ 22. The composition of claim 21, wherein the lipidoid is:

23. (a) R ｃ１ But, C ４ ~C １６ Alkyl or C ４ ~C １６ alkenyl, and / or (b) in formula (II B ), (II D ), or (II E ), R c2 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (c) in formula (II C ), (II D ), or (II E ), R c3 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (d) in formula (II E ), R c4 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (e) m1 is 1 or 2; and / or (f) in formula (II B ), (II D ), or (II E ), m2 is 1 or 2; and / or (g) in formula (II C ), (II D ), or (II E ), m3 is 1 or 2; and / or (h) in formula (II E ), m4 is 1 or 2; and / or (i) q1 is 1, 2, or 3; and / or (j) in formula (II B ), (II D ), or (II E ), q2 is 1, 2, or 3; and / or (k) in formula (II C ), (II D ), or (II E ), q3 is 1, 2, or 3; and / or (l) In formula (II E ), q4 is 1, 2, or 3; 22. The composition of claim 21.

24. The lipidoid is 【Chemistry 8】 and any pharma- ceutically acceptable salt thereof.

25. In the lipidoid of the formula (I) or a pharma- ceutically acceptable salt thereof, R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ are each independently H or 【Chemistry 9】 and R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ At least one of 【Chemistry 10】 2. The composition of claim 1 ,

26. R ｂ１ , R ｂ２ , R ｂ３ , and R ｂ４ At least two of the 【Chemistry 11】 26. The composition of claim 25, wherein

27. The lipidoid of formula (I) or a pharma- ceutically acceptable salt thereof is represented by the structural formula (III Ａ ), (III Ｂ ), (III Ｃ ), (III Ｄ ), or (III Ｅ ) a lipidoid having 【Chemistry 12】 or a pharma- ceutically acceptable salt thereof, m5, m6, m7, and m8 are each independently 1, 2, or 3; q5, q6, q7, and q8 are each independently 1, 2, 3, or 4; R ｃ５ , R ｃ６ , R ｃ７ , and R ｃ８ each independently, if present, ４ ~C ２０ Alkyl or C ４ ~C ２０ The composition of claim 25, wherein the alkyl group is alkenyl.

28. The lipidoid of the formula (I) or a pharma- ceutical acceptable salt thereof is Ｅ 28. The composition of claim 27, which is a lipidoid of the formula:

29. (a) R ｃ５ But, C ４ ~C １６ Alkyl or C ４ ~C １６ alkenyl, and / or (b) in formula (III B ), (III D ), or (III E ), R c6 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (c) in formula (III C ), (III D ), or (III E ), R c7 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (d) in formula (III E ), R c8 is C 4 -C 16 alkyl or C 4 -C 16 alkenyl; and / or (e) m5 is 1 or 2; and / or (f) in formula (III B ), (III D ), or (III E ), m6 is 1 or 2; and / or (g) in formula (III C ), (III D ), or (III E ), m7 is 1 or 2; and / or (h) in formula (III E ), m8 is 1 or 2; and / or (i) q5 is 1, 2, or 3; and / or (j) in formula (III B ), (III D ), or (III E ), q6 is 1, 2, or 3; and / or (k) in formula (III C ), (III D ), or (III E ), q7 is 1, 2, or 3; and / or (l) In formula (III E ), q8 is 1, 2, or 3; 28. The composition of claim 27.

30. The lipidoid is 【Chemistry 13】 【Chemistry 14】 and any pharma- ceutically acceptable salt thereof.

31. The composition of any one of claims 19 to 30, wherein the lipidoid or a pharma- ceutically acceptable salt thereof is present in the lipid composition in a molar percentage of about 20% to about 50%.

32. 32. The composition of any one of claims 19 to 31, wherein the lipid composition comprises a steroid or steroid derivative in a molar percentage of about 10% to about 50%.

33. 33. The composition of any one of claims 19 to 32, wherein the lipidoid or a pharma- ceutically acceptable salt thereof is present in the lipid composition in a mass or weight ratio to the pharmaceutical agent of about 5:1 to about 100:

1.

34. 34. The composition of any one of claims 19 to 33, for preferential delivery of the pharmaceutical agent to the liver or hepatic cells compared to delivery to non-liver organs or non-liver cells.

35. The composition of claim 34, wherein the pharmaceutical agent is configured to encode or regulate a target gene or its gene product that is specific to or found primarily in the liver or hepatocytes.

36. 36. The composition of claim 35, wherein the pharmaceutical agent is configured to provide an altered expression profile of the target gene or its gene product in the liver or hepatic cells.

37. 35. The composition of claim 34, wherein the pharmaceutical agent is associated with liver disease or damage.

38. 38. The composition of any one of claims 34 to 37, which provides a greater amount, expression or activity of the pharmaceutical agent in the liver or hepatic cells of the subject compared to a non-liver organ or non-hepatic cells of the subject.

Images