Lipid nanoparticle compositions and their uses

Abstract

Aerosolized pharmaceutical compositions comprising aerosol particles containing lipid nanoparticles (LNPs) comprising at least two selective organ-targeting (SORT) lipids and / or at least six liquids are provided herein. Methods for administering the aerosolized pharmaceutical compositions described herein are also provided herein.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 605,191, filed December 1, 2023, the entire contents of which are incorporated herein by reference.

[0002] By referencing and incorporating into the sequence list

[0003] This application contains a sequence list, which has been submitted via EFS-WEB in .XML format and is incorporated herein by reference in its entirety. The .XML file, created on November 25, 2024, is named 061529-509001WO.xml and is 20KB in size. Background Technology

[0004] Delivery of therapeutic agents to the lungs can be achieved by systemic administration of the agent to the subject or by direct administration to the lungs via the mouth or nose. In either case, a medium can be used to protect and facilitate the delivery of the agent. One type of medium used for therapeutic agents (such as proteins, nucleic acids, or small molecules) is lipid nanoparticles (LNPs). This type of medium is used, for example, in mRNA-based vaccines. LNP vaccines are typically administered subcutaneously, and like most LNPs, they are primarily delivered to the liver by default. In contrast, WO 2020 / 051220 A1 discloses compositions that preferentially target or deliver nucleic acid compositions to specific organs, such as the lungs. Therefore, one method of delivering LNPs to the lungs is to administer LNPs systemically as a pharmaceutical composition, which is injected into the subject for systemic distribution (e.g., intravenous injection) rather than by atomization to form an aerosol.

[0005] To deliver the drug to the lungs via the mouth or nose, a pharmaceutical composition containing a therapeutic agent or a vehicle containing a therapeutic agent can be atomized into fine particles (typically with an aerodynamic diameter of less than 10 micrometers). However, size control is an important consideration. Aerosol particles smaller than 2 micrometers can penetrate deep into the alveolar region. Atomizing a pharmaceutical composition containing a therapeutic agent in a manner that maintains therapeutic efficacy and produces aerosol particles with the desired physical properties for delivery to the appropriate areas of the lung remains challenging. Specifically, atomizing a pharmaceutical composition containing a LNP may result in LNP degradation, therapeutic agent unpacking, the formation of aerosol particles with physical properties that prevent targeted delivery to the desired areas of the lung, or other adverse effects on the LNP and its payload, or both.

[0006] Therefore, there is a long-standing but unmet need for aerosolized pharmaceutical compositions comprising lipid nanoparticles (LNPs) capable of delivering LNPs to the lungs of a subject (e.g., the tracheobronchial region of the subject) and / or possessing the desired physical properties. This disclosure provides such aerosolized pharmaceutical compositions, methods of their preparation and use, and further related compositions and methods. Summary of the Invention

[0007] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising at least two selective organ-targeting (SORT) lipids and / or at least six lipids.

[0008] In some embodiments, the LNP composition comprises an ionizable cationic lipid, optionally two or more ionizable lipids; and a permanent cationic lipid, optionally two or more permanent cationic lipids.

[0009] In some embodiments, the LNP composition comprises ionizable cationic lipids, permanent cationic lipids, optional phospholipids, optional polyethylene glycol (PEG)-lipids, and / or optional sterols.

[0010] In some embodiments, the LNP composition contains at least six lipids.

[0011] In some embodiments, the LNP composition comprises ionizable cationic lipids, permanent cationic lipids, optional phospholipids, optional polyethylene glycol (PEG)-lipids, and / or optional sterols.

[0012] In some embodiments, the LNP composition comprises at least two selective organ-targeting (SORT) lipids. In some embodiments, the ionizable cationic lipid is a dendritic lipid, optionally 4A3-SC7 or 5A2-SC8. In some embodiments, the ionizable cationic lipid is a SORT lipid; and / or the permanent cationic lipid is a SORT lipid. In some embodiments, the ionizable cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments, the permanent cationic lipid is trimethylammonium-propane, optionally 1,2-dimyristoyl-3-trimethylammonium-propane (14:0TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0TAP), or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the permanent cationic lipid is ethylphosphocholine (EPC), optionally 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), or 1,2-distearateyl-sn-glycerol-3-ethylphosphocholine (18:0 EPC).

[0013] In some embodiments, the LNP specifically transduces lung cells; and / or the LNP delivers the mRNA to lung cells in an amount that effectively increases the expression and / or function of the protein encoded by the mRNA. In some embodiments, the lung cells are ionocytes. In some embodiments, the lung cells are ciliated cells. In some embodiments, the lung cells are secretory cells.

[0014] In some embodiments, the LNP comprises an ionizable cationic lipid of greater than 15% molar percentage. In some embodiments, the ionizable cationic lipid is a dendritic lipid with a molar percentage between 10% and 30%. In some embodiments, the LNP comprises DODAP with a molar percentage between 5% and 40%. In some embodiments, the LNP comprises a permanent cationic lipid of less than 40% molar percentage. In some embodiments, the permanent cationic lipid has a molar percentage between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, and between 15% and 20%. In some embodiments, the permanent cationic lipid is trimethylammonium propane (TAP). In some embodiments, TAP is 1,2-dimyristoyl-3-trimethylammonium propane (14:0 TAP). In some embodiments, the permanent cationic lipid is 14:0 TAP with a molar percentage between 10% and 15%. In some embodiments, the permanent cationic lipid is ethylphosphocholine (EPC). In some embodiments, the TAP is 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC). In some embodiments, the permanent cationic lipid is 14:0 EPC with a molar percentage between 10% and 15%.

[0015] In some embodiments, the LNP contains phospholipids at a molar percentage between 10% and 30%. In some embodiments, the LNP contains cholesterol at a molar percentage greater than 25%. In some embodiments, the molar percentage of cholesterol is between 25% and 50%, between 30% and 50%, between 30% and 45%, between 30% and 40%, or between 30% and 35%. In some embodiments, the LNP contains polyethylene glycol (PEG) lipids at a molar percentage between 0.5% and 10% or between 1% and 4%.

[0016] In some embodiments, the LNP contains messenger RNA (mRNA). In some embodiments, the LNP contains mRNA at a lipid:mRNA ratio of less than 40:1. In some embodiments, the lipid:mRNA ratio is 36:1. In some embodiments, the lipid:mRNA ratio is 33:1. In some embodiments, the lipid:mRNA ratio is 30:1.

[0017] In some embodiments, the LNP contains a gene-editing payload. In some embodiments, the gene-editing payload contains a nuclease and / or one or more guide RNAs, and optionally a repair template. In some embodiments, the LNP contains a polypeptide or protein.

[0018] In some embodiments, the LNP comprises a cationic ionizable lipid of 5A2-SC8 or 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP or 14:0 EPC, a phospholipid of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), a sterol of cholesterol or sitosterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG, optionally DMG-PEG2000.

[0019] In some implementations, the LNP contains a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0020] In some implementations, the LNP contains a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 EPC, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0021] In some embodiments, the cationic ionizable lipid is 4A3-SC7, and the LNP contains between about 10% and about 30% 4A3-SC7 in molar percentage. In some embodiments, the cationic ionizable SORT lipid is DODAP, and the LNP contains between about 5% and about 40% DODAP in molar percentage. In some embodiments, the permanent cationic lipid is 14:0 TAP, and the LNP contains between about 5% and about 25% 14:0 TAP in molar percentage. In some embodiments, the permanent cationic lipid is 14:0 EPC, and the LNP contains between about 5% and about 25% 14:0 EPC in molar percentage.

[0022] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 TAP in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0023] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 TAP in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0024] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 EPC in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0025] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 EPC in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0026] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 TAP, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0027] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0028] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 EPC, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0029] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 EPC, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0030] In some implementations, the LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG, wherein the lipid:mRNA ratio is 33:1.

[0031] In some embodiments, the LNP contains a payload. In some embodiments, the payload is messenger RNA (mRNA). In some embodiments, the mRNA contains 100 bases to 8 kilobases (kb). In some embodiments, the mRNA contains 1 kb to 8 kb, or 2 kb to 8 kb, 3 kb to 8 kb, or 4 kb to 8 kb. In some embodiments, the mRNA contains (about) 2 kb. In some embodiments, the mRNA contains (about) 4.6 kb. In some embodiments, the mRNA encodes a cystic fibrosis transmembrane transduction regulator (CFTR) protein. In some embodiments, the mRNA encodes a dynein axonofilament intermediate chain 1 (DNAI1) protein. In some embodiments, the mRNA encodes a gene editing system or a component thereof. In some embodiments, the payload is shRNA or a polynucleotide encoding shRNA. In some embodiments, the payload is microRNA or a polynucleotide encoding microRNA. In some embodiments, the payload is a polypeptide. In some embodiments, the payload is a protein.

[0032] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is an aerosolized composition.

[0033] In some embodiments, the LNP has an encapsulation efficiency between 50% and 99%, 60% and 99%, 70% and 99%, or 80% and 99%. In some embodiments, the LNP has an encapsulation efficiency between 50% and 95%, 60% and 95%, 70% and 95%, or 80% and 95%. In some embodiments, the LNP composition is a liquid. In some embodiments, the LNP composition is an aerosol.

[0034] In another aspect, this disclosure provides a method for delivering a payload to cells, the method comprising contacting the cells with the LNP composition disclosed herein. In another aspect, this disclosure provides a method for expressing a protein or RNA in cells, the method comprising contacting the cells with the LNP composition disclosed herein. In some embodiments, the cells are lung cells. In some embodiments, the lung cells are secretory cells. In some embodiments, the lung cells are ionizing cells. In some embodiments, the lung cells are ciliated cells. In some embodiments, the method specifically transduces secretory cells compared to other lung cells. In some embodiments, the method specifically transduces ionizing cells compared to other lung cells. In some embodiments, the method specifically transduces ciliated cells compared to other lung cells. In some embodiments, the method comprises atomizing the LNP composition to generate an aerosolized composition, and then contacting the aerosolized composition with cells. In some embodiments, the LNP composition is an aerosolized composition, and the method comprises contacting the aerosolized composition with cells.

[0035] In another aspect, this disclosure provides a method for delivering a payload to the lungs of a subject, the method comprising administering the composition disclosed herein to the subject.

[0036] In another aspect, this disclosure provides a method for treating or preventing lung disease in a subject, the method comprising administering the composition disclosed herein to the subject.

[0037] In some embodiments, the method includes nebulizing the composition prior to the administration step. In some embodiments of the method, the LNP composition is administered by inhalation as an aerosolized composition. In some embodiments, the method delivers an effective amount of the LNP composition to the lungs. In some embodiments, the method delivers an effective amount of the LNP composition to the lungs to treat lung diseases.

[0038] On the other hand, this disclosure provides the use of the compositions described herein for the treatment of lung diseases.

[0039] On the other hand, this disclosure provides a composition described herein for treating lung diseases.

[0040] In another aspect, this disclosure provides a kit comprising the composition described herein and an atomizer mask and / or a mesh suitable for an atomizer.

[0041] In another aspect, this disclosure provides a method for preparing the LNP composition described herein, the method comprising mixing a lipid component and a payload under conditions of efficient assembly of an LNP comprising a payload. In some embodiments, the method includes atomizing the composition to produce an aerosolized LNP composition.

[0042] In another aspect, this disclosure provides an aerosolized pharmaceutical composition comprising the LNP composition described herein. In some embodiments of the aerosolized pharmaceutical composition, it comprises at least two selective organ-targeting (SORT) lipids and / or at least six lipids. In some embodiments of the aerosolized pharmaceutical composition, the LNP composition comprises ionizable cationic lipids, optionally two or more ionizable lipids; and permanent cationic lipids, optionally two or more permanent cationic lipids.

[0043] In some embodiments of the aerosolized pharmaceutical composition, the LNP composition comprises an ionizable cationic lipid, a permanent cationic lipid, an optional phospholipid, an optional polyethylene glycol (PEG)-lipid, and / or an optional sterol.

[0044] In some embodiments of the aerosolized pharmaceutical composition, the LNP composition contains at least six lipids.

[0045] In some embodiments of the aerosolized pharmaceutical composition, the LNP composition comprises an ionizable cationic lipid, a permanent cationic lipid, an optional phospholipid, an optional polyethylene glycol (PEG)-lipid, and / or an optional sterol.

[0046] In some embodiments of the aerosolized pharmaceutical composition, the LNP composition contains at least two selective organ-targeting (SORT) lipids.

[0047] In some embodiments, the ionizable cationic lipid is a dendritic lipid, optionally 4A3-SC7 or 5A2-SC8. In some embodiments of the aerosolized pharmaceutical composition, the ionizable cationic lipid is a SORT lipid; and / or the permanent cationic lipid is a SORT lipid. In some embodiments of the aerosolized pharmaceutical composition, the ionizable cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is trimethylammonium-propane, optionally 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is ethyl phosphocholine (EPC), optionally 1,2-dilauroyl-sn-glycerol-3-ethyl phosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethyl phosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethyl phosphocholine (16:0 EPC), or 1,2-distearateyl-sn-glycerol-3-ethyl phosphocholine (18:0 EPC).

[0048] In some embodiments of the aerosolized pharmaceutical composition, the LNP specifically transduces lung cells; and / or the LNP delivers the mRNA to lung cells in an amount that effectively enhances the expression and / or function of the protein encoded by the mRNA. In some embodiments of the aerosolized pharmaceutical composition, the lung cells are ionocytes. In some embodiments of the aerosolized pharmaceutical composition, the lung cells are ciliated cells. In some embodiments of the aerosolized pharmaceutical composition, the lung cells are secretory cells.

[0049] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises an ionizable cationic lipid of greater than 15% molar percentage. In some embodiments of the aerosolized pharmaceutical composition, the ionizable cationic lipid is a dendritic lipid with a molar percentage between 10% and 30%. In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises DODAP with a molar percentage between 5% and 40%. In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a permanent cationic lipid of less than 40% molar percentage. In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid has a molar percentage between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, and between 15% and 20%. In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is trimethylammonium propane (TAP). In some embodiments of the aerosolized pharmaceutical composition, the TAP is 1,2-dimyristoyl-3-trimethylammonium propane (14:0 TAP). In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is a 14:0 TAP with a molar percentage between 10% and 15%. In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is ethylphosphocholine (EPC). In some embodiments of the aerosolized pharmaceutical composition, the EPC is 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC). In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is a 14:0 EPC with a molar percentage between 10% and 15%. In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a phospholipid with a molar percentage between 10% and 30%. In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises cholesterol with a molar percentage greater than 25%. In some embodiments of the aerosolized pharmaceutical composition, the molar percentage of cholesterol is between 25% and 50%, between 30% and 50%, between 30% and 45%, between 30% and 40%, or between 30% and 35%. In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises polyethylene glycol (PEG) lipids in a molar percentage between 0.5% and 10% or between 1% and 4%.

[0050] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises messenger RNA (mRNA). In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises mRNA at a lipid:mRNA ratio of less than 40:1. In some embodiments of the aerosolized pharmaceutical composition, the lipid:mRNA ratio is 36:1. In some embodiments of the aerosolized pharmaceutical composition, the lipid:mRNA ratio is 33:1. In some embodiments of the aerosolized pharmaceutical composition, the lipid:mRNA ratio is 30:1.

[0051] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a gene-editing payload. In some embodiments of the aerosolized pharmaceutical composition, the gene-editing payload comprises Cas9, sgRNA, and / or ss DNA.

[0052] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a cationic ionizable lipid of 5A2-SC8 or 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP or 14:0 EPC, a phospholipid of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), a sterol of cholesterol or sitosterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG, optionally DMG-PEG2000.

[0053] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0054] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 EPC, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0055] In some embodiments of the aerosolized pharmaceutical composition, the cationic ionizable lipid is 4A3-SC7, and the LNP contains 4A3-SC7 in a molar percentage between about 10% and about 30%. In some embodiments of the aerosolized pharmaceutical composition, the cationic ionizable SORT lipid is DODAP, and the LNP contains DODAP in a molar percentage between about 5% and about 40%. In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is 14:0 TAP, and the LNP contains 14:0 TAP in a molar percentage between about 5% and about 25%. In some embodiments of the aerosolized pharmaceutical composition, the permanent cationic lipid is 14:0 EPC, and the LNP contains 14:0 EPC in a molar percentage between about 5% and about 25%.

[0056] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 TAP in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0057] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 TAP in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0058] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 EPC in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0059] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 EPC in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0060] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 TAP, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0061] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0062] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 EPC, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0063] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 EPC, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0064] In some embodiments of the aerosolized pharmaceutical composition, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG, wherein the lipid:mRNA ratio is 33:1.

[0065] In some embodiments of the aerosolized pharmaceutical composition, the LNP comprises a payload. In some embodiments of the aerosolized pharmaceutical composition, the payload is messenger RNA (mRNA). In some embodiments of the aerosolized pharmaceutical composition, the mRNA encodes a gene editing system or a component thereof. In some embodiments of the aerosolized pharmaceutical composition, the payload is shRNA or a polynucleotide encoding shRNA. In some embodiments of the aerosolized pharmaceutical composition, the payload is microRNA or a polynucleotide encoding microRNA. In some embodiments of the aerosolized pharmaceutical composition, the composition is a pharmaceutical composition.

[0066] In another aspect, this disclosure provides a liquid pharmaceutical composition for preparing the aerosolized pharmaceutical composition described herein.

[0067] In another aspect, this disclosure provides a method for delivering lipid nanoparticles (LNPs) to lung cells of a subject, the method comprising: atomizing the liquid pharmaceutical composition described herein to generate an aerosolized pharmaceutical composition, and administering the aerosolized pharmaceutical composition to the subject.

[0068] In another aspect, this disclosure provides a method for delivering a payload to the lung cells of a subject, the method comprising administering to the subject the aerosolized pharmaceutical composition described herein, wherein optionally the payload is a polynucleotide.

[0069] In another aspect, this disclosure provides a method for expressing a protein in the lungs of a subject, the method comprising administering to the subject the aerosolized pharmaceutical composition described herein.

[0070] In another aspect, this disclosure provides a method for treating a lung disease in a subject, the method comprising administering the aerosolized pharmaceutical composition described herein to the subject.

[0071] In another aspect, this disclosure provides a kit comprising a lipid nanoparticle composition containing at least two selective organ-targeting (SORT) lipids and / or at least six lipids, optionally containing polynucleotides.

[0072] Other aspects and embodiments of the invention are provided in the following detailed description. The scope of the invention is defined only by the claims. Those skilled in the art will be able to conceive of and implement many variations of the disclosed embodiments. Attached Figure Description

[0073] The patent or application document contains at least one color drawing. The Patent Office will provide a copy of the patent or patent application publication with the color drawing, as required and subject to payment of necessary fees.

[0074] Figure 1 A schematic diagram of the standards used to evaluate aerosolized pharmaceutical compositions is shown.

[0075] Figure 2 PARI was shown ® eFlow ® A graph showing the output rate when using various atomizer heads on an atomizer system.

[0076] Figure 3 PARI was shown ® eFlow ® A graph showing the output rate of composition A when using various atomizer heads on an atomizer system.

[0077] Figures 4A to 4B The predicted and actual atomization times for baseline or composition A / DNAI1 lipid nanoparticles are shown. Figure 4A The predicted and actual nebulization times for the baseline (PBS) are shown. Figure 4B The predicted and actual atomization times of the A / DNAI1 lipid nanoparticle composition are shown.

[0078] Figure 5 PARI was shown ® eFlow ® Encapsulation efficiency (%) of various atomizer heads in an atomizer system.

[0079] Figure 6 PARI was shown ® eFlow ® Encapsulation efficiency (%) data for the 40 HO V atomizer system.

[0080] Figure 7 PARI was shown ® eFlow ® mRNA integrity of the nebulizer system at 35 V or 40 HO V.

[0081] Figures 8A to 8C PARI was shown ® eFlow ® Encapsulation efficiency (%) of various heads in the atomizer system. Figure 8A PARI was shown ® eFlow ® Encapsulation efficiency of the atomizer at slow flow rates (%). Figure 8B PARI was shown ® eFlow ® Encapsulation efficiency (%) of the atomizer at medium flow rate. Figure 8C PARI was shown ® eFlow ®Encapsulation efficiency of the atomizer at high flow rates (%).

[0082] Figures 9A to 9C PARI was shown ® eFlow ® Hydrodynamic diameter (nm) of various heads in an atomizer system. Figure 9A PARI was shown ® eFlow ® Hydrodynamic diameter (nm) of the atomizer at slow flow rates. Figure 9B PARI was shown ® eFlow ® Hydrodynamic diameter (nm) of the atomizer at medium flow rates. Figure 9C PARI was shown ® eFlow ® Hydrodynamic diameter (nm) of the atomizer at high flow rates.

[0083] Figures 10A to 10C PARI was shown ® eFlow ® The polydispersity index of various heads in an atomizer system. Figure 10A PARI was shown ® eFlow ® The polydispersity index of an atomizer at slow flow rates. Figure 10B PARI was shown ® eFlow ® The polydispersity index of an atomizer at medium flow rates. Figure 10C PARI was shown ® eFlow ® The polydispersity index of an atomizer at high flow rates.

[0084] Figure 11 PARI was shown ® eFlow ® The hydrodynamic diameter (nm) of various heads in the atomizer system during the atomization process.

[0085] Figure 12 PARI was shown ® eFlow ® The polydispersity index of various heads in an atomizer system during the atomization process.

[0086] Figures 13A to 13D PARI was shown ® eFlow ® Characteristics of 40 HO V lipid nanoparticles in an atomizer system. Figure 13A The concentration of lipid nanoparticles is shown. Figure 13BThe encapsulation efficiency (%) of the lipid nanoparticles is shown. Figure 13C The hydrodynamic diameter (nm) of the lipid nanoparticles is shown. Figure 13D The polydispersity index of lipid nanoparticles is shown.

[0087] Figures 14A to 14B PARI was shown ® eFlow ® Drug deposition (μg) at various stages of nebulizer systems with 40 HO V or 30 HO V. Figure 14A The drug deposition (µg) at different stages at 40 HO V is shown. Figure 14B The drug deposition (µg) at different stages at 30 HOV is shown.

[0088] Figure 14C Aerosol characterization in a next-generation impactor (NGI) is shown.

[0089] Figures 15A to 15B Aerogen was shown ® Deposition fraction of multipath particle dosimetry (MPPD) using Solo or PDAP. Figure 15A Aerogen was shown ® Solo's deposition fraction based on multipath particle dosimetry (MPPD). Figure 15B The deposition fraction of PDAP multipath particle dosimetry (MPPD) is shown.

[0090] Figures 16A to 16E PARI was shown ® eFlow ® Deposition fraction of multipath particle dose measurement (MPPD) for various atomizer heads on the atomizer system. Figure 16A The deposition fraction of multipath particle dosimetry (MPPD) at 30 NV is shown. Figure 16B The deposition fraction of multipath particle dosimetry (MPPD) at 35 NV is shown. Figure 16C The deposition fraction of multipath particle dosimetry (MPPD) at 30V is shown. Figure 16D The deposition fraction of multipath particle dosimetry (MPPD) at 35V is shown. Figure 16E The deposition fraction of multipath particle dosimetry (MPPD) at 40 HO is shown.

[0091] Figures 17A to 17B The percentage of lipids recovered is shown when using different lipid extraction solutions and volumes. Figure 17A The percentage of lipids recovered is shown. Figure 17B Quantitative data on the percentage of lipid recovery are shown.

[0092] Figures 18A to 18C Comparative results of gravimetric analysis, RiboGeen, and high-performance liquid chromatography (HPLC-CAD) methods equipped with an electrosol detector are shown. Figure 18A Quantitative data showing the comparison results of different methods are presented. Figure 18B A comparison between gravimetric analysis and HPLC-CAD is shown. Figure 18C A comparison of RiboGreen and HPLC-CAD is shown.

[0093] Figures 19A to 19B The lipid weight fraction for various lipid extraction solutions and volumes is shown. Figure 19A The lipid weight fraction in the rat GLP-tox experiment is shown. Figure 19B The lipid weight fractions in the NHP GLP-tox experiment are shown.

[0094] Figures 20A to 20B The percentage of lipids recovered under different filter storage conditions is shown. Figure 20A The percentage of lipids recovered under different conditions is shown. Figure 20B Quantitative data on the percentage of lipid recovery are shown.

[0095] Figures 21A to 21E The gravimetric analysis, HPLC-CAD, and LC-MS comparison of total lipid mass for composition B are shown. Figure 21A The percentage of lipids recovered is shown. Figure 21B Quantitative data on the percentage of lipid recovery are shown. Figure 21C The total lipid mass comparisons by gravimetric analysis, HPLC-CAD, and LC-MS are shown. Figure 21D A comparison between LC-MS and gravimetric analysis is shown. Figure 21E A comparison of LC-MS and HPLC-CAD is shown.

[0096] Figure 22 The comparison of lipid fractions of composition B by HPCL-CAD and LC-MS is shown.

[0097] Figure 23 An image of the ATS-003 atomizing device is shown.

[0098] Figures 24A to 24C This is an image of the New Generation Impactor (NGI). Figure 24A An image of the next-generation impactor (NGI) is shown. Figure 24B The NGI sealing body (stage nozzle) is shown. Figure 24C The NIG cover (interstage channel) is shown.

[0099] Figures 25A to 25B This is an image of an NGI weight cup. Figure 25A An image of the NGI weight cup is shown. Figure 25B Images of the NGI weight cup components (NGI-specific cup, filter, and filter support) are shown.

[0100] Figures 26A to 26B This is an image of the NGI internal filter holder. Figure 26A Images of the NGI internal filter retainer (NGI-specific cup, filter support screen, and filter clamping ring) are shown. Figure 26B An image of the NGI internal filter holder is shown.

[0101] Figure 27 This is an image of the USP (United States Pharmacopeia) inhalation port.

[0102] Figure 28A An image of a laboratory stand with a claw atomizer is shown. Figure 28B The use of a frozen 50 mL conical tube with a cut cap suitable for an atomizer to collect mist is shown.

[0103] Figure 29 A schematic diagram of lipid nanoparticles (LNPs) encapsulating mRNA is shown.

[0104] Figure 30 The AUC / min for non-DOTAP LNP is shown.

[0105] Figure 31 The AUC / min of LNP based on DODAP is shown.

[0106] Figure 32 The AUC / min of composition B with different buffers is shown.

[0107] Figure 33A The screening results for compound B and composition R with different buffer solutions are shown. Figure 33B The screening results for compound B and composition U with different buffer solutions are shown.

[0108] Figures 34A to 34D A summary of lipid nanoparticle characterization is presented for various lipid nanoparticles in different pH buffers. Figure 34A The size of the lipid nanoparticles is shown. Figure 34B The polydispersity index of lipid nanoparticles is shown. Figure 34C The encapsulation efficiency (%) of the lipid nanoparticles is shown. Figure 34D The zeta potential (mV) of the lipid nanoparticles is shown.

[0109] Figure 35A The TNS determination of composition B in different buffer solutions is shown. Figure 35BThe TNS determination of composition C in different buffer solutions is shown. Figure 35C The TNS determination of composition A is shown.

[0110] Figures 36A to 36D A summary of the lipid nanoparticle characterization of compositions B and C in different pH buffers is presented. Figure 36A The size of the lipid nanoparticles is shown. Figure 36B The polydispersity index of lipid nanoparticles is shown. Figure 36C The encapsulation efficiency (%) of the lipid nanoparticles is shown. Figure 36D The zeta potential (mV) of the lipid nanoparticles is shown.

[0111] Figure 37A The TNS determination of composition B is shown. Figure 37B The TNS determination of composition C is shown.

[0112] Figures 38A to 38D The lipid nanoparticle characterization of composition B at pH 4, pH 5, or pH 6 is summarized. Figure 38A The size of the lipid nanoparticles is shown. Figure 38B The polydispersity index of lipid nanoparticles is shown. Figure 38C The encapsulation efficiency (%) of the lipid nanoparticles is shown. Figure 38D The zeta potential (mV) of the lipid nanoparticles is shown.

[0113] Figures 39A to 39D A summary of the lipid nanoparticle characterization of composition F at pH 4, pH 5, or pH 6 is presented. Figure 39A The size of the lipid nanoparticles is shown. Figure 39B The polydispersity index of lipid nanoparticles is shown. Figure 39C The encapsulation efficiency (%) of the lipid nanoparticles is shown. Figure 39D The zeta potential (mV) of the lipid nanoparticles is shown.

[0114] Figure 40 The AUC / min of composition variant B is shown.

[0115] Figure 41 The AUC / min values ​​for various compositions containing 50% cholesterol are shown.

[0116] Figure 42 The experimental conditions are shown.

[0117] Figures 43A to 43D The rescue of CFTR function in two unresponsive genotypes hBE is shown. Figure 43A This demonstrates the rescue of CFTR function in hBE with the R553X / W1282X genotype. Figure 43BThe results of transepithelial resistance (TEER) (top) and LDH release (bottom) measurements in the R553X / W1282X genotype hBE are shown. Figure 43C This demonstrates the rescue of CFTR function in hBE of the W1282X / W1282X genotype. Figure 43C The results of transepithelial resistance (TEER) (top) and LDH release (bottom) measurements in hBE of the W1282X / W1282X genotype are shown.

[0118] Figure 44 A summary of benchmark data for several CF genotypes and donors is presented.

[0119] Figure 45A shows the rescue of CFTR function in donor TXCF042716 cells. Figure 45B shows the measurement results of LDH release in donor TXCF042716 cells, used to detect the cytotoxicity of the aerosolized agent. Figure 45C shows the rescue of CFTR function in donor KKD012K cells. Figure 45D shows the measurement results of LDH release in donor KKD012K cells, used to detect the cytotoxicity of the aerosolized agent. Figure 45E shows the rescue of CFTR function in donor KKD025L cells. Figure 45F shows the measurement results of LDH release in donor KKD025L cells, used to detect the cytotoxicity of the aerosolized agent. Figure 45G shows the rescue of CFTR function in donor KKD003K cells. Figure 45H shows the measurement results of LDH release in donor KKD003K cells, used to detect the cytotoxicity of the aerosolized agent. Figure 45I shows the rescue of CFTR function in donor 20160524CF cells. Figure 45J shows the measurement results of LDH release in donor 20160524CF cells, used to detect the cytotoxicity of the aerosolized formulation. Figure 45K The rescue of CFTR function in donor KK017N cells is shown.

[0120] Figure 46A The rescue of CFTR function in donor KKD003K cells by various lipid nanoparticles at 24 h and 48 h is shown. Figure 46B The quantification of CFTR bands is shown. Figure 46C The expression of CFTR protein, detected by Western blot analysis, is shown.

[0121] Figure 47A Preliminary evaluation of lipid nanoparticles targeting secretory cells (e.g., goblet cells) in ΔF508 / ΔF508 (donor TXCF042716) hBE cells is presented. Figure 48B The quantification of TR-positive cells is shown.

[0122] Figures 48A to 48CThe correlation between CFTR function and CFTR protein levels was shown. Figure 48A The rescue of CFTR function in donor KKD003K or donor KKD012K cells is shown. Figure 48B The quantification of CFTR bands is shown. Figure 48C The expression of CFTR protein in donor KKD003K or donor KKD012K cells is shown by Western blot analysis.

[0123] Figure 49 The experimental conditions for the benchmark study are shown.

[0124] Figures 50A to 50B The effect of mucus on the transfection efficiency of aerosolized SORT lipid nanoparticles in W1282X / W1282X hBE is shown. Figure 50A The rescue of the CFTR function is shown. Figure 50B The results of the transepithelial resistance (TEER) measurement are shown.

[0125] Figures 51A to 51B The effect of mucus on the transfection efficiency of aerosolized SORT lipid nanoparticles in R553X / W1282X hBE is shown. Figure 51A The rescue of the CFTR function is shown. Figure 51B The results of the transepithelial resistance (TEER) measurement are shown.

[0126] Figures 52A to 52B The effect of CFTR activators on CFTR function after delivery of lipid nanoparticles is shown. Figure 52A The rescue of the CFTR function with or without Ivacator is shown in the R553X / W1282X hBE. Figure 52B The rescue of the CFTR function is shown with or without Ivacator in the W1282X / W1282X hBE.

[0127] Figures 53A to 53B In vivo studies of lipid nanoparticles are shown. Figure 53A The quantitative data of luminescence are shown. Figure 53B A full-body image of IVIS is shown.

[0128] Figures 54A to 54C It was shown that SORT LNP for PCD projects cannot salvage the CFTR function in ΔF508 / ΔF508 hBE when delivered via aerosol. Figure 54A The recovery of CFTR function after treatment with composition A or composition X is shown. Figure 54B The representative trace of chloride ion flux is shown. Figure 54C The results of the transepithelial resistance (TEER) measurement are shown.

[0129] Figures 55A to 55C illustrate the stability study of composition B over three weeks. Figure 55A shows the size of the lipid nanoparticles at week 1 and week 3. Figure 55B shows the polydispersity index of the lipid nanoparticles at week 1 and week 3. Figure 55C shows the encapsulation efficiency (%) of the lipid nanoparticles at week 1 and week 3.

[0130] Figures 56A to 56C illustrate the stability study of composition X over three weeks. Figure 56A shows the size of the lipid nanoparticles at week 1 and week 3. Figure 56B shows the polydispersity index of the lipid nanoparticles at week 1 and week 3. Figure 56C shows the encapsulation efficiency (%) of the lipid nanoparticles at week 1 and week 3.

[0131] Figures 57A to 57C A comparison of the characterization of lipid nanoparticles in pH 4 and pH 6 citrate buffers is shown. Figure 57A The size of the lipid nanoparticles is shown. Figure 57B The polydispersity index of lipid nanoparticles is shown. Figure 57C The encapsulation efficiency (%) of the lipid nanoparticles is shown.

[0132] Figures 58A to 58C The TNS assay of various lipid nanoparticles in pH 4 citrate buffer is shown. Figure 58A The TNS determination of composition B in different buffer solutions is shown. Figure 58B The TNS determination of composition X in different buffer solutions is shown. Figure 58C The TNS determination of composition Y in different buffer solutions is shown.

[0133] Figures 59A to 59C The TNS assay of various lipid nanoparticles in pH 6 citrate buffer is shown. Figure 59A The TNS determination of composition B in different buffer solutions is shown. Figure 59B The TNS determination of composition X in different buffer solutions is shown. Figure 59C The TNS determination of composition Y in different buffer solutions is shown.

[0134] Figure 60 The atomization characteristics of compositions B and X in a sucrose-containing citrate buffer are shown.

[0135] Figure 61 Characterization data of lipid nanoparticles during freeze-thaw storage are shown.

[0136] Figures 62A to 62G The optimization of composition X formulation is shown. Figure 62A The experimental protocol is shown. Figure 62B The buffer conditions for the experiment are shown. Figure 62CCharacterization of lipid nanoparticles after freeze-thaw cycles (particle size, polydispersity index, and encapsulation efficiency) is shown. Figure 62D Characterization of lipid nanoparticles under prolonged storage conditions is shown (particle size, polydispersity index, and encapsulation efficiency). Figure 62E The buffer conditions for the experiment are shown. Figure 62F Characterization of lipid nanoparticles after freeze-thaw cycles (particle size, polydispersity index, and encapsulation efficiency) is shown. Figure 62G Characterization of lipid nanoparticles under prolonged storage conditions is shown (particle size, polydispersity index, and encapsulation efficiency).

[0137] Figure 63 A pH titration study of the composition X lipid nanoparticle composition is shown.

[0138] Figure 64 A pH titration study of the X lipid nanoparticle composition alone is shown.

[0139] Figure 65 The CFTR function is shown when using composition X under different pH conditions.

[0140] Figure 66 A reference is shown for antibody selection based on immunofluorescence combinations.

[0141] Figure 67A The ΔF508 / ΔF508 hBE cell profile is shown. Figure 67B The genotypes and donor codes of hBE cells are shown. Figure 67C Antibody detection for each cell type is shown.

[0142] Figure 68 Immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with composition B / HA-CFTR are shown.

[0143] Figure 69 Immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with composition B / HA-CFTR are shown.

[0144] Figure 70 Immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with composition B / HA-CFTR are shown.

[0145] Figure 71A The expression level of HA-CFTR in F508del / F508del(TXCF042716) cells is shown. Figure 71B The expression level of HA-CFTR in F508del / F508del(20160524CF) cells is shown.

[0146] Figure 72 The relationship between CFTR function and its expression is shown.

[0147] Figure 73A The translocation of HA-CFTR protein to the apical membrane is shown in ΔF508 / ΔF508(TXCF042716)hBE cells. Figure 73B The rescue of the CFTR function in hBE is shown. Figure 73C The quantification of CFTR bands by Western blot analysis is shown.

[0148] Figure 74A The location of the HA-CFTR protein in ΔF508 / ΔF508 (20160524CF) is shown. Figure 74B The rescue of the CFTR function in hBE is shown. Figure 74C The quantification of CFTR bands by Western blot analysis is shown.

[0149] Figure 75 CFTR expression in W1282X / W1282X hBE cells treated with composition B or composition X is shown.

[0150] Figure 76A The localization of HA-CFTR protein in W1282X / W1282X (UI0014) cells administered with composition X is shown. Figure 76B The rescue of the CFTR function in hBE is shown. Figure 76C The quantification of CFTR bands by Western blot analysis is shown.

[0151] Figure 77A The localization of HA-CFTR protein in W1282X / W1282X (UI0014) cells administered with composition B is shown. Figure 77B The rescue of the CFTR function in hBE is shown. Figure 77C The quantification of CFTR bands by Western blot analysis is shown.

[0152] Figure 78 The research plan is shown.

[0153] Figure 79 The planned dose levels for the study are shown.

[0154] Figure 80 The event schedule is shown.

[0155] Figure 81 Cell tropism of composition B in dF#4 (KKD003K) is shown.

[0156] Figure 82Cell-mediated expression of HA-CFTR in dF#1, dF#4 and dF#4 is shown.

[0157] Figure 83A The highest expression and apical translocation of HA-CFTR were shown in F508del / F508del hBE (donor TXCF042716) cells administered with composition X. Figure 83B The rescue of chloride ion flux in hBE is shown. Figure 83C The quantification of CFTR bands by Western blot analysis is shown.

[0158] Figure 84A The expression of HA-CFTR in F508del / F508del hBE (donor 20160524CF) cells administered with composition X is shown. Figure 84B The rescue of chloride ion flux in hBE is shown. Figure 84C The quantification of CFTR bands by Western blot analysis is shown.

[0159] Figure 85A The expression and apical translocation of HA-CFTR in K710X / L467 (ND13816) hBE cells after exposure to aerosolized composition B are shown. Figure 85B The rescue of chloride ion flux in hBE is shown.

[0160] Figure 86 The HA-CFTR exhibits significant signs of fibrosis after exposure to aerosolized composition B in K710X / L467 (ND13816) culture.

[0161] Figure 87A The expression and translocation of HA-CFTR in K710X / L467(ND13816)hBE cells after exposure to aerosolized composition X are shown. Figure 87B The merged image is shown.

[0162] Figure 88 Translocation and granulation formation of HA-CFTR fibrosis were shown in K710X / L467 (ND13816) cells after exposure to aerosolized composition X.

[0163] Figure 89 The expression and translocation of HA-CFTR in F508del / F508del hBE (donor TXCF042716) cells after exposure to aerosolized composition X are shown.

[0164] Figure 90 Immunofluorescence images of F508del / F508del hBE (KKD017K) administered with composition B / HA-CFTR are shown.

[0165] Figure 91 The expression levels of HA-CFTR in different F508del / F508del hBE donor cells after administration of composition B, Y, or X are shown.

[0166] Figure 92 The expression levels of HA-CFTR and chloride flux are shown after administration of composition B, Y, or X to unresponsive genotypes.

[0167] Figures 93A to 93B It was shown that SORT LNP for PCD projects cannot salvage the CFTR function in F508del / F508del hBE when delivered via aerosol. Figure 93A The representative trace of chloride ion flux is shown. Figure 93B The results of the transepithelial resistance (TEER) measurements are shown (top) and the salvage of chloride ion flux is shown (bottom).

[0168] Figure 94 The results of LDH release measurements are shown, used to detect the cytotoxicity of aerosolized formulations.

[0169] Figure 95 This is a coordinate graph illustrating the DNAI1-HA mRNA levels in different lung regions 6 hours after administration.

[0170] Figure 96 This is a coordinate graph illustrating the time progression of DNAI1-HA mRNA levels in lung tissue after administration of the composition A-DNAI1-HA. The plot shows the mean ± standard deviation of values ​​from three sampled lung regions for each animal. N = 2 animals per group at each time point.

[0171] Figure 97 This is a coordinate graph illustrating the time course of 4A3-SC7 lipid levels in lung tissue following administration of the composition A-DNAI1-HA. The plot shows the mean ± standard deviation of values ​​from three sampled lung regions for each animal. N = 2 animals per group at each time point.

[0172] Figure 98 This is a coordinate graph illustrating the time course of 14:0 EPC lipid levels in lung tissue following administration of the composition A-DNAI1-HA. The plot shows the mean ± standard deviation of values ​​from three sampled lung regions for each animal. N = 2 animals per group at each time point.

[0173] Figure 99This is a coordinate graph illustrating the time course of DMG-PEG lipid levels in lung tissue following administration of the composition A-DNAI1-HA. The plot shows the mean ± standard deviation of values ​​from three sampled lung regions for each animal. N = 2 animals per group at each time point.

[0174] Figure 100 This is a coordinate graph illustrating the analysis of cell-specific expression of DNAI1-HA protein in the lungs 6 hours after administration by multiplex immunofluorescence. The DNAI1-HA+ population percentage for each cell type was calculated by combining cell counts from lung sections from all four examinations for each animal. The total number of cells counted per animal ranged from 225,419 to 319,654. Specific airway cell types were stained using the following markers: rod cells (SCGB1A1 / uterine globin), goblet cells (MUCSB), basal cells (cytokeratin 5), ciliated cells (acetylated tubulin), alveolar type II (ATII) cells (presuractive protein C), epithelial cells (EpCAM), and DNAI1-HA (HA epitope tag).

[0175] Figure 101 This is a coordinate graph illustrating the analysis of cell-specific expression of DNAI1-HA protein in the lungs 6 hours post-administration using multiplex immunofluorescence. Two tracheal sections, proximal and carina, were collected from each animal. For Group 1 (carrier) and Group 2 (low dose), carina tracheal sections from each animal were analyzed. For Group 3 (high dose), two tracheal sections were analyzed. For each tracheal section examined, the DNAI1-HA+ population percentage for each cell type was calculated. The total number of cells counted per section ranged from 5,604 to 25,436. Individual data points for each treated animal and mean ± standard deviation for each group are shown (N=2 for Groups 1 & 2; N=4 for Group 3). Specific airway cell types were stained using the following markers: rod cells (SCGB1A1 / uterine globin), goblet cells (MUC5B), basal cells (cytokeratin 5), ciliated cells (acetylated tubulin), epithelial cells (EpCAM), and DNAI1-HA (HA epitope tag).

[0176] Figure 102This is a coordinate graph illustrating the analysis of cell-specific expression of DNAI1-HA protein in the nasopharynx and oropharynx 6 hours post-administration by multiplex immunofluorescence. Two nasopharyngeal or oropharyngeal sections were collected from each animal. For Group 1 (solvent) and Group 2 (low dose), one section from each animal was analyzed. For Group 3 (high dose), two sections were analyzed. For each section examined, the DNAI1-HA+ population % for each cell type was calculated. The total number of cells counted for each section ranged from 58,993 to 145,142. Individual data points for each treated animal and mean ± standard deviation for each group are shown (N=2 for Groups 1 & 2; N=4 for Group 3). Specific airway cell types were stained using the following markers: rod cells (SCGB1A1 / uterine globin), goblet cells (MUC5B), basal cells (cytokeratin 5), ciliated cells (acetylated tubulin), epithelial cells (EpCAM), and DNAI1-HA (HA epitope tag).

[0177] Figure 103A This study describes Western blot analysis of lung samples from the high-dose group (0.34 mg / kg, Group 3) 6 hours post-exposure. For each case, 50 μg of total lung lysate protein was separated on an SDS-PAGE gel, transferred to a nitrocellulose membrane, and DNAI1-HA expression was detected using a rabbit anti-HA-HRP monoclonal antibody conjugate. Lung samples were collected from the right caudate lobe (Cd), cranial lobe (Cr), and middle lobe (Mi). As a positive control, 1.9 pg of recombinant human DNAI1-HA was added to each gel. The position of the DNAI1-HA band is indicated by arrows.

[0178] Figure 103B This study describes Western blot analysis of lung samples from the high-dose group (0.34 mg / kg, Group 3) 6 hours post-exposure. For each case, 50 μg of total lung lysate proteins were separated on an SDS-PAGE gel, transferred to a nitrocellulose membrane, and total endogenous monkey DNAI1 was detected using a rabbit anti-DNAI1 polyclonal antibody. Lung samples were collected from the right caudate lobe (Cd), cranial lobe (Cr), and median lobe (Mi). As a positive control, 1.9 pg of recombinant human DNAI1-HA was added to each gel. The positions of the DNAI1-HA bands are indicated by arrows.

[0179] Figure 104This is a coordinate graph illustrating hDNAI1 mRNA levels in lung tissue at 24 and 336 hours following administration of composition A-DNAI1. Individual values ​​for each sample, representing levels above the detection LOQ (limit of quantitation), are plotted. Two samples were tested from each of six animals in each treatment group (N=12). Solid lines indicate the median for each group. Dashed lines indicate the detection LOQ of 250 copies / μg total RNA. If no sample in a particular group exceeds the detection LOQ, a point at 10 copies / μg total RNA is shown on the graph.

[0180] Figure 105 This is a coordinate graph illustrating hDNAI1 mRNA levels in liver tissue at 24 and 336 hours following administration of composition A-DNAI1. Individual values ​​for each sample, representing levels above the detection LOQ (limit of quantitation), are plotted. Two samples were tested from each of six animals in each treatment group (N=12). Solid lines indicate the median for each group. Dashed lines indicate the detection LOQ of 250 copies / μg total RNA. If no sample in a particular group exceeds the detection LOQ, a point at 10 copies / μg total RNA is shown on the graph.

[0181] Figure 106 This is a coordinate graph illustrating hDNAI1 mRNA levels in spleen tissue at 24 and 336 hours following administration of composition A-DNAI1. Individual values ​​for each sample, representing levels above the detection LOQ (limit of quantitation), are plotted. One sample was taken from each of six animals in each treatment group (N=12). Solid lines indicate the median for each group. Dashed lines indicate the detection LOQ of 250 copies / μg total RNA. If no sample in a particular group exceeds the detection LOQ, a point at 10 copies / μg total RNA is shown on the graph.

[0182] Figure 107 This is a coordinate graph illustrating hDNAI1 mRNA levels in whole blood at 24 and 336 hours following administration of composition A-DNAI1. Individual values ​​above the assay LOQ are plotted for each sample. Samples were collected from six animals (3 males / 3 females; N=6) in each treatment group at each time point. Solid lines indicate the median for each group. Dashed lines indicate the assay LOQ of 250 copies / mL. If no sample in a particular group is above the assay LOQ, a point at 10 copies / mL is shown on the graph.

[0183] Figure 108These are immunofluorescence images of well-differentiated wild-type hBE cultures. Well-differentiated WT-hBE cultures (35 days post-ALI) were stained with cell type-specific antibodies against ciliated cells (acetylated α-tubulin, AT), rod cells (rod cell 10-kDa protein, CC10 or SCGB1A1), goblet cells (mucin 5AC, MUC5AC), and basal cells (cytokeratin 5 or CK5). Cell nuclei were stained using Hoechst. Each image is a collection of nine consecutive fields of view (FoV) stitched together. Each FoV was collected at 40x magnification.

[0184] Figure 109 This is a Western blot analysis showing the levels of DNAI1 (top) and DNAI2 (bottom) in WT-hBE and DNAI1-KD hBE cultures at 12, 15, and 22 days after ALI (air-liquid interface).

[0185] Figure 110 These are immunofluorescence images of WT-hBE and DNAI1-KD hBE cultures using ciliated cell-specific markers (acetylated α-tubulin and DNAI). Ciliated cells in WT-hBE colocalize with DNAI1, while DNAI1 protein is detected only in a few ciliated cells in DNAI1-KD hBE.

[0186] Figure 111 This is a coordinate plot illustrating ciliary activity in control and DNAI1-KD hBE cultures at 14, 18, and 21 days post-ALI. WT-hBE controls (untransduced) and WT-hBE transduced with TurboGFP or shRNA constructs were grown at ALI for 21 days under puromycin selection. Ciliary activity was measured using high-speed video microscopy and SAVA software (Ammons Engineering). The GFP-hBE control demonstrates that lentiviral transduction alone does not lead to loss of ciliary activity.

[0187] Figure 112 This is a coordinate graph (Western blot) illustrating the dose-dependent increase in DNAI1-HA protein expression 24 hours after treatment. Using 150 µg (0.9 µg / cm²) 2 ), 300µg dose (1.9µg / cm) 2 ) or 500µg (3.0µg / cm) 2 A single atomization treatment of well-differentiated DNAI1-KD hBE cultures (33 days after ALI) with composition A-DNAI1-HA.

[0188] Figure 113This is an immunofluorescence imaging study illustrating the incorporation of newly translated DNAI1-HA protein into the ciliary axons of DNAI1-KD hBE 72 hours after nebulization. Well-differentiated KD-hBE (33 days post-ALI) underwent two consecutive nebulization treatments with the A-DNAI1-HA composition at a dose of 300 μg (1.9 μg / cm²) for two consecutive days. The subplots show the DNAI1-HA protein in ciliated cells, acetylated α-tubulin in the ciliary axons, and their co-localization. Scale bar = 20 μm, 1x1 pixel merge; 25 ms exposure, z-stacked, deconvolutioned images. Untreated controls were stained using the same method as the treated samples.

[0189] Figure 114 This is a graph illustrating the dose-response study of DNAI1-HA protein incorporation into cilia 72 hours after nebulization. 150 µg (0.9 µg / cm²) was used. 2 ) or 300µg dose (1.9µg / cm) 2 ) or 500µg dose (3.0µg / cm) 2 The composition A-DNAI1-HA was used to treat well-differentiated DNAI1-KD hBE (33 days after ALI) with one or two atomizations.

[0190] Figure 115 This is an immunofluorescence imaging illustration demonstrating the persistent incorporation of DNAI1-HA protein into the axonils of ciliated cells following a single basolateral application of the composition A-DNAI1-HA. Differentiated hBEs were treated by adding the composition A-DNAI1 (10 μg / mL) to the basolateral culture medium. After a single treatment (the medium containing the formulation was replaced with fresh medium after 5 hours), the treated inserts were fixed at different time points (1d, 2d, 7d, 14d, and 24d refer to the days following the single basolateral treatment) for immunofluorescence localization. The images show the immunofluorescence localization of cilia (red, acetylated α-tubulin), the immunofluorescence localization of DNAI1-HA (green, HA), and the colocalization of DNAI1-HA protein with cilia (yellow).

[0191] Figure 116 This is a coordinate graph and Western blot illustrating the kinetics of newly translated DNAI1-HA protein in WT-hBE after nebulization of the A-DNAI1-HA composition. The nebulization was performed using 300 µg (1.9 µg / cm³) of [amount missing]. 2 The A-DNAI1-HA composition was used to treat well-differentiated WT-hBE (36 days after ALI). Inserts were collected at different time points after treatment, and 10 μg of total protein was analyzed on Western blot using an anti-HA antibody. NTC refers to untreated cultures.

[0192] Figure 117This is a coordinate graph illustrating the functional rescue of ciliary activity achieved by treatment with the nebulized composition A-DNAI1 in a DNAI1-KD HBE. Ciliary activity in the hBE was recorded and analyzed using Sisson-Amlys Video Analysis (SAVA) software, resulting in ciliary activity area % scores for approximately 700 individual non-overlapping fields of view (FOV). DNAI1 mRNA-treated cultures exhibited significantly higher levels of ciliary activity after 2, 4, and 6 treatments compared to tdTomato mRNA-treated cultures. The increased activity was statistically significant as determined by the Welch t-test. *=P<0.05, **=P=0.001, ****=P<0.0001, ns=not significant (P>0.05). Ciliary beating frequencies (right coordinate graph) of FOVs with an activity area % >29% (those with an activity area % greater than that observed in any tdTomato-treated cultures) fell within the normal range of 5 Hz to 15 Hz.

[0193] Figure 118 This is a coordinate graph illustrating the deposition distribution of aerosol characteristics simulated by Yeh / Schum, Weibel, and PNNL airway morphology measurement models in healthy adults using the eFlow nebulizer.

[0194] Figure 119 This is a coordinate graph illustrating model-dependent and generation-dependent MMPD sediment fraction predictions.

[0195] Figure 120 It is a schematic representation of a symmetrical airway model, illustrating the conduction airway (consisting of generations 0 to 8 representing the trachea, bronchi, and bronchioles, and generations 9 to 16 representing the terminal bronchioles) and the alveoli or lung airways (generations 17 to 23).

[0196] Figure 121 This is a coordinate graph illustrating simulated human deposition at generations 0 to 8 (trachea, bronchi, and bronchioles), generations 9 to 16 (bronchioles), and generations 0 to 16 (the entire TB region) as varied with nebulized composition A and a human airway model.

[0197] Figure 122 This is a coordinate graph illustrating the deposition distribution of NHP while wearing a mouth and nose mask, simulated using experimental aerosol characteristics.

[0198] Figure 123 A diagram of an exemplary embodiment having six lipid components is shown.

[0199] Figure 124 The rescue of CFTR function in hBE cells (G542X / F508del) derived from CF patients by various six-component lipid nanoparticles is demonstrated.

[0200] Figure 125 The encapsulation efficiency (%) of various six-component lipid nanoparticles before and after atomization is shown.

[0201] Figure 126A The CFTR function of CF patient-derived hBE cells (F508del / F508del) treated with composition 3D7 was demonstrated in a dose-dependent manner.

[0202] Figure 126B Protein expression analysis of CF patient-derived hBE cells (F508del / F508del) treated with composition 3D7 is shown in a dose-dependent manner.

[0203] Figure 127A The CFTR function of hBE cells (F508del / F508del) from CF patients treated with composition 3D7 at different time points is shown.

[0204] Figure 127B The results of transepithelial resistance (TEER) measurements of hBE cells (F508del / F508del) from CF patients treated with composition 3D7 at different time points are shown.

[0205] Figure 127C Protein expression analysis of hBE cells (F508del / F508del) derived from CF patients treated with composition 3D7 at different time points is shown.

[0206] Figure 128A In vivo images of rats nebulized with the composition 3D7 are shown.

[0207] Figure 128B Quantitative in vivo images of rats nebulized with the composition 3D7 are shown.

[0208] Figure 129A The mucociliary clearance of CF transgenic ferrets administered with composition 3D7 containing CFTR mRNA is shown.

[0209] Figure 129B The changes in mucociliary clearance in two CF transgenic ferrets administered with composition 3D7 containing CFTR mRNA are shown. One ferret was evaluated 24 hours after treatment, and the other ferret was evaluated at 1, 8, 15, and 48 days after treatment.

[0210] Figure 130A The proliferation of ionocytes in the apical region of the CF transgenic ferret is shown.

[0211] Figure 130B The study showed colocalization of cre-induced GFP with ion cells and CFTR at the apical cap of CF transgenic ferrets.

[0212] Figure 130C The study demonstrates the delivery of its target gene to ciliated cells in CF ferrets via the composition 3D7.

[0213] Figure 131A Gel images of the fragments before atomization analysis are shown. Figure 131B Gel images of fragment analysis in atomized LNPs are shown.

[0214] Figure 132A EGFP-positive cells after treatment with the composition 3D7 or cell control prepared with 2- or 3-gene editing groups are shown. Figure 132B BFP-positive cells after treatment with the composition 3D7 or cell control prepared with the 2- or 3-gene editing group are shown. Figure 132C The LDH levels in cells treated with the composition 3D7 or cell control prepared with 2- or 3-gene editing groups are shown. Detailed Implementation

[0215] This document provides a lipid nanoparticle (LNP) composition designed to exceed the traditional lipid count in LNPs, increasing it from five to six or more. This disclosure allows LNPs to incorporate blends of two or more selective organ-targeting (SORT) lipids. Notable aspects of these LNPs include reduced toxicity and improved encapsulation efficiency compared to their five-component counterparts. In some embodiments, the encapsulation efficiency is maintained to a greater extent after atomization compared to five-component LNPs. In some embodiments, the LNP composition comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or more lipids, wherein at least 1, 2, 3, 4, 5 or more lipids are SORT lipids.

[0216] Therefore, this document provides an LNP composition comprising at least two selective organ-targeting (SORT) lipids and / or at least six lipids. In some embodiments, the LNP composition may comprise ionizable cationic lipids, optionally two or more ionizable lipids; and permanent cationic lipids, optionally two or more permanent cationic lipids.

[0217] I. Definition

[0218] Specific features (including method steps) of this disclosure are mentioned in the summary and detailed description of the invention, as well as in the following claims and drawings. It should be understood that this disclosure includes all possible combinations of such specific features. For example, where a specific feature is disclosed in the context of a particular aspect or embodiment of this disclosure or in a particular claim, that feature may also be used to the extent possible in combination with and / or in the context of other specific aspects and embodiments of this disclosure, and is generally used in this disclosure.

[0219] In the case of a method comprising two or more qualifying steps mentioned herein, the qualifying steps may be performed in any order or simultaneously (unless the context precludes such a possibility), and the method may include one or more other steps that are performed before any qualifying step, between two qualifying steps, or after all qualifying steps (unless the context precludes such a possibility).

[0220] Unless expressly stated otherwise, the practice of this disclosure will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and / or cell biology, which are within the skill of a person skilled in the art, many of which will be described below for illustrative purposes. These techniques are explained in detail in the literature. It should be understood that this disclosure is not limited to any particular method, scheme, and reagent described, as these may vary depending on the context in which they are used by someone skilled in the art.

[0221] All publications and patents mentioned herein are incorporated herein by reference in their entirety, as if each individual publication or patent had been expressly and separately indicated as incorporated by reference. In case of conflict, this disclosure (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or in any way an implication that they constitute valid prior art or are part of common general knowledge in any country of the world.

[0222] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. Specifically, features described in one part of this disclosure may be combined with features in any other part.

[0223] While exemplary embodiments have been described and depicted, it should be understood that various changes may be made to these exemplary embodiments without departing from the spirit and scope of the invention.

[0224] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Various scientific dictionaries including those containing the terms included herein are well-known and available to those skilled in the art. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, some preferred methods and materials are described herein. Therefore, the terms defined immediately thereafter will be described more fully by referring to the entire specification.

[0225] The singular forms “a,” “a,” and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0226] As used herein, the term "about" means a range of values ​​that would be reasonably thought by one of ordinary skill in the art to be similar to the specified value. In embodiments, "about" means within the standard deviation of a measurement result generally acceptable in the art. In some embodiments, "about" means the range extends to + / -10%, + / -5%, + / -3%, or + / -1% of the specified value.

[0227] This article uses the term "at least" followed by a number to indicate the starting value of a range that begins with that number (which can be a range with or without an upper limit, depending on the variable being defined). For example, "at least 1" means 1 or greater than 1.

[0228] This document uses the term "at most" followed by a number to indicate the end value of a range that ends with that number (which can be a range with, for example, 1 or 0 as its lower limit, or a range without a lower limit, depending on the defined variable). For example, "at most 4" means 4 or less, and "at most 40%" means 40% or less. In this disclosure, when a range is given as "(first number) to (second number)" or "(first number) - (second number)", this means a range with the lower limit of the first number and the upper limit of the second number. For example, 25 to 100 mm means a range with the lower limit of 25 mm and the upper limit of 100 mm.

[0229] Throughout this specification, unless the context otherwise requires, the words “comprising” or “including” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements. For example, a composition “comprising” components A, B, and C may consist of components A, B, and C (i.e., containing only components A, B, and C), or may contain not only components A, B, and C, but also one or more other components.

[0230] As used herein, the term "consisting of" refers to, but is not limited to, anything following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements exist. The phrase "substantially constitutes" means to include all elements listed following that phrase, and is limited to other elements that do not interfere with or affect the activity or effect specified in this disclosure for the listed elements. Thus, the phrase "substantially constitutes" indicates that the listed elements are necessary or mandatory, but other elements are not optional, and may or may not exist depending on whether they affect the activity or effect of the listed elements.

[0231] As used herein, terms such as “specific / specifically / specificity” for a composition refer to the ability of a composition to have a specific effect (such as, but not limited to, inhibition) on a specific molecular target while having little or no effect on other proteins in the cell.

[0232] As used herein, the term "aerosol particle" refers to liquid or solid particles suspended in a gas (e.g., air). Aerosol particles include, but are not limited to, liquid aerosol droplets. Typically, aerosols have aerosol particles of, for example, between about 1 micrometer and about 100 micrometers, or in some cases between about 1 micrometer and about 20 micrometers, or between about 1 micrometer and about 10 micrometers.

[0233] As used herein, the term “selective delivery” refers to the delivery of an application amount of the composition to a target organ (e.g., lung), tissue, or cell at a rate of at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%).

[0234] As used herein, the term "contact" refers to the permission for two substances to interact, such as through chemical interactions (including ionic, nonionic, polar, hydrophobic, or hydrophilic interactions), or through physical contact recognized in the art, wherein the two substances can be lipid nanoparticles and cells, mucus, or tissue walls. In cell culture, LNPs can be contacted with cells by mixing the LNP composition with a suitable cell culture medium, or by contacting aerosol particles of the LNP composition with the cell culture, such that the aerosol particles dissolve into the liquid in the cell culture medium or the liquid or mucus surrounding the cells, thereby allowing the LNPs to contact the cells.

[0235] As used herein, “prevention” or any grammatical variation thereof means suppressing the development of the disease in a subject or patient who may be at risk of and / or susceptible to the disease but has not yet experienced or exhibited any or all of the pathology or symptoms of the disease, and / or mitigating the development of the pathology or symptoms of the disease in a subject or patient who may be at risk of and / or susceptible to the disease but has not yet experienced or exhibited any or all of the pathology or symptoms of the disease. Prevention can be complete (without detectable symptoms) or partial, such that fewer symptoms are observed than would be present without treatment.

[0236] Throughout this specification, references such as "an embodiment," "an embodiment," "another embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," "a further embodiment," or "some embodiments," or any combination thereof, mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of this disclosure. Therefore, the foregoing phrases appearing throughout this disclosure do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, any particular feature, structure, or characteristic may be combined in any suitable manner.

[0237] As used herein, the term "nebulizer" refers to a device that converts a liquid (e.g., a solution, dispersion, or suspension) into aerosol particles. This process is called "nebulization." The term "nebulization" refers to the process or state of converting a solution, aqueous dispersion, or suspension (such as a liquid pharmaceutical composition) into an aerosol. The terms "mesh nebulizer" and / or "vibrating mesh nebulizer" refer to nebulizers that achieve atomization by passing an input substance through a mesh. Using a piezoelectric element, the mesh can be vibrated, and these vibrations disperse the liquid into the surrounding air. The mesh of a mesh nebulizer can be characterized by its pore size and / or electrical charge. Mesh nebulizers are driven by a piezoelectric element and use ultrasonic frequencies to vibrate the mesh. When liquid passes through the mesh, the vibration of the mesh can lead to the generation of aerosol particles. In contrast, ultrasonic nebulizers generate ultrasound directly into the solution, thereby generating aerosol particles on the liquid surface. Other methods of generating aerosols include, but are not limited to, pressurized metered-dose inhalers, dry powder inhalers, jet nebulizers, soft fog inhalers, condensed aerosols, and aqueous nasal sprays, as described in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd edition, 2021). Nebulizers are also characterized as either ventilated or non-ventilated. Exemplary mesh nebulizers are described, for example, in U.S. Patent No. 9,061,303. Exemplary mesh nebulizers that can be used to practice the methods disclosed in this invention include, but are not limited to, those made by Aerogen. ® PARI ® Activaero ®and Omron ® Those that are manufactured and sold.

[0238] As used herein, the terms "aerosolization" or "aerosol" refer to a suspension of fine liquid and / or solid particles dispersed in a gas (e.g., air). A dispersion of liquid-containing particles in air or a gas is also included. An aerosol generated by an nebulizer can refer to a mixture of air and vaporized particles generated by an aerosol-generating material (such as any aerosolized pharmaceutical composition described herein). For example, an nebulizer can convert the liquid phase of any pharmaceutical composition described herein into a gas phase via, for example, ultrasonic vibration. In another example, an air jet mill can generate a dry powder aerosol from the dried lipid nanoparticles of this disclosure. Non-limiting examples of air jet mills include Jet-O-Mizer, Trost air jet mill, and Microjet. Generally, the aerosol particles of this disclosure have low settling velocities and relative airborne stability. In some embodiments, the nebulizer converts a liquid pharmaceutical composition into an aerosolized pharmaceutical composition.

[0239] As used herein, the term "output rate" refers to the rate at which a liquid is atomized into an aerosol, typically expressed as the volume of liquid converted into an aerosol per given time (e.g., milliliters per minute or mL / min). The output rate can be determined by measuring the decrease in the volume of the input liquid over time.

[0240] As used herein, the term "apparent pKa" refers to the total dissociation constant of all titratable groups in a lipid nanoparticle. Apparent pKa is an experimentally determined value for a molecule or nanoparticle. Apparent pKa can be expressed as the pH at which the number of ionized (protonated) and deionized groups in the system are equal. The surface charge and ionic interactions of the nanomaterial assembled in the nanoparticle can be estimated from the apparent pKa. The apparent pKa of a nanoparticle can be the result of the average ratio of all ionized groups to deionized groups in the nanoparticle. Therefore, apparent pKa is not the intrinsic pKa value of any single molecule. The apparent pKa of nanoparticles can be measured using various techniques. For example, the 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) acid-base titration fluorescence method is widely used to determine the apparent pKa of blank nanoparticles.

[0241] As used herein, the term "therapeutic effective dose" refers to an amount of therapeutic agent sufficient to treat a disease, symptom, or condition. For example, regarding the use of LNPs with mRNA payloads to treat conditions such as cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), a therapeutic effective dose is a dose or concentration of mRNA (e.g., CFTR or PCD mRNA) capable of eradicating, inhibiting, preventing, or slowing the progression of all or part of the respiratory symptoms of CF or PCD, or some combination thereof. For a given parameter, a therapeutic effective dose will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease in "folds." For example, a therapeutic effective dose can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to a control. "Therapeutic effective amount" can vary based on, for example, but not limited to, the compound, the disease or condition and / or its symptoms, the severity of the disease or condition and / or its symptoms, the age, weight and / or health status of the subject to be treated, and the judgment of the prescribing physician. The appropriate amount in any given situation can be determined by a person skilled in the art or can be determined through routine experiments.

[0242] The term "pharmaceutically acceptable" means, to a reasonable extent of medical judgment, those compounds, materials, compositions, carriers, mediators, diluents, excipients and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, in proportion to a reasonable benefit / risk ratio, and / or are generally chemically and / or physically compatible with the other components constituting the formulation.

[0243] The terms “lung disease,” “pulmonary disorder,” and “pulmonary symptom” broadly refer to diseases or conditions affecting the lungs. Lung diseases are characterized by symptoms including, but not limited to, dyspnea, cough, airway discomfort and inflammation, increased mucus, and / or pulmonary fibrosis. Non-limiting examples of lung diseases include primary ciliary dyskinesia (PCD) (also known as Katagone syndrome or immotile ciliary syndrome), cystic fibrosis, asthma, lung cancer, chronic obstructive pulmonary disease (COPD), bronchitis, emphysema, bronchiectasis, pulmonary edema, pulmonary fibrosis, sarcoidosis, pulmonary hypertension, pneumonia, tuberculosis, interstitial pulmonary fibrosis (IPF), interstitial lung disease (ILD), acute interstitial pneumonia (AIP), respiratory bronchiolitis-associated interstitial lung disease (RBILD), desquamative interstitial pneumonia (DIP), nonspecific interstitial pneumonia (NSIP), idiopathic interstitial pneumonia (IIP), bronchiolitis obliterans with organizing pneumonia (BOOP), restrictive lung disease, and pleurisy.

[0244] As used herein, the term "lipid nanoparticle" refers to a carrier or mediator formed of one or more lipid components for delivering a payload (e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide, or oligonucleotide) in the context of drug development. Lipid nanoparticles may have one or more lipids, with at least one dimension being nanoscale (e.g., 1 nm to 1000 nm). Typically, lipid nanoparticle compositions for delivery consist of one or more lipids, such as, but not limited to, synthetic ionizable or cationic lipids, phospholipids, structural lipids, and polyethylene glycol (PEG) lipids. These compositions may also contain other lipids. In some embodiments, at least one therapeutic agent (e.g., mRNA) may be trapped in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portions of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other adverse effects (e.g., adverse immune responses) triggered by biological mechanisms of the target subject, tissue, and / or cells. In some embodiments, the lipid nanoparticles contain at least one therapeutic agent (e.g., mRNA) which is either organized within reverse lipid micelles and encapsulated within a lipid monolayer membrane, or intercalated between adjacent lipid bilayers. In some embodiments, the lipid nanoparticles have a different morphology from traditional liposomes, which are characterized by a lipid bilayer surrounding an aqueous core. In some embodiments, the lipid nanoparticles are substantially non-toxic. In some embodiments, the therapeutic agent (e.g., mRNA) is resistant to degradation by intracellular or intercellular enzymes in aqueous solution.

[0245] As used herein, the term "neutral phospholipid" refers to a phospholipid that has little or no net charge at physiological pH. In some embodiments, neutral phospholipids are zwitterions, although other types of net neutral phospholipids are known and usable. In some embodiments, neutral phospholipids can be any vesicle-forming lipid having two hydrocarbon chain moieties (effectively producing a stable bilayer structure) and a polar head group (with no net charge between pH 5.5 and 8.5). Neutral phospholipids with a variety of hydrocarbon chain (e.g., acyl chain) groups of varying chain lengths and saturations are readily available or can be isolated or synthesized using well-known techniques.

[0246] As used herein, the term "PEG-lipid" refers to a lipid modified with a polyethylene glycol unit. In some embodiments, the PEG-lipid comprises dimyristoylglycerol (DMG). In some embodiments, the PEG-lipid comprises 1,2-distearate-sn-glycerol-3-phosphoethanolamine (DSPE).

[0247] As used herein, the term "sterol" refers to a subgroup of steroids having a hydroxyl group at the 3-position of the A ring in a sterane ring system. "Cholesterol" is a sterol with a structure of four fused hydrocarbon rings (sterane ring system), one end having a polar hydroxyl group and the other an eight-carbon branched aliphatic tail. Unbound by theory, the tetracyclic structure of cholesterol contributes to cell membrane fluidity because the molecule is in a trans conformation, making all parts except the cholesterol side chains rigid and planar. Cholesterol affects the fluidity, thickness, compressibility, water permeability, and intrinsic curvature of the lipid bilayer, for example, in LNPs. For example, "sterol" can refer to cholesterol or sitosterol.

[0248] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more reading frames or regions.

[0249] As used in this article, the term "shRNA" or "short hairpin RNA" refers to a short sequence of RNA that can form tight hairpin turns and can be used to silence gene expression.

[0250] As used in this article, the term “microRNA” refers to a non-coding RNA consisting of approximately 22 ribonucleotides that can silence messenger RNA by pairing with complementary sequence bases in its target mRNA, thereby regulating gene expression in the post-transcriptional stage.

[0251] As used herein, the phrase “N / P ratio” refers to the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide payload.

[0252] As used in this article, the phrase "lipid:RNA ratio" refers to the number of milligrams of lipids per milligram of mRNA drug substance, which affects the encapsulation efficiency of lipid nanoparticles.

[0253] As used herein, the phrase "lung cell" refers to lung airway cells. Examples of lung airway cells that can be targeted by delivering the compositions of this disclosure include, but are not limited to, basal cells, secretory cells (such as goblet cells and rod cells), ciliated cells, and any combination thereof.

[0254] As used herein, the term "goblet cell" refers to a type of secretory cell. Goblet cells are located in the epithelium of the conduction airway, and their apical surface typically protrudes into the lumen, a location suitable for their rapid response to inhaled airway injury.

[0255] As used herein, the phrase "ciliated cell" refers to a cell with a ciliated structure on its surface. Examples of ciliated cells include, but are not limited to, respiratory tract ciliated cells, fallopian tube ciliated cells, endometrial ciliated cells, reticulum testis ciliated cells, efferent tubule ciliated cells, and / or ciliated ependymal cells. Human respiratory tract ciliated cells may have 200 to 300 cilia on their surface. A cilia is a slender, movable cylindrical projection extending from the apical cell membrane, approximately 0.25 mm in diameter, containing microtubules and cytoplasm continuous with the cell's cytoplasm. Human tracheal cilia can be 5 mm to 8 mm long, gradually shortening in the more distal airways.

[0256] The term "subject" refers to a living organism to whom any composition as described herein may be administered. A subject may have a disease or condition that can be treated by administration of the aerosolized pharmaceutical composition provided herein, or be at risk of having such a disease or condition. Non-limiting examples of subjects include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammals. In some embodiments, the subject is a human.

[0257] The terms “identity,” “same,” and “sequence identity” refer to the degree to which two optimally aligned polynucleotide or polypeptide sequences remain unchanged throughout the alignment window of their components (e.g., nucleotides or amino acids). “Identity” can be readily calculated by known methods, including but not limited to those described in Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). Thus, a polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. The terms “sequence identity percentage,” “identity percentage,” or “same as…” refer to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) compared to the test (“test”) polynucleotide molecule (or its complementary strand) when two sequences are optimally aligned. In some embodiments, “identity percentage” may refer to the percentage of identical amino acids in an amino acid sequence. For sequence comparison, one sequence is used as a reference sequence and compared to the test sequence. The term “reference sequence” refers to the molecule compared to the test sequence. Sequence alignment methods used to compare and determine the percentage of sequence identity are well known in the art. The best sequence alignment for comparison can be performed, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol.Biol.48:443.

[0258] As used herein, in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, the phrase “substantially identical” or “substantially identical” means that two or more sequences or subsequences, when compared and aligned to achieve maximum correspondence, have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid residue identity, as measured by one of the following sequence comparison algorithms or by visual inspection. In some embodiments of this disclosure, substantial identity exists within continuous nucleotide regions of the nucleotide sequence of this disclosure, the length of which is about 16 nucleotides to about 30 nucleotides, about 18 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, about 150 nucleotides to about 200 nucleotides, about 250 nucleotides to about 400 nucleotides, about 500 nucleotides to about 750 nucleotides, about 700 nucleotides to about 1000 nucleotides, about 1250 nucleotides to about 2500 nucleotides, about 2000 nucleotides to about 4000 nucleotides or more, and any range therewith, up to the full length of the sequence. In some embodiments, the sequences are substantially identical over the entire length of the coding region. In some embodiments, substantially identical nucleotide or protein sequences perform substantially the same function (e.g., isopentenyltransferase activity).

[0259] The terms "fragment" or "variant" refer to any functional fragment, variant, derivative, or analog of a polynucleotide, polypeptide, or biomolecule that possesses the in vivo or in vitro activity characteristic of the polynucleotide, polypeptide, or therapeutic agent. In some embodiments, the length of the fragment, variant, or analog is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or greater of the length of the polynucleotide, polypeptide, or biomolecule. Those skilled in the art can readily determine the functional expression of the fragment or variant by testing enzyme activity and the ability to prepare products as described herein.

[0260] The term “treatment” means to alleviate, relieve, delay, reduce, improve, or manage one or more of the symptoms of a subject’s condition. The term “treatment” may also mean to prevent a condition, delay its onset (i.e., before the clinical manifestation of the condition), or reduce the risk of the condition occurring or worsening.

[0261] As used herein, the phrase “chloride flux” refers to the mass of ions incorporated into stimulated cells, or the release of ions from stimulated cells. Measurements of chloride flux are described, for example, in Moran et al., J. Cystic Fibrosis 7:483-494 (2008).

[0262] As used herein, the phrase “transepithelial resistance (TEER)” is a method for measuring the resistance across a cell monolayer to confirm the integrity and permeability of the monolayer. Measurements of TEER are described, for example, in Srinivasan et al., J. Lab. Automation 20:107-126 (2015).

[0263] II. Compositions disclosed herein

[0264] This document provides compositions and methods related to aerosolized pharmaceutical compositions, such as methods for treating lung diseases or pulmonary conditions, some of which are characterized by dyspnea, cough, airway discomfort and inflammation, increased mucus and / or pulmonary fibrosis, or other conditions that can be treated by administering an agent in conjunction with an aerosolized pharmaceutical composition. The compositions and methods disclosed herein provide aerosol particles comprising lipid nanoparticles (LNPs), and the composition can deliver LNPs to, for example, the tracheobronchial region of a subject.

[0265] The aerosolized pharmaceutical composition comprises a lactate nucleus (LNP). In some embodiments, the LNP may be selectively delivered to one or more of goblet cells, secretory cells, rod cells, basal cells, or ionized cells. In some embodiments, the LNP may be selectively delivered to one or more of ciliated cells, rod cells, or basal cells.

[0266] In one aspect, this document provides a lipid composition comprising: (i) an ionizable lipid; (ii) an accessory lipid; (iii) a PEG-lipid; and (iv) a sterol. In some embodiments, the lipid composition further comprises (v) an additional ionizable or permanent cationic lipid.

[0267] In some embodiments, the lipid nanoparticles described herein can be prepared according to any of the methods described in International Patent Publications No. WO2016094342, WO2017048789, WO2017201091, WO2017205767, WO201 / 246203, WO2020051220, WO2022169508, WO2022204053 and WO2022204215; the contents of each of these patents are incorporated herein by reference in their entirety.

[0268] A. Ionizable lipids

[0269] In some embodiments of the lipid compositions disclosed herein, the lipid compositions comprise ionizable lipids. In some embodiments, the ionizable lipids are ionizable cationic lipids. In some embodiments, the ionizable cationic lipids may contain one or more groups that are protonated at physiological pH but deprotonated and uncharged at pH values ​​above the lipid pKa. The ionizable cationic groups may contain one or more protonable amines capable of forming cationic groups at physiological pH. The cationic ionizable lipids may further comprise one or more lipid components, such as two or more C6-C6 lipids. 24 Fatty acids with alkyl or alkenyl carbon groups. One or more lipid components may be attached to a cationic ionizable lipid via an ester bond, or may be further added to a sulfur atom via Michael addition. In some embodiments, one or more lipid components may be dendrimers, dendrites, polymers, or combinations thereof.

[0270] Ionizable lipids have their common meaning in the art and can refer to lipids comprising one or more charged moieties. In some embodiments, ionizable lipids may be positively or negatively charged. For example, an ionizable lipid may be positively charged at a lower pH, in which case it may be referred to as a “cationic lipid.” In some embodiments, an ionizable lipid may contain an amine group and may be referred to as an ionizable amino lipid. A charged moiety is a chemical moiety that carries a formal electronic charge (e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc.). A charged moiety can be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Non-limiting examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In a particular embodiment, the charged moieties contain an amine group. Non-limiting examples of negatively charged groups or their precursors include carboxylic acid groups, sulfonic acid groups, sulfate groups, phosphonic acid groups, phosphate groups, hydroxyl groups, etc. In some cases, the charge of the charged portion can vary with environmental conditions; for example, changes in pH can alter the charge of the portion and / or cause it to become charged or uncharged. Generally, the charge density of the molecule can be selected as needed.

[0271] It should be understood that the terms "charged" or "charged portion" do not refer to "partial negative charge" or "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their common meaning in the art. A "partial negative charge" can arise when a functional group contains a polarized bond, in which case the electron density is drawn towards one atom of the bond, thus forming a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this manner.

[0272] The lipid nanoparticle compositions disclosed herein may comprise one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may be positively or partially positively charged at physiological pH). Ionizable lipids can be selected from the non-restricted group consisting of the following: 3-(bisdodecylamino)-N1,N1,4-tris(dodecyl)-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]N1,N4,N4-tris(dodecyl)-1,4-piperazine diethylamine (KL22), 14,25-bis(tetrazyl)-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxacyclopentane (Dlin-K-DMA), and heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate. (Dlin-MC3-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxacyclopentane (Dlin-KC2-DMA), 1,2-dioleenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8[(3(3)-cholest-5-en-3-yloxy]octoxy)N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-l-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-l-yloxy]prop-1-amine (octyl-CLinDMA) (2R) and (2S) 2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3–[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA (2S)), 4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate (ALC-0315) or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxy-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In addition to these, ionizable lipids can also be lipids containing cyclic amine groups.

[0273] Ionizable lipids can also be compounds disclosed in International Patent Publication No. WO 2017075531 A1, the entire contents of which are incorporated herein by reference. Ionizable lipids can also be compounds disclosed in International Patent Publication No. WO 2015199952 A1, the entire contents of which are incorporated herein by reference. In one embodiment, the ionizable lipid may be selected from, but is not limited to, International Publications No. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, and WO2013086373. Ionizable lipids as described in U.S. Patent Nos. WO2013086354, 7,893,302, 7,404,969, 8,283,333 and 8,466,122, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and S20130225836; the contents of each of these patents are incorporated herein by reference in their entirety.

[0274] As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosane-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexacosane-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentacosane-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyldocododecane-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyltetracosane-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyltetracosane-14,17-diene-6-amine, (15Z,18Z)-N,N-dimethyltetracosane- 15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylhexadecane-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracosane-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltricarane-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctadecane-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylhexadecane-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexadecane-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentadecane-16,1 9-Dien-6-amine, (22Z,25Z)-N,N-dimethylhexadecane-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriacontane-21,24-dien-9-amine, (18Z)-N,N-dimethylhexadecane-18-en-10-amine, (17Z)-N,N-dimethylhexadecane-17-en-9-amine, (19Z,22Z)-N,N-dimethylhexadecane-19,22-dien-7-amine, N,N-dimethylhexadecane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacontane-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonyleicosaecan-11, [14-dien-l-yl]pyrrolidine, (20Z)-N,N-dimethylhexadecane-20-en-10-amine, (15Z)-N,N-dimethylhexadecane-15-en-10-amine, (14Z)-N,N-dimethylnonadecane-14-en-10-amine, (17Z)-N,N-dimethylnonadecane-17-en-10-amine, (24Z)-N,N-dimethyltritriane-24-en-10-amine, (20Z)-N,N-dimethylnonadecane-20-en-10-amine, (22Z)-N,N-dimethyltrimonane-22-en-10-amine, (16Z)-N,N-dimethylpentadecane-16-en-8-amine, (12Z,15Z)-N,N-Dimethyl-2-nonyldocodecane-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocodecane-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]docodecane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl] [Methylcyclopropyl]nonadecano-10-amine, N,N-dimethyl-1–[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyldodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N-dimethyl-1-[(9Z,12Z)- Octadecyl-9,12-dien-l-oxy]-3-(octoxy)prop-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadecyl-9,12-dien-l-oxy]-3-(octoxy)prop-2-amine, 1-{2-[(9Z,12Z)-octadecyl-9,12-dien-l-oxy]-1-[(octoxy)methyl]ethylpyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadecyl-9,12-dien-l-oxy]-3-[(5Z)-oct-5-en-l-oxy]prop-2-amine, 1-{2-[(9Z,12Z)-octadecyl-9,12-dien-l-oxy]-1-[(octoxy)methyl]ethylpyrrolidine (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-l-oxy]prop-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-l-oxy]prop-2-amine, N,N-dimethyl-1-(nonoxy)-3-[(9Z,12Z)-octadecane-9,12-dien-l-oxy]prop-2-amine, N,N-dimethyl-1–[(9Z)-octadecane-9-en-1-oxy]-3–(octyloxy)prop-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadecane-6,9,12-trien-1-yloxy]-3–(octoxy)prop-2-amine, (2S)-1-[(11Z,14Z)-eicosano-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentoxy)prop-2-amine, (2S)-1-(hexoxy)-3–[(11Z,14Z)-eicosano-11,14-dien-1-yloxy]-N,N-dimethylprop-2-amine, 1-[(11Z,14Z)-eicosano-11,14-dien-1-yloxy]-N,N-dimethyl-3 –(octoxy)prop-2-amine, 1–[(13Z,16Z)-docosahexadecane-13,16-dien-l-yloxy]-N,N-dimethyl-3–(octoxy)prop-2-amine, (2S)-1-[(13Z,16Z)-docosahexadecane-13,16-dien-l-yloxy]-3–(hexoxy)-N,N-dimethylprop-2-amine, (2S)-1-[(13Z)-docosahexadecane-13-dien-l-yloxy]-3–(hexoxy)-N,N-dimethylprop-2-amine, 1–[(13Z) [-dodecano-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, 1-[(9Z)-hexadecano-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)prop-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadecano-9,12-dien-1-yloxy]prop-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-decano-1-yloxy]-3-[(9Z,12Z)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-1 ...decano-1-[(3, Octane-9,12-dien-1-yloxy]prop-2-amine, N,N-dimethyl-1–(octoxy)-3-({8-[(1S,2S)-2-[(1R,2R)-2-pentylcyclopropyl]methylcyclopropyl]octoxy)prop-2-amine, N,N-dimethyl-1–[8-(2-octylcyclopropyl)octyl]oxy}-3–(octoxy)prop-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacontane-11,20,2-trien-10-amine, or pharmaceutically acceptable salts or stereoisomers thereof.

[0275] In some embodiments of the lipid compositions of this application, ionizable cationic lipids refer to lipids and lipid-like molecules having a nitrogen atom that can acquire a positive charge. Ionizable cationic lipids may be referred to as cationic lipids in the literature. Ionizable cationic lipids having an amino group typically have 2 to 6 hydrophobic tails, usually alkyl or alkenyl, such as C6-C6. 24 Alkyl or alkenyl groups, but may have at least one, at least two, at least three, at least four, at least five or more than six tails.

[0276] 1. Dendritic polymers

[0277] In some embodiments, the cationic ionizable lipid is a dendritic polymer. A dendritic polymer is a polymer exhibiting regular dendritic branching, formed by adding branching layers sequentially or in generations to or from a core, and characterized by a core, at least one internal branching layer, and a surface branching layer. (See Petar R. Dvornic and Donald A. Tomalia, Chem. In Britain, 641-645, August 1994.) Dendritic polymers include, but are not limited to, molecular structures having an initiator core, repeating layers (or generations) of repeating units regularly connected to the initiator core, and an outer surface with terminal groups connected to the outermost generation. A dendrite is a dendritic polymer having branches emanating from a focal point that is directly or indirectly connected to or can be connected to the core, thereby forming a larger dendritic polymer. In some embodiments, the dendritic polymer structure has repeating groups radiating outward from a central core, with each repeating unit of each branch doubling the number of repeating groups. In some embodiments, the dendritic polymers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms can be used to describe compounds that have a dendritic appearance (e.g., molecules that radiate outward from a single focal point).

[0278] Although dendritic polymers are polymers, they may be superior to conventional polymers because they possess controllable structures, a single molecular weight, numerous and controllable surface functional groups, and traditionally exhibit a spherical conformation after reaching a certain generation. Dendritic polymers can be prepared through the sequential reaction of each repeating unit to produce monodisperse, dendritic, and / or generational polymer structures. Individual dendritic polymers consist of a central core molecule, with dendritic wedge structures attached to one or more functional sites on this central core. Depending on the assembly monomers used in the preparation process, the surface layer of the dendritic polymer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.

[0279] The physical properties of dendritic polymers can be tuned by modifying the functional groups and / or chemical properties of the core, repeating units, and surface or terminal groups. Some properties that can be altered include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment ability. Dendritic polymers are typically described according to their generation or the number of repeating units in their branches. A dendritic polymer consisting only of a core molecule is called generation 0, while each consecutive repeating unit along all branches is generation 1, generation 2, and so on, up to the terminal or surface groups. In some embodiments, half-generation is possible because only the first condensation reaction with an amine occurs, without the second condensation reaction with a thiol.

[0280] The preparation of dendritic polymers requires a certain level of synthetic control, achieved through a series of stepwise reactions, including the construction of dendritic polymers by each successive group. Dendritic polymer synthesis can be convergent or divergent. During divergent dendritic polymer synthesis, molecules are assembled from the core to the periphery in a stepwise process involving linking one generation to the previous generation and then altering functional groups to proceed to the next stage of the reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization can produce highly branched molecules that are not monodisperse, also known as hyperbranched polymers. Due to steric effects, the repeating units of a dendritic polymer continue to react, forming spherical or globular molecules until steric overcrowding prevents complete reaction of a particular generation and destroys the monodispersity of the molecule. Therefore, in some embodiments, dendritic polymers of generations G1–G10 are particularly considered. In some embodiments, the dendritic polymer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range thereof. In some embodiments, the dendritic polymers used herein are G0, G1, G2, or G3. However, by reducing the spacer units in the branched polymer, the possible generations (such as 11, 12, 13, 14, 15, 20 or 25) can be increased.

[0281] Furthermore, dendritic polymers possess two main chemical environments: one formed by specific surface groups on the terminal branches; and the interior of the dendritic structure, which, due to its higher-order structure, is shielded from the influence of the physical medium and surface groups. Because of these different chemical environments, dendritic polymers have demonstrated many different potential applications, including in therapeutic applications.

[0282] In some embodiments of the lipid compositions disclosed herein, dendritic polymers are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. The dendritic polymers may comprise secondary or tertiary amines formed by reacting acrylate groups with primary or secondary amines, and thioethers formed by reacting methacrylates with thiol groups. Additionally, the repeating units of the dendritic polymer may contain groups that are biodegradable under physiological conditions. In some embodiments, the repeating units may contain one or more geminal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine, which allows dendritic polymerization to proceed only in one direction. In other embodiments, the core molecule is a polyamine having multiple distinct dendritic branches, each dendritic branch containing one or more repeating units. The dendritic polymer is formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms (such as nitrogen atoms). In some embodiments, the terminal groups are lipophilic groups, such as long-chain alkyl or alkenyl groups. In other embodiments, the terminal groups are long-chain haloalkyl or haloalkenyl groups. In other embodiments, the terminal group is an aliphatic or aromatic group containing an ionizable group, such as an amine (-NH2) or a carboxylic acid (-CO2H). In other embodiments, the terminal group is an aliphatic or aromatic group containing one or more hydrogen bond donors, such as a hydroxyl group, an amide group, or an ester group.

[0283] The cationic ionizable lipids of this disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separable in optically active or racemic form. Therefore, unless a specific stereochemical or isomeric form is specifically specified, all chiral, diastereomeric, racemic, epimeric, and geometrical isomers of the chemical formula are referred to. The cationic ionizable lipids may exist as racemates and mixtures of racemates, as single enantiomers, mixtures of diastereomers, and single diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the cationic ionizable lipids of this disclosure may have an S or R configuration. Furthermore, it is contemplated that one or more cationic ionizable lipids may exist as structural isomers. In some embodiments, the compounds have the same formula but differ in the manner in which the nitrogen atom at the core is attached. Without being bound by theory, it is believed that such cationic ionizable lipids are possible because the starting monomer reacts first with a primary amine and then statistically with any secondary amine present. Thus, the structural isomers may present a mixture of a fully reacted primary amine and then a reacted secondary amine.

[0284] The chemical formulas used to represent the cationic ionizable lipids of this disclosure typically show only one of several possible tautomers. For example, many types of ketone groups are known to exist in equilibrium with their corresponding enol groups. Similarly, many types of imine groups can exist in equilibrium with enamine groups. Regardless of which tautomer is depicted in the given formula, and regardless of which tautomer is most common, all tautomers of the given chemical formula are referred to herein.

[0285] The cationic ionizable lipids disclosed herein may also have the following advantages: they may be more effective, less toxic, have a longer duration of action, be more potent, produce fewer side effects, be more easily absorbed and / or have better pharmacokinetic characteristics (e.g., higher oral bioavailability and / or lower clearance) than compounds known in the prior art, and / or have other useful pharmacological, physical or chemical properties superior to those of compounds known in the prior art, whether for the indications described herein or other indications.

[0286] Furthermore, the atoms constituting the cationic ionizable lipids of this application are intended to include all isotopic forms of these atoms. Isotopes include atoms with the same atomic number but different mass numbers. As a general example and not a limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include... 13 C and 14 C.

[0287] It should be recognized that the anions or cations that form part of any salt form of the cationic ionizable lipids presented herein are not important, as long as the salt as a whole is pharmaceutically acceptable. Further examples of pharmaceutically acceptable salts and their preparation and use are presented in the Handbook of Pharmaceutical Salts: Properties, and Use (2002), the full text of which is incorporated herein by reference.

[0288] In some embodiments of the lipid compositions disclosed herein, the ionizable lipid is a dendritic polymer or dendrite. In some embodiments, the ionizable lipid comprises a positively charged ammonium group at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the ionizable lipid is a dendritic polymer or dendrite. In some embodiments, the ionizable lipid comprises at least two C6-C... 24 Alkyl or alkenyl groups.

[0289] The physical properties of dendritic polymers can be tuned by modifying the functional groups and / or chemical properties of the core, repeating units, and surface or terminal groups. Some properties that can be altered include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment ability. Dendritic polymers are typically described according to their generation or the number of repeating units in their branches. A dendritic polymer consisting only of a core molecule is called generation 0, while each consecutive repeating unit along all branches is generation 1, generation 2, and so on, up to the terminal or surface groups. In some embodiments, half-generation is possible because only the first condensation reaction with an amine occurs, without the second condensation reaction with a thiol.

[0290] 2. Dendritic polymers of formula (I)

[0291] In some embodiments of the lipid compositions disclosed herein, the ionizable lipids comprise at least two C8-C atoms. 24 Alkyl groups. In some embodiments, the ionizable lipid is further defined as a dendritic polymer:

[0292] Core - (Repeating Unit) n -Terminal group (DI)

[0293] One or more core hydrogen atoms are replaced by repeating units, and in which:

[0294] The core has the following formula:

[0295] (D-II)

[0296] in:

[0297] X1 is an amino group or C1-C. 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 Heterocyclic alkyl, C5-C 12 heteroaryl groups or their substituted forms;

[0298] R1 represents amino, hydroxyl, mercapto, or C1-C. 12 Alkylamino or C1-C 12 Dialkylamino or a substituted form of any of these groups; and

[0299] a is 1, 2, 3, 4, 5, or 6; or

[0300] The core has the following formula:

[0301] (D-III)

[0302] in:

[0303] X2 is N(R5)y ;

[0304] R5 is hydrogen, C1-C 18 Alkyl or substituted C1-C 18 Alkyl; and

[0305] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0306] R2 represents amino, hydroxyl, thiol, or C1-C. 12 Alkylamino or C1-C 12 Dialkylamino or a substituted form of any of these groups;

[0307] b is 1, 2, 3, 4, 5, or 6; and

[0308] z can be 1, 2, or 3. The condition is that the sum of z and y is 3; or

[0309] The core has the following formula:

[0310] (D-IV)

[0311] in:

[0312] X3 is -NR6-, where R6 is hydrogen, C1-C8 alkyl or C1-C8 substituted alkyl, -O- or C1-C8 alkylamino diester, C1-C8 alkoxy diester, C6-C8 aromatic diester, C5-C8 heteroaromatic diester, C3-C8 heterocyclic alkane diester, or a substituted form of any of these groups;

[0313] R3 and R4 are each independently amino, hydroxyl, mercapto, or C1-C. 12 Alkylamino or C1-C 12 Dialkylamino or any of these groups in a substituted form; or a group of the following formula: −N(R f ) f (CH2CH2N(R c )) e R d , , or ;

[0314] in:

[0315] e and f are each independently 1, 2, or 3; the condition is that the sum of e and f is 3.

[0316] R c R d and R f Each is independently hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0317] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0318] The core is C1-C 18 Alkylamines, C1-C 36 Dialkylamines, C3-C 12 Heterocyclic alkanes or substituted forms of any of these groups;

[0319] The repeating unit contains a degradable diacyl group or a degradable diacyl group and a linker group;

[0320] The degradable diacyl group has the following formula:

[0321] (D-VII)

[0322] in:

[0323] A1 and A2 are each independently −O−, -S−, or −NR. a -, where:

[0324] R a It is hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0325] Y3 is C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups or substituted forms of any of these groups; or groups of the following formula:

[0326] or

[0327] in:

[0328] X3 and X4 are C1-C 12 Alkyl, C2-C 12 alkendiyl, C6-C 12 Aromatic dimethyl groups or substituted forms of any of these groups;

[0329] Y5 is a covalent bond, C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl or a substituted form of any of these groups; and

[0330] R9 is a C1-C8 alkyl or a substituted C1-C8 alkyl;

[0331] The linker group has the following formula:

[0332] (D-VI)

[0333] in:

[0334] Y1 is C1-C 12 Alkyl, C1-C 12 alkendiyl, C6-C 12 Aromatic dimethyl or a substituted form of any of these groups; and

[0335] Each of them Independently indicates the attachment point to another repeating unit or terminating group; and

[0336] The terminal group has the following formula:

[0337] (D-VIII)

[0338] in:

[0339] Y4 is an alkyldiyl or C1-C 18 Alkyl, wherein C1-C 18 One or more hydrogen atoms on the alkyl diel group have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or −OC(O)CH3;

[0340] R 10 Hydrogen, carboxyl, hydroxyl, C6-C 12 Aryl, C1-C 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 N-Heterocyclic alkyl groups, -C(O)N(R) 11 )-C1-C6 alkyldiyl-C3-C 12 Heterocyclic alkyl, -C(O)-C1-C 12 Alkyl-amino, -C(O)-C1-C 12 Dialkylamino or -C(O)-C3-C 12 N-Heterocyclic alkyl groups, wherein:

[0341] R 11 It is hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0342] The final degradable diacyl group in the chain is attached to the terminal group;

[0343] n is 0, 1, 2, 3, 4, 5, or 6;

[0344] Or its pharmaceutically acceptable salt.

[0345] In some implementations, the terminal group is further defined by the following formula:

[0346] (D-VIII)

[0347] in:

[0348] Y4 is C1-C 18 Alkyl; and

[0349] R 10 It is hydrogen. In some embodiments, A1 and A2 are each independently −O− or −NR−. a -.

[0350] In some embodiments of the dendritic polymer of formula (DI), the terminal groups are structures selected from those in Table 3.

[0351] In some embodiments of the dendritic polymer of formula (DI), the core is further defined by the following formula:

[0352] (D-III)

[0353] in:

[0354] X2 is N(R5) y ;

[0355] R5 is hydrogen or a C1-C8 alkyl or a substituted C1-C8 alkyl group. 18 Alkyl; and

[0356] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0357] R2 is an amino, hydroxyl, or thiol group, or a C1-C group. 12 Alkylamino, C1-C 12 Dialkylamino or a substituted form of any of these groups;

[0358] b is 1, 2, 3, 4, 5, or 6; and

[0359] z is 1, 2, or 3; the condition is that the sum of z and y is 3.

[0360] In some embodiments of the dendritic polymer of formula (DI), the core is further defined by the following formula:

[0361] (D-IV)

[0362] in:

[0363] X3 is -NR6-, where R6 is hydrogen, C1-C8 alkyl or substituted C1-C8 alkyl, -O- or C1-C8 alkylaminodiyl, C1-C8 alkoxydiyl, C1-C8 aromaticdiyl, C1-C8 heteroaromaticdiyl, C1-C8 heterocyclic alkanediyl or a substituted form of any of these groups;

[0364] R3 and R4 are each independently amino, hydroxyl, or thiol or C1-C. 12 Alkylamino or dialkylamino or any of these groups in a substituted form; or a group of the following formula: −N(R f ) f (CH2CH2N(R c )) e R d , , or ;

[0365] in:

[0366] e and f are each independently 1, 2, or 3; the condition is that the sum of e and f is 3.

[0367] R c R d and R f Each is independently hydrogen, C1-C6 alkyl, or substituted C1-C6 alkyl;

[0368] c and d can each be 1, 2, 3, 4, 5 or 6 independently.

[0369] In some embodiments of the dendritic polymer of formula (I), the terminal groups are represented by the following formula:

[0370] (D-VIII),

[0371] in:

[0372] Y4 is an alkyldiyl group. （C≤18） ;and

[0373] R 10 It is hydrogen.

[0374] In some embodiments of the dendritic polymer of formula (DI), the core of the structure of formula (D-IV) is:

[0375] , , , , , , , , , , , , , or Or its pharmaceutically acceptable salt.

[0376] In some embodiments of the dendritic polymer of formula (DI), the core comprises the structural formula shown in Table 2 and its pharmaceutically acceptable salt, where * indicates the connection point between the core and the repeating unit (i.e., the location where the hydrogen of the core is replaced by the repeating unit).

[0377] In some embodiments of the dendritic polymer of formula (DI), the degradable diacyl group is further defined as:

[0378] .

[0379] In some embodiments of the dendritic polymer of formula (DI), the linker is further defined as (D-VI),

[0380] Where Y1 is a C1-C8 alkyldiyl or substituted C1-C 12 Alkyl.

[0381] In some embodiments, R6 is H in the core of formula (D-IV). In some embodiments, R6 is a C1-C8 alkyl group in the core of formula (D-IV). In some embodiments, R6 is a substituted alkyl group (e.g., an alkyl group substituted with -NH2, an alkyl group substituted with -NHCH3, or an alkyl group substituted with -NHCH2CH3) in the core of formula (D-IV).

[0382] In some embodiments, one or two hydrogen atoms in the core are replaced by repeating units. In some embodiments, three or four hydrogen atoms in the core are replaced by repeating units. In some embodiments, five hydrogen atoms in the core are replaced by repeating units. In some embodiments, six hydrogen atoms in the core are replaced by repeating units.

[0383] In some implementations, the ionizable lipid is a compound having the structure of formula DA:

[0384] (DA),

[0385] in:

[0386] R D1 It is a C1-C4 alkyl group;

[0387] z1 and z2 are each independently 1, 2, or 3; and

[0388] z3 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0389] In some implementations, in compounds of formula DA, R D1 It is a methyl group.

[0390] In some implementations, in compounds of formula DA, z1 and z2 are each 2.

[0391] In some embodiments, the core of formula D-II, D-III, or D-IV has the structure shown in Table 2, and the terminal group of formula D-VII has the structure shown in Table 3. In each of the structures in Tables 3 and 4, Indicates the connection point with the following structures: The core group in Table 2 and the terminal group in Table 3 are connected to the opposite ends of the structure.

[0392] In some embodiments of the dendritic polymer of formula (DI), the dendritic polymer is selected from the group consisting of:

[0393] , , , ,

[0394] and ;

[0395] And its pharmaceutically acceptable salts.

[0396] 1. Dendritic polymers of formula (X)

[0397] In some embodiments of the lipid composition, the ionizable lipid is a dendritic polymer of the following formula: In some embodiments, the ionizable lipid is a dendritic polymer of the following formula.

[0398] .

[0399] In some embodiments of the lipid composition, the ionizable lipid is a dendritic polymer having the following structural formula:

[0400] ,

[0401] Or its pharmaceutically acceptable salt, wherein:

[0402] (VI) The core has a structural formula (X) 核心 ):

[0403] ,

[0404] in:

[0405] Q is independently a covalent bond, -O-, -S-, or -NR each time it appears. 2 -or-CR 3a R 3b -;

[0406] R 2 R is independent for each occurrence. 1g or -L 2 -NR 1e R 1f ;

[0407] R 3a and R 3b Each of these is independently hydrogen or optionally substituted (e.g., C1-C6, such as C1-C3) alkyl groups;

[0408] R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently of the junction with the branch, hydrogen, or optional substitution (e.g., C1-C). 12 )alkyl;

[0409] L 0 L 1 and L 2 Each occurrence is independently selected from covalent bonds, alkylene, heteroalkylene, [alkylene]-[heterocyclic alkyl]-[alkylene], [alkylene]-(aryl)-[alkylene], heterocyclic alkyl, and aryl; or

[0410] Alternatively, L 1 Part of R 1c and R 1d One of them forms (e.g., C4-C6) heterocyclic alkyl groups (e.g., containing one or two nitrogen atoms and optionally other heteroatoms selected from oxygen and sulfur); and

[0411] x 1 It can be 0, 1, 2, 3, 4, 5, or 6; and

[0412] (b) Each of the multiple (N) branches independently has a structure (X) 分支 ):

[0413] ,

[0414] in:

[0415] * indicates the connection point between the branch and the core;

[0416] g is 1, 2, 3, or 4;

[0417] Z=2 (g-1) ;

[0418] When g=1, G=0; or when g≠1, G= ;

[0419] Each diacyl group independently has a structural formula. ,in:

[0420] * indicates the junction of the diacyl group at its proximal end;

[0421] ** indicates the connection point of the diacyl group at its distal end;

[0422] Y 3 Independently substituted each time it appears (e.g., C1-C) 12 ) alkylene, optionally substituted (e.g., C1-C) 12 ) imide or optionally substituted (e.g., C1-C) 12 ) alkylene aryl;

[0423] A 1 and A 2 Each occurrence is independently -O-, -S-, or -NR. 4 -,in:

[0424] R 4 It is hydrogen or an optionally substituted (e.g., C1-C6) alkyl group;

[0425] m 1 and m 2 Each occurrence is independently 1, 2, or 3; and

[0426] R 3c R 3d R 3e and R 3f Each of these, in each instance, is independently hydrogen or an optionally substituted (e.g., C1-C8) alkyl group; and

[0427] (d) Each linker group independently has a structural formula ,

[0428] in:

[0429] ** indicates the connection point between the linker and the proximal diacyl group;

[0430] *** indicates the connection point between the linker and the distal diacyl group; and

[0431] Y1 is independently substituted each time it appears (e.g., C1-C). 12 ) alkylene, optionally substituted (e.g., C1-C) 12 ) imide or optionally substituted (e.g., C1-C) 12 ) alkylene aryl; and

[0432] Each terminal group is independently selected from those with optional substitutions (e.g., C1-C1). 18 Such as C4-C 18 ) alkyl thiols and optionally substituted (e.g., C1-C) 18 Such as C4-C 18 ) Alkenyl thiols.

[0433] In X 核心 In some implementations, Q is independently covalent, -O-, -S-, -NR each time it appears. 2 -or-CR 3a R 3b In X 核心 In some implementations, Q is a covalent bond independently each time it occurs. In X 核心 In some implementations, Q is independently -O- each time it occurs. In X 核心 In some implementations, Q is independently -S- each time it occurs. In X 核心 In some implementations, Q is independently -NR each time it occurs. 2 And R 2 R is independent for each occurrence. 1g or -L 2 -NR 1e R 1f In X 核心 In some implementations, Q is independently -CR each time it occurs. 3a R 3b R 3a And R 3a and R 3b Each of these is independently hydrogen or an optionally substituted alkyl group (e.g., C1-C6, such as C1-C3) each time it appears.

[0434] In X 核心 In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently a junction with a branch, a hydrogen atom, or an optionally substituted alkyl group. In X 核心In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently a connection point with the branch, and a hydrogen atom. In X 核心 In some implementation schemes, R 1a R 1b R 1c R 1d R 1e R 1f and R 1g (If present) each occurrence is independently a junction point with a branch, optionally substituted alkyl group (e.g., C1-C). 12 ).

[0435] In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from covalent bonds, alkylene, heteroalkylene, [alkylene]-[heterocyclic alkyl]-[alkylene], [alkylene]-(aryl)-[alkylene], heterocyclic alkyl, and aryl; or alternatively, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group (e.g., C4-C6, and containing one or two nitrogen atoms and optionally another heteroatom selected from oxygen and sulfur). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance can independently be a covalent bond. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it can independently be hydrogen. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each of these can independently be an alkylene group (e.g., C1-C) upon each occurrence. 12 (e.g., C1-C6 or C1-C3). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each of these can independently be a heteroalkylene group (e.g., C1-C) upon each occurrence. 12(e.g., C1-C8 or C1-C6). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each of these can independently be a heteroalkylene group (e.g., C2-C8 epoxides, such as oligomers (ethylene oxides)). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be [alkylene]-[heterocyclic alkyl]-[alkylene][(e.g., C1-C6)alkylene]-[(e.g., C4-C6)heterocyclic alkyl]-[(e.g., C1-C6)alkylene]. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be [alkylene]-(arylene)-[alkylene][(e.g., C1-C6)alkylene]-(arylene)-[(e.g., C1-C6)alkylene]. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence can independently be [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6)alkylene]-phenylene-[(e.g., C1-C6)alkylene]). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance can independently be a heterocyclic alkyl group (e.g., C4-C6 heterocyclic alkyl group). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each can be independently arylene (e.g., phenylene) in each occurrence. In X 核心 In some implementation schemes, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group. In X 核心 In some implementation schemes, L 1 Part of R 1c and R 1d One of them forms a heterocyclic alkyl group (e.g., a C4-C6 heterocyclic alkyl group), and the heterocyclic alkyl group may contain one or two nitrogen atoms and optional additional heteroatoms selected from oxygen and sulfur.

[0436] In X 核心In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from covalent bonds, C1-C6 alkylene groups (e.g., C1-C3 alkylene groups), C2-C 12 (e.g., C2-C8) epoxides (e.g., oligomeric (ethylene oxide), such as –(CH2CH2O) 1-4 -(CH2CH2)-), [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkylene]-[(C1-C4)alkylene] (e.g., ) and [(C1-C4)alkylene]-phenylene-[(C1-C4)alkylene] (e.g., ). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each occurrence is independently selected from C1-C6 alkylene groups (e.g., C1-C3 alkylene groups) and -(C1-C3 alkylene-O). 1-4 -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)- and –(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-. In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each instance is independently a C1-C6 alkylene group (e.g., C1-C3 alkylene groups). In some embodiments, L 0 L 1 and L 2 Each occurrence is independently C2-C. 12 (e.g., C2-C8) alkylene oxides (e.g., -(C1-C3 alkylene-O)) 1-4 -(C1-C3 alkylene)). In X 核心 In some implementation schemes, L 0 L 1 and L 2 Each time it appears, it is independently selected from [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-phenylene-(C1-C3alkylene)-) and [(C1-C4)alkylene]-[(C4-C6)heterocyclic alkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3alkylene)-piperazinyl-(C1-C3alkylene)-).

[0437] In X 核心 In some implementation schemes, x1 It can be 0, 1, 2, 3, 4, 5, or 6. In X 核心 In some implementation schemes, x 1 It is 0. In X 核心 In some implementation schemes, x 1 It is 1. In X 核心 In some implementation schemes, x 1 It is 2. In X Core In some implementation schemes, x 1 The values ​​are 0 and 3. In X 核心 In some implementation schemes, x 1 It is 4. In X 核心 In some implementation schemes, x 1 It is 5. In X 核心 In some implementation schemes, x 1 It is 6.

[0438] In X 核心 In some implementation schemes, the core has a structure: (For example, ). In X 核心 In some implementation schemes, the core has a structure: In X 核心 In some implementation schemes, the core has a structure: (For example, , , or ). In X 核心 In some implementation schemes, the core has a structure: (For example, Such as or ). In X 核心 In some implementation schemes, the core has a structure: Where Q' is -NR 2 -or-CR 3a R 3b -;q 1 and q 2 Each is independently 1 or 2. In X 核心 In some implementation schemes, the core has a structure: (For example, , , or ). In X 核心 In some implementation schemes, the core has a structural or (For example, , , or ), where ring A is an aryl group with optional substitution or optional substitution (e.g., C3-C). 12 Such as C3-C5) heteroaryl groups. In X 核心 In some implementation schemes, the core has a structural .

[0439] In X 核心 In some implementations, the core has the structural formula shown in Table 2 and its pharmaceutically acceptable salt, where * indicates the connection point between the core and one of the multiple branches.

[0440] In some implementations, the multiple (N) branches include at least 3 branches, at least 4 branches, and at least 5 branches. In some implementations, the multiple (N) branches include at least 3 branches. In some implementations, the multiple (N) branches include at least 4 branches. In some implementations, the multiple (N) branches include at least 5 branches.

[0441] In X 分支 In some implementations, g is 1, 2, 3, or 4. In X 分支 In some implementations, g is 1. In X 分支 In some implementations, g is 2. In X 分支 In some implementations, g is 3. In X 分支 In some implementations, g is 4.

[0442] In X 分支 In some implementation schemes, Z=2 (g-1) And when g=1, G=0. In X 分支 In some implementation schemes, Z=2 (g-1) And when g≠1, G= .

[0443] In X 分支 In some implementations, g=1, G=0, Z=1, and each of the multiple branches has a structure formula. .

[0444] In X 分支 In some implementations, g=2, G=1, Z=2, and each of the multiple branches has a structural formula. .

[0445] In X 分支 In some implementations, g=3, G=3, Z=4, and each of the multiple branches has a structural formula. .

[0446] In X 分支In some implementations, g=4, G=7, Z=8, and each of the multiple branches has a structural formula. .

[0447] In some embodiments, the dendritic polymer with generation (g)=1 described herein has the following structure: .

[0448] In some embodiments, the dendritic polymer with generation (g)=1 described herein has the following structure: .

[0449] Example designs of the dendritic polymers of generations 1 to 4 described herein are shown in Table 1. The number of diacid groups, linker groups, and terminal groups can be calculated based on g.

[0450] Table 1. Design based on dendritic polymer groups of generation (g)

[0451]

[0452] In some embodiments, the diacyl group independently has the structural formula * indicates the connection point of the diacyl group at its proximal end, and ** indicates the connection point of the diacyl group at its distal end.

[0453] In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted alkylene, optionally substituted alkenyl, or optionally substituted alkylaryl. In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted alkylene group (e.g., C1-C). 12 ). In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted alkenyl group (e.g., C1-C). 12 ). In X 分支 In some embodiments of the diacyl group, Y 3 Each time it appears, it is independently an optionally substituted alkylene aryl group (e.g., C1-C). 12 ).

[0454] In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -O-, -S-, or -NR. 4 -. In X 分支 In some embodiments of the diacyl group, A 1 and A 2Each occurrence is independently -O-. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -S-. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -NR 4 - and R 4 It is hydrogen or an optionally substituted alkyl group (e.g., C1-C6). In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 1, 2, or 3. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently set to 1. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 2. In X 分支 In some embodiments of the diacyl group, m 1 and m 2 Each occurrence is independently 3. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each of these is independently hydrogen or an optionally substituted alkyl group, each appearing independently. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each time it appears, it is independently hydrogen. In X 分支 In some embodiments of the diacyl group, R 3c R 3d R 3e and R 3f Each of these alkyl groups is independently, in each instance, an optionally substituted (e.g., C1-C8) alkyl group.

[0455] In some embodiments of the diacyl group, A 1 It is -O- or -NH-. In some embodiments of the diacyl group, A 1 For -O-. In some embodiments of the diacyl group, A 2 It is -O- or -NH-. In some embodiments of the diacyl group, A2 For -O-. In some embodiments of the diacyl group, Y 3 For C1-C 12 (e.g., C1-C6, such as C1-C3) alkylene groups.

[0456] In some embodiments of the diacyl group, the diacyl group independently has a structural formula each time it appears. (For example, Such as ), and optionally, R 3c R 3d R 3e and R 3f Each of them is independently hydrogen or C1-C3 alkyl.

[0457] In some implementations, the linking group independently has a structural formula. ** indicates the connection point between the linker and the proximal diacyl group, and *** indicates the connection point between the linker and the distal diacyl group.

[0458] In X 分支 In some embodiments of the linking group (if present), Y1 is independently, each time it appears, an optionally substituted alkylene, optionally substituted alkenyl, or optionally substituted alkylaryl. In X 分支 In some embodiments, the linking group (if present) is independently, each time it appears, an optionally substituted alkylene group (e.g., C1-C1). 12 ). In X 分支 In some embodiments, the linker group (if present) is independently, each time it appears, an optionally substituted alkenyl group (e.g., C1-C1). 12 ). In X 分支 In some embodiments, the linking group (if present) is independently, each time it appears, an optionally substituted alkylene aryl group (e.g., C1-C1). 12 ).

[0459] In X 分支 In some embodiments of the terminal groups, each terminal group is independently selected from optionally substituted alkyl thiols and optionally substituted alkenyl thiols. In X 分支 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkylthiol (e.g., C1-C1). 18 Such as C4-C 18 ). In X 分支 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkenyl thiol (e.g., C1-C1). 18 Such as C4-C 18 ).

[0460] In X 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 alkenyl thiols or C1-C 18 Alkyl thiols, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents, each of which is independently selected from halogens, C6-C... 12 Aryl, C1-C 12 Alkylamino, C4-C6 N-heterocyclic alkyl, -OH, -C(O)OH, −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C1-C 12 alkyl-amino), −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C4-C6 N-heterocyclic alkyl), −C(O)−(C1-C 12 Alkyl-amino) and −C(O)−(C4-C6 N-heterocyclic alkyl), and the C4-C6 N-heterocyclic alkyl portion of any of the aforementioned substituents is optionally replaced by C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0461] In X 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 (For example, C4-C) 18 ) alkenyl thiols or C1-C 18 (For example, C4-C) 18 C) Alkyl thiols, wherein the alkyl or alkenyl moiety is optionally substituted with one or more substituents, each of which is independently selected from halogens, C6-C... 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8)alkylamino (e.g., C1-C6 monoalkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (such as...) , , , C4-C6 N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) ), N-piperidinyl ( ), N-azacycloheptanyl ( ), -OH, -C(O)OH, −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C1-C 12 Alkyl-amino (e.g., mono- or dialkylamino) (e.g., ), −C(O)N(C1-C3 alkyl)−(C1-C6 alkylene)−(C4-C6 N-heterocyclic alkyl) (e.g., ), −C(O)−(C1-C 12 Alkyl-amino (e.g., mono- or dialkylamino) and −C(O)−(C4-C6 N-heterocyclic alkyl) (e.g., ), wherein the C4-C6 N-heterocyclic alkyl portion of any of the aforementioned substituents is optionally substituted with a C1-C3 alkyl or a C1-C3 hydroxyalkyl. In X 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 (For example, C4-C) 18 Alkyl thiols, wherein the alkyl moiety is optionally substituted with a substituent -OH. In X 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 (For example, C4-C) 18 ) alkyl thiols, wherein the alkyl moiety is optionally substituted by a substituent selected from the following: C1-C 12 (e.g., C1-C8)alkylamino (e.g., C1-C6 monoalkylamino (such as -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (such as...) , , , C4-C6 N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) and C4-C6 N-heterocyclic alkyl groups (e.g., N-pyrrolidinyl alkyl groups) ), N-piperidinyl ( ), N-azacycloheptanyl ( In X) 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 (For example, C4-C) 18 ) alkenyl thiols or C1-C 18 (For example, C4-C) 18 ) Alkyl thiols. In X 分支 In some embodiments of the terminal groups, each terminal group is independently C1-C1. 18 (For example, C4-C) 18 Alkyl thiols.

[0462] Table 2. Example Core Structure

[0463]

[0464]

[0465]

[0466] In X 核心In some implementation schemes, the core includes a structure selected from a group of the following: , , , , , , , , , , , , , , , , , , , , , And its pharmaceutically acceptable salts, where * indicates the connection point between the core and one of the multiple branches.

[0467] In X 分支 In some embodiments, each terminal group is independently a structure selected from those in Table 3. In some embodiments, the dendritic polymer described herein may comprise terminal groups selected in Table 3 or pharmaceutically acceptable salts thereof.

[0468] Table 3. Examples of terminal groups / peripheral structures

[0469]

[0470]

[0471] In some embodiments, the dendritic polymer of formula (X) is selected from those shown in Table 4 and their pharmaceutically acceptable salts.

[0472] Table 4. Examples of ionizable cationic lipid-dendritic polymers

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497] In some embodiments, the dendritic polymer is 2A2-SC14. In some embodiments, the dendritic polymer is 2A6-SC14. In some embodiments, the dendritic polymer is 2A9-SC14. In some embodiments, the dendritic polymer is 3A3-SC10. In some embodiments, the dendritic polymer is 3A3-SC14. In some embodiments, the dendritic polymer is 4A5-SC10. In some embodiments, the dendritic polymer is 3A5-SC14. In some embodiments, the dendritic polymer is 4A1-SC12. In some embodiments, the dendritic polymer is 4A3-SC12. In some embodiments, the dendritic polymer is 5A1-SC12. In some embodiments, the dendritic polymer is 5A1-SC8. In some embodiments, the dendritic polymer is 5A2-2-SC12. In some embodiments, the dendritic polymer is 5A3-1-SC12. In some embodiments, the dendritic polymer is 5A3-1-SC8. In some embodiments, the dendritic polymer is 5A4-1-SC12. In some embodiments, the dendritic polymer is 5A4-1-SC8. In some embodiments, the dendritic polymer is 5A5-SC8. In some embodiments, the dendritic polymer is 5A5-SC12. In some embodiments, the dendritic polymer is 5A2-4-SC12. In some embodiments, the dendritic polymer is 5A2-4-SC10. In some embodiments, the dendritic polymer is 5A3-2-SC8. In some embodiments, the dendritic polymer is 5A3-2-SC12. In some embodiments, the dendritic polymer is 5A4-2-SC8. In some embodiments, the dendritic polymer is 5A4-2-SC12. In some embodiments, the dendritic polymer is 6A4-SC8. In some embodiments, the dendritic polymer is 6A4-SC12. In some embodiments, the dendritic polymer is 2A2-g2-SC12. In some embodiments, the dendritic polymer is 2A2-g2-SC8. In some embodiments, the dendritic polymer is 2A11-g2-SC12. In some embodiments, the dendritic polymer is 2A11-g2-SC8. In some embodiments, the dendritic polymer is 3A3-g2-SC12. In some embodiments, the dendritic polymer is 3A3-g2-SC8. In some embodiments, the dendritic polymer is 3A5-g2-SC12. In some embodiments, the dendritic polymer is 2A11-g3-SC12. In some embodiments, the dendritic polymer is 2A11-g3-SC8. In some embodiments, the dendritic polymer is 1A2-g4-SC12. In some embodiments, the dendritic polymer is 4A1-g2-SC12. In some embodiments, the dendritic polymer is 1A2-g4-SC8.In some embodiments, the dendritic polymer is 4A1-g2-SC8. In some embodiments, the dendritic polymer is 4A3-g2-SC12. In some embodiments, the dendritic polymer is 4A3-g2-SC8. In some embodiments, the dendritic polymer is 1A2-g3-SC12. In some embodiments, the dendritic polymer is 1A2-g3-SC8. In some embodiments, the dendritic polymer is 2A2-g3-SC12. In some embodiments, the dendritic polymer is 2A2-g3-SC8. In some embodiments, the dendritic polymer is 5A2-4-SC8. In some embodiments, the dendritic polymer is 5A5-SC8. In some embodiments, the dendritic polymer is 5A2-6-SC8. In some embodiments, the dendritic polymer is 5A2-1-SC8. In some embodiments, the dendritic polymer is 5A2-2-SC8. In some embodiments, the dendritic polymer is 4A1-SC5. In some embodiments, the dendritic polymer is 4A1-SC8. In some embodiments, the dendritic polymer is 4A3-SC6. In some embodiments, the dendritic polymer is 4A3-SC7. In some embodiments, the dendritic polymer is 4A3-SC8. In some embodiments, the dendritic polymer is 5A4-2-SC5. In some embodiments, the dendritic polymer is 5A4-2-SC6. In some embodiments, the dendritic polymer is 5A2-4-SC8. In some embodiments, the dendritic polymer is 3A5-g2-SC8. In some embodiments, the dendritic polymer is 5A2-SC8.

[0498] 1. Other ionizable lipids

[0499] In some embodiments of the lipid composition, the cationic lipid has the structural formula (D-I'):

[0500] (D-I'),

[0501] in:

[0502] a is 1 and b is 2, 3 or 4; or alternatively, b is 1 and a is 2, 3 or 4;

[0503] m is 1 and n is 1; or alternatively, m is 2 and n is 0; or alternatively, m is 2 and n is 1; and

[0504] R 1 R 2 R 3 R 4 R 5 and R 6Each can be independently selected from the following groups: H, -CH2CH(OH)R 7 -CH(R) 7 )CH2OH、-CH2CH2C(=O)OR 7 -CH2CH2C(=O)NHR 7 and -CH2R 7 , where R 7 Independently selected from C3-C 18 Alkyl group, C3-C with one C=C double bond 18 Protecting groups of alkenyl and amino groups, -C(=NH)NH2, poly(ethylene glycol) chains, and acceptor ligands;

[0505] The condition is R 1 To R 6 At least two parts of it are independently selected from -CH2CH(OH)R 7 -CH(R) 7 )CH2OH、-CH2CH2C(=O)OR 7 -CH2CH2C(=O)NHR 7 or -CH2R 7 , where R 7 Independently selected from C3-C 18 Alkyl groups or C3-C groups with one C=C double bond 18 alkenyl; and

[0506] One or more nitrogen atoms indicated in formula (D-I') may be protonated to provide cationic lipids.

[0507] In some embodiments of the cationic lipid of formula (D-I'), a is 1. In some embodiments of the cationic lipid of formula (D-I'), b is 2. In some embodiments of the cationic lipid of formula (D-I'), m is 1. In some embodiments of the cationic lipid of formula (D-I'), n is 1. In some embodiments of the cationic lipid of formula (D-I'), R... 1 R 2 R 3 R 4 R 5 and R 6 Each is independently H or -CH2CH(OH)R 7 In some embodiments of the cationic lipid of formula (D-I'), R 1 R 2 R 3 R 4 R 5 and R 6 Each independently is H or In some embodiments of the cationic lipid of formula (D-I'), R 1 R 2 R 3 R 4 R 5 and R 6 Each independently is H or In some embodiments of the cationic lipid of formula (D-I'), R 7 For C3-C 18 Alkyl (e.g., C6-C) 12 alkyl).

[0508] In some embodiments, the cationic lipid of formula (D-I') is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentadecane-11,25-diol: .

[0509] In some embodiments, the cationic lipid of formula (D-I') is (11R,25R)-13,16,20-tris(1-2-hydroxydodecyl)-13,16,20,23-tetraazapentadecane-11,25-diol: .

[0510] Other cationic lipids that may be used in the compositions and methods of this application include those described in the following literature: J. McClellan, MCKing, Cell 2010, 141, References 210-217, and international patent publications WO2010144740, WO2013149140, WO2016118725, WO2016118724, WO2013063468, WO2016205691, WO2015184256, WO2016004202, WO2015199952, WO2017004143, WO2017075531, WO2017117528, WO2017049245, WO2017173054, and WO2015095340, are incorporated herein by reference for all purposes. Examples of ionizable cationic lipids include, but are not limited to, those shown in Table 5.

[0511] Table 5. Examples of ionizable cationic lipids

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524] In some embodiments of the lipid compositions disclosed herein, the ionizable lipids are present in an amount of about 20 mol% to about 23 mol%. In some embodiments, the ionizable lipids are present in an amount of about 20 mol%, about 20.5 mol%, about 21 mol%, about 21.5 mol%, about 22 mol%, about 22.5 mol%, or about 23 mol%. In other embodiments, the ionizable lipids are present in an amount of about 7.5 mol% to about 20 mol%. In some embodiments, the ionizable lipids are present in an amount of about 7.5 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, or about 20 mol%.

[0525] In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises about 5 mol% to about 30 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises about 10 mol% to about 25 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises about 15 mol% to about 20 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises about 10 mol% to about 20 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises about 20 mol% to about 30 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid composition comprises at least (about) 5 mol%, at least (about) 10 mol%, at least (about) 15 mol%, at least (about) 20 mol%, at least (about) 25 mol%, or at least (about) 30 mol% of an amount of ionizable lipids. In some embodiments of the lipid compositions disclosed herein, the lipid compositions comprise an amount of up to (about) 5 mol%, up to (about) 10 mol%, up to (about) 15 mol%, up to (about) 20 mol%, up to (about) 25 mol%, or up to (about) 30 mol%.

[0526] B. Support lipids

[0527] In some embodiments, the accessory lipid is a phospholipid. Phospholipids, as defined herein, are any lipid containing a phosphate group. The lipid component of the lipid nanoparticle may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Typically, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group of lauric acid, myristic acid, myristone acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0528] Non-natural substances were also considered, including natural substances with modifications and substitutions (including branching, oxidation, cyclization, and alkynes). For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkyne groups can undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions can be used to functionalize nanoparticles to facilitate membrane permeation or cell recognition, or to conjugate nanoparticles to other components, such as targeting or imaging components (e.g., dyes).

[0529] In some embodiments, the LNP described herein comprises about 5 mol% to about 30 mol% of phospholipids. In some embodiments, the LNP comprises about 10 mol% to about 30 mol%, or about 12 mol% to about 30 mol%, or about 14 mol% to about 30 mol%, or about 16 mol% to about 30 mol%, or about 18 mol% to about 30 mol%, or about 20 mol% to about 30 mol%, or about 22 mol% to about 30 mol%, or about 24 mol% to about 30 mol%, or about 26 mol% to about 30 mol%, or about 28 mol% to about 30 mol%. In some implementations, LNP comprises about 10 mol%, or about 11 mol%, or about 12 mol%, or about 13 mol%, or about 14 mol%, or about 15 mol%, or about 16 mol%, or about 17 mol%, or about 18 mol%, or about 19 mol%, or about 20 mol%, or about 21 mol%, or about 22 mol%, or about 23 mol%, or about 24 mol%, or about 25 mol%, or about 26 mol%, or about 27 mol%, or about 28 mol%, or about 29 mol%, or about 30 mol%.

[0530] In some embodiments, the LNP contains about 5% to about 30% by weight of phospholipids. In some embodiments, the LNP contains about 5% by weight, or 10%, or 12%, or 15%, or 18%, or 20%, or 25%, or 30% by weight of phospholipids.

[0531] In some embodiments of the lipid component of this disclosure, the lipid component may also have a molar percentage of phospholipids to total lipid composition of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derived therefrom. In other embodiments, the molar percentage is about 7.5 to about 60. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derived therefrom.

[0532] In some embodiments of the lipid components disclosed herein, the lipid components comprise a molar percentage of about 8% to about 23% phospholipids. In some embodiments of the lipid components disclosed herein, the lipid components comprise a molar percentage of about 10% to about 20% phospholipids. In some embodiments of the lipid components of this application, the lipid components comprise a molar percentage of about 15% to about 20% phospholipids. In some embodiments of the lipid components disclosed herein, the lipid components comprise a molar percentage of about 8% to about 15% phospholipids. In some embodiments of the lipid components disclosed herein, the lipid components comprise a molar percentage of about 10% to about 15% phospholipids. In some embodiments of the lipid components disclosed herein, the lipid components comprise a molar percentage of about 12% to about 18% phospholipids. In some embodiments of the lipid components disclosed herein, the lipid composition comprises a molar percentage of at least (about) 8%, at least (about) 10%, at least (about) 12%, at least (about) 15%, at least (about) 18%, at least (about) 20%, or at least (about) 23% phospholipids. In some embodiments of the lipid component disclosed herein, the lipid component comprises phospholipids in a molar percentage of up to (about) 8%, up to (about) 10%, up to (about) 12%, up to (about) 15%, up to (about) 18%, up to (about) 20%, or up to (about) 23%.

[0533] Phospholipids that may be used or potentially used in the compositions and methods described in this invention may be selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DIPC), and others. 1,2-Diundecyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-Diundecyl-sn-glycerol-3-phosphate choline (DUPC), 1-Palmyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-Di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:O diether PC), 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OchemsPC), 1-Hexadecyl-sn-glycerol-3-phosphate choline (C16) LysoPC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-diphydanoyl-sn-glycerol-3-phosphate choline (4ME 16:0 PC), 1,2-diphydanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphydanoyl-sn-glycerol-3-phosphate-L-serine (sodium salt) (4ME 16:0 PG). PS), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, and sodium 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DOPG) and sphingomyelin.

[0534] In some implementations, phospholipids may contain one or two long chains (e.g., C6-C). 24The phospholipid may contain an alkyl or alkenyl group, glycerol or sphingosine, one or two phosphate groups, and optionally a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein zwitterionic lipids are defined as lipids and lipid-like molecules that simultaneously possess both positive and negative charges.

[0535] In some embodiments of the lipid components disclosed herein, the phospholipids are not ethyl phosphocholine.

[0536] C. Polymer-conjugated lipids

[0537] The lipid components disclosed herein may include lipids conjugated to polymers, such as lipids conjugated to polyethylene glycol (PEG-lipids). Exemplary methods for preparing and using PEG-lipids are described, for example, in International Patent Publication No. WO2012099755 and U.S. Patent Publication No. 2014 / 0200257.

[0538] In one embodiment, the PEG-lipids used in this disclosure may be the PEG-lipids described in International Patent Publication No. WO 2012 / 099755, the entire contents of which are incorporated herein by reference. Any of these exemplary PEG-lipids described herein may be modified to include hydroxyl groups on the PEG chain. In some embodiments, the PEG-lipid is a PEG-OH lipid. A PEG-OH lipid is a PEG-lipid having one or more hydroxyl (-OH) groups on its lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, the PEG-OH or hydroxy-PEG-lipid includes -OH groups at the ends of the PEG chain. Each possibility represents a separate embodiment of this disclosure.

[0539] In some embodiments of the lipid component disclosed herein, the lipid component further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG-lipid. In some embodiments, the PEG-lipid is a diglyceride that further comprises a PEG chain linked to a glycerol group. In other embodiments, the PEG-lipid is a compound containing one or more C6-C6 groups. 24 Long-chain alkyl or alkenyl groups or C6-C 24Fatty acid groups are linked to the PEG chain via linker groups. Some non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified 1,2-diacyloxypropyl-3-amine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG-lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE. In some embodiments, PEG-modified distearylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol is used. In some embodiments, PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 Da to about 15,000 Da. In some embodiments, the molecular weight is about 200 Da to about 500 Da, about 400 Da to about 5,000 Da, about 500 Da to about 3,000 Da, or about 1,200 Da to about 3,000 Da. PEG-modified molecules have molecular weights of about 100 Da, 200 Da, 400 Da, 500 Da, 600 Da, 800 Da, 1,000 Da, 1,250 Da, 1,500 Da, 1,750 Da, 2,000 Da, 2,250 Da, 2,500 Da, 2,750 Da, 3,000 Da, 3,500 Da, 4,000 Da, 4,500 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 12,500 Da to about 15,000 Da. Some non-limiting examples of lipids that may be used in this disclosure are taught in U.S. Patent No. 5,820,873, International Patent Publication No. WO 2010 / 141069, or U.S. Patent No. 8,450,298, the entire contents of which are incorporated herein by reference.

[0540] In some embodiments of the lipid compositions of this application, the PEG-lipid has the following structural formula: , where: R 12 and R 13 Each is independently an alkyl group （C≤24） alkenyl （C≤24） Or a substituted form of any of these groups; R e Hydrogen, alkyl （C≤8） or substituted alkyl （C≤8） And x is between 1 and 250. In some implementations, R e alkyl （C≤8） Such as methyl. R 12 and R 13Each is independently an alkyl group （C≤4-20） In some embodiments, x is 5 to 250. In one embodiment, x is 5 to 125, or x is 100 to 250. In some embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol.

[0541] In some embodiments of the lipid components disclosed herein, the PEG-lipid has the following structural formula: Where: n1 is an integer between 1 and 100, and n2 and n3 are each independently selected from integers between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derived therefrom. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0542] In some embodiments of the lipid component of this disclosure, the component may further comprise about 4.0% to about 4.6% of the PEG-lipid composition in molar percentage to the total lipid composition. In some embodiments, the molar percentage is about 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% to about 4.6% or any range derived therefrom. In other embodiments, the molar percentage is about 1.5% to about 4.0%. In some embodiments, the molar percentage is about 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75% to about 4.0% or any range derived therefrom.

[0543] In some embodiments of the lipid components disclosed herein, the lipid component comprises polymer-conjugated lipids at a molar percentage of about 0.5% to about 10%. In some embodiments of the lipid components disclosed herein, the lipid composition comprises polymer-conjugated lipids at a molar percentage of about 1% to about 8%. In some embodiments of the lipid components disclosed herein, the lipid composition comprises polymer-conjugated lipids at a molar percentage of about 2% to about 7%. In some embodiments of the lipid components of this application, the lipid component comprises polymer-conjugated lipids at a molar percentage of about 3% to about 5%. In some embodiments of the lipid components disclosed herein, the lipid component comprises polymer-conjugated lipids at a molar percentage of about 5% to about 10%. In some embodiments of the lipid component disclosed herein, the lipid component comprises a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10% of a polymer-conjugated lipid. In some embodiments of the lipid component of this disclosure, the lipid component comprises a molar percentage of up to (about) 0.5%, up to (about) 1%, up to (about) 1.5%, up to (about) 2%, up to (about) 2.5%, up to (about) 3%, up to (about) 3.5%, up to (about) 4%, up to (about) 4.5%, up to (about) 5%, up to (about) 5.5%, up to (about) 6%, up to (about) 6.5%, up to (about) 7%, up to (about) 7.5%, up to (about) 8%, up to (about) 8.5%, up to (about) 9%, up to (about) 9.5%, or up to (about) 10% of a polymer-conjugated lipid.

[0544] D. Structural lipids

[0545] Lipid nanoparticles may comprise one or more structured lipids. The structured lipids may be steroids or steroid derivatives. In some embodiments of the lipid components disclosed herein, the lipid components further comprise steroids or steroid derivatives. In some embodiments, the steroids or steroid derivatives include any steroid or steroid derivative. Steroids are a class of compounds having a tetracyclic 17-carbon ring structure, which may also contain one or more substituents, including alkyl groups, alkoxy groups, hydroxyl groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms. In one aspect, the cyclic structure of the steroid comprises three fused cyclohexyl rings and one fused cyclopentyl ring, as shown in the following formula: In some embodiments, the steroid derivative comprises the aforementioned cyclic structure having one or more non-alkyl substituted components. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as: In some embodiments of this disclosure, the steroid or steroid derivative is a cholesterane or a cholesterane derivative. In cholesteranes, the cyclic structure is further defined by the following formula: As described above, the cholesterane derivative comprises one or more non-alkyl substitutions in the aforementioned ring system. In some embodiments, the cholesterane or cholesterane derivative is cholesterene or a cholesterene derivative. In some embodiments, the steroid or steroid derivative is a ring-opening steroid or a ring-opening steroid derivative. In some embodiments, the steroid or steroid derivative is a cardiac steroid or a cardiac steroid derivative. In some embodiments, the steroid or steroid derivative is a sapogenin or a sapogenin derivative. In some embodiments, the steroid or steroid derivative is a saponin or a saponin derivative. In some embodiments, the steroid or steroid derivative is an arachidic acid or an arachidic acid derivative. In some embodiments, the steroid or steroid derivative is an alkaloid or an alkaloid derivative. In some embodiments, the steroid or steroid derivative is a sterol or a sterol derivative.

[0546] The sterols that can be used in the compositions and methods described herein may be selected from, but are not limited to, cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, tomatine, ursolic acid, and α-tocopherol.

[0547] In some embodiments of the lipid components, these components may further comprise about 40% to about 46% of the steroid relative to the total lipid composition in molar percentage. In some embodiments, the molar percentage is about 40%, 41%, 42%, 43%, 44%, 45% to about 46%, or any range derived therefrom. In other embodiments, the steroid relative to the total lipid composition in molar percentage is about 15% to about 40%. In some embodiments, the molar percentage is 15%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, or 40%, or any range derived therefrom.

[0548] In some embodiments, the lipid component comprises a molar percentage of about 15% to about 60% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 15% to about 55% of a steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of about 15% to about 50% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 15% to about 46% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 20% to about 40% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 25% to about 35% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 30% to about 40% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a molar percentage of about 20% to about 30% of a steroid or steroid derivative. In some embodiments, the lipid component comprises a steroid or steroid derivative in a molar percentage of at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, or at least (about) 46%. In some embodiments, the lipid component comprises a steroid or steroid derivative in a molar percentage of up to (about) 15%, up to (about) 20%, up to (about) 25%, up to (about) 30%, up to (about) 35%, up to (about) 40%, up to (about) 45%, or up to (about) 46%.

[0549] In some embodiments, the cationic lipid is a sterol amine. The hydrophobic portion of the sterol amine has a sterol, and the hydrophilic portion has an amine group. The sterol portion is selected from, but not limited to, cholesterol, sitosterol, campesterol, stigmasterol, or derivatives thereof. The amine group may contain one to five primary, secondary, tertiary amines, or mixtures thereof. At least one of these amines has a pKa of 8 or greater and is charged at physiological pH. The primary, secondary, or tertiary amine may be part of a larger amine containing a functional group selected from, but not limited to, -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N-. The amine group may be contained in a three- to eight-membered heteroalkyl or heteroaryl ring.

[0550] E. Other lipids

[0551] The lipid composition may comprise additional anionic lipids, ionizable cationic lipids, or permanent cationic lipids. In some embodiments, the lipid nanoparticles are preferentially delivered to a target organ. In some embodiments, the target organ is the lung, lung tissue, or lung cells. “Preferential delivery” means that at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the administered amount of the composition is delivered to the target organ (e.g., lung), tissue, or cells. Additional lipids may support the selective delivery of the compositions of this disclosure to specific organs. In some embodiments, the additional lipids may be selective organ-targeting (SORT) lipids. In some embodiments, SORT lipids may allow LNP delivery to the target organ, tissue, or cells.

[0552] SORT lipids are lipids that, when added to an LNP composition, enable the LNP to selectively and predictably target organs, cell types, or tissues (e.g., as described in Cheng et al., Nature 15:313-320 (2020); Wang et al., Nat. Protoc.18(1):265-291; and U.S. Patent Publications US 2022 / 0071916 A1 and US2021 / 0259980 A1, the entire contents of which are incorporated herein by reference). For example, adding a specific SORT lipid to an LNP can retarget the LNP from the liver to the lungs. SORT lipids include, but are not limited to, permanently cationic lipids, anionic lipids, zwitterionic lipids, and ionizable cationic lipids. Without being bound by theory, anionic SORT lipids generally favor delivery to the spleen, at least when administered intravenously; ionizable cationic SORT lipids generally favor delivery to the liver; permanently cationic SORT lipids generally favor delivery to the lungs; and zwitterionic SORT lipids favor delivery to the spleen.

[0553] In some embodiments, the additional lipid comprises a permanently positively charged portion (i.e., a permanently cationic lipid). The permanently positively charged portion can be positively charged at physiological pH, such that the additional lipid (e.g., SORT lipid) is positively charged when delivering the payload (e.g., a polynucleotide) to the cell. In some embodiments, the positively charged portion is a quaternary ammonium or quaternary ammonium ion. In some embodiments, the additional lipid (e.g., SORT lipid) comprises an antiion, or otherwise co-exists with or interacts with an antiion.

[0554] In some embodiments, the additional lipid is a permanently cationic lipid (i.e., comprising one or more hydrophobic components and a permanently cationic group). The permanently cationic lipid may contain a group that carries a positive charge regardless of pH. One type of permanently cationic group that can be used in permanently cationic lipids is a quaternary ammonium group. The permanently cationic lipid may have the following structural formula: (SI), where:

[0555] Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5;

[0556] The condition is that at least one of Y1, Y2, and Y3 is X2N. + R3R4R5;

[0557] R1 is C1-C 24 Alkyl, C1-C 24 Substituted alkyl groups, C1-C 24 alkenyl, C1-C 24 Substituted alkenyl groups;

[0558] X1 is either 0 or NR a , where R a It is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl;

[0559] X2 is a C1-C6 alkyldiyl or a C1-C6 substituted alkyldiyl;

[0560] R3, R4, and R5 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 alkenyl, C1-C 24 Substitute alkenyl groups; and

[0561] A1 is an anion with a charge equal to that of X2N in the compound. + The number of R3R4R5 groups.

[0562] In some embodiments, additional permanent cationic lipids (e.g., SORT lipids) have the following structural formula: (S-II), where:

[0563] R6-R9 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 alkenyl, C1-C 24 Substitute alkenyl groups; the condition being that at least one of R6-R9 is C8-C. 24 The group; and

[0564] A2 is a monovalent anion.

[0565] In some embodiments, the permanent cationic lipids are 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), 1,2-distearatel-sn-glycerol-3-ethylphosphocholine (18:0 EPC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1 EPC), and dimethyl dioctadecylammonium (18:0 EPC). DDAB), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP, DOTAP) or 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA).

[0566] In some embodiments, SORT lipids are ionizable cationic lipids (i.e., comprising one or more hydrophobic components and an ionizable cationic group, such as a tertiary amine group). The ionizable cationic group can carry a positive charge at physiological pH. One type of ionizable cationic group that can be used in ionizable lipids is a tertiary amine group. In some embodiments, additional lipids (e.g., SORT lipids) have the following structural formula: (S-I'a), where:

[0567] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other substituted form; and

[0568] R3 and R3' are each independently C1-C6 alkyl or substituted C1-C6 alkyl.

[0569] In some implementations of formula (S-I'a), R1 and R2 are each independently C8-C 24 Alkenyl (e.g., hexadecene, heptadecanene, or octadecene). In some embodiments of formula (S-I'a), R3 and R3' are each independently C1-C6 alkyl (e.g., methyl or ethyl). In some embodiments of formula (S-I'a), R1 and R2 are each independently C8-C6 alkyl. 24The alkenyl group (e.g., hexadecene, heptadecanene, or octadecene) and R3 and R3' are each independently C1-C6 alkyl (e.g., methyl or ethyl).

[0570] In some embodiments, the ionizable cationic lipid is 1,2-distearyl-3-dimethylammonium-propane (18:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dioleoyl-3-dimethylammonium-propane (18:1 DAP, DODAP), or 1,2-diolenoyloxy-3-dimethylaminopropane (DODMA).

[0571] In some embodiments of the lipid composition, the additional ionizable lipid or permanent cationic lipid comprises a head group with a specific structure. In some embodiments, the additional lipid (e.g., SORT lipid) comprises a head group having the following structural formula: Where L is the linker base; Z + It is the positively charged part, and X - It is a counterion. In one embodiment, the linker is a biodegradable linker. The biodegradable linker can be degraded at physiological pH and temperature. The biodegradable linker can be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged portion is a quaternary ammonium ion or a quaternary ammonium.

[0572] In some implementations, SORT (another ionizable lipid or permanent cationic) lipids have the following structural formula: , where R 1 and R 2 Each of the C6-Cs can be substituted independently. 24 Alkyl or optionally substituted C6-C 24 Alkenyl group.

[0573] In some implementations, additional lipids (e.g., SORT lipids) have the following structural formula: .

[0574] In some embodiments, the additional lipid (e.g., SORT lipid) contains a linker (L). In some embodiments, L is... ,in:

[0575] p and q are each independently 1, 2, or 3; and

[0576] R 4 It is an optional substituted C1-C6 alkyl group.

[0577] In some implementations, additional lipids (e.g., SORT lipids) have the following structural formula: (IA), where:

[0578] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0579] R3, R3' and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl;

[0580] R4 is a C1-C6 alkyl or a substituted C1-C6 alkyl; and

[0581] X − It is a monovalent anion.

[0582] In some embodiments, the additional lipid (e.g., SORT lipid) is phosphatidylcholine (e.g., 14:0EPC). In some embodiments, the phosphatidylcholine compound is further defined as: (IA), where:

[0583] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0584] R3, R3', and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl; and

[0585] X − It is a monovalent anion.

[0586] In some embodiments, the additional lipid (e.g., SORT lipid) is a phosphorocholine lipid. In some embodiments, the additional lipid (e.g., SORT lipid) is ethylphosphocholine. Ethylphosphocholine can be, for example, but not limited to, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1 EPC), 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (18:1 EPC), 1,2-distearate-sn-glycerol-3-ethylphosphocholine (18:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine (16:0-18:0 EPC).

[0587] In some implementations, SORT lipids have the following structural formula: (S-I'), where:

[0588] R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 Alkenyl or any other group substitution form;

[0589] R3, R3' and R3'' are each independently a C1-C6 alkyl or a substituted C1-C6 alkyl;

[0590] X − It is a monovalent anion.

[0591] For example (but not limited to), another lipid (e.g., SORT lipid) immediately following the previous structure is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

[0592] In some implementations, additional lipids (e.g., SORT lipids) have the following structural formula: (S-II'), where:

[0593] R4 and R4' are each independently an alkyl group. (C6-C24) alkenyl (C6-C24) Or a substituted form of any group;

[0594] R4'' is an alkyl group. （C≤24） alkenyl （C≤24） Or a substituted form of any group;

[0595] R4''' is an alkyl group (C1-C8) alkenyl (C2-C8) Or any substituted form of a group; and

[0596] X2 is a monovalent anion.

[0597] For example (but not limited to), another lipid in the structure immediately following the previous paragraph (e.g., SORT lipid) is dimethyl dioctadecyl ammonium (DDAB).

[0598] In some implementations, additional lipids (e.g., SORT lipids) are

[0599] 1,2-Dioleoyl-sn-glycerol-3-phosphate (18:1PA).

[0600] In some embodiments of the lipid composition, the additional lipids are selected from the lipids shown in Table 6.

[0601] Table 6. Examples of other lipids (e.g., SORT lipids)

[0602]

[0603]

[0604] X- is a counter ion (e.g., Cl-, Br-, etc.).

[0605] In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 20% to about 65%. In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 25% to about 60%. In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 30% to about 55%. In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 20% to about 50%. In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 30% to about 60%. In some embodiments, the lipid component comprises an additional lipid (e.g., SORT lipids) at a molar percentage of about 25% to about 60%. In some embodiments, the lipid component comprises at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, at least (about) 60%, or at least (about) 65% of other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at most (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65% of other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises additional lipids (e.g., SORT lipids) in a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or any two of the aforementioned values ​​(including the extreme values).

[0606] Table 7 provides non-limiting illustrative LNP compositions.

[0607] Table 7. Non-limiting illustrative LNP compositions

[0608]

[0609] 1. Six-component lipid nanoparticles

[0610] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising at least two selective organ-targeting (SORT) lipids and / or at least six lipids.

[0611] In some embodiments, the LNP composition comprises an ionizable cationic lipid, optionally two or more ionizable lipids; and a permanent cationic lipid, optionally two or more permanent cationic lipids.

[0612] In some embodiments, the LNP composition comprises ionizable cationic lipids, permanent cationic lipids, optional phospholipids, optional polyethylene glycol (PEG)-lipids, and / or optional sterols.

[0613] In some embodiments, the LNP composition contains at least six lipids.

[0614] In some embodiments, the LNP composition comprises ionizable cationic lipids, permanent cationic lipids, optional phospholipids, optional polyethylene glycol (PEG)-lipids, and / or optional sterols.

[0615] In some embodiments, the LNP composition comprises at least two selective organ-targeting (SORT) lipids. In some embodiments, the ionizable cationic lipid is a dendritic lipid, optionally 4A3-SC7 or 5A2-SC8. In some embodiments, the ionizable cationic lipid is a SORT lipid; and / or the permanent cationic lipid is a SORT lipid. In some embodiments, the ionizable cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). In some embodiments, the permanent cationic lipid is trimethylammonium-propane, optionally 1,2-dimyristoyl-3-trimethylammonium-propane (14:0TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0TAP), or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the permanent cationic lipid is ethylphosphocholine (EPC), optionally 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (16:0 EPC), or 1,2-distearateyl-sn-glycerol-3-ethylphosphocholine (18:0 EPC).

[0616] In some embodiments, the LNP specifically transduces lung cells; and / or the LNP delivers the mRNA to lung cells in an amount that effectively increases the expression and / or function of the protein encoded by the mRNA. In some embodiments, the lung cells are ionocytes. In some embodiments, the lung cells are ciliated cells. In some embodiments, the lung cells are secretory cells.

[0617] In some embodiments, the LNP comprises an ionizable cationic lipid of greater than 15% molar percentage. In some embodiments, the ionizable cationic lipid is a dendritic lipid with a molar percentage between 10% and 30%. In some embodiments, the LNP comprises DODAP with a molar percentage between 5% and 40%. In some embodiments, the LNP comprises a permanent cationic lipid of less than 40% molar percentage. In some embodiments, the permanent cationic lipid has a molar percentage between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, and between 15% and 20%. In some embodiments, the permanent cationic lipid is trimethylammonium propane (TAP). In some embodiments, TAP is 1,2-dimyristoyl-3-trimethylammonium propane (14:0 TAP). In some embodiments, the permanent cationic lipid is 14:0 TAP with a molar percentage between 10% and 15%. In some embodiments, the permanent cationic lipid is ethylphosphocholine (EPC). In some embodiments, the TAP is 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:0 EPC). In some embodiments, the permanent cationic lipid is 14:0 EPC with a molar percentage between 10% and 15%.

[0618] In some embodiments, the LNP contains phospholipids at a molar percentage between 10% and 30%. In some embodiments, the LNP contains cholesterol at a molar percentage greater than 25%. In some embodiments, the molar percentage of cholesterol is between 25% and 50%, between 30% and 50%, between 30% and 45%, between 30% and 40%, or between 30% and 35%. In some embodiments, the LNP contains polyethylene glycol (PEG) lipids at a molar percentage between 0.5% and 10% or between 1% and 4%.

[0619] In some embodiments, the LNP contains messenger RNA (mRNA). In some embodiments, the LNP contains mRNA at a lipid:mRNA ratio of less than 40:1. In some embodiments, the lipid:mRNA ratio is 36:1. In some embodiments, the lipid:mRNA ratio is 33:1. In some embodiments, the lipid:mRNA ratio is 30:1.

[0620] In some embodiments, the LNP contains a gene-editing payload. In some embodiments, the gene-editing payload contains a nuclease and / or one or more guide RNAs, and optionally a repair template. In some embodiments, the LNP contains a polypeptide or protein.

[0621] In some embodiments, the LNP comprises a cationic ionizable lipid of 5A2-SC8 or 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP or 14:0 EPC, a phospholipid of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), a sterol of cholesterol or sitosterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG, optionally DMG-PEG2000.

[0622] In some implementations, the LNP contains a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 TAP, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0623] In some implementations, the LNP contains a cationic ionizable lipid of 4A3-SC7, a cationic ionizable SORT lipid of DODAP, a permanent cationic lipid of 14:0 EPC, a phospholipid of DOPE, a sterol of cholesterol, and / or a polyethylene glycol (PEG)-lipid of DMG-PEG.

[0624] In some embodiments, the cationic ionizable lipid is 4A3-SC7, and the LNP contains between about 10% and about 30% 4A3-SC7 in molar percentage. In some embodiments, the cationic ionizable SORT lipid is DODAP, and the LNP contains between about 5% and about 40% DODAP in molar percentage. In some embodiments, the permanent cationic lipid is 14:0 TAP, and the LNP contains between about 5% and about 25% 14:0 TAP in molar percentage. In some embodiments, the permanent cationic lipid is 14:0 EPC, and the LNP contains between about 5% and about 25% 14:0 EPC in molar percentage.

[0625] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 TAP in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0626] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 TAP in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0627] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 30%, DODAP in a molar percentage between about 5% and about 40%, 14:0 EPC in a molar percentage between about 5% and about 25%, DOPE in a molar percentage between about 10% and about 30%, cholesterol in a molar percentage between about 30% and about 50%, and DMG-PEG in a molar percentage between about 0.5% and about 10%.

[0628] In some implementations, LNP comprises 4A3-SC7 in a molar percentage between about 10% and about 15%, DODAP in a molar percentage between about 5% and about 20%, 14:0 EPC in a molar percentage between about 10% and about 20%, DOPE in a molar percentage between about 15% and about 25%, cholesterol in a molar percentage between about 30% and about 40%, and DMG-PEG in a molar percentage between about 1% and about 5%.

[0629] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 TAP, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0630] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0631] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 15% 14:0 EPC, about 22% DOPE, about 30% cholesterol, and about 3% DMG-PEG.

[0632] In some implementations, LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 EPC, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG.

[0633] In some implementations, the LNP comprises about 15% 4A3-SC7, about 15% DODAP, about 12% 14:0 TAP, about 22% DOPE, about 32% cholesterol, and about 3% DMG-PEG, wherein the lipid:mRNA ratio is 33:1.

[0634] In some embodiments, the LNP contains a payload. In some embodiments, the payload is messenger RNA (mRNA). In some embodiments, the mRNA contains 100 bases to 8 kilobases (kb). In some embodiments, the mRNA contains 1 kb to 8 kb, or 2 kb to 8 kb, 3 kb to 8 kb, or 4 kb to 8 kb. In some embodiments, the mRNA contains (about) 2 kb. In some embodiments, the mRNA contains (about) 4.6 kb. In some embodiments, the mRNA encodes a cystic fibrosis transmembrane transduction regulator (CFTR) protein. In some embodiments, the mRNA encodes a dynein axonofilament intermediate chain 1 (DNAI1) protein. In some embodiments, the mRNA encodes a gene editing system or a component thereof. In some embodiments, the payload is shRNA or a polynucleotide encoding shRNA. In some embodiments, the payload is microRNA or a polynucleotide encoding microRNA. In some embodiments, the payload is a polypeptide. In some embodiments, the payload is a protein.

[0635] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is an aerosolized composition.

[0636] In some embodiments, the LNP has an encapsulation efficiency between 50% and 99%, 60% and 99%, 70% and 99%, or 80% and 99%. In some embodiments, the LNP has an encapsulation efficiency between 50% and 95%, 60% and 95%, 70% and 95%, or 80% and 95%. In some embodiments, the LNP composition is a liquid. In some embodiments, the LNP composition is an aerosol.

[0637] In some embodiments, the lipid nanoparticles comprise a composition of six lipids.

[0638] In some embodiments of the six-component lipid nanoparticle, the LNP comprises DODAP in a molar percentage between 12.5% ​​and 17.5%, permanent cationic lipids in a molar percentage between 2% and 12.5%, cholesterol in a molar percentage between 30% and 40%, and polyethylene glycol (PEG) lipids in a molar percentage between 1.5% and 3.5%.

[0639] Key benchmarks for lipid nanoparticle delivery include maximizing cellular uptake and facilitating efficient release of mRNA from endosomes. In one embodiment, the lipid nanoparticles (LNPs) disclosed herein exhibit enhanced cellular uptake or endosome release. Each component of the LNP plays a crucial role in ensuring payload delivery and particle stability. For example, cholesterol and PEG-lipids contribute to stability, while phospholipids enhance fusion, thereby facilitating endosome escape and enabling the bioavailability of nucleic acids in the cytosol.

[0640] While LNPs offer several advantages in improving drug stability, enhancing bioavailability, and facilitating targeted delivery, their safety profile is a critical consideration. Understanding the relationship between LNP composition, dosage, route of administration, and potential side effects may be essential for developing safe and effective LNP-based therapies. The selection of biocompatible lipids may be crucial. For example, coating LNPs with polyethylene glycol (PEG) or other polymers can improve their stability, reduce aggregation, and minimize interactions with the immune system. PEGylation can also prolong the circulation time of LNPs in the bloodstream, potentially reducing immune system activation. The size of nanoparticles can also play a role in their toxicity. Controlling particle size within a specific range can affect biodistribution, cellular uptake, and potential adverse reactions. Smaller particles may be more readily cleared by the kidneys, while larger particles may be more readily uptaken by the liver. Furthermore, the surface charge of LNPs can influence their interactions with biological systems. Neutral or slightly negatively charged LNPs are generally considered less toxic than highly positively charged LNPs. Charge modification can also affect cellular uptake and stability. Ensuring high encapsulation efficiency of the therapeutic payload within the LNP reduces the amount of free, unencapsulated drug or nucleic acid (which may cause toxicity). Furthermore, careful consideration of the administration dose may be crucial. This may involve determining the maximum tolerated dose through dose-escalation studies.

[0641] F. Payload

[0642] The payload may encompass bioactive molecules, including small molecules, biomolecules, nucleic acids (e.g., DNA, RNA, siRNA, shRNA), proteins, or peptides, all of which are part of the LNP composition. The payload may be covalently or non-covalently linked to the LNP, encapsulated within the LNP, linked to the LNP, or bound to an LNP within the LNP composition. In some embodiments, the LNP contains the payload. In some embodiments, the payload comprises a polynucleotide, a protein, or an antibody. In some embodiments, the payload comprises a polynucleotide, wherein the polynucleotide is mRNA.

[0643] In some embodiments, the length of the mRNA molecule is greater than 2000 nucleotides, greater than 2500 nucleotides, greater than 3000 nucleotides, greater than 3500 nucleotides, greater than 4000 nucleotides, greater than 4500 nucleotides, or greater than 5000 nucleotides. In some embodiments, the length of the mRNA molecule is about 2000 nucleotides. In some embodiments, the length of the mRNA molecule is about 2500 nucleotides. In some embodiments, the length of the mRNA molecule is about 3000 nucleotides. In some embodiments, the length of the mRNA molecule is about 3500 nucleotides. In some embodiments, the length of the mRNA molecule is about 4000 nucleotides. In some embodiments, the length of the mRNA molecule is about 4500 nucleotides. In some embodiments, the length of the mRNA molecule is about 5000 nucleotides.

[0644] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length.

[0645] In some embodiments, the polynucleotide has a concentration of 0.5 mg / mL to 3.0 mg / mL, 1.0 mg / mL to 3.0 mg / mL, or 2.0 mg / mL to 3.0 mg / mL or 1.0 mg / mL. In some embodiments, the polynucleotide has a concentration of 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL. In some embodiments, the polynucleotide has a concentration of 1.0 mg / mL.

[0646] In some embodiments, the concentration of polynucleotides in the LNP composition is from about 0.5 mg / mL to about 3 mg / mL (i.e., about 0.5 mg to about 3.0 mg of polynucleotides per mL of LNP composition). For example, in some embodiments, the concentration of polynucleotides in the LNP composition is from about 0.5 mg / mL to about 2.0 mg / mL, from about 1.0 mg / mL to about 3.0 mg / mL, or from about 2.0 mg / mL to about 3.0 mg / mL. In some embodiments, the concentration of polynucleotides in the LNP composition is from about 0.5 mg / mL, from about 0.6 mg / mL, from about 0.7 mg / mL, from about 0.8 mg / mL, from about 0.9 mg / mL, from about 1.0 mg / mL, from about 1.1 mg / mL, from about 1.2 mg / mL, from about 1.3 mg / mL, from about 1.4 mg / mL, from about 1.5 mg / mL, from about 1.6 mg / mL, from about 1.7 mg / mL, from about 1.8 mg / mL, from about 1.9 mg / mL, or from about 2.0 mg / mL. In some embodiments, the concentration of polynucleotides in the LNP composition is about 1.0 mg / mL.

[0647] In some embodiments, the concentration of polynucleotides in the aerosol formulation is from about 0.5 mg / mL to about 3 mg / mL (i.e., about 0.5 mg to about 3.0 mg of polynucleotides per mL of LNP composition). For example, in some embodiments, the concentration of polynucleotides in the aerosol formulation is from about 0.5 mg / mL to about 2.0 mg / mL, from about 1.0 mg / mL to about 3.0 mg / mL, or from about 2.0 mg / mL to about 3.0 mg / mL. In some embodiments, the concentration of the polynucleotide in the aerosol formulation is about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of the polynucleotide in the aerosol formulation is about 1.0 mg / mL.

[0648] In some embodiments, the concentration of polynucleotides in the aerosol particles is from about 0.5 mg / mL to about 3 mg / mL (i.e., about 0.5 mg to about 3.0 mg of polynucleotides per mL of LNP composition). For example, in some embodiments, the concentration of polynucleotides in the aerosol particles is from about 0.5 mg / mL to about 2.0 mg / mL, from about 1.0 mg / mL to about 3.0 mg / mL, or from about 2.0 mg / mL to about 3.0 mg / mL. In some embodiments, the concentration of polynucleotides in the aerosol particles is about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of polynucleotides in the aerosol particles is about 1.0 mg / mL.

[0649] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length and has a concentration of 1.0 mg / mL.

[0650] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length and has a concentration of 0.9 mg / mL.

[0651] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length and has a concentration of 0.8 mg / mL.

[0652] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length and has a concentration of 0.7 mg / mL.

[0653] In some embodiments, the polynucleotide is 2000 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is 2500 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is 3000 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is 3500 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is 4000 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL. In some embodiments, the polynucleotide is 4500 to 5000 nucleotides in length and has a concentration of 0.6 mg / mL.

[0654] In some embodiments, the polynucleotide is mRNA. In some embodiments, the mRNA encodes the dynein axonofilament intermediate strand 1 (DNAI1) protein. In other embodiments, the mRNA encodes the cystic fibrosis transmembrane transduction regulator (CFTR).

[0655] In some embodiments, the mRNA comprises the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises at least 80% of the polynucleotide sequence identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises at least 85% of the polynucleotide sequence identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises at least 90% of the polynucleotide sequence identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises at least 95% of the polynucleotide sequence identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises at least 99% of the polynucleotide sequence identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises the same polynucleotide sequence as SEQ ID NO: 1.

[0656] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises at least 80% of the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises at least 85% of the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises at least 90% of the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises at least 95% of the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises at least 99% of the same polynucleotide sequence as SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises the same polynucleotide sequence as SEQ ID NO: 4.

[0657] In some embodiments, the payload has an average molecular weight of up to 20,000,000 Da. In some embodiments, the payload may have an average molecular weight of up to 2,000,000 Da. In some embodiments, the payload may have an average molecular weight of up to 150,000 Da. In further embodiments, the payload has an average molecular weight of up to 15,000 Da, 5,000 Da, or 1,000 Da.

[0658] In one aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising LNP, wherein the LNP comprises less than 25% or less than 20% of 1,2-dioleoyl-3-dimethylammonium propane (DODAP); more than 40% of cholesterol; and / or messenger RNA (mRNA) with a lipid:mRNA ratio of less than 40:1.

[0659] In another aspect, this disclosure provides a lipid nanoparticle (LNP) composition comprising LNPs, wherein the LNPs specifically transduce secretory cells and / or ionizing cells; and / or the LNPs deliver mRNA to lung cells in an amount that effectively enhances the expression and / or function of polypeptides or polynucleotides encoded by mRNA.

[0660] In some embodiments, LNPs specifically transduce secretory cells and / or ionizing cells; and / or wherein LNPs deliver mRNA to lung cells in an amount that effectively enhances the expression and / or function of polypeptides or polynucleotides encoded by mRNA.

[0661] In some embodiments, the LNP comprises an ionizable cationic lipid; neutral phospholipids; polyethylene glycol (PEG)-lipids; and / or cholesterol. In some embodiments, the LNP comprises a second ionizable cationic lipid. In some embodiments, the LNP comprises anionic lipids. In some embodiments, the LNP comprises permanent cationic lipids.

[0662] In some embodiments, the LNP contains less than 25% or less than 20% of DODAP in molar percentage. In some embodiments, the LNP contains less than 5%, less than 10%, less than 15%, less than 16%, less than 17%, less than 18%, less than 19%, less than 20%, less than 21%, less than 22%, less than 22%, less than 23%, less than 24%, or less than 25% of DODAP in molar percentage.

[0663] In some implementations, LNP comprises DODAP with a molar percentage between 5% and 25%, between 7.5% and 25%, between 10% and 25%, between 15% and 25%, between 20% and 25%, between 5% and 20%, between 7.5% and 20%, between 10% and 20%, between 15% and 20%, between 5% and 15%, between 7.5% and 15%, between 10% and 15%, between 5% and 10%, or between 7.5% and 10%.

[0664] In some implementations, the LNP contains DODAP in molar percentages between 17.5% and 20%, between 17.5% and 22.5%, between 17.5% and 25%, between 5% and 17.5%, between 7.5% and 17.5%, between 10% and 17.5%, between 12.5% ​​and 17.5%, or between 15% and 17.5%.

[0665] In some implementations, LNP contains approximately 15% DODAP in molar percentage.

[0666] In some implementations, LNP contains approximately 16% DODAP in molar percentage.

[0667] In some implementations, LNP contains cholesterol with a molar percentage greater than 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0668] In some embodiments, LNP contains cholesterol in molar percentages between 40% and 60%, between 45% and 60%, between 50% and 60%, between 55% and 60%, between 40% and 55%, between 40% and 50%, between 40% and 45%, between 45% and 55%, between 45% and 50%, or between 50% and 55%. In some embodiments, LNP contains cholesterol in molar percentages of 50%.

[0669] Table 8. Exemplary Formulations

[0670]

[0671]

[0672] In some implementations, the LNP contains messenger RNA (mRNA).

[0673] In some implementations, LNPs contain mRNA at a lipid:mRNA ratio of less than 40:1.

[0674] In some implementations, the lipid:mRNA ratio is between 20:1 and 40:1, between 25:1 and 40:1, between 30:1 and 40:1, between 35:1 and 40:1, between 20:1 and 35:1, between 25:1 and 35:1, between 30:1 and 35:1, between 20:1 and 30:1, between 25:1 and 30:1, between 20:1 and 25:1, between 25:1 and 30:1, between 25:1 and 35:1, between 20:1 and 36:1, or between 25:1 and 36:1.

[0675] In some implementations, the lipid:mRNA ratio is 36:1.

[0676] In some implementations, the lipid:mRNA ratio is 25:1.

[0677] In some embodiments, the ionizable cationic lipid is 5A2-SC8 or 4A3-SC7; the neutral phospholipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC); and / or the polyethylene glycol (PEG)-lipid is DMG-PEG, optionally DMG-PEG2000. In some embodiments, the ionizable cationic lipid is 4A3-SC7; the neutral phospholipid is DOPE; and the polyethylene glycol (PEG)-lipid is DMG-PEG.

[0678] In some implementations, the LNP comprises a second cationic lipid, and the second cationic lipid is DODAP.

[0679] In some embodiments, the LNP comprises a second cationic lipid, and the second cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

[0680] In some embodiments, the ionizable cationic lipid is 4A3-SC7, and the LNP contains 4A3-SC7 in molar percentages between 13% and 15%, between 13.5% and 15%, between 14% and 15%, between 14.5% and 15%, between 13% and 14.5%, between 13.5% and 14.5%, between 14% and 14.5%, between 13% and 14%, between 13.5% and 14%, or between 13% and 13.5%.

[0681] In some implementations, the LNP contains PEG-lipids in molar percentages between 2% and 8%, between 4% and 8%, between 6% and 8%, between 2% and 6%, between 4% and 6%, between 2% and 4%, between 2% and 3%, between 3% and 4%, between 2.5% and 3.5%, between 2.5% and 3%, or between 3% and 3.5%.

[0682] In some implementations, the molar percentage of PEG-lipids is (approximately) 3%.

[0683] In some implementations, the LNP contains neutral phospholipids, and the neutral phospholipids are DOPE.

[0684] In some implementations, the LNP contains DOPE with a molar percentage between 10% and 25%, between 10% and 20%, between 10% and 15%, between 10% and 12.5%, between 15% and 25%, between 15% and 20%, or between 20% and 25%.

[0685] In some implementations, the LNP contains approximately 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% DOPE.

[0686] In some implementations, the LNP contains 11% or 22% DOPE in molar percentage.

[0687] In some implementations, the LNP contains about 11% or about 22% DOPE in molar percentage.

[0688] In some implementations, the LNP contains a payload.

[0689] In some implementations, the payload is messenger RNA (mRNA).

[0690] In some implementations, the mRNA contains 100 to 8 kilobases (kb).

[0691] In some implementations, the mRNA comprises 1kb to 8kb, 2kb to 8kb, 3kb to 8kb, 4kb to 8kb, 5kb to 8kb, 6kb to 8kb, 7kb to 8kb, 1kb to 7kb, 2kb to 7kb, 3kb to 7kb, 4kb to 7kb, 5kb to 7kb, or 6kb to 7kb, 1kb to 6kb, 2kb to 6kb, 3kb to 6kb, 4kb to 6kb, or 5kb to 6kb.

[0692] In some implementations, the mRNA contains approximately 2 kb.

[0693] In some implementations, the mRNA contains approximately 4.6 kb.

[0694] In some implementations, the mRNA encodes the cystic fibrosis transmembrane transduction regulator (CFTR) protein.

[0695] In some implementations, the mRNA encodes the dynamin axonofilament intermediate chain 1 (DNAI1) protein.

[0696] In some implementations, an mRNA-encoding gene editing system or a component thereof.

[0697] In some implementations, the mRNA encodes shRNA or microRNA.

[0698] In some embodiments, the LNP composition is a pharmaceutical composition.

[0699] In some embodiments, the LNP composition is an aerosolized composition.

[0700] In some embodiments, the LNP composition has an encapsulation efficiency between 50% and 99%, between 60% and 99%, between 70% and 99%, between 80% and 99%, between 90% and 99%, between 95% and 99%, between 50% and 95%, between 60% and 95%, between 70% and 95%, between 80% and 95%, between 85% and 95%, or between 90% and 95%.

[0701] In some embodiments, the LNP composition is substantially free of any anionic lipids, any permanent cationic lipids, or any combination of both. In some embodiments, the LNP composition is substantially free of any ionizable cationic lipids.

[0702] 1. Gene editing payload

[0703] The LNP disclosed herein may contain one or more components for gene editing, such as, but not limited to, guide RNA, tracr RNA, sgRNA, mRNA encoding gene editing or base editing proteins, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly spaced short palindromic repeat (CRISPR) nucleases (e.g., Cas9), DNA templates for gene editing, or combinations thereof. In some embodiments, the payload of the LNP may be suitable for genome editing technologies. In some embodiments, the genome editing technology may be CRISPR or TALEN. In some embodiments, the LNP may contain one or more mRNAs that encode gene editing or base editing proteins. In some embodiments, the LNP may contain both mRNA encoding gene editing or base editing proteins and one or more guide RNAs. In some embodiments, the LNP may contain at least one nucleic acid suitable for genome editing technologies, such as CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA), and DNA repair template. In some embodiments, the CRISPR nuclease may have altered activity; for example, the nuclease may be modified to act as a nicking enzyme instead of performing double-strand cleavage, or to bind to a sequence specified by the guide RNA but without enzymatic activity. In some embodiments, the base-editing protein may be a fusion protein comprising a deaminase domain and a sequence-specific DNA-binding domain, such as an inactive CRISPR nuclease.

[0704] (a) Gene editing methods

[0705] The LNP or pharmaceutical composition described herein may contain a payload of any conventional gene editing method. In some embodiments, the gene editing component may be selectively delivered to cells of a target organ. In some embodiments, the target organ may be the lung. In some embodiments, the cells of the target organ may be lung cells. In some embodiments, the cells may be ciliated cells, goblet cells, secretory cells, rod cells, basal cells, or ionized cells.

[0706] In some implementations, gene editing can be targeted editing. Targeted editing can be achieved through nuclease-independent or nuclease-dependent methods.

[0707] Nuclease-independent targeted editing, such as base editing and / or leader editing, can involve precise modifications to DNA sequences without producing double-strand breaks. Homologous recombination can be guided by homologous sequences flanking a foreign polynucleotide, introducing the foreign polynucleotide into an endogenous sequence via enzymatic mechanisms within the target organ's cells.

[0708] Base editing allows the conversion of one DNA base pair to another at a specific target site. In some embodiments, the nuclease may be a fusion of a deaminase with a modified Cas9 protein (dCas9) or other engineered Cas variants. In some embodiments, base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in endogenous DNA. Guide RNA can be programmed to target specific genomic locations of interest in target organ cells.

[0709] Leader editing allows for more complex and precise DNA modifications, including insertions, deletions, and all 12 possible base-to-base transitions (A, C, G, T) without double-strand breaks. A leader editing guide RNA can be designed, consisting of a guide sequence and a template for the desired editing. A primer-editing protein (PE2), which combines reverse transcriptase and a Cas9 variant, can be guided to the target site via the leader editing guide RNA. The Cas9 variant creates a single-strand break (nick) in the DNA. The reverse transcriptase can then use the template sequence of the leader editing guide RNA to copy the desired change into the nicked strand of the DNA. Subsequently, the target organ's cellular repair mechanisms repair the nick via homology-directed repair (HDR), integrating the edited sequence.

[0710] Nuclease-dependent methods can achieve higher-frequency targeted editing by specifically introducing double-strand breaks (DSBs) using rare cleaving nucleases (e.g., endonucleases). This nuclease-dependent targeted editing can also utilize DNA repair mechanisms, such as non-homologous end joining (NHEJ), which occur in response to DSBs. In some embodiments, DNA repair via NHEJ can result in the random insertion or deletion of a small number of endogenous nucleotides (insertion / deletion). In contrast to NHEJ-mediated repair, repair can also be performed via homology-directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homologous arms is available, the exogenous genetic material can be introduced into the genome via HDR, thereby achieving targeted integration of the exogenous genetic material. In some embodiments, the nucleases used for nuclease-dependent targeted editing may include, but are not limited to, CRISPR-Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo, and / or TALEN.

[0711] The use of CRISPR-Cas gene editing technology to generate genomic deletions (e.g., gene knockout in cells) is a well-known technique. See, for example, Bauer et al., J Vis Exp. 95:e52118 (2015). Available endonucleases capable of introducing specific, targeted DSBs include, but are not limited to, ZFN, TALEN, and CRISPR / Cas9.

[0712] In some implementations, targeted gene editing can be achieved by utilizing a dual integrase box exchange (DICE) system of phiC31 and Bxb1 integrases.

[0713] i. (i) CRISPR-Cas9 gene editing system

[0714] The CRISPR-Cas9 system, a naturally occurring defense mechanism in prokaryotes, has been engineered as an RNA-guided DNA-targeting platform for gene editing. The CRISPR-Cas9 system relies on the DNA nuclease Cas9 and two non-coding RNAs (crisprRNA (crRNA) and trans-activating RNA (tracrRNA)) to target and cleave DNA. CRISPRs are DNA sequences found in the genomes of bacteria and archaea that contain DNA fragments (spacer regions) similar to foreign DNA previously exposed to the cell (e.g., introduced by viruses infecting or attacking prokaryotes). Prokaryotes can use these DNA fragments to detect and destroy similar foreign DNA that is reintroduced, such as DNA from similar viruses during subsequent attacks. Transcription of the CRISPR locus forms an RNA molecule containing the spacer region sequence, which binds to and targets a Cas (CRISPR-associated) protein capable of recognizing and cleaving foreign DNA. For example, Koonin et al., Curr OpinMicrobiol 37:67-78 (2017) have described many types and categories of CRISPR / Cas systems.

[0715] crRNA drives the sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing (typically pairing with about 20 nucleotides in the target DNA). Changing the sequence of the 5' 20 nucleotides in crRNA allows the CRISPR-Cas9 complex to target specific loci. If the target sequence is followed by a specific short DNA motif (with the sequence NGG) called the preinterstitial sequence adjacent motif (PAM), the CRISPR-Cas9 complex will only bind to DNA sequences containing a sequence that matches the first 20 nucleotides of the crRNA.

[0716] tracrRNA can hybridize with the 3' end of crRNA to form an RNA double-stranded structure. This structure can be bound by Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave target DNA.

[0717] Once the CRISPR-Cas9 complex binds to DNA at the target site, the two independent nuclease domains within the Cas9 enzyme cleave one of the DNA strands upstream of the PAM site, resulting in a double-strand break (DSB), in which both strands of DNA terminate with a single base pair (flat end).

[0718] After the CRISPR-Cas9 complex binds to DNA at a specific target site and forms a site-specific DSB, cells can repair the DSB using two main DNA repair pathways: non-homologous end joining (NHEJ) and homologous directed repair (HDR). NHEJ is a highly active repair mechanism in most cell types, including non-dividing cells. NHEJ can be error-prone and often results in the removal or addition of one to several hundred nucleotides at the DSB site, although such modifications are usually less than 20 nucleotides. The resulting insertions and deletions (insertions and deletions) can disrupt either the coding or non-coding regions of a gene. Alternatively, HDR can repair DSBs with high fidelity using long segments of homologous donor DNA provided endogenously or exogenously. HDR is active only in dividing cells and occurs relatively infrequently in most cell types.

[0719] ii. CRISPR endonuclease

[0720] In some embodiments, the Cas9 endonuclease can be used in a CRISPR approach to genetically engineer cells of the target organ of the LNP described herein. In some embodiments, the Cas9 enzyme may be derived from *Streptococcus pyogenes*, although other Cas9 homologs may also be used. In some embodiments, the Cas9 enzyme may be wild-type Cas9. In some embodiments, the Cas9 enzyme may be a modified form of Cas9 (e.g., an evolved form of Cas9, or a Cas9 ortholog or variant). In some embodiments, Cas9 may be replaced by another RNA-guided endonuclease, such as Cpf1 (a class II CRISPR / Cas system).

[0721] In some implementations, the CRISPR / Cas system may contain components derived from type I, type II, or type III systems. In some implementations, the CRISPR / Cas system may contain components derived from type 1 and type 2 CRISPR / Cas systems, having types I to V or types II, V, and VI, respectively (Makarova et al., Nat Rev Microbiol 13(11):722-36 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0722] Class 2 CRISPR / Cas systems can have single-protein effectors. Type II, V, and VI Cas proteins can be single-protein RNA-guided endonucleases, referred to herein as class 2 Cas nucleases. Class 2 Cas nucleases may include, for example, but not limited to, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0723] In some embodiments, the Cas nuclease may be derived from a type II CRISPR / Cas system (e.g., the Cas9 protein from the CRISPR / Cas9 system). In some embodiments, the Cas nuclease may be derived from a class II CRISPR / Cas system (a single-protein Cas nuclease, such as the Cas9 protein or the Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity and can be directed to cleave desired nucleic acid targets by designing appropriate guide RNAs, which will be explained further below.

[0724] In some embodiments, the Cas nuclease may contain more than one nuclease domain. In some embodiments, the Cas9 nuclease may contain at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease may introduce a DSB into the target sequence. In some embodiments, the Cas9 nuclease may be modified to contain only one functional nuclease domain. For example, the Cas9 nuclease may be modified such that one of the nuclease domains is mutated or completely or partially deleted, thereby reducing its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease may be modified to exclude the functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease may be modified to exclude the functional HNH-like nuclease domain. In some embodiments where only one nuclease domain is functional, the Cas9 nuclease may be a cleavage enzyme that can introduce single-strand breaks (nicks) into the target sequence. In some embodiments, conserved amino acids within the Cas9 nuclease domain may be replaced to reduce or alter nuclease activity. In some embodiments, the Cas nuclease nicking enzyme may contain amino acid substitutions in the RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC-like nuclease domain may include D10A (based on *Streptococcus pyogenes* Cas9 nuclease). In some embodiments, the nicking enzyme may contain amino acid substitutions in the HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH-like nuclease domain may include, but are not limited to, E762A, H840A, N863A, H983A, and D986A (based on *Streptococcus pyogenes* Cas9 nuclease).

[0725] In some embodiments, the Cas nuclease may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a type I CRISPR / Cas system. For example, the Cas nuclease may be a Cas3 nuclease. In some embodiments, the Cas nuclease may be derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type IV CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type V CRISPR / Cas system. In some embodiments, the Cas nuclease may be derived from a type VI CRISPR / Cas system.

[0726] Type I CRISPR / Cas systems utilize a large effector complex called Cascade (a CRISPR-associated complex for antiviral defense) for target binding and interference. The Cascade complex may contain multiple Cas proteins, including Cas3, which is responsible for disrupting the target DNA. Type II CRISPR / Cas systems, particularly the CRISPR-Cas9 system, utilize a single Cas9 protein, guided by synthetic guide RNA (sgRNA), to introduce double-strand breaks into the target DNA for subsequent repair or modification. Type III CRISPR / Cas systems can utilize the Csm (CRISPR-Cas isoform multiprotein) or Cmr (CRISPR-Cas isoform ribonucleoprotein) complexes for interference. Type III CRISPR / Cas systems can target RNA molecules in addition to DNA. Type V CRISPR / Cas systems, including Cpf1 (also known as Cas12) and C2c2 (also known as Cas13), utilize a single effector protein for interference. Type VI CRISPR / Cas systems can utilize a single Cas protein, such as C2c2 (also known as Cas13), to target and cleave RNA molecules, making them usable for RNA editing and manipulation.

[0727] iii. Guide RNA (gRNA)

[0728] CRISPR technology may involve using a genome-targeting nucleic acid that guides one or more endonucleases to a specific target sequence within the target gene, thereby performing gene editing at that specific target sequence. The genome-targeting nucleic acid may be RNA. The genome-targeting RNA is referred to herein as "guide RNA" or "gRNA". The guide RNA may contain at least one spacer sequence and a CRISPR repeat sequence, the at least one spacer sequence being capable of hybridizing with a target nucleic acid sequence within the target gene for editing.

[0729] In type II gRNAs, the gRNA may also contain a second RNA sequence called tracrRNA. In type II gRNAs, the CRISPR repeat sequence and the tracrRNA sequence can hybridize to form a double strand. In type V gRNAs, the crRNA can form a double strand. In both systems, the double strand can bind a site-directing peptide, allowing the guide RNA and the site-directing peptide to form a complex. In some implementations, the nucleic acid targeting the genome can provide target specificity to the complex through its binding to the site-directing peptide. Therefore, the nucleic acid targeting the genome can guide the activity of the site-directing peptide.

[0730] As will be understood by those skilled in the art, each guide RNA may be designed to contain a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science 337:816-821 (2012); Deltcheva et al., Nature 471:602-607 (2011).

[0731] In some implementations, the nucleic acid targeting the genome (e.g., gRNA) can be a double-stranded guide RNA comprising two RNA strands. The first strand may contain an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand may contain a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0732] In some implementations, the nucleic acid targeting the genome (e.g., gRNA) can be a single-molecule guide RNA (sgRNA). In type II systems, the sgRNA may include, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single-molecule guide adapter, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension sequence may include elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide adapter may connect the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension sequence may include one or more hairpins. In type V systems, the single-molecule guide RNA may include a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0733] The spacer sequence in a gRNA is a sequence (e.g., a 20-nucleotide sequence) that defines a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) for a target gene of interest (e.g., DNAI1 or CFTR). In some embodiments, the spacer sequence can be in the range of 15 to 30 nucleotides. For example, the spacer sequence can contain 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence can contain 20 nucleotides.

[0734] The target sequence is located in a target gene (e.g., DNAI1 or CFTR), which may be adjacent to a PAM sequence and may be the sequence to be modified by an RNA-guided nuclease (e.g., Cas9). The target sequence is located on the PAM strand of a target nucleic acid, which is a double-stranded molecule containing a PAM strand and a complementary non-PAM strand. Those skilled in the art will recognize that the gRNA spacer region sequence can hybridize with a complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Therefore, the gRNA spacer region sequence can be an RNA equivalent of the target sequence. The gRNA spacer region can interact with the target nucleic acid of interest in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer region can vary depending on the target sequence of the target nucleic acid of interest.

[0735] In the CRISPR / Cas system, the spacer region sequence can be programmed to hybridize to a region on the target nucleic acid located at the 5' of the PAM that the Cas9 enzyme used in the system can recognize. The spacer region can be a perfect match to the target sequence or can have a mismatch. Each Cas9 enzyme can have a specific PAM sequence that it can recognize in the target DNA. For example, Streptococcus pyogenes can recognize a PAM containing the sequence 5'-NRG-3' in the target nucleic acid, where R can contain A or G, N can be any nucleotide, and N can be immediately adjacent to the 3' of the target nucleic acid sequence targeted by the spacer region sequence.

[0736] In some embodiments, the target nucleic acid sequence may be about 20 nucleotides in length. In some embodiments, the target nucleic acid may be less than about 20 nucleotides in length. In some embodiments, the target nucleic acid may be more than about 20 nucleotides in length. In some embodiments, the target nucleic acid may be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid may be at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence may have 20 bases at the 5' of the first nucleotide immediately adjacent to the PAM. For example, in a sequence containing 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleotide may be a sequence corresponding to Ns, where N can be any nucleotide, and the underlined NRG sequence may be Streptococcus pyogenes PAM.

[0737] The guide RNA can target any sequence of interest via the spacer region sequence in the crRNA. In some embodiments, the complementarity between the spacer region sequence of the guide RNA and the target sequence in the target gene can be approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer region sequence of the guide RNA and the target sequence in the target gene can be 100% complementary. In other embodiments, the spacer region sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, such as up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch.

[0738] The length of the spacer sequence in the gRNA can depend on the CRISPR / Cas9 system and the components used to edit any target gene (e.g., DNAI1 or CFTR). For example, different Cas9 proteins from different bacterial species may have different optimal spacer sequence lengths. Therefore, the spacer sequence length can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides. In some embodiments, the spacer sequence length can be 18 to 24 nucleotides. In some embodiments, the target sequence length can be 19 to 21 nucleotides. In some embodiments, the spacer sequence length can be 20 nucleotides.

[0739] In some embodiments, the gRNA may be sgRNA, which may include a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may include a variable-length spacer sequence of about 17 to 30 nucleotides at the 5' end of the sgRNA sequence.

[0740] In some embodiments, the gRNA may comprise unmodified ribonucleic acid. In some embodiments, the gRNA may comprise modified ribonucleic acid. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic production of RNA, such as modifications that enhance stability, reduce the likelihood or extent of innate immune responses, and / or enhance other properties, as described in the art. In some embodiments, non-naturally modified nucleobases can be introduced into any gRNA during or after synthesis. In some embodiments, modifications can occur at nucleoside internucleotide bonds, purine or pyrimidine bases, or sugars. In some embodiments, modifications can be introduced at the ends of the gRNA using chemical synthesis or polymerases.

[0741] In some implementations, more than one guide RNA may be used with a CRISPR / Cas nuclease system. Each guide RNA may contain a different target sequence, allowing the CRISPR / Cas system to cleave more than one target nucleic acid. In some implementations, one or more guide RNAs may have the same or different properties, such as activity or stability within the Cas9 RNP complex. When more than one guide RNA can be used, each guide RNA may be encoded on the same or different vectors. The promoters used to drive the expression of more than one guide RNA may be the same or different.

[0742] In some implementations, enzymatic or chemical linking methods can be used to conjugate polynucleotides or regions thereof to different functional parts, such as targeting or delivery agents, fluorescent tags, liquids, nanoparticles, etc.

[0743] In some embodiments, the CRISPR / Cas nuclease system may contain multiple gRNAs, such as 2, 3, or 4 gRNAs. These multiple gRNAs can target different sites within the same target gene. Alternatively, the multiple gRNAs can target different genes. In some embodiments, the guide RNA and the Cas protein can form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex. The guide RNA guides the Cas protein to a target sequence on one or more target genes (e.g., DNAI1 and CFTR), whereby the Cas protein cleaves the target gene at the target site. In some embodiments, the CRISPR / Cas complex may be a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex may be a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein may be the Cas9 protein. In some embodiments, the CRISPR / Cas9 complex may be a Cas9 / guide RNA complex.

[0744] In some embodiments, the insertion / deletion frequency (editing frequency) of a specific CRISPR / Cas nuclease system containing one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify highly efficient gRNA molecules for editing target genes. In some embodiments, highly efficient gRNAs produce gene editing frequencies higher than 80%. For example, a gRNA is considered highly efficient if it produces a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0745] iv. Other gene editing methods

[0746] In addition to the CRISPR system disclosed herein, other gene editing systems known in the art can also be used as payloads for the LNPs described herein. In some embodiments, other gene editing systems may include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, broad-spectrum nucleases, homing endonucleases, etc.

[0747] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA-binding domain (ZFBD). This ZFBD can be a polypeptide domain that binds to DNA in a sequence-specific manner via one or more zinc fingers. The zinc finger can be a domain of approximately 30 amino acids within the ZFBD, and its structure can be stabilized by coordination with zinc ions. Examples of zinc fingers include, but are not limited to, C2H2, C3H, and C4 zinc fingers. The designed ZFBD can be a domain not found in nature, and its design / composition is primarily derived from rational criteria, such as applying substitution rules and computerized algorithms to process information in a database storing existing ZFP designs and binding data. The selected ZFBD can be a domain not found in nature, and its generation can be primarily derived from empirical processes such as phage display, interaction traps, or hybridization selection. In some embodiments, the ZFN can be a fusion of a FokI nuclease and a zinc finger DNA-binding domain.

[0748] TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA-binding domain. A “transcription activator-like effector DNA-binding domain,” “TAL effector DNA-binding domain,” or “TALE DNA-binding domain” is a polypeptide domain of a TAL effector protein responsible for binding the TAL effector protein to DNA. Xanthomonas plant pathogens secrete TAL effector proteins during infection. These proteins enter the nucleus of plant cells, bind to effector-specific DNA sequences via their DNA-binding domains, and activate gene transcription at these sequences via their transactivation domains. The specificity of the TAL effector DNA-binding domain can depend on a variable number of incomplete 34-amino acid repeat sequences in the effector, which may contain polymorphisms at selected repeat positions, known as variable double residue repeats (RVDs). In some embodiments, TALEN can be a fusion polypeptide of a FokI nuclease and a TAL effector DNA-binding domain.

[0749] Other examples of suitable targeted nucleases include, but are not limited to, Bxb1, phiC31, PhiBT1, and Wβ / SPBc / TP901-1, whether used alone or in combination. Bxb1 nuclease, also known as Bxb1 integrase, is a site-specific recombinase derived from the mycobacterial phage Bxb1. Bxb1 integrase catalyzes site-specific recombination between two specific DNA sequences, called the att site. Bxb1 integrase recognizes a specific 48-base pair sequence within the att site. phiC31 nuclease (also known as phiC31 integrase) is derived from the phage phiC31. phiC31 nuclease catalyzes site-specific recombination between two specific DNA sequences called attB (the att site in the phage) and attP (the att site in the phage). phiC31 nuclease promotes the integration of DNA fragments flanked by attB and attP into the genome of cells in target organs. phiBT1 nuclease integrates into a different att site than phiC31. Wβ / SPBc / TP901-1 nuclease, also known as phage P2 Bxb1 Cre nuclease, is a site-specific recombinase derived from temperate phage P2.

[0750] Physical properties and characteristics of G. LNP

[0751] This disclosure relates in part to aerosolized pharmaceutical compositions comprising lipid nanoparticles (LNPs). In some embodiments, the LNPs may be delivered to the tracheobronchial region of a subject. In some embodiments, the LNPs have one or more of the following characteristics: encapsulation efficiency (EE) greater than 50%; mRNA integrity greater than 50%; diameter from 20 nm to 600 nm; and polydispersity less than 0.6.

[0752] 1. Encapsulation efficiency

[0753] Encapsulation efficiency (EE) refers to the fraction of the payload encapsulated within or otherwise incorporated into the lipid nanoparticle composition during LNP formation. Encapsulation efficiency can be determined by comparing the amount of input payload to the amount of payload encapsulated in the LNP sample, or by comparing the amount of payload in the LNP to the free excess payload not encapsulated in the sample. In some embodiments, fluorescence detection (e.g., RiboGreen) is used for determination. ™ To determine encapsulation efficiency, the method involves measuring free RNA in samples with intact LNPs and total RNA in samples treated to destroy LNPs.

[0754] Encapsulation refers to the process of confining a payload within a lipid nanoparticle (LNP). In some embodiments, encapsulation refers to confining one or more mRNA molecules within the LNP. In some embodiments, the payload may be captured within the lipid portion of the LNP or within an aqueous space encapsulated by some or all of the lipid components of the LNP. In some embodiments, the payload may be encapsulated within a lipid monolayer or bilayer membrane. In some embodiments, the payload may be intercalated between lipid components. In some embodiments, the payload may be encapsulated within the aqueous core of the LNP.

[0755] In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency ranging from about 75% to about 95%.

[0756] In various embodiments, the LNP has an encapsulation efficiency greater than 50%. For example, the EE of the LNP is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the LNP has an EE of 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, or 90% to 95%. In some embodiments, the LNP has an EE of 91%, 92%, 93%, 94%, or 95%. In some embodiments, the LNP has an EE of 95%.

[0757] In various embodiments, this disclosure provides a composition having a polynucleotide (e.g., mRNA molecule) incorporated into the LNP described herein. In some embodiments, the mRNA molecule is incorporated into a majority of the LNP. In some embodiments, more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the mRNA molecule is incorporated into the LNP. In some embodiments, at least 70% of the total mRNA molecule encoding CFTR is incorporated into the composition. In various embodiments, at least 70% of the total mRNA molecule encoding DNAI1 is incorporated into the LNP composition.

[0758] 2. mRNA integrity

[0759] mRNA integrity can refer to the quality of mRNA. In some embodiments, mRNA integrity refers to, for example, the percentage of undegraded mRNA after a purification process. In some embodiments, mRNA integrity refers to the quality of mRNA within the LNP after nebulization.

[0760] mRNA integrity can be determined by any method well known in the art, such as RNA agarose gel electrophoresis (e.g., Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology). The gel can be analyzed to determine whether the banding pattern and apparent nucleotide lengths conform to analytical reference standards. Other methods for assessing RNA integrity include, for example, using capillary gel electrophoresis (CGE) to evaluate purified mRNA.

[0761] In some implementations, mRNA integrity is measured by multiplex capillary electrophoresis (CE), for example using an Agilent Advanced Analytical 5200 fragment analyzer. The integrity of the mRNA transcript can be quantified by analyzing the electrophoretic pattern using software such as ProSize, through tailing analysis.

[0762] In some implementations, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the mRNA product is full-length.

[0763] In various embodiments, the LNPs described herein have greater than 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% mRNA integrity. In other embodiments, the LNPs have 75% to 99%, 80% to 95%, 85% to 90%, or 90% to 95% mRNA integrity.

[0764] 3. LNP diameter

[0765] In various embodiments, the LNP has a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNP has a diameter of about 80 nm to 150 nm. In some embodiments, the LNP has a diameter of about 20 nm to 600 nm, about 40 nm to 600 nm, about 60 nm to 600 nm, about 80 nm to 600 nm, about 100 nm to 600 nm, or 150 nm to 600 nm, or 200 nm to 600 nm, or 250 nm to 600 nm, or 300 nm to 600 nm, or 350 nm to 600 nm.

[0766] In various embodiments, the LNP has a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNP has a diameter of about 80 nm to 150 nm. In some embodiments, the LNP has a diameter of about 20 nm to 400 nm, about 40 nm to 400 nm, about 60 nm to 400 nm, about 80 nm to 400 nm, about 100 nm to 400 nm, or 150 nm to 400 nm, or 200 nm to 400 nm, or 250 nm to 400 nm, or 300 nm to 400 nm, or 350 nm to 400 nm.

[0767] In some embodiments, the LNP has a size distribution in which the average size (e.g., diameter) is from about 70 nm to about 200 nm, and more typically the average size is about 100 nm or less. In some embodiments, the LNP has a diameter of 50 nm to 90 nm.

[0768] In various embodiments, the LNP has a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNP has a size distribution in which the average size (e.g., diameter) is from about 70 nm to about 200 nm, and more typically the average size is about 100 nm or less.

[0769] In some embodiments, the LNP has a diameter of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, the LNP has an average size of about 150 nm or less (e.g., between 75 nm and 150 nm, particularly between 100 nm and 150 nm).

[0770] In various implementations, the atomized LNP has a diameter of 20 nm to 180 nm, 30 nm to 180 nm, 40 nm to 180 nm, 50 nm to 180 nm, 60 nm to 180 nm, 70 nm to 180 nm, 80 nm to 180 nm, 90 nm to 180 nm, 100 nm to 180 nm, or 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, or 180 nm.

[0771] In some implementations, the LNP has a diameter of 100nm to 400nm, 120nm to 400nm, 140nm to 400nm, 160nm to 400nm, 180nm to 400nm, 200nm to 400nm, 220nm to 400nm, 240nm to 400nm, 260nm to 400nm, 280nm to 400nm, 300nm to 400nm, 320nm to 400nm, 340nm to 400nm, 360nm to 400nm, or 380nm to 400nm.

[0772] In various implementations, the diameter of the LNP is determined by various techniques known in the art, including but not limited to dynamic light scattering (DLS).

[0773] 4. Polydispersity Index (PDI)

[0774] In some implementations, the LNP is characterized by having a polydispersity (or polydispersity index) of less than 0.5. In various implementations, the LNP has a polydispersity of less than 0.5. The polydispersity index (PDI) is the standard deviation of the LNP diameter distribution divided by the average LNP diameter. The PDI is commonly used as an indicator of the quality of the LNP in terms of its size distribution.

[0775] In various embodiments, dynamic light scattering (DLS) can be used to characterize the polydispersity index and size of the LNPs of this disclosure. DLS measures the light scattering produced by the sample under the influence of a light source. The PDI determined from the DLS measurement represents the distribution of LNP size in the population (at or near the average LNP diameter), where the PDI is zero for a completely homogeneous LNP population.

[0776] In various embodiments, the LNP has a polydispersity of less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1. In some embodiments, the LNP has a polydispersity of 0.5. In some embodiments, the LNP has a polydispersity of 0.4. In some embodiments, the LNP has a polydispersity of 0.3. In some embodiments, the LNP has a polydispersity of 0.2. In some embodiments, the LNP has a polydispersity of 0.1.

[0777] H. Physical properties and characteristics of aerosol particles

[0778] This disclosure relates in part to aerosolized pharmaceutical compositions comprising lipid nanoparticles (LNPs). In some embodiments, the LNPs may be delivered to the tracheobronchial region of a subject. In some embodiments, the aerosol particles of this disclosure have one or more of the following properties: a median mass aerodynamic diameter (MMAD) between 1 μm and 10 μm, a geometric standard deviation (GSD) of 1 to 5, and a fine particle fraction (FPF) percentage of at least 50%.

[0779] 1. Mass Median Aerodynamic Diameter (MMAD)

[0780] The size distribution of aerosol particles in an aerosol can be measured by determining their mass median aerodynamic diameter (MMAD). The MMAD of an aerosol is the median diameter of the aerosol particles within the aerosol. In some embodiments, a next-generation impactor (NGI) is used to measure the MMAD. As described herein, when administered to a subject, the MMAD of an aerosol can influence the deposition site of the aerosol in the lungs.

[0781] Unbound by theory, aerosols with a median mass aerodynamic diameter (MMAD) of 5 μm to 10 μm can primarily deposit in the large conduction airways and oropharyngeal region. Aerosol particles with an MMAD range of 1 μm to 5 μm can primarily deposit in the small lung airways and alveoli, while over 50% of aerosol particles with an MMAD of 3 μm can deposit in the alveolar region. In the case of systemic drug delivery via the pulmonary route, aerosols with small average particle size are required to ensure peripheral drug penetration.

[0782] In some implementations, MMAD is calculated in accordance with the USP section. <601> In some embodiments, 50% of the aerosol particles by mass are larger than this diameter, and 50% of the aerosol particles are smaller than this diameter. In some embodiments, the intercept of a straight line is plotted on a log-normal distribution plot, and this line is determined by using the entire distribution but focusing more on the points that have the majority of the mass.

[0783] After atomization, an aerosolized drug composition comprises aerosol particles of the drug composition. Aerosol particles refer to particles of a solution (or solid) that have been atomized. Each aerosol particle may contain a certain amount of LNP suspended in the solution (or solid). The size of the LNP is substantially smaller than the size of the aerosol particle. An aerosolized drug composition can be characterized by a number of parameters, including the particle size of the aerosol (e.g., diameter), for example by measuring the mass median aerodynamic diameter or fine particle fraction associated with the aerosol particles of the aerosolized drug composition. MMAD can be determined by impactor measurements, such as the Anderson Cascade Impactor (ACI) or the Next Generation Impactor (NGI).

[0784] In some embodiments, the aerosol particles, after atomization, have a MMAD of about 1 μm to about 9 μm, about 1 μm to about 8 μm, about 1 μm to about 7 μm, about 1 μm to about 6 μm, about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, about 1 μm to about 2 μm, or about 3 μm to about 5 μm. In various embodiments, the aerosol particles, after atomization, have a MMAD of about 1 μm to about 9 μm, or about 1 μm to about 8 μm, or about 1 μm to about 7 μm, or about 1 μm to about 6 μm, or about 1 μm to about 5 μm, or about 1 μm to about 4 μm, or about 1 μm to about 3 μm, or about 1 μm to about 2 μm, or 2 μm to 5 μm, or about 3 μm to about 5 μm, or 2 μm to 6 μm, or 3 μm to 6 μm.

[0785] In some embodiments, the aerosol particles have MMAD of about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, or about 6 μm.

[0786] In some embodiments, the aerosol particles have MMAD of about 3.0 µm, about 3.1 µm, about 3.2 µm, about 3.3 µm, about 3.4 µm, about 3.5 µm, about 3.6 µm, about 3.7 µm, about 3.8 µm, about 3.9 µm, 4.0 µm, about 4.1 µm, about 4.2 µm, about 4.3 µm, about 4.4 µm, about 4.5 µm, about 4.6 µm, about 4.7 µm, about 4.8 µm, about 4.9 µm, about 5.0 µm, about 5.1 µm, about 5.2 µm, about 5.3 µm, about 5.4 µm, about 5.5 µm, about 5.6 µm, about 5.7 µm, about 5.8 µm, about 5.9 µm, or about 6 µm.

[0787] 2. Geometric Standard Deviation (GSD)

[0788] The uniformity of the particle size distribution of aerosols (such as the aerosolized pharmaceutical compositions of this disclosure) can be quantified as the geometric standard deviation (GSD) of the aerosol particle size. GSD is a measure of the variability in aerosol particle diameter. The GSD of an aerosol can be calculated as the square root of the ratio of the observed droplet size at the 84th percentile of the cumulative percentage mass sieve distribution to the observed droplet size at the 16th percentile. A low GSD reflects a narrower droplet size distribution (i.e., uniform droplet size), which may be advantageous for targeting the aerosol to the respiratory system.

[0789] Monodispersity and polydispersity are related to the uniformity of the particle size distribution of aerosols. The lower the GSD of an aerosol, the higher the monodispersity of its particle size distribution. Similarly, the higher the GSD of an aerosol, the higher the polydispersity of its particle size distribution. For example, monodisperse particle size distributions typically include aerosols with a GSD of about 2 or less, while polydisperse particle size distributions typically include aerosols with a GSD of about 3 or greater.

[0790] In various embodiments, the aerosol particles have a GSD of about 1 to about 4, about 1 to about 3, or about 1 to about 2, or about 1, about 1.5, about 2, about 2.5, or about 3. In various embodiments, the aerosol particles, after atomization, have a GSD of about 1 to about 4, or about 1 to about 3, or about 1 to about 2, or about 1, about 1.5, about 2, about 2.5, or about 3. In some embodiments, the aerosol particles have a GSD of about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.

[0791] In other implementations, the GSD is determined by the formula provided below:

[0792]

[0793] In some implementation schemes, D 16 (Probit = -1) means that 84% of the particles by mass are larger than this diameter, and 16% of the particles are smaller than this diameter. In some implementations, D 84 (Probit = 1) means that 16% of the particles by mass are larger than this diameter, and 84% of the particles are smaller than this diameter. The intercept of a straight line is plotted on a log-normal distribution plot, and this line is determined using the entire distribution on both sides of Probit = 1.

[0794] The aerosolized pharmaceutical compositions provided herein may have an average droplet size of less than about 5 μm, or from about 1 μm to about 5 μm. The aerosolized pharmaceutical compositions may have a GSD in the range of 1.0 to 2.2, or from about 1.0 to about 2.2, or from 1.5 to 2.2, or from about 1.5 to about 2.2.

[0795] 3. Fine particle fraction

[0796] Fine particulate fraction (FPF) represents the mass percentage of aerosol particles with an aerodynamic diameter less than 5 μm and is used for in vitro evaluation of the aerosol's aerodynamic properties. In some embodiments, FPF represents the mass percentage of LNPs with an aerodynamic diameter less than 5 μm. In some embodiments, FPF is used for in vitro evaluation of the aerosol's aerosol's aerodynamic properties.

[0797] In some implementations, the fine particle dose (FPD) is defined as the total mass of drug (e.g., polynucleotide payload) with a size <5.0 μm, specifically by measuring the amount of drug (e.g., polynucleotide payload) with an equivalent circle diameter (ECD) <5.0 μm collected at all stages.

[0798] In various embodiments, the aerosol particles of this disclosure have a fine particle fraction of about 50% to about 80% (e.g., about 60% to about 70%). In various embodiments, the aerosol particles of this disclosure have a fine particle fraction of about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, or about 90%. In various embodiments, the aerosol particles of this disclosure, after atomization, have a fine particle fraction of about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, or about 90%.

[0799] III. The Method of This Disclosure

[0800] In one aspect, this disclosure provides a method for treating a lung disease or lung condition in a subject, the method comprising administering the aerosolized pharmaceutical composition described herein. This disclosure particularly provides a method and composition for treating cystic fibrosis, comprising administering to a subject an aerosolized LNP composition comprising mRNA encoding a cystic fibrosis transmembrane transduction regulator (CFTR) protein. In other embodiments, this disclosure provides a method and composition for treating primary ciliary dyskinesia (PCD), comprising administering to a subject an aerosolized LNP composition comprising mRNA encoding a dynein axonoderm intermediate chain 1 (DNAI1) protein.

[0801] Cystic fibrosis, also known as mucocutaneous cystic fibrosis, is an autosomal recessive genetic disorder that most severely affects the lungs, as well as the pancreas, liver, and intestines (Gibson et al., Am J Respir Crit Care Med. (2003) 168(8):918-951; Ratjen et al., Lancet Lond Engl. (2003) 361(9358):681-689; O'Sullivan et al., Lancet Lond Engl. (2009) 373(9678):1891-1904). Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane transduction regulator (CFTR) protein. This protein acts as a channel for transporting chloride ions across the cell membrane and is essential for regulating components of mucus, sweat, saliva, tears, and digestive enzymes. Pathogenic mutations in the CFTR protein impair its channel activity, leading to abnormal transepithelial transport of chloride and sodium ions, resulting in thick, viscous secretions in the lungs, pancreas, and other organs (O'Sulliven et al., Lancet Lond Engl. (2009) 373(9678):1891-1904; Rowe et al., N Engl J Med. (2005) 352(19):1992-2001). Most CF patients develop severe chronic lung disease associated with airway obstruction, partly due to elevated levels of sulfated mucin, inflammation, and recurrent infections, which ultimately lead to death; the median predicted lifespan in the United States is 40.7 years. Cystic fibrosis is the most common fatal genetic disease in the white population.

[0802] In individuals with CF, the lungs become colonized and infected by bacteria from childhood. This leads to chronic airway infection and inflammation, which further develops into bronchiectasis, air trapping, hypoxemia, and hypercapnia. In the United States, lung failure accounts for 68.1% of CF-related deaths. In the early stages, common bacteria such as Staphylococcus aureus and Hemophilus influenzae colonize and infect the lungs. Eventually, Pseudomonas aeruginosa (and sometimes Burkholderia cepacia) becomes dominant. By age 18, 80% of classic CF patients carry Pseudomonas aeruginosa, and 3.5% carry Burkholderia cepacia. Once in the lungs, these bacteria adapt and develop resistance to commonly used antibiotics.

[0803] Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder characterized by abnormal cilia and flagella in the lining of the airways, reproductive system, and other organs and tissues. The incidence of PCD is approximately 1 in 16,000. Symptoms can appear as early as birth, manifesting as difficulty breathing, and affected individuals experience frequent respiratory infections from early childhood. PCD patients also present with perennial nasal congestion and chronic cough. Chronic respiratory infections can lead to a condition called bronchiectasis, which damages the passageways called bronchi and can cause life-threatening breathing problems. Some individuals with PCD also experience infertility, recurrent ear infections, and abnormal positioning of organs in the thoracic and abdominal cavities.

[0804] Among the several genes confirmed to be directly involved in the pathogenesis of PCD, a large number of mutations occur in two genes: DNAI1 and DNAH5, which encode the intermediate and heavy chains of axonoderm dynamin, respectively. Mutations in other genes encoding proteins involved in axonoderm ultrastructure (DNAH11, DNAI2, TXRDC3, RSPH9, RSPH4A) or assembly (KTU, CRRC50) have also been reported, as well as mutations in the RPGR gene in some PCD cases. It is estimated that mutations in DNAI1 and DNAH5 (both associated with a defective phenotype of the ciliary extra-derived dynamin arm (ODA)) account for nearly 40% of PCD cases combined.

[0805] A. Treatment methods

[0806] In some implementations, patients requiring treatment are men or women aged 2 or older, 3 or older, 6 or older, 7 or older, 12 or older, 13 or older, 18 or older, 19 or older, 25 or older, 30 or older, 35 or older, 40 or older, 45 or older, or 50 or older. In some implementations, patients requiring treatment are men or women under 50 years of age, under 45 years of age, under 40 years of age, under 35 years of age, under 30 years of age, under 25 years of age, under 20 years of age, under 19 years of age, under 18 years of age, under 13 years of age, under 12 years of age, under 7 years of age, under 6 years of age, under 3 years of age, or under 2 years of age. In some implementations, the patients requiring treatment are males or females aged 2 to 18, 2 to 12, 2 to 6, 6 to 12, 6 to 18, 12 to 16, 2 to 50, 6 to 50, 12 to 50, or 18 to 50. In some implementations, the patients requiring treatment are pregnant or potentially pregnant women.

[0807] Patients with CF have higher chloride ion levels in their sweat than those without CF. For children with CF, a high chloride ion level in sweat is sufficient for diagnosis. Infants must sweat sufficiently for this test to be performed. Full-term infants typically produce enough sweat by 2 weeks of age. Therefore, in some implementations, patients requiring treatment have sweat chloride ion levels ≥60 mmol / L, ≥65 mmol / L, ≥70 mmol / L, ≥75 mmol / L, ≥80 mmol / L, ≥85 mmol / L, ≥90 mmol / L, ≥95 mmol / L, ≥100 mmol / L, ≥110 mmol / L, ≥120 mmol / L, ≥130 mmol / L, ≥140 mmol / L, or ≥150 mmol / L, as measured by quantitative pilocarpine iontophoresis (recorded in the subject's medical record). In some implementations, patients requiring treatment have chronic sinus lung disease and / or gastrointestinal / nutritional abnormalities consistent with CF.

[0808] In some implementations, forced expiratory volume in one second (FEV1) is an established indicator of cystic fibrosis (CF) disease progression, used to capture clinical course and assess treatment efficacy. Therefore, in various implementations, based on the patient's age, sex, and height, patients requiring treatment are required to achieve an FEV1 ≥50% and ≤90% of the predicted normal value (i.e., the average FEV1 of non-CF patients) (e.g., ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, or ≤55%). In some implementations, patients requiring treatment have a resting oxygen saturation ≥92% in indoor air (pulse oximetry). In some implementations, patients requiring treatment have a body mass index ≥17.5 kg / m². 2 And their weight is ≥40kg.

[0809] In some embodiments, any CF treatment method disclosed herein generates CFTR protein in the subject. In some embodiments, any PCD treatment method disclosed herein generates DNAI1 protein in the subject. In some embodiments, any treatment method disclosed herein increases CFTR protein or DNAI1 protein in the subject by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to baseline.

[0810] In some embodiments, an increase in CFTR protein or DNAI1 protein can be detected approximately 6, 8, 12, 24, 36, or 48 hours after administration of the pharmaceutical composition. In some embodiments, an increase in CFTR protein can be detected by qPCR of RNA purified from tissue samples. In some embodiments, an increase in DNAI1 protein can be detected by qPCR of RNA purified from tissue samples.

[0811] In some implementation plans, the patient requiring treatment has already received or is receiving other lung disease medications concurrently. For example, the patient requiring treatment may be receiving the rumacapto / ivacapto combination drug (ORKAMBI). ® Or, the patient may have received treatment for at least 28 days prior to the initiation of treatment according to this disclosure. The structures of Rumacapto and Ivacapto are provided as follows:

[0812]

[0813] Other CF medications may include, but are not limited to, routine inhaled therapy for airway clearance and respiratory infection management, such as bronchodilators, rhDNase (Pulmozyme). ® (Alfa chain enzyme), hypertonic saline, antibiotics and steroids; and other routine CF-related therapies, such as systemic antibiotics, pancreatic enzymes, multivitamins and diabetes and liver medications.

[0814] Specifically, the treatment method comprises: (1) providing: a) a nebulizer, and b) a container containing an LNP formulation for nebulization in a pharmaceutically acceptable carrier; and (2) administering the LNP formulation using the nebulizer. In some embodiments, the volume of the LNP formulation in the container has a volume of (about) 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, or 1 mL. In some embodiments, the formulation and composition typically contain a pharmaceutically acceptable carrier. The carrier is preferably a liquid carrier. Furthermore, the carrier preferably contains water and may contain other components. In some embodiments, the composition containing the LNP formulation is stored in ampoules, vials, or disposable vials prior to administration. In some embodiments, the composition is stored in disposable vials prior to administration.

[0815] In some embodiments, administration of the aerosolized pharmaceutical composition of this disclosure results in the expression of a protein (e.g., CFTR or DNAI1) in the lungs of a subject. In other embodiments, administration of the aerosolized pharmaceutical composition of this disclosure results in the detection of a protein (e.g., CFTR or DNAI1) in the lungs of a subject between 6 and 12 hours after delivery. In some embodiments, the protein is detected in the lungs at 6, 7, 8, 9, 10, 11, or 12 hours. In various embodiments, any technique known in the art (including, but not limited to, Western blot analysis) can be used to detect the protein (e.g., CFTR or DNAI1).

[0816] In some embodiments, the mRNA delivered according to this disclosure increases the protein level or activity in the upper, central, or peripheral airways of a subject's lungs, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, or 1500 times compared to a control (e.g., endogenous protein level or activity before or after treatment according to this disclosure, or a historical reference level).

[0817] In some embodiments, the CFTR mRNA delivered according to this disclosure increases the level or activity of CFTR protein in the upper, central, or peripheral airways of a subject's lungs, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, or 1500 times compared to a control (e.g., endogenous protein level or activity before or after treatment according to this disclosure, or a historical reference level).

[0818] In some embodiments, the DNAI mRNA delivered according to this disclosure increases the level or activity of DNAI protein in the upper, central, or peripheral airways of a subject's lungs, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, or 1500 times compared to a control (e.g., endogenous protein level or activity before or after treatment according to this disclosure, or a historical reference level).

[0819] In various embodiments, mRNA expression can be detected or quantified by qPCR of RNA purified from tissue samples. Protein expression can be determined by measuring the immune response to the protein (e.g., CFTR or DNAI1). Qualitative assessment of the protein can also be performed, for example, by Western blot analysis. Protein activity can be measured by appropriate activity assays. Various other methods are known in the art and can be used to determine protein expression or activity.

[0820] CFTR mRNA expression can be detected or quantified by qPCR of RNA purified from tissue samples. In some embodiments, DNAI mRNA expression can be detected or quantified by qPCR of RNA purified from tissue samples. CFTR protein expression can be determined by measuring the immune response to CFTR protein. In some embodiments, IgG antibodies against CFTR protein are measured in collected serum samples by enzyme-linked immunosorbent assay (ELISA). In some embodiments, CFTR-specific T cell responses are assessed using collected peripheral blood mononuclear cells. In some embodiments, T cell responses to CFTR protein are measured by human interferon-γ ELISA as described by Calcedo et al. (Calcedo et al., HumGene Ther Clin Dev. (2013) 24:108-15). Qualitative assessment of CFTR protein or DNAI can also be performed, for example, by Western blot analysis. CFTR protein activity can be measured by CFTR chloride channel activity in appropriate tissue cells. The steady-state potential corresponding to the average of the 10-second scoring intervals after perfusion is recorded. CFTR activity can be estimated by measuring the change in potential difference after perfusion of chloride-free isoproterenol. Various other methods are known in the art and can be used to determine CFTR mRNA and CFTR protein expression or activity.

[0821] In another aspect, this disclosure provides a method for delivering a payload to a cell, the method comprising contacting the cell with the LNP composition of this disclosure.

[0822] In another aspect, this disclosure provides a method for expressing proteins or RNA in cells, the method comprising contacting cells with the LNP composition of this disclosure.

[0823] In another aspect, this disclosure provides a method for increasing chloride ion flux in cells, the method comprising contacting cells with the LNP composition of this disclosure, wherein optionally, the cells contain a homozygous inactivating mutation in the CFTR gene.

[0824] On the other hand, this disclosure provides methods for maintaining transepithelial resistance (TEER) or reducing TEER by up to 10%, up to 20%, or up to 30%.

[0825] In some embodiments, the cells are lung cells. In some embodiments, the lung cells are secretory cells and / or ionizing cells.

[0826] In some implementations, the method specifically transduces secretory cells and / or ionizing cells compared to other lung cells.

[0827] In some implementations, lung cells are ciliated cells.

[0828] In some implementations, this method specifically transduces ciliated cells compared to other lung cells.

[0829] In some embodiments, any treatment method disclosed herein includes atomizing the composition to produce an aerosolized composition, and then contacting the aerosolized composition with cells.

[0830] In some embodiments, the compositions disclosed herein are aerosolized compositions, and any treatment methods disclosed herein involve contacting the aerosolized composition with cells.

[0831] In another aspect, this disclosure provides a method for delivering a payload to the lungs of a subject, the method comprising administering the composition of this disclosure to the subject.

[0832] In one aspect, this disclosure provides a method for treating or preventing lung disease in a subject, the method comprising administering the composition of this disclosure to the subject.

[0833] In some embodiments, any treatment method disclosed herein includes nebulizing the composition disclosed prior to the application step.

[0834] In some embodiments, the compositions of this disclosure are administered by inhalation in the form of an aerosolized composition.

[0835] In some embodiments, any of the treatments disclosed herein deliver an effective amount of the composition to the lungs.

[0836] In some implementations, any treatment method disclosed herein delivers an amount of medication to the lungs that is effective in treating lung disease.

[0837] In some embodiments, any treatment method disclosed herein is more effective than contacting cells with elecato, tezacto, rumacto, ivacato, or any combination thereof, or administering elecato, tezacto, rumacto, ivacato, or any combination thereof to a subject. In some embodiments, any treatment method disclosed herein is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% more effective than administering elecato, tezacto, rumacto, ivacato, or any combination thereof. In some embodiments, any method disclosed herein is 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, or 60% to 70% more effective than administering elecato, tezacto, rumacto, ivacato, or any combination thereof. The structure of ivacato is:

[0838]

[0839] The structure of Rumacato is:

[0840]

[0841] In one aspect, this disclosure provides the use of the compositions disclosed herein for the treatment of lung diseases.

[0842] On the other hand, this disclosure provides various LNP compositions for treating lung diseases, which are described in more detail above.

[0843] In another aspect, this disclosure provides a method for treating or preventing lung disease in a subject, the method comprising administering the composition disclosed herein to the subject.

[0844] In some embodiments, the method includes atomizing the composition prior to the application step. In some embodiments of the method, the LNP composition is administered by inhalation as an aerosolized composition.

[0845] In another aspect, this disclosure provides the use of the compositions described herein for treating lung diseases. In yet another aspect, this disclosure provides a composition described herein for treating lung diseases.

[0846] In another aspect, this disclosure provides a method for treating a lung disease in a subject, the method comprising administering the aerosolized pharmaceutical composition described herein to the subject.

[0847] B. Nebulization and Lung Delivery

[0848] The compositions disclosed herein, whether used alone or in combination with other suitable components, can be formulated into aerosol formulations (i.e., they can be nebulized). In various embodiments, the compositions disclosed herein can be formulated for administration by inhalation (e.g., intranasal or intratracheal) (see Brigham et al., Am. J. Sci., 298:278 (1989)). In some embodiments, delivery is pulmonary delivery, such as nebulization.

[0849] In some aspects, this document discloses aerosol formulations (i.e., formulations comprising the LNP composition of this disclosure and, for example, a buffer solution, which can be nebulized). In some embodiments, the aerosol formulation comprises the LNP composition of this disclosure and a buffer solution. In some embodiments, the buffer solution is a citrate buffer, an acetate buffer, or a tris(hydroxymethyl)aminomethane (Tris) buffer. For example, in some embodiments, the buffer solution comprises Tris and phosphate-buffered saline (PBS), such as 1×PBS, 15 mM Tris buffer. In some embodiments, the buffer solution is a citrate buffer.

[0850] In some embodiments, the buffer contains citrate at a concentration of about 5 mM to about 20 mM. For example, in some embodiments, the buffer contains citrate at concentrations of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0851] In some embodiments, the buffer contains tris at a concentration of about 5 mM to about 20 mM. For example, in some embodiments, the buffer contains citrate at concentrations of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0852] In some embodiments, the buffer also contains sucrose. For example, in some embodiments, the buffer also contains about 5% to about 15% sucrose (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15%).

[0853] Payloads, such as CFTR mRNA or DNAI mRNA, can be incorporated into lipid nanoparticles for delivery via various routes of administration. In some embodiments, CFTR mRNA or DNAI mRNA is incorporated into the LNP for pulmonary delivery. As used herein, pulmonary delivery refers to delivery to the lungs via, for example, the nasal cavity, trachea, bronchi, bronchioles, and / or other pulmonary systems. In some embodiments, CFTR mRNA is incorporated into the LNP for nebulization. In a particular embodiment, DNAI1 mRNA is incorporated into the LNP for nebulization. In these embodiments, the delivery medium may be in an inhalable aerosolized pharmaceutical composition.

[0854] In some embodiments, the compositions disclosed herein are atomized before inhalation.

[0855] In some implementations, the aerosol formulation may be placed in a pressurized, acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, etc.

[0856] In various embodiments, this document provides a method for targeting aerosolized pharmaceutical compositions to lung cells of a subject, the method comprising administering the aerosolized pharmaceutical composition to the subject. For example, the method comprises delivering a polynucleotide to the lung cells of the subject, including administering the aerosolized pharmaceutical composition described herein. In other examples, this disclosure provides for expressing a protein in the lungs of a subject, including administering the aerosolized pharmaceutical composition described herein to the subject. In some embodiments, this document provides a method for expressing a protein in the lungs of a subject, the method comprising administering the aerosolized pharmaceutical composition to the subject.

[0857] In various implementation schemes, an aerosolized drug composition is administered to the subject using a nebulizer.

[0858] In some embodiments, the nebulizer is applied at an output rate of 0.1 mL / min to 1.0 mL / min. In other embodiments, the nebulizer is applied at an output rate of 0.5 mL / min.

[0859] In some embodiments, the aerosolized drug composition is applied for less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. In some embodiments, the aerosolized drug composition is applied for less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. In some embodiments, the aerosolized drug composition is applied for less than 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. In some embodiments, the aerosolized drug composition is applied for less than 1 minute.

[0860] In some embodiments, the duration of atomization ranges from 1 minute to 60 minutes. In some embodiments, the duration of atomization is less than or equal to 1 minute. In some embodiments, the duration of atomization is less than or equal to 2 minutes. In some embodiments, the duration of atomization is less than or equal to 3 minutes. In some embodiments, the duration of atomization is less than or equal to 6 minutes. In some embodiments, the duration of atomization is less than or equal to 9 minutes. In some embodiments, the duration of atomization is less than or equal to 12 minutes. In some embodiments, the duration of atomization is less than or equal to 15 minutes. In some embodiments, the duration of atomization is less than or equal to 18 minutes. In some embodiments, the duration of atomization is less than or equal to 21 minutes. In some embodiments, the duration of atomization is less than or equal to 24 minutes. In some embodiments, the duration of atomization is less than or equal to 27 minutes. In some embodiments, the duration of atomization is less than or equal to 30 minutes. In some embodiments, the duration of atomization is less than or equal to 33 minutes. In some embodiments, the duration of atomization is less than or equal to 36 minutes. In some embodiments, the duration of atomization is less than or equal to 40 minutes. In some embodiments, the duration of atomization is less than or equal to 45 minutes. In some embodiments, the atomization duration is less than or equal to 50 minutes. In some embodiments, the atomization duration is less than or equal to 55 minutes. In some embodiments, the atomization duration is less than or equal to 60 minutes.

[0861] In various embodiments, the volume of the composition administered by atomization is from 1 mL to 10 mL. In some embodiments, the volume of the composition administered by atomization is at most about 1 mL. In some embodiments, the volume of the composition administered by atomization is at most about 4 mL. In some embodiments, the volume of the composition administered by atomization is at most about 8 mL.

[0862] In another aspect, this disclosure provides a method for delivering a payload to cells, the method comprising contacting the cells with the LNP composition disclosed herein. In another aspect, this disclosure provides a method for expressing a protein or RNA in cells, the method comprising contacting the cells with the LNP composition disclosed herein. In some embodiments, the cells are lung cells. In some embodiments, the lung cells are secretory cells. In some embodiments, the lung cells are ionizing cells. In some embodiments, the lung cells are ciliated cells. In some embodiments, the method specifically transduces secretory cells compared to other lung cells. In some embodiments, the method specifically transduces ionizing cells compared to other lung cells. In some embodiments, the method specifically transduces ciliated cells compared to other lung cells. In some embodiments, the method comprises nebulizing the LNP composition to generate an aerosolized composition, and then contacting the aerosolized composition with cells. In some embodiments, the LNP composition is an aerosolized composition, and the method comprises contacting the aerosolized composition with cells. In another aspect, this disclosure provides a method for delivering a payload to the lungs of a subject, the method comprising administering the composition disclosed herein to the subject.

[0863] In some embodiments, the method delivers an e...

Claims

1. A lipid nanoparticle (LNP) composition comprising at least six lipids.

2. The LNP composition according to claim 1, wherein the LNP composition comprises: (l) First ionizable cationic lipid; (m) Second ionizable cationic lipid; (n) Permanent cationic lipids; (o) Optional phospholipids; (p) Optional polyethylene glycol (PEG)-lipids; and / or (q) Optional sterols.

3. The LNP composition according to any one of claims 1 or 2, wherein the first ionizable cationic lipid is a dendritic lipid, optionally 4A3-SC7 or 5A2-SC8.

4. The LNP composition according to any one of claims 1 to 3, wherein the second ionizable cationic lipid is 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

5. The LNP composition according to any one of claims 1 to 4, wherein the permanent cationic lipid is trimethylammonium-propane, optionally 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

6. The LNP composition according to any one of claims 1 to 5, wherein the permanent cationic lipid is ethyl phosphocholine (EPC), optionally 1,2-dilauroyl-sn-glycerol-3-ethyl phosphocholine (12:0 EPC), 1,2-dimyristoyl-sn-glycerol-3-ethyl phosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycerol-3-ethyl phosphocholine (16:0 EPC) or 1,2-distearateyl-sn-glycerol-3-ethyl phosphocholine (18:0 EPC).

7. The LNP composition according to any one of claims 1 to 6, wherein the LNP composition specifically transduces lung cells; and / or the LNP delivers the mRNA to lung cells in an amount that effectively increases the expression and / or function of the protein encoded by the mRNA, optionally wherein the lung cells are ionocytes, ciliated cells, or secretory cells.

8. The LNP composition according to any one of claims 1 to 7, wherein the LNP comprises the first ionizable cationic lipid in a molar percentage between 10% and 30%.

9. The LNP composition according to any one of claims 1 to 8, wherein the LNP comprises DODAP in a molar percentage between 5% and 40%.

10. The LNP composition according to any one of claims 1 to 9, wherein the LNP comprises the permanent cationic lipid in a molar percentage between 5% and 25%, between 5% and 20%, between 5% and 15%, between 5% and 10%, between 10% and 20%, and between 15% and 20%.

11. The LNP composition according to any one of claims 1 to 10, wherein the sterol is cholesterol, and wherein the LNP contains a molar percentage of cholesterol greater than 25%.

12. The LNP composition of claim 11, wherein the LNP comprises cholesterol in a molar percentage between 25% and 50%, between 30% and 50%, between 30% and 45%, between 30% and 40%, or between 30% and 35%.

13. The LNP composition according to any one of claims 1 to 12, wherein the LNP comprises polyethylene glycol (PEG) lipids in a molar percentage between 0.5% and 10% or between 1% and 4%.

14. The LNP composition according to any one of claims 1 to 13, wherein: (g) The cationic ionizable lipid is 5A2-SC8 or 4A3-SC7; (h) The cationic ionizable SORT lipid is DODAP; (i) The permanent cationic lipid is 14:0 TAP or 14:0 EPC; (j) The phospholipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC). (k) The sterols mentioned are cholesterol or sitosterol; and / or (l) The polyethylene glycol (PEG) lipid is DMG-PEG, optionally DMG-PEG2000.

15. The LNP composition according to any one of claims 1 to 13, wherein: (g) The cationic ionizable lipid is 4A3-SC7; (h) The cationic ionizable SORT lipid is DODAP; (i) The permanent cationic lipid is 14:0 TAP; (j) The phospholipid mentioned is DOPE; (k) The sterols mentioned are cholesterol; and / or (l) The polyethylene glycol (PEG) lipid is DMG-PEG.

16. The LNP composition according to any one of claims 1 to 13, wherein: (g) The cationic ionizable lipid is 4A3-SC7; (h) The cationic ionizable SORT lipid is DODAP; (i) The permanent cationic lipid is 14:0 EPC; (j) The phospholipid mentioned is DOPE; (k) The sterols mentioned are cholesterol; and / or (l) The polyethylene glycol (PEG) lipid is DMG-PEG.

17. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage between approximately 10% and approximately 30%; (h) DODAP with a molar percentage between approximately 5% and approximately 40%; (i) 14:0 TAP with a molar percentage between approximately 5% and approximately 25%; (j) DOPE with a molar percentage between approximately 10% and approximately 30%; (k) Cholesterol with a molar percentage between approximately 30% and approximately 50%; and (l) DMG-PEG with a molar percentage between about 0.5% and about 10%.

18. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage between approximately 10% and approximately 15%; (h) DODAP with a molar percentage between approximately 5% and approximately 20%; (i) 14:0 TAP with a molar percentage between approximately 10% and approximately 20%; (j) DOPE with a molar percentage between approximately 15% and approximately 25%; (k) Cholesterol with a molar percentage between approximately 30% and approximately 40%; and (l) DMG-PEG with a molar percentage between approximately 1% and approximately 5%.

19. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage between approximately 10% and approximately 30%; (h) DODAP with a molar percentage between approximately 5% and approximately 40%; (i) 14:0 EPC with a molar percentage between approximately 5% and approximately 25%; (j) DOPE with a molar percentage between approximately 10% and approximately 30%; (k) Cholesterol with a molar percentage between approximately 30% and approximately 50%; and (l) DMG-PEG with a molar percentage between about 0.5% and about 10%.

20. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage between approximately 10% and approximately 15%; (h) DODAP with a molar percentage between approximately 5% and approximately 20%; (i) 14:0 EPC with a molar percentage between approximately 10% and approximately 20%; (j) DOPE with a molar percentage between approximately 15% and approximately 25%; (k) Cholesterol with a molar percentage between approximately 30% and approximately 40%; and (l) DMG-PEG with a molar percentage between approximately 1% and approximately 5%.

21. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage of approximately 15%; (h) DODAP with a molar percentage of approximately 15%; (i) 14:0 TAP with a molar percentage of approximately 15%; (j) DOPE with a molar percentage of approximately 22%; (k) cholesterol, with a molar percentage of approximately 30%; and (l) DMG-PEG with a molar percentage of approximately 3%.

22. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage of approximately 15%; (h) DODAP with a molar percentage of approximately 15%; (i) 14:0 TAP with a molar percentage of approximately 12%; (j) DOPE with a molar percentage of approximately 22%; (k) cholesterol, with a molar percentage of approximately 32%; and (l) DMG-PEG with a molar percentage of approximately 3%.

23. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage of approximately 15%; (h) DODAP with a molar percentage of approximately 15%; (i) 14:0 EPC with a molar percentage of approximately 15%; (j) DOPE with a molar percentage of approximately 22%; (k) cholesterol, with a molar percentage of approximately 30%; and (l) DMG-PEG with a molar percentage of approximately 3%.

24. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage of approximately 15%; (h) DODAP with a molar percentage of approximately 15%; (i) 14:0 EPC with a molar percentage of approximately 12%; (j) DOPE with a molar percentage of approximately 22%; (k) cholesterol, with a molar percentage of approximately 32%; and (l) DMG-PEG with a molar percentage of approximately 3%.

25. The LNP composition according to any one of claims 1 to 13, wherein the LNP comprises: (g) 4A3-SC7 with a molar percentage of approximately 15%; (h) DODAP with a molar percentage of approximately 15%; (i) 14:0 TAP with a molar percentage of approximately 12%; (j) DOPE with a molar percentage of approximately 22%; (k) cholesterol, with a molar percentage of approximately 32%; and (l) DMG-PEG with a molar percentage of about 3%, wherein the lipid:mRNA ratio is 33:

1.

26. The LNP composition according to any one of claims 1 to 25, wherein the LNP composition comprises a payload.

27. The LNP composition of claim 26, wherein the payload is messenger RNA (mRNA).

28. The LNP composition of claim 27, wherein the mRNA comprises 100 bases to 8 kilobases (kb).

29. The LNP composition according to any one of claims 27 or 28, wherein the LNP composition comprises mRNA at a lipid:mRNA ratio of less than 40:1, optionally, the ratio being 36:1, 33:1 or 30:

1.

30. The LNP composition according to any one of claims 27 to 29, wherein the mRNA encodes cystic fibrosis transmembrane transduction regulator (CFTR) protein or dynein axonoderm intermediate chain 1 (DNAI1) protein.

31. The LNP composition according to any one of claims 27 to 30, wherein the mRNA-encoding gene editing system or a component thereof.

32. The LNP composition according to any one of claims 26 to 31, wherein the payload is or further comprises shRNA or a polynucleotide encoding shRNA.

33. The LNP composition according to any one of claims 26 to 32, wherein the payload is or further comprises microRNA or a polynucleotide encoding microRNA.

34. The LNP composition according to any one of claims 26 to 33, wherein the payload is or further comprises a polypeptide.

35. The LNP composition according to any one of claims 1 to 34, wherein the LNP composition has an encapsulation efficiency between 50% and 99%, between 60% and 99%, between 70% and 99%, or between 80% and 99%.

36. A method for delivering a payload to a cell, the method comprising contacting the cell with an LNP composition according to any one of claims 1 to 35.

37. A method for expressing a protein or RNA in a cell, the method comprising contacting the cell with an LNP composition according to any one of claims 1 to 35.

38. The method according to claim 36 or 37, wherein the cell is a lung cell, optionally wherein the lung cell is a secretory cell, an ionizing cell, or a ciliated cell.

39. The method of claim 37, wherein the method specifically transduces the secretory cells compared to other lung cells.

40. The method of claim 37, wherein the method specifically transduces the ion cells compared to other lung cells.

41. The method of claim 37, wherein the method specifically transduces the ciliated cells compared to other lung cells.

42. The method according to any one of claims 36 to 41, wherein the method comprises atomizing the LNP composition to produce an aerosolized composition, and then contacting the aerosolized composition with the cells.

43. The method according to any one of claims 36 to 42, wherein the LNP composition is an aerosolized composition, and the method comprises contacting the aerosolized composition with the cells.

44. A method of delivering a payload to the lungs of a subject, the method comprising administering to the subject the composition according to any one of claims 1 to 35.

45. A method for treating or preventing lung disease in a subject, the method comprising administering to the subject the composition according to any one of claims 1 to 35.

46. ​​The method according to claim 44 or 45, wherein the method comprises atomizing the composition prior to the application step.

47. The method according to any one of claims 36 to 46, wherein the LNP composition is administered by inhalation in the form of an aerosolized composition.

48. The method according to any one of claims 36 to 47, wherein the method delivers an effective amount of the LNP composition to the lungs.

49. The method according to any one of claims 36 to 48, wherein the method delivers an effective amount of the LNP composition to the lungs to treat the lung disease.

50. Use of the LNP composition according to any one of claims 1 to 35 for the treatment of lung diseases.

51. The composition according to any one of claims 1 to 35, wherein the composition is used to treat lung diseases.

52. A kit comprising the composition according to any one of claims 1 to 35 and an atomizer mask and / or a mesh suitable for an atomizer.

53. A method for preparing an LNP composition according to any one of claims 1 to 35, the method comprising mixing the lipid component and the payload under conditions of effective assembly of the LNP containing the payload.

54. The method of claim 53, wherein the method comprises atomizing the composition to produce an aerosolized LNP composition.

55. An aerosolized pharmaceutical composition comprising the LNP composition according to any one of claims 1 to 35.

56. A liquid pharmaceutical composition for preparing the aerosolized pharmaceutical composition according to claim 55.

57. A method for delivering lipid nanoparticles (LNPs) to lung cells of a subject, the method comprising: The liquid pharmaceutical composition according to claim 56 is atomized to produce an aerosolized pharmaceutical composition, and the aerosolized pharmaceutical composition is administered to the subject.

58. A method of delivering a payload to lung cells of a subject, the method comprising administering to the subject the aerosolized pharmaceutical composition according to claim 55, wherein optionally the payload is a polynucleotide.

59. A method for expressing a protein in the lungs of a subject, the method comprising administering to the subject the aerosolized pharmaceutical composition according to claim 55.

60. A method for treating a lung disease in a subject, the method comprising administering to the subject the aerosolized pharmaceutical composition according to claim 55.

61. A kit comprising a lipid nanoparticle composition containing at least two selective organ-targeting (SORT) lipids and / or at least six lipids, optionally containing polynucleotides.

62. The aerosolized pharmaceutical composition of claim 55, wherein the aerosolized composition comprises aerosol particles.

63. The aerosolized pharmaceutical composition according to claim 62, wherein the aerosol particles have a median mass aerodynamic diameter (MMAD) of about 3 µm to about 5 µm.

64. The aerosolized pharmaceutical composition according to claim 62 or 63, wherein the aerosol particles have a geometric standard deviation (GSD) of about 1 to about 4.

65. The aerosolized pharmaceutical composition according to any one of claims 62 to 64, wherein the aerosol particles have a fine particle fraction (FPF) of about 50% to about 80%.

66. The aerosolized pharmaceutical composition according to any one of claims 62 to 65, wherein the concentration of the polynucleotide in the aerosol particles is from about 0.5 mg / mL to about 3 mg / mL.

67. The aerosolized pharmaceutical composition according to any one of claims 62 to 66, wherein the aerosolized pharmaceutical composition further comprises a buffer solution.

68. An aerosol formulation comprising the LNP composition according to any one of claims 1 to 70 and a buffer solution.

69. The liquid pharmaceutical composition according to claim 56, wherein the liquid pharmaceutical composition further comprises a buffer solution.

70. The liquid pharmaceutical composition of claim 56, the aerosolized pharmaceutical composition of claim 67, or the aerosol formulation of claim 68, wherein the buffer solution is a citrate buffer, an acetate buffer, or a tris (hydroxymethyl)aminomethane (Tris) buffer.

71. The liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation according to claim 70, wherein the buffer solution is a Tris buffer solution.

72. The liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation according to claim 70, wherein the buffer solution is a citrate buffer solution.

73. The liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation according to any one of claims 70 to 72, wherein the buffer solution further comprises sucrose.

74. The liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation according to any one of claims 70 to 73, wherein the buffer solution has a pH of about 4 or about 7.

5.

75. The liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation according to any one of claims 70 to 72, wherein the concentration of the polynucleotide in the liquid pharmaceutical composition, aerosolized pharmaceutical composition, or aerosol formulation is from about 0.5 mg / mL to about 3 mg / mL.

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