Lipid nanoparticle compositions and uses thereof

Abstract

An aerosolized pharmaceutical composition is provided, the aerosolized pharmaceutical composition comprising an aerosol particle, the aerosol particle comprising a lipid nanoparticle (LNP). Also provided herein are liquid pharmaceutical compositions for the preparation of aerosolized pharmaceutical compositions. Also provided herein are methods of administering the aerosolized pharmaceutical compositions. Also provided herein are kits comprising a lipid nanoparticle composition comprising one or more of a phospholipid, an ionizable lipid, a PEG-lipid, and a sterol.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 431,166, filed December 8, 2022, and U.S. Provisional Patent Application No. 63 / 485,863, filed February 17, 2023, each of which is incorporated herein by reference in its entirety. Incorporation by Reference into the Sequence Listing

[0002] This application contains a sequence listing submitted via EFS-WEB in .XML format and hereby incorporated by reference in its entirety. The .xml file was created on November 8, 2023, is named 06529-503001WO_SeqList_ST26.xml, and is 16 kilobytes in size. Background Art

[0003] The delivery of therapeutic agents to the lungs can be achieved by systemically administering the agent to the subject or by directly administering it to the lungs via the mouth or nose. In either case, a vehicle can be used to protect and help the delivery of the agent. A type of vehicle for therapeutic agents (such as proteins, nucleic acids or small molecules) is lipid nanoparticles (LNPs). This type of vehicle is, for example, used in vaccines based on mRNA. LNP vaccines are usually administered subcutaneously, and like most LNPs, they are mainly transported to the liver by default. In contrast, WO 2020 / 051220A1 discloses compositions that nucleic acid compositions are preferentially targeted or delivered to specific organs (such as the lungs). Therefore, a method for delivering LNP to the lungs is to administer the LNP in a pharmaceutical composition systemically, wherein the pharmaceutical composition is not atomized to form an aerosol, but is injected into the subject for systemic distribution (e.g., intravenous injection).

[0004] For being delivered to lung via mouth or nose, the pharmaceutical composition containing therapeutic agent or the vehicle containing therapeutic agent can be atomized to form fine particles (aerodynamic diameter is usually less than 10 microns). However, size control is an important consideration. Aerosol particles less than 2 microns can go deep into the alveolar region. It is still challenging to atomize the pharmaceutical composition containing therapeutic agent in a manner that keeps therapeutic efficacy and produces aerosol particles with the required physical characteristics of the appropriate region of the lung. Especially, the atomization of the pharmaceutical composition containing LNP can cause the degraded of LNP, the unsealing of therapeutic agent, the formation of aerosol particles with the physical characteristics that hinder targeted delivery to the desired region of the lung or to LNP and its payload or other undesirable effects of the two.

[0005] Therefore, there is a long-standing unmet need for aerosolized pharmaceutical compositions comprising aerosol particles containing lipid nanoparticles (LNPs) that are capable of delivering the LNPs to the lungs of a subject, for example, to the tracheobronchial region of a subject, and / or that have desirable physical characteristics. The present disclosure provides such aerosolized pharmaceutical compositions, methods of making and using the same, and other related compositions and methods. Summary of the Invention

[0006] In one aspect, the present disclosure provides an aerosolized pharmaceutical composition comprising aerosol particles comprising lipid nanoparticles (LNPs),

[0007] wherein the composition is capable of delivering the LNP to the lungs and / or tracheobronchial regions of a subject, and / or wherein the LNP has one or more of the following: an encapsulation efficiency (EE) greater than 50%, mRNA integrity greater than 50%, a diameter of 20 nm to 600 nm, a polydispersity less than 0.6, and / or wherein the aerosol particles have one or more of the following: a mass median 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%.

[0008] In some embodiments, the composition comprises LNPs for selective delivery to one or more of the following: goblet cells, secretory cells, club cells, basal cells, intermediate cells, serous cells, precursor cells, ionocytes, or ciliated cells. In some embodiments, the composition is capable of delivering the LNPs to the tracheobronchial region of a subject. In some embodiments, the composition is capable of delivering the LNPs to the upper airways, central airways, or peripheral airways of the lungs of the subject.

[0009] In some embodiments, the EE of the LNP is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or wherein the EE of the LNP is 50%-95%, 60%-95%, 70%-95%, 80%-95%, or 90%-95%. In some embodiments, the mRNA integrity of the LNP is greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or wherein the mRNA integrity of the LNP is 75%-99%, 80%-95%, 85%-90%, or 90%-95%. In some embodiments, the mRNA integrity loss of the LNP is less than 20%, less than 15%, less than 10%, less than 5%, or less than 5%-20%, less than 5%-15%, or less than 5%-10%. In some embodiments, the polydispersity of the LNP is 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 diameter of the LNP is 20 to 180 nm, 30 to 180 nm, 40 to 180 nm, 50 to 180 nm, 60 to 180 nm, 70 to 180 nm, 80 to 180 nm, 90 to 180 nm, 100 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. In some embodiments, the MMAD of the aerosol particles is 1 μm to 9 μm, or 1 μm to 8 μm, or 1 μm to 7 μm, or 1 μm to 6 μm, or 1 μm to 5 μm, or 1 μm to 4 μm, or 1 μm to 3 μm, or 1 μm to 2 μm, or 3 μm to 5 μm. In some embodiments, the GSD of the aerosol particles is 1 to 4, or 1 to 3, or 1 to 2, or 1, 1.5, 2, 2.5, or 3. In some embodiments, the FPF of the aerosol particles is 55%, 60%, 70%, 75%, 80%, 85%, or 90%.

[0010] In some embodiments, the LNP comprises one or more, two or more, or three or more of a phospholipid, an ionizable lipid, a polyethylene glycol (PEG)-lipid, and a sterol. In some embodiments, the composition further comprises one or more of a PEG-lipid, a sucrose, and a buffer, wherein the buffer comprises a citrate buffer, an acetate buffer, or a Tris buffer. In some embodiments, the molar percentage of the PEG-lipid is between 2% and 8%. In some embodiments, the concentration of the sucrose is 1% to 15% w / v, 5% to 15% w / v, 1% to 10% w / v, or 5% to 10% w / v. In some embodiments, the buffer is a citrate buffer, optionally at a pH of 4 to 8; an acetate buffer, optionally at a pH of 4 to 8; or a Tris buffer, optionally at a pH of 6 to 9.

[0011] In some embodiments, the LNP comprises a payload. In some embodiments, the payload comprises oligonucleotides, polynucleotides, peptides or proteins, such as nucleases and antibodies or antibody chains. In some embodiments, the polynucleotides comprise mRNA. In some embodiments, the length of the mRNA is about 1000 nucleotides (nt) to about 5000 nucleotides (nt), about 2000 nucleotides (nt) to about 5000 nt, about 2500 nt to about 5000 nt, about 3000 nt to about 5000 nt, about 3500 nt to about 5000, about 4000 nt to about 5000 nt or about 4500 nt to about 5000 nt. In some embodiments, the mRNA encoding dynein axoneme intermediate chain 1 (DNAI1) protein or cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments, the concentration of the polynucleotide is 0.5-3.0 mg / mL, or 1.0-3.0 mg / mL, or 2.0-3.0 mg / mL, or 1.0 mg / mL.

[0012] In some embodiments, the composition has a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the composition has an apparent pKa of 4 to 9.

[0013] In some embodiments, the composition results in expression of a protein from the mRNA in the lungs of the subject. In some embodiments, the composition results in detection of a protein from the mRNA in the lungs of the subject between 6 and 12 hours after delivery to the subject.

[0014] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a PEG-lipid; and / or a sterol. In some embodiments, the LNP comprises a second ionizable lipid. In some embodiments, the LNP comprises 1,2-dioleoyl-3-dimethylammonium propane (DODAP). In some embodiments, the LNP comprises 1,2-dioleoyl-3-dimethylammonium propane (DODAP) at a molar percentage of between about 5% and about 50%, between about 5% and about 35%, between about 20% and about 50%, between about 20% and about 50%, or about 20%.

[0015] In some embodiments, the LNP comprises a permanent cationic lipid. In some embodiments, the permanent cationic lipid comprises a trimethylammonium group, optionally wherein the permanent cationic lipid is 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0EPC). In some embodiments, the permanent ionizable lipid comprises a trimethylammonium group, optionally wherein the permanent cationic lipid is dioleoyl-3-trimethylammonium propane (DOTAP).

[0016] In some embodiments, the ionizable lipid is a dendritic lipid, optionally a dendritic lipid of Formula (I) or Formula (X), optionally 4A3-SC7 or 5A2-SC8.

[0017] In some embodiments, the LNP comprises about 19%, about 20%, about 19%, about 39%, and about 3.8% 4A3-SC7, 14:0EPC, DOPE, cholesterol, and DMG-PEG in molar percentages, respectively; and / or wherein the LNP has a lipid to RNA (weight / weight) ratio of about 30.

[0018] In another aspect, the present disclosure provides a liquid pharmaceutical composition for use in preparing the aerosolized pharmaceutical composition described herein.

[0019] On the other hand, the present disclosure provides a method for delivering lipid nanoparticles (LNP) to lung cells of a subject, the method comprising atomizing a liquid pharmaceutical composition as described herein to produce an aerosolized pharmaceutical composition, and administering the aerosolized pharmaceutical composition to the subject. On the other hand, the present disclosure provides a method for delivering a payload to lung cells of a subject, the method comprising administering an aerosolized pharmaceutical composition as described herein to the subject, wherein optionally the payload is a polynucleotide. On the other hand, the present disclosure provides a method for expressing a protein in the lungs of a subject, the method comprising administering an aerosolized pharmaceutical composition as described herein to the subject. On the other hand, the present disclosure provides a method for treating a lung disease in a subject, the method comprising administering an aerosolized pharmaceutical composition as described herein to the subject.

[0020] In some embodiments, the method comprises the aerosol particles, the aerosol particles comprising lipid nanoparticles (LNPs), the lipid nanoparticles comprising ionizable lipids, phospholipids, polyethylene glycol (PEG)-lipids; and / or sterols. In some embodiments, the method comprises the particles, wherein the LNPs comprise a second ionizable lipid. In some embodiments, the method comprises the particles, wherein the LNPs comprise 1,2-dioleoyl-3-dimethylammonium propane (DODAP).

[0021] In some embodiments, the method comprises the particle, wherein the LNP is stored in a buffer. In some embodiments of the method, the buffer is a citrate buffer, optionally at a pH of 4 to 8; wherein the buffer is an acetate buffer, optionally at a pH of 4 to 8; or wherein the buffer is a Tris buffer, optionally at a pH of 4 to 8.

[0022] In some embodiments, the method comprises the particles, wherein the particles are used In some embodiments, the method includes atomizing the particles using a sieve having a pore size of 40 HO V. The device aerosolizes the LNPs.

[0023] In some embodiments of the methods, the lung disease comprises primary ciliary dyskinesia (PCD) or cystic fibrosis (CF).

[0024] In some embodiments of the method, the aerosolized particles are selectively delivered to the tracheobronchial region of the lungs of the subject. In some embodiments of the method, the aerosolized pharmaceutical composition of claim 1 is administered to the subject using a nebulizer, wherein the nebulizer is administered at an output rate of 0.1 to 1 mL / min. In some embodiments of the method, the aerosolized pharmaceutical composition is administered at an output rate of 0.5 mL / min. In some embodiments of the method, the aerosolized pharmaceutical composition is administered for less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes.

[0025] In some embodiments of the methods, the administering comprises administering via intranasal administration, intratracheal administration, or oral administration, wherein the administering step delivers the aerosolized pharmaceutical composition to the tracheobronchial region (TB).In some embodiments of the methods, the subject is a human subject.

[0026] In another aspect, the present disclosure provides a method for preparing an aerosolized pharmaceutical composition according to claim 1, comprising introducing a liquid pharmaceutical composition comprising the aerosolized pharmaceutical composition into a nebulizer, wherein the nebulizer operates at an output rate of 0.1 to 1 mL / min or at an output rate of 0.5 mL / min.

[0027] In another aspect, the present disclosure provides a kit comprising a lipid nanoparticle composition and a screen, optionally comprising a polynucleotide, wherein the lipid nanoparticle composition comprises one or more of a phospholipid, an ionizable lipid, a PEG-lipid, and a sterol. In some embodiments of the kit, the phospholipid, the ionizable lipid, the PEG-lipid, and the sterol are in separate containers from the polynucleotide, or the phospholipid, the ionizable lipid, the PEG-lipid, and the sterol are in the same container as the polynucleotide.

[0028] In another aspect, the present disclosure provides a method for determining the amount of lipid nanoparticles (LNPs) in an aerosolized pharmaceutical composition, the method comprising contacting the aerosolized pharmaceutical composition with a filter comprising glass fibers, and extracting lipids from the filter with an extraction solution comprising ammonium ions, optionally ammonium acetate.

[0029] In some embodiments, the method comprises measuring the amount of at least one lipid in the extraction solution. In some embodiments of the method, the measuring is performed by high performance liquid chromatography (HPLC).

[0030] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising LNPs, wherein the LNPs comprise less than 25% or less than 20% 1,2-dioleoyl-3-dimethylammonium propane (DODAP) by mole, greater than 40% sterol by mole, and / or a messenger RNA (mRNA) having a lipid:mRNA ratio of less than 40:1.

[0031] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising LNPs, wherein the LNPs specifically transduce secretory cells and / or ionocytes, and / or the LNPs deliver mRNA to lung cells in an amount effective to increase expression and / or function of a protein encoded by the mRNA.

[0032] In some embodiments of the composition, the LNP composition specifically transduces secretory cells and / or ionocytes, and / or wherein the LNP composition delivers mRNA to lung cells in an amount effective to increase expression and / or function of a protein encoded by the mRNA.

[0033] In some embodiments of the compositions, the LNP composition comprises an ionizable lipid, a phospholipid, a polyethylene glycol (PEG)-lipid, and / or a sterol.

[0034] In some embodiments of the composition, the LNP comprises a second ionizable lipid. In some embodiments of the composition, the LNP comprises an anionic lipid. In some embodiments of the composition, the LNP comprises a permanent cationic lipid.

[0035] In some embodiments of the composition, the LNP comprises less than 25% or less than 20% DODAP by mole. In some embodiments of the composition, the LNP comprises between 5% and 25%, between 7.5% and 25%, between 10% and 25%, between 5% and 20%, between 7.5% and 20%, or between 10% and 20% DODAP by mole. In some embodiments of the composition, the LNP comprises between 5% and 17.5%, between 7.5% and 17.5%, or between 10% and 17.5% DODAP by mole. In some embodiments of the composition, the LNP comprises about 16% DODAP by mole.

[0036] In some embodiments of the compositions, the LNP comprises a cholesterol content greater than 40% by mole. In some embodiments of the compositions, the LNP comprises a cholesterol content between 40% and 60%, between 45% and 60%, or between 50% and 60% by mole. In some embodiments of the compositions, the LNP comprises a cholesterol content between 40% and 55%, between 45% and 55%, or between 50% and 55% by mole. In some embodiments of the compositions, the LNP comprises a cholesterol content between 40% and 50% or between 45% and 50% by mole. In some embodiments of the compositions, the LNP comprises a cholesterol content of about 50% by mole.

[0037] In some embodiments of the compositions, wherein the LNP comprises messenger RNA (mRNA). In some embodiments of the compositions, the LNP comprises an mRNA having a lipid:mRNA ratio of less than 40:1. In some embodiments of the compositions, the lipid:mRNA ratio is between 20:1 and 40:1, between 25:1 and 40:1, between 35:1 and 40:1, or between 30:1 and 40:1. In some embodiments of the compositions, the lipid:mRNA ratio is between 20:1 and 36:1, or between 25:1 and 36:1. In some embodiments of the compositions, the lipid:mRNA ratio is 36:1. In some embodiments of the compositions, the lipid:mRNA ratio is 25:1.

[0038] In some embodiments of the composition, the ionizable lipid is 5A2-SC8 or 4A3-SC7, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and / or the polyethylene glycol (PEG)-lipid is DMG-PEG, optionally DMG-PEG2000.

[0039] In some embodiments of the composition, the ionizable lipid is 4A3-SC7, the phospholipid is DOPE, and the polyethylene glycol (PEG)-lipid is DMG-PEG.

[0040] In some embodiments of the composition, the LNP comprises a second ionizable lipid and the second ionizable lipid is DODAP. In some embodiments of the composition, the LNP comprises a second ionizable lipid and the second ionizable lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

[0041] In some embodiments of the composition, the ionizable lipid is 4A3-SC7 and the LNP comprises between about 13% and about 15% 4A3-SC7 by mole. In some embodiments of the composition, the ionizable lipid is 4A3-SC7 and the LNP comprises between about 13% and about 15% 4A3-SC7 by mole.

[0042] In some embodiments of the composition, the LNP comprises a molar percentage of between about 2% and about 8% PEG-lipid. In some embodiments of the composition, the molar percentage of the PEG-lipid is about 3%.

[0043] In some embodiments of the composition, the LNP comprises a neutral phospholipid and the neutral phospholipid is DOPE. In some embodiments of the composition, the LNP comprises between about 10% and about 25% DOPE by mole. In some embodiments of the composition, the LNP comprises about 11% or about 22% DOPE by mole.

[0044] In some embodiments of the composition, the LNP comprises about 15 mol% 4A3-SC7, about 16 mol% DODAP, about 22 mol% DOPE, about 3 mol% DMG-PEG2000, and about 44 mol% cholesterol.

[0045] In some embodiments of the composition, the LNP comprises about 14 mole% 4A3-SC7, about 22 mole% DODAP, about 11 mole% DOPE, about 3 mole% DMG-PEG2000, and about 50 mole% cholesterol.

[0046] In some embodiments of the compositions, the LNP comprises a payload. In some embodiments of the compositions, the payload is messenger RNA (mRNA). In some embodiments of the compositions, the mRNA is comprised between 100 bases and 8 kilobases (kb). In some embodiments of the compositions, the mRNA is comprised between 1kb and 8kb, or between 2kb and 8kb, between 3kb and 8kb, or between 4kb and 8kb. In some embodiments of the compositions, the mRNA is comprised between 1kb and 7kb, or between 2kb and 7kb, between 3kb and 7kb, or between 4kb and 7kb. In some embodiments of the compositions, the mRNA is comprised between 1kb and 6kb, or between 2kb and 6kb, between 3kb and 6kb, or between 4kb and 6kb. In some embodiments of the compositions, the mRNA comprises (approximately) 2kb. In some embodiments of the compositions, the mRNA comprises (approximately) 4.6kb. In some embodiments of the composition, the mRNA encodes cystic fibrosis transmembrane conductance regulator (CFTR) protein. In some embodiments of the composition, the mRNA encodes dynein axoneme intermediate chain 1 (DNAI1) protein. In some embodiments of the composition, the mRNA encodes a gene editing system or a component thereof.

[0047] In some embodiments of the composition, the payload is shRNA or a polynucleotide encoding shRNA. In some embodiments of the composition, the payload is microRNA or a polynucleotide encoding microRNA.

[0048] In some embodiments of the composition, the composition is a pharmaceutical composition. In some embodiments of the composition, the composition is an aerosolized composition.

[0049] In some embodiments of the composition, the LNP has an encapsulation efficiency of between 50% and 99%, between 60% and 99%, between 70% and 99%, or between 80% and 99%. In some embodiments of the composition, the LNP has an encapsulation efficiency of between 50% and 95%, between 60% and 95%, between 70% and 95%, or between 80% and 95%.

[0050] In another aspect, the present disclosure provides a method for delivering a payload to a cell, the method comprising contacting the cell with an LNP composition as described herein. In another aspect, the present disclosure provides a method for delivering an expressed protein or RNA in a cell, the method comprising contacting the cell with an LNP composition as described herein. In another aspect, the present disclosure provides a method for increasing chloride ion flux in a cell, the method comprising contacting the cell with an LNP composition according to any one of claims 105, 107-113, wherein optionally the cell comprises a homozygous inactivating mutation in the CFTR gene.

[0051] In some embodiments, the method maintains transepithelial electrical resistance (TEER) or reduces TEER by at most 10%, at most 20%, or at most 30%.

[0052] In some embodiments of the methods, the cell is a lung cell. In some embodiments of the methods, the lung cell is a secretory cell and / or an ionocyte.

[0053] In some embodiments of the method, the method specifically transduces the secretory cells and / or the ionocytes compared to other lung cells. In some embodiments of the method, wherein the lung cells are ciliated cells. In some embodiments, the method specifically transduces the ciliated cells compared to other lung cells.

[0054] In some embodiments, the method comprises atomizing the LNP composition to produce an aerosolized composition, and then contacting the aerosolized composition with the cells. In some embodiments of the method, the LNP composition is an aerosolized composition, and the method comprises contacting the aerosolized composition with the cells.

[0055] On the other hand, the present disclosure provides a method for delivering a payload to the lungs of a subject, the method comprising administering a composition as described herein to the subject. On the other hand, the present disclosure provides a method for treating or preventing a lung disease in a subject, the method comprising administering a composition as described herein to the subject. In some embodiments, the method comprises atomizing the composition before the administering step. In some embodiments, 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 amount effective for treating the lung disease to the lungs. In some embodiments, the method is more effective than contacting the cells with or administering to the subject: elexacaftor, tezacaftor, lumacaftor, ivacaftor, or a combination thereof.

[0056] In another aspect, the present disclosure provides uses of the compositions described herein for treating lung disease. In another aspect, the present disclosure provides compositions described herein for treating lung disease. In another aspect, the present disclosure provides a kit comprising a composition described herein and a nebulizer mask and / or screen suitable for use in a nebulizer. In another aspect, the present disclosure provides a method for preparing an LNP composition as described herein, comprising mixing a lipid component and the payload under conditions effective to assemble the LNP comprising the payload. In some embodiments, the method comprises atomizing the composition to produce an aerosolized LNP composition.

[0057] Other aspects and embodiments of the present invention are provided by the following detailed description. The scope of the present invention is limited only by the claims. Those skilled in the art will be able to conceive and implement many variations of the disclosed embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0059] Figure 1 A schematic diagram showing the criteria used to evaluate aerosolized pharmaceutical compositions.

[0060] Figure 2 Shown in A graph of the output rates of various nebulizer heads used on the nebulizer system.

[0061] Figure 3Shown in Graph of the output rate of Composition A on the nebulizer system using various nebulizer heads.

[0062] Figure 4A-4B Predicted and actual nebulization times for baseline or composition A / DNAI1 lipid nanoparticles are shown. Figure 4A Predicted and actual nebulization times for the baseline (PBS) are shown. Figure 4B The predicted and actual nebulization times for composition A / DNAI1 lipid nanoparticles are shown.

[0063] Figure 5 Shown in Encapsulation efficiency (%) of various nebulizer heads on the nebulizer system.

[0064] Figure 6 Shown Encapsulation efficiency (%) of 40HO V of the nebulizer system.

[0065] Figure 7 Shown mRNA integrity of the nebulizer system at 35 V or 40 HO V.

[0066] Figures 8A-8C Shown Encapsulation efficiency (%) of various heads of the nebulizer system. Figure 8A Shown Encapsulation efficiency (%) of the nebulizer at slow flow rate. Figure 8B Shown Encapsulation efficiency (%) of the nebulizer at medium flow rate. Figure 8C Shown Encapsulation efficiency (%) of the nebulizer at fast flow rate.

[0067] Figures 9A-9C Shown Hydrodynamic diameters (nm) of various heads of the nebulizer system. Figure 9A Shown Hydrodynamic diameter of the nebulizer at slow flow rates (nm). Figure 9B Shown Hydrodynamic diameter of the nebulizer at medium flow rate (nm). Figure 9C Shown Hydrodynamic diameter of the nebulizer at fast flow rates (nm).

[0068] Figures 10A-10C Shown Polydispersity index of various heads of the nebulizer system. Figure 10A Shown Polydispersity index of the nebulizer at slow flow rates. Figure 10B Shown Polydispersity index of the nebulizer at medium flow rates. Figure 10C Shown Polydispersity index of the nebulizer at fast flow rates.

[0069] Figure 11 Shown The hydrodynamic diameter (nm) of various tips of the nebulizer system during the nebulization process.

[0070] Figure 12 Shown Polydispersity index of various heads of the nebulizer system during the atomization process.

[0071] Figures 13A-13D Shown Characterization of lipid nanoparticles with the 40HO V nebulizer system. Figure 13A The concentrations of lipid nanoparticles are shown. Figure 13B The encapsulation efficiency (%) of lipid nanoparticles is shown. Figure 13C The hydrodynamic diameters (nm) of the lipid nanoparticles are shown. Figure 13D The polydispersity index of lipid nanoparticles is shown.

[0072] Figures 14A-14B Shown Drug deposition (μg) in each stage for 40 HO V or 30 HO V of the nebulizer system. Figure 14A Drug deposition (μg) in each stage at 40 HO V is shown. Figure 14B Drug deposition (μg) in each stage at 30 HO V is shown.

[0073] Figure 14C Aerosol characterization in the Next Generation Impactor (NGI) is shown.

[0074] Figures 15A-15B Shown Deposition fraction of multi-path particle dosimetry (MPPD) from Solo or PDAP. Figure 15A Shown Deposition fraction of Solo's multi-path particle dosimetry (MPPD). Figure 15B The deposition fraction of the multi-path particle dosimetry (MPPD) of the PDAP is shown.

[0075] Figures 16A-16E Shown in Deposition fraction of multipath particle dosimetry (MPPD) for various nebulizer heads on a nebulizer system. Figure 16A Deposition fractions from multi-path particle dosimetry (MPPD) at 30 NV are shown. Figure 16BDeposition fractions from multi-path particle dosimetry (MPPD) at 35 NV are shown. Figure 16C The deposition fractions from multi-path particle dosimetry (MPPD) at 30 V are shown. Figure 16D The deposition fractions from multi-path particle dosimetry (MPPD) at 35V are shown. Figure 16E The deposition fraction of multi-path particle dosimetry (MPPD) at 40 HO is shown.

[0076] Figures 17A-17B The percentage of lipid recovery using different lipid extraction solutions and volumes is shown. Figure 17A Percent lipid recovery is shown. Figure 17B Quantitative data of percentage lipid recovery are shown.

[0077] Figures 18A-18C Comparative results of the gravimetric, RiboGeen, and high performance liquid chromatography with charged aerosol detector (HPLC-CAD) methods are shown. Figure 18A Quantitative data showing comparative results of different methods are shown. Figure 18B A comparison of gravimetric and HPLC-CAD is shown. Figure 18C A comparison of RiboGreen and HPLC-CAD is shown.

[0078] Figures 19A-19B The lipid weight fractions for various lipid extraction solutions and volumes are shown. Figure 19A Lipid weight fractions in rat GLP-tox experiments are shown. Figure 19B Lipid weight fractions in NHP GLP-tox experiments are shown.

[0079] Figure 20A-Figure 20B The percentage of lipid recovery through the different filter deposits is shown. Figure 20A The percentage of lipid recovery under different conditions is shown. Figure 20B Quantitative data of percentage lipid recovery are shown.

[0080] Figures 21A-21E A comparison of total lipid mass by gravimetric, HPLC-CAD, and LC-MS for Composition B is shown. Figure 21A Percent lipid recovery is shown. Figure 21B Quantitative data of percentage lipid recovery are shown. Figure 21C Comparison of total lipid mass by gravimetric, HPLC-CAD, and LC-MS is shown. Figure 21D A comparison of LC-MS and gravimetric methods is shown. Figure 21E Comparison of LC-MS and HPLC-CAD is shown.

[0081] Figure 22Comparison of lipid fractions by HPCL-CAD and LC-MS for composition B is shown.

[0082] Figure 23 An image of the ATS-003 atomization device is shown.

[0083] Figures 24A-24C This is an image from the Next Generation Impactor (NGI). Figure 24A An image of the Next Generation Impactor (NGI) is shown. Figure 24B An NGI seal (staged nozzle) is shown. Figure 24C The NIG cover (interstage channel) is shown.

[0084] Figures 25A-25B This is an image of an NGI gravimetric cup. Figure 25A An image of an NGI weight cup is shown. Figure 25B An image of the NGI weight cup components (NGI specific cup, filter, and filter holder) is shown.

[0085] Figures 26A-26B Here is an image of the filter holder inside NGI. Figure 26A Shown is an image of the NGI internal filter holder (NGI specialty cup, filter holder grid, and filter hold down ring). Figure 26B An image showing the filter holder inside the NGI.

[0086] Figure 27 This is an image of a USP (United States Pharmacopeia) sensing port.

[0087] Figure 28A An image of an experimental stand with a claw-type nebulizer is shown. Figure 28B Shown is the collection of aerosol nebula using a pre-chilled 50 mL conical tube with an open cap to fit a nebulizer.

[0088] Figure 29 Schematic diagram of lipid nanoparticles (LNPs) encapsulating mRNA is shown.

[0089] Figure 30 AUC / min of non-DOTAP LNPs are shown.

[0090] Figure 31 The AUC / min of the DODAP-based LNPs are shown.

[0091] Figure 32 Shown is the AUC / min of Composition B with different buffers.

[0092] Figure 33AShown are the screening results of Compound B and Composition R with different buffers. Figure 33B Shown are the screening results of Compound B and Composition U with different buffers.

[0093] Figures 34A-34D A summary of lipid nanoparticle characterization is shown for various lipid nanoparticles in different pH buffers. Figure 34A The sizes of lipid nanoparticles are shown. Figure 34B The polydispersity index of lipid nanoparticles is shown. Figure 34C The encapsulation efficiency (%) of lipid nanoparticles is shown. Figure 34D Zeta potential (mV) of lipid nanoparticles is shown.

[0094] Figure 35A Shown are TNS assays of composition B in different buffers. Figure 35B Shown are TNS assays of composition C in different buffers. Figure 35C The TNS assay for Composition A is shown.

[0095] Figures 36A-36D A summary of lipid nanoparticle characterization of Composition B and Composition C in different pH buffers is shown. Figure 36A The sizes of lipid nanoparticles are shown. Figure 36B The polydispersity index of lipid nanoparticles is shown. Figure 36C The encapsulation efficiency (%) of lipid nanoparticles is shown. Figure 36D Zeta potential (mV) of lipid nanoparticles is shown.

[0096] Figure 37A The TNS assay for composition B is shown. Figure 37B The TNS assay for composition C is shown.

[0097] Figures 38A-38D A summary of lipid nanoparticle characterization of composition B at pH 4, pH 5, or pH 6 is shown. Figure 38A The sizes of lipid nanoparticles are shown. Figure 38B The polydispersity index of lipid nanoparticles is shown. Figure 38C The encapsulation efficiency (%) of lipid nanoparticles is shown. Figure 38D Zeta potential (mV) of lipid nanoparticles is shown.

[0098] Figures 39A-39D A summary of lipid nanoparticle characterization of composition F at pH 4, pH 5, or pH 6 is shown. Figure 39A The sizes of lipid nanoparticles are shown. Figure 39B The polydispersity index of lipid nanoparticles is shown. Figure 39C The encapsulation efficiency (%) of lipid nanoparticles is shown. Figure 39D Zeta potential (mV) of lipid nanoparticles is shown.

[0099] Figure 40 AUC / min for Composition B variants are shown.

[0100] Figure 41 The AUC / min of each with 50% cholesterol is shown.

[0101] Figure 42 Experimental conditions are shown.

[0102] Figures 43A-43D Rescue of CFTR function in two non-responsive hBE genotypes is shown. Figure 43A Shown is the rescue of CFTR function in hBE of the R553X / W1282X genotype. Figure 43B Shown are the results of measurements of transepithelial electrical resistance (TEER) (top) and LDH release (bottom) in hBE of the R553X / W1282X genotype. Figure 43C Shown is the rescue of CFTR function in hBE of the W1282X / W1282X genotype. Figure 43C Shown are the results of measurements of transepithelial electrical resistance (TEER) (top) and LDH release (bottom) in hBE of the W1282X / W1282X genotype.

[0103] Figure 44 A summary of benchmarking data across several CF genotypes and donors is shown.

[0104] Figure 45A Shown is the rescue of CFTR function in donor TXCF042716 cells. Figure 45B Shown are the results of measurements of LDH release in donor TXCF042716 cells to detect cytotoxicity from aerosolized formulations. Figure 45C Shown is the rescue of CFTR function in donor KKD012K cells. Figure 45D Shown are the results of measurements of LDH release in donor KKD012K cells to detect cytotoxicity from aerosolized formulations. Figure 45E Shown is the rescue of CFTR function in donor KKD025L cells. Figure 45F Shown are the results of measurements of LDH release in donor KKD025L cells to detect cytotoxicity from aerosolized formulations. Figure 45G Shown is the rescue of CFTR function in donor KKD003K cells. Figure 45H Shown are the results of measurements of LDH release in donor KKD003K cells to detect cytotoxicity from aerosolized formulations. Figure 45IShown is the rescue of CFTR function in donor 20160524CF cells. Figure 45J Shown are the results of measurements of LDH release in donor 20160524CF cells to detect cytotoxicity from aerosolized formulations. Figure 45K Shown is the rescue of CFTR function in donor KK017N cells.

[0105] Figure 46A Shown are the rescue of CFTR function in donor KKD003K cells by various lipid nanoparticles at 24h and 48h. Figure 46B Quantification of CFTR bands is shown. Figure 46C Shown is the expression of CFTR protein analyzed by Western blot.

[0106] Figure 47A Shown are preliminary evaluations of lipid nanoparticles targeting secretory cells (eg, goblet cells) in ΔF508 / ΔF508 (donor TXCF042716) hBE cells. Figure 48B Quantification of TR-positive cells is shown.

[0107] Figures 48A-48C The correlation of CFTR function and CFTR protein levels is shown. Figure 48A Shown is the rescue of CFTR function in donor KKD003K or donor KKD012K cells. Figure 48B Quantification of CFTR bands is shown. Figure 48C Shown is the expression of CFTR protein in donor KKD003K or donor KKD012K cells by Western blot analysis.

[0108] Figure 49 The experimental conditions of the benchmark study are shown.

[0109] Figures 50A-50B Shown is the effect of mucus on the transfection efficiency of aerosolized SORT lipid nanoparticles in W1282X / W1282X hBE. Figure 50A Rescue of CFTR function is shown. Figure 50B Transepithelial electrical resistance (TEER) measurements are shown.

[0110] Figures 51A-51B Shown is the effect of mucus on the transfection efficiency of aerosolized SORT lipid nanoparticles in R553X / W1282X hBE. Figure 51A Rescue of CFTR function is shown. Figure 51B Transepithelial electrical resistance (TEER) measurements are shown.

[0111] Figure 52A-52BShown are the effects of CFTR activators on CFTR function following lipid nanoparticle delivery. Figure 52A Shown is the rescue of CFTR function in R553X / W1282X hBE with or without ivacaftor. Figure 52B Shown is the rescue of CFTR function in W1282X / W1282X hBE with or without ivacaftor.

[0112] Figure 53A-Figure 53B In vivo studies of lipid nanoparticles are shown. Figure 53A Quantification of luminescence is shown. Figure 53B Whole body IVIS imaging is shown.

[0113] Figures 54A-54C It was shown that in ΔF508 / ΔF508 hBE, SORT LNPs used for the PCD procedure did not rescue CFTR function when delivered by aerosol. Figure 54A Rescue of CFTR function with Composition A or Composition X treatment is shown. Figure 54B Representative traces of chloride ion flux are shown. Figure 54C Transepithelial electrical resistance (TEER) measurements are shown.

[0114] Figures 55A-55C Shown is the stability study of Composition B over three weeks. Figure 55A The sizes of lipid nanoparticles at 1 and 3 weeks are shown. Figure 55B The polydispersity index of lipid nanoparticles at week 1 and week 3 is shown. Figure 55C The encapsulation efficiency (%) of lipid nanoparticles at 1 and 3 weeks is shown.

[0115] Figures 56A-56C Shown is a stability study of Composition X over three weeks. Figure 56A The sizes of lipid nanoparticles at 1 and 3 weeks are shown. Figure 56B The polydispersity index of lipid nanoparticles at week 1 and week 3 is shown. Figure 56C The encapsulation efficiency (%) of lipid nanoparticles at 1 and 3 weeks is shown.

[0116] Figures 57A-57C Shown is a comparison of lipid nanoparticle characterization in pH 4 and pH 6 citrate buffer. Figure 57A The sizes of lipid nanoparticles are shown. Figure 57B The polydispersity index of lipid nanoparticles is shown. Figure 57C The encapsulation efficiency (%) of lipid nanoparticles is shown.

[0117] Figures 58A-58CShown are TNS assays of various lipid nanoparticles in pH 4 citrate buffer. Figure 58A Shown are TNS assays of composition B in different buffers. Figure 58B Shown are TNS assays of composition X in different buffers. Figure 58C Shown are TNS assays of composition Y in different buffers.

[0118] Figures 59A-59C Shown are TNS assays of various lipid nanoparticles in pH 6 citrate buffer. Figure 59A Shown are TNS assays of composition B in different buffers. Figure 59B Shown are TNS assays of composition X in different buffers. Figure 59C Shown are TNS assays of composition Y in different buffers.

[0119] Figure 60 Shown are the post-nebulization characteristics of Composition B and Composition X in citrate buffer containing sucrose.

[0120] Figure 61 Lipid nanoparticle characterization data on freeze-thaw storage are shown.

[0121] Figures 62A-62G Optimization of the Composition X formulation is shown. Figure 62A The experimental protocol is shown. Figure 62B Buffer conditions for the experiments are shown. Figure 62C Characterization of lipid nanoparticles (particle size, polydispersity index, and encapsulation efficiency) after freeze-thaw cycles is shown. Figure 62D Characterization of lipid nanoparticles (particle size, polydispersity index, and encapsulation efficiency) for longer storage conditions is shown. Figure 62E Buffer conditions for the experiments are shown. Figure 62F Characterization of lipid nanoparticles (particle size, polydispersity index, and encapsulation efficiency) after freeze-thaw cycles is shown. Figure 62G Characterization of lipid nanoparticles (particle size, polydispersity index, and encapsulation efficiency) for longer storage conditions is shown.

[0122] Figure 63 Shown are pH titration studies of Composition X lipid nanoparticle compositions.

[0123] Figure 64 Shown is a pH titration study of composition X lipid nanoparticle composition on Solo.

[0124] Figure 65 CFTR function for composition X in different pH conditions is shown.

[0125] Figure 66References for antibodies selected for immunofluorescence panels are shown.

[0126] Figure 67A The ΔF508 / ΔF508 hBE cell profile is shown. Figure 67B The genotype and donor code of hBE cells are shown. Figure 67C Antibody detection for each cell type is shown.

[0127] Figure 68 Shown are immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with Composition B / HA-CFTR.

[0128] Figure 69 Shown are immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with Composition B / HA-CFTR.

[0129] Figure 70 Shown are immunofluorescence images of ΔF508 / ΔF508 hBE (KKD003K) administered with Composition B / HA-CFTR.

[0130] Figure 71A Shown are the expression levels of HA-CFTR in F508del / F508del (TXCF042716) cells. Figure 71B Shown are the expression levels of HA-CFTR in F508del / F508del (20160524CF) cells.

[0131] Figure 72 The relationship between CFTR function and its expression is shown.

[0132] Figure 73A Shown is the translocation of HA-CFTR protein to the apical membrane in ΔF508 / ΔF508(TXCF042716) hBE cells. Figure 73B Rescue of CFTR function in hBE is shown. Figure 73C Quantification of CFTR bands by Western blot analysis is shown.

[0133] Figure 74A The localization of HA-CFTR protein in ΔF508 / ΔF508(20160524CF) is shown. Figure 74B Rescue of CFTR function in hBE is shown. Figure 74C Quantification of CFTR bands by Western blot analysis is shown.

[0134] Figure 75 CFTR expression in W1282X / W1282X hBE cells treated with Composition B or Composition X is shown.

[0135] Figure 76A The localization of HA-CFTR protein in W1282X / W1282X(UI0014) cells dosed with Composition X is shown. Figure 76B Rescue of CFTR function in hBE is shown. Figure 76C Quantification of CFTR bands by Western blot analysis is shown.

[0136] Figure 77A The localization of HA-CFTR protein in W1282X / W1282X(UI0014) cells dosed with Composition B is shown. Figure 77B Rescue of CFTR function in hBE is shown. Figure 77C Quantification of CFTR bands by Western blot analysis is shown.

[0137] Figure 78 The study protocol is shown.

[0138] Figure 79 The planned dose levels for the study are indicated.

[0139] Figure 80 A timeline of events is shown.

[0140] Figure 81 The cell tropism of composition B in dF#4 (KKD003K) is shown.

[0141] Figure 82 Cell-mediated expression of HA-CFTR in dF#1, dF#4, and dF#4 is shown.

[0142] Figure 83A Highest expression, apical translocation of HA-CFTR in F508del / F508del hBE (donor TXCF042716) cells dosed with Composition X is shown. Figure 83B Shown is the rescue of chloride flux in hBE. Figure 83C Quantification of CFTR bands by Western blot analysis is shown.

[0143] Figure 84A The expression of HA-CFTR in F508del / F508del hBE (donor 20160524CF) cells dosed with Composition X is shown. Figure 84B Shown is the rescue of chloride flux in hBE. Figure 84C Quantification of CFTR bands by Western blot analysis is shown.

[0144] Figure 85AShown are the expression and apical translocation of HA-CFTR in K710X / L467 (ND13816) hBE cells following exposure to aerosolized composition B. Figure 85B Shown is the rescue of chloride flux in hBE.

[0145] Figure 86 Shown is HA-CFTR after exposure to aerosolized Composition B in K710X / L467 (ND13816) cultures with significant signs of fibrosis.

[0146] Figure 87A Shown are the expression and translocation of HA-CFTR in K710X / L467 (ND13816) hBE cells following exposure to aerosolized composition X. Figure 87B The merged image is shown.

[0147] Figure 88 Shown are the translocation and particulation of HA-CFTR fibrillar cells following exposure to aerosolized composition X in K710X / L467 (ND13816) cells.

[0148] Figure 89 Shown are the expression and translocation of HA-CFTR in F508del / F508del hBE (donor TXCF042716) cells following exposure to aerosolized composition X.

[0149] Figure 90 Shown are immunofluorescence images of F508del / F508del hBE (KKD017K) dosed with Composition B / HA-CFTR.

[0150] Figure 91 Shown are the expression levels of HA-CFTR in different F508del / F508del hBE donor cells after administration with compositions B, Y, or X.

[0151] Figure 92 Shown are the expression levels of HA-CFTR and chloride flux after administration of f508 with compositions B, Y, or X in the non-responder genotype.

[0152] Figures 93A-93B It was shown that SORT LNPs used for the PCD procedure did not rescue CFTR function when delivered by aerosol in F508del / F508del hBE. Figure 93A Representative traces of chloride ion flux are shown. Figure 93B Shown are measurements of transepithelial electrical resistance (TEER) (top) and rescue of chloride flux (bottom).

[0153] Figure 94Shown are the results of measurements of LDH release to detect cytotoxicity from aerosolized formulations.

[0154] Figure 95 is a graph showing the levels of DNAI1-HA mRNA in different lung regions 6 hours after administration.

[0155] Figure 96 is a graph showing the time course of DNAI1-HA mRNA levels in lung tissue after administration of composition A-DNAI1-HA. The mean ± standard deviation of values ​​from three sampled lung areas per animal are plotted. N = 2 animals / group / time point.

[0156] Figure 97 Graph showing the time course of 4A3-SC7 lipid levels in lung tissue following administration of composition A-DNAI1-HA. Mean ± standard deviation of values ​​from three sampled lung regions per animal are plotted. N = 2 animals / group / time point.

[0157] Figure 98 Graph showing the time course of 14:0EPC lipid levels in lung tissue following administration of composition A-DNAI1-HA. Mean ± standard deviation of values ​​from three sampled lung regions per animal are plotted. N = 2 animals / group / time point.

[0158] Figure 99 Graph showing the time course of DMG-PEG lipid levels in lung tissue following administration of composition A-DNAI1-HA. Mean ± standard deviation of values ​​from three sampled lung regions per animal are plotted. N = 2 animals / group / time point.

[0159] Figure 100 Figure 11 is the figure of the analysis of the cell-specific expression of DNAI1-HA protein in lung after administration for 6 hours by multiple immunofluorescence.The DNAI1-HA+ colony % of every cell type is calculated by combining the cell counting of the lung sections of all four inspections from every animal. The total range of the cell counted by every animal is 225,419 to 319,654.The following markers are used to dye specific airway cell types: rod-shaped cells (SCGB1A1 / uterine globin), goblet cells (MUCSB), basal cells (cytokeratin 5), ciliated cells (acetylated tubulin), alveolar type II cells (ATII) (front surfactant protein C (prosurfactant protein C)), epithelial cells (EpCAM) and DNAI1-HA (HA epitope tag).

[0160] Figure 101: It is a figure showing the analysis of cell-specific expression of DNAI1-HA protein in the lung 6 hours after administration by multiple immunofluorescence. Two tracheal sections, i.e., proximal sections and carina sections, were collected from each animal. For Group 1 (vehicle) 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% of each cell type was calculated. The total number of cells counted in each section ranged from 5,604 to 25,436. The individual data points for each treated animal and the mean ± standard deviation of each group are shown (Group 1, Group 2, N=2; Group 3, N=4). The following markers were used to stain for specific airway cell types: club cells (SCGB1A1 / uteroglobin), goblet cells (MUC5B), basal cells (cytokeratin 5), ciliated cells (acetylated tubulin), epithelial cells (EpCAM), and DNAI1-HA (HA epitope tag).

[0161] Figure 102 It is a figure showing the analysis of the cell-specific expression of DNAI1-HA protein in the nasopharynx and oropharynx 6 hours after administration by multiple immunofluorescence. Two nasopharyngeal or oropharyngeal sections were collected from each animal. For Group 1 (vehicle) 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 percentage of each cell type was calculated. The total number of cells counted in each section ranged from 58,993 to 145,142. The individual data points of each treated animal and the mean ± standard deviation of each group are shown (Group 1, Group 2, N=2; Group 3, N=4). 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).

[0162] Figure 103A 6h after exposure, Western blot analysis of lung samples from high dose (0.34mg / kg, group 3). For each sample, 50μg of total lung lysate protein was separated on 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 taken from the right caudal lobe (Cd), cranial lobe (Cr) and median lobe (Mi). As a positive control, 1.9pg of recombinant human DNAI1-HA was included on each gel. The position of the DNAI1-HA band is indicated by an arrow.

[0163] Figure 103B6h after exposure, Western blot analysis of lung samples from high dose (0.34mg / kg, Group 3). For each sample, 50μg of total lung lysate protein was separated on SDS-PAGE gel, transferred to a nitrocellulose membrane, and total endogenous monkey DNAI1 was detected using rabbit anti-DNAI1 polyclonal antibody. Lung samples were taken from the right caudal lobe (Cd), cranial lobe (Cr), and median lobe (Mi). As a positive control, 1.9pg of recombinant human DNAI1-HA was included on each gel. The position of the DNAI1-HA band is indicated by an arrow.

[0164] Figure 104 Graph showing hDNAI1 mRNA levels in lung tissue 24h and 336h after administration of composition A-DNAI1. Individual values ​​for each sample whose levels were above the assay LOQ (limit of quantitation) are plotted. Two samples were tested per animal in six animals in each treatment group (N=12). The solid line indicates the median of each group. The dotted line indicates the assay LOQ of 250 copies / μg total RNA. If no sample in a particular group was above the assay LOQ, a single point of 10 copies / μg total RNA is shown in the graph.

[0165] Figure 105 Graph showing hDNAI1 mRNA levels in liver tissue 24h and 336h after administration of composition A-DNAI1. Individual values ​​for each sample whose levels were above the assay LOQ (limit of quantitation) are plotted. Two samples were tested per animal in six animals in each treatment group (N=12). The solid line indicates the median of each group. The dotted line indicates the assay LOQ of 250 copies / μg total RNA. If no sample in a particular group was above the assay LOQ, a single point of 10 copies / μg total RNA is shown in the graph.

[0166] Figure 106 1 is a graph showing the hDNAI1 mRNA levels in spleen tissue 24h and 336h after composition A-DNAI1 administration. The individual values ​​for each sample whose levels were higher than the assay LOQ (limit of quantitation) were plotted. In the six animals in each treatment group, one sample was tested per animal (N-12). The solid line indicates the median value of each group. The dotted line indicates the assay LOQ of 250 copies / μg of total RNA. If no sample in a particular group was higher than the assay LOQ, a single point of 10 copies / μg of total RNA was displayed in the figure.

[0167] Figure 107Graph showing the hDNAI1 mRNA levels in whole blood 24h and 336h after composition A-DNAI1 administration. Individual values ​​for each sample whose levels were higher than the assay LOQ were plotted. Samples were collected from six animals in each treatment group at each time point (3 males / 3 females; N=6). The solid line indicates the median value of each group. The dotted line indicates the assay LOQ of 250 copies / mL. If no sample in a particular group was higher than the assay LOQ, a single point of 10 copies / mL is shown in the graph.

[0168] Figure 108 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), club cells (club cell 10 kDa protein, CC10 or SCGB1A1), goblet cells (mucin 5AC, MUC5AC), and basal cells (cytokeratin 5 or CK5). Nuclei were stained with Hoechst. Each image is a collection of nine consecutive fields of view (FoVs) stitched together. Each FoV was collected at 40x magnification.

[0169] Figure 109 Western blot analysis showing DNAI1 (top) and DNAI2 (bottom) levels in WT-hBE and DNAI1-KD hBE cultures 12, 15, and 22 days after ALI (air-liquid interface).

[0170] Figure 110 Immunofluorescence images of WT-hBE and DNAI1-KD hBE cultures using specific markers for ciliated cells (acetylated α-tubulin and DNAI). In WT-hBE, ciliated cells colocalize with DNAI1, whereas DNAI1 protein is detected in only a few ciliated cells in DNAI1-KD hBE.

[0171] Figure 111 Figure 2 shows ciliary activity in control and DNAI1-KD hBE cultures 14, 18, and 21 days after 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 shows that lentiviral transduction alone does not result in loss of ciliary activity.

[0172] Figure 112is a graph showing a dose-dependent increase in DNAI1-HA protein expression 24 h after treatment (Western blot). Well-differentiated DNAI1-KD hBE cultures (33 days after ALI) were treated with 150 μg (0.9 μg / cm 2 ), 300 μg dose (1.9 μg / cm 2 ) or 500 μg (3.0 μg / cm 2 ) of the composition A-DNAI1-HA.

[0173] Figure 113 Figure 1 is an immunofluorescence image showing the incorporation of newly translated DNAI1-HA protein into the ciliary axoneme of DNAI1-KD hBE as of 72 h after nebulization. Highly differentiated KD-hBE (33 days after ALI) were treated with two nebulizations of composition A-DNAI1-HA at a dose of 300 μg (1.9 μg / cm2) on two consecutive days. The inset shows DNAI1-HA protein in ciliated cells, acetylated α-tubulin in the ciliary axoneme, and their colocalization. Scale bar = 20 μm pixel binning 1 x 1; 25 ms exposure, z-stacked deconvoluted images. Staining of untreated controls was performed as for treated samples.

[0174] Figure 114 Figure 2 is a graph showing a dose response study of DNAI1-HA protein incorporation into cilia 72 h after nebulization. Highly differentiated DNAI1-KD hBE (33 days after ALI) were treated with 150 μg (0.9 μg / cm 2 ) or 300 μg dose (1.9 μg / cm 2 ) or 500 μg dose (3.0 μg / cm 2 ) single or double atomization treatment of the composition A-DNAI1-HA.

[0175] Figure 115 Figure 1 is an immunofluorescence image showing the persistent incorporation of DNAI1-HA protein into the axonemes of ciliated cells after a single basolateral administration of composition A-DNAI1-HA. Differentiated hBE were treated by adding composition A-DNAI1 (10 μg / mL) to the basolateral culture medium. After a single treatment (the culture medium containing the preparation was replaced with fresh culture medium after 5 h), the treated inserts were fixed for immunofluorescence localization at different time points (1d, 2d, 7d, 14d and 24d refer to the days after a single basolateral treatment). The image shows the immunofluorescence localization of cilia in red (acetylated α-tubulin), the immunofluorescence localization of DNAI1-HA in green (HA), and the co-localization of DNAI1-HA protein with cilia in yellow.

[0176] Figure 116 Figure 2 is a graph and Western blot showing the kinetics of newly translated DNAI1-HA protein in WT-hBE after nebulization of composition A-DNAI1-HA. 2 ) were treated with a composition of A-DNAI1-HA by nebulization. Inserts were collected at different time points after treatment, and 10 μg of total protein was analyzed on a Western blot using an anti-HA antibody. NTC refers to untreated cultures.

[0177] Figure 117 : is a graph showing the functional rescue of ciliary activity in DNAI1-KD hBE by treatment with aerosolized composition A-DNAI1. Ciliary activity in hBE was recorded and analyzed by Sisson-Ammons Video Analysis (SAVA) software to generate ciliary activity area % scores for approximately 700 separate non-overlapping fields of view (FoV). Compared to cultures treated with tdTomato mRNA, cultures treated with DNAI1 mRNA had higher levels of ciliary activity after 2, 4, and 6 treatments. The increased activity was statistically significant as determined by Welch's t-test. * = P < 0.05, ** = P = 0.001, **** = P < 0.0001, ns = not significant (P> 0.05). The ciliary beat frequency (right graph) for FoVs with activity area %> 29% (activity area % greater than those observed in any tdTomato-treated cultures) fell within the normal range of 5-15 Hz.

[0178] Figure 118 is a graph showing simulated deposition distributions in healthy adults using the Yeh / Schum, Weibel, and PNNL airway morphometry models using the eFlow nebulizer aerosol profile.

[0179] Figure 119 is a graph showing model-dependent and generation-dependent MMPD deposition fraction predictions.

[0180] Figure 120 is a schematic diagram of a symmetrical airway model showing the conducting airways (composed of generations 0-8 representing the trachea, bronchi, and bronchioles, and generations 9-16 representing the terminal bronchioles) and the alveolar or pulmonary airways (generations 17-23).

[0181] Figure 121 Graphs showing simulated human deposition at passages 0-8 (trachea, bronchi, and bronchioles), passages 9-16 (bronchioles), and passages 0-16 (entire TB region) as a function of aerosolized composition A and a human airway model.

[0182] Figure 122 is a graph showing the simulated deposition distribution of NHPs wearing an oronasal mask using experimental aerosol characterization. DETAILED DESCRIPTION

[0183] Provided herein are aerosolized pharmaceutical compositions of lipid nanoparticles (LNPs). In some embodiments, the disclosure provides aerosolized pharmaceutical compositions wherein the LNPs have one or more of the following: (a) an encapsulation efficiency (EE) greater than 50%, (b) mRNA integrity greater than 50%, (c) a diameter of 20 nm to 200 nm, (d) a polydispersity less than 0.6, and / or the aerosolized particles have one or more of the following: (a) a mass median aerodynamic diameter (MMAD) between 1 μm and 10 μm, (b) a geometric standard deviation (GSD) of 1 to 5, and (c) a fine particle fraction (FPF) percentage of at least 50%. In embodiments, the aerosolized pharmaceutical composition can deliver the LNPs to the tracheobronchial region of a subject. I. Definition

[0184] In the Summary of the Invention and Detailed Description of the Invention of the present disclosure and in the claims above, as well as in the drawings, reference is made to specific features (including method steps) of the present disclosure. It should be understood that the present disclosure includes all possible combinations of such specific features. For example, when a specific feature is disclosed in the context of a particular aspect or embodiment of the present disclosure or a particular claim, that feature may also be used in combination with and / or in the context of other specific aspects and embodiments of the present disclosure to the greatest extent possible, and in the present disclosure as a whole.

[0185] Where reference is made herein to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the description excludes this possibility), and the method may include one or more other steps that are performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the description excludes this possibility).

[0186] Unless otherwise indicated, the practice of this disclosure will employ conventional methods within the capabilities of those skilled in the art of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and / or cell biology, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. It should be understood that this disclosure is not limited to any particular method, protocol, or reagent described, as these may vary depending on the circumstances in which they are used by those skilled in the art.

[0187] All publications and patents mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present disclosure, including any definitions herein, will control. However, reference to any reference, article, publication, patent, patent disclosure, or patent application cited herein is not, and should not be taken as, an admission or any form of representation that such reference constitutes valid prior art or forms part of the common general knowledge in any country in the world.

[0188] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. In particular, features described in one section can be combined with features in any other section of this disclosure.

[0189] While illustrative embodiments have been described and depicted, it will be understood that various changes can be made in these illustrative embodiments without departing from the spirit and scope of the invention.

[0190] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Various scientific dictionaries including the terms included herein are well known and available to those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, some preferred methods and materials are described herein. Therefore, the terms defined immediately below are more fully described by reference to the specification as a whole.

[0191] The singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0192] As used herein, the term "about" means a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In embodiments, about means within the standard deviation of the range using art-recognized measurements. In some embodiments, about means a range that extends to + / - 10%, + / - 5%, + / - 3%, or + / - 1% of the specified value.

[0193] The term "at least" followed by a number is used herein to indicate the starting point of a range starting from that number (the range may 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.

[0194] The term "up to" and a number following it are used herein to indicate the endpoint of a range ending at that number (the range can be a range with, for example, 1 or 0 as its lower limit, or a range with no lower limit, depending on the variable being defined). For example, "up to 4" means 4 or less than 4, and "up to 40%" means 40% or less than 40%. In this disclosure, when a range is given in the form of "(first number) to (second number)" or "(first number) - (second number)", the range means a range with a lower limit of the first number and an upper limit of the second number. For example, 25 to 100 mm means a range with a lower limit of 25 mm and an upper limit of 100 mm.

[0195] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. For example, a composition that "comprises" (or that it comprises) components A, B, and C may consist of components A, B, and C (i.e., contain only those components), or may contain not only components A, B, and C, but also one or more other components.

[0196] As used herein, the term "consisting of is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. The phrase "consisting essentially of is intended to include any element listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action detailed herein for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0197] As used herein, the terms "specific," "specifically," "specific," and the like of a composition refer to the ability of the composition to elicit a specific effect (such as, but not limited to, inhibition) on a specific molecular target with minimal or no effect on other proteins in the cell.

[0198] As used herein, the term "aerosol particles" refers to liquid or solid particles suspended in a gas (e.g., air). Aerosol particles include, but are not limited to, aerosol droplets of liquid. Typically, aerosols have a size of, for example, between about 1 micron and about 100 microns, or in some cases between about 1 micron and about 20 microns, or between about 1 micron and about 10 microns.

[0199] As used herein, the term "selectively deliver" is used to refer to a composition that, when delivered, at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%) of the administered amount is delivered to the target organ (e.g., lung), target tissue or target cell.

[0200] As used herein, the term "contacting" refers to allowing two species to interact, such as through chemical interactions, including ionic, nonionic, polar, hydrophobic or hydrophilic interactions, or to physically contact as is accepted in the art, wherein the two species can be a lipid nanoparticle and a cell, mucus or tissue lining. In cell culture, the LNP can be contacted with the cells by mixing the LNP composition with a suitable cell culture medium, or by allowing aerosol particles of the LNP composition to contact the cell culture, thereby dissolving the aerosol particles into the liquid of the cell culture medium or into the liquid or mucus surrounding the cells, thereby allowing the LNP to contact the cells.

[0201] As used herein, "prevent," "prevent," or any grammatical variation thereof, refers to inhibiting the onset of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or symptoms of the disease; and / or slowing the onset of symptoms or symptoms of a disease in a subject or patient who may be at risk for and / or susceptible to the disease but who does not yet experience or display any or all of the symptoms or symptoms of the disease. Prevention may be complete (i.e., no detectable symptoms), or partial, such that fewer symptoms are observed than would be the case in the absence of treatment.

[0202] References throughout this specification to, for example, "one embodiment," "an embodiment," "another embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," "other embodiments," 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 the present disclosure. Thus, the appearances of the aforementioned phrases in various places throughout this disclosure are not necessarily all referring to the same embodiment. Furthermore, any particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0203] As used herein, the term "nebulizer" refers to a device that can convert a liquid (e.g., a solution, dispersion, or suspension) into aerosol particles. This process is called "atomization." The term "atomization" refers to the process or state of converting a solution, aqueous dispersion, or suspension (such as a liquid pharmaceutical composition) into an aerosol. The term "mesh nebulizer" and / or "vibrating mesh nebulizer" refers to a nebulizer that achieves atomization by passing the 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 mesh aperture and / or charge. The mesh nebulizer is driven by a piezoelectric element and uses ultrasonic frequency to vibrate the mesh. When the liquid passes through the mesh, the vibration of the mesh can cause the generation of aerosol particles. In contrast, an ultrasonic nebulizer generates ultrasonic waves directly into the solution, thereby causing aerosol particles to be generated on the surface of the liquid. Other means of generating aerosols include, but are not limited to, pressurized metered dose inhalers, dry powder inhalers, jet nebulizers, soft mist inhalers, condensation aerosols, and aqueous nasal sprays, as described in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd edition, 2021). Nebulizers are also described as ventilated or non-ventilated. Illustrative mesh nebulizers are described, for example, in U.S. Patent No. 9,061,303. Illustrative mesh nebulizers that can be used in the practice of the methods disclosed herein include, but are not limited to, those made of and Those prepared and sold.

[0204] As used herein, the term "aerosolization" or "aerosol" refers to a suspension in which fine liquid and / or solid particles are dispersed in a gas (e.g., air). A dispersion of particles containing liquid in air or gas. The aerosol generated from a nebulizer can refer to a mixture of air and vaporized particles produced from the material that produces the aerosol, such as any of the aerosolized pharmaceutical compositions described herein. For example, a nebulizer can convert any of the liquid phases of the pharmaceutical compositions described herein into a gas phase by, for example, ultrasonic vibration. In another example, an air jet mill can produce a dry powder aerosol from the dry lipid nanoparticles of the present disclosure. Non-limiting examples of air jet mills include Jet-O-Mizer, Trost jet mill, and Microjet. Typically, the aerosol particles of the present disclosure have a low settling velocity and relative airborne stability. In some embodiments, a nebulizer converts a liquid pharmaceutical composition into an aerosolized pharmaceutical composition.

[0205] 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 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.

[0206] As used herein, the term "apparent pKa" refers to the total dissociation constant of all titratable groups in a lipid nanoparticle. The apparent pKa is an experimentally determined value for a molecule or nanoparticle. The apparent pKa can be expressed as the pH when the number of ionized (protonated) groups and deionized groups in the system is equal. The surface charge and ionic interactions of the nanomaterials assembled in the nanoparticles can be estimated based on 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, the apparent pKa is not the intrinsic pKa value of any individual molecule. The apparent pKa of a nanoparticle can be measured by a variety of techniques. For example, acid-base titration of 2-(p-toluylamino)-6-naphthalenesulfonic acid (TNS) fluorescence is widely used to determine the apparent pKa of blank nanoparticles.

[0207] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to treat a disease, disorder, or condition. For example, with respect to the use of LNPs with an mRNA payload for the treatment of, for example, cystic fibrosis (CF) or primary ciliary dyskinesia (PCD), a therapeutically effective amount is a dose or concentration of mRNA (e.g., CFTR or PCD mRNA) that is capable of eradicating, inhibiting, preventing, or slowing the progression of, for example, all or part of the respiratory symptoms of CF or PCD, or some combination thereof. For a given parameter, a therapeutically effective amount 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 a "fold" increase or decrease. For example, a therapeutically effective amount can have an effect that is at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more greater than a control. A "therapeutically effective amount" may vary depending on, for example, but not limited to, the compound, the disease or its conditions and / or symptoms, the severity of the disease or its conditions and / or symptoms, the age, weight, and / or health of the subject to be treated, and the judgment of the prescribing physician. In any given case, the appropriate amount may be determined by one skilled in the art or can be ascertained by routine experimentation.

[0208] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers, vehicles, diluents, excipients and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and / or are generally chemically and / or physically compatible with the other ingredients making up the formulation.

[0209] The terms "pulmonary disease," "lung disease," and "pulmonary disorder" broadly refer to diseases or disorders of the lungs. Pulmonary disease may be characterized by symptoms including, but not limited to, difficulty breathing, coughing, airway discomfort and inflammation, increased mucus, and / or pulmonary fibrosis. Non-limiting examples of lung diseases include primary ciliary dyskinesia (PCD) (also known as Kartagener syndrome or immotile cilia syndrome), cystic fibrosis, asthma, lung cancer, chronic obstructive pulmonary disease (COPD), bronchitis, emphysema, bronchiectasis, pulmonary edema, pulmonary fibrosis, sarcoidosis, pulmonary hypertension, pneumonia, tuberculosis, pulmonary fibrosis (IPF), interstitial lung disease (ILD), acute interstitial pneumonia (A1P), respiratory bronchiolitis-associated interstitial lung disease (RBILD), desquamative interstitial pneumonia (DIP), nonspecific interstitial pneumonia (NSIP), idiopathic interstitial pneumonia (IIP), bronchiolitis obliterans organizing pneumonia (BOOP), restrictive lung disease, and pleurisy.

[0210] As used herein, the term "lipid nanoparticle" refers to a carrier or vehicle formed by one or more lipid components for delivery of a payload (e.g., nucleic acid, protein, peptide, polypeptide, polynucleotide or oligonucleotide) in the context of drug development. Lipid nanoparticles can have one or more lipids with at least one dimension being nanoscale (e.g., 1-1000 nm). Typically, the lipid nanoparticle compositions for delivery are composed of one or more lipids, such as, but not limited to, synthetic ionizable lipids or cationic lipids, phospholipids, structural lipids and polyethylene glycol (PEG) lipids. These compositions may also include other lipids. In some embodiments, at least one therapeutic agent (e.g., mRNA) may be captured in the lipid portion of a lipid nanoparticle or in an aqueous space encapsulated by part or all of the lipid portions of the lipid nanoparticle, thereby preventing the enzymatic degradation of other undesirable effects (e.g., adverse immune responses) induced by the biological mechanisms of the target subject, tissue and / or cell. In some embodiments, the lipid nanoparticles comprise at least one therapeutic agent (e.g., mRNA) that is organized in reverse lipid micelles and encapsulated in lipid monolayer encapsulations, or inserted between adjacent lipid bilayers. In some embodiments, the morphology of the lipid nanoparticles is different from traditional liposomes characterized by lipid bilayers 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.

[0211] As used herein, the term "neutral phospholipid" refers to a phospholipid that has almost no net charge at physiological pH. In some embodiments, neutral phospholipids are zwitterions, but other types of net neutral phospholipids are known and can be used. In some embodiments, neutral phospholipids can be any lipid that forms a vesicle, having two hydrocarbon chain moieties that can effectively produce a stable double layer and a polar head group that does not have a net charge at a pH between about 5.5-8.5. Neutral phospholipids with a variety of hydrocarbon chains (such as acyl chains) groups having different chain lengths and degrees of saturation can be easily obtained, or can be separated or synthesized by known technology.

[0212] As used herein, the term "PEG-lipid" refers to a lipid modified with polyethylene glycol units. In some embodiments, the PEG-lipid comprises dimyristoylglycerol (DMG). In some embodiments, the PEG-lipid comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0213] As used herein, the term "sterol" refers to a subgroup of steroids having a hydroxyl group at position 3 of the A ring of a sterane ring system. "Cholesterol" is a sterol with a structure of four fused hydrocarbon rings (sterane ring system) having a polar hydroxyl group at one end and an eight-carbon branched aliphatic tail at the other end. Without being bound by theory, the four-ring structure of cholesterol contributes to the fluidity of the cell membrane because the molecule is in a trans conformation, making cholesterol rigid and planar except for the side chain. Cholesterol affects the fluidity, thickness, compressibility, water permeability and intrinsic curvature of the lipid bilayer, for example, in LNPs. For example, "sterol" can be cholesterol or sitosterol.

[0214] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. An mRNA may contain one or more reading frames or regions.

[0215] As used herein, the term "shRNA" or "short hairpin RNA" refers to a short RNA sequence that can be turned into a tight hairpin and can be used to silence gene expression.

[0216] As used herein, the term "microRNA" refers to a non-coding RNA composed of about 22 ribonucleotides that can silence messenger RNA by base pairing with complementary sequences in its targeted mRNA, thereby regulating gene expression in the post-transcriptional stage.

[0217] 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.

[0218] As used herein, the phrase "lipid:RNA ratio" refers to milligrams of lipid per milligram of mRNA drug substance, which affects the encapsulation efficiency of lipid nanoparticles.

[0219] As used herein, the phrase "pneumocytes" refers to lung airway cells. Examples of lung airway cells that can be targeted by delivering the compositions of the present disclosure include, but are not limited to, basal cells, secretory cells (such as goblet cells and club cells), ciliated cells, and any combination thereof.

[0220] As used herein, the term "goblet cell" refers to a type of secretory cell. Goblet cells are located in the epithelium of the conducting airways, with their apical surface typically projecting into the lumen, a position that makes them well-suited for rapid responses to inhalational airway injury.

[0221] As used herein, the phrase "ciliated cell" refers to a cell that has ciliary structures on its cell surface. Examples of ciliated cells include, but are not limited to, respiratory ciliated cells, fallopian tube ciliated cells, endometrial ciliated cells, rete testis ciliated cells, efferent tubule ciliated cells, and / or ciliated ependymal cells. Human respiratory ciliated cells may have 200 to 300 cilia on their surface. Cilia are slender, motile, cylindrical projections that arise from the apical cell membrane, are approximately 0.25 mm in diameter, and contain microtubules and a cytoplasm that is continuous with the cell's cytoplasm. Cilia in human trachea may be 5 to 8 mm long, becoming shorter in more distal airways.

[0222] The term "subject" refers to a living organism to which any of the compositions described herein can be administered. The subject may be suffering from, or at risk for, a disease or condition that can be treated by administering an aerosolized pharmaceutical composition as provided herein. Non-limiting examples of subjects include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the subject is a human.

[0223] The terms "identity," "identical," and "sequence identity" refer to the degree to which two optimally aligned polynucleotide or polypeptide sequences are constant over the entire alignment window of components (e.g., nucleotides or amino acids). "Identity" can be easily 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 "percentage of sequence identity," "percentage of identity," or "identical to..." refer to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary chain) compared to a test ("subject") polynucleotide molecule (or its complementary chain) when two sequences are optimally aligned. In some embodiments, "percentage of identity" can refer to the percentage of identical amino acids in an amino acid sequence. For sequence comparison, a sequence is used as a reference sequence for comparison with a test sequence. The term "reference sequence" refers to a molecule to which a test sequence is compared. Sequence alignment methods for comparing and determining percentages of sequence identity are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443.

[0224] As used herein, the phrases "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences, means that two or more sequences or subsequences 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 when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the present disclosure, there is substantial identity in a contiguous nucleotide region of a nucleotide sequence of the present disclosure having a length of 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 therein, 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., prenyltransferase activity).

[0225] The term "fragment" or "variant" refers to any functional fragment, variant, derivative, or analog of a polynucleotide, polypeptide, or biomolecule that has an in vivo or in vitro activity specific to 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 more of the length of the polynucleotide, polypeptide, or biomolecule. One of ordinary skill in the art can readily determine the functional expression of a fragment or variant by testing for enzymatic activity and the ability to produce a product as described herein.

[0226] The term "treat" means to alleviate, relieve, delay, reduce, improve or manage one or more of at least one symptom of a condition in a subject. The term "treat" may also mean to arrest a condition, delay the onset of a condition (i.e., the stage before clinical manifestation of a condition), or reduce the risk of a condition developing or worsening.

[0227] As used herein, the phrase "chloride flux" refers to the mass of ions incorporated into, or released from, a stimulated cell. Measurement of chloride flux is described, for example, in Moran et al. J. Cystic Fibrosis 7:483-494 (2008).

[0228] As used herein, the phrase "transepithelial electrical resistance (TEER)" is a measurement of the electrical resistance across a cell monolayer, used to confirm the integrity and permeability of the monolayer. Measurement of TEER is described, for example, in Srinivasan et al. J. Lab. Automation 20: 107-126 (2015). II. Compositions of the Disclosure

[0229] Provided herein are compositions and methods related to aerosolized pharmaceutical compositions, such as methods for treating lung diseases or lung disorders, 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 a medicament having an aerosolized pharmaceutical composition. The compositions and methods of the present disclosure provide aerosol particles, wherein the aerosol particles include lipid nanoparticles (LNPs), and the compositions can deliver the LNPs, for example, to the tracheobronchial region of a subject.

[0230] The aerosolized pharmaceutical composition comprises LNP. In some embodiments, the LNP can be selectively delivered to one or more of goblet cells, secretory cells, club cells, basal cells, or ionocytes. In some embodiments, the LNP can be selectively delivered to one or more of ciliated cells, club cells, or basal cells.

[0231] In one aspect, provided herein are lipid compositions comprising: (i) an ionizable lipid; (ii) a helper lipid; (iii) a PEG-lipid; and (iv) a sterol. In some embodiments, the lipid composition further comprises (v) an additional ionizable lipid or a permanent cationic lipid.

[0232] In some embodiments, the lipid nanoparticles described herein may be prepared according to any of the methods described in International Publication Nos. WO 2016094342, WO 2017048789, WO 2017201091, WO2017205767, WO201 / 246203, WO 2020051220, WO 2022169508, WO 2022204053, and WO2022204215; the contents of each of which are incorporated herein by reference in their entirety. A. Ionizable lipids

[0233] In some embodiments of the lipid compositions of the present disclosure, 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 may be deprotonated and uncharged at a pH higher than the pKa of the lipid. The ionizable cationic groups may contain one or more protonated amines that can form cationic groups at physiological pH. The cationic ionizable lipids may further comprise one or more lipid components, such as two or more C6-C 24 The one or more lipid components can be attached to the cationically ionizable lipid via an ester bond, or can be further added to the sulfur atom by Michael addition. In some embodiments, the one or more lipid components can be a dendrimer, a dendrimer, a polymer, or a combination thereof.

[0234] Ionizable lipid has its common meaning in this area, and can refer to the lipid comprising one or more charged moieties. In some embodiments, ionizable lipid can be positively charged or negatively charged. For example, ionizable lipid can be positively charged under lower pH, in which case it can be referred to as "cationic lipid". In certain embodiments, ionizable lipid can comprise amine groups and can be referred to as ionizable amino lipids. Charged moiety is the chemical part that carries formal electronic charge, for example, monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3) etc. Charged moiety can be anionic (i.e., negatively charged) or cationic (i.e., positively charged). The non-limiting example of positively charged moiety includes amine groups (e.g., primary amine, secondary amine and / or tertiary amine), ammonium group, pyridinium group, guanidine group and imidazolium group. In specific embodiments, charged moiety includes amine groups. The non-limiting example of negatively charged group or its precursor includes carboxylic acid group, sulfonic acid group, sulfuric acid group, phosphonic acid group, phosphoric acid group, hydroxyl group etc. In some cases, the charge of a charged moiety can vary with environmental conditions, for example, a change in pH can alter the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule can be selected as desired.

[0235] It should be understood that the terms "charged" or "charged moiety" do not refer to a "partial negative charge" or "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meanings in the art. A "partial negative charge" can be obtained when a functional group contains a bond that becomes polarized, such that electron density is drawn toward one atom of the bond, thereby generating a partial negative charge on the atom. One of ordinary skill in the art will generally recognize bonds that can be polarized in this manner.

[0236] The lipid nanoparticle compositions of the present disclosure can comprise one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that can have a positive charge or a partial positive charge at physiological pH). The ionizable lipids can be selected from the non-limiting group consisting of: 3-(didodecylamino)-N1,N1,4-tri(dodecyl)-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tri(dodecyl)-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-triacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2, 2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), heptahistriacontac-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (Dlin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8[(3(3)-cholest-5-en-3-yloxy)- yloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3(3)-cholest-5-en-3-yloxy]

[00155] octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate (ALC-0315), or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In addition to these, the ionizable lipid may also be a lipid containing a cyclic amine group.

[0237] Ionizable lipids may also be compounds disclosed in International Publication No. WO 2017075531 A1, which is hereby incorporated by reference in its entirety. Ionizable lipids may also be compounds disclosed in International Publication No. WO 2015199952 A1, which is hereby incorporated by reference in its entirety. In one embodiment, the ionizable lipids may be selected from, but are not limited to, the ionizable lipids described in International Publication Nos. WO 2012040184, WO 2011153120, WO 2011149733, WO 2011090965, WO 2011043913, WO 2011022460, WO 2012061259, WO2012054365, WO 2012044638, WO 2010080724, WO201021865, WO 2008103276, WO2013086373 and WO 2013086354, U.S. Patent Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122, and U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, and US20130225836; the contents of each of which are incorporated herein by reference in their entirety.

[0238] As non-limiting examples, the cationic lipids may be selected from (20Z,23Z)-N,N-dimethylnonacos-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacos-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocos-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhexicos-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien- -7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z) -N,N-dimethyltriacontria-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriacontria-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacos-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonyltriacontria-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethyltriacontria-1 Heptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltricaria-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethyltricontacos-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhexadione-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-l-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadeca-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadeca-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadeca-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneca-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadeca-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadeca-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadeca-10-amine -8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradec-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyldodec-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadec-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadeca-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadeca-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)- Octadecta-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadecta-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethylpyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadecta-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadecta-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethylazetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecta-9,12-dien-1-yloxy] l-[(6Z,9Z,12Z)-octadeca-9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-eicos-13,16-dien-1 - yloxy] -N, N-dimethyl-3- (octyloxy) propan-2-amine, (2S) -1- [(13Z, 16Z) - docos-13,16-dien-1-yloxy] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, (2S) -1- [(13Z) - docos-13-en-1-yloxy] -3- (hexyloxy) -N, N-dimethylpropan-2-amine, 1- [(13Z) - docos-13-en-1-yloxy] -N, N-dimethyl-3- (Octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine [(11E,20Z,23Z)-N,N-dimethylnonacos-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0239] In some embodiments of the lipid compositions of the present application, ionizable cationic lipids refer to lipids and lipid-like molecules having nitrogen atoms that can obtain a positive charge. Ionizable cationic lipids may be referred to as cationic lipids in the literature. Ionizable cationic lipids having amino groups typically have between 2 and 6 hydrophobic chains, typically alkyl or alkenyl groups, such as C6-C 24 Alkyl or alkenyl, but may have at least 1, at least 2, at least 3, at least 4, at least 5, or more than 6 tails. 1. Dendrimers

[0240] In some embodiments, the cationically ionizable lipid is a dendrimer. Dendrimers are polymers that exhibit regular tree-like branches, which are formed by continuously adding or adding branching layers to or from a core, and are characterized in that a core, at least one internal branching layer, and a surface branching layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. In Britain, 641-645, August 1994.) Dendrimers include but are not limited to having the following molecular architecture: a core is initiated, a repeating layer (or generation) of repeating units regularly attached to this core is initiated, and the outer surface of the terminal groups attached to the outermost generation. A dendrimer is a dendrimer species with branches emanating from a focus, and the focus is or can be joined to the core directly or via a connecting portion to form a larger dendrimer. In some embodiments, the dendrimer structure has a repeating group extending from a central core, and the central core is multiplied with each repeating unit of each branch. In some embodiments, dendrimers as described herein can be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms may be used to describe compounds that have a dendrimer-like appearance (eg, molecules extending radially from a single focal point).

[0241] Although dendrimers are polymers, dendrimers may be preferred over traditional polymers because they have a controllable structure, a single molecular weight, a number of controllable surface functional groups, and traditionally adopt a globular configuration after reaching a specific generation. Dendrimers can be prepared by sequentially reacting each repeating unit to produce a monodisperse, tree-like and / or intergenerational structured polymeric structure. An individual dendrimer consists of a central core molecule and a tree-like wedge attached to one or more functional sites on the central core. Depending on the assembly monomers used during preparation, the dendrimer surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic or lipophilic groups.

[0242] By changing the functional groups and / or chemical properties of the core, repeating units and surface or terminating groups, their physical properties can be regulated. Some characteristics that can be changed include but are not limited to solubility, toxicity, immunogenicity and biological adhesion ability. Dendrimers are usually described by the number of repeating units in their generations or branches. The dendrimers that are only composed of the core molecule are called the 0th generation, and each continuous repeating unit along all branches is the 1st generation, the 2nd generation, etc., until termination or surface groups. In some embodiments, half a generation is possible, by only carrying out the first condensation reaction with amine and not carrying out the second condensation reaction with mercaptan to obtain.

[0243] The preparation of dendrimers requires a certain level of synthetic control, which is achieved through a stepwise reaction series that includes building up the dendrimer through each successive group. Dendrimer synthesis can be convergent or divergent. During divergent dendrimer synthesis, molecules are assembled from the core to the periphery in a stepwise process that involves attaching one generation to the previous generation and then changing the functional group for the next reaction stage. Functional group conversion is necessary to prevent uncontrolled polymerization. This polymerization will result in highly branched molecules, which are not monodisperse and are also known as hyperbranched polymers. Due to steric effects, continuing to react the dendrimer repeat units will produce spherical or globular molecules until steric overcrowding prevents complete reaction in a particular generation and destroys the monodispersity of the molecule. Therefore, in some embodiments, dendrimers of generations G1-G10 are particularly contemplated. In some embodiments, the dendrimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeat units or any range that can be derived therefrom. In some embodiments, the dendrimer used herein is G0, G1, G2 or G3. However, the number of possible generations can be increased (eg, 11, 12, 13, 14, 15, 20, or 25) by reducing the spacer unit in the branched polymer.

[0244] Furthermore, dendrimers possess two primary chemical environments: one created by specific surface groups on the terminating generation, and the interior of the dendritic structure, which, due to its higher-order structure, can be isolated from the bulk medium and surface groups. Due to these distinct chemical environments, dendrimers have found many different potential uses, including in therapeutic applications.

[0245] In some embodiments of the lipid compositions of the present disclosure, the different reactivities of acrylic acid and methacrylic acid groups with amines and thiols are used to assemble dendrimers. Dendrimers can include secondary or tertiary amines and thioethers formed by the reaction of acrylic acid groups with primary or secondary amines and the reaction of methacrylic acid with sulfhydryl groups. In addition, the repeating units of dendrimers can contain groups that are degradable under physiological conditions. In some embodiments, the repeating units can contain one or more geminal diethers, esters, amides, or disulfide groups. In some embodiments, the core molecule is a monoamine, which allows dendrimerization to be carried out only in one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches, each of which can contain one or more repeating units. Dendrimers can be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are located on heteroatoms (such as nitrogen atoms). In some embodiments, the terminating group is a lipophilic group, such as a long-chain alkyl or alkenyl group. In other embodiments, the terminating group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the terminating group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In still other embodiments, the terminating 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.

[0246] The cation ionizable lipids of the present disclosure can contain one or more asymmetrically substituted carbon or nitrogen atoms and can be separated in optically active form or racemic form. Therefore, unless specific stereochemistry or isomeric forms are clearly indicated, all chiral forms, diastereomeric forms, racemic forms, epimeric forms and all geometric isomeric forms of the chemical formula are expected. The cation ionizable lipids can exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the cation ionizable lipids of the present disclosure can have an S or R configuration. In addition, it is contemplated that one or more of the cation ionizable lipids can exist as structural isomers. In some embodiments, the compounds have the same chemical formula, but are different from the connectivity of the nitrogen atom in the core. Without being bound by theory, it is believed that such cation ionizable lipids can exist because the starting monomers first react with primary amines and then statistically react with any secondary amines present. Thus, the constitutional isomers may present a mixture of fully reacted primary amines followed by reacted secondary amines.

[0247] Chemical formulas used to represent the cationic ionizable lipids of the present disclosure will typically show only one of several possible different tautomers. For example, many types of keto groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups can exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given formula, and regardless of which is most common, all tautomers of a given chemical formula are contemplated herein.

[0248] The cationic ionizable lipids of the present disclosure may also have the following advantages: they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more readily absorbed, and / or have better pharmacokinetic characteristics (e.g., greater oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties, whether for use in the indications described herein or otherwise, compared to compounds known in the prior art.

[0249] Furthermore, the atoms comprising the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C.

[0250] It will be appreciated that the anion or cation forming part of any salt form of the cationically ionizable lipids provided herein is not critical, so long as the salt as a whole is pharmaceutically acceptable. Additional examples of pharmaceutically acceptable salts and methods of their preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference in its entirety.

[0251] In some embodiments of the lipid compositions of the present disclosure, the ionizable lipid is a dendrimer or dendrimer. In some embodiments, the ionizable lipid comprises an ammonium group that is positively charged 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 dendrimer or dendrimer. In some embodiments, the ionizable lipid comprises at least two C6-C 24 Alkyl or alkenyl.

[0252] By changing the functional groups and / or chemical properties of the core, repeating units and surface or terminating groups, their physical properties can be regulated. Some characteristics that can be changed include but are not limited to solubility, toxicity, immunogenicity and biological adhesion ability. Dendrimers are usually described by the number of repeating units in their generations or branches. The dendrimers that are only composed of the core molecule are called the 0th generation, and each continuous repeating unit along all branches is the 1st generation, the 2nd generation, etc., until termination or surface groups. In some embodiments, half a generation is possible, by only carrying out the first condensation reaction with amine and not carrying out the second condensation reaction with mercaptan to obtain. 2. Dendrimers of formula (I)

[0253] In some embodiments of the lipid compositions of the present disclosure, the ionizable lipid comprises at least two C8-C 24 In some embodiments, the ionizable lipid is a dendrimer further defined by the formula: Core-(repeating unit) n -Terminator group (DI) wherein one or more hydrogen atoms of the core are replaced by repeating units, and wherein: The core has the following formula: in: X1 is amino or C1-C 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 Heterocycloalkyl, C5-C 12 heteroaryl or a substituted form thereof; R1 is amino, hydroxyl, thiol, C1-C 12 Alkylamino or C1-C 12 dialkylamino or a substituted form of any of these groups; and a is 1, 2, 3, 4, 5, or 6; or The core has the following formula: in: X2 is N(R5) y ; R5 is hydrogen, C1-C 18 Alkyl or substituted C1-C 18 alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3; R2 is amino, hydroxyl, thiol, C1-C 12 Alkylamino or C1-C 12 dialkylamino or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3; provided that the sum of z and y is 3; or The core has the following formula: in: X3 is -NR6-, wherein R6 is hydrogen, C1-C8 alkyl or C1-C8 substituted alkyl, -O- or C1-C8 alkylaminodiyl, C1-C8 alkoxydiyl, C6-C8 arenediyl, C5-C8 heteroarenediyl, C3-C8 heterocycloalkanediyl, or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxyl, thiol, C1-C 12 Alkylamino or C1-C 12 dialkylamino or a substituted form of any of these groups; or a group of the formula: -N(R f ) f (CH2CH2N(R c )) e R d 、 in: e and f are each independently 1, 2 or 3; provided that the sum of e and f is 3; R c 、R d and R f are each independently hydrogen, C1-C6 alkyl or substituted C1-C6 alkyl; c and d are each independently 1, 2, 3, 4, 5 or 6; or The core is C1-C 18 Alkylamine, C1-C 36 Dialkylamine, C3-C 12 heterocycloalkane or a substituted form of any of these groups; wherein the repeating unit comprises a degradable diacyl group or a degradable diacyl group and a linker; The degradable diacyl group has the formula: in: A1 and A2 are each independently -O-, -S- or -NR a -,in: R a is hydrogen, C1-C6 alkyl or substituted C1-C6 alkyl; Y3 is C1-C 12 Alkanediyl, C1-C 12 Alkenediyl, C6-C12 arenediyl or a substituted form of any of these groups; or a group having the formula: in: X3 and X4 are C1-C 12 Alkanediyl, C2-C 12 Alkenediyl, C6-C 12 arenediyl or a substituted form of any of these groups; Y5 is a covalent bond, C1-C 12 Alkanediyl, C1-C 12 Alkenediyl, C6-C 12 arenediyl or a substituted form of any of these groups; and R9 is C1-C8 alkyl or substituted C1-C8 alkyl; The linker group has the formula: in: Y1 is C1-C 12 Alkanediyl, C1-C 12 Alkenediyl, C6-C 12 arenediyl or a substituted form of any of these groups; and Each of these independently represent the point of attachment to another repeat unit or a terminating group; and The terminating group has the formula: in: Y4 is an alkanediyl or a C1-C 18 Alkanediyl, wherein C1-C 18 One or more of the hydrogen atoms on the alkanediyl group are replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3; R 10 It is hydrogen, carboxyl, hydroxyl, C6-C 12 Aryl, C1-C 12 Alkylamino, C1-C 12 Dialkylamino, C3-C 12 N-heterocycloalkyl, -C(O)N(R 11 )-C1-C6 alkanediyl-C3-C 12 Heterocycloalkyl, -C(O)-C1-C 12 Alkylamino, -C(O)-C1-C 12 dialkylamino or -C(O)-C3-C 12N-heterocycloalkyl, wherein: R 11 is hydrogen, C1-C6 alkyl or substituted C1-C6 alkyl; wherein the final degradable diacyl group in the chain is attached to a terminating group; n is 0, 1, 2, 3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, the terminating group is further defined by the formula: in: Y4 is C1-C 18 alkanediyl; and R 10 In some embodiments, A1 and A2 are each independently -O- or -NR a -.

[0255] In some embodiments of the dendrimer of Formula (DI), the terminating group is a structure selected from the structures in Table 3.

[0256] In some embodiments of the dendrimer of formula (DI), the core is further defined by the formula: in: X2 is N(R5) y ; R5 is hydrogen or C1-C8 alkyl or substituted C1-C 18 alkyl; and y is 0, 1, or 2, provided that the sum of y and z is 3; R2 is amino, hydroxyl, or thiol, or C1-C 12 Alkylamino, C1-C 12 dialkylamino or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3; the condition is that the sum of z and y is 3.

[0257] In some embodiments of the dendrimer of formula (DI), the core is further defined by the formula: in: X3 is -NR6-, wherein R6 is hydrogen, C1-C8 alkyl or substituted C1-C8 alkyl, -O- or C1-C8 alkylaminodiyl, C1-C8 alkoxydiyl, C1-C8 arenediyl, C1-C8 heteroarenediyl, C1-C8 heterocycloalkanediyl, or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxyl, thiol, or C1-C 12 Alkylamino, dialkylamino, or a substituted form of any of these groups; or a group of the formula: -N(R f ) f (CH2CH2N(R c )) e R d 、 in: e and f are each independently 1, 2 or 3; provided that the sum of e and f is 3; R c 、R d and R f are each independently hydrogen, C1-C6 alkyl or substituted C1-C6 alkyl; c and d are each independently 1, 2, 3, 4, 5 or 6.

[0258] In some embodiments of the dendrimer of formula (I), the terminating group is represented by the formula: in: Y4 is an alkanediyl group (C≤18) ;and R 10 It's hydrogen.

[0259] In some embodiments of the dendrimer of formula (DI), the core of the structure of formula (D-IV) is: or a pharmaceutically acceptable salt thereof.

[0260] In some embodiments of the dendrimer of Formula (DI), the core comprises the structural formula shown in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates the point of attachment of the core to the repeat unit (i.e., where a hydrogen of the core is replaced by a repeat unit).

[0261] In some embodiments of the dendrimer of formula (DI), the degradable diacyl group is further defined as:

[0262] In some embodiments of the dendrimer of formula (DI), the linker is further defined as Wherein Y1 is C1-C8 alkanediyl or substituted C1-C 12 Alkanediyl.

[0263] In some embodiments, in the core of formula (D-IV), R6 is H. In some embodiments, in the core of formula (D-IV), R6 is C1-C8 alkyl. In some embodiments, in the core of formula (D-IV), R6 is substituted alkyl (e.g., alkyl substituted with -NH2, alkyl substituted with -NHCH3, or alkyl substituted with -NHCH2CH3).

[0264] In some embodiments, one or two hydrogen atoms of the core are replaced by repeating units. In some embodiments, three or four hydrogen atoms of the core are replaced by repeating units. In some embodiments, five hydrogen atoms of the core are replaced by repeating units. In some embodiments, six hydrogen atoms of the core are replaced by repeating units.

[0265] In some embodiments of the dendrimer of formula (DI), the dendrimer is selected from: and pharmaceutically acceptable salts thereof. 3. Dendrimer of formula (X)

[0266] In some embodiments of the lipid composition, the ionizable lipid is a dendrimer of the formula: In some embodiments, the ionizable lipid is a dendrimer of the formula:

[0267] In some embodiments of the lipid composition, the ionizable lipid is a dendrimer of generation (g) having the following structural formula: or a pharmaceutically acceptable salt thereof, wherein: (VI) The core comprises the structural formula (X 核心 ): in: Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 -or-CR 3a R 3b -; R 2 R independently at each occurrence 1g or -L2 -NR 1e R 1f ; R 3a and R 3b is independently at each occurrence hydrogen or optionally substituted (e.g., C1-C6, such as C1-C3) alkyl; R 1a 、R 1b 、R 1c 、R 1d 、R 1e 、R 1f and R 1g (if present) is independently at each occurrence the point of attachment to the branch, hydrogen, or an optionally substituted (e.g., C1-C 12 )alkyl; L 0 , L 1 and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, Alternatively, L 1 Part of R 1c and R 1d one of which forms a (e.g., C4-C6)heterocycloalkyl (e.g., containing one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur); and x 1 is 0, 1, 2, 3, 4, 5, or 6; and (b) Each of the plurality (N) branches independently comprises the structural formula (X 分支 ): in: *Indicates the attachment points of the branches to the core; g is 1, 2, 3, or 4; Z=2(g- 1 ); When g=1, G=0; or when g≠1, Each diacyl group independently comprises the formula in: * indicates the point of attachment of the diacyl group at its proximal end; ** indicates the attachment point of the diacyl group at its distal end; Y 3 is independently at each occurrence optionally substituted (e.g., C1-C 12 ) alkylene, optionally substituted (e.g., C1-C12 ) alkenylene or optionally substituted (e.g., C1-C 12 ) arene groups; A 1 and A 2 is independently -O-, -S- or -NR at each occurrence 4 -,in: R 4 is hydrogen or optionally substituted (e.g., C1-C6) alkyl; m 1 and m 2 is independently 1, 2, or 3 at each occurrence; and R 3c 、R 3d 、R 3e and R 3f is independently at each occurrence hydrogen or optionally substituted (e.g., C1-C8) alkyl; and (d) Each linker group independently comprises the formula in: **Indicates the point of attachment of the linker to the proximal diacyl group; *** indicates the point of attachment of the linker to the distal diacyl group; and Y1 is independently optionally substituted at each occurrence (e.g., C1-C 12 ) alkylene, optionally substituted (e.g., C1-C 12 ) alkenylene or optionally substituted (e.g., C1-C 12 ) an arylene group; and Each terminating group is independently selected from optionally substituted (e.g., C1-C 18 , such as C4-C 18 ) alkylthiol and optionally substituted (e.g., C1-C 18 , such as C4-C 18 )Alkenyl thiol.

[0268] In X 核心 In some embodiments, Q at each occurrence is independently a covalent bond, -O-, -S-, -NR 2 -or-CR 3a R 3b In X 核心 In some embodiments of , Q is independently a covalent bond at each occurrence. 核心 In some embodiments, Q is independently -O- at each occurrence. 核心 In some embodiments, Q is independently -S- at each occurrence. 核心 In some embodiments, Q at each occurrence is independently -NR2 , and R 2 R independently at each occurrence 1g or -L 2 -NR 1e R 1f In X 核心 In some embodiments, Q is independently -CR at each occurrence 3a R 3b R 3a , and R 3a and R 3b and R is independently at each occurrence hydrogen or optionally substituted alkyl (eg, C1-C6, such as C1-C3).

[0269] In X 核心 In some embodiments, R 1a 、R 1b 、R 1c 、R 1d 、R 1e 、R 1f and R 1g (if present) is independently at each occurrence the point of attachment to the branch, hydrogen or optionally substituted alkyl. 核心 In some embodiments, R 1a 、R 1b 、R 1c 、R 1d 、R 1e 、R 1f and R 1g (if present) is independently at each occurrence the point of attachment to the branch, a hydrogen. 核心 In some embodiments, R 1a 、R 1b 、R 1c 、R 1d 、R 1e 、R 1f and R 1g (if present) is independently at each occurrence the point of attachment to the branch, an optionally substituted alkyl group (e.g., C1-C 12 ).

[0270] In X 核心 In some embodiments, L 0 , L 1 and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or, alternatively, L 1 Part of R 1c and R1d In X 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of is independently a covalent bond. 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of is independently hydrogen. 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence is independently an alkylene group (e.g., C1-C 12 , such as C1-C6 or C1-C3). In X 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence independently can be heteroalkylene (e.g., C1-C 12 , such as C1-C8 or C1-C6). In X 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of is independently heteroalkylene (e.g., C2-C8 alkylene oxide, such as oligo(ethylene oxide)). 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of X is independently [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g. C1-C6)alkylene]-[(e.g. C4-C6)heterocycloalkyl]-[(e.g. C1-C6)alkylene]. 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of X is independently [alkylene]-(arylene)-[alkylene] [(e.g. C1-C6)alkylene]-(arylene)-[(e.g. C1-C6)alkylene]. 核心 In some embodiments, L 0 , L 1 and L 2Each occurrence of X can independently be [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6)alkylene]-phenylene-[(e.g., C1-C6)alkylene]). 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of is independently heterocycloalkyl (e.g., C4-C6 heterocycloalkyl). 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence of is independently an arylene group (e.g., a phenylene group). 核心 In some embodiments, L 1 Part of R 1c and R 1d In X 核心 In some embodiments, L 1 Part of R 1c and R 1d One of the forms a heterocycloalkyl group (e.g., C4-C6 heterocycloalkyl group), and the heterocycloalkyl group may contain one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur.

[0271] In X 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence is independently selected from a covalent bond, a C1-C6 alkylene group (e.g., a C1-C3 alkylene group), a C2-C 12 (e.g., C2-C8)alkylene oxides (e.g., oligo(ethylene oxides), such as -(CH2CH2O) 1-4 -(CH2CH2)-), [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene] (e.g., ) and [(C1-C4)alkylene]-phenylene-[(C1-C4)alkylene] (e.g., ). 核心 In some embodiments, L 0 , L 1 and L 2 Each occurrence is independently selected from C1-C6 alkylene (e.g., C1-C3 alkylene), -(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 embodiments, L 0 , L 1 and L 2 Each occurrence is independently C1-C6 alkylene (e.g., C1-C3 alkylene). In some embodiments, L 0 , L 1 and L 2 Each occurrence is independently C2-C 12 (e.g., C2-C8)alkylene oxide (e.g., -(C1-C3 alkylene-O) 1-4 -(C1-C3 alkylene)). In X 核心 In some embodiments, L 0 , L 1 and L 2 and [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-) and [(C1-C4)alkylene]-[(C4-C6)heterocycloalkyl]-[(C1-C4)alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).

[0272] In X 核心 In some embodiments, x 1 is 0, 1, 2, 3, 4, 5, or 6. 核心 In some embodiments, x 1 is 0. 核心 In some embodiments, x 1 is 1. In X 核心 In some embodiments, x 1 is 2. In X 核心 In some embodiments, x 1 0, 3. 核心 In some embodiments, x 1 is 4. In X 核心 In some embodiments, x 1 is 5. In X 核心 In some embodiments, x 1 is 6.

[0273] In X 核心 In some embodiments, the core comprises the structural formula: (For example, ). 核心 In some embodiments, the core comprises the structural formula: In X 核心 In some embodiments, the core comprises the structural formula: (For example, ). 核心 In some embodiments, the core comprises the structural formula: (For example, like ). 核心 In some embodiments, the core comprises the structural formula: Where Q' is -NR 2 -or-CR 3a R 3b -;q 1 and q 2 Each independently is 1 or 2. 核心 In some embodiments, the core comprises the structural formula: (For example, ). 核心 In some embodiments of the present invention, the core comprises the structural formula (For example, ), wherein Ring A is optionally substituted aryl or optionally substituted (e.g., C3-C 12 , such as C3-C5) heteroaryl. In X 核心 In some embodiments, the core comprises a

[0274] In X 核心 In some embodiments, the core comprises the structural formula shown in Table 2 and pharmaceutically acceptable salts thereof, wherein * indicates the point of attachment of the core to one of the plurality of branches.

[0275] In some embodiments, the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality (N) of branches comprises at least 3 branches. In some embodiments, the plurality (N) of branches comprises at least 4 branches. In some embodiments, the plurality (N) of branches comprises at least 5 branches.

[0276] In X 分支 In some embodiments of , g is 1, 2, 3, or 4. 分支 In some embodiments of X, g is 1. 分支 In some embodiments of X, g is 2. 分支 In some embodiments of X, g is 3. 分支 In some embodiments, g is 4.

[0277] In X 分支 In some embodiments, Z=2 (g-1), and when g=1, G=0. 分支 In some embodiments, Z=2 (g-1) , and when g≠1,

[0278] In X 分支 In some embodiments of the present invention, g=1, G=0, Z=1, and each of the plurality of branches comprises the structural formula, each of the plurality of branches comprises the structural formula

[0279] In X 分支 In some embodiments of the present invention, g=2, G=1, Z=2, and each of the plurality of branches comprises the formula

[0280] In X 分支 In some embodiments of the present invention, g=3, G=3, Z=4, and each of the plurality of branches comprises the formula

[0281] In X 分支 In some embodiments of the present invention, g=4, G=7, Z=8, and each of the plurality of branches comprises the formula

[0282] In some embodiments, the dendrimers described herein with generation (g) = 1 have the following structure:

[0283] In some embodiments, the dendrimers described herein with generation (g) = 1 have the following structure:

[0284] Exemplary formulations of dendrimers of generations 1-4 described herein are shown in Table 1. The number of diacyl groups, linker groups, and terminating groups can be calculated based on g. Table 1. Formulations of dendrimer groups based on generation (g) g＝1 g＝2 g＝3 g＝4 The number of diacyl groups 1 1+2＝3 <![CDATA[1+2+2 2 =7]]> <![CDATA[1+2+2 2 +2 3 =15]]> <![CDATA[1+2+...+2 g-1 ]]> Number of linker groups 0 1 1+2 <![CDATA[1+2+2 2 ]]> <![CDATA[1+2+...+2 g-2 ]]> Number of terminating groups 1 2 <![CDATA[2 2 ]]> <![CDATA[2 3 ]]> <![CDATA[2 (g-1) ]]>

[0285] In some embodiments, the diacyl groups independently comprise the formula * indicates the point of attachment of the diacyl group at its proximal end, and ** indicates the point of attachment of the diacyl group at its distal end.

[0286] In X 分支 In some embodiments of the diacyl group, Y 3 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arylene. 分支 In some embodiments of the diacyl group, Y 3Each occurrence is independently optionally substituted alkylene (e.g., C1-C 12 ). 分支 In some embodiments of the diacyl group, Y 3 Each occurrence is independently optionally substituted alkenylene (e.g., C1-C 12 ). 分支 In some embodiments of the diacyl group, Y 3 Each occurrence is independently an optionally substituted arylene group (e.g., C1-C 12 ).

[0287] In X 分支 In some embodiments of the diacyl group, A 1 and A 2 is independently -O-, -S- or -NR at each occurrence 4 -. In X 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -O-. 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -S-. 分支 In some embodiments of the diacyl group, A 1 and A 2 Each occurrence is independently -NR 4 -, and R 4 is hydrogen or an optionally substituted alkyl group (e.g., C1-C6). 分支 In some embodiments of the diacyl group, m 1 and m 2 is independently 1, 2, or 3 at each occurrence. 分支 In some embodiments of the diacyl group, m 1 and m 2 is 1 independently at each occurrence. 分支 In some embodiments of the diacyl group, m 1 and m 2 is 2 independently at each occurrence. 分支 In some embodiments of the diacyl group, m 1 and m 2 is 3 independently at each occurrence. 分支 In some embodiments of the diacyl group, R 3c 、R 3d 、R 3e and R 3fis independently hydrogen or optionally substituted alkyl at each occurrence. 分支 In some embodiments of the diacyl group, R 3c 、R 3d 、R 3e and R 3f is independently hydrogen at each occurrence. 分支 In some embodiments of the diacyl group, R 3c 、R 3d 、R 3e and R 3f and is independently at each occurrence an optionally substituted (eg, C1-C8) alkyl group.

[0288] In some embodiments of the diacyl group, A 1 Is -O- or -NH-. In some embodiments of the diacyl group, A 1 Is -O-. In some embodiments of the diacyl group, A 2 Is -O- or -NH-. In some embodiments of the diacyl group, A 2 In some embodiments of the diacyl group, Y 3 It is C1-C 12 (For example, C1-C6, such as C1-C3)alkylene.

[0289] In some embodiments of the diacyl group, the diacyl group independently comprises, at each occurrence, the structural formula (For example, like ), and optionally R 3c 、R 3d 、R 3e and R 3f Each occurrence is independently hydrogen or C1-C3 alkyl.

[0290] In some embodiments, the linker groups independently comprise the formula ** indicates the point of attachment of the linker to the proximal diacyl group, and *** indicates the point of attachment of the linker to the distal diacyl group.

[0291] In X 分支 In some embodiments of the linker group (if present), Y 1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted arylene. 分支 In some embodiments of the linker group (if present), Y1 is independently at each occurrence an optionally substituted alkylene (e.g., C1-C 12 ). 分支In some embodiments of the linker group (if present), Y1 is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C 12 ). 分支 In some embodiments of the linker group (if present), Y1 is independently at each occurrence an optionally substituted arylene group (e.g., C1-C 12 ).

[0292] In X 分支 In some embodiments of the terminating group of X, each terminating group is independently selected from optionally substituted alkylthiols and optionally substituted alkenylthiols. 分支 In some embodiments of the terminating groups of 18 , such as C4-C 18 ). 分支 In some embodiments of the terminating groups of 18 , such as C4-C 18 ).

[0293] In X 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 Alkenyl thiol or C1-C 18 Alkylthiol, and the alkyl or alkenyl moiety is optionally substituted by one or more substituents each independently selected from the group consisting of halogen, C6-C 12 Aryl, C1-C 12 Alkylamino, C4-C6 N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocycloalkyl), -C(O)-(C1-C 12 The C4-C6 N-heterocycloalkyl portion of any of the foregoing substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0294] In X 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 (For example, C4-C 18 ) alkenyl thiol or C1-C 18 (For example, C4-C 18) alkylthiol, wherein the alkyl or alkenyl portion is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, C6-C 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (e.g., -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (e.g., )), C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl N-piperidinyl N-azepanyl ), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 Alkylamino (e.g., monoalkylamino or dialkylamino)) (e.g., ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6N-heterocycloalkyl) (e.g., )、-C(O)-(C1-C 12 Alkylamino (e.g., monoalkylamino or dialkylamino)) and -C(O)-(C4-C6 N-heterocycloalkyl) (e.g., ), wherein the C4-C6N-heterocycloalkyl portion of any of the aforementioned substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl. 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 (For example, C4-C 18 ) alkylthiol, wherein the alkyl portion is optionally substituted with one substituent -OH. In X 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 (For example, C4-C 18 ) alkylthiol, wherein the alkyl portion is optionally substituted with a substituent selected from the group consisting of: C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (e.g., -NHCH2CH2CH2CH3) or C1-C8 dialkylamino (e.g., )) and C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl N-piperidinyl N-azepanyl ). 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 (For example, C4-C 18) alkenyl thiol or C1-C 18 (For example, C4-C 18 ) alkylthiol. In X 分支 In some embodiments of the terminating group, each terminating group is independently C1-C 18 (For example, C4-C 18 ) alkylthiols. Table 2. Exemplary core structures

[0295] In X 核心 In some embodiments, the core comprises a structural formula selected from the group consisting of: A pharmaceutically acceptable salt thereof, wherein * indicates the point of attachment of the core to one of the multiple branches.

[0296] In X 分支 In some embodiments of the terminating group, each terminating group is independently a structure selected from the structures in Table 3. In some embodiments, the dendrimers described herein can include a terminating group selected from Table 3 or a pharmaceutically acceptable salt thereof. Table 3. Exemplary Termination Groups / Peripheral Structures

[0297] In some embodiments, the dendrimer of formula (X) is selected from those shown in Table 4 and pharmaceutically acceptable salts thereof. Table 4. Exemplary ionizable cationic lipid dendrimers

[0298] In some embodiments, the dendrimer is 2A2-SC14. In some embodiments, the dendrimer is 2A6-SC14. In some embodiments, the dendrimer is 2A9-SC14. In some embodiments, the dendrimer is 3A3-SC10. In some embodiments, the dendrimer is 3A3-SC14. In some embodiments, the dendrimer is 4A5-SC10. In some embodiments, the dendrimer is 3A5-SC14. In some embodiments, the dendrimer is 4A1-SC12. In some embodiments, the dendrimer is 4A3-SC12. In some embodiments, the dendrimer is 5A1-SC12. In some embodiments, the dendrimer is 5A1-SC8. In some embodiments, the dendrimer is 5A2-2-SC12. In some embodiments, the dendrimer is 5A3-1-SC12. In some embodiments, the dendrimer is 5A3-1-SC8. In some embodiments, the dendrimer is 5A4-1-SC12. In some embodiments, the dendrimer is 5A4-1-SC8. In some embodiments, the dendrimer is 5A5-SC8. In some embodiments, the dendrimer is 5A5-SC12. In some embodiments, the dendrimer is 5A2-4-SC12. In some embodiments, the dendrimer is 5A2-4-SC10. In some embodiments, the dendrimer is 5A3-2-SC8. In some embodiments, the dendrimer is 5A3-2-SC12. In some embodiments, the dendrimer is 5A4-2-SC8. In some embodiments, the dendrimer is 5A4-2-SC12. In some embodiments, the dendrimer is 6A4-SC8. In some embodiments, the dendrimer is 6A4-SC12. In some embodiments, the dendrimer is 2A2-g2-SC12. In some embodiments, the dendrimer is 2A2-g2-SC8. In some embodiments, the dendrimer is 2A11-g2-SC12. In some embodiments, the dendrimer is 2A11-g2-SC8. In some embodiments, the dendrimer is 3A3-g2-SC12. In some embodiments, the dendrimer is 3A3-g2-SC8. In some embodiments, the dendrimer is 3A5-g2-SC12. In some embodiments, the dendrimer is 2A11-g3-SC12. In some embodiments, the dendrimer is 2A11-g3-SC8. In some embodiments, the dendrimer is 1A2-g4-SC12. In some embodiments, the dendrimer is 4A1-g2-SC12. In some embodiments, the dendrimer is 1A2-g4-SC8. In some embodiments, the dendrimer is 4A1-g2-SC8.In some embodiments, the dendrimer is 4A3-g2-SC12. In some embodiments, the dendrimer is 4A3-g2-SC8. In some embodiments, the dendrimer is 1A2-g3-SC12. In some embodiments, the dendrimer is 1A2-g3-SC8. In some embodiments, the dendrimer is 2A2-g3-SC12. In some embodiments, the dendrimer is 2A2-g3-SC8. In some embodiments, the dendrimer is 5A2-4-SC8. In some embodiments, the dendrimer is 5A5-SC8. In some embodiments, the dendrimer is 5A2-6-SC8. In some embodiments, the dendrimer is 5A2-1-SC8. In some embodiments, the dendrimer is 5A2-2-SC8. In some embodiments, the dendrimer is 4A1-SC5. In some embodiments, the dendrimer is 4A1-SC8. In some embodiments, the dendrimer is 4A3-SC6. In some embodiments, the dendrimer is 4A3-SC7. In some embodiments, the dendrimer is 4A3-SC8. In some embodiments, the dendrimer is 5A4-2-SC5. In some embodiments, the dendrimer is 5A4-2-SC6. In some embodiments, the dendrimer is 5A2-4-SC8. In some embodiments, the dendrimer is 3A5-g2-SC8. In some embodiments, the dendrimer is 5A2-SC8. 4. Other ionizable lipids

[0299] In some embodiments of the lipid composition, the cationic lipid comprises structural formula (DI'): in: a is 1 and b is 2, 3 or 4; or, alternatively, b is 1 and a is 2, 3 or 4; 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 R 1 、R 2 、R 3 、R 4 、R 5 and R 6 Each independently selected from H, -CH2CH(OH)R 7 、-CH(R 7 )CH2OH, -CH2CH2C(=O)OR 7 、-CH2CH2C(=O)NHR 7 and -CH2R 7 , where R7 Independently selected from C3-C 18 Alkyl, C3-C with one C=C double bond 18 an alkenyl group, a protecting group for an amino group, -C(=NH)NH2, a poly(ethylene glycol) chain, and a receptor ligand; The condition is R 1 to R 6 At least two of the parts 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 or C3-C with one C=C double bond 18 alkenyl; and One or more of the nitrogen atoms indicated in formula (DI') may be protonated to provide a cationic lipid.

[0300] In some embodiments of the cationic lipid of Formula (DI'), a is 1. In some embodiments of the cationic lipid of Formula (DI'), b is 2. In some embodiments of the cationic lipid of Formula (DI'), m is 1. In some embodiments of the cationic lipid of Formula (DI'), n is 1. In some embodiments of the cationic lipid of Formula (DI'), R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are each independently H or -CH2CH(OH)R 7 In some embodiments of the cationic lipid of formula (DI'), 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 (DI'), 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 (DI'), R 7 It is C3-C18 Alkyl (e.g., C6-C 12 alkyl).

[0301] In some embodiments, the cationic lipid of formula (DI') is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatriacontane-11,25-diol:

[0302] In some embodiments, the cationic lipid of formula (DI') is (11R,25R)-13,16,20-tris(1-2-hydroxydodecyl)-13,16,20,23-tetraazapentatriacontane-11,25-diol:

[0303] Additional cationic lipids that can be used in the compositions and methods of the present application include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and International Patent Publications WO2010144740, WO 2013149140, WO 2016118725, WO 2016118724, WO 2013063468, WO2016205691, WO 2015184256, WO 2016004202, WO 2015199952, WO 2017004143, WO2017075531, WO 2017117528, WO 2017049245, WO 2017173054, and WO 2015095340, which is incorporated herein by reference for all purposes. Examples of those ionizable cationic lipids include, but are not limited to, those shown in Table 5. Table 5. Exemplary ionizable cationic lipids

[0304] In some embodiments of the lipid compositions of the present disclosure, ionizable lipid exists with the amount of about 20mol% to about 23mol%. In some embodiments, ionizable lipid exists with the amount of about 20mol%, about 20.5mol%, about 21mol%, about 21.5mol%, about 22mol%, about 22.5mol% or about 23mol%. In other embodiments, ionizable lipid exists with the amount of about 7.5mol% to about 20mol%. In some embodiments, ionizable lipid exists with the amount of about 7.5mol%, about 8mol%, about 9mol%, about 10mol%, about 11mol%, about 12mol%, about 13mol%, about 14mol%, about 15mol%, about 16mol%, about 17mol%, about 18mol%, about 19mol% or about 20mol%.

[0305] In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of about 5 mol% to about 30 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of about 10 mol% to about 25 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of about 15 mol% to about 20 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of about 10 mol% to about 20 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of about 20 mol% to about 30 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid compositions include an amount of at least (approximately) 5 mol%, at least (approximately) 10 mol%, at least (approximately) 15 mol%, at least (approximately) 20 mol%, at least (approximately) 25 mol% or at least (approximately) 30 mol% ionizable lipids. In some embodiments of the lipid compositions of the present disclosure, the lipid composition comprises ionizable lipids in an amount of at most (about) 5 mol%, at most (about) 10 mol%, at most (about) 15 mol%, at most (about) 20 mol%, at most (about) 25 mol%, or at most (about) 30 mol%. B. Helper lipids

[0306] In some embodiments, the helper lipid is a phospholipid. As defined herein, a phospholipid is any lipid comprising a phosphate group. The lipid component of the lipid nanoparticle can include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can be assembled into one or more lipid bilayers. Generally, a phospholipid can include a phospholipid portion and one or more fatty acid portions. The phospholipid portion can be selected from the following non-limiting groups: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid portion can be selected from the following non-limiting groups: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0307] Also contemplated are non-natural species, which include natural species with modification and substitution (including branching, oxidation, cyclization, and alkynes). For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds) or cross-linked with one or more alkynes. Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloadditions when exposed to azide. Such reactions may be used to functionalize nanoparticles to promote membrane penetration or cell recognition, or for coupling nanoparticles to other components such as targeting moieties or imaging moieties (e.g., dyes).

[0308] In some embodiments, LNP as described herein comprises about 5 mol% to about 30 mol% phospholipids. In some embodiments, 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 embodiments, the LNPs comprise 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%.

[0309] In some embodiments, LNP comprises about 5% to about 30% phospholipids by weight.In some embodiments, LNP comprises about 5% by weight, or 10% by weight, or 12% by weight, or 15% by weight, or 18% by weight, or 20% by weight, or 25% by weight, or 30% by weight phospholipids.

[0310] In some embodiments of the lipid components of the present disclosure, the lipid component can further comprise a mole percentage of phospholipids to the total lipid composition of about 20 to about 23. In some embodiments, the mole percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the mole percentage is about 7.5 to about 60. In some embodiments, the mole percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0311] In some embodiments of the lipid components of the present disclosure, the lipid components comprise phospholipids at a molar percentage of about 8% to about 23%. In some embodiments of the lipid components of the present disclosure, the lipid components comprise phospholipids at a molar percentage of about 10% to about 20%. In some embodiments of the lipid components of the present application, the lipid components comprise phospholipids at a molar percentage of about 15% to about 20%. In some embodiments of the lipid components of the present disclosure, the lipid components comprise phospholipids at a molar percentage of about 8% to about 15%. In some embodiments of the lipid components of the present disclosure, the lipid components comprise phospholipids at a molar percentage of about 10% to about 15%. In some embodiments of the lipid components of the present disclosure, the lipid components comprise phospholipids at a molar percentage of about 12% to about 18%. In some embodiments of the lipid components of the present disclosure, the lipid composition comprises phospholipids at 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%. In some embodiments of the lipid component of the present disclosure, the lipid component comprises a molar percentage of at most (about) 8%, at most (about) 10%, at most (about) 12%, at most (about) 15%, at most (about) 18%, at most (about) 20% or at most (about) 23% phospholipids.

[0312] The phospholipids that can be used or potentially used in the compositions and methods of the present invention can be selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-dicondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

[0313] In some embodiments, the phospholipids may contain one or two long chains (e.g., C6-C 24) alkyl or alkenyl, glycerol or sphingosine, one or two phosphate groups and optionally a small organic molecule. The small organic molecule can be an amino acid, a sugar, or an amino-substituted alkoxy group (such as choline or ethanolamine). In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is a distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein the zwitterionic lipid definition has lipids and lipid-like molecules that have both a positive charge and a negative charge.

[0314] In some embodiments of the lipid component of the present disclosure, the phospholipid is not ethylphosphocholine. C. Polymer-conjugated lipids

[0315] The lipid component of the present disclosure can include a lipid conjugated to a polymer, such as a lipid conjugated to polyethylene glycol (PEG-lipid). Illustrative methods for preparing and using PEG-lipids are described in, for example, International Patent Publication No. WO2012099755 and U.S. Patent Publication No. 2014 / 0200257.

[0316] In one embodiment, the PEG-lipid that can be used for the present disclosure can be the PEG-lipid described in International Patent Publication No. WO2012 / 099755 (its content is incorporated herein by reference in its entirety).Any of these exemplary PEG-lipids described herein can be modified to include a hydroxyl group on the PEG chain.In certain embodiments, the PEG-lipid is a PEG-OH lipid. The PEG-OH lipid is a PEG-lipid with one or more hydroxyl groups (-OH) on the lipid. In certain embodiments, the PEG-OH lipid is included in one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH or hydroxyl-PEG-lipid is included in the-OH group at the end of the PEG chain.Every possibility represents a separate embodiment of the present disclosure.

[0317] In some embodiments of the lipid component of the present disclosure, 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 also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C6-C6-D1 attached to a linker group having a PEG chain. 24 Long chain alkyl or alkenyl or C6-C 24Fatty acid 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-diacyloxypropane-3-amine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, the PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE. In some embodiments, PEG-modified distearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. 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. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000 Da. Some non-limiting examples of lipids useful in the present disclosure are taught in US Pat. No. 5,820,873, International Patent Publication No. WO 2010 / 141069, or US Pat. No. 8,450,298, which are incorporated herein by reference in their entireties.

[0318] In some embodiments of the lipid compositions of the present application, the PEG-lipid has the structural formula: Where: R 12 and R 13 are each independently an alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any of these groups; R e Is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) ; and x is 1-250. In some embodiments, R e is an alkyl group (C≤8) , such as methyl. R 12 and R 13 are each independently an alkyl (C≤4-20)In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0319] In some embodiments of the lipid component of the present disclosure, the PEG-lipid has the structural formula: wherein: n1 is an integer between 1 and 100 and n2 and n3 are each independently selected from an integer 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 derivable therein. In some embodiments, n1 is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is from 11 to about 17. In some embodiments, n3 is from 5 to 23. In some embodiments, n3 is from 11 to about 17.

[0320] In some embodiments of the lipid component of the present disclosure, the component can further include the molar percentage of about 4.0% to about 4.6% PEG-lipid and 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 scope that can be 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 scope that can be derived therefrom.

[0321] In some embodiments of the lipid components of the present disclosure, the lipid component comprises a polymer-conjugated lipid at a molar percentage of about 0.5% to about 10%. In some embodiments of the lipid components of the present disclosure, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 1% to about 8%. In some embodiments of the lipid components of the present disclosure, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 2% to about 7%. In some embodiments of the lipid components of the present application, the lipid component comprises a polymer-conjugated lipid at a molar percentage of about 3% to about 5%. In some embodiments of the lipid components of the present disclosure, the lipid component comprises a polymer-conjugated lipid at a molar percentage of about 5% to about 10%. In some embodiments of the lipid component of the present disclosure, 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% polymer-conjugated lipid. In some embodiments of the lipid component of the present disclosure, the lipid component comprises a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5% or at most (about) 10% polymer-conjugated lipid. D. Structural lipids

[0322] Lipid nanoparticles can comprise one or more structural lipids. The structural lipid can be a steroid or a steroid derivative. In some embodiments of the lipid component of the present disclosure, the lipid component further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. Steroids are a class of compounds with a tetracyclic 17-carbon ring structure, and the ring structure can further comprise one or more substitutions, the substitutions comprising alkyl, alkoxy, hydroxyl, oxo, acyl or a double bond between two or more carbon atoms. On the one hand, the ring structure of the steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula: In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as: In some embodiments of the present disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the formula: As described above, cholestane derivatives comprise one or more non-alkyl substitutions of the above-mentioned ring systems. In some embodiments, cholestane or cholestane derivatives are cholestene or cholestene derivatives. In some embodiments, steroids or steroid derivatives are open-ring steroids or open-ring steroid derivatives. In some embodiments, steroids or steroid derivatives are cardiac glycosides or cardiac glycoside derivatives. In some embodiments, steroids or steroid derivatives are sapogenins or sapogenin derivatives. In some embodiments, steroids or steroid derivatives are saponins or saponin derivatives. In some embodiments, steroids or steroid derivatives are eicosanoids or eicosanoid derivatives. In some embodiments, steroids or steroid derivatives are alkaloids or alkaloid derivatives. In some embodiments, steroids or steroid derivatives are sterols or sterol derivatives.

[0323] Sterols useful in the compositions and methods described herein can be selected from, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and alpha-tocopherol.

[0324] In some embodiments of lipid components, the component can further include the molar percentage of about 40% to about 46% steroid and total lipid composition. In some embodiments, the molar percentage is about 40%, 41%, 42%, 43%, 44%, 45% to about 46% or any scope that can be derived therefrom. In other embodiments, the molar percentage of steroid relative to total lipid composition 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 scope that can be derived therefrom.

[0325] In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 15% to about 60%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 15% to about 55%. In some embodiments, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 15% to about 50%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 15% to about 46%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 20% to about 40%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 25% to about 35%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 30% to about 40%. In some embodiments, the lipid component comprises a steroid or steroid derivative at a molar percentage of about 20% to about 30%. In some embodiments, the lipid component comprises a steroid or steroid derivative at 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 at a molar percentage of at most (about) 15%, at most (about) 20%, at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at most (about) 45%, or at most (about) 46%.

[0326] In some embodiments, the cationic lipid is a sterolamine. A sterolamine has a sterol for its hydrophobic portion and an amine group for its hydrophilic portion. The sterol portion is selected from, but not limited to, cholesterol, sitosterol, campesterol, stigmasterol, or a derivative thereof. The amine group may comprise one to five primary, secondary, or tertiary amines, or a mixture thereof. At least one of the 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 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. E. Additional lipids

[0327] In some embodiments, lipid nanoparticles are preferentially delivered to target organs. In some embodiments, the target organ is lung, lung tissue or lung cell. "Preferential delivery" means that at least 25% (for example, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%) of the amount administered by the composition is delivered to the target organ (for example, lung), target tissue or target cell. Other lipids can support the selective delivery of the composition of the present disclosure to specific organs. In some embodiments, other lipids can be selective organ targeting (SORT) lipids. In some embodiments, SORT lipids can allow LNP to be delivered to the target organ, target tissue or target cell.

[0328] SORT lipids refer to lipids that, when included in an LNP composition, enable the LNP to selectively and predictably target an organ, cell type, or tissue (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 Publication Nos. US2022 / 0071916 A1 and US2021 / 0259980A1, the entire contents of which are incorporated herein in their entirety). 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, permanent 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; permanent cationic SORT lipids generally favor delivery to the lungs; and zwitterionic SORT lipids favor delivery to the spleen.

[0329] In some embodiments, the additional lipid comprises a permanently positively charged moiety (i.e., is a permanently cationic lipid). The permanently positively charged moiety can be positively charged at physiological pH such that when the payload (e.g., polynucleotide) is delivered to the cell, the additional lipid (e.g., SORT lipid) comprises a positive charge. In some embodiments, the positively charged moiety is a quaternary amine or quaternary ammonium ion. In some embodiments, the additional lipid (e.g., SORT lipid) comprises a counterion or otherwise complexes or interacts with a counterion.

[0330] In some embodiments, the additional lipid is a permanent cationic lipid (i.e., comprising one or more hydrophobic components and a permanent cationic group). The permanent cationic lipid can contain a group that has a positive charge regardless of pH. One permanent cationic group that can be used for the permanent cationic lipid is a quaternary ammonium group. The permanent cationic lipid can comprise the structural formula: in: Y1, Y2 or Y3 are each independently X1C(O)R1 or X2N + R3R4R5; The condition is that at least one of Y1, Y2 and Y3 is X2N + R3R4R5; R1 is C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl; X1 is O or NR a , where R a is hydrogen, C1-C4 alkyl or C1-C4 substituted alkyl; X2 is a C1-C6 alkanediyl or a C1-C6 substituted alkanediyl; R3, R4 and R5 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl; and A1 is equal to the charge of X2N in the compound + The number of R3R4R5 groups in the anion.

[0331] In some embodiments, the additional permanent cationic lipid (e.g., a SORT lipid) has the structural formula: in: R6-R9 are each independently C1-C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 Substituted alkenyl; provided that at least one of R6-R9 is C8-C 24 and A2 is a monovalent anion.

[0332] In some embodiments, the permanent cationic lipid is 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16: 0-18:0EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1EPC), dimethyldioctadecyl ammonium (18:0DDAB), 1,2-dimyristoyl-3-trimethylammonium-propane (14:0TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0TAP), 1,2-stearoyl-3-trimethylammonium-propane (18:0TAP), 1,2-dioleoyl-3-trimethylammonium-propane (18:1TAP, DOTAP), or 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

[0333] In some embodiments, the SORT lipid is an ionizable cationic lipid (i.e., comprising one or more hydrophobic components and an ionizable cationic group, e.g., a tertiary amino group). The ionizable cationic group can be positively charged at physiological pH. One ionizable cationic group that can be used for the ionizable lipid is a tertiary amine group. In some embodiments, the additional lipid (e.g., a SORT lipid) has the structural formula: in: R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of either group; and R3 and R3' are each independently a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

[0334] In some embodiments of Formula (S-I'a), R1 and R2 are each independently C8-C 24 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 (e.g., methyl or ethyl). 24 R3 and R3' are each independently a C1-C6 alkyl group (eg, methyl or ethyl).

[0335] In some embodiments, the ionizable cationic lipid is 1,2-distearoyl-3-dimethylammonium-propane (18:0DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0DAP), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0DAP), 1,2-dioleoyl-3-dimethylammonium-propane (18:1DAP, DODAP), or 1,2-dioleyloxy-3-dimethylaminopropane (DODMA).

[0336] In some embodiments of lipid compositions, other ionizable lipids or permanent cationic lipids include a head group of specific structure. In some embodiments, other lipids (e.g., SORT lipids) include a head group with following structural formula: Where L is the linker; Z + is the positively charged part and X - is a counterion. In some embodiments, the linker is a biodegradable linker. The biodegradable linker can be degradable at physiological pH and temperature. The biodegradable linker can be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or a quaternary amine.

[0337] In some embodiments, the SORT (Additional Ionizable Lipids or Permanent Cations) lipid has the structural formula: where R 1 and R 2 are each independently an optionally substituted C6-C 24 Alkyl or optionally substituted C6-C 24 Alkenyl.

[0338] In some embodiments, the additional lipid (e.g., a SORT lipid) has the structural formula:

[0339] In some embodiments, the additional lipid (e.g., a SORT lipid) comprises a linker (L). In some embodiments, L is in: p and q are each independently 1, 2 or 3; and R 4 is an optionally substituted C1-C6 alkyl group.

[0340] In some embodiments, the additional lipid (e.g., a SORT lipid) has the structural formula: in: R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of any group; R3, R3' and R3" are each independently C1-C6 alkyl or substituted C1-C6 alkyl; R4 is C1-C6 alkyl or substituted C1-C6 alkyl; and X - It is a monovalent anion.

[0341] In some embodiments, the additional lipid (e.g., a SORT lipid) is a phosphatidylcholine (e.g., 14:0EPC). In some embodiments, the phosphatidylcholine compound is further defined as: in: R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of any group; R3, R3' and R3" are each independently C1-C6 alkyl or substituted C1-C6 alkyl; and X - It is a monovalent anion.

[0342] In some embodiments, the additional lipid (e.g., SORT lipid) is a phosphocholine lipid. In some embodiments, the additional lipid (e.g., SORT lipid) is ethylphosphocholine. For example, the ethylphosphocholine can be, but is not limited to, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0EPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:0EPC).

[0343] In some embodiments, the SORT lipid has the structural formula: (S-I'), where: R1 and R2 are each independently C8-C 24 Alkyl, C8-C 24 alkenyl or a substituted form of any group; R3, R3' and R3" are each independently C1-C6 alkyl or substituted C1-C6 alkyl; X - It is a monovalent anion.

[0344] By way of example, but not limitation, an additional lipid of the formula of the preceding paragraph (eg, a SORT lipid) is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (eg, chloride salt).

[0345] In some embodiments, the additional lipid (e.g., a SORT lipid) has the structural formula: in: R4 and R4' are each independently an alkyl group (C6-C24) , alkenyl (C6-C24) or a substituted form of any group; R4" is an alkyl group (C≤24) , alkenyl (C≤24) , or a substituted form of any group; R4"' is an alkyl group (C1-C8) , alkenyl (C2-C8) , or a substituted form of any group; and X2 is a monovalent anion.

[0346] By way of example, but not limitation, an additional lipid of the formula of the preceding paragraph (eg, a SORT lipid) is dimethyldioctadecyl ammonium (DDAB).

[0347] In some embodiments, the additional lipid (e.g., a SORT lipid) is 1,2-Dioleoyl-sn-glycero-3-phosphate (18:1PA).

[0348] In some embodiments of the lipid composition, the additional lipid is selected from the lipids shown in Table 6. Table 6. Exemplary additional lipids (e.g., SORT lipids) X- is a counter ion (eg, Cl-, Br-, etc.).

[0349] In some embodiments, the lipid component comprises a molar percentage of about 20% to about 65% other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of about 25% to about 60% other lipids (e.g., SORT lipids). In some, the lipid component comprises a molar percentage of about 30% to about 55% other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of about 20% to about 50% other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of about 30% to about 60% other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of about 25% to about 60% other lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of 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% additional lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises a molar percentage of at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at least (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65% additional lipids (e.g., SORT lipids). In some embodiments, the lipid component comprises additional lipids (e.g., SORT lipids) at a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or a range between (inclusive of) any two of the foregoing values.

[0350] Non-limiting illustrative LNP compositions are provided in Table 7. Table 7. Non-limiting illustrative LNP compositions F. Payload

[0351] Payload can encompass bioactive molecules, including small molecules, biomolecules, nucleic acids (e.g., DNA, RNA, siRNA, shRNA), proteins or peptides, which are part of LNP compositions. In LNP compositions, payload can be attached to, encapsulated in, connected to or combined with LNP via covalent or non-covalent bonds with LNP. In some embodiments, the LNP comprises a 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 an mRNA.

[0352] 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.

[0353] In some embodiments, the length of the polynucleotide is 2000 nucleotides to 5000 nucleotides. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides.

[0354] In some embodiments, the concentration of the polynucleotide is 0.5-3.0 mg / mL, 1.0-3.0 mg / mL, or 1.0 mg / mL of 2.0-3.0 mg / mL. In some embodiments, the concentration of the polynucleotide is 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 concentration of the polynucleotide is 1.0 mg / mL.

[0355] In some embodiments, the length of the polynucleotide is 2000 to 5000 nucleotides and the concentration is 1.0 mg / mL. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides and the concentration is 1.0 mg / mL. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides and the concentration is 1.0 mg / mL. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides and the concentration is 1.0 mg / mL. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides and the concentration is 1.0 mg / mL. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides and the concentration is 1.0 mg / mL.

[0356] In some embodiments, the length of the polynucleotide is 2000 to 5000 nucleotides and the concentration is 0.9 mg / mL. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides and the concentration is 0.9 mg / mL. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides and the concentration is 0.9 mg / mL. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides and the concentration is 0.9 mg / mL. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides and the concentration is 0.9 mg / mL. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides and the concentration is 0.9 mg / mL.

[0357] In some embodiments, the length of the polynucleotide is 2000 to 5000 nucleotides and the concentration is 0.8 mg / mL. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides and the concentration is 0.8 mg / mL. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides and the concentration is 0.8 mg / mL. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides and the concentration is 0.8 mg / mL. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides and the concentration is 0.8 mg / mL. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides and the concentration is 0.8 mg / mL.

[0358] In some embodiments, the length of the polynucleotide is 2000 to 5000 nucleotides and the concentration is 0.7 mg / mL. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides and the concentration is 0.7 mg / mL. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides and the concentration is 0.7 mg / mL. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides and the concentration is 0.7 mg / mL. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides and the concentration is 0.7 mg / mL. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides and the concentration is 0.7 mg / mL.

[0359] In some embodiments, the length of the polynucleotide is 2000 to 5000 nucleotides and the concentration is 0.6 mg / mL. In some embodiments, the length of the polynucleotide is 2500 to 5000 nucleotides and the concentration is 0.6 mg / mL. In some embodiments, the length of the polynucleotide is 3000 to 5000 nucleotides and the concentration is 0.6 mg / mL. In some embodiments, the length of the polynucleotide is 3500 to 5000 nucleotides and the concentration is 0.6 mg / mL. In some embodiments, the length of the polynucleotide is 4000 to 5000 nucleotides and the concentration is 0.6 mg / mL. In some embodiments, the length of the polynucleotide is 4500 to 5000 nucleotides and the concentration is 0.6 mg / mL.

[0360] In some embodiments, the mRNA encodes dynein axoneme intermediate chain 1 (DNAI1) protein. In other embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR).

[0361] In some embodiments, the mRNA comprises the polynucleotide sequence of SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is at least 99% identical to SEQ ID NO: 1. In some embodiments, the mRNA encoding the CFTR protein comprises a polynucleotide sequence that is identical to SEQ ID NO: 1.

[0362] In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is at least 99% identical to SEQ ID NO: 4. In some embodiments, the mRNA encoding the DNAI1 protein comprises a polynucleotide sequence that is identical to SEQ ID NO: 4.

[0363] In some embodiments, the average molecular weight of the payload is at most 20,000,000 Da. In some embodiments, the average molecular weight of the payload can be at most 2,000,000 Da. In some embodiments, the average molecular weight of the payload can be at most 150,000 Da. In other practices, the average molecular weight of the payload is at most 15,000 Da, 5,000 Da, or 1,000 Da.

[0364] In one aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising LNPs, wherein the LNPs comprise less than 25% or less than 20% 1,2-dioleoyl-3-dimethylammonium propane (DODAP) by mole, greater than 40% cholesterol by mole; and / or messenger RNA (mRNA) having a lipid:mRNA ratio of less than 40:1.

[0365] In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition comprising LNPs, wherein the LNPs specifically transduce secretory cells and / or ionocytes; and / or the LNPs deliver mRNA to lung cells in an amount effective to increase expression and / or function of a polypeptide or polynucleotide encoded by the mRNA.

[0366] In some embodiments, the LNP specifically transduces secretory cells and / or ionocytes; and / or wherein the LNP delivers mRNA to lung cells in an amount effective to increase expression and / or function of a polypeptide or polynucleotide encoded by the mRNA.

[0367] In some embodiments, the LNP comprises an ionizable cationic lipid; a neutral phospholipid; a polyethylene glycol (PEG)-lipid; and / or cholesterol. In some embodiments, the LNP comprises a second ionizable cationic lipid. In some embodiments, the LNP comprises an anionic lipid. In some embodiments, the LNP comprises a permanent cationic lipid.

[0368] In some embodiments, the LNP comprises less than 25% or less than 20% DODAP by mole. In some embodiments, the LNP comprises 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% DODAP by mole.

[0369] In some embodiments, the LNP comprises 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% DODAP.

[0370] In some embodiments, the LNP comprises a mole percentage of 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% DODAP.

[0371] In some embodiments, the LNP comprises 16 mol% DODAP.

[0372] In some embodiments, the LNP comprises greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% cholesterol by mole percentage.

[0373] In some embodiments, the LNP comprises 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% cholesterol by mole. In some embodiments, the LNP comprises 50% cholesterol by mole. Table 8. Exemplary formulations

[0374] In some embodiments, the LNP comprises messenger RNA (mRNA).

[0375] In some embodiments, the LNP comprises mRNA at a lipid:mRNA ratio of less than 40:1.

[0376] In some embodiments, 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.

[0377] In some embodiments, the lipid:mRNA ratio is 36:1.

[0378] In some embodiments, the lipid:mRNA ratio is 25:1.

[0379] In some embodiments, the ionizable cationic lipid is 5A2-SC8 or 4A3-SC7; the neutral phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and / or the polyethylene glycol (PEG)-lipid is DMG-PEG, optionally DMG-PEG 2000. In some embodiments, the ionizable cationic lipid is 4A3-SC7; the neutral phospholipid is DOPE; and the polyethylene glycol (PEG)-lipid is DMG-PEG.

[0380] In some embodiments, the LNP comprises a second cationic lipid, and the second cationic lipid is DODAP.

[0381] In some embodiments, the LNP comprises a second cationic lipid, and the second cationic lipid is 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA).

[0382] In some embodiments, the ionizable cationic lipid is 4A3-SC7 and the LNP comprises a molar percentage of 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% 4A3-SC7.

[0383] In some embodiments, the LNP comprises a molar percentage of 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% PEG-lipid.

[0384] In some embodiments, the molar percentage of PEG-lipid is (about) 3%.

[0385] In some embodiments, the LNP comprises a neutral phospholipid and the neutral phospholipid is DOPE.

[0386] In some embodiments, the LNP comprises 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% DOPE by mole percentage.

[0387] In some embodiments, the LNP comprises 11% or 22% DOPE by mole.

[0388] In some embodiments, the LNP comprises a payload.

[0389] In some embodiments, the payload is messenger RNA (mRNA).

[0390] In some embodiments, the mRNA comprises between 100 bases and 8 kilobases (kb).

[0391] In some embodiments, the mRNA comprises between 11 kb and 8 kb, between 2 kb and 8 kb, between 3 kb and 8 kb, between 4 kb and 8 kb, between 5 kb and 8 kb, between 6 kb and 8 kb, between 7 kb and 8 kb, between 1 kb and 7 kb, between 2 kb and 7 kb, between 3 kb and 7 kb, between 4 kb and 7 kb, between 5 kb and 7 kb, or between 6 kb and 7 kb, between 1 kb and 6 kb, between 2 kb and 6 kb, between 3 kb and 6 kb, between 4 kb and 6 kb, or between 5 kb and 6 kb.

[0392] In some embodiments, the mRNA comprises (approximately) 2 kb.

[0393] In some embodiments, the mRNA comprises (approximately) 4.6 kb.

[0394] In some embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0395] In some embodiments, the mRNA encodes dynein axonemal intermediate chain 1 (DNAI1) protein.

[0396] In some embodiments, the mRNA encodes a gene editing system or a component thereof.

[0397] In some embodiments, the mRNA encodes a shRNA or a microRNA.

[0398] In some embodiments, the LNP composition is a pharmaceutical composition.

[0399] In some embodiments, the LNP composition is an aerosolized composition.

[0400] In some embodiments, the encapsulation efficiency of the LNP composition is 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%.

[0401] In some embodiments, the LNP is a composition that is substantially free of any anionic lipid, any permanent cationic lipid, or any anionic lipid and any permanent cationic lipid. In some embodiments, the LNP is a composition that is substantially free of any ionizable cationic lipid. 1. Gene Editing Payload

[0402] The LNP of the present disclosure can include one or more components for gene editing, such as, but not limited to, guide RNA, tracr RNA, sgRNA, mRNA encoding a gene or base editing protein, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) nuclease (e.g., Cas9), DNA template for gene editing, or a combination thereof. In some embodiments, the payload of LNP is applicable to genome editing technology. In some embodiments, genome editing technology can be CRISPR or TALEN. In some embodiments, LNP can include one or more mRNAs that can encode gene editing proteins or base editing proteins. In some embodiments, LNP can include both mRNA encoding gene editing proteins or base editing proteins and one or more guide RNAs. In some embodiments, LNP can include at least one nucleic acid suitable for genome editing technology, such as CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), guide RNA (gRNA) and DNA repair template. In some embodiments, the CRISPR nuclease can have an altered activity, for example, modifying the nuclease so that it can be a nickase instead of performing double-stranded cleavage, or so that it can bind to a sequence specified by a guide RNA but has no enzymatic activity. In some embodiments, the base editing protein can be a fusion protein comprising a deaminase domain and a sequence-specific DNA binding domain, such as an inactive CRISPR nuclease. (a) Gene editing methods

[0403] The LNP or pharmaceutical composition of the present invention can contain any payload of conventional gene editing methods. In some embodiments, the gene editing component can be selectively delivered to cells of the target organ. In some embodiments, the target organ can be the lung. In some embodiments, the cells of the target organ can be pneumocytes. In some embodiments, the cells can be ciliated cells, goblet cells, secretory cells, club cells, basal cells, or ionocytes.

[0404] In some embodiments, gene editing can be targeted editing. Targeted editing can be achieved by nuclease-independent methods or by nuclease-dependent methods.

[0405] Nuclease-independent targeted editing (such as base editing and / or prime editing) can involve precise modification of DNA sequences without generating double-strand breaks. Homologous recombination can be directed by homologous sequences flanking exogenous polynucleotides, which are introduced into endogenous sequences by enzymatic mechanisms in target organ cells.

[0406] Base editing can allow one DNA base pair to be converted into another at a specific target site. In some embodiments, the nuclease can be a fusion of a deaminase and a modified Cas9 protein (dCas9) or other engineered Cas variants. In some embodiments, base editing can change the C (cytosine) in endogenous DNA to T (thymine) or A (adenine) to G (guanine). Guide RNA can be designed to target the specific genomic location of the target organ cell.

[0407] Lead editing can allow more complex and accurate DNA modifications, including insertions, deletions, and all 12 possible base conversions (A, C, G, T) without double-strand breaks. Lead editing guide RNA can be designed, which can be composed of a guide sequence and a template for the desired editing. The lead editor protein (PE2) that can combine reverse transcriptase and Cas9 variants can be guided to the target site by the lead editing guide RNA. Cas9 variants can produce single-strand breaks (nicks) in DNA. Then, reverse transcriptase can use the template sequence of the lead editing guide RNA to copy the desired changes into the nicked chain of DNA. Subsequently, the cellular repair mechanism of the cells of the target organ can repair the nick via homology-directed repair (HDR), thereby incorporating the edited sequence.

[0408] Nuclease-dependent methods can achieve targeted editing with a higher frequency by specifically introducing double-strand breaks (DSBs) with specific rare cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing can also utilize DNA repair mechanisms that can be performed in response to DSBs, such as non-homologous end joining (NHEJ). In some embodiments, the DNA repair performed by NHEJ can result in random insertions or deletions (insertion deletions (indels)) of a small amount of endogenous nucleotides. Compared to the repair mediated by NHEJ, repair can also be performed by homology-directed repair (HDR). When there is a donor template containing exogenous genetic material flanking a pair of homology arms, exogenous genetic material can be introduced into the genome by HDR, which can result in targeted integration of exogenous genetic material. In some embodiments, the nuclease of nuclease-dependent targeted editing can include but is not limited to CRISPR-Cas9, CRISPR-Cas12 (Cpf1), CRISPR-Cas13, C2c2, C2c6, NgAgo and / or TALEN.

[0409] Methods for producing genomic deletions (e.g., knocking out genes in cells) using CRISPR-Cas gene editing technology are well-known techniques. See, for example, Bauer et al., J Vis Exp. 95: e52118 (2015). Available nucleases capable of introducing specifically targeted DSBs may include, but are not limited to, ZFNs, TALENs, and CRISPR / Cas9.

[0410] In some embodiments, targeted gene editing can be achieved via the dual integrase cassette exchange (DICE) system utilizing phiC31 and Bxb1 integrases.

[0411] The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has been reused as a platform for the RNA-guided targeted DNA of gene editing. It can rely on the DNA nuclease Cas9 and two non-coding RNAs (crisprRNA (crRNA) and trans-activating RNA (tracrRNA)) to target DNA cutting. CRISPR is a family of DNA sequences found in the genomes of bacteria and archaea that contains DNA fragments (spacer DNA) similar to foreign DNA previously exposed to cells (e.g., by viruses that have infected or attacked the prokaryotes). These DNA fragments can be used by prokaryotes to detect and destroy similar foreign DNA (e.g., from similar viruses during subsequent attacks) when reintroduced. Transcription of the CRISPR locus can result in the formation of an RNA molecule comprising a spacer sequence, which can associate and target the Cas (CRISPR-associated) protein that can recognize and cut the foreign exogenous DNA. Many types and classes of CRISPR / Cas systems have been described, for example, in Koonin et al., Curr Opin Microbiol 37:67-78 (2017).

[0412] crRNA can drive the sequence recognition and specificity of CRISPR-Cas9 complex by Watson-Crick base pairing with about 20 nucleotide sequences in target DNA. Changing the sequence of the 20 nucleotides at 5' in crRNA can allow the CRISPR-Cas9 complex to target a specific locus. The CRISPR-Cas9 complex can only bind to DNA sequences containing sequences that match the first 20 nucleotides of crRNA if the target sequence is followed by a specific short DNA motif (having the sequence NGG) called a protospacer adjacent motif (PAM).

[0413] The tracrRNA can hybridize to the 3' end of the crRNA to form an RNA-duplex structure, which can be bound by the Cas9 endonuclease to form a catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0414] Once the CRISPR-Cas9 complex binds to the DNA at the target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) in which both strands of the DNA terminate with base pairs (blunt end).

[0415] After the CRISPR-Cas9 complex binds to the DNA at a specific target site and forms a site-specific DSB, the cell can use the following two major DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ is a repair mechanism that is highly active in most cell types, including non-dividing cells. NHEJ can be error-prone and can typically result in the removal or addition of between one and hundreds of nucleotides at the DSB site, but such modifications can typically be less than 20 nucleotides. The resulting insertions and deletions (indels) may damage the coding or non-coding regions of the gene. Alternatively, HDR can use a long segment of homologous donor DNA provided endogenously or exogenously to repair DSBs with high fidelity. HDR is only active in dividing cells and can occur at a relatively low frequency in most cell types. CRISPR endonucleases

[0416] In some embodiments, the Cas9 endonuclease can be used in the CRISPR method to genetically engineer cells of the target organ of the LNP described herein. In some embodiments, the Cas9 enzyme can be from Streptococcus pyogenes (Streptococcus pyogenes), but other Cas9 homologs can also be used. In some embodiments, the Cas9 enzyme can be wild-type Cas9. In some embodiments, the Cas9 enzyme can be a modified form of Cas9 (e.g., an evolved form of Cas9 or a Cas9 ortholog or variant). In some embodiments, Cas9 can be replaced by another RNA-guided endonuclease (such as Cpf1) (Class II CRISPR / Cas system).

[0417] In some embodiments, the CRISPR / Cas system can include components derived from a type I, type II, or type III system. In some embodiments, the CRISPR / Cas system can include components derived from class 1 and class 2 CRISPR / Cas systems (types I to V or type II, type V, and type VI, respectively) (Makarova et al., Nat Rev Microbiol 13(11):722-36 (2015); Shmakov et al., Mol Cell 60:385-397 (2015)).

[0418] Class 2 CRISPR / Cas systems can have a single protein effector. 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 can include, for example, but are not limited to, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. The Cpf1 nuclease is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0419] In some embodiments, the Cas nuclease can be from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease can be from a class 2 CRISPR / Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity, and they can be guided to cut a desired nucleic acid target by designing appropriate guide RNAs, as further explained below.

[0420] In some embodiments, the Cas nuclease may include more than one nuclease domain. In some embodiments, the Cas9 nuclease may include 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 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 so that one of the nuclease domains may mutate or be completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease may be modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease may be modified to contain no functional HNH-like nuclease domain. In some embodiments, only one nuclease domain may be functional, and the Cas9 nuclease may be a nickase capable of introducing a single-strand break (nick) into the target sequence. In some embodiments, the conserved amino acids within the Cas9 nuclease domain may be substituted to reduce or change nuclease activity. In some embodiments, the Cas nuclease nickase may include an amino acid replacement in a RuvC-like nuclease domain. Exemplary amino acid replacements in a RuvC-like nuclease domain may include D10A (based on Streptococcus pyogenes (S. pyogenes) Cas9 nuclease). In some embodiments, the nickase may include an amino acid replacement in a HNH-like nuclease domain. Exemplary amino acid replacements in an HNH-like nuclease domain may include, but are not limited to, E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes Cas9 nuclease).

[0421] In some embodiments, the Cas nuclease may be 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.

[0422] Type I CRISPR / Cas systems can utilize a large effector complex called Cascade (CRISPR-associated complex for antiviral defense) for target binding and interference. The Cascade complex can contain multiple Cas proteins, including Cas3, which can be responsible for the destruction of target DNA. Type II CRISPR / Cas systems (particularly CRISPR-Cas9 systems) can utilize a single Cas9 protein guided by a synthetic guide RNA (sgRNA) to introduce double-strand breaks in the target DNA for subsequent repair or modification. Type III CRISPR / Cas systems can utilize Csm (CRISPR-Cas subtype polyprotein) or Cmr (CRISPR-Cas subtype ribonucleoprotein) complexes for interference. In addition to DNA, type III CRISPR / Cas systems can also target RNA molecules. Type V CRISPR / Cas systems (including Cpf1 (also known as Cas12) and C2c2 (also known as Cas13)) can 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 useful for RNA editing and manipulation. Guide RNA (gRNA)

[0423] CRISPR technology can involve the use of nucleic acids targeting the genome, which can guide one or more nucleases to a specific target sequence in a target gene for gene editing at a specific target sequence. The nucleic acid targeting the genome can be RNA. The RNA targeting the genome is referred to herein as "guide RNA" or "gRNA". The guide RNA can include at least one spacer sequence and CRISPR repeats, which can hybridize with the target nucleic acid sequence for editing in the target gene.

[0424] In the type II system, the gRNA may further comprise a second RNA, called a tracrRNA sequence. In the type II gRNA, the CRISPR repeat sequence and the tracrRNA sequence may hybridize to form a duplex. In the type V gRNA, the crRNA may form a duplex. In both systems, the duplex may bind to the site-directed polypeptide, allowing the guide RNA and the site-directed polypeptide to form a complex. In some embodiments, the nucleic acid targeting the genome may provide target specificity for the complex by virtue of its association with the site-directed polypeptide. Thus, the nucleic acid targeting the genome may guide the activity of the site-directed polypeptide.

[0425] As will be appreciated by those of ordinary skill in the art, each guide RNA can be designed to include 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).

[0426] In some embodiments, the nucleic acid (e.g., gRNA) of the targeting genome can be a double-stranded guide RNA comprising two RNA molecule chains. The first chain can include an optional spacer extension sequence, a spacer sequence, and a minimum CRISPR repeat sequence in the 5' to 3' direction. The second chain can include a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0427] In some embodiments, the nucleic acid (e.g., gRNA) of the targeting genome can be a single molecule guide RNA (sgRNA). The sgRNA in the II type system can include an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single molecule guide joint, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension can include an element that can contribute other functionality (e.g., stability) to the guide RNA. The single molecule guide joint can connect the minimum CRISPR repeat sequence and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can include one or more hairpins. The single molecule guide RNA in the V type system can include a minimum CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction.

[0428] The spacer sequence in gRNA is a sequence (e.g., a sequence of 20 nucleotides) that can define a target sequence (e.g., a DNA target sequence, such as a genomic target sequence) of a target gene of interest (e.g., DNAI1 or CFTR). In some embodiments, the range of the spacer sequence can be 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.

[0429] The target sequence is located in the target gene (e.g., DNAI1 or CFTR), and the target sequence can be adjacent to the PAM sequence and can be a sequence to be modified by the nuclease (e.g., Cas9) guided by RNA. The target sequence is located on the PAM chain in the target nucleic acid, and the target nucleic acid is a double-stranded molecule containing a PAM chain and a complementary non-PAM chain. Those skilled in the art will recognize that the gRNA spacer sequence can hybridize with the complementary sequence in the non-PAM chain of the target nucleic acid of interest. Therefore, the gRNA spacer sequence can be the RNA equivalent of the target sequence. The spacer of gRNA can interact with the target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the spacer can change according to the target sequence of the target nucleic acid of interest.

[0430] In the CRISPR / Cas system, a spacer sequence can be designed to hybridize to a region in the target nucleic acid located 5' of a PAM that can be recognized by the Cas9 enzyme used in the system. The spacer can completely match the target sequence or can have mismatches. 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 in a target nucleic acid that contains the sequence 5'-NRG-3', where R can contain A or G, where N can be any nucleotide, and where N can be immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0431] In some embodiments, the length of the target nucleic acid sequence can be about 20 nucleotides. In some embodiments, the length of the target nucleic acid can be less than about 20 nucleotides. In some embodiments, the length of the target nucleic acid can be greater than about 20 nucleotides. In some embodiments, the length of the target nucleic acid can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the length of the target nucleic acid can be at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. In some embodiments, the target nucleic acid sequence can have 20 bases 5' of the first nucleotide immediately adjacent to the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid can be a sequence corresponding to the multiple Ns, wherein N can be any nucleotide, and the underlined NRG sequence can be a Streptococcus pyogenes PAM.

[0432] The guide RNA can target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can be 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene can contain up to 10 mismatches, such as up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 mismatch.

[0433] The length of the spacer sequence in gRNA can depend on the CRISPR / Cas9 system and component for editing any target gene (for example, DNAI1 or CFTR).For example, different Cas9 proteins from different bacterial species can have different optimal spacer sequence lengths.Therefore, the length of the spacer sequence 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 length of the spacer sequence can be 18-24 nucleotides.In some embodiments, the length of the targeting sequence can be 19-21 nucleotides.In some embodiments, the length of the spacer sequence can include 20 nucleotides.

[0434] In some embodiments, the gRNA can be an sgRNA that can include a 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA can include a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA can include a spacer sequence of more than 20 nucleotides at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA can include a spacer sequence of variable length of about 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0435] In some embodiments, gRNA can comprise unmodified ribonucleic acid. In some embodiments, gRNA can comprise modified ribonucleic acid. Various types of RNA modifications can be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, for example, the modification of stability, the possibility or degree of reducing innate immune response and / or enhancing other attributes can be enhanced, as described in the art. In some embodiments, non-natural modified core bases can be introduced into any gRNA during or after synthesis. In some embodiments, modification can be located between nucleosides, purine or pyrimidine bases or sugars. In some embodiments, modification can be introduced at the end of gRNA by chemical synthesis or by polymerase.

[0436] In some embodiments, more than one guide RNA can be used with the CRISPR / Cas nuclease system. Each guide RNA can contain a different targeting sequence so that the CRISPR / Cas system can cut more than one target nucleic acid. In some embodiments, one or more guide RNAs can have the same or different properties, such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA can be used, each guide RNA can be encoded on the same or different vectors. The promoters used to drive the expression of more than one guide RNA can be the same or different.

[0437] In some embodiments, enzymatic or chemical ligation methods can be used to conjugate a polynucleotide or region thereof to various functional moieties (eg, targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.).

[0438] In some embodiments, the CRISPR / Cas nuclease system can contain multiple gRNAs, such as 2, 3 or 4 gRNAs. Such multiple gRNAs can target different sites in the same target gene. Alternatively, multiple gRNAs can target different genes. In some embodiments, one or more guide RNAs and Cas proteins can form ribonucleoproteins (RNPs), such as CRISPR / Cas complexes. Guide RNA can guide Cas proteins to one or more target sequences on one or more target genes (e.g., DNAI1 and CFTR), where Cas proteins can cut the target gene at the target site. In some embodiments, the CRISPR / Cas complex can be a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex can be a type II CRISPR / Cas9 complex. In some embodiments, the Cas protein can be a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex can be a Cas9 / guide RNA complex.

[0439] In some embodiments, the insertion and deletion frequency (editing frequency) of a specific CRISPR / Cas nuclease system comprising one or more specific gRNAs can be determined using TIDE analysis, which can be used to identify efficient gRNA molecules for editing target genes. In some embodiments, an efficient gRNA can produce a gene editing frequency greater than 80%. For example, if a gRNA can produce a gene editing frequency of at least 80%, at least 85%, at least 90%, at least 95%, or 100%, the gRNA can be considered efficient.

[0440] In addition to the CRISPR system disclosed herein, other gene editing systems known in the art can also be used as the payload of the LNP described herein. In some embodiments, the other gene editing system can include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), restriction endonucleases, mega nucleases, homing endonucleases, etc.

[0441] ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain (ZFBD), which can be a polypeptide domain that can bind to DNA in a sequence-specific manner via one or more zinc fingers. A zinc finger can be a domain of about 30 amino acids within a zinc finger binding domain, the structure of which can be stabilized by the coordination of zinc ions. Examples of zinc fingers include, but are not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. The designed zinc finger domain can be a domain that does not exist in nature, the design / composition of which is primarily derived from rational criteria, such as substitution rules and the application of computerized algorithms for processing information in databases storing existing ZFP design information and binding data. The selected zinc finger domain can be a domain that is not found in nature, and the generation of the domain may be primarily derived from empirical processes such as phage display, interaction traps, or hybrid selection. In some embodiments, a ZFN can be a fusion of a FokI nuclease and a zinc finger DNA binding domain.

[0442] 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 that is responsible for the binding of the TAL effector protein to DNA. TAL effector proteins can be secreted by plant pathogens of the genus Xanthomonas during infection. These proteins can 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. TAL effector DNA binding domain specificity can depend on the effector-variable number of an incomplete 34 amino acid repeat sequence that can contain polymorphisms at selected repeat positions called repeat variable diresidues (RVDs). In some embodiments, TALEN can be a fusion polypeptide of a FokI nuclease and a TAL effector DNA binding domain.

[0443] Additional examples of targeted nucleases suitable for use may 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 mycobacteriophage Bxb1. Bxb1 integrase can catalyze site-specific recombination between two specific DNA sequences, called attachment (att) sites. Bxb1 integrase can recognize a specific 48 base pair sequence within the attachment site. phiC31 nuclease (also known as phiC31 integrase) is derived from the bacteriophage phiC31. phiC31 nuclease can catalyze site-specific recombination between two specific DNA sequences, called attB (attachment site in phage) and attP (attachment site in phage). phiC31 nuclease can promote the integration of DNA fragments flanked by attB and attP into the genome in the cells of the target organ. phiBT1 nuclease can be integrated into an attachment site different from phiC31. Wβ / SPBc / TP901-1 nuclease (also known as bacteriophage P2 Bxb1 Cre nuclease) is a site-specific recombinase derived from the temperate bacteriophage P2. G. Physical properties and characteristics of LNP

[0444] The present disclosure relates in part to aerosolized pharmaceutical compositions of aerosol particles having lipid nanoparticles (LNPs). In some embodiments, the LNPs can be delivered to the tracheobronchial region of a subject. In some embodiments, the LNPs have one or more of the following: an encapsulation efficiency (EE) greater than 50%; an mRNA integrity greater than 50%; a diameter of 20 nm to 600 nm; and a polydispersity less than 0.6. 1. Encapsulation efficiency

[0445] Encapsulation efficiency (EE) refers to the fraction of the payload that is encapsulated in the lipid nanoparticle composition or otherwise incorporated into the lipid nanoparticle composition when forming LNP. Encapsulation efficiency can be determined by comparing the amount of the input payload with the amount of the payload encapsulated in the LNP sample, or by comparing the amount of the payload in the LNP with the free excess payload that is not encapsulated in the sample. In some embodiments, the free RNA in the sample is measured, and the complete LNP with total RNA in the sample treated to destroy the LNP is measured using fluorescence detection assays (e.g., RiboGreen TM ) to determine the encapsulation efficiency.

[0446] Encapsulation refers to the process by which payload is limited to LNP. In some embodiments, encapsulation refers to that one or more mRNA molecules are limited to LNP. In some embodiments, payload can be captured in the lipid portion of LNP or in the aqueous space encapsulated by some or all of the lipid components of LNP. In some embodiments, payload can be wrapped in lipid monolayer or double layer encapsulation. In some embodiments, payload can be embedded between lipid components. In some embodiments, payload can be wrapped in the aqueous core of LNP.

[0447] In some embodiments, the LNP is formed with an average encapsulation efficiency in the range of about 50% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency in the range of about 50% to about 70%. In some embodiments, the LNP is formed with an average encapsulation efficiency in the range of about 70% to about 90%. In some embodiments, the LNP is formed with an average encapsulation efficiency in the range of about 90% to about 100%. In some embodiments, the LNP is formed with an average encapsulation efficiency in the range of about 75% to about 95%.

[0448] In various embodiments, the encapsulation efficiency of the LNP is 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 EE of the LNP is 50%-95%, 60%-95%, 70%-95%, 80%-95%, or 90%-95%. In some embodiments, the EE of the LNP is 91%, 92%, 93%, 94% or 95%. In some embodiments, the EE of the LNP is 95%.

[0449] In various embodiments, present disclosure provides a kind of compositions, described compositions has the polynucleotide (for example, mRNA molecule) that is mixed in LNP as herein described.In some embodiments, mRNA molecule is mixed in most of LNP.In some embodiments, greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% mRNA molecule is mixed in LNP.In some embodiments, at least 70% total mRNA molecule of coding CFTR is mixed in compositions.In various embodiments, at least 70% total mRNA molecule of coding DNAI1 is mixed in LNP compositions. 2. mRNA integrity

[0450] mRNA integrity can refer to the quality of the mRNA. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after, for example, a purification process. In some embodiments, mRNA integrity refers to the quality of the mRNA within the LNP after nebulization.

[0451] mRNA integrity can be determined by any method well known in the art, for example, by 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 length are consistent with an analytical reference standard. Additional methods for assessing RNA integrity include, for example, assessment of purified mRNA using capillary gel electrophoresis (CGE).

[0452] In some embodiments, mRNA integrity is measured via multiplex capillary electrophoresis (CE), e.g., using an Agilent Advanced Analytical 5200 fragment analyzer. Electropherograms can be analyzed, e.g., using ProSize data analysis software, to quantify mRNA transcript integrity using smear analysis.

[0453] In some embodiments, 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.

[0454] In various embodiments, the mRNA integrity of the LNPs described herein is greater than 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In other embodiments, the mRNA integrity of the LNPs is 75%-99%, 80%-95%, 85%-90%, or 90%-95%. 3.LNP diameter

[0455] In various embodiments, the diameter of the LNP is 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 diameter of the LNP is about 80 to 150 nm. In some embodiments, the diameter of the LNP is about 20 to 600 nm, about 40 to 600 nm, about 60 to 600 nm, about 80 to 600 nm, about 100 to 600 nm, or 150 to 600 nm, or 200 to 600 nm, or 250 to 600 nm, or 300 to 600 nm, or 350 to 600 nm.

[0456] In various embodiments, the diameter of the LNP is 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 diameter of the LNP is about 80 to 150 nm. In some embodiments, the diameter of the LNP is about 20 to 400 nm, about 40 to 400 nm, about 60 to 400 nm, about 80 to 400 nm, about 100 to 400 nm, or 150 to 400 nm, or 200 to 400 nm, or 250 to 400 nm, or 300 to 400 nm, or 350 to 400 nm.

[0457] In some embodiments, the LNPs have 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 LNPs have a diameter of 50-90 nm.

[0458] 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 about 70 nm to about 200 nm, and more typically the average size is about 100 nm or less.

[0459] In some embodiments, the diameter of the LNPs is 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 average size of the LNPs is about 150 nm or less, for example, between 75 nm and 150 nm, more particularly, between 100 nm and 150 nm.

[0460] In various embodiments, after aerosolization, the LNPs have a diameter of 20 to 180 nm, 30 to 180 nm, 40 to 180 nm, 50 to 180 nm, 60 to 180 nm, 70 to 180 nm, 80 to 180 nm, 90 to 180 nm, 100 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.

[0461] In some embodiments, the LNP has a diameter of 100 to 400 nm, 120 to 400 nm, 140 to 400 nm, 160 to 400 nm, 180 to 400 nm, 200 to 400 nm, 220 to 400 nm, 240 to 400 nm, 260 to 400 nm, 280 to 400 nm, 300 to 400 nm, 320 to 400 nm, 340 to 400 nm, 360 to 400, or 380 to 400 nm.

[0462] In various embodiments, the diameter of the LNP is determined by various techniques known in the art including, but not limited to, dynamic light scattering (DLS). 4. Polydispersity Index (PDI)

[0463] In some embodiments, the LNPs are characterized by a polydispersity (or polydispersity index) of less than 0.5. In various embodiments, the polydispersity of the LNPs is 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 generally used as an indicator of the quality of the LNPs with respect to size distribution.

[0464] In various embodiments, dynamic light scattering (DLS) can be used to characterize the polydispersity index and size of the LNPs of the present disclosure. DLS measures the light scattering produced by placing a sample under a light source. The PDI (as determined from the DLS measurements) represents the distribution of LNP sizes in a population (at or near the average LNP diameter), and a completely uniform LNP population would have a PDI of zero.

[0465] In various embodiments, the polydispersity of the LNPs is 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 polydispersity of the LNPs is 0.5. In some embodiments, the polydispersity of the LNPs is 0.4. In some embodiments, the polydispersity of the LNPs is 0.3. In some embodiments, the polydispersity of the LNPs is 0.2. In some embodiments, the polydispersity of the LNPs is 0.1. H. Physical Properties and Characteristics of Aerosol Particles

[0466] The present disclosure relates in part to aerosolized pharmaceutical compositions comprising aerosol particles of lipid nanoparticles (LNPs). In some embodiments, the LNPs can be delivered to the tracheobronchial region of a subject. In some embodiments, the aerosol particles of the present disclosure have one or more of the following characteristics: a mass median 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%. 1. Mass Median Aerodynamic Diameter (MMAD)

[0467] The size distribution of aerosol particles in an aerosol can be measured by determining its mass median aerodynamic diameter (MMAD). The MMAD of an aerosol is the median of the diameters of the aerosol particles in the aerosol. In some embodiments, MMAD is measured using a next generation impactor (NGI). As described herein, when administered to a subject, the MMAD of an aerosol can affect the position at which the aerosol is deposited in the lungs.

[0468] Without wishing to be bound by theory, aerosols with a mass median aerodynamic diameter (MMAD) of 5-10 μm may be deposited primarily in the large conducting airways and oropharyngeal region. Aerosol particles with an MMAD range of 1-5 μm may be deposited primarily in the small lung airways and alveoli, while more than 50% of aerosol particles with an MMAD of 3 μm may be deposited in the alveolar region. In the case of systemic drug delivery using the pulmonary route, aerosols with a small average particle size are desired to ensure peripheral penetration of the drug.

[0469] In some embodiments, the calculation of MMAD complies with USP No. <601> In some embodiments, the diameter of the aerosol particles is such that 50% by mass are larger and 50% are smaller. In some embodiments, the intercept of a line drawn on a lognormal distribution graph and the line are determined by using the entire distribution more concentrated on points having the majority of mass.

[0470] During atomization, the aerosolized pharmaceutical composition comprises aerosol particles of the pharmaceutical composition. Aerosol particles refer to particles of a solution (or solid) that has been atomized. Each aerosol particle can comprise a certain amount of LNP suspended in the solution (or solid). The size of the LNP is significantly smaller than the size of the aerosol particles. The aerosolized pharmaceutical composition can be characterized by multiple parameters, including the particle size (e.g., diameter) of the aerosol, for example, by measuring the mass median aerodynamic diameter or the fine particle fraction associated with the aerosol particles of the aerosolized pharmaceutical composition. MMAD can be determined by impactor measurement results (e.g., Anderson cascade impactor (ACI) or next generation impactor (NGI)).

[0471] In some embodiments, the MMAD of the aerosol particles is 1 μm to 9 μm, 1 μm to 8 μm, 1 μm to 7 μm, 1 μm to 6 μm, 1 μm to 5 μm, 1 μm to 4 μm, 1 μm to 3 μm, 1 μm to 2 μm, or 3 μm to 5 μm. In various embodiments, the MMAD of the aerosol particles after atomization is 1 μm to 9 μm, or 1 μm to 8 μm, or 1 μm to 7 μm, or 1 μm to 6 μm, or 1 μm to 5 μm, or 1 μm to 4 μm, or 1 μm to 3 μm, or 1 μm to 2 μm, or 3 μm to 5 μm. 2. Geometric Standard Deviation (GSD)

[0472] The uniformity of the particle size distribution of an aerosol (such as the aerosolized pharmaceutical composition of the present disclosure) can be quantified as the geometric standard deviation (GSD) of the particle size of the aerosol particles. GSD is a measure of the variability of the diameter of the aerosol particles. The GSD of an aerosol can be calculated as the square root of the ratio of the droplet size observed at the 84th percentile divided by the droplet size observed at the 16th percentile based on the cumulative percent mass undersized distribution. A low GSD reflects a narrow droplet size distribution (i.e., droplets of uniform size), which may be advantageous for targeting the aerosol to the respiratory system.

[0473] Monodispersity and polydispersity relate to the uniformity of the particle size distribution of an aerosol. The lower the GSD of an aerosol, the more monodisperse the particle size distribution. Similarly, the higher the GSD of an aerosol, the more polydisperse the particle size distribution. For example, a monodisperse particle size distribution typically includes aerosols with a GSD of about 2 or less, and a polydisperse particle size distribution typically includes aerosols with a GSD of about 3 or greater.

[0474] In various embodiments, the GSD of the aerosol particles is 1 to 4, 1 to 3, or 1 to 2, or 1, 1.5, 2, 2.5, or 3. In various embodiments, the GSD of the aerosol particles after atomization is 1 to 4, or 1 to 3, or 1 to 2, or 1, 1.5, 2, 2.5, or 3.

[0475] In other embodiments, GSD is determined by the formula provided below:

[0476] In some embodiments, D 16 (Probit = -1) is the diameter at which 84% of the particles by mass have a larger diameter and 16% have a smaller diameter. In some embodiments, D 84 (Probit=1) is the diameter at which 16% of the particles by mass are larger and 84% are smaller. A line drawn on a lognormal distribution graph and its intercept are determined using the entire distribution on both sides of Probit=1.

[0477] The mean droplet size of the aerosolized pharmaceutical compositions provided herein can be less than about 5 μm or about 1 μm to about 5 μm. The GSD of the aerosolized pharmaceutical compositions can be in the range of 1.0 to 2.2, or about 1.0 to about 2.2, or 1.5 to 2.2, or about 1.5 to about 2.2. 3. Fine particle fraction

[0478] The fine particle fraction (FPF) represents the mass percentage of aerosol particles with an aerodynamic diameter below 5 μm and is used for in vitro assessment of the aerodynamic properties of an aerosol. In some embodiments, FPF represents the mass percentage of LNPs with an aerodynamic diameter below 5 μm. In some embodiments, FPF is used for in vitro assessment of the aerodynamic properties of an aerosol.

[0479] In some embodiments, the fine particle dose (FPD) is determined as the total mass of drug (e.g., polynucleotide payload) with a size <5.0 μm, determined by measuring the amount of drug (e.g., polynucleotide payload) collected on all fractions with an equivalent circular diameter (ECD) <5.0 μm.

[0480] In various embodiments, the fine particle fraction of the aerosol particles of the present disclosure is 55%, 60%, 70%, 75%, 80%, 85% or 90%. In various embodiments, the fine particle fraction of the aerosol particles of the present disclosure after atomization is 55%, 60%, 70%, 75%, 80%, 85% or 90%. III. Methods of the Disclosure

[0481] In one aspect, the present disclosure provides a method for treating a lung disease or lung disorder in a subject, the method comprising administering an aerosolized pharmaceutical composition as described herein. Among other things, the present disclosure provides methods and compositions for treating cystic fibrosis, comprising administering to a subject an aerosolized LNP composition comprising mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In other embodiments, the present disclosure provides methods and compositions for treating primary ciliary dyskinesia (PCD), comprising administering to a subject an aerosolized LNP composition comprising mRNA encoding a dynein axoneme intermediate chain 1 (DNAI1) protein.

[0482] Cystic fibrosis (also known as mucoviscidosis) 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 conductance regulator (CFTR) protein. This protein acts as a channel for transporting chloride ions across cell membranes and is essential for regulating components of mucus, sweat, saliva, tears, and digestive enzymes. Pathogenic mutations in the CFTR protein cause dysfunction of its channel activity, leading to abnormal transport of chloride and sodium ions across the epithelium, resulting in the production of 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 increased levels of sulfated mucins, inflammation, and ultimately fatal recurrent infections; the median predicted survival in the United States is 40.7 years. Cystic fibrosis is the most common fatal genetic disease in the white population.

[0483] The lungs of individuals with CF become colonized and infected with bacteria from an early age. This leads to chronic airway infection and inflammation, progressing to bronchiectasis, air trapping, hypoxemia, and hypercapnia. In the United States, pulmonary valve regurgitation causes 68.1% of CF-related deaths. Initially, common bacteria such as Staphylococcus aureus and Haemophilus influenzae colonize and infect the lungs. Eventually, Pseudomonas aeruginosa (and sometimes Burkholderia cepacia) become dominant. By age 18, 80% of patients with classic CF have P. aeruginosa, and 3.5% have B. cepacia. Once in the lungs, these bacteria adapt and develop resistance to commonly used antibiotics.

[0484] Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder characterized by abnormal cilia and flagella found in the lining of the airways, reproductive system, and other organs and tissues. PCD occurs in approximately 1 in 16,000 people. Symptoms are present as early as birth, with breathing problems, and affected individuals suffer from frequent upper respiratory infections starting in early childhood. People with PCD also suffer from year-round nasal congestion and a chronic cough. Chronic respiratory infections may lead to a condition called bronchiectasis, which damages the passages called bronchi and can cause life-threatening breathing problems. Some individuals with PCD also suffer from infertility, recurrent ear infections, and abnormal positioning of their organs within their chest and abdomen.

[0485] Among the several genes directly implicated in the pathogenesis of PCD, a significant number of mutations have been found in two genes: DNAI1 and DNAH5, which encode the intermediate and heavy chains of axonemal dynein, respectively. Mutations have also been reported in other genes encoding proteins involved in axonemal ultrastructure (DNAH11, DNAI2, TXNDC3, RSPH9, RSPH4A) or assembly (KTU, CRRC50), as well as in RPGR genes in some cases of PCD. It is estimated that mutations in DNAI1 and DNAH5 (both of which are associated with a phenotype of defects in the outer dynein arm (ODA) of the cilia) together account for nearly 40% of PCD cases. A. Treatment methods

[0486] In some embodiments, the patient in need of treatment is a male or female 2 years of age or older, 3 years of age or older, 6 years of age or older, 7 years of age or older, 12 years of age or older, 13 years of age or older, 18 years of age or older, 19 years of age or older, 25 years of age or older, 30 years of age or older, 35 years of age or older, 40 years of age or older, 45 years of age or older, or 50 years of age or older. In some embodiments, the patient in need of treatment is less than 50 years of age, less than 45 years of age, less than 40 years of age, less than 35 years of age, less than 30 years of age, less than 25 years of age, less than 20 years of age, less than 19 years of age, less than 18 years of age, less than 13 years of age, less than 12 years of age, less than 7 years of age, less than 6 years of age, less than 3 years of age, or less than 2 years of age. In some embodiments, the patient in need of treatment is a male or female between the ages of 2 and 18, 2 and 12, 2 and 6, 6 and 12, 6 and 18, 12 and 16, 2 and 50, 6 and 50, 12 and 50, or 18 and 50. In some embodiments, the patient in need of treatment is a female who is pregnant or may become pregnant.

[0487] Patients with CF have more chloride ions in their sweat than people who do not have CF. For children with CF, a sweat chloride test result will confirm the diagnosis by showing a high chloride ion level. The infant must sweat enough for the test to be done. Full-term infants generally do not produce enough sweat until they are 2 weeks old. Therefore, in some embodiments, the sweat chloride ion value of a patient in need of treatment is ≥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 embodiments, the patient in need of treatment has chronic sinus lung disease and / or gastrointestinal / nutritional abnormalities consistent with CF disease.

[0488] In some embodiments, forced expiratory volume in 1 second (FEV1) is an established marker of cystic fibrosis (CF) disease progression that is used to capture the clinical course and evaluate treatment efficacy. Thus, in various embodiments, the FEV1 of a patient requiring treatment is ≥50% and ≤90% (e.g., ≤85%, ≤80%, ≤75%, ≤70%, ≤65%, ≤60%, or ≤55%) of the predicted normal value (i.e., the average FEV1 of a non-CF patient) based on the patient's age, sex, and height. In some embodiments, the patient requiring treatment has a resting oxygen saturation of ≥92% (pulse oximeter) on room air. In some embodiments, the patient requiring treatment has a body mass index ≥17.5 kg / m 2and weigh ≥40kg.

[0489] In some embodiments, any of the CF treatment methods disclosed herein result in the production of CFTR protein in a subject. In some embodiments, any of the PCD treatment methods disclosed herein result in the production of DNAI1 protein in a subject. In some embodiments, any of the treatment methods disclosed herein result in an increase in CFTR protein or DNAI1 protein in a 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.

[0490] In some embodiments, an increase in CFTR protein or DNAI1 protein is detectable within about 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, or 48 hours of administering the pharmaceutical composition. In some embodiments, an increase in CFTR protein can be detected by qPCR of RNA purified from a tissue sample. In some embodiments, an increase in DNAI1 protein can be detected by qPCR of RNA purified from a tissue sample.

[0491] In some embodiments, the patient in need of treatment has received or is currently receiving other pulmonary disease medications. For example, the patient in need of treatment may be receiving the combination drug Lumacaftor / Ivacaftor. Alternatively, the treatment may have been carried out for at least 28 days before starting treatment according to the present disclosure. The structures of lumacaftor and ivacaftor are provided below:

[0492] Other CF medications may include, but are not limited to, conventional inhaled therapies for airway clearance and control of respiratory infections, such as bronchodilators, rhDNase ( (dornase alfa), hypertonic saline, antibiotics, and steroids; and other conventional CF-related therapies, such as systemic antibiotics, pancreatic enzymes, multivitamins, and diabetes and liver medications.

[0493] Specifically, the method of treatment consists of: (1) providing: a) a nebulizer, and b) a container containing an LNP formulation for aerosolization 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 (approximately) 10 mL, 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, or 1 mL. In some embodiments, formulations and compositions generally comprise a pharmaceutically acceptable carrier. The carrier is preferably a liquid carrier. In addition, the carrier preferably comprises water and may include other components. In some embodiments, the composition comprising the LNP formulation is stored in an ampoule, vial, or disposable vial prior to administration. In some embodiments, the composition is stored in a disposable vial prior to administration.

[0494] In some embodiments, the administration of the aerosolized pharmaceutical composition of the present disclosure results in the expression of a protein (e.g., CFTR or DNAI1) in the lungs of a subject. In still other embodiments, the administration of the aerosolized pharmaceutical composition of the present 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 being delivered to the subject. In some embodiments, the protein is detected in the lungs at 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In various embodiments, the protein (e.g., CFTR or DNAI1) can be detected using any technology known in the art (including but not limited to Western blot analysis).

[0495] In some embodiments, the mRNA delivered in accordance with the present disclosure results in an increase in protein levels or activity in the upper, central, or peripheral airways of the lungs of a subject, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 1500-fold, as compared to a control (endogenous levels of the protein or activity in the absence of or prior to treatment in accordance with the present disclosure, or a historical reference level).

[0496] In some embodiments, CFTR mRNA delivered in accordance with the present disclosure results in an increase in CFTR protein levels or activity in the upper, central, or peripheral airways of the lungs of a subject, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 1500-fold, as compared to a control (endogenous levels of protein or activity in the absence of or prior to treatment in accordance with the present disclosure, or a historical reference level).

[0497] In some embodiments, DNAI mRNA delivered in accordance with the present disclosure results in an increase in DNAI protein levels or activity in the upper, central, or peripheral airways of the lungs of a subject, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or 1500-fold, as compared to a control (endogenous levels of the protein or activity in the absence of or prior to treatment in accordance with the present disclosure, or a historical reference level).

[0498] In various embodiments, mRNA expression can be detected or quantified by performing qPCR on RNA purified from tissue samples. Protein expression (e.g., CFTR or DNAI1) can be determined by measuring the immune response to the protein. Qualitative assessment of protein can also be performed by, for example, 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.

[0499] CFTR mRNA expression can be detected or quantified by performing qPCR on RNA purified from tissue samples. In some embodiments, DNAI mRNA expression can be detected or quantified by performing qPCR on RNA purified from tissue samples. CFTR protein expression can be determined by measuring the immune response to the CFTR protein. In some embodiments, IgG antibodies against the CFTR protein are measured by performing enzyme-linked immunosorbent assay in collected serum samples. In some embodiments, CFTR-specific T cell responses are assessed using collected peripheral blood mononuclear cells. In some embodiments, the T cell response to the CFTR protein is measured by human interferon gamma enzyme-linked immunospot assay, as described by Calcedo et al. (Calcedo et al., Hum Gene Ther Clin Dev. (2013) 24: 108-15). Qualitative assessment of CFTR protein or DNAI can also be performed by, for example, Western blot analysis. CFTR protein activity can be measured by CFTR chloride channel activity in appropriate tissue cells. After solution perfusion, stable potentials are recorded at scoring intervals with an average value of 10 seconds. CFTR activity is estimated by the change in electrical potential difference following perfusion with isoproterenol without chloride ions. Various other methods are known in the art and can be used to determine CFTR mRNA and CFTR protein expression or activity.

[0500] In another aspect, the present disclosure provides a method of delivering a payload to a cell, the method comprising contacting the cell with an LNP composition of the present disclosure.

[0501] In another aspect, the present disclosure provides a method of delivering expressed protein or RNA in a cell, the method comprising contacting the cell with an LNP composition of the present disclosure.

[0502] In another aspect, the present disclosure provides a method of increasing chloride ion flux in a cell, the method comprising contacting the cell with an LNP composition of the present disclosure, wherein optionally the cell comprises a homozygous inactivating mutation in the CFTR gene.

[0503] In another aspect, the present disclosure provides methods of maintaining transepithelial electrical resistance (TEER) or reducing TEER by up to 10%, up to 20%, or up to 30%.

[0504] In some embodiments, the cell is a pneumocyte. In some embodiments, the pneumocyte is a secretory cell and / or an ionocyte.

[0505] In some embodiments, the method specifically transduces secretory cells and / or ionocytes as compared to other lung cells.

[0506] In some embodiments, the lung cells are ciliated cells.

[0507] In some embodiments, the method specifically transduces ciliated cells as compared to other lung cells.

[0508] In some embodiments, any of the treatment methods disclosed herein comprises nebulizing the composition to produce an aerosolized composition, and then contacting the aerosolized composition with the cells.

[0509] In some embodiments, the composition of the present disclosure is an aerosolized composition, and any of the treatment methods disclosed herein comprises contacting the aerosolized composition with a cell.

[0510] In another aspect, the present disclosure provides a method of delivering a payload to the lungs of a subject, the method comprising administering to the subject a composition of the present disclosure.

[0511] In one aspect, the present disclosure provides a method of treating or preventing a lung disease in a subject, the method comprising administering to the subject a composition of the present disclosure.

[0512] In some embodiments, any of the treatment methods disclosed herein comprises aerosolizing a composition of the present disclosure prior to the administering step.

[0513] In some embodiments, the compositions of the present disclosure are administered by inhalation as aerosolized compositions.

[0514] In some embodiments, any of the treatment methods disclosed herein delivers an effective amount of the composition to the lung.

[0515] In some embodiments, any of the treatment methods disclosed herein delivers to the lung an amount effective to treat a pulmonary disease.

[0516] In some embodiments, any of the treatment methods disclosed herein is more effective than contacting a cell with, or administering to a subject, elexadol, tizcator, lumacator, ivacaftor, or any combination thereof. In some embodiments, any of the treatment methods 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 elexadol, tizcator, lumacator, ivacaftor, or any combination thereof. In some embodiments, any of the methods disclosed herein is 10%-70%, 20%-70%, 30%-70%, 40%-70%, 50%-70%, or 60%-70% more effective than administering elexadol, tizcator, lumacator, ivacaftor, or any combination thereof. The structure of ivacaftor is:

[0517] The structure of Luma Kato is:

[0518] In one aspect, the present disclosure provides use of the compositions disclosed herein for treating lung diseases.

[0519] In another aspect, the present disclosure provides various LNP compositions for treating lung disease, which are described in more depth above. B. Nebulization and Pulmonary Delivery

[0520] The compositions of the present disclosure can be formulated into aerosol formulations (i.e., they can be aerosolized) alone or in combination with other suitable components. In various embodiments, the compositions of the present disclosure can be formulated for administration via inhalation (e.g., intranasally or intratracheally) (see Brigham et al., Am. J. Sci., 298:278 (1989)). In some embodiments, delivery is pulmonary delivery, e.g., including aerosolization.

[0521] Payload (such as CFTR mRNA or DNA1 mRNA) can be incorporated into lipid nanoparticles for delivery via different routes of administration. In some embodiments, CFTR mRNA or DNA1 mRNA is incorporated into LNP for pulmonary delivery. As used herein, pulmonary delivery refers to delivery to the lung via, for example, the nasal cavity, trachea, bronchi, bronchioles and / or other pulmonary systems. In some embodiments, CFTR mRNA is incorporated into LNP for aerosolization. In specific embodiments, DNA11 mRNA is incorporated into LNP for aerosolization. In these embodiments, the delivery vehicle can be in an aerosolized pharmaceutical composition that can be inhaled.

[0522] In some embodiments, the compositions of the present disclosure are aerosolized prior to inhalation.

[0523] In some embodiments, aerosol formulations can be placed into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0524] In various embodiments, provided herein is a method for targeting an aerosolized pharmaceutical composition to lung cells of a subject, the method comprising administering an aerosolized pharmaceutical composition to the subject. For example, the method comprises delivering a polynucleotide to lung cells of a subject, comprising administering an aerosolized pharmaceutical composition as described herein. In other examples, the disclosure provides for expressing a protein in the lungs of a subject, comprising administering an aerosolized pharmaceutical composition as described herein to the subject. In some embodiments, provided herein is a method for expressing a protein in the lungs of a subject, the method comprising administering an aerosolized pharmaceutical composition to the subject.

[0525] In various embodiments, an aerosolized pharmaceutical composition is administered to a subject using a nebulizer.

[0526] In some embodiments, the nebulizer is administered at an output rate of 0.1 to 1.0 mL / min. In other embodiments, the nebulizer is administered at an output rate of 0.5 mL / min.

[0527] In some embodiments, the aerosolized pharmaceutical composition is administered 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 pharmaceutical composition is administered 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 pharmaceutical composition is administered for less than 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute. In some embodiments, the aerosolized pharmaceutical composition is administered for less than 1 minute.

[0528] In some embodiments, the duration of nebulization ranges from 1 minute to 60 minutes. In some embodiments, the duration of nebulization is less than or equal to 1 minute. In some embodiments, the duration of nebulization is less than or equal to 2 minutes. In some embodiments, the duration of nebulization is less than or equal to 3 minutes. In some embodiments, the duration of nebulization is less than or equal to 6 minutes. In some embodiments, the duration of nebulization is less than or equal to 9 minutes. In some embodiments, the duration of nebulization is less than or equal to 12 minutes. In some embodiments, the duration of nebulization is less than or equal to 15 minutes. In some embodiments, the duration of nebulization is less than or equal to 18 minutes. In some embodiments, the duration of nebulization is less than or equal to 21 minutes. In some embodiments, the duration of nebulization is less than or equal to 24 minutes. In some embodiments, the duration of nebulization is less than or equal to 27 minutes. In some embodiments, the duration of nebulization is less than or equal to 30 minutes. In some embodiments, the duration of nebulization is less than or equal to 33 minutes. In some embodiments, the duration of nebulization is less than or equal to 36 minutes. In some embodiments, the duration of nebulization is less than or equal to 40 minutes. In some embodiments, the duration of nebulization is less than or equal to 45 minutes. In some embodiments, the nebulization duration is less than or equal to 50 minutes. In some embodiments, the nebulization duration is less than or equal to 55 minutes. In some embodiments, the nebulization duration is less than or equal to 60 minutes.

[0529] In various embodiments, the volume of the composition administered by aerosolization is 1 mL to 10 mL. In some embodiments, the volume of the composition administered by aerosolization is at most about 1 mL. In some embodiments, the volume of the composition administered by aerosolization is at most about 4 mL. In some embodiments, the volume of the composition administered by aerosolization is at most about 8 mL. C. Formulation of Pharmaceutical Compositions

[0530] The present disclosure also provides pharmaceutical compositions comprising the LNPs described herein (e.g., in liquid form prior to aerosolization). Such compositions can be used to treat lung diseases in patients or subjects. The pharmaceutical compositions of the present disclosure may include pharmaceutically acceptable carriers, and a detailed discussion of such carriers can be found in Chapter 30 of Remington: The Science and Practice of Pharmacy (23rd edition, 2021).

[0531] In some embodiments, the compositions of the present disclosure comprise LNPs for selective delivery to one or more of the following: goblet cells, secretory cells, club cells, basal cells, or ionocytes. In other embodiments, the aerosolized pharmaceutical compositions comprise LNPs for selective delivery to one or more of the following: ciliated cells, club cells, or basal cells.

[0532] In some embodiments, the pharmaceutical compositions of the present disclosure include one or more of: a poloxamer (e.g., poloxamer 188), polyethylene glycol ("PEG"), sucrose, and a buffer, wherein the buffer includes citrate buffer, acetate buffer, or Tris buffer.

[0533] In some embodiments, the LNPs of the present disclosure comprise PEG at a concentration ranging from 1% to 4% (w / v). In other embodiments, the concentration of PEG is from 1% to 5%, or from 2% to 4%.

[0534] In some embodiments, the pharmaceutical compositions of the present disclosure include Poloxamer 188 at a concentration between about 0.001% w / v and 0.5% v / w.

[0535] In some embodiments, the pharmaceutical compositions of the present disclosure comprise sucrose. In some embodiments, the concentration of sucrose is 1% to 15% w / v, 5% to 15% w / v, 1% to 10% w / v, or 5% to 10% w / v.

[0536] In some embodiments, the pharmaceutical composition of the present disclosure comprises a citrate buffer. For example, the pH of the citrate buffer is 4 to 8. In various examples, the buffer is an acetate buffer and has a pH of 4 to 8. In still other embodiments, the composition comprises a Tris buffer, and the pH of the Tris buffer is 4 to 8.

[0537] In some embodiments, the pH of the pharmaceutical composition is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pharmaceutical composition has an apparent pKa of 4 to 7.

[0538] In some embodiments, the pharmaceutical composition comprising the LNPs described herein has at least one pharmaceutically acceptable excipient or carrier. In some embodiments, the pharmaceutically acceptable excipient or carrier is used for aerosolization of the composition described herein.

[0539] In some embodiments, pharmaceutical composition can also include excipients and / or additives. Non-limiting examples of excipients and / or additives are surfactants, stabilizers, chelating agents, antioxidants or preservatives, flavorings, vitamins or other additives known in the art that extend the duration of use of finished pharmaceutical formulations. Chelating agents include but are not limited to ethylenediaminetetraacetic acid (EDTA) or its salts (such as disodium salt), citric acid, nitrilotriacetic acid and its salts. In some embodiments, preservatives include but are not limited to those that protect solution from pathogen particle contamination, including benzalkonium chloride or benzoic acid or benzoate (such as sodium benzoate). Antioxidants include but are not limited to vitamins, provitamins, ascorbic acid, vitamin E, its salts or esters.

[0540] In some embodiments, one or more tonicity agents may be added to the pharmaceutical compositions of the present invention to provide the desired ionic strength. Tonicity agents used herein include those that exhibit no pharmacological activity or only negligible pharmacological activity after administration. Both inorganic and organic tonicity modifiers may be used.

[0541] In some embodiments, the methods described herein comprise atomizing a composition disclosed herein to produce an aerosolized LNP composition. IV. Device

[0542] In various aspects of the present disclosure, pulmonary drug delivery involves the inhalation of a drug (e.g., for nasal, tracheal, or bronchial delivery). In some embodiments, an aerosol is a stable suspension of fine liquid particles (droplets) dispersed in a gas or vapor. In some embodiments, an LNP composition of the present disclosure with a payload is atomized before the subject inhales. In other embodiments, an LNP composition of the present disclosure with a polynucleotide as a payload is atomized before the subject inhales. In various embodiments, an LNP composition of the present disclosure with an mRNA molecule as a payload is atomized before the subject inhales. In some embodiments, an LNP composition comprising CFTR mRNA is atomized before inhalation. In some embodiments, an LNP composition comprising DNA1 mRNA is atomized before inhalation.

[0543] A nebulizer is a device used to produce an aerosolized pharmaceutical composition for pulmonary drug delivery. A nebulizer converts a liquid into a mist so that it can be more easily inhaled into the lungs. Nebulizers are effective for infants, children, and adults. Nebulizers can atomize large doses of inhaled medication. One type of nebulizer is a jet nebulizer, which contains a tube connected to a compressor so that compressed air or oxygen flows through the liquid medication at high speed to turn it into an aerosol, which is then inhaled by the patient.

[0544] In other embodiments, the nebulizer is an ultrasonic nebulizer, which comprises an electronic oscillator that generates high-frequency ultrasonic waves, thereby causing mechanical vibrations of a piezoelectric element in contact with a liquid reservoir. The high-frequency vibrations of the liquid are sufficient to generate a vapor mist. Non-limiting exemplary ultrasonic nebulizers include the Omron NE-U17 and the Beurer Nebulizer IH30.

[0545] In various embodiments, the nebulizer comprises vibrating mesh technology (VMT). VMT comprises a mesh / membrane with 1000-7000 holes that vibrates on top of a liquid reservoir, thereby forcing out a mist of very fine droplets through the holes in the mesh / membrane. Non-limiting exemplary VMT nebulizers comprise Respironics i-Neb, Beurer nebulizer IH50, Aerogen Aeroneb, HC Med deepro TM , Pulmotree Kolibri mesh atomizer and Philips InnoSpireGo.

[0546] The nebulizers described herein include those that provide an increased amount of aerosol during inhalation while minimizing both aerosol loss during exhalation and residual drug in the nebulizer reservoir (see U.S. Patent No. 9,061,303, the contents of which are incorporated herein by reference in their entirety). The nebulizer includes an aerosol generator that aerosolizes a liquid through a diaphragm into a particle size effective for delivery to the lungs. This nebulizer is currently marketed under the trade name Commercialization. U.S. Patent Application Nos. US2005 / 0006359; US2008 / 0311648; US2008 / 0060640 and U.S. Patent No. 5,518,179 disclose other aspects of the technology, and are incorporated herein by reference in their entirety.

[0547] The principle of atomization generally involves exposing a solution (e.g., an aqueous solution) to shear stress, which may negatively affect the delicate properties of polynucleotides (e.g., mRNA). However, the present disclosure provides that The nebulizer can maintain the integrity of mRNA and LNP and is therefore suitable for pulmonary administration of LNP compositions.

[0548] In one aspect, a device suitable for pulmonary delivery can contain and be used to deliver a single dose of an LNP composition of the present disclosure. In another aspect, a device suitable for pulmonary delivery can contain and be used to deliver multiple doses of an LNP composition of the present disclosure.

[0549] Nebulizer-type inhalation delivery devices can contain the LNP compositions of the present disclosure as solutions (typically aqueous solutions), suspensions, or micellar suspensions. For example, various embodiments of the LNP compositions of the present invention can be suspended in saline or a buffer solution and loaded into an inhalation delivery device. In producing an aerosolized spray of the LNP composition for inhalation, the nebulizer delivery device can be driven ultrasonically, by compressed air, by other gases, electronically, or mechanically (e.g., a vibrating mesh or orifice plate). Vibrating mesh nebulizers produce fine particle, low velocity aerosols and atomize therapeutic solutions and suspensions at a faster rate than conventional jet nebulizers or ultrasonic nebulizers. Vibrating mesh nebulizers suitable for use with the methods described herein include the Philips Respironics Omron MicroAir, Nektar or Solo.

[0550] The nebulizer can be portable and handheld in design and can be equipped with a self-contained electrical unit. The nebulizer device can include a nozzle having two overlapping outlet channels of defined apertures through which the liquid formulation can be accelerated. This results in the collision of the two streams and atomization of the formulation (e.g., any of the LNP compositions described herein). The nebulizer can use a mechanical actuator to force the liquid formulation through one or more porous nozzles of defined apertures to produce an aerosol of the formulation for inhalation. V. Kit

[0551] The present disclosure provides a variety of kits for conveniently and / or effectively performing the methods or using the compositions described herein. Typically, the kit will contain sufficient amounts and / or quantities of components to allow the user to perform multiple treatments and / or experiments on a subject. In some embodiments, the kit comprises one or more containers comprising any of the LNP compositions described herein or their pharmaceutical compositions.

[0552] In one aspect, the present disclosure provides a kit comprising the LNPs of the present disclosure. The present disclosure also provides a kit that can be used to prepare an aerosolized pharmaceutical composition. In some embodiments, the kit comprises a lipid nanoparticle composition and a sieve, wherein the lipid nanoparticle composition comprises one or more of a phospholipid, an ionizable lipid, a PEG-lipid, or cholesterol. In some embodiments, the kit may further comprise packaging and instructions and / or a delivery agent to form a liquid formulation comprising any of the LNP compositions described herein before atomization. The delivery agent may comprise sucrose, saline, a buffer (such as, but not limited to, a citrate buffer, an acetate buffer, or a Tris buffer) or any other known delivery agent for atomization. In some embodiments, the delivery agent may be in a lyophilized form. The included instructions may include a description of administering the LNP composition of the present invention to treat a target disease (e.g., CF and / or PCD), delay the onset of a target disease, or alleviate a target disease. In some embodiments, the instructions may include a description of administering the LNP composition of the present invention to a subject at risk of a target disease (e.g., CF and / or PCD). In some embodiments, the kit provides a product comprising the contents of the kit described herein.

[0553] In some embodiments, the instructions include dosage information, dosing schedule, and route of administration. In some embodiments, the kit includes one or more containers, and the one or more containers are unit doses, bulk packaging (e.g., multi-dose packaging), or subunit doses. In some embodiments, the instructions are written instructions on a label or package insert (e.g., a paper sheet included in the kit). In some embodiments, the instructions are machine-readable instructions (e.g., instructions on a magnetic storage disk or optical storage disk).

[0554] In some embodiments, the label or package insert of the kit indicates that the LNP or any pharmaceutical composition disclosed herein is used to treat, delay the onset of, and / or alleviate a lung-related disease or disorder (e.g., CF and / or PCD). Instructions for practicing any of the treatment methods described herein can be provided.

[0555] In some embodiments, the kits described herein are in suitable packaging. In some embodiments, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed or plastic bag) or a combination thereof. In some embodiments, the packaging includes packaging for use in combination with a specific device, such as a nebulizer, inhaler, and / or nasal administration device (e.g., a sprayer).

[0556] In one aspect, the present disclosure provides a kit comprising a composition and a nebulizer mask and / or screen suitable for use in a nebulizer. VI. Embodiment A. LNP Compositions

[0557] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 38 mol% cholesterol, about 4 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40, and / or wherein the N / P ratio is about 13.

[0558] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 38 mol% cholesterol, about 4 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 10.

[0559] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 28 mol% DODAP, about 17 mol% DOPE, about 33 mol% cholesterol, about 3 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0560] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 28 mol% DODAP, about 17 mol% DOPE, about 33 mol% cholesterol, about 3 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25, and / or wherein the N / P ratio is about 9.

[0561] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 37 mol% DODAP, about 14 mol% DOPE, about 28 mol% cholesterol, about 3 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 12.

[0562] In some embodiments, the LNP composition comprises about 24 mol% 4A3-SC7, about 19 mol% DODAP, about 18 mol% DOPE, about 36 mol% cholesterol, about 4 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0563] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 38 mol% cholesterol, about 4 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0564] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 36 mol% cholesterol, about 6 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0565] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 34 mol% cholesterol, about 8 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0566] In some embodiments, the LNP composition comprises about 19 mol% 4A3-SC7, about 20 mol% DODAP, about 19 mol% DOPE, about 32 mol% cholesterol, about 10 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0567] In some embodiments, the LNP composition comprises about 16 mol% 4A3-SC7, about 16 mol% DODAP, about 16 mol% DOPE, about 50 mol% cholesterol, about 4 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0568] In some embodiments, the LNP composition comprises about 14 mol% 4A3-SC7, about 22 mol% DODAP, about 11 mol% DOPE, about 50 mol% cholesterol, about 3 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25.

[0569] In some embodiments, the LNP composition comprises about 15 mol% 4A3-SC7, about 16 mol% DODAP, about 22 mol% DOPE, about 44 mol% cholesterol, about 3 mol% DMG-PEG, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 36.

[0570] In some embodiments, the LNP composition comprises about 13% to about 15% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 40% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1.

[0571] In some embodiments, the LNP composition comprises about 10% to about 20% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 30% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1. B. CFTR Payload

[0572] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 38% cholesterol by mole, about 4% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40, and / or wherein the N / P ratio is about 13.

[0573] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 38% cholesterol by mole, about 4% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 10.

[0574] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 28% DODAP by mole, about 17% DOPE by mole, about 33% cholesterol by mole, about 3% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0575] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 28% DODAP by mole, about 17% DOPE by mole, about 33% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25, and / or wherein the N / P ratio is about 9.

[0576] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 37% DODAP by mole, about 14% DOPE by mole, about 28% cholesterol by mole, about 3% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 12.

[0577] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 24% 4A3-SC7 by mole, about 19% DODAP by mole, about 18% DOPE by mole, about 36% cholesterol by mole, about 4% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0578] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 38% cholesterol by mole, about 4% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0579] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 36% cholesterol by mole, about 6% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0580] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 34% cholesterol by mole, about 8% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0581] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 32% cholesterol by mole, about 10% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0582] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 16% 4A3-SC7 by mole, about 16% DODAP by mole, about 16% DOPE by mole, about 50% cholesterol by mole, about 4% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0583] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 14% 4A3-SC7 by mole, about 22% DODAP by mole, about 11% DOPE by mole, about 50% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25.

[0584] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 15% 4A3-SC7 by mole, about 16% DODAP by mole, about 22% DOPE by mole, about 44% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 36.

[0585] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 13% to about 15% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 40% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1.

[0586] In some embodiments, the LNP composition comprises RNA encoding a CFTR protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 1), about 10% to about 20% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 30% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1. C. DNAI1 payload

[0587] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 at a molar percentage, about 20% DODAP at a molar percentage, about 19% DOPE at a molar percentage, about 38% cholesterol at a molar percentage, about 4% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40, and / or wherein the N / P ratio is about 13.

[0588] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 38% cholesterol by mole, about 4% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 10.

[0589] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 28% DODAP by mole, about 17% DOPE by mole, about 33% cholesterol by mole, about 3% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0590] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 28% DODAP by mole, about 17% DOPE by mole, about 33% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25, and / or wherein the N / P ratio is about 9.

[0591] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 37% DODAP by mole, about 14% DOPE by mole, about 28% cholesterol by mole, about 3% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 12.

[0592] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 24% 4A3-SC7 by mole, about 19% DODAP by mole, about 18% DOPE by mole, about 36% cholesterol by mole, about 4% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0593] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 38% cholesterol by mole, about 4% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0594] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 36% cholesterol by mole, about 6% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0595] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 34% cholesterol by mole, about 8% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0596] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 19% 4A3-SC7 by mole, about 20% DODAP by mole, about 19% DOPE by mole, about 32% cholesterol by mole, about 10% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0597] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 16% 4A3-SC7 by mole, about 16% DODAP by mole, about 16% DOPE by mole, about 50% cholesterol by mole, about 4% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0598] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 14% 4A3-SC7 by mole, about 22% DODAP by mole, about 11% DOPE by mole, about 50% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25.

[0599] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 15% 4A3-SC7 by mole, about 16% DODAP by mole, about 22% DOPE by mole, about 44% cholesterol by mole, about 3% DMG-PEG by mole, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 36.

[0600] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 13% to about 15% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 40% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1.

[0601] In some embodiments, the LNP composition comprises RNA encoding DNAI1 protein (optionally having a polynucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4), about 10% to about 20% 4A3-SC7 by mole, about 15% to about 25% DODAP by mole, about 10% to about 25% DOPE by mole, about 30% to about 60% cholesterol by mole, about 2% to about 6% DMG-PEG by mole, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 20:1 to about 40:1. D. Gene Editing Payload

[0602] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 20% DODAP at a molar percentage, about 19% DOPE at a molar percentage, about 38% cholesterol at a molar percentage, about 4% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40, and / or wherein the N / P ratio is about 13.

[0603] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 20% DODAP at a molar percentage, about 19% DOPE at a molar percentage, about 38% cholesterol at a molar percentage, about 4% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 10.

[0604] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 28% DODAP at a molar percentage, about 17% DOPE at a molar percentage, about 33% cholesterol at a molar percentage, about 3% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0605] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 28% DODAP at a molar percentage, about 17% DOPE at a molar percentage, about 33% cholesterol at a molar percentage, about 3% DMG-PEG at a molar percentage, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 25, and / or wherein the N / P ratio is about 9.

[0606] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 37% DODAP at a molar percentage, about 14% DOPE at a molar percentage, about 28% cholesterol at a molar percentage, about 3% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 12.

[0607] In some embodiments, the LNP composition comprises one or more components for gene editing, about 24% 4A3-SC7 at a molar percentage, about 19% DODAP at a molar percentage, about 18% DOPE at a molar percentage, about 36% cholesterol at a molar percentage, about 4% DMG-PEG at a molar percentage, wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 30, and / or wherein the N / P ratio is about 11.

[0608] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 20% DODAP at a molar percentage, about 19% DOPE at a molar percentage, about 38% cholesterol at a molar percentage, about 4% DMG-PEG at a molar percentage, and / or wherein the total lipid to RNA (weight / weight) ratio of the LNP is about 40.

[0609] In some embodiments, the LNP composition comprises one or more components for gene editing, about 19% 4A3-SC7 at a molar percentage, about 20% DODAP at a molar percentage, about 19% DOPE at a molar percent...

Claims

1. An aerosolized pharmaceutical composition, comprising aerosol particles, wherein the aerosol particles comprise lipid nanoparticles (LNPs), wherein the composition is capable of delivering the LNP to the lungs and / or tracheobronchial regions of a subject; and / or Wherein the LNP has one or more of the following: (a) Encapsulation efficiency (EE) greater than 50%, (b) greater than 50% mRNA integrity, (c) a diameter of 20 nm to 600 nm, (d) a polydispersity of less than 0.6; and / or The aerosol particles have one or more of the following: (a) Mass median aerodynamic diameter (MMAD) between 1 μm and 10 μm, (b) Geometric standard deviation (GSD) from 1 to 5, and (c) a fine particle fraction (FPF) percentage of at least 50%.

2. The aerosolized pharmaceutical composition of claim 1, wherein the composition comprises LNPs for selective delivery to one or more of the following: goblet cells, secretory cells, club cells, basal cells, intermediate cells, plasma cells, precursor cells, ionocytes, or ciliated cells.

3. The aerosolized pharmaceutical composition of any one of claims 1-2, wherein the composition is capable of delivering the LNP to the tracheobronchial region of a subject.

4. The aerosolized pharmaceutical composition of any one of claims 1-2, wherein the composition is capable of delivering the LNP to the upper, central, or peripheral airways of the lungs of the subject.

5. The aerosolized pharmaceutical composition of any one of claims 1-3, wherein the EE of the LNP is greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or wherein the EE of the LNP is 50%-95%, 60%-95%, 70%-95%, 80%-95% or 90%-95%.

6. The aerosolized pharmaceutical composition of any one of claims 1-4, wherein the mRNA integrity of the LNP is greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or wherein the mRNA integrity of the LNP is 75%-99%, 80%-95%, 85%-90% or 90%-95%.

7. The aerosolized pharmaceutical composition of any one of claims 1-6, wherein the LNP has a loss of mRNA integrity of less than 20%, less than 15%, less than 10%, less than 5%, or less than 5%-20%, less than 5%-15%, or less than 5%-10%.

8. The aerosolized pharmaceutical composition of any one of claims 1-7, wherein the polydispersity of the LNPs is less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.

1.

9. The aerosolized pharmaceutical composition of any one of claims 1-8, wherein the LNP has a diameter of 20 to 180 nm, 30 to 180 nm, 40 to 180 nm, 50 to 180 nm, 60 to 180 nm, 70 to 180 nm, 80 to 180 nm, 90 to 180 nm, 100 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.

10. An aerosolized pharmaceutical composition according to any one of claims 1 to 9, wherein the MMAD of the aerosol particles is 1 μm to 9 μm, or 1 μm to 8 μm, or 1 μm to 7 μm, or 1 μm to 6 μm, or 1 μm to 5 μm, or 1 μm to 4 μm, or 1 μm to 3 μm, or 1 μm to 2 μm, or 3 μm to 5 μm.

11. The aerosolized pharmaceutical composition according to any one of claims 1 to 10, wherein the GSD of the aerosol particles is 1 to 4, or 1 to 3, or 1 to 2, or 1, 1.5, 2, 2.5 or 3.

12. The aerosolized pharmaceutical composition according to any one of claims 1 to 11, wherein the FPF of the aerosol particles is 55%, 60%, 70%, 75%, 80%, 85% or 90%.

13. The aerosolized pharmaceutical composition according to any one of claims 1 to 12, wherein the LNP comprises one or more, two or more, or three or more of a phospholipid, an ionizable lipid, a polyethylene glycol (PEG)-lipid, and a sterol.

14. The aerosolized pharmaceutical composition according to any one of claims 1 to 13, wherein the composition further comprises one or more of PEG-lipid, sucrose, and a buffer, wherein the buffer comprises citrate buffer, acetate buffer, or Tris buffer.

15. The aerosolized pharmaceutical composition of claim 14, wherein the molar percentage of the PEG-lipid is between 2% and 8%.

16. The aerosolized pharmaceutical composition of claim 14, wherein the concentration of sucrose is 1% to 15% w / v, 5% to 15% w / v, 1% to 10% w / v, or 5% to 10% w / v.

17. The aerosolized pharmaceutical composition according to claim 14, wherein the buffer is citrate buffer, optionally at a pH of 4 to 8; acetate buffer, optionally at a pH of 4 to 8; or Tris buffer, optionally at a pH of 6 to 9.

18. The aerosolized pharmaceutical composition of any one of claims 1-17, wherein the LNP comprises a payload.

19. The aerosolized pharmaceutical composition according to any one of claims 1 to 18, wherein the payload comprises an oligonucleotide, a polynucleotide, a peptide or a protein, such as a nuclease and an antibody or an antibody chain.

20. The aerosolized pharmaceutical composition of claim 19, wherein the polynucleotide comprises mRNA.

21. The aerosolized pharmaceutical composition of claim 20, wherein the mRNA has a length of about 1000 nucleotides (nt) to about 5000 nucleotides (nt), about 2000 nucleotides (nt) to about 5000 nt, about 2500 nt to about 5000 nt, about 3000 nt to about 5000 nt, about 3500 nt to about 5000, about 4000 nt to about 5000 nt, or about 4500 nt to about 5000 nt.

22. The aerosolized pharmaceutical composition of claim 21, wherein the mRNA encodes dynein axonemal intermediate chain 1 (DNAI1) protein or cystic fibrosis transmembrane conductance regulator (CFTR) protein.

23. The aerosolized pharmaceutical composition of any one of claims 19 to 22, wherein the concentration of the polynucleotide is 0.5-3.0 mg / mL, or 1.0-3.0 mg / mL, or 2.0-3.0 mg / mL or 1.0 mg / mL.

24. The aerosolized pharmaceutical composition of any one of claims 1-23, wherein the pH of the composition is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

0.

25. The aerosolized pharmaceutical composition of any one of claims 1-24, wherein the composition has an apparent pKa of 4 to 9.

26. The aerosolized pharmaceutical composition of any one of claims 1-25, wherein the composition results in expression of a protein from the mRNA in the lungs of the subject.

27. The aerosolized pharmaceutical composition of any one of claims 1-26, wherein the composition results in detection of protein from the mRNA in the lungs of the subject between 6 and 12 hours after delivery to the subject.

28. The aerosolized pharmaceutical composition of any one of claims 1-27, wherein the LNP comprises an ionizable lipid, a phospholipid, a PEG-lipid; and / or a sterol.

29. The aerosolized pharmaceutical composition of claim 28, wherein the LNP comprises a second ionizable lipid.

30. An aerosolized pharmaceutical composition according to claim 28 or claim 29, wherein the LNP comprises 1,2-dioleoyl-3-dimethylammonium propane (DODAP).

31. An aerosolized pharmaceutical composition according to claim 28 or claim 29, wherein the LNP comprises a mole percentage of between about 5% and about 50%, between about 5% and about 35%, between about 20% and about 50%, between about 20% and about 50%, or about 20% 1,2-dioleoyl-3-dimethylammonium propane (DODAP).

32. The aerosolized pharmaceutical composition of claim 28, wherein the LNP comprises a permanently cationic lipid.

33. The aerosolized pharmaceutical composition of claim 32, wherein the permanent cationic lipid comprises a trimethylammonium group, optionally wherein the permanent cationic lipid is 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0EPC).

34. The aerosolized pharmaceutical composition of claim 32, wherein the permanently ionizable lipid comprises a trimethylammonium group, optionally wherein the permanently cationic lipid is dioleoyl-3-trimethylammonium propane (DOTAP).

35. An aerosolized pharmaceutical composition according to any one of claims 28 to 34, wherein the ionizable lipid is a dendritic lipid, optionally a dendritic lipid of formula (I) or formula (X), optionally 4A3-SC7 or 5A2-SC8.

36. An aerosolized pharmaceutical composition according to claim 35, wherein the LNP comprises about 19%, about 20%, about 19%, about 39% and about 3.8% 4A3-SC7, 14:0EPC, DOPE, cholesterol and DMG-PEG in molar percentages, respectively; and / or wherein the LNP has a lipid to RNA (weight / weight) ratio of about 30.

37. A liquid pharmaceutical composition for use in preparing an aerosolized pharmaceutical composition according to any one of claims 1 to 36.

38. A method for delivering lipid nanoparticles (LNPs) to lung cells of a subject, the method comprising nebulizing the liquid pharmaceutical composition of claim 37 to produce an aerosolized pharmaceutical composition, and administering the aerosolized pharmaceutical composition to the subject.

39. A method of delivering a payload to lung cells of a subject, the method comprising administering to the subject an aerosolized pharmaceutical composition according to any one of claims 1-36, wherein optionally the payload is a polynucleotide.

40. A method for expressing a protein in the lungs of a subject, the method comprising administering to the subject an aerosolized pharmaceutical composition according to any one of claims 1-36.

41. A method for treating a lung disease in a subject, the method comprising administering to the subject an aerosolized pharmaceutical composition according to any one of claims 1-36.

42. The method of any one of claims 38-41, comprising the aerosol particles comprising lipid nanoparticles (LNPs) comprising ionizable lipids, phospholipids, polyethylene glycol (PEG)-lipids; and / or sterols.

43. The method of any one of claims 38-42, comprising the particle, wherein the LNP comprises a second ionizable lipid.

44. The method of any one of claims 38-43, comprising the particle, wherein the LNP comprises 1,2-dioleoyl-3-dimethylammonium propane (DODAP).

45. The method of any one of claims 38-44, comprising the particle, wherein the LNP is stored in a buffer.

46. ​​The method of any one of claims 38-45, wherein the buffer is citrate buffer, optionally at a pH of 4 to 8; wherein the buffer is acetate buffer, optionally at a pH of 4 to 8; or wherein the buffer is Tris buffer, optionally at a pH of 4 to 8.

47. The method according to any one of claims 38 to 46, comprising the particles, wherein the particles are The device aerosolizes the LNPs.

48. The method according to any one of claims 38 to 47, comprising the particles being screened using a screen having a mesh size of 40 HO V. The device aerosolizes the LNPs.

49. The method of any one of claims 38-48, wherein the lung disease comprises primary ciliary dyskinesia (PCD) or cystic fibrosis (CF).

50. The method of any one of claims 38-49, wherein the aerosolized particles are selectively delivered to the tracheobronchial region of the subject's lungs.

51. The method of claims 38-50, wherein the aerosolized pharmaceutical composition of claim 1 is administered to the subject using a nebulizer, wherein the nebulizer is administered at an output rate of 0.1 to 1 mL / min.

52. The method of claims 38-51, wherein the aerosolized pharmaceutical composition is administered at an output rate of 0.5 mL / min.

53. The method of claims 38-52, wherein the aerosolized pharmaceutical composition is administered for less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes.

54. The method of claims 38-53, wherein the administering comprises administering via intranasal administration, intratracheal administration, or oral administration, wherein the administering step delivers the aerosolized pharmaceutical composition to the tracheobronchial region (TB).

55. The method of any one of claims 38 to 54, wherein the subject is a human subject.

56. A method for preparing an aerosolized pharmaceutical composition according to any one of claims 1 to 37, the method comprising introducing a liquid pharmaceutical composition comprising the aerosolized pharmaceutical composition into a nebulizer, wherein the nebulizer is operated at an output rate of 0.1 to 1 mL / min or at an output rate of 0.5 mL / min.

57. A kit comprising a lipid nanoparticle composition and a screen, optionally comprising a polynucleotide, wherein the lipid nanoparticle composition comprises one or more of a phospholipid, an ionizable lipid, a PEG-lipid, a sterol.

58. The kit of claim 57, wherein the phospholipid, the ionizable lipid, the PEG-lipid, and the sterol are in separate containers from the polynucleotide, or wherein the phospholipid, the ionizable lipid, the PEG-lipid, and the sterol are in the same container as the polynucleotide.

59. A method for determining the amount of lipid nanoparticles (LNPs) in an aerosolized pharmaceutical composition, the method comprising: (a) contacting the aerosolized pharmaceutical composition with a filter comprising glass fibers; (b) extracting lipids from the filter with an extraction solution comprising ammonium ions, optionally ammonium acetate.

60. The method of claim 58, wherein the method comprises measuring the amount of at least one lipid in the extraction solution.

61. The method of claim 58, wherein the measuring is performed by high performance liquid chromatography (HPLC).

62. A lipid nanoparticle (LNP) composition comprising LNP, wherein the LNP comprises: Less than 25% or less than 20% by mole of 1,2-dioleoyl-3-dimethylammonium propane (DODAP); greater than 40 mole percent sterols; and / or Messenger RNA (mRNA) with a lipid:mRNA ratio of less than 40:

1.

63. A lipid nanoparticle (LNP) composition comprising LNP, wherein: The LNP specifically transduces secretory cells and / or ionocytes; and / or The LNPs deliver mRNA to lung cells in an amount effective to increase the expression and / or function of the protein encoded by the mRNA.

64. The composition of claim 62, wherein: The LNP composition specifically transduces secretory cells and / or ionocytes; and / or wherein the LNP composition delivers mRNA to lung cells in an amount effective to increase the expression and / or function of the protein encoded by the mRNA.

65. The composition of any one of claims 62 to 64, wherein the LNP composition comprises: (a) Ionizable lipids; (b) phospholipids; (c) polyethylene glycol (PEG)-lipids; and / or (d) Sterols.

66. The composition of any one of claims 62 to 65, wherein the LNP comprises a second ionizable lipid.

67. The composition of any one of claims 62 to 66, wherein the LNP comprises an anionic lipid.

68. The composition of any one of claims 62 to 67, wherein the LNP comprises a permanent cationic lipid.

69. The composition of any one of claims 62 to 68, wherein the LNP comprises less than 25% or less than 20% DODAP by mole.

70. The composition of claim 69, wherein the LNP comprises between 5% and 25%, between 7.5% and 25%, between 10% and 25%, between 5% and 20%, between 7.5% and 20%, or between 10% and 20% DODAP in a molar percentage.

71. The composition of claim 69, wherein the LNP comprises between 5% and 17.5%, between 7.5% and 17.5%, or between 10% and 17.5% DODAP in a molar percentage.

72. The composition of claim 69, wherein the LNP comprises about 16 mol% DODAP.

73. The composition of any one of claims 62 to 68, wherein the LNP comprises greater than 40% cholesterol by mole.

74. The composition of claim 73, wherein the LNP comprises between 40% and 60%, between 45% and 60%, or between 50% and 60% cholesterol by mole percentage.

75. The composition of claim 73, wherein the LNP comprises between 40% and 55%, between 45% and 55%, or between 50% and 55% cholesterol by mole percentage.

76. The composition of claim 73, wherein the LNP comprises between 40% and 50% or between 45% and 50% cholesterol in a molar percentage.

77. The composition of claim 73, wherein the LNP comprises about 50 mole% cholesterol.

78. The composition of any one of claims 62 to 77, wherein the LNP comprises messenger RNA (mRNA).

79. The composition of claim 78, wherein the LNP comprises mRNA at a lipid:mRNA ratio of less than 40:

1.

80. The composition of claim 79, wherein the lipid:mRNA ratio is between 20:1 and 40:1, between 25:1 and 40:1, between 35:1 and 40:1, or between 30:1 and 40:

1.

81. The composition of claim 79, wherein the lipid:mRNA ratio is between 20:1 and 36:1, or between 25:1 and 36:

1.

82. The composition of claim 79, wherein the lipid:mRNA ratio is 36:

1.

83. The composition of claim 79, wherein the lipid:mRNA ratio is 25:

1.

84. The composition of any one of claims 62 to 83, wherein: (a) the ionizable lipid is 5A2-SC8 or 4A3-SC7; (b) the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and / or (c) The polyethylene glycol (PEG)-lipid is DMG-PEG, optionally DMG-PEG2000.

85. The composition of any one of claims 62 to 83, wherein: (a) the ionizable lipid is 4A3-SC7; (b) the phospholipid is DOPE; and (c) The polyethylene glycol (PEG)-lipid is DMG-PEG.

86. The composition of claim 84 or claim 85, wherein the LNP comprises a second ionizable lipid and the second ionizable lipid is DODAP.

87. The composition of claim 84 or claim 85, wherein the LNP comprises a second ionizable lipid and the second ionizable lipid is 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA).

88. The composition of any one of claims 62 to 87, wherein the ionizable lipid is 4A3-SC7 and the LNP comprises between about 13% and about 15% 4A3-SC7 by mole.

89. The composition of any one of claims 62 to 87, wherein the ionizable lipid is 4A3-SC7 and the LNP comprises about 13% to about 15% 4A3-SC7 by mole.

90. The composition of any one of claims 62 to 87, wherein the LNP comprises between about 2% and about 8% PEG-lipid in a molar percentage.

91. The composition of claim 90, wherein the mole percentage of the PEG-lipid is about 3%.

92. The composition of claim 84 or claim 85, wherein the LNP comprises a neutral phospholipid and the neutral phospholipid is DOPE.

93. The composition of claim 92, wherein the LNP comprises between about 10% and about 25% DOPE by mole.

94. The composition of claim 92, wherein the LNP comprises about 11% or about 22% DOPE by mole.

95. The composition of any one of claims 62 to 94, wherein the LNP comprises: (a) about 15% by mole of 4A3-SC7; (b) DODAP with a molar percentage of about 16% (c) about 22 mol% DOPE; (d) about 3 mol% DMG-PEG2000; and (e) approximately 44 mole percent cholesterol.

96. The composition of any one of claims 62 to 94, wherein the LNP comprises: (a) about 14% by mole of 4A3-SC7; (b) DODAP with a molar percentage of about 22% (c) about 11 mol% DOPE; (d) about 3 mol% DMG-PEG2000; and (e) about 50 mole percent cholesterol.

97. The composition of any one of claims 62 to 96, wherein the LNP comprises a payload.

98. The composition of claim 97, wherein the payload is messenger RNA (mRNA).

99. The composition of claim 98, wherein the mRNA comprises between 100 bases and 8 kilobases (kb).

100. The composition of claim 98, wherein the mRNA is comprised between 1 kb and 8 kb, or between 2 kb and 8 kb, between 3 kb and 8 kb, or between 4 kb and 8 kb.

101. The composition of claim 98, wherein the mRNA is comprised between 1 kb and 7 kb, or between 2 kb and 7 kb, between 3 kb and 7 kb, or between 4 kb and 7 kb.

102. The composition of claim 98, wherein the mRNA is comprised between 1 kb and 6 kb, or between 2 kb and 6 kb, between 3 kb and 6 kb, or between 4 kb and 6 kb.

103. The composition of claim 98, wherein the mRNA comprises (approximately) 2 kb.

104. The composition of claim 98, wherein the mRNA comprises (approximately) 4.6 kb.

105. The composition of any one of claims 98 to 104, wherein the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein.

106. The composition of any one of claims 98 to 104, wherein the mRNA encodes dynein axoneme intermediate chain 1 (DNAI1) protein.

107. The composition of any one of claims 98 to 104, wherein the mRNA encodes a gene editing system or a component thereof.

108. The composition of any one of claims 97 to 107, wherein the payload is shRNA or a polynucleotide encoding shRNA.

109. The composition of any one of claims 97 to 108, wherein the payload is a microRNA or a polynucleotide encoding a microRNA.

110. The composition of any one of claims 62 to 109, wherein the composition is a pharmaceutical composition.

111. The composition of any one of claims 62 to 110, wherein the composition is an aerosolized composition.

112. The composition of any one of claims 62 to 111, wherein the LNP has an encapsulation efficiency between 50% and 99%, between 60% and 99%, between 70% and 99%, or between 80% and 99%.

113. The composition of any one of claims 62 to 111, wherein the LNP has an encapsulation efficiency between 50% and 95%, between 60% and 95%, between 70% and 95%, or between 80% and 95%.

114. A method of delivering a payload to a cell, the method comprising contacting the cell with the LNP composition of any one of claims 62 to 113.

115. A method of delivering expressed protein or RNA in a cell, the method comprising contacting the cell with the LNP composition of any one of claims 62 to 113.

116. A method of increasing chloride ion flux in a cell, the method comprising contacting the cell with the LNP composition of any one of claims 105, 107-113, wherein optionally the cell comprises a homozygous inactivating mutation in the CFTR gene.

117. The method of claim 116, wherein the method maintains transepithelial electrical resistance (TEER) or reduces TEER by at most 10%, at most 20%, or at most 30%.

118. The method of any one of claims 114 to 117, wherein the cell is a lung cell.

119. The method of claim 118, wherein the lung cells are secretory cells and / or ionocytes.

120. The method of claim 119, wherein the method specifically transduces the secretory cells and / or the ionocytes as compared to other lung cells.

121. The method of claim 118, wherein the lung cells are ciliated cells.

122. The method of claim 119, wherein the method specifically transduces the ciliated cells as compared to other lung cells.

123. The method of any one of claims 114 to 122, wherein the method comprises atomizing the LNP composition to produce an aerosolized composition and then contacting the aerosolized composition with the cells.

124. The method of any one of claims 114 to 122, wherein the LNP composition is an aerosolized composition, and the method comprises contacting the aerosolized composition with the cells.

125. A method of delivering a payload to the lungs of a subject, the method comprising administering to the subject a composition according to any one of claims 62 to 113.

126. A method of treating or preventing a lung disease in a subject, the method comprising administering to the subject a composition according to any one of claims 62 to 113.

127. The method of claim 125 or claim 126, comprising atomizing the composition prior to the administering step.

128. The method of any one of claims 114 to 127, wherein the LNP composition is administered by inhalation as an aerosolized composition.

129. The method of any one of claims 114 to 126, wherein the method delivers an effective amount of the LNP composition to the lung.

130. The method of any one of claims 114 to 128, wherein the method delivers to the lung an amount effective to treat the lung disease.

131. The method of any one of claims 114 to 129, wherein the method is more effective than contacting the cell with or administering to the subject: elexadol, tizancator, lumacaftor, ivacaftor, or a combination thereof.

132. Use of a composition according to any one of claims 62 to 113 for the treatment of a lung disease.

133. A composition according to any one of claims 62 to 113 for use in treating a lung disease.

134. A kit comprising a composition according to any one of claims 62 to 113 and a nebuliser mask and / or screen suitable for use in a nebuliser.

135. A method of preparing an LNP composition according to any one of claims 62 to 113, comprising mixing a lipid component and the payload under conditions effective to assemble the LNP comprising the payload.

136. The method of claim 135, wherein the method comprises atomizing the composition to produce an aerosolized LNP composition.

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