Aerosolized lipid nanoparticles and uses thereof

Abstract

Provided is a lipid nanoparticle (LNP) that contains specific cationic ionizable lipids with biologically active polynucleotides (such as RNA).In some aspects, LNP complexes are provided as aerosols and / or dry powders, for example, for delivery to the lungs.Provided are methods for making and using such compositions. TIFF2025538217000033.tif72128

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of priority to U.S. Provisional Application No. 63 / 425,129, filed November 14, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates generally to the fields of pharmaceutical formulations, biologics and their manufacture, and more particularly to lipid nanoparticles and methods for using same to deliver nucleic acid molecules. [Background technology]

[0003] 2. Description of Related Art Messenger RNA (mRNA) is a promising therapeutic approach for treating lung diseases. mRNA delivery has the potential to both treat and cure genetic diseases such as cystic fibrosis and primary ciliary dyskinesia (Translate Bio Inc., 2020; Woo et al., 2022). Preclinical results of mRNA delivery are promising, but more effective carriers are needed to see clinical improvement (Translate Bio Inc., 2020).

[0004] Lipid nanoparticles (LNPs) have emerged as a powerful technology for delivering mRNA. For example, LNPs were the first drug carriers clinically approved in the United States to mitigate the COVID-19 pandemic (Baden et al., 2020; Polack et al., 2020). This versatile platform has been successfully used to deliver mRNA for many other applications, such as cancer, genetic disorders, and other infectious diseases (Zhang et al., 2020a; Qiu et al., 2021). However, because these formulations are inherently liver-targeted (Jayaraman et al., 2012; Akinc et al., 2019), specifically designed to deliver siRNA to the liver via the intravenous route, targeting the lung has been challenging. More recently, LNPs have demonstrated efficacy in lung-targeted systemic delivery. For example, positively charged components possess lung-targeting properties during systemic delivery (Cheng et al., 2020). Furthermore, modifications to the lipidoid head group have shifted targeting from the liver to the lungs (Qiu et al., 2022). While these therapies are promising, inhaled pulmonary delivery increases concentrations at the site of action and reduces off-target effects compared to systemic delivery (Rudokas et al., 2016). Furthermore, local delivery via aerosolization is attractive because it avoids toxic effects and delivers high concentrations of mRNA (Cipolla et al., 2013; Garbuzenko et al., 2009). To date, only one clinical trial has been completed for pulmonary delivery of mRNA LNPs (Translate Bio Inc., 2020). A Phase 1 / 2 clinical trial (RESTORE-CF) evaluating LNPs encapsulating CFTR mRNA in CF patients demonstrated safety and tolerability after repeated dosing, but no significant improvement in lung function was observed. Therefore, translatable mRNA LNPs for pulmonary delivery are needed.

[0005] Delivery efficacy is highly dependent on the route of administration. Therefore, formulations for local administration must overcome several different barriers. To create an inhalable drug, the formulation must be aerosolized. During this process, LNPs are subjected to shear forces, causing mRNA degradation and subsequent loss of delivery. Furthermore, LNPs must deposit in the pulmonary tree at a clinically relevant location and successfully escape endosomes to achieve therapeutic efficacy. Subsequently, LNPs must penetrate lung mucus, release transporters into the cytoplasm, and be translated by endosomal escape within lung epithelial cells. Although there are studies on stability during aerosolization and mRNA delivery (Lokugamage et al., 2021; Kim et al., 2022), new compounds, compositions, and methods that overcome these barriers for local pulmonary delivery are needed. Summary of the Invention

[0006] The present disclosure provides pharmaceutical compositions comprising cationic ionizable lipids that can be used to deliver nucleic acids to therapeutically relevant locations.

[0007] In some embodiments, the present disclosure provides: a) Lipid nanoparticles (LNPs), comprising: i) a cationic ionizable lipid of the formula: TIFF2025538217000002.tif25128 (in the formula: m, n, and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X2 and X3 are each independently -O- or -N-; R1 is hydroxy, amino, halo, or mercapto; or Alkoxy (C≦8) , alkylamino (C≦8) , dialkylamino (C≦12) or a substituted form of any of these groups; and R2 and R3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl(C≦24) ), or a pharmaceutically acceptable salt thereof; ii) phospholipids; iii) PEG-lipids; and iv) Sterols the LNP comprising: b) Nucleic acid and wherein the cationic ionizable lipid is further defined as: TIFF2025538217000003.tif25128 (in the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X2 and X3 are each independently -O- or -N-; R1 is hydroxy, amino, halo, or mercapto; or Alkoxy (C≦8) , alkylamino (C≦8) , dialkylamino (C≦12) or a substituted form of any of these groups; and R2 and R3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) ), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic ionizable lipid is further defined as: TIFF2025538217000004.tif25128(in the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and X2 and X3 are each independently -O- or -N-; and R2 and R3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) ), or a pharmaceutically acceptable salt thereof. In some embodiments, the cationic ionizable lipid is further defined as: TIFF2025538217000005.tif25128 (in the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R2 and R3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) ), or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the present disclosure provides pharmaceutical compositions wherein m is 1, 2, 3, or 4. In some embodiments, m is 2 or 3. In further embodiments, m is 2. In other embodiments, m is 3. In some embodiments, n is 3, 4, 5, 6, 7, 8, or 9. In further embodiments, n is 4, 5, 6, 7, or 8. In further embodiments, n is 5 or 7. In further embodiments, n is 5. In other embodiments, n is 7. In some embodiments, o is 3, 4, 5, 6, 7, 8, or 9. In further embodiments, o is 4, 5, 6, 7, or 8. In further embodiments, o is 5 or 7. In further embodiments, o is 5. In other embodiments, o is 7. In some embodiments, n is 5 and o is 7. In other embodiments, n and o are each 5. In other embodiments, n is 7 and o is 5. In other embodiments, n and o are each 7.

[0009] In some embodiments, R2 is alkyl (C≦24) or substituted alkyl (C≦24) In a further embodiment, R2 is alkyl (C≦24) In a further embodiment, R2 is alkyl (C6~20) In a further embodiment, R2 is n-alkyl (C6~20)In some embodiments, R2 is n-alkyl (C6~12) In further embodiments, R2 is n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, or n-dodecane. In some embodiments, the carbon atom of R2 bonded to X2 is a secondary carbon. In some embodiments, R2 is heptadecan-9-yl.

[0010] In some embodiments, R3 is alkyl (C≦24) or substituted alkyl (C≦24) In some embodiments, R3 is alkyl (C≦24) In a further embodiment, R3 is alkyl (C6~20) In a further embodiment, R3 is n-alkyl (C6~20) In some embodiments, R3 is n-alkyl (C6~12) In further embodiments, R3 is n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, or n-dodecane. In some embodiments, the carbon atom of R3 bonded to X3 is a secondary carbon. In some embodiments, R3 is heptadecan-9-yl. In some embodiments, R2 is n-alkyl (C6~20) and the carbon of R3 bonded to X3 is a secondary carbon. In other embodiments, R2 and R3 are n-alkyl (C6~20) In other embodiments, the carbon of R2 bonded to X2 is a secondary carbon and the carbon of R3 bonded to X3 is a secondary carbon.

[0011] In some embodiments, the present disclosure provides that the cationic ionizable lipid is further defined as: TIFF2025538217000006.tif215145, or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the LNPs of the compositions of the present disclosure comprise a molar ratio of cationic ionizable lipid to LNP of about 0.3 to 0.7. In further embodiments, the LNPs comprise a molar ratio of cationic ionizable lipid to LNP of about 0.4 to 0.6. In further embodiments, the LNPs comprise a molar ratio of cationic ionizable lipid to LNP of about 0.45.

[0013] In some embodiments, the phospholipid comprises DOPE, DSPC, or DPPC. In further embodiments, the phospholipid is DPPC. In some embodiments, the LNPs comprise a molar ratio of phospholipid to LNP of about 0.02 to 0.4. In further embodiments, the LNPs comprise a molar ratio of phospholipid to LNP of about 0.05 to 0.3. In further embodiments, the LNPs comprise a molar ratio of phospholipid to LNP of about 0.2.

[0014] In some embodiments, the PEG-lipid comprises DMG-PEG, DMPE-PEG, or DSPE-PEG. In further embodiments, the PEG-lipid is DMPE-PEG. In some embodiments, the LNP comprises a molar ratio of PEG-lipid to LNP of about 0.005 to 0.03. In further embodiments, the LNP comprises a molar ratio of PEG-lipid to LNP of about 0.01 to 0.015. In further embodiments, the LNP comprises a molar ratio of PEG-lipid to LNP of about 0.01. In some embodiments, the LNP comprises DOPE and DMG-PEG, DOPE and DMPE-PEG, DOPE and DSPE-PEG, DSPC and DMG-PEG, DSPC and DMPE-PEG, DSPC and DSPE-PEG, DPPC and DMG-PEG, DPPC and DMPE-PEG, or DPPC and DSPE-PEG. In further embodiments, the LNP comprises DPPC and DMPE-PEG.

[0015] In some embodiments, the sterol is cholesterol. In some embodiments, the LNPs comprise a molar ratio of sterol to LNP of about 0.1 to 0.6. In further embodiments, the LNPs comprise a molar ratio of sterol to LNP of about 0.15 to 0.5. In further embodiments, the LNPs comprise a molar ratio of sterol to LNP of about 0.34.

[0016] In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). In some embodiments, the nucleic acid is RNA. In further embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises siRNA. In some embodiments, the nucleic acid is encapsulated in LNPs. In some embodiments, the weight ratio of CIL to nucleic acid is about 20:1 to about 1:5. In further embodiments, the weight ratio of CIL to nucleic acid is about 15:1 to about 1:3. In a further embodiment, the weight ratio of CIL to nucleic acid is about 11.3:1.

[0017] In some embodiments, the N / P ratio of the composition is about 4 to 7. In further embodiments, the N / P ratio is about 5 to 6. In further embodiments, the N / P ratio is about 5.7. In some embodiments, the LNPs have an average particle size of about 50 nm to about 250 nm. In further embodiments, the LNPs have an average particle size of about 50 nm to about 150 nm. In further embodiments, the composition has an average particle size of about 100 nm. In other embodiments, the composition has an average particle size of about 150 nm. In some embodiments, the composition has a polydispersity index (PDI) of about 0.01 to about 0.5. In further embodiments, the composition has a polydispersity index (PDI) of about 0.02 to about 0.4. In further embodiments, the composition has a polydispersity index (PDI) of about 0.05. In other embodiments, the composition has a polydispersity index (PDI) of about 0.3. In some embodiments, the composition has a zeta potential of about -0.5 mV to about -40 mV. In further embodiments, the composition has a zeta potential of about -0.5 mV to about -20 mV. In further embodiments, the composition has a zeta potential of about -10 mV. In other embodiments, the composition has a zeta potential of about -15 mV.

[0018] In one aspect, the present disclosure provides a nebulized composition using the pharmaceutical composition described herein. In another aspect, the present disclosure provides a method for treating a disease, disorder, injury, or infection, comprising administering an effective amount of the pharmaceutical composition described herein. In some embodiments, the disease is a genetic disease. In some embodiments, the disease, injury, or infection is a lung disease, lung injury, or lung infection. In further embodiments, the disease is a lung disease. In further embodiments, the lung disease is interstitial lung disease, chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis (CF), pulmonary fibrosis, alpha-1 antitrypsin deficiency, or primary ciliary dyskinesia (PCD). In some embodiments, the pharmaceutical composition is formulated for administration by inhalation. In some embodiments, the subject is a mammal. In further embodiments, the subject is a human.

[0019] In another aspect, the present disclosure provides a method for regulating gene expression, comprising delivering nucleic acid to cells, and comprising contacting cells with the pharmaceutical composition disclosed herein under conditions sufficient to cause the nucleic acid to be incorporated into cells.In some embodiments, the cells are contacted in vitro or ex vivo.In some embodiments, the cells are contacted in vitro.In some embodiments, the regulation of gene expression is sufficient to treat disease or disorder.In some embodiments, the nucleic acid is mRNA.

[0020] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating certain aspects of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0021] The following drawings form part of the present specification and are provided to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] [Figure 1] FIG. 1 shows a block diagram detailing one embodiment of the lipid nanoparticle composition of the present disclosure. [Figure 2A]In vitro delivery in ALI Calu-3 cells is shown. Figure 2A: Composition of Set 1 LNPs. Figure 2B: Quantification of luminescence from ALI Calu-3 cells 24 hours after transfection with aerosolized Set 1 LNPs delivering 1000 ng of NLuc mRNA. Significance is relative to NLuc F11 (n = 3; mean ± standard deviation, ** p < 0.01, *** p < 0.001; Student's t-test, two-tailed). Figure 2C: Structure of ionizable lipids used in Sets 1 and 2 LNPs. Figure 2D: Composition of Set 2 LNPs. Figure 2E: Quantification of luminescence from ALI Calu-3 cells 24 hours after transfection with aerosolized Set 2 and A-1 LNPs delivering 1000 ng of NLuc mRNA (n = 3; mean ± standard deviation, ** p < 0.01, *** p < 0.001; Student's t test, two-tailed). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 3] Figure 3 shows the physicochemical properties of non-aerosolized and aerosolized LNPs of the present disclosure. Figure 3A: Size (nm) by DLS (n = 3; mean ± standard deviation). Figure 3B: PDI by DLS (n = 3; mean ± standard deviation). Figure 3C: Zeta potential (mV) by DLS (n = 3; mean ± standard deviation). Figure 3D: Encapsulation efficiency (%) by Ribogreen (n = 2; mean ± standard deviation). [Figure 4]TEM images of NLuc F11, A-1, B-1, and NLuc Onpattro visualized at 43,000x magnification before (top) and after (bottom) aerosolization. Before aerosolization, A-1 had a uniform distribution of small, spherical particles. Before aerosolization, B-1 had a uniform distribution of small, spherical particles. After aerosolization, A-1 was still spherical but more polydisperse and larger in size. After aerosolization, B-1 was more polydisperse, larger in size, and had more surface defects than A-1. [Figure 5] We present evidence that B-1 enables significantly higher transfection efficiency in Balb / c mouse lungs compared to the baseline NLuc F11. Figure 5A: View of five mouse lung lobes in vivo and after harvest. Created with BioRender.com. Figure 5B: Brightness of each of the five Balb / c mouse lung lobes 24 hours after intratracheal administration of aerosolized LNPs delivering 750 ng of NLuc mRNA. Figure 5C: Brightness quantification of Figure 5A (n = 4; mean ± standard deviation, ** p < 0.01, *** p < 0.001; Student's t-test, two-tailed). [Figure 6] Next-generation impaction (NGI) results at a flow rate of 15 L / min are shown. Figure 6A: Droplet distribution in the human airway as a function of NGI stage and particle size. Generated with BioRender.com. Figure 6B: Deposition profile across the device, throat, stages 1-7, and MOC (n = 2; mean ± standard deviation). Figure 6C: Aerodynamic performance (n = 2; mean ± standard deviation). [Figure 7]The effect of SM-102 analogs on LNP transfection is shown. Figure 7A: Schematic of SM-102 structure relative to the analog. Figure 7B: Structural changes of SM-102 analogs compared to SM-102. Figure 7C: Quantification of luminescence from ALI Calu-3 cells 24 hours after transfection with aerosolized SM-102 analog LNPs delivering 1000 ng of NLuc mRNA. Significance is relative to B-1 (n = 3; mean ± standard deviation, * p < 0.05, ** p < 0.01, *** p < 0.001; ns = not statistically significant; Student's t-test, two-tailed). Figure 7D: Deposition profile of C-1 compared to B-1 across the device, throat, stages 1-7, and MOC (n = 2; mean ± standard deviation). Figure 7E: Aerodynamic performance of C-1 compared to B-1 (n = 2; mean ± standard deviation). [Figure 8] Figure 8 shows in vitro delivery of non-aerosolized LNPs in ALI Calu-3 cells. Quantification of luminescence from ALI Calu-3 cells 24 hours after transfection with non-aerosolized (Figure 8A) Set 1 and (Figure 8B) Set 2 LNPs delivering 1000 ng of NLuc mRNA (n = 3; mean ± standard deviation). [Figure 9] Assessment of cell types in the lungs for in vivo delivery is shown. Figure 9A: Schematic of the delivery schedule for the Rosa26 locus and Cre B-1 LNP in Ai9 mice. Successful Cre-mediated recombination results in deletion of the stop cassette, and the CAG promoter drives tdTomato expression. Aerosolized Cre B-1 LNP was delivered at 0.5 mg / kg every other day for 4 days. Lungs were harvested 3 days after the last dose. Figure 9B: Quantification of tdTomato brightness from Ai9 mouse lungs after Cre B-1 delivery compared to PBS controls. Figure 9C: Assessment of tdTomato cells in endothelial, epithelial, and immune cell populations using flow cytometry (n = 3; mean ± standard deviation, ** p < 0.01; Student's t-test, two-tailed). [Figure 10] Quantification of brightness in each individual lung lobe from Figure 5B is shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Provided herein are lipid nanoparticle (LNP) carriers for biologically active nucleic acid molecules. In some embodiments, the LNPs can contain single-stranded or double-stranded RNA or DNA. Such polynucleotides can be encapsulated in lipid nanoparticles or complexed with lipid nanoparticles. For example, in some cases, polynucleotides such as mRNA are provided complexed with LNPs. In certain aspects, the LNPs comprise at least one cationic ionizable lipid, at least one phospholipid, at least one PEGylated lipid, and cholesterol, as depicted in FIG. 1. In certain aspects, the polynucleotide-LNPs can be used for gene replacement and / or gene editing. In some aspects, the mRNA-LNP complexes can encode therapeutically active proteins (e.g., for gene replacement therapy) or antigens (e.g., for vaccination). In a preferred aspect, the LNP carrier and the biologically active polynucleotide can be formulated as an aerosol (e.g., by nebulization), such as for delivery to the lungs. In a further aspect, the LNP complex is provided as a dry powder, such as by ultra-rapid freezing (URF). Preferably, such a dry powder composition further comprises at least a first excipient. For example, an RNA-LNP powder further comprising at least a first excipient, such as a sugar or an amino acid, is provided. In some aspects, the dry powder can be directly administered to a subject (e.g., by dispersion into the lungs) to treat a disease or stimulate an immune response. In some embodiments, the pharmaceutical compositions of the present disclosure may exhibit improved nucleic acid delivery. The pharmaceutical compositions may demonstrate less degradation by natural RNAses, less targeting or tagging by antibodies, and therefore less clearance by alveolar macrophages, or less susceptibility to being trapped in mucus and removed via mucociliary clearance. The pharmaceutical compositions of the present disclosure may have favorable transfection efficiency, particularly in lung cells, in addition to or in combination with any of the above properties.

[0024] I. Nanoparticles and Nanoparticle Compositions As used herein, the term "nanoparticle" refers to any substance having dimensions in the range of 1 to 1,000 nm. In some embodiments, nanoparticles have dimensions in the range of 50 to 500 nm. Nanoparticles used in this embodiment include nanoscale substances such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes (Ferrari, 2005). Conjugation of polypeptides or nucleic acids to nanoparticles provides structures with potential applications in targeted delivery, controlled release, enhanced cellular uptake and trafficking, and molecular imaging of therapeutic peptides in vitro and in vivo (West, 2004; Stayton et al., 2000; Ballou et al., 2004; Frangioni, 2003; Dubertret et al., 2002; Michalet et al., 2005; Dwarakanath et al., 2004).

[0025] (1) Lipid nanoparticles (LNPs) Lipid-based nanoparticles include liposomes, lipid preparations, and lipid-based vesicles. Lipid-based nanoparticles may be positively charged, negatively charged, or neutral. In a preferred embodiment of the present disclosure, the lipid-based nanoparticles of the present disclosure comprise cationic ionizable lipids.

[0026] "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the creation of an enclosed lipid bilayer or aggregate. Liposomes can be characterized as having a vesicular structure with a bilayer generally comprising phospholipids and an internal medium generally comprising an aqueous composition. Liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged.

[0027] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilic molecules, or molecules with lipophilic regions, can also be dissolved in or associated with the lipid bilayer.

[0028] In certain aspects, the polypeptide or nucleic acid may be, for example, encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, encapsulated within the liposome, or complexed with the liposome, etc.

[0029] The size of liposomes varies depending on the method of synthesis. Liposomes in this embodiment can be of various sizes. In certain embodiments, the liposomes are small, e.g., having an outer diameter of less than about 200 nm, about 190 nm, about 180 nm, about 170 nm, about 160 nm, about 150 nm, about 140 nm, about 130 nm, about 120 nm, about 110 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or about 50 nm. For example, generally, prior to nucleic acid uptake, liposomes for use in this embodiment comprise a size of about 50-250 nm. Such liposome formulations may also be defined by particle charge (zeta potential) and / or optical density (OD). For example, liposome formulations typically have an OD of less than 0.45 prior to nucleic acid uptake. 400 Similarly, the overall charge of such particles in solution can be defined by a zeta potential of about 50-80 mV. In other embodiments, the liposomes or lipid nanoparticles may have a larger diameter, such as about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1000 nm, or any range derivable therein.

[0030] Liposomes provided by the present disclosure are shown, for example, above, in the Summary of the Invention section, and in the appended claims. They can be produced using the methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry applied by those skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated herein by reference.

[0031] Furthermore, any protocol described herein or known to those skilled in the art can be used in preparing such liposomes. Further non-limiting examples of liposome preparations are described in WO02 / 100435A1, WO03 / 015757A1, WO04029213A2, U.S. Patent Application No. 2004 / 0208921, U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; 5,030,453, 5,962,016, 6,680,068, International Application Nos. PCT / US85 / 01161 and PCT / US89 / 05040; UK Patent Application No. GB ​​2193095 A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al. 1987; Mayhew et al., 1984; Cheng et al., 1987; and Liposome Technology, 1984, each of which is incorporated herein by reference. A process for making liposomes is also described in WO04 / 002453A1.

[0032] In certain embodiments, the lipid-based nanoparticles are positive liposomes. As used herein, "positive liposomes" or "cationic liposomes" are defined as liposomes having one or more lipid components that result in an essentially positive net charge (substantially positive). "Essentially positive" means that the lipid components within a given population (e.g., a population of liposomes) as a whole contain a positive charge that is not offset by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1%). In certain embodiments, positive liposomes may primarily contain lipids and / or phospholipids that are themselves positive under physiological conditions (i.e., at about pH 7). As used herein, lipid components that result in the essentially positive net charge of positive liposomes, i.e., lipids that are themselves positive under physiological conditions, may also be known as cationic ionizable lipids. In some embodiments, cationic ionizable lipids are neutral at physiological pH and may be positively charged at acidic pH or in an acidic environment. The local microenvironment surrounding the cationic ionizable lipid can affect the protonation state of the cationic ionizable lipid, resulting in a positively charged cationic ionizable lipid under conditions that would not otherwise result in a positively charged cationic ionizable lipid.In some embodiments, the cationic ionizable lipid is an amino lipid.In some embodiments, the cationic ionizable lipid comprises a tertiary amine.In some embodiments, the alkyl group attached to the tertiary amine can be independently substituted with a functional group such as an ester or a hydroxyl group.In some embodiments, the cationic ionizable lipid is SM-102, MC3 or ALC-0315.

[0033] The cationic ionizable lipid component of the lipid nanoparticle composition of the present disclosure can be present in various molar ratios relative to the composition. In some embodiments of the present invention, the cationic ionizable lipid is present in a molar ratio of about 0.2 to about 1.0 relative to the lipid nanoparticle composition. In some embodiments, the molar ratio of cationic ionizable lipid to the lipid nanoparticle composition is about 0.3 to about 0.7, or about 0.4 to about 0.6. The molar ratio of cationic lipid to the lipid nanoparticle composition can be about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0, or any range derivable therein. In some embodiments, the molar ratio of cationic lipid to the lipid nanoparticle composition is about 0.45.

[0034] The cationic ionizable lipids and other lipids of the present disclosure contain one or more asymmetrically substituted carbon or nitrogen atoms and can be isolated in optically active or racemic form.Therefore, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric and all geometric isomeric forms of the chemical formula are intended.The cationic ionizable lipids can occur 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 cationic ionizable lipids of the present disclosure can have an S or R configuration.Furthermore, it is contemplated that one or more of the cationic ionizable lipids can exist as structural isomers.In some embodiments, the compounds have the same formula but different connectivity.

[0035] The chemical formula used to represent the cationic ionizable lipid of the present disclosure will typically only show one of several different tautomers.For example, it is known that many types of ketone groups exist in equilibrium with corresponding enol groups.Similarly, many types of imine groups exist in equilibrium with enamine groups.No matter which tautomer is depicted for a given formula, all tautomers of a given chemical formula are intended, no matter which one is the most common.

[0036] The cationic ionizable lipids of the present disclosure, whether used in the indications described herein or otherwise, may also be advantageous in that they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be readily absorbed, metabolically stable, lipophilic, hydrophilic, and / or have a favorable pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance) than compounds known in the prior art, and / or have other useful pharmacological, physical, or chemical properties compared to compounds known in the prior art.

[0037] Furthermore, the atoms that make up the cationic ionizable lipids of the present disclosure are intended to include all isotopic forms of such atoms.Isotopes as used herein include atoms with the same atomic number but different mass numbers.By way of general example, and without limitation, hydrogen isotopes include tritium and deuterium, and carbon isotopes include tetrahydrogen and tetrahydrofuran. 13 C and 14 Contains C.

[0038] It should be recognized that the specific anion or cation that forms part of any salt form of cationic ionizable lipid provided herein is not important, as long as the salt as a whole is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0039] (2) Lipids In some aspects of the present disclosure, one or more types of additional lipids are mixed with the cationic ionizable lipid of the present disclosure to create nanoparticle composition.In some embodiments, cationic ionizable lipid is mixed with 1, 2, 3, 4 or 5 different lipids.It is contemplated that cationic ionizable lipid can be mixed with multiple different lipids of a single type.

[0040] In some embodiments, at least one of the additional lipids can be a steroid or steroid derivative. In some embodiments, the additional lipid can be a PEG-lipid. In some embodiments, the additional lipid can be a phospholipid. In some embodiments, the nanoparticle composition comprises a steroid or steroid derivative, a PEG-lipid and a phospholipid, or any combination thereof. Further details of the types of lipids that can be used to form the nanoparticle composition are provided in the following sections.

[0041] Steroids and steroid derivatives In the lipid nanoparticle compounds of the present disclosure, a cationic ionizable lipid (or compound) is mixed with one or more steroids or steroid derivatives and other ingredients described below to form a nanoparticle composition. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" refers to a class of compounds having a tetracyclic 17-carbon ring structure that may further include one or more substitutions, including alkyl, alkoxy, hydroxy, oxo, acyl, or double bonds between two or more carbon atoms. In one aspect, the ring structure of the steroid includes three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula: TIFF2025538217000007.tif17128.

[0042] 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, the formula of which is further defined as follows: TIFF2025538217000008.tif18128.

[0043] Another steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the following formula: TIFF2025538217000009.tif35128.

[0044] As described above, the cholestane derivative contains one or more non-alkyl substitutions of the ring system. The cholestane or cholestane derivative can be cholestene or a cholestene derivative or a sterol or a sterol derivative. The cholestane or cholestane derivative can be both cholestene and a sterol or a derivative thereof. In a preferred embodiment, the nanoparticle composition comprises cholesterol.

[0045] In some embodiments, the composition comprises a molar ratio of steroid or steroid derivative to lipid nanoparticle composition of about 0.05 to about 0.12 or about 0.1 to about 0.6. The molar ratio can be about 0.15 to about 0.5, such as a molar ratio of steroid or steroid derivative to lipid nanoparticle composition of about 0.34. In some embodiments, the molar ratio of steroid or steroid derivative to lipid nanoparticle composition is about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.4, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about 0.85, about 0.90, about 1.0, or about 1.2, or any range derivable therein.

[0046] PEG or PEGylated lipids In the lipid nanoparticle compositions of the present disclosure, a cationic ionizable lipid (or compound) is mixed with one or more PEGylated lipids (or PEG lipids) and other ingredients described above and below to form a nanoparticle composition. In some embodiments, the present disclosure includes using any lipid with a PEG group attached. In some embodiments, the PEG lipid is a diglyceride that also contains a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group by a PEG chain. In some embodiments, the PEG-lipid has advantages such as preventing aggregation or reducing uptake of the composition by immune cells. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide-conjugated PEG-modified dialkylamine and PEG-modified 1,2-diacyloxypropan-3-amine, PEG-modified diacylglycerol and dialkylglycerol. In some embodiments, the PEG lipid is PEG-modified diastearoylphosphatidylethanolamine. In some embodiments, the PEG lipid comprises a PEG-modified phospholipid, such as any of the lipids mentioned in the following section. In some embodiments, the PEG lipid is a PEG-modified dimyristoyl phosphatidylethanolamine or a PEG-modified myristoyl diglyceride.

[0047] In some embodiments, the 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 to about 5,000. In some embodiments, the molecular weight is about 200 to about 500 or about 1,200 to about 3,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught by U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0048] In some embodiments, the composition comprises a molar ratio of PEG-lipid to lipid nanoparticle composition of about 0.001 to about 0.04 or about 0.005 to about 0.03. The molar ratio can be about 0.01 to about 0.015. In some embodiments, the molar ratio of PEG-lipid to lipid nanoparticle composition can be about 0.01. In some embodiments, the ratio is about 0.001, about 0.005, about 0.006, about 0.007, about 0.008, about 0.009, about 0.01, about 0.011, about 0.012, about 0.013, about 0.014, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035 to about 0.04, or any range derivable therein.

[0049] phospholipids In the lipid nanoparticle compositions of the present disclosure, a cationic ionizable lipid (or compound) is mixed with one or more phospholipids and other ingredients described above and below to form a nanoparticle composition. Phospholipids may also be referred to herein as "helper lipids." In some embodiments, the compositions disclosed herein include a helper lipid containing a phosphate group. In some embodiments, two or more phospholipids may be used to form the composition. In some embodiments, the phospholipid is a structure comprising one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and, optionally, an organic small molecule. In some embodiments, the organic small molecule is 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 distearoylphosphatidylcholine, dioleoylphosphatidylcholine, or dipalmitoylphosphatidylcholine. In some embodiments, the helper lipid can be neutral under physiological conditions (i.e., at about pH 7). In some embodiments, the helper lipid has the advantage of improving structure or enhancing endosomal escape.

[0050] Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine, because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), and these compounds may be particularly useful for creating cationic liposomes. In certain embodiments, the phospholipid DPPC is used to produce cationic liposomes.

[0051] Phospholipids that can be components of the compositions disclosed herein include glycerophospholipids and certain sphingolipids. Phospholipids include dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("DSPC"). 1-Palmitoyl-2-stearoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG") "), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylethanolamine ("DM ... phatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoylsphingomyelin ("DPSP"), dimyristylphosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,Examples of suitable phosphatidylcholine include, but are not limited to, 2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyleoylphosphatidylcholine ("POPC"), palmitoyleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0052] In some embodiments, the composition comprises a molar ratio of phospholipid to lipid nanoparticle composition of about 0.01 to about 0.5 or about 0.02 to about 0.4. The molar ratio can be about 0.05 to about 0.3, such as a molar ratio of about 0.2. In some embodiments, the molar ratio of phospholipid to lipid nanoparticle composition is about 0.01, about 0.03, about 0.05, about 0.07, about 0.09, about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.2, about 0.22, about 0.24, about 0.26, about 0.28, about 0.3, about 0.35 to about 0.4, or any range derivable therein.

[0053] Phospholipid can be derived from natural or synthetic sources.However, in some embodiments, the phospholipid from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and plant or bacterial phosphatidylethanolamine, is not used as the main phosphatide (i.e., constitutes more than 50% of the total phosphatide composition), because it may cause the resulting liposome to be unstable and prone to leaking.

[0054] II. Biologically Active Polynucleotides The methods and compositions of the embodiments relate to biologically active polynucleotides. In some cases, these may comprise single-stranded or double-stranded RNA or DNA. It should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. However, those skilled in the art can easily identify related homologues in various other sources of nucleic acids, including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat and other species), so the present disclosure is not limited in scope to any particular source, sequence or type of nucleic acid. It is intended that the nucleic acid used in the present disclosure may include sequences based on naturally occurring sequences.

[0055] The amount of nucleic acid encapsulated by or located within the lipid nanoparticles can vary based on the intended use. The amount of nucleic acid can be calculated as a ratio (w / w) to the lipid nanoparticle composition or to any of the individual components of the lipid nanoparticle composition. For example, the ratio of cationic ionizable lipid to nucleic acid can be about 50:1 (w / w), about 20:1 (w / w), about 15:1 (w / w), about 14:1 (w / w), about 13:1 (w / w), about 12:1 (w / w), about 11:1 (w / w), about 10:1 (w / w), about 9:1 (w / w), about 8:1 (w / w), about 7:1 (w / w), about 6:1 (w / w) to about 5:1 (w / w), or any range derivable therein. In some embodiments, the ratio of cationic ionizable lipid to nucleic acid is about 11.33 (w / w).The length of the nucleic acid that is encapsulated by or located within lipid nanoparticles can also vary based on intended use.The length of nucleic acid can be about 20bp, about 50bp, about 75bp, about 100bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, about 500bp, about 550bp, about 600bp, about 650bp, about 700bp, about 750bp, about 800bp, about 850bp, about 900bp, about 950bp, about 1000bp, or any range that can be derived therein. Longer nucleic acids are also contemplated, such as nucleic acids of about 1500 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 3500 bp, about 4000 bp, about 4500 bp, about 5000 bp, about 5500 bp, about 6000 bp, about 6500 bp, about 7000 bp, about 7500 bp, about 8000 bp, about 8500 bp, about 9000 bp, about 9500 bp, about 10,000 bp, or any range derivable therein.

[0056] In some aspects, nucleic acid is a sequence that silences, is complementary to, or replaces another sequence that exists in vivo.A sequence of 17 bases in length should only occur once in human genome, and therefore is sufficient to specify a unique target sequence.Although shorter oligomers are easier to create and increase in vivo accessibility, many other factors are involved in determining the specificity of hybridization.Both the binding affinity and sequence specificity of oligonucleotide to its complementary target increase with increasing length.8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100 or more base pairs are used as exemplary oligonucleotides, but other ones are also contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or more are similarly contemplated.

[0057] The nucleic acids used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. However, in preferred embodiments, the nucleic acids will comprise complementary DNA (cDNA). cDNAs containing natural introns or introns derived from another gene are also contemplated; such engineered molecules are sometimes referred to as "minigenes." At a minimum, these and other nucleic acids of the present disclosure can be used, for example, as molecular weight standards in gel electrophoresis.

[0058] The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, unprocessed, or partially processed RNA template, is that the cDNA contains primarily the coding sequence of the corresponding protein. There may be cases where a complete or partial genomic sequence is preferred, such as when non-coding regions are required for optimal expression or when non-coding regions such as introns are targeted in antisense strategies.

[0059] In some embodiments, the nucleic acid comprises one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methodology utilizes the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that the polynucleotides are capable of base pairing according to the standard Watson-Crick complementarity rules. That is, larger purines will base pair with smaller pyrimidines to form combinations such as guanine paired with cytosine (G:C), adenine paired with thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. The inclusion of less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others, in the hybridizing sequence does not interfere with pairing.

[0060] Targeting double-stranded (ds) DNA with a polynucleotide will result in triple helix formation; targeting RNA will result in double helix formation. When introduced into a target cell, an antisense polynucleotide specifically binds to its target polynucleotide and interferes with transcription, RNA processing, transport, translation, and / or stability. Antisense RNA constructs, or DNA encoding such antisense RNA, can be used to inhibit gene transcription or translation, or both, in host cells, either in vitro or in vivo, for example, in host animals, including human subjects.

[0061] Antisense constructs can be designed to bind to promoters and other control regions, exons, introns, or even exon-intron boundaries of genes. It is believed that the most effective antisense constructs contain regions complementary to intron / exon splice junctions. Therefore, preferred embodiments are proposed to include antisense constructs with complementarity to regions within 50-200 bases of intron-exon splice junctions. It has been observed that some exon sequences can be included in the construct without significantly affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. It is easy to test whether too much exon DNA has been included by simply testing the construct in vitro to determine whether normal cellular function is affected or whether expression of the associated gene bearing the complementary sequence is affected.

[0062] As mentioned above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary over their entire length and have few base mismatches. For example, a sequence 15 bases in length can be called complementary if it has complementary nucleotides at 13 or 14 positions. Of course, a fully complementary sequence would be one that is completely complementary over its entire length and has no base mismatches. Other sequences with lower degrees of homology are also contemplated. For example, antisense constructs (e.g., ribozymes; see below) can be designed that contain limited regions of high homology but also non-homologous regions. These molecules have less than 50% homology but will bind to the target sequence under appropriate conditions.

[0063] It may be advantageous to combine part of genomic DNA with cDNA or synthetic sequence to form siRNA or create specific construct.For example, if intron is desired in final construct, it is necessary to use genomic clone.cDNA, siRNA or synthetic polynucleotide can provide more convenient restriction site for the rest of construct, and therefore be used for the rest of sequence.Other embodiments include dsRNA or ssRNA that can be used to target genomic sequence or coding / non-coding transcript.

[0064] In other embodiments, the nanoparticles may contain nucleic acids comprising one or more expression vectors used in gene therapy. Expression requires the provision of appropriate signals in the vector, including various regulatory elements, such as enhancers / promoters from both viral and mammalian sources, that drive the expression of the gene of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells are also defined. Conditions for the use of several key drug selection markers to establish permanent, stable cell clones expressing the product are also provided, as are elements that link the expression of drug selection markers to the expression of polypeptides.

[0065] Throughout this application, the term "expression construct" is intended to include any type of genetic construct that includes a nucleic acid encoding a gene product in which part or all of the nucleic acid coding sequence can be transcribed. The transcription product may, but need not, be translated into a protein. In certain embodiments, expression includes both transcription of the gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding the gene of interest.

[0066] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but is in a location within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the art would be knowledgeable to construct vectors using standard recombinant techniques as described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0067] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of a gene product capable of being transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription, and optionally translation, of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions as well, as described below.

[0068] mRNA In some aspects, the compounds and compositions of the present invention can be used to deliver mRNA to cells. Messenger RNA, or mRNA, is a short RNA strand that transmits genetic code from DNA to ribosomes, where it can be translated into therapeutic proteins or peptides or antigens. The mRNA described herein can be unprocessed, processed to add a poly(A) tail, edited in vivo, or 5'-capped. mRNA molecules can include a 5' UTR or 3' UTR. The compositions are contemplated for the delivery of a variety of different mRNAs, including unprocessed and further processed ones. Furthermore, these nucleic acids can be used therapeutically, to produce antibodies in vivo, or in vaccine formulations. mRNA molecules can provide a more direct method of expressing a polypeptide of interest in target cells. However, such molecules are typically very unstable and rapidly degraded. In some aspects, LNP processing according to embodiments can be used to substantially stabilize mRNA. In a preferred aspect, the mRNA is provided encapsulated in or complexed with LNPs.

[0069] As noted above, in some aspects, the nucleic acid molecules of the embodiments encode a therapeutic polypeptide. For example, the therapeutic protein can be a protein, such as an enzyme that is non-functional or disrupted in a particular disease state (e.g., CFTR in cystic fibrosis).

[0070] In a further aspect, the polynucleotide of the embodiment encodes an antigen, for example, an antigen derived from a pathogen or a cancer cell-associated antigen. For example, the cancer-associated antigen can be CD19, CD20, ROR1, CD22, carcinoembryonic antigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, folate-binding protein, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2. In some specific aspects, the antigen is GP240, 5T4, HER1, CD-33, CD-38, VEGFR-1, VEGFR-2, CEA, FGFR3, IGFBP2, IGF-1R, BAFF-R, TACI, APRIL, Fn14, ERBB2, or ERBB3.

[0071] Antigens useful in the present disclosure include Arenaviridae (e.g., lymphocytic choriomeningitis virus), Arteriviridae (e.g., equine arteritis virus), Astroviridae (human astrovirus 1), Birnaviridae (e.g., infectious pancreatic necrosis virus, infectious bursal disease virus), Bunyaviridae (e.g., California encephalitis virus group), Caliciviridae (e.g., calicivirus), Coronaviridae (e.g., human coronaviruses 299E and OC43), Deltaviruses (e.g., hepatitis delta virus), Filoviridae (e.g., Marburg virus, Ebola virus), Flaviviridae (e.g., yellow fever virus group, hepatitis C virus), Hepadnaviridae (e.g., hepatitis B virus), Herpesviridae (e.g., Epstein-Barr virus, simplex virus group), , Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g., influenza viruses A, B, and C), Parvoviridae (e.g., Papillomavirus), Paramyxoviridae (e.g., Paramyxovirus, e.g., human parainfluenza virus 1, Morbillivirus, e.g., measles virus, Rubulavirus, e.g., mumps virus, Pneumovirus, e.g., human respiratory syncytial virus), Picornaviridae (e.g., Rhinovirus, e.g., human rhinovirus 1A, Hepatovirus, e.g., human hepatitis A virus, human poliovirus, Cardiovirus, e.g., encephalomyocarditis virus, Aphthovirus, e.g., foot-and-mouth disease virus O, Coxsackievirus), Poxyiridae(e.g., Orthopoxvirus, such as smallpox virus or monkeypox virus), Reoviridae (e.g., Rotavirus, such as groups A to F rotavirus), Retroviridae (primate lentivirus group, such as human immunodeficiency virus types 1 and 2), Rhabdoviridae (e.g., rabies virus), Togaviridae (e.g., Rubivirus, such as rubella virus), human T-cell leukemia virus, murine leukemia virus, vesicular stomatitis virus, wart virus, bluetongue virus, Sendai virus, feline leukemia virus, Simian virus 40, mouse mammary tumor virus, dengue virus, HIV-1 and HIV-2, West Nile, H1N1, These may include, but are not limited to, those derived from viruses such as SARS, 1918 influenza, tick-borne encephalitis group viruses (Absettarov, Hanzalova, Hypr), Russian spring-summer encephalitis virus, Congo-Crimean hemorrhagic fever virus, Junin virus, Kumlinge virus, Marburg virus, Machupo virus, Kyasanur Forest disease virus, Lassa virus, Omsk hemorrhagic fever virus, FIV, SIV, herpes simplex types 1 and 2, varicella-zoster, human parvovirus (B19), respiratory syncytial virus, poxvirus (all types and serotypes), Coltivirus, reovirus - all types, and / or Rubivirus (rubella).

[0072] Antigens useful in the present disclosure include bacteria, such as, but not limited to, Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis, Streptococcus pneumoniae, Hemophilis influenzae type B, Treponema pallidum, the causative spirochete of Lyme disease, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, and the like. tuberculosis, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis spiralis), Theileria parva, Taenia hydatigena, ovine tapeworm (Taenia ovis), Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Aspergillus fumigatus fumigatus, Penicillium marneffei, Bacillus anthracis, Bartonella spp., Bordetella pertussis, Brucella - all serotypes, Chlamydia trachomatis, Chlamydia pneumoniae, Clostridium botulinum - any from the Clostridium serotypes, Haemophilus influenzae, Helicobacter pylori, Klebsiella spp. - all serotypes, Legionella spp. - all serotypes, Listeria monocytogenes, Mycobacterium spp. - all serotypes, Mycoplasma spp. - human and animal serotypes, Rickettsia spp. - all serotypes, Shigella spp. - all serotypes, Staphylococcus aureus aureus, Streptococcus - S. pneumoniae, S. pyogenes, Vibrio cholera, Yersinia enterocoliticaenterocolitica and / or Yersinia pestis.

[0073] Antigens useful in the present disclosure may include those derived from parasites such as, but not limited to, human hookworms of the genus Ancylostoma, Leishmania - all strains, Microsporidium, human hookworms of the genus Necator, Onchocerca, Plasmodium - all human strains and simian species, Toxoplasma - all strains, Trypanosoma - all serotypes and / or Wuchereria bancrofti.

[0074] siRNA As noted above, the present disclosure contemplates the use of one or more inhibitory nucleic acids to reduce the expression and / or activation of a gene or gene product. Examples of inhibitory nucleic acids include, but are not limited to, molecules that target nucleic acid sequences, such as siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and molecules that target genes or gene products, such as aptamers.

[0075] Inhibitory nucleic acids can inhibit transcription of a gene or prevent translation of a gene transcript in a cell. Inhibitory nucleic acids can be 16 to 1000 nucleotides in length, and in certain embodiments, 18 to 100 nucleotides in length.

[0076] Inhibitory nucleic acid is well known in the art.For example, siRNA, shRNA and double-stranded RNA are described in United States Patent (USP) No. 6,506,559 and United States Patent (USP) No. 6,573,099, and United States Patent Application Publication No. 2003 / 0051263, United States Patent Application Publication No. 2003 / 0055020, United States Patent Application Publication No. 2004 / 0265839, United States Patent Application Publication No. 2002 / 0168707, United States Patent Application Publication No. 2003 / 0159161 and United States Patent Application Publication No. 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0077] Since the discovery of RNAi by Fire and colleagues in 1998, the biochemical mechanism has been rapidly characterized. Double-stranded RNA (dsRNA) is cleaved by Dicer, a ribonuclease of the RNAase III family. This process yields siRNAs approximately 21 nucleotides long. These siRNAs are incorporated into the multiprotein RNA-induced silencing complex (RISC), which is guided to the target mRNA. RISC cleaves the target mRNA in the center of the complementary region. In mammalian cells, related microRNAs (miRNAs), which are short RNA fragments (approximately 22 nucleotides), are found. miRNAs are generated after Dicer-mediated cleavage of longer (approximately 70 nucleotides) precursors with imperfect hairpin RNA structures. miRNAs are incorporated into miRNA-protein complexes (miRNPs), which result in translational repression of the target mRNA.

[0078] When designing a nucleic acid capable of producing an RNAi effect, several factors must be considered, such as the nature of the siRNA, the duration of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA introduced into an organism will typically contain exon sequences. Furthermore, because the RNAi process depends on homology, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. In particular, siRNAs exhibit greater than 80, 85, 90, 95, 98%, or even 100% identity between the siRNA sequence and a portion of the EphA nucleotide sequence. Sequences with less than about 80% identity to the target gene are substantially less effective. Therefore, the higher the identity between the siRNA and the gene to be inhibited, the less likely it is that the expression of unrelated genes will be affected.

[0079] Furthermore, the size of the siRNA is an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that contain at least about 19 to 25 nucleotides and can regulate gene expression. In the context of the present disclosure, siRNAs are particularly less than 500, 200, 100, 50, 25, or 20 nucleotides in length. In some embodiments, siRNAs are about 25 to about 35 nucleotides in length or about 19 to about 25 nucleotides in length.

[0080] To improve the efficacy of siRNA-mediated gene silencing, guidelines for selecting target sites on mRNA have been developed for optimal siRNA design (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies may enable a rational approach to selecting siRNA sequences to achieve maximum gene knockdown. To facilitate the entry of siRNA into cells and tissues, various vectors, including plasmids and viral vectors such as adenoviruses, lentiviruses, and retroviruses, have been used (Wadhwa et al., 2004).

[0081] Within an inhibitory nucleic acid, the components of the nucleic acid need not be of the same type or species throughout (e.g., an inhibitory nucleic acid can contain nucleotides and nucleic acid or nucleotide analogs). Typically, an inhibitory nucleic acid forms a double-stranded structure; the double-stranded structure can result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present disclosure, an inhibitory nucleic acid contains only a single nucleic acid (polynucleotide) or nucleic acid analog, which can form a double-stranded structure by becoming complementary with itself (e.g., forming a hairpin loop). The double-stranded structure of an inhibitory nucleic acid can contain from 16 to 500 or more consecutive nucleobases, including all derivable ranges. The inhibitory nucleic acid may comprise 17 to 35 consecutive nucleobases, more particularly 18 to 30 consecutive nucleobases, more particularly 19 to 25 nucleobases, more particularly 20 to 23 consecutive nucleobases, or 20 to 22 consecutive nucleobases, or 21 consecutive nucleobases, that hybridize with a complementary nucleic acid (which may be another portion of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[0082] siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of several techniques known to those skilled in the art.For example, commercial sources of pre-designed siRNA include Invitrogen's Stealth™ Select technology (Carlsbad, CA), Ambion® (Austin, TX), and Qiagen® (Valencia, CA).The inhibitory nucleic acid applicable to the compositions and methods of the present disclosure can be any nucleic acid sequence found by any source to be a verified down-regulator of gene or gene product.

[0083] In some embodiments, the present disclosure features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least 10 but not more than 30 consecutive nucleotides of a nucleic acid encoding a gene, and that reduces expression of the gene or gene product. In one embodiment of the present disclosure, the siRNA molecule has at least one strand that is substantially complementary to at least 10 but not more than 30 consecutive nucleotides of an mRNA encoding the gene or gene product.

[0084] In one embodiment, the siRNA molecule is at least 75, 80, 85, or 90% homologous, particularly at least 95%, 99%, or 100% similar or identical, or any percentage therebetween, to at least 10 consecutive nucleotides of any of the nucleic acid sequences encoding the target therapeutic protein (e.g., the present disclosure contemplates 75% and more, 80% and more, 85% and more, etc., and the ranges are intended to include all integers therebetween).

[0085] siRNAs may also contain modifications of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, such as to the end or internal (one or more nucleotide positions) of the 19-25 nucleotide RNA. In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the RNA molecules of the present disclosure may also include non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Double-stranded oligonucleotides may contain modified backbones, such as phosphorothioates, phosphorodithioates, or other modified backbones known in the art, or non-natural internucleoside linkages. Further modifications of siRNA (such as 2'-O-methylribonucleotide, 2'-deoxy-2'-fluororibonucleotide, " universal base " nucleotide, 5-C-methyl nucleotide, one or more phosphorothioate internucleotide linkages, and inverted deoxybasic residues) can be found in US Patent Application Publication No. 2004 / 0019001 and US Patent No. 6,673,611 (each of which is incorporated by reference in its entirety).Collectively, all of the nucleic acid or RNA described above that are modified in this way are referred to as modified siRNA.

[0086] In one embodiment, the siRNA can reduce expression of a particular gene product by at least 10%, at least 20%, at least 30%, or at least 40%, at least 50%, at least 60%, or at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or more, or any range therebetween.

[0087] III. Definition When used in the context of chemical groups: "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also written as -COOH or -COH); "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH; "hydroxyamino" means -NHOH; "nitro" means -NO; imino means -NH; "cyano" means -CN; "isocyanato" means -N=C=O; "azido" means -N; in the monovalent context, "phosphate" means -OP(O)(OH) or its deprotonated form; in the divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated form; "mercapto" means -SH; "thio" means =S; "Sulfonyl" means -S(O)2-; "hydroxysulfonyl" means -S(O)2OH; "sulfonamido" means -S(O)2NH2; and "sulfinyl" means -S(O)-.

[0088] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. TIFF2025538217000010.tif4128 represents any bond, which, if present, may be either a single or double bond. TIFF2025538217000011.tif4128 indicates a single or double bond. Thus, for example, the formula TIFF2025538217000012.tif10128 TIFF2025538217000013.tif10128. It is understood that such ring atoms do not form part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereogenic atoms. Rather, it encompasses all stereoisomers and mixtures thereof. TIFF2025538217000014.tif4128 when drawn perpendicularly across the bond (For example, for methyl, TIFF2025538217000015.tif6128) , indicates the point of attachment of the group. Note that points of attachment are typically only identified in this manner on larger groups to aid the reader in clearly identifying the point of attachment. TIFF2025538217000016.tif4128 indicates a single bond in which the group attached to the thick end of the wedge is "off the page." TIFF2025538217000017.tif4128 indicates a single bond in which the group attached to the thick end of the wedge is "inside the page." TIFF2025538217000018.tif4128 refers to a single bond where the geometry around the double bond (e.g., either E or Z) is undefined. Therefore, both options, and combinations thereof, are contemplated. Any undefined valence on an atom of a structure shown in this application implies a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented out of the plane of the paper.

[0089] The group "R" may, for example, be of the formula: In TIFF2025538217000019.tif13128, if depicted as a "floating group" on the ring system, R may replace any hydrogen atom attached to any ring atom, including drawn, implied, or explicitly defined hydrogens, so long as a stable structure is formed. When depicted as a "floating group" on a fused ring system, as in TIFF2025538217000020.tif16128, R may replace any hydrogen bonded to any ring atom of any of the fused rings, unless otherwise specified. Substitutable hydrogens include drawn hydrogens (e.g., hydrogens bonded to nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens bonded to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present on either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the parenthesized group "R" represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substitutable hydrogen atoms in the ring or ring system.

[0090] In the case of chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, the minimum number being as small as possible for the group / class in question, e.g., the group "alkenyl (C≦8) " or class "Alkene (C≦8) " is understood to be the minimum number of carbon atoms in "alkoxy" which refers to an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. (C2~10) " denotes an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class that they modify, and may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "Olefins C5The terms " and " are all synonymous.

[0091] The term "saturated," when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. If such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that the substance is no longer soluble in the solution.

[0092] The term "aliphatic," when used without the "substituted" modifier, indicates that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms can be linked together in a straight chain, a branched chain, or a non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) linked by a single carbon-carbon bond, or unsaturated by one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0093] The term "aromatic," when used to modify an atom of a compound or chemical group, means a compound or chemical group that contains a planar, unsaturated ring of atoms stabilized by the interaction of bonds that form the ring.

[0094] The term "alkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. Examples include the groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu), and -CHC(CH)(neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH- (methylene), -CHCH-, -CHC(CH)CH-, and -CHCHCH- are non-limiting examples of alkanediyl groups. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl, as that term is defined above. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The following groups are non-limiting examples of substituted alkyl groups: -CHOH, -CHCl, -CF, -CHCN, -CHC(O)OH, -CHC(O)OCH, -CHC(O)NH, -CHC(O)CH, -CHOCH, -CHOC(O)CH, -CHNH, -CHN(CH), and -CHCHCl. ​​The term "haloalkyl" is a subgroup of substituted alkyl limited to the replacement of hydrogen atoms by halo (i.e., -F, -Cl, -Br, or -I) so that no other atoms other than carbon, hydrogen, and halogen are present. The group -CHCl is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subgroup of substituted alkyl limited to the replacement of hydrogen atoms by fluoro so that no other atoms other than carbon, hydrogen, and fluorine are present. The groups -CHF, -CF, and -CHCF are non-limiting examples of fluoroalkyl groups.

[0095] The term "cycloalkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having two carbon atoms as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Group TIFF2025538217000021.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula H-R, where R is cycloalkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0096] The term "alkenyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as a point of attachment, a straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCHCH3, and -CH=CHCH=CH2. The term "alkenediyl," when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, a straight-chain or branched, straight-chain or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is noted that although alkenediyl groups are aliphatic, when joined at both ends, this does not preclude the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene that has only one carbon-carbon double bond, which bond is part of a vinyl group at the end of the molecule. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0097] The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH, and -CHC≡CCH are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, where R is alkynyl. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0098] The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more six-membered aromatic ring structures, all ring atoms being carbon, and the group consisting of atoms other than carbon and hydrogen. When two or more rings are present, the rings may be fused or unfused. As used herein, this term does not exclude the presence of one or more alkyl or aralkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and biphenyl. The term "arenediyl," when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms as attachment points, the carbon atoms forming part of one or more six-membered aromatic ring structures, the ring atoms being all carbon, and the monovalent group consisting of no atoms other than carbon and hydrogen. As used herein, this term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limitations permitting) attached to the first aromatic ring or any additional aromatic rings present. When two or more rings are present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (carbon number limitations permitting). Non-limiting examples of arenediyl groups include: TIFF2025538217000022.tif32128 is an example.

[0099] "Arene" refers to the class of compounds having the formula H-R, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0100] The term "aralkyl," when used without the "substituted" modifier, refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the above definitions. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-eth-1-yl.

[0101] The term "heteroaryl," when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the heteroaryl group consisting of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. The heteroaryl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a point of attachment. The term "heteroarenediyl", when used without the modifier "substituted", refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, which atoms form one or more aromatic ring structures, at least one ring atom is nitrogen, oxygen, or sulfur, and the divalent group does not consist of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (where the carbon number limit permits). As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where the carbon number limit permits) attached to an aromatic ring or aromatic ring system.Non-limiting examples of heteroarenediyl groups include: TIFF2025538217000023.tif15128 is an example. "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0102] The term "heterocycloalkyl," when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the heterocycloalkyl group consisting of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. A heterocycloalkyl ring can contain one, two, three, or four ring atoms selected from nitrogen, oxygen, or sulfur. When more than one ring is present, the rings can be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number limitations permitting) attached to the ring or ring system. Similarly, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the attachment point. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl," when used without the modifier "substituted," refers to a divalent ring group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two attachment points, which form part of one or more ring structures, at least one ring atom being nitrogen, oxygen, or sulfur, and the divalent group consisting of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked through one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (carbon number limit permitting). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limit permitting) attached to the ring or ring system.Likewise, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: TIFF2025538217000024.tif13128. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0103] The term "acyl," when used without the "substituted" modifier, refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as those terms are defined above. The groups -CHO, -C(O)CH(acetyl, Ac), -C(O)CHCH, -C(O)CHCHCH, -C(O)CH(CH), -C(O)CH(CH), -C(O)CH(CH), -C(O)CHH, -C(O)CHCHCH, -C(O)CHCH, -C(O)(imidazolyl) are non-limiting examples of acyl groups. A "thioacyl" is defined in a similar manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, and is -C(S)R. The term "aldehyde" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly bonded to the carbon atom of the carbonyl or thiocarbonyl group, if any) are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0104] The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, where R is alkyl, as that term is defined above. Non-limiting examples include -OCH(methoxy), -OCHCH(ethoxy), -OCHCHCH, -OCH(CH)(isopropoxy), -OC(CH)(tert-butoxy), -OCH(CH), -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy," "alkenyloxy," "alkynyloxy," "aryloxy," "aralkoxy," "heteroaryloxy," "heterocycloalkoxy," and "acyloxy," when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio," when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH.

[0105] The term "alkylamino," when used without the "substituted" modifier, refers to the group -NHR, where R is alkyl, as that term is defined above. Non-limiting examples include -NHCH and -NHCHCH. The term "dialkylamino," when used without the "substituted" modifier, refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' together can represent an alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH) and -N(CH)(CHCH). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocycloalkylamino," "alkoxyamino," and "alkylsulfonylamino," when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH3. The term "alkylimino," when used without the "substituted" modifier, refers to the divalent group =NR, where R is alkyl, as that term is defined above.When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms bonded to a carbon atom are independently replaced with -OH, -F, -Cl, -Br, -I, -NH, -NO, -COH, -COCH, -CN, -SH, -OCH, -OCHCH, -C(O)CH, -NHCH, -NHCHCH, -N(CH), -C(O)NH, -C(O)NHCH, -C(O)N(CH), -OC(O)CH, -NHC(O)CH, -S(O)OH, or -S(O)NH. The groups NHC(O)OCH and -NHC(O)NHCH are non-limiting examples of substituted amide groups.

[0106] All compounds of the present invention can be used in some embodiments for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more compounds characterized or exemplified herein as intermediates, metabolites, and / or prodrugs may also be useful for the prevention and treatment of one or more diseases or disorders. Therefore, unless expressly stated otherwise, all compounds of the present invention are considered "active compounds" and "therapeutic compounds" intended for use as active pharmaceutical ingredients (APIs). Actual suitability for use in humans or animals is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting public health by ensuring the safety, effectiveness, quality, and assurance of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0107] The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." As used herein, "another" may mean at least a second or more.

[0108] As used herein, the terms "drug," "pharmaceutical," "therapeutic agent," and "therapeutically active agent" are used interchangeably to refer to compounds that produce a therapeutic or pharmacological effect in humans or animals and are used to treat diseases, disorders, or other conditions. In some embodiments, these compounds have received regulatory approval for administration to an organism.

[0109] As generally used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0110] "Pharmaceutically acceptable salts" refers to salts of the compounds disclosed herein that are pharmaceutically acceptable as defined above and possess the desired pharmacological activity. Such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or salts with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, etc. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be understood that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.Further examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0111] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to only alternatives or unless the alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more.

[0112] As used in this specification and the claims, the words "comprising" (and any form of compris- ing, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0113] As used herein, the term "significant" (and any form of significant, such as "significantly") is not meant to imply a statistical difference between two values, but simply the importance or extent of the difference in parameters.

[0114] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists between study subjects or experimental studies. Unless another definition applies, the term "about" refers to ±10% of the stated value.

[0115] As used herein, the terms "substantially free of" or "substantially free" in connection with a specified ingredient are used herein to mean that none of the specified ingredients have been intentionally formulated into the composition and / or are present as contaminants or in trace amounts only. The total amount of all contaminants, by-products, and other materials is present in the composition in an amount of less than 2%. The terms "more substantially free of" or "more substantially free" are used to indicate that the composition contains less than 1% of the specified ingredient. The terms "essentially free of" or "essentially free" mean that the specified ingredient is present in less than 0.5%.

[0116] As used herein, the term "nanoparticle" has its conventional and usual definition and refers to a discrete particle that behaves as a whole unit rather than as individual molecules within a particle. Nanoparticles can have a size of about 1 to about 10,000 nm, with ultrafine nanoparticles having a size of 1 nm to 100 nm, fine particles having a size of 100 nm to 2,500 nm, and coarse particles having a size of 2,500 nm to 10,000 nm. In some embodiments, the nanoaggregates described herein comprise a composition of multiple nanoparticles and can have a size of about 10 nm to about 100 μm.

[0117] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements and parameters. [Example]

[0118] IV. Working Examples To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are provided. Those skilled in the art will recognize that the techniques disclosed in the following examples represent techniques found by the inventors to work well in the practice of the disclosure and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure. The following examples should in no way be read as limiting or defining the overall scope of the disclosure.

[0119] In certain embodiments, provided herein are lipid nanoparticle formulations in which the ratio of components can be varied. The components that can be varied in the lipid nanoparticle formulation can independently include cationic ionizable lipids, phospholipids, helper lipids such as PEG lipids, or sterols such as cholesterol. As disclosed herein, the lipid nanoparticle formulation or pharmaceutical composition can have different transfection efficiencies depending on the ratio of components. In some embodiments, the length of the alkyl chain present in the cationic ionizable lipid is modified to improve advantageous features or characteristics of the composition, such as biodegradability or pulmonary delivery. Provided herein are compositions that demonstrate increased mRNA delivery in air-liquid interface human lung cell models and healthy mouse lungs, and high distribution in clinically relevant locations in the human lung in next-generation impactor experiments. These and other details are described below.

[0120] Example 1 - Stably aerosolized mRNA lipid nanoparticles for pulmonary delivery A. Materials and Methods In vitro transcription (IVT) NLuc mRNA was codon-optimized using GenScript's GenSmart™ Codon Optimization online tool. Next, a custom gene block encoding the T7 promoter, 5' UTR, codon-optimized NLuc, and 3' UTR sequences, ordered from Twist Biosciences, was PCR-amplified to generate the NLuc mRNA template. All mRNA was synthesized and purified as previously described. Briefly, amplicons were transcribed using the AmpliScribe™ T7-Flash Transcription Kit (Lucigen, ASF-3507) according to the manufacturer's instructions. After transcription, mRNA was purified using RNA Clean & Concentrator-100 (Zymo, R1019). After purification, cap1 structures were added using the Vaccinia Capping System (NEB, M2080S) and mRNA Cap 2'-O-methyltransferase (NEB, M0366S). Subsequently, a 3'-poly(A) tail approximately 100 bp long was enzymatically added using Escherichia coli (E. coli) poly(A) polymerase (NEB, M0276L). After polyadenylation, the mRNA was purified again using RNA Clean & Concentrator-100. The mRNA concentration was determined using a Nanodrop 1000 (Thermo Fisher Scientific Inc.), and aliquots were stored at -80°C until use.

[0121] lipids The ionizable lipid MC3 ((6Z,9Z,28Z,31Z)-heptatriacon-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was purchased from BioFine International Inc. The lipids SM-102 (heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), C-1 (BP-26399), C-2 (BP-26367), C-3 (BP-26361), C-4 (BP-26371), ALC-0135 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)), and ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide) were purchased from BroadPharm. The helper lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) were purchased from Avanti Polar Lipids. The PEG-lipids DMG-PEG 2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) and DMPE-PEG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) were purchased from NOF America Corporation. Cholesterol was purchased from Sigma-Aldrich.

[0122] LNP formulation LNPs were formulated using a microfluidic setup previously described (Zhang et al. 2020). Briefly, 10 mg / mL aliquots of each lipid in 100% ethanol were combined at the appropriate molar ratio. NLuc mRNA was diluted in 50 mM sodium citrate buffer at pH 3.0. Using a NanoAssemblr microfluidic mixer (Precision Nanosystems), the organic and aqueous phases were combined at a 3:1 ratio with a total flow rate of 9 mL / min. NLuc LNPs were formulated at an N / P ratio of 5.67 with an mRNA concentration of 15 ng / μL for in vitro and in vivo experiments and 100 ng / μL for TEM and NGI experiments. Cre B-1 was formulated at a concentration of 150 ng / μL with an N / P ratio of 5.67. After formulation, the LNPs were dialyzed overnight against 1× PBS pH = 7.4 using Slide-A-Lyzer™ G2 Dialysis Cassettes (87730, Thermo Fisher Scientific) with a molecular weight cutoff of 10 kDa, with a sample volume of 500 times greater. The formulations were stored at 4°C until use.

[0123] Aerosol generation Each formulation was aerosolized using an Aerogen Solo vibrating mesh nebulizer (Aerogen Ltd.) After aerosol generation, the formulation was collected in a 1.5 mL Eppendorf tube for use.

[0124] LNP characterization The size, polydispersity (PDI), and zeta potential of LNPs were measured using a Zetasizer Nano-ZS (Malvern Instruments). All samples were diluted with 0.1x PBS to a final mRNA concentration of 0.75 ng / μL. Size and PDI were measured using a UV-Cuvette micro (759200, BrandTech), and zeta potential was measured using a Folded Capillary Zeta Cell (DTS1070, Malvern).

[0125] Encapsulation efficiency was assessed using the Quant-IT RiboGreen RNA Assay Kit (R11490, Thermo Fisher). LNP samples were prepared in 1x Tris-EDTA (TE) to measure unencapsulated mRNA and in 1% Triton to measure total mRNA, reaching a final concentration of 0.6 ng / μL. A wide-range standard curve was prepared using ribosomal RNA from the kit in both 1x TE and 1% Triton. Both samples and standards were added to a 96-well black clear-bottom plate (3631, Corning) in a volume of 100 μL. After 10 minutes of incubation at 37°C, 100 μL of 200-fold diluted RiboGreen RNA reagent was added to each well. Sample fluorescence was then measured using a SpectraMax M3 plate reader (Molecular Devices) at excitation 480 nm and emission 520 nm. Encapsulation efficiency was calculated using the following formula: (1-[unencapsulated mRNA] / [total mRNA]). * 100.

[0126] Air-liquid interface (ALI) cell culture Calu-3 cells (HTB-55, American Type Culture Collection) at passages 12–15 were grown in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1× penicillin / streptomycin. After reaching approximately 80% confluence in the flask, cells were passaged and plated at 300,000 cells / cm in 200 μL onto the apical side of ThinCert™ CellCoat™ 24-Well Cell Culture Inserts (662641, Greiner Bio-One). 2 After 3 days, the apical medium was removed and the cells were cultured at the ALI. The cells were incubated at 37°C in a 5% CO atmosphere, and the basolateral medium was replaced with fresh medium every 2–3 days.

[0127] TEER values ​​were measured using a Millicell ERS-2 with a silver / silver chloride electrode (MERS00002, Millipore Sigma). Prior to TEER measurements, transwells were incubated on both the apical and basolateral sides in HBSS at 37°C in a 5% CO2 atmosphere for 15 minutes. Values ​​were calculated by subtracting the resistance of a blank transwell and then multiplying by the surface area. Cells reached a TEER value of 400 Ω after 1 week in ALI culture. * cm 2 was used for the experiment.

[0128] In vitro transfection efficiency The transfection efficiency of LNP was evaluated in ALI Calu-3 cells. LNP was added to the apical side of ALI Calu-3 cells in a final volume of 100 μL. After 24 hours, cells were harvested by scraping each transwell and transferring them to a white plate containing 100 μL of furimazine from the Nano-Glo® Luciferase Assay System (N1110, Promega) using a multichannel scanner. Samples were incubated at room temperature for 3 minutes before analysis using a SpectraMax M3 plate reader (Molecular Devices).

[0129] In vivo transfection efficiency using Balb / c mice The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas at Austin. Balb / c mice (female, 6–8 weeks old) were purchased from Charles River. Mice were allowed to acclimate for at least 1 week before testing.

[0130] Mice were anesthetized under continuous 2% isoflurane and administered 750 ng of LNP intratracheally in a volume of approximately 50 μL. 24 hours later, mice were sacrificed, and lungs were harvested and separated into five lobes (left, anterior, accessory, median, and posterior lobes). Lung lobes were immersed in 400 μL of Nano-Glo® Luciferase Assay System substrate for 5 minutes. Luminescence was measured using an In Vivo Imaging System (IVIS) with a 1-second exposure, medium binning, and an F-stop of 1, and quantified using Living Image (PerkinElmer).

[0131] In vivo delivery using Ai9 mice Ai9 mice (female, 6-8 weeks) were purchased from Jackson (007909). Mice were allowed to acclimate for at least 1 week before testing. Mice were anesthetized under continuous 2% isoflurane and intratracheally dosed with LNPs delivering 0.5 mg / kg Cre mRNA (L-7211, TriLink) in approximately 50 μL. Mice were dosed every other day for 4 days. Lungs were harvested 3 days after the final dose.

[0132] To visualize tdTomato fluorescence, lungs were imaged using an In Vivo Imaging System (IVIS) with an exposure time of 5 seconds, excitation at 53.5 nm and emission at 580 nm, medium binning, and an F-stop of 1.

[0133] To prepare lung cells for single-cell suspension, the tissue was minced and incubated for 1 hour at 37°C in digestion medium consisting of 90 units / mL collagenase type I (SCR103, Millipore Sigma), 50 units / mL DNase I (11284932001, Sigma-Aldrich), and 60 units / mL hyalurinidase (H3506, Sigma-Aldrich) in DMEM, as previously described (Cheng et al., 2020). After incubation, the digestion medium was quenched with DMEM + 20% FBS, and the tissue was filtered through a 70 μm filter. The cells were washed once with 1x PBS and then lysed in 5 mL of ACK lysis buffer (A1049201, ThermoFisher) for 3 minutes at room temperature. The cells were washed with 1x PBS and resuspended in 50 μL of 1x PBS for staining.

[0134] Each sample was stained with 1 μL of a 1:10 dilution of Zombie NIR (423105, BioLegend) and incubated for 30 minutes at 4°C. Cells were then washed twice with 1 mL of 1x PBS and resuspended in 50 μL of 1x PBS + 2% FBS + 0.05% sodium azide. Cells were then stained with 1 μL each of the following antibodies: Pacific Blue anti-mouse CD45 (103126, BioLegend), Alexa Fluor 488 anti-mouse CD31 (102414, BioLegend), and Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (118212, BioLegend). Cells were analyzed using an Attune NxT flow cytometer.

[0135] Next Generation Impactor (NGI) Aerosol distribution of LNPs was evaluated through an NGI (MSP Corporation). An Aerogen Solo vibrating mesh nebulizer was connected to the induction port using a T-plug (device). Prior to each run, all NGI components were transferred to a refrigerated room and pre-cooled to 4°C for at least 90 minutes (Berg et al., 2007). 1 mL of LNP per formulation was loaded into the nebulizer. The flow rate was 15 L / min. At this flow rate, the cutoff diameters for each stage were 14.1 μm for stage 1, 8.61 μm for stage 2, 5.39 μm for stage 3, 3.30 μm for stage 4, 2.08 μm for stage 5, 1.36 μm for stage 6, and 0.98 μm for stage 7.

[0136] The LNPs were first collected by wrapping the induction port and device in parafilm. Then, 3 mL of 1x TE was added to these components, as well as stages 1–7 and the MOC. The buffer was swirled around each component approximately 10 times. After collection, the amount of mRNA in each stage was determined by RiboGreen assay.

[0137] EF, MMAD, GSD and FPF 5μm Calculation of The cutoff diameters used for each stage at a flow rate of 15 L / min were 14.1 μm for stage 1, 8.61 μm for stage 2, 5.39 μm for stage 3, 3.30 μm for stage 4, 2.08 μm for stage 5, 1.36 μm for stage 6, and 0.98 μm for stage 7. Using the concentrations calculated by the above method, the aerodynamic diameters of stages 1–7 were determined using Equation 1 (Patel et al., 2020; Yanez Arteta et al., 2018). TIFF2025538217000025.tif7128

[0138] In this equation, Q is the flow velocity through the impactor and D 50, Q is the aerodynamic diameter at the flow velocity, Q, and A * and B* is an empirical constant determined for Q = 15 L / min for use with flow rates of 15-30 L / min. EF was calculated by dividing the mass deposited in the throat, stages 1-7, and MOC by the mass deposited in all components of the NGI. FPF 5 μm was interpolated from a graph of the cumulative fraction of emitted dose plotted against particle cutoff size. MMAD was calculated by plotting the log cumulative fraction of drug against aerodynamic diameter. GSD was calculated at aerodynamic diameters corresponding to 15.87% and 84.13%, determined by plotting the cumulative fraction of mass below a specified aerodynamic diameter against aerodynamic diameter (log).

[0139] Transmission electron microscope (TEM) LNP structures were visualized using an FEI Tecnai TEM. For each sample, 5 μL of LNP was loaded onto a Carbon Type-A 300 mesh copper grid (01820, Ted Pella). After a 5-minute incubation, the sample was wiped off with filter paper, and the grid was washed with 5 μL of HO. Negative staining was performed by adding 5 μL of 2% uranyl acetate. Images were taken at 43,000x magnification while operating at 80 kV.

[0140] statistical analysis All in vitro experiments were completed in triplicate, and all in vivo experiments were completed in quadruplicate. All P values ​​were calculated using Student's t-test. * p-value < 0.05; ** p-value < 0.01, *** p-value < 0.001 and **** A p-value < 0.0001 was considered statistically significant. Data values ​​are presented as mean ± standard deviation. All analyses were performed in GraphPad Prism (version 8.4.3). For Figures 2 and 3, simple linear regression was used to determine statistical significance.

[0141] B. Results and Discussion (1) Results Screening of aerosolized LNPs for increased transfection efficiency in air-liquid interface (ALI) lung cells. We evaluated the effect of varying the component ratio of LNPs based on the known formulation F11 (Zhang et al., 2020b). This LNP, with its base composition of MC3, DPPC, DMPE-PEG, and cholesterol (0.6 / 0.2 / 0.01 / 0.19), was a lead candidate for delivering eGFP mRNA to plated lung cells and Fluc mRNA to the lungs of Balb / c mice. From F11, we developed the first set of two LNPs (Figure 2A) with varying component ratios of MC3, DPPC, and cholesterol to a fixed amount of PEG-lipid (0.01 molar ratio). The mRNA LNP delivery of these LNPs was compared with F11 and Onpattro, a clinically approved siRNA LNP also based on MC3. All formulations encapsulated NanoLuc mRNA, a reporter molecule 100-fold more sensitive than conventional firefly luciferase. The comparative LNP compositions F11 and Onpattro encapsulating NanoLuc mRNA are also referred to herein as "NLuc F11" and "NLuc Onpattro," respectively.

[0142] We then evaluated transfection efficiency by delivering 1000 ng of each aerosolized LNP to Calu-3 cells cultured in an ALI, a physiologically relevant cell culture model of the human lung. At the ALI, Calu-3 cells produce mucus and form tight junctions, thereby acquiring in vivo-like barrier properties, such as tight junction formation and secretion of mucus components, making it a useful model for screening formulations for inhalation delivery (Grainger et al., 2006; Fiegel et al., 2003; Bivas-Benita et al., 2004; Amidi et al., 2006; Grenha et al., 2007). To establish the ALI, Calu-3 cells were exposed to air on the apical side of the transwell and maintained in contact with the medium on the basolateral side. To quantify the integrity of the tight junctions, we measured transepithelial electrical resistance (TEER). After 1 week of culture at the ALI, Calu-3 cells maintained a TEER value of 400 Ω. * cm 2 The LNPs demonstrated complete tight junctions exceeding 1000 ng / mL. After aerosolization, 1000 ng of each LNP was delivered to the apical side of the transwell and incubated for 24 hours. Twenty-four hours after administration, cells were harvested and luminescence intensity was measured. Decreasing the MC3 ratio from 0.6 to 0.45 resulted in significantly higher luminescence in both A-1 and A-2 compared to NLuc F11 (Figure 2B). The best-performing formulation, A-1, had a greater than 2.6-fold increase compared to NLuc F11 and a 1.3-fold increase compared to NLuc Onpattro. A-1 had the highest luminescence and was therefore selected for further study.

[0143] Without being bound by theory, we hypothesized that varying the type of cationic ionizable lipid could enhance efficacy. To date, there are two other approved cationic ionizable lipids besides MC3: SM-102 for the Moderna mRNA LNP COVID-19 vaccine and ALC-0315 for the Pfizer / BioNTech COVID-19 vaccine. Although A-1 is an MC3-based LNP, its component ratios and component types are similar to those of these vaccines (Baden et al., 2020; Polack et al., 2020). Therefore, a second set of two LNPs was formulated containing the cationic ionizable lipids SM-102 (B-1) and ALC-0315 (B-2), with the component ratios, helper lipid type, PEG-lipid type, and cholesterol type remaining constant as provided by composition A-1 (Figures 2C and 2D).

[0144] The COVID-19 vaccines Spikevax and Comirnaty were formulated to serve as comparative LNPs for B-1 and B-2, respectively. Because these LNPs encapsulate NLuc rather than the full-length SARS-CoV-2 spike protein, the comparative formulations are appropriately referred to herein as "NLuc Spikevax" and "NLuc Comirnaty." These formulations were screened in the same manner as the LNPs in Set 1. After aerosolization, B-1 had the highest transfection efficiency, a more than three-fold increase compared to A-1. However, B-2 did not exhibit significantly higher luminescence than A-1 (Figure 2E). This result was unexpected, given the similar structures of SM-102 and ALC-0315, and the significant structural differences between SM-102 and ALC-0315 relative to MC3 when considered together or separately. More specifically, both SM-102 and ALC-0315 have one hydroxyl head group, one tertiary amine, two esters, and a saturated hydrocarbon, while MC3 has a dimethylamine head group and an unsaturated dilinoleic acid lipid tail. Without being bound by theory, the structure of the cationic ionizable lipid had a greater impact on transfection efficiency after aerosolization than the component ratio. Furthermore, both B-1 and B-2 LNP formulations had higher luminescence compared to the corresponding vaccine formulation, although this increase was not statistically significant for B-1. Together, both screens identified B-1 as a lead candidate, demonstrating the importance of CIL in enhancing mRNA LNP delivery to ALI Calu-3 cells.

[0145] To determine whether improved transfection before aerosolization correlated with improved transfection after aerosolization, we compared non-aerosolized LNPs with aerosolized LNPs, Sets 1 and 2 (Figure 8). Particles that showed lower transfection compared to A-1 before aerosolization also showed lower transfection after aerosolization. For example, NLuc F11 showed reduced luciferase expression compared to A-1 both before and after aerosolization.

[0146] However, this was not the case for highly transfected particles. Despite all three LNPs having similarly high luciferase expression before aerosolization, both B-1 and NLuc Spikebax had significantly higher luciferase expression after aerosolization compared to A-1. These results were consistent with previous findings that some formulations, identified as lead candidates before aerosolization, exhibited significantly reduced delivery after aerosolization (Zhang et al., 2020). Overall, these data, without being bound by theory, indicate that improved transfection before aerosolization does not necessarily guarantee improved transfection after aerosolization. As can be seen, post-aerosolization transfection efficiency may not be predictable based on composition or pre-aerosolization transfection efficiency. Furthermore, size, polydispersity index, zeta potential, and encapsulation efficiency before and after aerosolization were not correlated with transfection efficiency (Figures 3A-3D). Importantly, all particles were stable before aerosolization, with sizes ranging from 62.38 ± 0.49 to 120.46 ± 1.11 and PDIs less than 0.2.

[0147] Characterization of LNP morphology before and after aerosolization We used TEM to evaluate the morphology of A-1 and B-1 before and after aerosolization. This allowed us to compare the effect of the type of cationic ionizable lipid (MC3 vs. SM-102) on aerosolization stability. Before aerosolization, A-1 and B-1 had the same spherical structure as noted by other TEM images of mRNA LNPs (Figure 4) (Kim et al., 2021; Kulkarni et al., 2019; Patel et al., 2020; Carrasco et al., 2021; Eygeris et al., 2020). After aerosolization, both had a roughly spherical shape and a larger size distribution, as shown in DLS data (Figure 3A and Figure 3B). However, B-1 was observed to have more defects on the surface of the aerosolized LNPs compared to A-1. Other studies of LNPs have shown, without being bound by theory, that multifaceted surfaces, such as those of B-1, are associated with more efficient membrane fusion and therefore increased escape from endosomes (Patel et al., 2020). Without being bound by theory, the four double bonds in MC3 may play a role in limiting LNP structural flexibility during aerosolization, whereas the less constrained SM-102 structure, lacking double bonds, may be more susceptible to structural changes under shear forces. Without being bound by theory, these differences demonstrate the importance of CIL for aerosolized mRNA LNPs and indicate that the most morphologically intact particles do not necessarily have the highest transfection efficiency. Thus, the transfection efficiency of the LNPs disclosed herein cannot be predicted or determined from particle morphology.

[0148] In vivo delivery of mRNA LNPs to mouse lungs To evaluate the transfection efficiency of NanoLuc mRNA LNPs in lung epithelial cells, 750 ng each of aerosolized F11, NLuc F11, A-1, B-1, and NLuc Onpattro were aerosolized using a vibrating mesh nebulizer and then delivered intratracheally to Balb / c mice. Unencapsulated NLuc mRNA (free mRNA) and PBS served as negative controls. Mouse lungs were harvested and separated into five lung lobes (left, anterior, accessory, middle, and posterior) 24 hours after administration and stained with furimazine (Figure 5A). All formulations showed similar distribution throughout each of the five lung lobes. Furthermore, all LNPs achieved higher mRNA expression compared to free mRNA throughout all lung lobes (Figure 10). Overall, B-1 showed the highest intensity. The transfection efficiency of B-1 was 8.1-fold, 5.6-fold, 5.8-fold, and 103.2-fold higher than that of NLuc F11, A-1, NLuc Onpattro, and free mRNA, respectively (Figure 5B, Figure 5C). Although A-1 and NLuc Onpattro showed increased transfection compared to NLuc F11, the difference was not statistically significant. Therefore, B-1 was selected as a lead candidate based on its delivery efficacy in both ALI Calu-3 cells and healthy mouse lungs.

[0149] Assessment of B-1 transfected cell types in the lung To track the delivery of B-1 to therapeutically relevant cell types in the lungs after aerosolization and quantify transfection capacity, we utilized the Ai9 reporter mouse model (Figure 9A) (Madisen et al., 2010). These mice contain a stop cassette flanked by LoxP sites in the Rosa26 locus, which silences transcription of the fluorescent tdTomato protein. Cre-mediated recombination of the LoxP sites deletes the stop cassette, allowing expression of tdTomato. tdTomato-positive cells can then be isolated by flow cytometry along with surface markers of choice to identify which cell types have been transfected with our formulations.

[0150] Cre mRNA-loaded B-1 LNPs were delivered, and lungs were harvested 7 days later for analysis (Figure 9A). Fluorescence imaging revealed that B-1 had significantly higher brightness than the PBS control (Figure 9B). Flow cytometry revealed that 8.9% of epithelial cells, 1.9% of immune cells, and 0.6% of endothelial cells were tdTomato-positive (Figure 9C). Upon local delivery, relative uptake in epithelial cells was greater than in other cell types, which is important in CF, where the epithelium is a primary target for gene editing therapy. It has been shown that approximately 5–50% of epithelial cells need to express functional CFTR to improve lung function in CF patients (Chu et al., 2015); these findings suggest the promising cell transfection properties of aerosolized B-1.

[0151] B-1 LNPs target a relatively higher number of desired epithelial cells compared to lung-targeted LNPs (Cheng et al., 2020). Compared to intravenously delivered five-component LNPs with lung targeting, B-1 exhibits higher relative uptake in epithelial cells than in immune cells (4.7-fold vs. 1.9-fold). Furthermore, transfection of B-1 in endothelial cells is significantly lower (0.6% vs. 66%). The route of administration may affect delivery. The LNP formulations disclosed herein are intended for localized pulmonary delivery to reach the epithelium, whereas other formulations are delivered intravenously and must cross the vascular endothelium before gaining access to target epithelial cells. For at least this reason, the present invention is advantageous for localized delivery for LNP uptake in lung epithelial cells.

[0152] Next-generation impact testing for evaluating aerodynamic characteristics Successful inhalation therapy requires droplets between 1 and 5 μm in size to avoid expulsion or entrapment in the upper airways (Chow et al., 2020). To assess aerosol distribution throughout the human lung, we used a next-generation impactor (NGI). This device uses airflow to deposit the aerosolized formulation along the throat, seven stages, and microorifice collector (MOC). The airflow velocity through the NGI determines the aerodynamic diameter of each component particle and therefore its distribution within the lung. At a flow rate of 15 L / min, particles collected from stages 1–3 deposit in the nasal cavity, while particles collected from stages 4–7 and the MOC deposit throughout therapeutically relevant regions of the lung (Figure 6A) (Marple et al., 2004). Using this device, we measured the aerodynamic diameter and lung deposition of NLuc F11, A-1, B-1, and NLuc Onpattro upon aerosolization (Figure 6B and Figure 6C). To mimic tidal respiration, the flow rate was operated at 15 L / min. Therefore, the cutoff diameters for each stage were 14.1 μm for stage 1, 8.61 μm for stage 2, 5.39 μm for stage 3, 3.30 μm for stage 4, 2.08 μm for stage 5, 1.36 μm for stage 6, and 0.98 μm for stage 7. After each run, LNPs were collected from the NGI component and quantified using the RiboGreen assay. These values ​​were used to calculate the released fraction (EF), the fine particle fraction <5 μm (FPF), and the fraction <5 μm (FPF). 5μm The FPF, mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) (US Pharmacopeia, 2003) were calculated. EF is the percentage of particles recovered from the throat, stages 1-7, and MOC out of the total particles deposited in the NGI components. 5μmThe FPF is the percentage of emitted particles with a diameter less than 5 μm. MMAD is the benchmark value for the deposition limit in the lower respiratory tract (5 μm), with 50% of the aerosol droplets having a diameter larger or smaller than this. GSD represents the spread of the aerosol particle size distribution. The majority of A-1, B-1, and NLuc F11 and NLuc Onpattro controls deposited within the NGI component were collected from stages 4-7 and MOC, indicating that they may be deposited throughout the small and large airways (Figure 6B). This correlates with a high FPF (77.04%-82.00%) (Figure 6C). Importantly, the FPF 5μm The range of FPF was 72.63% to 79.18%, highlighting that the majority of particles are within the respirable range. In comparison, this percentage is higher than that of Arikayce (FPF), the only clinically approved liposomal drug developed for inhalation. 5μm (50.3-53.5%) (Li et al., 2021; Leong and Ge, 2022). Therefore, the present invention represents an improvement over solutions known in the art. Compared to Arikayce, the MMAD was also less than 5 μm for all LNPs except A-1. Furthermore, the GSD was also less than 2 for all LNPs.

[0153] Structure-function analysis of SM-102 While SM-102 has received clinical approval for Moderna's COVID-19 vaccine, identifying structural components that improve transfection efficiency after aerosolization would be beneficial for pulmonary delivery. In a previous study, cationic ionizable lipids with structural similarity to SM-102 significantly improved transfection efficiency in CD-1 mice (Sabnis et al., 2018). The structure-function of SM-102 was probed by screening four analogs with different carbon numbers in the head, tail, and carbon chain from the primary nitrogen to both esters (Figure 7A, Figure 7B). Each analog was formulated into LNPs (C1–C4) and compared with B-1 while maintaining the A-1 component ratio. NLuc Spikebax was used as a benchmark formulation. After 24 hours of aerosolized LNP delivery of 1000 ng to ALI Calu-3 cells, cells were harvested and luminescence intensity was measured.

[0154] Most analogs produced significant effects compared to SM-102 (Figure 7C). Without being bound by theory, increasing the carbon number in the head structure of C-1 resulted in a 2-fold increase in emission compared to B-1 and the highest delivery. Furthermore, changing the carbon number from amine to two esters in C-2 and C-3 also increased delivery, as evidenced by a 1.3-fold increase in emission of LNPs formed from each. However, shortening the tail in C-4 resulted in a 1.5-fold decrease in delivery.

[0155] Similar to B-1, the majority of C-1 was deposited in stages 4–7 and within the MOC (Figure 7D). Furthermore, C-1 exhibited high EF (83.47 ± 1.67%) and FPF. 5μm (78.77 ± 0.52%), as well as an MMAD of less than 5 μm (4.79 ± 0.08 μm) and a GSD of less than 2 (1.23 ± 0.00) (Figure 7E). This chemistry may improve the transfection efficiency of SM-102 analogs, but this is not consistent with any particular effect on their deposition potential in the human lung.

[0156] (2) Discussion The present disclosure provides compositions with reduced degradation during aerosolization and improved particle deposition in therapeutically relevant locations. As an example, B-1 was stable upon aerosolization and achieved the highest transfection efficiency in ALI human lung cells and healthy mouse lungs. Furthermore, Cre B-1 demonstrated increased transfection efficiency in epithelial cells (8.9%) compared with lung immune cells (1.9%) or endothelial cells (0.6%). The high transfection rate of aerosolized Cre B-1 in epithelial cells is promising for lung diseases such as CF (Marquez et al., 2021). Furthermore, B-1 also possessed desirable droplet properties in simulated human airways. Through NGI, B-1 demonstrated a higher FPF than Arikayce, the only clinically approved liposomal drug for inhalation using a mesh nebulizer. 5μm (Li et al., 2021; Leong and Ge, 2022). What is important is that FPF 5μm A higher β indicates a higher percentage of therapeutically active mRNA. While transfection experiments and NGI assess different drug properties, each method offers valuable criteria for aerosolized formulations. Transfection demonstrates the ability of cells to translate and express the protein of interest after delivery of mRNA, while NGI demonstrates the potential of the formulation to deposit in clinically relevant locations in simulated human airways. Clinically relevant LNPs must be able to do both. The LNPs of the present disclosure have improved transfection or improved deposition in clinically relevant locations, as described elsewhere in the Examples. The present disclosure also provides more potent LNPs, such as LNPs with three carbons in the head structure instead of two, as exemplified by analog C-1 above, as one non-limiting example. Therefore, the compositions of the present disclosure represent a novel compositional solution for pulmonary delivery.

[0157] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods described herein, as well as to the steps or sequence of steps of the methods, without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein while the same or similar results are achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure, as defined by the appended claims.

[0158] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025538217000026.tif184142TIFF2025538217000027.tif77140

Claims

1. a) Lipid nanoparticles (LNPs), comprising: i) A cationic ionizable lipid (CIL) of the formula: (In the formula, m, n, and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X 2 and X 3 are each independently -O- or -N-; R 1 is hydroxy, amino, halo, or mercapto; or Alkoxy (C≦8) , alkylamino (C≦8) , dialkylamino (C≦12) or a substituted form of any of these groups; and R 2 and R 3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) (which is or a pharmaceutically acceptable salt thereof; ii) phospholipids; iii) PEG-lipids; and iv) Sterols the LNP comprising: b) Nucleic acid 1. A pharmaceutical composition formulated for aerosolization, comprising:

2. The cationic ionizable lipid is further defined as: (In the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; X 2 and X 3 are each independently -O- or -N-; R 1 is hydroxy, amino, halo, or mercapto; or Alkoxy (C≦8) , alkylamino (C≦8) , dialkylamino (C≦12) or a substituted form of any of these groups; and R 2 and R 3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) (which is or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition of claim 1.

3. The cationic ionizable lipid is further defined as: (In the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and X 2 and X 3 are each independently -O- or -N-; and R 2 and R 3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) (which is or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition of claim 2.

4. The cationic ionizable lipid is further defined as: (In the formula: m is 1, 2, 3, 4 or 5; n and o are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and R 2 and R 3 are each independently alkyl (C≦24) , alkenyl (C≦24) or alkynyl (C≦24) (which is or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition of claim 3.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein m is 1, 2, 3 or 4.

6. 6. The pharmaceutical composition of claim 5, wherein m is 2 or 3.

7. 7. The pharmaceutical composition of claim 6, wherein m is 2.

8. 7. The pharmaceutical composition of claim 6, wherein m is 3.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein n is 3, 4, 5, 6, 7, 8 or 9.

10. 10. The pharmaceutical composition of claim 9, wherein n is 4, 5, 6, 7 or 8.

11. 11. The pharmaceutical composition of claim 10, wherein n is 5 or 7.

12. 12. The pharmaceutical composition of claim 11, wherein n is 5.

13. 12. The pharmaceutical composition of claim 11, wherein n is 7.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein o is 3, 4, 5, 6, 7, 8 or 9.

15. 15. The pharmaceutical composition of claim 14, wherein o is 4, 5, 6, 7 or 8.

16. 16. The pharmaceutical composition of claim 15, wherein o is 5 or 7.

17. 17. The pharmaceutical composition of claim 16, wherein o is 5.

18. 17. The pharmaceutical composition of claim 16, wherein o is 7.

19. 19. The pharmaceutical composition of any one of claims 1-12 and 14-16 and 18, wherein n is 5 and o is 7.

20. 18. The pharmaceutical composition of any one of claims 1 to 12 and 14 to 17, wherein n and o are each 5.

21. 18. The pharmaceutical composition of any one of claims 1 to 11, 13 and 14 to 17, wherein n is 7 and o is 5.

22. 19. The pharmaceutical composition of any one of claims 1-11, 13, 14-16 and 18, wherein n and o are each 7.

23. R 2 is alkyl (C≦24) or substituted alkyl (C≦24) 23. The pharmaceutical composition of any one of claims 1 to 22, wherein

24. R 2 is alkyl (C≦24) 24. The pharmaceutical composition of any one of claims 1 to 23, wherein

25. R 2 is alkyl (C6～20) 25. The pharmaceutical composition of any one of claims 1 to 24, wherein

26. R 2 is n-alkyl (C6～20) 26. The pharmaceutical composition of any one of claims 1 to 25, wherein

27. R 2 is n-alkyl (C6～12) 27. The pharmaceutical composition of any one of claims 1 to 26, wherein

28. R 2 28. The pharmaceutical composition of any one of claims 1-27, wherein is n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, or n-dodecane.

29. X 2 R bonded to 2 26. The pharmaceutical composition of any one of claims 1 to 25, wherein the carbon atom in is a secondary carbon.

30. R 2 30. The pharmaceutical composition of any one of claims 1-25 and 29, wherein is heptadecan-9-yl.

31. R 3 is alkyl (C≦24) or substituted alkyl (C≦24) 31. The pharmaceutical composition of any one of claims 1 to 30, wherein

32. R 3 is alkyl (C≦24) 32. The pharmaceutical composition of any one of claims 1 to 31, wherein

33. R 3 is alkyl (C6～20) 33. The pharmaceutical composition of any one of claims 1 to 32, wherein

34. R 3 is n-alkyl (C6～20) 34. The pharmaceutical composition of any one of claims 1 to 33, wherein

35. R 3 is n-alkyl (C6～12) 35. The pharmaceutical composition of any one of claims 1 to 34, wherein

36. R 3 36. The pharmaceutical composition of any one of claims 1-35, wherein is n-hexane, n-heptane, n-octane, n-nonane, n-decane, n-undecane, or n-dodecane.

37. X 3 R bonded to 3 34. The pharmaceutical composition of any one of claims 1 to 33, wherein the carbon atom in is a secondary carbon.

38. R 3 38. The pharmaceutical composition of any one of claims 1-33 and 37, wherein is heptadecan-9-yl.

39. R 2 is n-alkyl (C6～20) and X 3 R bonded to 3 40. The pharmaceutical composition of any one of claims 1-28, 31-33, 37 and 38, wherein the carbon is a secondary carbon.

40. R 2 and R 3 is n-alkyl (C6～20) 37. The pharmaceutical composition of any one of claims 1 to 28 and 31 to 36, wherein

41. X 2 R bonded to 2 The carbon of X is a secondary carbon, 3 R bonded to 3 39. The pharmaceutical composition of any one of claims 1-25, 29-33, 37 and 38, wherein the carbon is a secondary carbon.

42. The cationic ionizable lipid is further defined as: or a pharmaceutically acceptable salt thereof; 10. The pharmaceutical composition of claim 1.

43. 43. The pharmaceutical composition of any one of claims 1-42, wherein the LNP comprises a molar ratio of cationic ionizable lipid to LNP of about 0.3 to 0.

7.

44. 44. The pharmaceutical composition of any one of claims 1-43, wherein the LNPs comprise a molar ratio of cationic ionizable lipid to LNPs of about 0.4 to 0.

6.

45. 45. The pharmaceutical composition of any one of claims 1-44, wherein the LNPs comprise a molar ratio of cationic ionizable lipid to LNPs of about 0.

45.

46. 46. ​​The pharmaceutical composition of any one of claims 1 to 45, wherein the phospholipid comprises DOPE, DSPC or DPPC.

47. 47. The pharmaceutical composition of any one of claims 1 to 46, wherein the phospholipid is DPPC.

48. 48. The pharmaceutical composition of any one of claims 1-47, wherein the LNPs comprise a molar ratio of phospholipid to LNPs of about 0.02 to 0.

4.

49. 49. The pharmaceutical composition of any one of claims 1-48, wherein the LNPs comprise a molar ratio of phospholipid to LNPs of about 0.05 to 0.

3.

50. 50. The pharmaceutical composition of any one of claims 1-49, wherein the LNPs comprise a molar ratio of phospholipid to LNPs of about 0.

2.

51. 51. The pharmaceutical composition of any one of claims 1 to 50, wherein the PEG-lipid comprises DMG-PEG, DMPE-PEG, or DSPE-PEG.

52. 52. The pharmaceutical composition of any one of claims 1 to 51, wherein the PEG-lipid is DMPE-PEG.

53. 53. The pharmaceutical composition of any one of claims 1-52, wherein the LNP comprises a molar ratio of PEG-lipid to LNP of about 0.005 to 0.

03.

54. 54. The pharmaceutical composition of any one of claims 1-53, wherein the LNP comprises a molar ratio of PEG-lipid to LNP of about 0.01 to 0.

015.

55. 55. The pharmaceutical composition of any one of claims 1-54, wherein the LNPs comprise a molar ratio of PEG-lipid to LNPs of about 0.

01.

56. 56. The pharmaceutical composition of any one of claims 1-55, wherein the LNP comprises DOPE and DMG-PEG, DOPE and DMPE-PEG, DOPE and DSPE-PEG, DSPC and DMG-PEG, DSPC and DMPE-PEG, DSPC and DSPE-PEG, DPPC and DMG-PEG, DPPC and DMPE-PEG, or DPPC and DSPE-PEG.

57. 57. The pharmaceutical composition of any one of claims 1-56, wherein the LNP comprises DPPC and DMPE-PEG.

58. 56. The pharmaceutical composition of any one of claims 1 to 55, wherein the sterol is cholesterol.

59. 59. The pharmaceutical composition of any one of claims 1-58, wherein the LNPs comprise a molar ratio of sterol to LNPs of about 0.1 to 0.

6.

60. 60. The pharmaceutical composition of any one of claims 1-59, wherein the LNPs comprise a molar ratio of sterol to LNPs of about 0.15 to 0.

5.

61. 61. The pharmaceutical composition of any one of claims 1-60, wherein the LNPs comprise a molar ratio of sterol to LNPs of about 0.

34.

62. 62. The pharmaceutical composition of any one of claims 1 to 61, wherein the nucleic acid is a therapeutic nucleic acid.

63. 63. The composition of claim 62, wherein the nucleic acid is an siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA).

64. 64. The pharmaceutical composition of any one of claims 1 to 63, wherein the nucleic acid is RNA.

65. 65. The pharmaceutical composition of any one of claims 1 to 64, wherein the nucleic acid comprises mRNA.

66. 66. The pharmaceutical composition of any one of claims 1 to 65, wherein the nucleic acid comprises an siRNA.

67. 67. The pharmaceutical composition of any one of claims 1 to 66, wherein the nucleic acid is encapsulated in an LNP.

68. 68. The pharmaceutical composition of any one of claims 1-67, wherein the weight ratio of CIL to nucleic acid is from about 20:1 to about 1:

5.

69. 69. The pharmaceutical composition of any one of claims 1-68, wherein the weight ratio of CIL to nucleic acid is from about 15:1 to about 1:

3.

70. 70. The pharmaceutical composition of any one of claims 1-69, wherein the weight ratio of CIL to nucleic acid is about 11.3:

1.

71. 71. The pharmaceutical composition of any one of claims 1 to 70, wherein the N / P ratio is about 4 to 7.

72. 72. The pharmaceutical composition of any one of claims 1-71, wherein the N / P ratio is about 5-6.

73. 73. The pharmaceutical composition of any one of claims 1-72, wherein the N / P ratio is about 5.

7.

74. 74. The pharmaceutical composition of any one of claims 1-73, wherein the LNPs have an average particle size of about 50 nm to about 250 nm.

75. 75. The pharmaceutical composition of any one of claims 1-74, wherein the LNPs have an average particle size of about 50 nm to about 150 nm.

76. 76. The pharmaceutical composition of any one of claims 1 to 75, having an average particle size of about 100 nm.

77. 76. The pharmaceutical composition of any one of claims 1 to 75, having an average particle size of about 150 nm.

78. 78. The pharmaceutical composition of any one of claims 1-77, having a polydispersity index (PDI) of about 0.01 to about 0.

5.

79. 79. The pharmaceutical composition of any one of claims 1-78, having a polydispersity index (PDI) of about 0.02 to about 0.

4.

80. 80. The pharmaceutical composition of any one of claims 1-79, having a polydispersity index (PDI) of about 0.

05.

81. 80. The pharmaceutical composition of any one of claims 1-79, having a polydispersity index (PDI) of about 0.

3.

82. 82. The pharmaceutical composition of any one of claims 1-81, having a zeta potential of about -0.5 mV to about -40 mV.

83. 83. The pharmaceutical composition of any one of claims 1-82, having a zeta potential of about -0.5 mV to about -20 mV.

84. 84. The pharmaceutical composition of any one of claims 1-83, having a zeta potential of about -10 mV.

85. 84. The pharmaceutical composition of any one of claims 1-83, having a zeta potential of about -15 mV.

86. 86. A spray composition according to any one of claims 1 to 85.

87. 87. A method of treating a disease, disorder, injury or infection, comprising administering to a subject an effective amount of the pharmaceutical composition of any one of claims 1-86.

88. 88. The method of claim 87, wherein the disease is a genetic disease.

89. 89. The method of any one of claims 87 or 88, wherein the disease, injury or infection is a lung disease, lung injury or lung infection.

90. 90. The method of any one of claims 87-89, wherein the disease is a pulmonary disease.

91. 91. The method of any one of claims 87-90, wherein the pulmonary disease is interstitial lung disease, chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis (CF), pulmonary fibrosis, alpha-1 antitrypsin deficiency, or primary ciliary dyskinesia (PCD).

92. 92. The method of any one of claims 87-91, wherein the pharmaceutical composition is formulated for administration by inhalation.

93. The method of any one of claims 87 to 92, wherein the subject is a mammal.

94. The method of any one of claims 87 to 93, wherein the subject is a human.

95. 95. A method of modulating expression of a gene comprising delivering a nucleic acid to a cell, said method comprising contacting said cell with the pharmaceutical composition of any one of claims 1 to 94 under conditions sufficient to cause uptake of the nucleic acid into the cell.

96. 96. The method of claim 95, wherein the cell is contacted in vitro or ex vivo.

97. 96. The method of claim 95, wherein the cells are contacted in vitro.

98. The method of any one of claims 95-97, wherein modulation of gene expression is sufficient to treat the disease or disorder.

99. 99. The method of any one of claims 95 to 98, wherein the nucleic acid is mRNA.

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