Compositions and methods for targeted delivery to cells

Abstract

Described herein are compositions, kits and methods for potent delivery to cells of a subject. The cells can be specific cell types, such as basal cells, ciliated cells, or secretory cells. In some cases, the cells can be specific cell types of lung cells. Also described herein are pharmaceutical compositions that include therapeutic or prophylactic agents combined with lipid compositions. The lipid compositions can include ionizable cationic lipids, phospholipids, and selective organ targeting lipids. Additionally, described herein are high potency dosage forms of therapeutic or prophylactic agents formulated with lipid compositions.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 164,523, filed March 22, 2021, U.S. Provisional Application No. 63 / 229,497, filed August 4, 2021, and U.S. Provisional Application No. 63 / 305,426, filed February 1, 2022, each of which is incorporated by reference in its entirety for all purposes.

[0002] Background of the Invention Therapeutic approaches such as CRISPR / Cas (clustered regularly interspaced short palindromic repeat / CRISPR-associated protein (Cas)) technology often require precise and potent delivery of therapeutic agents to target organs or cells, sometimes in a sequence-dependent manner. To date, there remains a clear need to achieve therapeutically safe and effective lipid-based carriers to achieve clinical outcomes in the context of genetic disorders and many other applications, particularly respiratory diseases.

[0003] Summary of the Invention This application relates generally to the safe, efficient, and potent delivery of therapeutic or prophylactic agents, such as polynucleotides, polypeptides, or small molecule compounds, in lipid nanoparticles to target cells.

[0004] In certain aspects, the present application provides methods for potent delivery to cells (e.g., lung) of a subject, the methods comprising administering to the subject a (e.g., aerosol) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of SORT lipid) results in delivery of the therapeutic agent to the cells of the subject characterized by a greater therapeutic effect (e.g., about 1.1-fold or 10-fold) compared to the therapeutic effect achieved with a reference lipid composition (e.g., no amount of SORT lipid). In some embodiments, the lipid composition further comprises a phospholipid.

[0005] In another aspect, the present application provides a method for potent delivery to (e.g., lung) cells of a subject, the method comprising administering to the subject a (e.g., aerosol) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of SORT lipid) results in delivery of the therapeutic agent to the subject's cells characterized by a greater therapeutic effect in a plurality of (e.g., lung) cells (e.g., about 1.1-fold or 10-fold) compared to the therapeutic effect achieved with a reference lipid composition (e.g., no amount of SORT lipid). In some embodiments, the lipid composition further comprises a phospholipid.

[0006] In another aspect, the present application provides methods for targeted delivery to (e.g., lung) cells of a subject, the methods comprising administering to a subject a (e.g., aerosol) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to a greater proportion of cell types compared to that achieved with a reference lipid composition. In some embodiments, the lipid composition further comprises a phospholipid.

[0007] In another aspect, the present application provides a method for targeted delivery to (e.g., lung) cells of a subject, the method comprising administering to the subject a (e.g., aerosol) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to the subject's cells, characterized by a therapeutic effect in a first plurality of (e.g., lung) cells of a first cell type and a (e.g., about 1.1-fold or 10-fold) greater second plurality of (e.g., lung) cells of a second cell type. In some embodiments, the lipid composition further comprises a phospholipid.

[0008] In another aspect, the present application provides a method for targeted delivery to (e.g., lung) cells of a subject, the method comprising administering to the subject a (e.g., aerosol) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to the subject's cells characterized by a greater therapeutic effect (e.g., about 1.1-fold or 10-fold) in first (e.g., lung) cells of a first cell type in the subject compared to the therapeutic effect in second (e.g., lung) cells of a second cell type in the subject, wherein the first cell type is different from the second cell type. In some embodiments, the lipid composition further comprises a phospholipid.

[0009] In another aspect, the present application provides a pharmaceutical composition comprising a therapeutic agent in combination with a lipid composition, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid, wherein the SORT lipid is configured to provide delivery of the therapeutic agent characterized by one or more of the following: (a) a greater therapeutic effect (e.g., 1.1-fold or 10-fold) in a subject's (e.g., lung) cells compared to the therapeutic effect achieved using a reference lipid composition; (b) a therapeutic effect achieved using a reference lipid composition; (c) a therapeutic effect in a first plurality of (e.g., lung) cells of a first cell type and a (e.g., 1.1-fold or 10-fold) greater second plurality of (e.g., lung) cells of a second cell type; and (d) a (e.g., 1.1-fold or 10-fold) greater therapeutic effect in first (e.g., lung) cells of a first cell type in the subject compared to the therapeutic effect in second (e.g., lung) cells of a second cell type in the subject. In some embodiments, the lipid composition further comprises a phospholipid.

[0010] In another aspect, the present disclosure provides a high-potency dosage form of a therapeutic agent formulated with a selective organ targeting (SORT) lipid, the dosage form comprising the therapeutic agent in combination with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a SORT lipid that is separate from the ionizable cationic lipid, wherein the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of the therapeutic agent that is lower (e.g., at least about 1.1-fold or 10-fold) than the dose required for a reference lipid composition. In some embodiments, the lipid composition further comprises a phospholipid.

[0011] In another aspect, the present disclosure provides a high-potency dosage form of a therapeutic agent formulated with a selective organ targeting (SORT) lipid, the dosage form comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a SORT lipid that is separate from the ionizable cationic lipid, wherein the therapeutic agent (e.g., a heterologous polynucleotide) is present in the dosage form at a dose not exceeding about 2 milligrams per kilogram of body weight (mg / kg, or mpk). In some embodiments, the lipid composition further comprises a phospholipid.

[0012] In another aspect, the present application provides a method for delivery to basal cells of a subject (e.g., lung), the method comprising administering (e.g., systemically) to a subject a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid, thereby delivering the therapeutic agent to an organ or tissue (e.g., lung) of the subject, wherein the therapeutic agent results in a detectable therapeutic effect in at least about 5%, 10%, or 15% of basal cells in the organ or tissue of the subject.

[0013] In some embodiments, provided herein are methods for nebulization delivery to lung cells of a subject, the method comprising administering to the subject a (e.g., pharmaceutical) composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, thereby delivering the therapeutic agent to the lung cells in the subject's lungs. In some embodiments, the method provides an (e.g., therapeutically) effective amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung epithelial cells. In some embodiments, the method provides an (e.g., therapeutically) effective amount or activity of the therapeutic agent in at least about 2%, 5%, or 10% of the subject's lung ciliated cells. In some embodiments, the method provides an (e.g., therapeutically) effective amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung secretory cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung club cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung goblet cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung basal cells. In some embodiments, the lipid composition comprises a phospholipid. In some embodiments, the (e.g., pharmaceutical) composition comprising the therapeutic agent in combination with the lipid composition is an aerosol composition.

[0014] Additional aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present application are shown and described. As will be realized, the present application is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature, and not as restrictive.

[0015] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting content. [Brief explanation of the drawings]

[0016] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"). [Figure 1] FIG. 1 shows the chemical structures of lipids. [Figure 2] FIG. 1 shows the chemical structure of a dendrimer or dendron lipid. [Figure 3] FIG. 1 shows a chart of cell types and expression levels of delivered mRNA using LNPs of different compositions. [Figure 4]FIG. 1 shows images using in vivo imaging of bioluminescence in mice after inhalation aerosol delivery of Luc mRNA / LNPs using LNPs of multiple compositions. [Figure 5] FIG. 1 shows a chart of the cytotoxicity of various LNP compositions in human bronchial epithelial (hBE) cells. [Figure 6] FIG. 1 shows the stability and general properties of various LNP compositions. [Figure 7] FIG. 1 shows a chart of tissue-specific radiance over time in mice for an LNP composition (“Lung-SORT”; 5A2-SC8 DOTAP). [Figure 8] FIG. 1 shows images of tissue-specific radiance over time in mice of an LNP composition (“Lung-SORT”; 5A2-SC8 DOTAP). [Figure 9A] FIG. 1 illustrates the workflow of a human safety and tolerability study using the compositions described herein. [Figure 9B] FIG. 1 shows an ex vivo model in which ciliated epithelial cells (mouse tracheal epithelial cells or MTECs) were cultured at an air-liquid interface (ALI) to test the efficacy of rescue by DNAI1 mRNA treatment as described herein. [Figure 9C] FIG. 1 shows that ciliary activity in KO mouse cells was rescued by DNAI1 mRNA treatment, and the effect of the treatment remained stable for several weeks after discontinuation of the drug. [Figure 10A] FIG. 1 shows a summary of experiments performed to measure the properties of the lipid compositions described herein. [Figure 10B] FIG. 1 shows the results of experiments performed to measure the properties of the lipid compositions described herein. [Figure 10C] FIG. 1 shows positive lung labeling (red) of DNAI1 mRNA (left) and DNAI1 protein (right) in non-human primates using a lipid composition as described herein (e.g., including SORT lipids). [Figure 10D]FIG. 1 shows that 100% replacement of U in mRNA with modified nucleotide m1Ψ minimized the cytokine response. [Figure 10E] FIG. 1 shows data collected from experiments demonstrating the cilia function restoration, tolerability, and selectivity of the lipid compositions described herein. [Figure 11A] Figure 1 shows immunofluorescence images of human DNAI1 knockdown cells treated with LNPs containing DNAI-HA mRNA. Well-differentiated human DNAI1 knockdown cells were treated with a single dose of the DNAI1-HA mRNA formulation described herein and immunostained with anti-acetylated tubulin and anti-HA. Accumulation of newly expressed DNAI1-HA at ciliary axonemes peaked between 48 and 72 hours after treatment, DNAI1-HA was detected in ciliary axonemes for more than 24 days after a single dose, and repeated administration resulted in rescue of ciliary activity, which persisted for several weeks after discontinuation of medication. [Figure 11B] This figure shows that newly produced HA-tagged DNAI1 was rapidly incorporated into the cilia of human bronchial epithelial cells (hBE). Well-differentiated human DNAI1 knockdown cells were treated with a single dose of LNP-formulated DNAI1-HA (10 μg in 2 ml of medium) (basal administration). 72 hours after administration, cells were immunostained with anti-acetylated tubulin and anti-HA, and more than 90% of ciliated cells were DNAI1-HA positive. [Figure 12] FIG. 1 shows a multiplexed immunofluorescence panel of NHP respiratory epithelia that can be used to distinguish cell types that exhibit newly translated DNAI1 protein. [Figure 13A] FIG. 1 shows the cell tropism signatures of the LNP formulations described herein. [Figure 13B]This figure shows that aerosol administration of formulated DNAi1 mRNA rescued ciliary activity in knockdown primary hBE ALI cultures. Well-differentiated human DNAi1 knockdown cells (hBE) were treated twice weekly with LNP-formulated DNAi1 (300 μg per Vitrocell nebulized inhalation) starting on day 25 (culture age) after ALI, with the final dose administered on day 50 after ALI. Increased ciliary activity in the treated DNAi1 knockdown cultures was first detected 7 days after the start of dosing, and the rescued ciliary activity exhibited a normal beating frequency (9-17 Hz) and appeared synchronous. [Figure 14A] FIG. 1 shows the cell-tropic signature of a lipid composition containing 20% ​​ionizable cationic lipid (e.g., DODAP) at certain concentrations and conditions. [Figure 14B] FIG. 1 shows the cell-tropic signature of a lipid composition containing 20% ​​ionizable cationic lipid (e.g., DODAP) at certain concentrations and conditions. [Figure 14C] FIG. 1 shows the cell-tropic signature of a lipid composition containing 20% ​​ionizable cationic lipid (e.g., DODAP) at certain concentrations and conditions. [Figure 14D] FIG. 1 shows the cell tropism signature of a lipid composition containing 20% ​​permanent cationic lipid (e.g., 14:0 EPC) at certain concentrations and conditions. [Figure 14E] FIG. 1 shows the cell tropism signature of a lipid composition containing 20% ​​permanent cationic lipid (e.g., 14:0 TAP) under certain concentrations and conditions. [Figure 15] FIG. 1 shows cell type-associated DNAI1 protein expression in target cells of the respiratory epithelium of NHPs. [Figure 16A] FIG. 1 shows cell type-associated DNAI1 protein expression in target cells of the respiratory epithelium of NHPs. [Figure 16B] FIG. 1 shows cell type-associated DNAI1-HA protein expression in target cells of the respiratory epithelium of NHPs (left panel: lung, right panel: bronchi). [Figure 17] FIG. 1 shows biodistribution, potency, and tolerability studies of the LNP formulations described herein. [Figure 18A] FIG. 1 shows aerosol concentrations administered to NHPs. [Figure 18B] FIG. 1 shows exemplary measurements of dose delivered to NHPs. [Figure 18C] Figure 1 shows the characterization of aerosol composition droplets (MMAD: mass median aerodynamic diameter; GSDL: geometric standard deviation). Droplet characterization results were within the recommended ranges of the Organization for Economic Cooperation and Development (OECD) Guidance 433 for inhalation toxicity studies, with MMAD ≤ 4 μm and GSD 1.0-3.0. [Figure 19A] FIG. 1 shows measurements of LNP lipids (derived from aerosol droplets) in the lungs in low-dose and high-dose NHP groups (ionizable lipids in the lung). [Figure 19B] FIG. 1 shows measurement of LNP lipids (from aerosol droplets) in the lungs in low-dose and high-dose NHP groups (DMG-PEG in lungs). [Figure 19C] FIG. 1 shows measurements of LNP lipids (derived from aerosol droplets) in the lungs in low- and high-dose NHP groups (SORT lipids). [Figure 20A] FIG. 1 shows DNAI1-HA protein expression in the lung by Western blotting. [Figure 20B] FIG. 1 shows DNAI1-HA protein expression in the lung by ELISA. [Figure 21A] FIG. 1 shows clinical chemistry measurements of AST, ALT, and ALP; no significant changes in AST, ALT, or ALP were observed after treatment with the lipid compositions described herein. [Figure 21B] FIG. 1 shows hematological counts of white blood cells and neutrophils; some increase in neutrophils was observed in post-treatment measurements in both the vehicle and RTX0052 groups. [Figure 21C]FIG. 1 shows BAL cell differentiation. For cytokine and complement analysis, cytokine levels were measured in NHP serum and BAL; analytes measured included IFN-α2a, IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-17A, IP-10, MCP-1, and TNFα; all cytokine levels were in the same range as normal reported levels. [Figure 21D] FIG. 1 shows exemplary measurements of cytokines in serum. [Figure 21E] FIG. 1 shows exemplary measurements of cytokines in BAL. [Figure 21F] FIG. 1 shows exemplary complement measurements of C3a and sC5b-9 in plasma and serum, respectively. [Figure 21G] FIG. 1 shows exemplary complement measurements of C3a and sC5b-9 measurements in BAL. [Figure 22A] FIG. 1 shows the aerosol concentrations administered to rats. [Figure 22B] FIG. 1 shows exemplary measured aerosol homogeneity over three stages. [Figure 22C] FIG. 1 shows the dosage delivered to rats. [Figure 22D] FIG. 1 shows the characterization of aerosol composition droplets (MMAD: mass median aerodynamic diameter; GSDL: geometric standard deviation). [Figure 23A] FIG. 1 shows measurements of LNP lipids (derived from aerosol droplets) in the lungs in low, medium, and high dose rat groups (ionizable lipids in the lung). [Figure 23B] FIG. 1 shows measurements of LNP lipids (derived from aerosol droplets) in the lungs in low, medium, and high dose rat groups (DMG-PEG in lungs). [Figure 23C] FIG. 1 shows the measurement of LNP lipids (derived from aerosol droplets) in the lungs in low, medium, and high dose rat groups (SORT lipids). [Figure 24A]This figure shows DNAI1-HA protein expression in rat lungs by Western blotting. Six out of ten lung samples from the 1.2 mg / kg, 6-hour group were DNAI1-HA positive. [Figure 24B] FIG. 1 shows DNAI1-HA protein expression in rat lungs by ELISA. [Figure 25A] FIG. 1 shows clinical chemistry measurements of AST, ALT and ALP in treated rats. [Figure 25B] FIG. 1 shows hematological counts of leukocytes and neutrophils in treated rats. [Figure 25C] FIG. 1 shows BAL cell differentiation in treated rats. [Figure 25D] FIG. 1 shows exemplary measurements of alpha-2-macroglobulin in treated rats. [Figure 26A] FIG. 1 shows information on four groups of mice repeatedly treated with nebulized inhalation of LNP / DNAI1-HA mRNA. [Figure 26B] FIG. 1 shows the dosing, imaging, and necropsy protocol for repeat-dose mice. [Figure 27A] Figure 27 shows whole-body in vivo imaging (IVIS) of mice with repeated administration. Animals (B6 albino, male, approximately 7 weeks old, naive) were administered 4.0 mg of LNP-formulated DNAI1-HA / luciferase via nebulization inhalation at 66.6 μL / min with a zero-grade dry airflow of 2 L / min over 2 hours. Four hours after dosing, two mice were administered 2 mL of luciferin (30 mg / mL) via nebulization inhalation. Images were taken by IVIS within 1 to 15 minutes of luciferin administration. All images were pseudocolored to the same scale, and the lung signal is plotted on the graph in Figure 27A. [Figure 27B]Figure 27 shows whole-body in vivo imaging (IVIS) of repeated-dose mice. Animals (B6 albino, male, approximately 7 weeks old, naive) were administered 4.0 mg of LNP-formulated DNAI1-HA / luciferase via nebulization inhalation at 66.6 μL / min with a zero-grade dry airflow of 2 L / min over 2 hours. Four hours after dosing, two mice were administered 2 mL of luciferin (30 mg / mL) via nebulization inhalation. Images were taken by IVIS within 1 to 15 minutes of luciferin administration. All images were pseudocolored to the same scale, and the whole-body signal is plotted on the graph in Figure 27B. [Figure 27C] FIG. 1 shows histopathological results of mice administered repeatedly. [Figure 27D] Figure 1 shows qPCR results showing relative abundance of DNAI1-HA mRNA. Two mice per group were perfused after the last imaging of the last dose (dose 8). [Figure 27E] FIG. 1 shows Western blotting results showing protein expression of DNAI1-HA. [Figure 28A] FIG. 1 shows delivery of 0.4 mg / kg of DNAI1 mRNA formulated in LNPs by inhalation in intubated and mechanically ventilated NHPs administered in less than 30 minutes. [Figure 28B] FIG. 1 shows the aerosol properties of LNP formulations. The aerosol particle size range for all three formulations was appropriate for deposition in the conducting airways. [Figure 28C] FIG. 1 shows the biodistribution of DNAI1-HA mRNA in target cells. [Figure 28D] FIG. 1 shows the ISH results of DNAI1-HA mRNA by H-Score. The ISH results demonstrated that high levels of DNAI1-HA mRNA were delivered to lung cells, with lower levels in the bronchi and trachea. [Figure 29A]Figure 1 shows high levels of DNAI1-HA mRNA delivery to the lung without exposure to the liver, spleen, or blood. After a single dose of 0.4 mg / kg, DNAI1-HA mRNA levels were measured in whole blood, lung, liver, and spleen tissue using digital PCR. High levels of DNAI1-HA mRNA were detected in all three lung regions sampled 6 hours after exposure to RTX0051 and RTX0052. [Figure 29B] Figure 29B shows high levels of DNAI1-HA mRNA delivery to the lung without exposure to the liver, spleen, or blood. After a single dose of 0.4 mg / kg, DNAI1-HA mRNA levels were measured in whole blood, lung, liver, and spleen tissue using digital PCR. High levels of DNAI1-HA mRNA were detected in all three lung regions sampled 6 hours after exposure to RTX0051 and RTX0052, with no DNAI1-HA mRNA detected above background in the spleen (6 hours, Figure 29B). [Figure 29C] Figure 29C shows high levels of DNAI1-HA mRNA delivery to the lung without exposure to the liver, spleen, or blood. After a single dose of 0.4 mg / kg, DNAI1-HA mRNA levels were measured in whole blood, lung, liver, and spleen tissue using digital PCR. High levels of DNAI1-HA mRNA were detected in all three lung regions sampled 6 hours after exposure to RTX0051 and RTX0052, with no DNAI1-HA mRNA detected above background in the liver (6 hours, Figure 29C). [Figure 29D] Figure 29D shows high levels of DNAI1-HA mRNA delivery to the lung without exposure to the liver, spleen, or blood. After a single dose of 0.4 mg / kg, DNAI1-HA mRNA levels were measured in whole blood, lung, liver, and spleen tissue using digital PCR. High levels of DNAI1-HA mRNA were detected in all three lung regions sampled 6 hours after exposure to RTX0051 and RTX0052, with no DNAI1-HA mRNA detected above background in whole blood (30 or 60 minutes, Figure 29D). [Figure 30A]FIG. 1 shows multiplex immunofluorescence (IF) images of epithelial cell types. [Figure 30B] Figure 1 shows multiplex IF analysis demonstrating DNAI1-HA protein expression in lung target cells. [Figure 30C] Figure 1 shows multiplex IF analysis demonstrating DNAI1-HA protein expression in lung target cells using RTX0051. [Figure 30D] FIG. 1 shows multiplex IF analysis demonstrating DNAI1-HA protein expression in bronchial target cells. [Figure 30E] Figure 1 shows multiplex IF analysis demonstrating DNAI1-HA protein expression in bronchial target cells using RTX0051. [Figure 30F] FIG. 1 shows multiplex IF analysis demonstrating DNAI1-HA protein expression in tracheal target cells. [Figure 31A] FIG. 1 shows BAL cytokine and complement results. [Figure 31B] FIG. 1 shows BAL cytokine and complement results. [Figure 31C] FIG. 1 shows BAL cytokine and complement results. [Figure 31D] FIG. 1 shows BAL cytokine and complement results. [Figure 31E] FIG. 1 shows BAL cytokine and complement results. [Figure 32A] FIG. 1 shows the results of plasma cytokines. [Figure 32B] FIG. 1 shows the results of plasma cytokines. [Figure 32C] FIG. 1 shows the results of plasma cytokines. [Figure 32D] FIG. 1 shows the results of plasma cytokines. [Figure 32E] FIG. 1 shows the results of plasma cytokines. [Figure 33] FIG. 1 shows the transient increase in neutrophils observed in the BAL and blood 6 hours after exposure. [Figure 34]FIG. 1 shows selected clinical chemistry results; slight increases in AST, LDH, and creatine kinase were observed in individual animals following treatment. [Figure 35] FIG. 1 shows a summary of tolerability as determined by clinical observations and organ weights in a single-dose inhalation study. [Figure 36A] Diagram of the aerosol delivery system. The amount of aerosolized drug delivered through the endotracheal tube was estimated using the test setup shown on the left, where pre-weighed glass fiber and MCE filters were attached directly to the outlet of the endotracheal tube, multiple collections were made before, during and after treatment of the animals, the glass filters were dried and quantified using both gravimetric analysis, and the MCE filters were analyzed for mRNA content using the RiboGreen assay. [Figure 36B] Figure 1 shows the results of aerosol particle size measurements. Particle sizes of test article exposures were measured for deposition in the conducting airways (human branching generations 0-15). [Figure 37] Figure 1 shows DBAI1-HA mRNA doses present in NHPs. The black horizontal dashed line represents the target proposed dose of 0.1 mg / kg. The open yellow circles represent filter captures (GF, n=2) before and after dosing with RTX0001 / DNAI1-HA mRNA. The open yellow squares represent filter captures (GF, n=2) before and after dosing with RTX0004 / DNAI1-HA mRNA. Similar results were obtained for mRNA using MCE filters and the RiboGreen assay. [Figure 38A] Figure 1 shows DNAI1-HA mRNA ISH results for lung tissue. Assay data: one out of four samples per animal was analyzed, and DNAI1-HA mRNA was detected in all animals. [Figure 38B] FIG. 1 shows that a significant proportion of lung cells contained DNAI1-HA mRNA after treatment with RTX0001, as determined by ISH and bin scoring. [Figure 38C]FIG. 1 shows imaging of lung tissue used in ISH analysis. [Figure 39A] Figure 1 shows that high levels of DNAI1-HA delivery to the lung did not result in similar delivery to the liver or spleen. Following a single dose of 0.1 mg / kg, DNAI1-HA mRNA levels were measured in whole blood, lung, liver, and spleen tissue using digital PCR. High levels of DNAI1-HA mRNA were detected in all three lung regions sampled 6 hours after RTX0001 exposure, while in the spleen and liver, DNAI1-HA mRNA was measured only at or below the assay's LLOQ. [Figure 39B] Figure 1 shows positive staining of DNAI1-HA tagged protein in NHPs. For RTX0001, DNAI1-HA was detected 6 or 24 hours after administration, with areas of higher mRNA levels correlating with areas showing the highest levels of DNAI1-HA protein. DNAI1-HA mRNA was present in all eight treated animals, with no signal detected in vehicle-treated animals, and mRNA levels were highest at 6 hours and lowest at 24 hours. [Figure 39C] Figure 1 shows a multiplex IF panel for major epithelial cell types. Ten NHP FFPE lung tissue blocks (one from each animal) were used for the mIF assay. Two slides from each block were double-stained, and the cell counts of single marker-positive cells, double-positive cells with DNAI1 expression, and DNAI1 MFI in double-positive cells were reported. [Figure 40A] Figure 1 shows the results of a multiplex IF panel for NHP lung samples. DNAI1-HA is expressed in cells of the respiratory epithelium. The percentage of DNAI1-HA positive cells was calculated by combining cell counts from one examined lung section per animal. DNAI1-HA expression was detected in lung samples from NHPs treated with RTX0001, and co-localized with markers of epithelial cells, including club cells, basal cells, and ciliated cells (club cells and basal cells are precursors of ciliated cells). No staining was detected in lung samples from NHPs treated with RTX0004. [Figure 40B] Figure 1 shows multiplex IF analysis of DNAI1-HA protein expression in lung target cells. A single dose of 0.1 mg / kg of RTX0001 / DNAI1-HA mRNA was administered by inhalation, and lung sections were collected from two NHPs at 6 and 24 hours post-dose. The percentage of DNAI1-HA positive cells was calculated by combining cell counts from all four lung sections examined for each animal. The total number of cells counted per animal was approximately 690,000-1,100,000. Individual data points for each treated animal and each group (N=2) are shown, along with the mean ± standard deviation.

[0017] Detailed Description Before describing embodiments of the present disclosure, it is understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. Numerous variations, modifications, and substitutions will readily occur to those skilled in the art without departing from the present invention.

[0019] In the context of this application, the following terms have the meanings ascribed to them unless otherwise specified.

[0020] As used throughout this specification and claims, the terms "a," "an," and "the" are generally used in the sense of meaning "at least one," "at least the first," "one or more," or "a plurality" of the referenced component or step, unless an upper limit is specifically stated thereafter. For example, as used herein, "cleavage sequence" means "at least the first cleavage sequence," but includes multiple cleavage sequences. Operable limits and parameters of combinations, as well as the amounts of any single agent, will be known to those skilled in the art in light of this application.

[0021] The terms "polypeptide," "peptide," and "protein" are generally used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The terms also encompass amino acid polymers modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0022] As used herein in reference to a polypeptide structure, the terms "N-terminus" (or "amino terminus") and "C-terminus" (or "carboxyl terminus") generally refer to the extreme amino and carboxyl ends of a polypeptide, respectively.

[0023] The term "N-terminal sequence" as used herein with respect to a polypeptide or polynucleotide sequence of interest generally means that there are no other amino acid or nucleotide residues at the N-terminus before the N-terminal sequence in the polypeptide or polynucleotide sequence of interest. The term "C-terminal sequence" as used herein with respect to a polypeptide or polynucleotide sequence of interest generally means that there are no other amino acid or nucleotide residues at the C-terminus after the C-terminal sequence in the polypeptide or polynucleotide sequence of interest.

[0024] The terms "non-naturally occurring" and "non-naturally occurring" are used interchangeably herein. As used herein with respect to a therapeutic or prophylactic agent, the term "non-naturally occurring" or "non-naturally occurring" generally means that the agent is not biologically derived from a mammal (including, but not limited to, a human). As applied to a sequence, the terms "non-naturally occurring" and "non-naturally occurring" as used herein refer to a polypeptide or polynucleotide sequence that is not paired with, complementary to, or highly homologous to a wild-type or naturally occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, or less amino acid sequence identity with a native sequence when properly aligned.

[0025] "Physiological conditions" refers to a set of conditions found in a living host and in vitro conditions that mimic those of a living subject, including temperature, salt concentration, and pH. Numerous physiologically relevant conditions have been established for use in in vitro assays. Generally, physiological buffers contain physiological concentrations of salt and are adjusted to a neutral pH ranging from about 6.5 to about 7.8, preferably from about 7.0 to about 7.5. Various physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperatures range from about 25°C to about 38°C, preferably from about 35°C to about 37°C.

[0026] As used herein, the terms "treatment" or "treating," or "palliating" or "ameliorating" are used interchangeably herein. These terms generally refer to an approach to obtaining a beneficial or desired result, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological symptoms or improvement of one or more clinical parameters associated with the underlying disease, such that an improvement is observed in the subject, even though the subject may still be suffering from the underlying disease. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease or who reports one or more physiological symptoms of the disease, even if the disease may not have been diagnosed.

[0027] As used herein, "therapeutic effect" or "therapeutic benefit" generally refers to a physiological effect resulting from administration of a polypeptide of the present disclosure, other than the ability to induce the production of antibodies against an antigenic epitope possessed by the biologically active protein, including, but not limited to, the alleviation, amelioration, or prevention of disease in a human or other animal, or the improvement of one or more clinical parameters associated with an underlying disease, or otherwise promoting the physical or mental well-being of a human or animal. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, a symptom or symptom of a disease, or a subject reporting one or more physiological symptoms of a disease, even if the disease may not have been diagnosed.

[0028] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" generally refer to an amount of a drug or biologically active protein, alone or as part of a polypeptide composition, that, when administered to a subject in one or multiple doses, is capable of having any detectable beneficial effect on the symptoms, aspects, measured parameters, or characteristics of a disease state or condition. Such an effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0029] The term "equimolar dose" generally means that the amount of a material administered to a subject has an equivalent molar amount based on the molecular weight of the material used in the dose.

[0030] The term "therapeutically effective and non-toxic dose" as used herein generally refers to a tolerable dose of a composition as defined herein that is high enough to cause tumor or cancer cell depletion, tumor elimination, tumor shrinkage, or disease stabilization in a subject without or essentially without significant toxic effects. Such a therapeutically effective and non-toxic dose can be determined by dose escalation studies as described in the art, and should be below the dose that induces serious side effects.

[0031] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0032] 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, and "isocyanato" means -F, -Cl, -Br, or -I. , -N=C=O, "azido" means -N3, in the monovalent context "phosphate" means -OP(O)(OH)2 or a deprotonated form thereof, and in the divalent context "phosphate" means -OP(O)(OH)O- or a deprotonated form thereof, "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)-.

[0033] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. [ka] represents any bond, which, if present, is either a single or double bond. [ka] represents a single or double bond. Thus, for example, the formula [ka] teeth, [ka] and it is understood that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate a preferred stereochemistry when linking one or two stereoatoms. Instead, it covers all stereoisomers and mixtures thereof. [ka] is the bond (e.g., for methyl [ka] ) indicates the point of attachment of the group. Note that points of attachment are typically identified in this way only for larger groups, to allow the reader to clearly identify the point of attachment. [ka] denotes a single bond in which the group attached to the thick end of the wedge is "off the page." [ka] denotes a single bond in which the group attached to the thick end of the wedge is "into the page". [ka] means a single bond with no defined geometry around the double bond (e.g., either E or Z). Thus, both options and combinations thereof are contemplated. An undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is pointing out of the plane of the paper.

[0034] The group "R" may be, for example, a group of the formula [ka] When depicted as a "floating group" on the ring system, R may replace any hydrogen atom attached to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is formed. [ka] When depicted as a "floating group" on a fused ring system, as in R may replace any hydrogen bonded to any ring atom of the fused ring, unless otherwise specified. Replaceable hydrogens include depicted hydrogens (e.g., hydrogen bonded to a 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 present depending on the identity of the ring atoms (e.g., hydrogen 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 in 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 may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0035] In 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 may be present in the group / class, with the minimum number being as small as possible for that group / class, e.g., the group "alkenyl (C≦8) " or class "Alkenes (C≦8) " is understood to be 2. " " designates 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)" designates an alkyl group having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class 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 C5 " are all synonymous.

[0036] When the term "saturated" is used to modify a compound or chemical group, it means that the compound or chemical group has no carbon-carbon double bonds or 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 the case of substituents of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. When such bonds are present, it does not exclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerization. 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.

[0037] 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 joined in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated (alkane / alkyl) joined by carbon-carbon single bonds, or unsaturated with one or more carbon-carbon double bonds (alkene / alkenyl), or unsaturated with one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0038] The term "aromatic," when used to modify an atom of a compound or chemical group, means that the compound or chemical group contains a planar, unsaturated ring of atoms stabilized by the interaction of bonds that form the ring.

[0039] The term "alkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, and containing 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 with 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 subset of substituted alkyl where hydrogen atom replacement is limited to halo (i.e., -F, -Cl, -Br, or -I) and no other atoms are present except carbon, hydrogen, and halogen. The group -CHCl is a non-limiting example of haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl where hydrogen atom replacement is limited to fluoro and no other atoms are present except carbon, hydrogen, and fluorine. The groups -CHF, -CF, and -CHCF are non-limiting examples of fluoroalkyl groups.

[0040] The term "cycloalkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group with 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 with two carbon atoms as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group [ka] is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula HR, 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.

[0041] The term "alkenyl," 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 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=CHCH3, 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 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 should be noted that although alkenediyl groups are aliphatic, this does not preclude the group from forming part of an aromatic structure when attached at both termini. 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 having 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.

[0042] 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≡CCH3, and -CH2C≡CCH3 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.

[0043] The term "aryl," when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group in which two aromatic carbon atoms are the points of attachment, the carbon atoms form one or more six-membered aromatic ring structures, and the ring atoms are all carbon, with the group consisting of atoms only of carbon and hydrogen. When two or more rings are present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitations permitted) 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 in which two aromatic carbon atoms are the points of attachment, the carbon atoms form one or more six-membered aromatic ring structures, and the ring atoms are all carbon, with the monovalent group consisting of atoms only of carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limitations permitted) 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 a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitations permitted). Non-limiting examples of arenediyl groups include: [ka] Examples include:

[0044] "Arene" refers to the class of compounds having the formula HR, 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.

[0045] 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 consistent with the definitions provided above. 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.

[0046] 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 is composed 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 (carbon number limitations permitted) 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 with a nitrogen atom as the point of attachment. The term "heteroarenediyl," when used without the "substituted" modifier, refers to a divalent aromatic group with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structures, and at least one ring atom being nitrogen, oxygen, or sulfur, and the divalent group is composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. 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 a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitations permitted). As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number limitations permitted) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyl groups include: [ka] Examples include:

[0047] "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.

[0048] 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 is composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. A heterocycloalkyl ring may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or 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 groups (carbon number limitations permitted) attached to a ring or ring system. The term also does not exclude the presence of one or more double bonds in a 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 with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl," when used without the "substituted" modifier, refers to a divalent cyclic group with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structures, and at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group is composed 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 via one or more of a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitations permitted). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitations permitted) attached to the ring or ring system.The term also 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 heterocycloalkanediyl groups include: [ka] When 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.

[0049] 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 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. "Thioacyl" is defined in an analogous manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom -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 any hydrogen atoms directly bonded to the carbon atom of a carbonyl or thiocarbonyl 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. 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.

[0050] 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" refers 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" refers 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.

[0051] 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 --NHCH.

[0052] 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 alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH)2 and -N(CH3)(CH2CH3). 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, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.The groups -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amide groups.

[0053] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present application. In general, the term "about" as used herein in reference to a measurable value, such as weight, time, dose, and the like, is meant to encompass variations of, in one example, ±20% or ±10%, in another example, ±5%, in another example, ±1%, and in yet another example, ±0.1% from the stated amount, where such variations are appropriate for carrying out the disclosed methods.

[0054] As used in this application, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have a different degree of polymerization, and therefore a different molar mass. Average molecular weight can be used to represent the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number-average molar mass, weight-average (mass) molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, average molecular weight refers to either the number-average molar mass or weight-average molar mass of the formula. In some embodiments, average molecular weight is number-average molar mass. In some embodiments, average molecular weight can be used to represent the PEG component present in lipids.

[0055] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs is also open-ended, such as "comprises," "comprising," "has," "having," "includes," or "including." For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other unlisted steps.

[0056] The term "effective," as used herein and / or in the claims, means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to effectively effect such treatment for the disease.

[0057] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximal response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit half of a given biological, biochemical, or chemical process (or component of that process, i.e., enzyme, cell, cell receptor, or microorganism).

[0058] An "isomer" of a first compound is a distinct chemical compound in which each molecule contains the same constituent atoms as the first compound, but differs in the arrangement of those atoms in three dimensions.

[0059] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., a non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0060] The terms "assemble" or "assembled," as used herein in connection with delivery of a payload to a target cell, generally refer to a covalent or non-covalent interaction or association, e.g., where a therapeutic or prophylactic agent is complexed with or encapsulated in a lipid composition.

[0061] As used herein, the term "lipid composition" generally refers to a composition comprising lipid compounds, including, but not limited to, lipoplexes, liposomes, lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

[0062] For example, as used herein, "RTX0001" refers to an exemplary lipid composition tested herein. RTX0001 is a five-component lipid nanoparticle composition containing approximately 19.05% 4A3-SC7 (an ionizable cationic lipid), approximately 20% DODAP (a SORT lipid), approximately 19.05% DOPE, approximately 38.9% cholesterol, and approximately 3.81% DMG-PEG (a PEG-conjugated lipid), where each lipid component is defined as a mole percent of the total lipid composition.

[0063] As another example, as used herein, "RTX0004" refers to an exemplary lipid composition tested herein. RTX0004 is a four-component lipid nanoparticle composition containing approximately 23.81% 5A2-SC8 (an ionizable cationic lipid), approximately 23.81% DOPE, approximately 47.62% cholesterol, and approximately 4.76% DMG-PEG (a PEG-conjugated lipid), where each lipid component is defined as a mole percent of the total lipid composition.

[0064] As another example, "RTX0051" as used herein refers to an example lipid composition tested herein. RTX0051 is a five-component lipid nanoparticle composition containing approximately 19.05% 4A3-SC7 (an ionizable cationic lipid), approximately 20% 14:0 EPC (a SORT lipid), approximately 19.05% DOPE, approximately 38.9% cholesterol, and approximately 3.81% DMG-PEG (a PEG-conjugated lipid), where each lipid component is defined as a mole percent of the total lipid composition.

[0065] As yet another example, as used herein, "RTX0052" refers to an exemplary lipid composition tested herein. RTX0052 is a five-component lipid nanoparticle composition comprising approximately 19.05% 4A3-SC7 (an ionizable cationic lipid), approximately 20% 14:0 TAP (a SORT lipid), approximately 19.05% DOPE, approximately 38.9% cholesterol, and approximately 3.81% DMG-PEG (a PEG-conjugated lipid), where each lipid component is defined as a mole percent of the total lipid composition.

[0066] As used herein, the term "detectable" refers to the occurrence or change of a signal that can be detected directly or indirectly by observation or instrumentation. Typically, the detectable response is the occurrence of a signal in which the fluorophore is inherently fluorescent and does not undergo a signal change upon binding to a metal ion or biological compound. Alternatively, the detectable response is an optical response that results in a change in the wavelength distribution pattern or intensity of absorbance or fluorescence, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0067] The term "potent" or "potency" as used herein in relation to the delivery of a therapeutic agent generally refers to a higher ability of a delivery system (e.g., a lipid composition) to achieve or cause a desired amount, activity, or effect (such as a desired level of translation, transcription, production, expression, or activity of a protein or gene) of a therapeutic or prophylactic agent in a cell (e.g., a target cell) to any measurable extent, for example, compared to a reference delivery system. For example, a lipid composition with higher potency may achieve a desired therapeutic effect in a larger proportion of relevant cell populations, within a shorter response time, or for a longer duration.

[0068] 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 excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0069] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present application that are pharmaceutically acceptable as defined above and have the desired pharmacological activity. Such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or salts of 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, and ethanesulfonic acid. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as benzoic acid, fumaric acid, glucoheptonic acid, 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-butyl acetate, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present can react 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, etc. It should be recognized that the particular anion or cation forming a part of any salt of the present disclosure is not critical, so long as the salt, as a whole, is pharmaceutically acceptable.Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are found in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0070] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting a chemical agent.

[0071] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptoms of the disease, and / or (2) delaying the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptoms of the disease.

[0072] A "repeating unit" is the simplest structural entity of a material, e.g., a framework and / or polymer, whether organic, inorganic, or organometallic. In a polymer chain, repeating units are linked together sequentially along the chain like beads on a necklace. For example, the -[-CH2CH2-] repeat unit in polyethylene nIn -, the repeat unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units connected to each other. If the value of "n" is left undefined or is absent, it simply indicates the repetition of the formula within the parentheses and the polymeric nature of the material. The concept of a repeat unit equally applies when the connectivity between repeat units extends three-dimensionally, such as in metal-organic frameworks, modified polymers, and thermosetting polymers. Within the context of dendrimers or dendrons, repeat units are sometimes described as branching units, internal layers, or generations. Similarly, terminal groups are sometimes described as surface groups.

[0073] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the three-dimensional arrangement of those atoms. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain chiral centers, also called stereocenters or stereoisomeric centers, which are any points within a molecule that have groups such that any two groups, when interchanged, result in a stereoisomer, but not necessarily atoms. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, but other atoms can also be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, resulting in many stereoisomers. In compounds where stereoisomerism is due to a tetrahedral stereocenter (e.g., a tetrahedral carbon), the total number of hypothetical possible stereoisomers is 2. nn is the number of tetrahedral stereocenters. The number of stereoisomers of symmetric molecules is often less than the maximum possible number. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched, with one enantiomer present in greater than 50%. Typically, enantiomers and / or diastereomers can be separated or resolved using techniques known in the art. For any stereocenter or axis of chirality with undefined stereochemistry, it is contemplated that the stereoisomer or axis of chirality may exist as its R-, S-, or mixtures of R- and S-isomers, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of another stereoisomer.

[0074] "Treatment" or "treating" includes (1) inhibiting the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease (e.g., preventing further progression of the symptoms and / or symptoms), (2) ameliorating the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease (e.g., reversing the symptoms and / or symptoms), and / or (3) causing any measurable decrease in the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease.

[0075] The above definitions take precedence over any conflicting definitions in any reference incorporated herein by reference. However, the fact that a particular term is defined should not be taken as indicating that the undefined term is unclear. Rather, all terms used are believed to describe the present disclosure in terms that allow one skilled in the art to understand the scope and practice the present application.

[0076] composition lipid composition In one aspect, provided herein is a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0077] Ionizable cationic lipids In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid contains one or more groups that are protonated at physiological pH but may be deprotonated and uncharged at pHs above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines that can form cationic groups at physiological pH. Cationic ionizable lipid compounds may also contain C6-C6 24 The compound may further comprise one or more lipid components, such as two or more fatty acids having alkyl or alkenyl carbon groups. These lipid groups may be linked via ester bonds or may be further attached via Michael addition to sulfur atoms. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0078] In some embodiments of the lipid compositions of the present application, ionizable cationic lipids refer to lipids and lipid-like molecules that have a nitrogen atom capable of acquiring an electric charge (pKa). These lipids are sometimes known in the literature as cationic lipids. These molecules, which have an amino group, typically have two to six hydrophobic chains, often C6 to C8. 24The cationic ionizable lipids may have an alkyl or alkenyl group, such as an alkyl or alkenyl group, and may have at least one or more than six tails. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers exhibiting regular dendritic branching, formed by the sequential or generational addition of branches to or from a core, characterized by a core, at least one internal branching layer, and surface branching layers. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, pp. 641-645, August 1994.) In other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular structure having an internal core, internal layers (or "generations") of repeating units regularly attached to this initiator core, and an external surface of terminal groups attached to the outermost generation. A "dendron" is a dendrimer species with branches emanating from a central core that are, or can be, tethered to a core directly or via a linking moiety to form a larger dendrimer. In some embodiments, the dendrimer structure has repeating groups radiating from a central core that double with each repeat unit on each branch. In some embodiments, the dendrimers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms may be used to describe compounds described herein that have a dendron-like appearance (e.g., molecules radiating from a single center).

[0079] Dendrimers are polymers that may be considered preferable to conventional polymers because they have a controllable structure, a single molecular weight, numerous controllable surface functional groups, and traditionally adopt a globular conformation after reaching a certain generation. Dendrimers can be prepared by sequentially reacting each repeating unit, creating monodisperse, tree-like, and / or generationally structured polymer structures. Individual dendrimers consist of a central core molecule with dendritic wedges attached to one or more functional sites on the core. Depending on the assembly monomers used during preparation, the dendrimer surface layer can have a variety of functional groups disposed on it, including anionic, cationic, hydrophilic, or lipophilic groups.

[0080] The functional groups and / or chemical properties of the core, repeating units, and surface or terminal groups can be modified to adjust their physical properties. Some properties that can be varied include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment capability. Dendrimers are often described by the generation and number of repeating units within a branch. A dendrimer consisting only of the core molecule is called generation 0, and each successive repeating unit along all branches is generation 1, generation 2, and so on, continuing up to the terminal or surface groups. In some embodiments, half-generations are possible that result from only the first condensation reaction with an amine and not the second condensation reaction with a thiol.

[0081] The preparation of dendrimers requires a level of synthetic control achieved through a series of stepwise reactions involving the construction of the dendrimer by each successive group. Dendrimer synthesis can be convergent or divergent. During divergent dendrimer synthesis, molecules are assembled from the core to the periphery in a stepwise process that connects one generation to the previous generation and alters the functional groups for the next step of reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerizations result in highly branched molecules that are not monodisperse, also known as hyperbranched polymers. Due to steric effects, continued reaction of dendrimer repeat units results in spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation, destroying the monodispersity of the molecules. Thus, in some embodiments, dendrimers of generations G1 to G10 are specifically contemplated. In some embodiments, dendrimers contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units, or any range derivable therefrom. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3, however, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by decreasing the spacing units in the branched polymer.

[0082] Furthermore, dendrimers have two main chemical environments: the environment created by the specific surface groups of the terminal generations, and the interior of the dendritic structure, which can be shielded from the bulk medium and surface groups by the higher-order structure. Because of these different chemical environments, dendrimers have found many different potential applications, including therapeutic applications.

[0083] In some embodiments of the lipid compositions of the present application, dendrimers or dendrons are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers or dendrons may contain secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylates with mercapto groups. Furthermore, the repeating units of the dendrimer or dendron may contain groups that are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine, allowing for dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple distinct dendritic branches, each of which may contain one or more repeating units. Dendrimers or dendrons can be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are located on heteroatoms, such as nitrogen atoms. In some embodiments, the terminal groups are lipophilic groups, such as long-chain alkyl or alkenyl groups. In other embodiments, the terminal group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the terminal group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH) or a carboxylic acid (-COH). In still other embodiments, the terminal group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0084] The cationic ionizable lipids of the present application may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic forms. 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 may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the cationic ionizable lipids of the present application may have an S or R configuration. Furthermore, it is contemplated that one or more of the cationic ionizable lipids may exist as structural isomers. In some embodiments, the compounds have the same formula but differ in the connectivity to the core nitrogen atom. Without wishing to be bound by theory, it is believed that such cationic ionizable lipids exist because the starting monomer first reacts with a primary amine and then with a statistically present secondary amine. Thus, a structural isomer may represent a mixture of fully reacted primary amines and subsequently reacted secondary amines.

[0085] The chemical formula used to represent the cationic ionizable lipids of this application will typically only show one of several different tautomers.For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups.Similarly, many types of imine groups exist in equilibrium with enamine groups.Regardless of which tautomer is depicted for a given chemical formula, and regardless of which tautomer is most common, all tautomers of a given chemical formula are intended.

[0086] The cationic ionizable lipids of the present application, whether for use in the indications described herein or otherwise, may also have the advantage that they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art.

[0087] Furthermore, the atoms constituting the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms with the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include tetrahydrofuran and tetrahydrofuran. 13 C and 14 Contains C.

[0088] 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 pharmaceutically acceptable.Additional examples of pharmaceutically acceptable salts and their preparation and use are described in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0089] In some embodiments of the lipid compositions of the present application, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C 24 It contains an alkyl or alkenyl group.

[0090] Dendrimers or dendrons of formula (I) In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C 24 In some embodiments, the ionizable cationic lipid comprises an alkyl group. Core-Repeat Unit-End Group (DI) or a pharmaceutically acceptable salt thereof; wherein the core is linked to the repeat unit by removing one or more hydrogen atoms from the core and replacing said atoms with the repeat unit; The core has the formula: [ka] and During the ceremony X1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) , heterocycloalkyl (C≦12) , heteroaryl (C≦12) or a substitution thereof, R1 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substitution of any of these groups, a is 1, 2, 3, 4, 5, or 6; or The core has the formula: [ka] and During the ceremony, X2 is N(R5) y and R5 is hydrogen, alkyl (C≦18) , or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substitution of any of these groups, b is 1, 2, 3, 4, 5, or 6; z is 1, 2, or 3, with the proviso that the sum of z and y is 3; or The core has the formula: [ka] and During the ceremony, X3 is -NR6-, where R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or a substitution of any of these groups, R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) or a substituent of any of these groups; or a group of the formula: -N(R f ) f (CH2CH2N(R c )) e Rd , [ka] is the basis of During the ceremony, e and f are each independently 1, 2, or 3, with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) , or substituted alkyl (C≦6) and c and d are each independently 1, 2, 3, 4, 5, or 6; or The core is an alkylamine (C≦18) , dialkylamine (C≦36) , heterocycloalkane (C≦12) or a substituted version of these groups, The repeat unit comprises a degradable diacyl and a linker, The degradable diacyl group has the formula: [ka] and During the ceremony, A1 and A2 each independently represent -O-, -S-, or -NR a - in which R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and Y3 is alkanediyl (C≦12) , Alkendiyl (C≦12) , Arendjiil (C≦12) or a substitution of any of these groups; or a group of the formula: [ka] is the basis of During the ceremony, X3 and X4 are alkanediyl (C≦12) , Alkendiyl (C≦12) , Arendjiil(C≦12) or a substitution of any of these groups, Y5 is a covalent bond, alkanediyl (C≦12) , Alkendiyl (C≦12) , Arendjiil (C≦12) or a substitution of any of these groups, R9 is alkyl (C≦8) or substituted alkyl (C≦8) and The linker group may have the formula: [ka] and During the ceremony, Y1 is alkanediyl (C≦12) , Alkendiyl (C≦12) , Arendjiil (C≦12) or a substitution of any of these groups, When the repeat unit comprises a linker group, the linker group comprises independent degradable diacyl groups bonded to both the nitrogen atom and the sulfur atom of the linker group when n is greater than 1, the first group in the repeat unit is a degradable diacyl group, and for each linker group, the next repeat unit comprises two degradable diacyl groups bonded to nitrogen atoms of the linker group, and n is the number of linker groups present in the repeat unit; The terminal group has the formula [ka] and During the ceremony, Y4 is alkanediyl (C≦18) , or alkanediyl (C≦18) Alkanediyl in which one or more of the above hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≦18) and R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12), N-heterocycloalkyl (C≦12) , -C(O)N(R 11 )-Alkanediyl (C≦6) -heterocycloalkyl (C≦12) , -C(O)-alkylamino (C≦12) , -C(O)-dialkylamino (C≦12) , —C(O)—N-heterocycloalkyl (C≦12) wherein: R 11 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and The last degradable diacyl in the chain is attached to the terminal group, n is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the terminal group has the formula [ka] is further defined in During the ceremony, Y4 is alkanediyl (C≦18) and R 10 In some embodiments, A1 and A2 are each independently -O- or -NR a -It is.

[0091] In some embodiments of a dendrimer or dendron of formula (DI), the core is of formula [ka] is further defined in During the ceremony, X2 is N(R5) y and R5 is hydrogen or alkyl (C≦8) , or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12)or a substitution of any of these groups, b is 1, 2, 3, 4, 5, or 6; z is 1, 2, or 3, with the proviso that the sum of z and y is 3.

[0092] In some embodiments of dendrimers or dendrons of formula (DI), the core is [ka] is further defined in During the ceremony, X3 is -NR6-, where R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or a substitution of any of these groups, R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) or a substituent of any of these groups; or a group of formula: -N(R f ) f (CH2CH2N(R c )) e R d , [ka] is the basis of During the ceremony, e and f are each independently 1, 2, or 3, with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) , or substituted alkyl (C≦6) and c and d are each independently 1, 2, 3, 4, 5, or 6.

[0093] In some embodiments of dendrimers or dendrons of formula (I), the terminal group is of the formula [ka] is expressed as During the ceremony, Y4 is alkanediyl (C≦18) and R 10 is hydrogen.

[0094] In some embodiments of dendrimers or dendrons of formula (DI), the core is [ka] It is further defined as:

[0095] In some embodiments of dendrimers or dendrons of formula (DI), the decomposable diacyl is [ka] It is further defined as:

[0096] In some embodiments of a dendrimer or dendron of formula (DI), the linker is [ka] is further defined as In the formula, Y1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8) is.

[0097] In some embodiments of a dendrimer or dendron of formula (DI), the dendrimer or dendron is [ka] [ka] and pharmaceutically acceptable salts thereof.

[0098] a dendrimer or dendron of formula (X) In some embodiments of the lipid composition, the ionizable cationic lipid has the formula: [ka] In some embodiments, the ionizable cationic lipid is a dendrimer or dendron of the formula [ka] The dendrimer or dendron is

[0099] In some embodiments of the lipid composition, the ionizable cationic lipid has the structural formula [ka] Dendrimers or dendrons of generation (g) having or a pharmaceutically acceptable salt thereof, wherein: (a) The core has the structural formula (X コア ): [ka] Including, During the ceremony, Q, independently in each occurrence, is a covalent bond, -O-, -S-, or -NR 2 - or -CR 3a R 3b - and R 2 independently at each occurrence, R 1g or -L 2 -NR 1e R 1f and R 3a and R 3bare each independently at each occurrence hydrogen or optionally substituted alkyl (e.g., C1-C6, such as C1-C3), R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) independently at each occurrence represent a point of attachment to a branch, hydrogen, or an optionally substituted (e.g., C1-C 12 ) alkyl, L 0 , L 1 , and L 2 are each independently at each occurrence selected from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; Alternatively, L 1 Part of R 1c and R 1d with one of the following to form a (e.g., C4-C6) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur); x 1 is 0, 1, 2, 3, 4, 5, or 6, (b) each branch of the plurality (N) of branches independently has the structural formula (X 分岐 ): [ka] Including, During the ceremony, * indicates the attachment point of the branch to the core; g is 1, 2, 3, or 4; Z=2 (g-1) and If g = 1 then G = 0; or if g ≠ 1 then G =

number

[0100] X コア In some embodiments of the formula (I), Q is independently at each occurrence a covalent bond, —O—, —S—, —NR 2 - or -CR 3a R 3b X コア In some embodiments, Q is independently at each occurrence a covalent bond. コア In some embodiments of X, Q is independently at each occurrence -O-. コア In some embodiments of X, Q is independently at each occurrence -S-. コア In some embodiments, Q is independently at each occurrence -NR 2 and R 2 independently at each occurrence, R 1g or -L 2 -NR 1e R 1f X コア In some embodiments, Q is independently at each occurrence -CR 3a R 3b R 3a and R 3a and R 3b is independently at each occurrence hydrogen or optionally substituted alkyl (eg, C1-C6, such as C1-C3).

[0101] X コア In some embodiments, R 1a , R 1b , R1c , R 1d , R 1e , R 1f , and R 1g (when present) is independently at each occurrence a connection point to a branch, hydrogen, or optionally substituted alkyl. コア In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (if present) is, independently at each occurrence, a connection point to a branch, hydrogen. コア In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present), independently at each occurrence, represent the point of attachment to the branch, optionally substituted alkyl (e.g., C1-C 12 )

[0102] X コア In some embodiments, L 0 , L 1 , and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or alternatively, L 1 Part of R 1c and R 1d and one of X to form a heterocycloalkyl (e.g., C4-C6, containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur). コア In some embodiments, L 0 , L 1 , and L 2 X may each independently at each occurrence be a covalent bond. コアIn some embodiments, L 0 , L 1 , and L 2 X may each independently be hydrogen at each occurrence. コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents alkylene (e.g., C1-C 12 , C1 to C6, or C1 to C3, etc. コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents heteroalkylene (e.g., C1-C 12 , for example, C1 to C8 or C1 to C6). コア In some embodiments, L 0 , L 1 , and L 2 X at each occurrence can independently be heteroalkylene (e.g., C2-C8 alkylene oxide, such as oligo(ethylene oxide)). コア In some embodiments, L 0 , L 1 , and L 2 X may each independently at each occurrence be [alkylene]-[heterocycloalkyl]-[alkylene][(e.g., C1-C6) alkylene]-[(e.g., C4-C6) heterocycloalkyl]-[(e.g., C1-C6) alkylene]. コア In some embodiments, L 0 , L 1 , and L 2 X may each independently at each occurrence be [alkylene]-(arylene)-[alkylene][(e.g., C1-C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene]. コア In some embodiments, L 0 , L 1 , and L 2X may each independently at each occurrence be [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6) alkylene]-phenylene-[(e.g., C1-C6) alkylene]). コア In some embodiments, L 0 , L 1 , and L 2 At each occurrence, X can independently be heterocycloalkyl (e.g., C4-C6 heterocycloalkyl). コア In some embodiments, L 0 , L 1 , and L 2 Each occurrence of X can independently be arylene (e.g., phenylene). コア In some embodiments, L 1 Part of R 1c and R 1d and one of X to form a heterocycloalkyl. コア In some embodiments, L 1 Part of R 1c and R 1d and one of the following to form a heterocycloalkyl (e.g., a C4-C6 heterocycloalkyl), which may contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.

[0103] X コア In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C 12 (e.g., C2-C8) alkylene oxide (e.g., oligo(ethylene oxide, -(CH2CH2O) 1~4 -(CH2CH2)-, etc.), [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., [ka] ), and [(C1-C4) alkylene]-phenylene-[(C1-C4) alkylene] (e.g., [ka] ) is selected from X コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents C1-C6 alkylene (e.g., C1-C3 alkylene), -(C1-C3 alkylene-O) 1~4 -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-, and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-. X コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence C1-C6 alkylene (e.g., C1-C3 alkylene). 0 , L 1 , and L 2 are independently generated at each occurrence, C2 to C 12 (e.g., C2-C8) alkylene oxide (e.g., -(C1-C3 alkylene-O) 1~4 -(C1-C3 alkylene)). X コア In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).

[0104] X コア In some embodiments, x 1is 0, 1, 2, 3, 4, 5, or 6. X コア In some embodiments, x 1 is 0. X コア In some embodiments, x 1 is 1. X コア In some embodiments, x 1 is 2. X コア In some embodiments, x 1 is 0, 3. X コア In some embodiments, x 1 is 4. X コア In some embodiments, x 1 is 5. X コア In some embodiments, x 1 is 6.

[0105] X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula: [ka] wherein Q′ is —NR 2 -or-CR 3a R 3b - and q 1 and q 2 are each independently 1 or 2. コア In some embodiments, the core has the structural formula: [ka] Contains X コア In some embodiments, the core has the structural formula [ka] wherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C3-C 12 and is heteroaryl (C3 to C5, etc.). X コア In some embodiments, the core has the structural formula [ka] Includes.

[0106] X コア In some embodiments, the core comprises a structural formula set forth in Table 1, and pharmaceutically acceptable salts thereof, where * indicates the point of attachment of the core to one branch of the multiple branches. In some embodiments, the exemplary cores in Table 1 are not limited to the listed stereoisomers (i.e., enantiomers, diastereomers).

[0107] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0108] X コア In some embodiments, the core is [ka] [ka] and pharmaceutically acceptable salts thereof, wherein * denotes the point of attachment of the core to one branch of the plurality of branches or H. In some embodiments, * denotes the point of attachment of the core to one branch of the plurality of branches.

[0109] X コア In some embodiments, the core has the structure [ka] where * indicates the point of attachment of the core to one of the branches or to H. In some embodiments, at least two branches are attached to the core. In some embodiments, at least three branches are attached to the core. In some embodiments, at least four branches are attached to the core.

[0110] X コア In some embodiments, the core has the structure [ka] where * indicates the point of attachment of the core to one of the branches or to H. In some embodiments, at least four branches are attached to the core. In some embodiments, at least five branches are attached to the core. In some embodiments, at least six branches are attached to the core.

[0111] In some embodiments, the plurality (N) of branches comprises at least three branches, at least four branches, or at least five branches. In some embodiments, the plurality (N) of branches comprises at least three branches. In some embodiments, the plurality (N) of branches comprises at least four branches. In some embodiments, the plurality (N) of branches comprises at least five branches.

[0112] X 分岐 In some embodiments, g is 1, 2, 3, or 4. 分岐 In some embodiments, g is 1 and X 分岐 In some embodiments, g is 2 and X 分岐 In some embodiments, g is 3 and X 分岐 In some embodiments of the formula, g is 4.

[0113] X 分岐 In some embodiments, Z=2 (g-1) and when g=1, G=0. X 分岐 In some embodiments, Z=2 (g-1) and if g≠1, then G=

number

[0114] X 分岐 In some embodiments of the invention, g=1, G=0, Z=1, and each branch of the plurality of branches has the structural formula [ka] Includes.

[0115] X 分岐 In some embodiments of the invention, g=2, G=1, Z=2, and each branch of the plurality of branches has the structural formula [ka] Includes.

[0116] X 分岐 In some embodiments of the invention, g=3, G=3, Z=4, and each branch of the plurality of branches is represented by the structural formula [ka] Includes.

[0117] X 分岐 In some embodiments of the invention, g=4, G=7, and Z=8, and each branch of the plurality of branches has the structural formula [ka] Includes.

[0118] In some embodiments, a dendrimer or dendron described herein of generation (g)=1 has the structure: [ka] It has.

[0119] In some embodiments, a dendrimer or dendron described herein of generation (g)=1 has the structure: [ka] It has.

[0120] Examples of formulations of dendrimers or dendrons described herein of generations 1 to 4 are shown in Table 2. The number of diacyl groups, linker groups, and terminal groups can be calculated based on g.

[0121] [Table 2]

[0122] In some embodiments, the diacyl groups independently have the structural formula [ka] where * indicates the point of attachment of the diacyl group at its proximal end and ** indicates the point of attachment of the diacyl group at its distal end.

[0123] X 分岐 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. 分岐 In some embodiments of the diacyl group of Y 3 independently at each occurrence represents an optionally substituted alkylene (e.g., C1-C 12 ) X 分岐 In some embodiments of the diacyl group of Y 3 independently at each occurrence represents an optionally substituted alkenylene (e.g., C1-C 12 ) X 分岐 In some embodiments of the diacyl group of Y 3 independently at each occurrence represents an optionally substituted allenylene (e.g., C1-C 12 )

[0124] X 分岐 In some embodiments of the diacyl group of A 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -X 分岐 In some embodiments of the diacyl group of A 1 and A 2 is independently -O- at each occurrence. 分岐In some embodiments of the diacyl group of A 1 and A 2 is independently -S- at each occurrence. 分岐 In some embodiments of the diacyl group of A 1 and A 2 are each independently, at each occurrence, -NR 4 - and R 4 is hydrogen or optionally substituted alkyl (e.g., C1-C6). X 分岐 In some embodiments of the diacyl group of m 1 and m 2 is independently 1, 2, or 3 at each occurrence. In some embodiments of the X branched diacyl group, m 1 and m 2 are each independently 1 in each occurrence. 分岐 In some embodiments of the diacyl group of m 1 and m 2 is independently 2 in each occurrence. 分岐 In some embodiments of the diacyl group of m 1 and m 2 is independently 3 in each occurrence. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f X is independently at each occurrence hydrogen or optionally substituted alkyl. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f X is independently hydrogen at each occurrence. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f is independently at each occurrence an optionally substituted (eg, C1-C8) alkyl.

[0125] In some embodiments of the diacyl group, A 1 is -O- or -NH-. In some embodiments of a diacyl group, A 1 In some embodiments of the diacyl group, A 2 is -O- or -NH-. In some embodiments of a diacyl group, A 2 In some embodiments of the diacyl group, Y 3 is C1~C 12 It is alkylene (for example, C1 to C6, such as C1 to C3).

[0126] In some embodiments of a diacyl group, the diacyl group may be independently represented at each occurrence by the structural formula [ka] and optionally, wherein R 3c , R 3d , R 3e , and R 3f is independently at each occurrence hydrogen or C1-C3 alkyl.

[0127] In some embodiments, the linker groups independently have the structural formula [ka] where ** indicates the point of attachment of the linker to the proximal diacyl group and *** indicates the point of attachment of the linker to the distal diacyl group.

[0128] X 分岐 In some embodiments when a linker group of the formula 1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. 分岐 In some embodiments when a linker group of the formula 1 independently at each occurrence represents an optionally substituted alkylene (e.g., C1-C 12 ) X分岐 In some embodiments when a linker group of the formula 1 independently at each occurrence represents an optionally substituted alkenylene (e.g., C1-C 12 ) X 分岐 In some embodiments when a linker group of the formula 1 independently at each occurrence represents an optionally substituted allenylene (e.g., C1-C 12 )

[0129] X 分岐 In some embodiments of the terminal groups of X, each terminal group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol. 分岐 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkylthiol (e.g., C1-C 18 And C4~C 18 etc.) X 分岐 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkenyl thiol (e.g., C1-C 18 And C4~C 18 etc.).

[0130] X 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 Alkenyl thiol or C1-C 18 Alkylthiol, the alkyl or alkenyl moiety is halogen, C6-C 12 Aryl, C1-C 12 Alkylamino, C4-C6N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6N-heterocycloalkyl), -C(O)-(C1-C 12-C(O)-(C-C N-heterocycloalkyl), and -C(O)-(C-C N-heterocycloalkyl), wherein the C-C N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C-C alkyl or C-C hydroxyalkyl.

[0131] X 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 (For example, C4~C 18 ) Alkenyl thiol or C1-C 18 (For example, C4~C 18 ) alkylthiol, wherein the alkyl or alkenyl portion is halogen, C6-C 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.), C4-C6 N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino)) (e.g., [ka] ), —C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocycloalkyl) (e.g., [ka] ), -C(O)-(C1~C 12 alkylamino (e.g., mono- or dialkylamino)), and —C(O)—(C4-C6N-heterocycloalkyl) (e.g., [ka] ) optionally substituted with one or more substituents each independently selected from the following: wherein the C4-C6 N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl. 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion of which is optionally substituted with one substituent -OH. X 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion is C1 to C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (e.g., -NHCH2CH2CH2CH3 etc.) or C1-C8 dialkylamino (e.g., [ka] and C4-C6 N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )) and optionally substituted with one substituent selected from 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 (For example, C4~C 18 ) Alkenyl thiol or C1-C 18 (For example, C4~C 18 ) alkylthiol. X 分岐 In some embodiments of the terminal group, each terminal group is independently C1 to C 18 (For example, C4~C 18 ) alkylthiol.

[0132] X 分岐 In some embodiments of the terminal groups, each terminal group is independently a structure set forth in Table 3. In some embodiments, a dendrimer or dendron described herein can include a terminal group or a pharmaceutically acceptable salt thereof selected in Table 3. In some embodiments, the exemplary terminal groups in Table 3 are not limited to the listed stereoisomers (i.e., enantiomers, diastereomers).

[0133] [Table 3-1] [Table 3-2]

[0134] In some embodiments, the dendrimer or dendron of formula (X) is selected from those set forth in Table 4, and pharmaceutically acceptable salts thereof.

[0135] [Table 4-1] [Table 4-2]

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

[0136] Other ionizable cationic lipids In some embodiments of the lipid composition, the cationic lipid has structural formula (D-I'): [ka] Including, During the ceremony, a is 1 and b is 2, 3, or 4, or alternatively, b is 1 and a is 2, 3, or 4; m is 1 and n is 1, or alternatively, m is 2 and n is 0, or alternatively, m is 2 and n is 1; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H, -CHCH(OH)R 7 , -CH(R 7 )CH2OH, -CH2CH2C(=O)OR 7 , -CH2CH2C(=O)NHR 7 , and -CHR 7 wherein R 7 are independently C3 to C 18 Alkyl, C3-C with one C=C double bond 18selected from alkenyl, an amino protecting group, -C(=NH)NH, a poly(ethylene glycol) chain, and an acceptor ligand; However, R 1 ~R 6 At least two moieties of are independently —CHCH(OH)R 7 , -CH(R 7 )CH2OH, -CH2CH2C(=O)OR 7 , -CH2CH2C(=O)NHR 7 , or -CH2R 7 wherein R 7 are independently C3 to C 18 Alkyl or C3-C with one C=C double bond 18 alkenyl, One or more of the nitrogen atoms shown in formula (D-I') may be protonated to provide a cationic lipid.

[0137] In some embodiments of the cationic lipid of formula (D-I'), a is 1. In some embodiments of the cationic lipid of formula (D-I'), b is 2. In some embodiments of the cationic lipid of formula (D-I'), m is 1. In some embodiments of the cationic lipid of formula (D-I'), n is 1. In some embodiments of the cationic lipid of formula (D-I'), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or -CHCH(OH)R 7 In some embodiments of the cationic lipid of formula (D-I′), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or [ka] In some embodiments of the cationic lipid of formula (D-I′), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or [ka] In some embodiments of the cationic lipid of formula (D-I′), R 7 is C3~C 18 Alkyl (e.g., C6-C 12 alkyl).

[0138] In some embodiments, the cationic lipid of formula (D-I′) is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: [ka] is.

[0139] In some embodiments, the cationic lipid of formula (D-I′) is (11R,25R)-13,16,20-tris((R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: [ka] is.

[0140] Additional cationic lipids that can be used in the compositions and methods of the present application include those described in J. McClellan, MC King, Cell 2010, 141, 210-217, and International Patent Publications WO2010 / 144740, WO2013 / 149140, WO2016 / 118725, WO2016 / 118724, WO2013 / 063468, WO2016 / 205691, WO2015 / 184256, WO2016 / 004202, WO2015 / 199952, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2017 / 049245, WO2017 / 173054, and WO2015 / 095340, which are incorporated herein by reference for all purposes. Examples of such ionizable cationic lipids include, but are not limited to, those shown in Table 5.

[0141] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14]

[0142] In some embodiments of the lipid compositions of the present application, the ionizable cationic lipid is present in an amount of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is about 7.5 to about 20. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0143] In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 5% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 10% to about 25%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30% molar percentage of ionizable cationic lipids. In some embodiments of the lipid composition of the present application, the lipid composition comprises at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30% molar percentage of ionizable cationic lipids.

[0144] Selective Organ Targeting (SORT) Lipids In some embodiments of the lipid compositions of the present application, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ. In some embodiments, the target organ is the lung, lung tissue, or lung cells. As used herein, the term "preferentially delivered" refers to a composition that, upon delivery, delivers at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the administered dose to the target organ (e.g., lung), tissue, or cell.

[0145] In some embodiments of the lipid composition, the lipid composition comprises one or more selective organ targeting (SORT) lipids that provide for selective delivery of the composition to a particular organ. In some embodiments, the SORT lipid is a C6-C 24 The alkyl or alkenyl group may have two or more alkyl or alkenyl chains.

[0146] In some embodiments of the lipid composition, the SORT lipid comprises a permanently positively charged moiety. The permanently positively charged moiety may be positively charged at physiological pH, such that the SORT lipid comprises a positive charge upon delivery of polynucleotides to cells. In some embodiments, the positively charged moiety is a quaternary amine or a quaternary ammonium ion. In some embodiments, the SORT lipid comprises a counterion or otherwise complexes with or interacts with a counterion.

[0147] In some embodiments of the lipid composition, the SORT lipid is a permanent cationic lipid (i.e., comprises one or more hydrophobic components and a permanent cationic group). The permanent cationic lipid may contain a group that has a positive charge regardless of pH. One permanent cationic group that can be used in the permanent cationic lipid is a quaternary ammonium group. The permanent cationic lipid has the structural formula: [ka] wherein Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5, However, at least one of Y1, Y2, and Y3 is X2N + R3R4R5, R1 is C1~C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; X1 is O or NR a where R ais hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl; X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl, R3, R4, and R5 are each independently selected from C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; A1 is the X2N in the compound + It is an anion with a charge equal to the number of R3R4R5 groups.

[0148] In some embodiments of the SORT lipid, the permanent cationic SORT lipid has the structural formula: [ka] wherein R6 to R9 are each independently C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl, provided that at least one of R6 to R9 is C8 to C 24 is the basis of A2 is a monovalent anion.

[0149] In some embodiments of the lipid composition, the SORT lipid is an ionizable cationic lipid (i.e., comprises one or more hydrophobic components and an ionizable cationic group). The ionizable positively charged moiety may be positively charged at physiological pH. One ionizable cationic group that may be used in the ionizable cationic lipid is a tertiary amine group. In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl(C8~C24) or a substitution of either group, R3 and R3' are each independently alkyl (C≦6) or substituted alkyl (C≦6) is.

[0150] In some embodiments of the lipid composition, the SORT lipid comprises a head group of a particular structure. In some embodiments, the SORT lipid has the structural formula: [ka] wherein L is a linker and Z + is the positively charged part, and X - is a counterion. In some embodiments, the linker is a biodegradable linker. The biodegradable linker may be degradable under physiological pH and temperature. The biodegradable linker may be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or a quaternary amine.

[0151] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R 1 and R 2 are each independently an optionally substituted C-C 24 Alkyl or optionally substituted C-C 24 It is alkenyl.

[0152] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] It has.

[0153] In some embodiments of the lipid composition, the SORT lipid comprises a linker (L). In some embodiments, L is [ka] wherein: p and q are each independently 1, 2, or 3; R 4 is an optionally substituted C1-C6 alkyl.

[0154] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of either group, R3, R3' and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and R4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0155] In some embodiments of the lipid composition, the SORT lipid is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is [ka] wherein: R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of either group, R3, R3' and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6)and X - is a monovalent anion.

[0156] In some embodiments of the lipid composition, the SORT lipid is a phosphocholine lipid. In some embodiments, the SORT lipid is an ethylphosphocholine. The ethylphosphocholine can be, by way of example and not limitation, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, or 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

[0157] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of either group, R3, R3' and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0158] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

[0159] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R4 and R4' are each independently alkyl (C6~C24) , alkenyl (C6~C24) or a substitution of either group, R4'' is alkyl (C≦24) , alkenyl (C≦24) or a substitution of either group R4''' is alkyl (C1~C8) , alkenyl (C2~C8) or a substitution of either group, X2 is a monovalent anion.

[0160] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB) (e.g., the bromide salt).

[0161] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of either group, R3, R3' and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0162] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0163] In some embodiments of the lipid composition, the SORT lipid is an anionic lipid. In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of either group, R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) or -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is.

[0164] In some embodiments of the lipid composition, the SORT lipid comprises one or more selected from the lipids listed in Table 6.

[0165] [Table 6-1] [Table 6-2]

[0166] In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 20% to about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 25% to about 60%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a molar percentage of SORT lipids of at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, at least (about) 60%, or at least (about) 65%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a molar percentage of SORT lipids of at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at least (about) 45%, at most (about) 50%, at most (about) 55%, at most (about) 60%, or at most (about) 65%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises SORT lipids at a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or a molar percentage range between and including any two of the foregoing values.

[0167] Additional fats In some embodiments of the lipid compositions of the present application, the lipid composition further comprises additional lipids, including, but not limited to, steroids or steroid derivatives, PEG lipids, and phospholipids.

[0168] Phospholipids or other zwitterionic lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises a phospholipid. In some embodiments, the phospholipid is a phospholipid having one or two long chains (e.g., C6-C 24 The phospholipid may contain an alkyl or alkenyl group, glycerol or sphingosine, one or two phosphate groups, and optionally a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, which define lipids and lipid-like molecules that have both positive and negative charges.

[0169] In some embodiments of the lipid composition, the phospholipid is not ethylphosphocholine.

[0170] In some embodiments of the lipid compositions of the present application, the composition may further comprise a molar percentage of phospholipids relative to the total lipid composition of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is about 7.5 to about 60. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0171] In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 8% to about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 8% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 10% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises phospholipids at a molar percentage of about 12% to about 18%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at least (about) 8%, at least (about) 10%, at least (about) 12%, at least (about) 15%, at least (about) 18%, at least (about) 20%, or at least (about) 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at most (about) 8%, at most (about) 10%, at most (about) 12%, at most (about) 15%, at most (about) 18%, at most (about) 20%, or at most (about) 23%.

[0172] steroids or steroid derivatives In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" refers to a class of compounds having a four-ring, 17-carbon ring structure that may further comprise one or more substituents, including alkyl, alkoxy, hydroxy, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid is represented by the formula [ka] In some embodiments, the steroid or steroid derivative comprises the above ring structure with one or more non-alkyl substitutions, as shown in the formula: [ka] In some embodiments of the present application, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is of the formula [ka] As described above, the cholestane derivative contains one or more non-alkyl substitutions of the ring system. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative, or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both cholestene and a sterol or a derivative thereof.

[0173] In some embodiments of the lipid composition, the composition may further comprise a molar percentage of steroid relative to the total lipid composition of about 40 to about 46. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45 to about 46, or any range derivable therein. In other embodiments, the molar percentage of steroid relative to the total lipid composition is about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.

[0174] In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 15% to about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 25% to about 35%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 30% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 30%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, or at least (about) 46%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of at most (about) 15%, at most (about) 20%, at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at most (about) 45%, or at most (about) 46%.

[0175] Polymer-Conjugated Lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG-lipid. In some embodiments, the PEG-lipid is a diglyceride that also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG-lipid is one or more C6-C glycerol groups attached to a linker group bearing a PEG chain. 24 Long chain alkyl or alkenyl groups or C6-C 24A compound containing a fatty acid group. Some non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamines and phosphatidic acids, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoylphosphatidylethanolamines or PEG-modified dimyristoyl-sn-glycerols. 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 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present application 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.

[0176] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein R 12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or a substitution of any of these groups, and R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8)and x is 1 to 250. In some embodiments, R e is an alkyl group such as methyl. (C≦8) R 12 and R 13 are each independently an alkyl (C≦4~20) In some embodiments, x is 5 to 250. In some embodiments, x is 5 to 125, or x is 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0177] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein n1 is an integer from 1 to 100, and n2 and n3 are each independently selected from integers from 1 to 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, n1 is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is from 11 to about 17. In some embodiments, n3 is from 5 to 23. In some embodiments, n3 is from 11 to about 17.

[0178] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of PEG lipid relative to the total lipid composition of about 4.0 to about 4.6. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range derivable therein. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0, or any range derivable therein.

[0179] In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 1% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 2% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 1% to about 8%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 2% to about 7%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 3% to about 5%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10% molar percentage of polymer-conjugated lipids. In some embodiments of the lipid compositions of the present application, the lipid composition comprises at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10% molar percentage of polymer-conjugated lipids.

[0180] Pharmaceutical Composition Therapeutic or preventive agents In another aspect, provided herein is a pharmaceutical composition comprising a therapeutic agent (or prophylactic agent) in combination with a lipid composition described herein.

[0181] In some embodiments of the pharmaceutical composition, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof. In some embodiments, the compound, polynucleotide, polypeptide, or a combination thereof is exogenous or heterologous to the cell or subject being treated with the pharmaceutical composition described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polynucleotide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polypeptide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof.

[0182] In some embodiments, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) for treating a pulmonary disease, such as asthma, COPD, or lung cancer. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a steroid, such as prednisone, hydrocortisone, prednisolone, methylprednisolone, or dexamethasone. In some embodiments, the therapeutic agent (or prophylactic agent) comprises Abraxane, afatinib dimaleate, Afinitor, Afinitor dispersible tablets, or other steroids. Disperz), Alecensa, alectinib, Alimta, Alunbrig, atezolizumab, Avastin, bevacizumab, brigatinib, capmatinib hydrochloride, carboplatin, ceritinib, crizotinib, Cyramza, dabrafenib mesylate, dacomitinib, docetaxel, doxorubicin hydrochloride, durvalumab, entrectinib, erlotinib hydrochloride, everolimus, Gavreto, gefitinib, Gilotrif, gemcitabine, ipilimumab, Iressa, Keytruda, Lorbrena, Mekinist, methotrexate sodium, necitumumab, nivolumab, osimertinib mesylate, paclitaxel, These include pembrolizumab, pemetrexed disodium, pralsetinib, ramucirumab, LetVimo, selpercatinib, Tabrecta, Tafinlar, Tagrisso, trametinib dimethyl sulfoxide, vidinpro, vinorelbine tartrate, Xalkori, Yervoy, Zirabev, Zykadia, carboplatin, gemcitabine-cisplatin, Afinitor, atezolizumab, durvalumab, etopofos, etoposide, Hycamtin, Imfinzi, Keytruda, lurbinectedin, methotrexate sodium, nivolumab, Opdivo, pembrolizumab, Tecentriq, topotecan hydrochloride, Trexol, or Xepzelca. Other non-limiting examples of therapeutic (or prophylactic) agents that contain compounds include 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acetretin, acrivastine, adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac,Aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol, all-trans retinoic acid (ATRA), aloxiprine, alprazolam, alprenolol, altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone hydrochloride, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphotericin, amphotericin B, ampicillin, amp Lenavir, amsacrine, amyl nitrate, amylobarbitone, anastrozole, amrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbitone, BCG Live, beclamide, beclomethasone, bendroflumethiazide, benezepril, benidipine, benorilate, benperidol, bentazepam, benzamide, benzanthrazene, benzathine penicillin, benzahexol HCl, benznidazole, benzodiazepine, benzoic acid, bephenium hydroxynaphthoate, betamethasone, bevacizumab (Avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizola , budesonide, bumetanide, bupropion, busulfan, butalbital, butamben, butenafine HCl, butobarbitone, butobarbitone (butetal), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calciprotriene, calcitriol, calsterone, campbendazole, camphor, camptothecin, camptothecin analogs, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, carbofuran, carboplatin, carbromal, kalimazole, carmustine, cefamandole, cefazolin, cefiximeCeftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol, chrysene, cilostazol, cimetidine, cinaridine, cinoxacin, ciprofibrate, Ciprofloxacin HCl, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estrogens, corticosterone, cortisone, cortisone acetate, cyclizine, cyclobarbital, cycloheximide Lobenzaprine, cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopropane-spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthron, dantrolene sodium, dapsone, darbepoetin alfa, dalodipine, daunorubicin, decoquinate , dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycorticosterone, desoximetasone, dexamethasone, dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine, diatrizoic acid, diazepam, diazoxide, dichlorophen, dichloroprop, diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequin,Dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxanide furoate, dimenhydrinate, dimorpholamine, dinitrilmide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin, doxycycline, dromostanolone propionate, droperidol, dyphylline, echinocandin, econazo ol, econazole nitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone, epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetin alfa, eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate, erlotinib, erythromycin, estradiol, estramustine, estriol, estrone, ethacrynic acid, ethambutol, etinamate, ethionamide, ethopropazine HCl, 4-aminobenzoic acid ethyl ester (benzocaine) ), ethylparaben, ethinyl estradiol, etodolac, etomidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecamyd acetate, floxacin Lysine, fludarabine, fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid, flunanisone, flunarizine HCl, flunisolide, flunitrazepam, fluocortolone, fluometuron, fluorene, fluorouracil, fluoxetine HCl, fluoxymesterone, flupentixol decanoate, flufentixol decanoate, flurazepam, flurbiprofen, fluticasone propionate, fluvastatin, Folic acid, fosinopril, fosphenytoin sodium, frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC (lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine, glibenclamide, gliclazide, glimepiride, glipizide, glutethimide, glyburide, glyceryl trinitrate (nitroglycerin), goserelin acetate, grepafloxacin, griseofulvin, guaifenesin, guanabenz acetate, guanine, halofantrine HCl, haloperidol, hydrochloride Lochlorothiazide, heptabarbital, heroin, hesperetin, hexachlorobenzene, hexetal, histrelin acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine, hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interferon alpha-2a, interferon alpha-2b, iodine amide, iopanoic acid, iprodione, irbesartan, irinotecan, isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid, isopropyl barbiturate, isoproturon, isosorbide dinitrate, isosorbide mononitrate, izradipine, itraconazole, itraconazole, itraconazole (Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin, labetalol, lamivudine, lamotrigine, lanatoside C, lanospragle, L-DOPA, leflunomide, lenalidomide, Letrozole, leucovorin, leuprolide acetate, levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lorefloxacin, lormetazepam, losartan mesylate, lovastatin, lisuride maleate, maprotiline HCl, mazindol, mebendazole, meclizine HCl, meclofenamic acid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid, mefloquine HCl, megestrol acetate, melphalan,Mepenzolate bromide, meprobamate, meptazinol, mercaptopurine, mesalazine, mesna, mesoridazine, mestranol, methadone, methaqualone, methocarbamol, methoine, methotrexate, methoxsalen, mesuximide, methyclothiazide, methylphenidate, methylphenobarbitone, methyl p-hydroxybenzoate, methylprednisolone, methyltestosterone, methyprylon, methylsergide maleate, metoclopramide, metolazone, metoprolol, metronidazole, mianserin HCl, miconazole, midazolam Zolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mofetil mycophenolate, molindone, montelukast, morphine, moxifloxacin HCl, nabumetone, nadolol, nalbuphine, nalidixic acid, nandrolone, naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotinamide, nicotinic acid, nicoumarone, nifedipine, nilutamide, nimodipine, nimorazol fluticasone, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron HCl, oprelvequin, ornidazole, oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide, oxazepam, oxcarbazepine, oxfendazole , oxiconazole, oxyprenolol, oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin, pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione, paroxetine HCl, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, penicillamine, pentaerythritol tetranitrate, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline, perphenazine, perphenazine pimozide, perylene,Phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital, phenol barbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamine HCl, phenoxymethylpenicillin, phenximide, phenylbutazone, phenytoin, pindolol, pioglitazone, pipobroman, piroxicam, pizotifen maleate, platinum compounds, plicamycin, polyenes, polymyxin B, porfimer sodium, posaconazole (Posa), pramipexole, prasterone, pravastatin, prazi Cantel, prazosin, prazosin HCl, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanil HCl, promethazine, propofol, propoxur, propranolol, propylparaben, propylthiouracil, prostaglandins, pseudoephedrine, pteridine-2-methylthiol, pteridine-2-thiol, pteridine-4-methylthiol, pteridine-4-thiol, pteridine-7-methylthiol, pteridine Lysine-7-thiol, pyrantelembonate, pyrazinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal, reserpine, retinoids, rifabutin, rifampicin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib, Pinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital, sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone, stanozolol, stavudine, stilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine, sulfamerazine,Sulfamethazine, sulfamethoxazole, sulfanilamide, sulfathiazole, sulindac, sulfabenzamide, sulfaacetamide, sulfadiazine, sulfadoxine, sulfafurazole, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfasalazine, sulfinpyrazone, sulpiride, sulthiame, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate, tamulosin, targretin, Taxanes, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole, terfenadine, testolactone, testosterone, tetracycline, tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine, theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, thotoin, thimipenem , tiagabine HCl, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone, triamterene, triazolam, triazole, triflupromazine, trimethoprim, trimipramine maleate, triphenylene, troglitazone, tromethamine, tropicamide, and small molecules selected from lobafloxacin, thibamate, ubidecarenone (coenzyme Q10), undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, barbicine, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirkust, zidovudine, zileton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, or zopiclone.

[0183] Polynucleotides In some embodiments of the pharmaceutical compositions of the present application, the therapeutic agent (or prophylactic agent) combined with the lipid composition comprises one or more polynucleotides. The present application is not limited in scope to any particular source, sequence, or type of polynucleotide. However, those skilled in the art will readily be able to identify related homologs in various other sources of polynucleotides, 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). It is contemplated that the polynucleotides used in the present application may include sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, sequences with at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90%, and most preferably about 95% of the nucleotides are identical to the nucleotide sequence of a naturally occurring sequence. In another embodiment, the polynucleotide comprises a nucleic acid sequence that is complementary to a naturally occurring sequence, or that is 75%, 80%, 85%, 90%, 95%, and 100% complementary to the naturally occurring sequence. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or more are contemplated herein.

[0184] In some embodiments, the polynucleotides used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. However, in preferred embodiments, the polynucleotide comprises complementary DNA (cDNA). Also contemplated are cDNAs with the addition of natural introns or introns from other genes; such artificial molecules are sometimes referred to as "minigenes." At a minimum, these and other nucleic acids of the present application can be used, for example, as molecular weight standards in gel electrophoresis. 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 unprocessed or partially processed genomic RNA templates, is that it contains primarily the coding sequence of the corresponding protein. In some cases, a complete or partial genomic sequence may be preferred, such as when non-coding regions are required for optimal expression or when targeting non-coding regions such as introns in antisense strategies.

[0185] In some embodiments, the polynucleotide comprises one or more segments comprising a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a microribonucleic acid (miRNA), a primary microribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), or a double-stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic agents (or prophylactic agents) described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide, such as a guide RNA (gRNA) or a guide DNA (gDNA), for forming a complex with a guide RNA-guided nuclease described herein. In some embodiments, the polynucleotide encodes a heterologous nuclease guided by at least one guide polynucleotide. The nuclease may be an endonuclease.Non-limiting examples of heterologous endonucleases guided by a guide polynucleotide include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and extremely halophilic archaea (Natronobacterium gregoryi Argonaute (NgAgo); adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof; any derivative thereof; any variant thereof; and any fragment thereof.

[0186] In some embodiments, the therapeutic (or prophylactic) agent is a transfer ribonucleic acid (tRNA) that introduces an amino acid into the growing peptide chain of a protein of a target gene. The target gene can be one listed in Table 7.

[0187] [Table 7-1] [Table 7-2]

[0188] Some embodiments of the therapeutic (or prophylactic) agents provided herein comprise a heterologous polypeptide comprising an actuator moiety. The actuator moiety can be configured to form a complex with a target polynucleotide corresponding to a target gene. In some embodiments, administration of the therapeutic (or prophylactic) agent results in altered expression or activity of the target gene. Altered expression or activity of a target gene may be detectable, for example, in at least about 5%, 10%, 15%, or 20% of the subject's lung epithelial cells, at least about 2%, 5%, or 10% of the subject's lung ciliated cells, at least about 5%, 10%, 15%, or 20% of the subject's lung secretory cells, at least about 5%, 10%, 15%, or 20% of the subject's lung club cells, at least about 5%, 10%, 15%, or 20% of the subject's lung goblet cells, or at least about 5%, 10%, 15%, or 20% of the subject's lung basal cells, or a combination thereof. Biomarkers for characterizing cell types may be known in the art, such as those described in the Examples section herein below. The therapeutic agent (or prophylactic agent) may comprise a heterologous polynucleotide encoding an actuator portion. The actuator portion may be configured to form a complex with a target polynucleotide corresponding to the target gene. The heterologous polynucleotide may encode a guide polynucleotide configured to direct the actuator portion to a target polynucleotide. The actuator portion may include a heterologous endonuclease or a fragment thereof (e.g., directed by the guide polynucleotide to specifically bind to the target polynucleotide). The heterologous endonuclease may (1) be part of a ribonucleoprotein (RNP) or (2) form a complex with the guide polynucleotide. The heterologous endonuclease may be part of a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) protein complex.The heterologous endonuclease may be a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease. The heterologous endonuclease may comprise an inactivating endonuclease. The inactivating endonuclease may be fused to a regulatory moiety. The regulatory moiety may comprise a transcriptional activator, a transcriptional repressor, an epigenetic modifier, or a fragment thereof.

[0189] In some embodiments, the polynucleotide encodes at least one guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA))-guided heterologous endonuclease. In some embodiments, the polynucleotide encodes at least one guide polynucleotide and at least one heterologous endonuclease, and the guide polynucleotide is capable of forming a complex with the at least one heterologous endonuclease and directing it to cleave a genetic locus of any one of the genes described herein. In some embodiments, the polynucleotide is a heterologous endonuclease guided by at least one guide polynucleotide, such as Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, Cas14, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, Casl Encodes Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cul966; any derivative thereof; any variant thereof; or any fragment thereof. In some embodiments, Cas13 may include, but is not limited to, Cas13a, Cas13b, Cas13c, and Cas13d (e.g., CasRx).

[0190] In some embodiments, the heterologous endonuclease comprises an inactivating endonuclease, optionally fused to a regulatory moiety, such as an epigenetic modifier, for remodeling the epigenome to mediate expression of a selected gene of interest. In some cases, epigenetic modifiers can include methyltransferases, demethylases, dismutases, alkylating enzymes, depurinases, oxidases, photolyases, integrases, transposases, recombinases, polymerases, ligases, helicases, glycosylases, acetyltransferases, deacetylases, kinases, phosphatases, ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitin ligases, deubiquitinating enzymes, adenylate-forming enzymes, AMP-transferases, deAMP-transferases, SUMO-transferases, deSUMO-transferases, ribosylases, deribosylases, N-myristoyltransferases, chromatin remodeling enzymes, proteases, oxidoreductases, transferases, hydrolases, lyases, isomerases, synthases, synthetases, or demyristoylating enzymes. In some embodiments, the epigenetic modifier may include one or more selected from the group consisting of p300, TET1, LSD1, HDAC1, HDAC8, HDAC4, HDAC11, HDT1, SIRT3, HST2, CobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV39H1, DIM5, KYP, SUVR4, Set4, Set1, SETD8, and TgSET8.

[0191] In some embodiments, the polynucleotide encodes a guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA)) that is at least partially complementary to a genomic region of a gene, such that when the guide polynucleotide binds to the gene, the guide polynucleotide recruits a nuclease guided by the guide polynucleotide to cleave the region and genetically modify it. Examples of genes that can be modified by nucleases guided by guide polynucleotides include CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0192] In some embodiments, the polynucleotide comprises or encodes at least one mRNA, the expression of which restores the function of a defective gene in a subject treated with the pharmaceutical composition described herein. For example, the polynucleotide comprises or encodes an mRNA that expresses a wild-type CFTR protein, which can be used to rescue a subject suffering from a congenital mutation of the CFTR protein. Other examples of mRNAs that can be expressed from the polynucleotides include mRNAs encoding DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0193] In some embodiments, the polynucleotides of the present application comprise at least one chemical modification of one or more nucleotides. In some embodiments, the chemical modification increases the specificity of a guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA)) binding to a complementary genomic locus (e.g., the genomic locus of any one of the genes described herein). In some embodiments, the at least one chemical modification increases resistance to nuclease digestion when the polynucleotide is administered to a subject in need thereof. In some embodiments, the at least one chemical modification reduces immunogenicity when the polynucleotide is administered to a subject in need thereof. In some embodiments, the at least one chemical modification stabilizes a scaffold, such as a tRNA scaffold. Such chemical modifications may have desirable properties, such as increased resistance to nuclease digestion or increased binding affinity to a target genomic locus relative to a polynucleotide lacking the at least one chemical modification.

[0194] In some embodiments, at least one chemical modification comprises a modification to the sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including, but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is allyl, amino, azido, thio, O-allyl, O-C1-C1-C2-C3 ... 10 Alkyl, O--C1~C 10 Substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ), and O--CH2--C(=O)--N(R m )(R n ) and each R mand Rn are independently H or substituted or unsubstituted C1-C 10 and alkyl. Examples of 5'-position sugar substituents include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises two or more non-bridging sugar substituents, such as TF-5'-methyl sugar moieties.

[0195] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, a 2'-substituted nucleoside is a nucleoside that is substituted with halo, allyl, amino, azido, SH, CN, OCN, CF, OCF, O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S, or N(R m )-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ) or O--CH2--C(=O)--N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0196] In some embodiments, the 2'-substituted nucleoside is selected from the group consisting of F, NH, N, OCF, O--CH, O(CH)NH, CH-CH=CH, O--CH-CH=CH, OCHCHOCH, O(CH)SCH, O--(CH)-O--N(R m )(R n), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O--CH2--C(=O)--N(R m )(R n ), wherein each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is alkyl.

[0197] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0198] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0199] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4'-2' furanose ring atoms. Examples of such 4'-2' sugar substituents include -[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O-- or --C(R a R b)--O--N(R)--; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2'(LNA); 4'-(CH2)--S-2'; 4'-(CH2)2--O-2'(ENA); 4'-CH(CH3)--O-2'(cEt) and 4'-CH(CHOCH3)--O-2', and analogs thereof; 4'-C(CH3)(CH3)--O-2' and analogs thereof; 4'-CH2--N(OCH3)-2' and analogs thereof; 4'-CH2--O--N(CH3)-2'; 4'-CH2--O--N(R)-2', and 4'-CH2--N(R)--O-2'-, where each R is independently H, a protecting group, or a C1-C 12 alkyl; 4'-CH2--N(R)--O-2', where R is H, C1-C 12 alkyl, or those which are protecting groups; 4'-CH2--C(H)(CH3)-2'; and 4'-CH2--C(=CH2)-2' and analogs thereof.

[0200] In some embodiments, such 4' to 2' bridges are independently —[C(R a )(R b )] n --, --C(R a )=C(R b )--, --C(R a )=N--, ​​--C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a )2--, --S(=O) x --, and --N(R a )—, x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where each J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0201] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include: (A) α-L-methyleneoxy (4'-CH2-O-2') BNAs, (B) β-D-methyleneoxy (4'-CH2-O-2') BNAs (also known as locked nucleic acids or LNAs), (C) ethyleneoxy (4'-(CH2)2-O-2') BNAs, (D) aminooxy (4'-CH2-O-N(R)-2') BNAs, (E) oxyamino (4'-CH2-N(R)-O-2') BNAs, and (F) methyl(methyleneoxy) (4'-CH(CH3)-O-2'). (R) methylene-thio (4'-CH--S-2') BNA, (H) methyleneamino (4'-CH-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH--CH(CH)-2') BNA, (J) propylene carbocyclic (4'-(CH)-2') BNA, and (K) methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also called constrained MOE or cMOE).

[0202] In some embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by their isomeric configuration. For example, nucleosides containing a 4'-2' methyleneoxy bridge may be in the α-L or β-D configuration. Previously, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that exhibit antisense activity.

[0203] In some embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., a 5'-substituted sugar and a 4'-2' bridged sugar, e.g., an LNA substituted with a 5'-methyl group or a 5'-vinyl group).

[0204] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also contain bridging and / or non-bridging substituents as described above. For example, some sugar surrogates contain a 4'-sulfur atom and substituents at the 2' and / or 5' positions. As an additional example, carbocyclic bicyclic nucleosides having a 4'-2' bridge are described.

[0205] In some embodiments, the sugar surrogate comprises a ring having more than five atoms. For example, in some embodiments, the sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA), and fluoro-HNA (F-HNA).

[0206] In some embodiments, modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H; in some embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In some embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro, R2 is H, R1 is methoxy, R2 is H, or R1 is methoxyethoxy, and R2 is H.

[0207] Many other bicyclic and tricyclic sugar surrogate ring systems are known in the art that can be used to modify nucleosides and incorporated into antisense compounds.

[0208] Combinations of modifications, such as, but not limited to, 2'-F-5'-methyl substituted nucleosides, replacement of the ribosyl ring oxygen atom with S, and further substitutions at the 2' position, alternatively 5'-substitution of bicyclic nucleic acids, are also provided. In some embodiments, 4'-CH2--O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group. The synthesis and preparation of carbocyclic bicyclic nucleosides, as well as their oligomerization and biochemical studies, are also described.

[0209] In some embodiments, the present application provides polynucleotides comprising modified nucleosides. These modified nucleotides may comprise modified sugars, modified nucleobases, and / or modified linkages. The particular modifications are selected so that the resulting polynucleotide has desirable properties. In some embodiments, the polynucleotide comprises one or more RNA-like nucleosides. In some embodiments, the polynucleotide comprises one or more DNA-like nucleotides.

[0210] In some embodiments, the nucleosides of the present application comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present application comprise one or more modified nucleobases.

[0211] In some embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, and 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0212] In some embodiments, the present application provides a polynucleotide comprising linked nucleosides. In such embodiments, the nucleosides can be linked using any internucleoside linkage. Two major classes of internucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linkage groups include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to native phosphodiester linkages. In some embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0213] The polynucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), α or β, as in the case of sugar anomers, or (D) or (L), as in the case of amino acids, etc. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0214] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2--N(CH3)--O-5'), amide-3 (3'-CH2--C(=O)--N(H)-5'), amide-4 (3'-CH2--N(H)--C(=O)-5'), formacetal (3'-O--CH2--O-5'), and thioformacetal (3'-S--CH2--O-5'). Additional neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and amides (see, e.g., "Carbohydrate Modifications in Antisense Research"; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed component parts of N, O, S, and CH.

[0215] Additional modifications may be made to other positions of the oligonucleotide, particularly the 3'-position of the sugar of the 3'-terminal nucleotide and the 5'-position of the 5'-terminal nucleotide. For example, one additional modification of the ligand-conjugated polynucleotides of the present application involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0216] In some embodiments, the polynucleotides described herein comprise or encode at least one tRNA described herein. In some embodiments, the tRNA expressed from the polynucleotide restores the function of at least one defective tRNA in a subject treated with the pharmaceutical composition described herein. In some embodiments, the at least one tRNA expressed by the polynucleotides described herein may comprise a tRNA encoding alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylaniline, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the at least one tRNA expressed by the polynucleotides described herein may comprise a tRNA encoding arginine, tryptophan, glutamic acid, glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine. In some embodiments, the tRNA encoded by the polynucleotides described herein can restore the expression of any one of the genes described herein.In some embodiments, the tRNA encoded by the polynucleotides described herein can restore the expression of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0217] Polypeptides In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent combined with the lipid composition comprises one or more polypeptides. Some polypeptides may comprise an enzyme, such as any one of the nuclease enzymes described herein. For example, the nuclease enzyme can be a CRISPR-associated (Cas) protein or Cas nuclease, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and extremely halophilic archaea (Natronobacterium gregoryi Argonaute (NgAgo); adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof; any derivative thereof; any variant thereof; and any fragment thereof. In some embodiments, the nuclease enzyme may be selected from Cas proteins, such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, These may include Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.In some embodiments, a Cas protein can complex with a guide polynucleotide described herein to form a CRISPR ribonucleoprotein (RNP).

[0218] The nuclease in the compositions described herein may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence, of any one of the genes described herein. For example, CRISPR enzymes can be directed to and cleave the genomic locus of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0219] CRISPR enzyme may be mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence.For example, the aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of S. pyogenes Cas9 converts Cas9 from a nuclease that cleaves both strands into a nickase (cleaves a single strand).In some embodiments, Cas9 nickase can be used in combination with a guide sequence, for example, two guide sequences that target the sense strand and antisense strand of DNA target, respectively.This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0220] In some embodiments, the present application provides polypeptides containing one or more therapeutic proteins. Therapeutic proteins that can be included in the compositions include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, clotting factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase α, tissue plasminogen activator (TPA) activase, glucocerebrosidase, interferon (IF) beta-1a, interferon beta-1b, interferon gamma, interferon α, TNF-α, IL-1 through IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle-stimulating hormone (FSH), and Factor VIII.

[0221] In some embodiments, the polypeptide comprises a peptide sequence at least partially identical to any therapeutic (or prophylactic) agent that comprises the peptide sequence. For example, the polypeptide may comprise a peptide sequence at least partially identical to an antibody (e.g., a monoclonal antibody) for treating a lung disease, such as lung cancer.

[0222] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject treated with the pharmaceutical composition described herein. For example, the polynucleotide comprises a peptide or protein that restores the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which can be used to rescue subjects suffering from congenital anomalies that lead to the expression of mutated CFTR proteins. Other examples of rescue include administering to a subject in need thereof wild-type dynein axonemal heavy chain 5, dynein axonemal heavy chain 11, bone morphogenetic protein receptor type 2, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, α-L-iduronidase, type IV collagen α3 chain, type IV collagen α4 chain, type IV collagen α5 chain, polycystin 1, polycystin 2, fibrocystin (or polyductin), solute carrier family 3 member 1, solute carrier family 7 member 9, paired box gene 9, myosin VIIA, cadherin-related 23, ussharin, clarin 1, gap junction β2 protein, gap junction β6 protein, rhodopsin, myotonic dystrophy myotonin kinase (MDK), ... kinase), dystrophin, sodium voltage-gated channel alpha subunit 1, sodium voltage-gated channel beta subunit 1, coagulation factor VIII, coagulation factor IX, N-glycanase 1, tumor protein p53, palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, Kv11.1 (potassium ion channel alpha subunit), palmitoyl protein thioesterase 1, ATM serine / threonine kinase, and fibrillin 1.

[0223] In some embodiments, the pharmaceutical composition of the present application includes multiple payloads combined with (e.g., encapsulated within) a lipid composition. The multiple payloads combined with the lipid composition may be configured for gene editing or gene expression modification. The multiple payloads combined with the lipid composition may include a polynucleotide encoding an actuator portion (e.g., including a heterologous endonuclease such as Cas) or a polynucleotide encoding an actuator portion. The multiple payloads combined with the lipid composition may further include one or more (e.g., one or two) guide polynucleotides. The multiple payloads combined with the lipid composition may further include one or more donor or template polynucleotides. The multiple payloads combined with the lipid composition may include ribonucleoproteins (RNPs).

[0224] In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is (about) 20:1 or less, (about) 15:1 or less, (about) 10:1 or less, or (about) 5:1 or less. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is less than (about) 20:1, (about) 15:1, (about) 10:1, or (about) 5:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 10:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 15:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 10:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 15:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 15:1 to about 20:1.

[0225] In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 1:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 1:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 50:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 1:1 to about 1:20. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 20:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 50:1 to about 1:70. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 70:1 to about 1:100. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipids in the lipid composition is (about) 1:1 or less, (about) 1:5 or less, (about) 1:10 or less, (about) 1:15 or less, (about) 1:20 or less, (about) 1:25 or less, (about) 1:30 or less, (about) 1:35 or less, (about) 1:40 or less, (about) 1:45 or less, (about) 1:50 or less, (about) 1:60 or less, (about) 1:70 or less, (about) 1:80 or less, (about) 1:90 or less, or (about) 1:100 or less. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipids in the lipid composition is less than (about) 1:1, less than (about) 1:5, less than (about) 1:10, less than (about) 1:15, less than (about) 1:20, less than (about) 1:25, less than (about) 1:30, less than (about) 1:35, less than (about) 1:40, less than (about) 1:45, less than (about) 1:50, less than (about) 1:60, less than (about) 1:70, less than (about) 1:80, less than (about) 1:90, or less than (about) 1:100.

[0226] In some embodiments of the pharmaceutical compositions of the present application, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% of the therapeutic agent is encapsulated in particles of the lipid composition.

[0227] In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles characterized by one or more of the following properties: (1) a (e.g., average) size of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of about 0.2 or less; and (3) a zeta potential of -10 millivolts (mV) to 10 mV. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 70 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 60 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of at most about 100 nanometers (nm), at most about 90 nanometers (nm), at most about 85 nanometers (nm), at most about 80 nanometers (nm), at most about 75 nanometers (nm), at most about 70 nanometers (nm), at most about 65 nanometers (nm), at most about 60 nanometers (nm), at most about 55 nanometers (nm), or at most about 50 nanometers (nm). In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of at least about 100 nanometers (nm), at least about 90 nanometers (nm), at least about 85 nanometers (nm), at least about 80 nanometers (nm), at least about 75 nanometers (nm), at least about 70 nanometers (nm), at least about 65 nanometers (nm), at least about 60 nanometers (nm), at least about 55 nanometers (nm), or at least about 50 nanometers (nm). The (e.g., average) size can be determined by size exclusion chromatography (SEC).The (eg, average) size can be determined by spectroscopic or image-based methods, such as dynamic light scattering, static light scattering, multi-angle light scattering, laser light scattering, or dynamic image analysis, or a combination thereof.

[0228] In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.05 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.3. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.2 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 or less, or about 0.05 or less.

[0229] In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or less. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or less. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or less. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or less. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -30 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 0 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 5 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 10 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of 15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of 20 millivolts (mV) or less.

[0230] In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -30 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 0 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 5 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 10 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a zeta potential of 15 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a zeta potential of 20 millivolts (mV) or greater.

[0231] In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, or about 13 or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 13. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 10. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 9 to about 11. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 10 to about 13. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 12. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 10 to about 12.

[0232] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipids in the pharmaceutical composition result in delivery of a therapeutic agent characterized by one or more of the following: (a) a greater therapeutic effect in a cell of the subject compared to the therapeutic effect achieved with a reference lipid composition; (b) a greater therapeutic effect in a larger plurality of cells of the subject compared to the therapeutic effect achieved with a reference lipid composition; (c) a greater therapeutic effect in a first plurality of cells of a first cell type and a second plurality of cells of a second cell type; and (d) a greater therapeutic effect in a first cell of a first cell type in a subject compared to the therapeutic effect in a second cell of a second cell type in the subject. In some embodiments, the first cell type is different from the second cell type.

[0233] In some embodiments of the pharmaceutical composition of the present application, the cells are lung cells. In some embodiments, the lung cells are lung airway cells. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof. The cells can be located in the trachea, bronchi, bronchioles, or other parts or related areas of the lung.

[0234] In some embodiments of the pharmaceutical compositions of the present application, the therapeutic effect is characterized by a therapeutically effective amount of the therapeutic agent, e.g., in the subject's lungs, lung cells, multiple lung cells, or lung cell type. In some embodiments, the therapeutic effect is characterized by activity of the therapeutic agent, e.g., in the subject's lungs, lung cells, multiple lung cells, or lung cell type. In some embodiments, the therapeutic effect is characterized by effect of the therapeutic agent, e.g., in the subject's lungs, lung cells, multiple lung cells, or lung cell type. In some embodiments, a greater therapeutic effect is characterized by a greater therapeutic amount of the therapeutic agent. In some embodiments, a greater therapeutic effect is characterized by greater activity of the therapeutic agent. In some embodiments, a greater therapeutic effect is characterized by greater effect of the therapeutic agent.

[0235] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG lipid.

[0236] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments, the SORT lipids achieve a therapeutic effect that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater than the therapeutic effect achieved with a reference lipid composition.

[0237] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipids in the pharmaceutical compositions achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof, that is about 1.1-fold to about 20-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof, that is about 1.1-fold to about 10-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof, that is about 5-fold to about 10-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof, that is about 10-fold to about 20-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments, the SORT lipids achieve at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof, compared to the therapeutic effect achieved with a reference lipid composition.

[0238] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in multiple cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG lipid.

[0239] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition achieves a therapeutic effect in about 1.1 to about 20 times more cells than the therapeutic effect achieved using a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 1.1 to about 10 times more cells than the therapeutic effect achieved using a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 5 to about 10 times more cells than the therapeutic effect achieved using a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 10 to about 20 times more cells than the therapeutic effect achieved using a reference lipid composition. In some embodiments, the SORT lipids achieve a therapeutic effect in cells that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater than the therapeutic effect achieved with a reference lipid composition.

[0240] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipids in the pharmaceutical composition achieve a therapeutic effect in about 1.1-fold to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments, the SORT lipids achieve a therapeutic effect in about 1.1-fold to about 10-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments, the SORT lipids achieve a therapeutic effect in about 5-fold to about 10-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments, SORT lipids achieve a therapeutic effect in about 10- to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, where the cells are selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof. In some embodiments, SORT lipids achieve a therapeutic effect in about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, where the cells are selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof.

[0241] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent to cells of a subject characterized by a therapeutic effect in a first plurality of cells of a first cell type and a greater therapeutic effect in a second plurality of cells of a second cell type, in some embodiments, the first cell type is different from the second cell type.

[0242] In some embodiments of the pharmaceutical composition of the present application, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cells, secretory cells, such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0243] In some embodiments of the pharmaceutical composition of the present application, the second cell type is a lung cell. In some embodiments, the second cell type is a lung airway cell. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof. The cells can be located in the trachea, bronchi, bronchioles, or other parts or related areas of the lung.

[0244] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 20-fold larger than the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 10-fold larger than the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 5-fold to about 10-fold larger than the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 10-fold to about 20-fold larger than the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than the first plurality of cells of the first cell type.

[0245] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in a first cell of a first cell type compared to a therapeutic effect in a second cell of a second cell type, in some embodiments, the first cell type is different from the second cell type.

[0246] In some embodiments of the pharmaceutical composition of the present application, the first cell type is a lung cell. In some embodiments, the first cell type is a lung airway cell. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof. The cells can be located in the trachea, bronchi, bronchioles, or other parts or related areas of the lung.

[0247] In some embodiments of the pharmaceutical composition of the present application, the second cell type is lung cell.In some embodiments, the second cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cells, secretory cells, such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0248] In some embodiments of the pharmaceutical compositions of the present application, the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect in a first cell of a first cell type compared to the therapeutic effect achieved in a second cell of a second cell type.

[0249] In some embodiments, the pharmaceutical composition is an aerosol composition. In some embodiments, the aerosol composition is generated by a nebulizer at a nebulization inhalation rate of 70 mL / min or less. In some embodiments, the aerosol composition is generated by a nebulizer at a nebulization inhalation rate of 50 mL / min or less. In some embodiments, the aerosol composition is generated by a nebulizer at a nebulization inhalation rate of 30 mL / min or less.

[0250] In some embodiments, the aerosol composition has an average droplet size of about 0.5 microns (μm) to about 10 μm. In some embodiments, the aerosol composition has an average droplet size of about 0.5 microns (μm) to about 10 μm. In some embodiments, the aerosol composition has an average droplet size of about 1 micron (μm) to about 10 μm. In some embodiments, the aerosol composition has an average droplet size of about 0.5 microns (μm) to about 5 μm. In some embodiments, the aerosol droplets are generated by a nebulizer at a nebulization inhalation rate of 70 mL / min or less. In some embodiments, the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 microns (μm) to about 10 μm. In some embodiments, the droplet size varies by less than about 50% over a sustained period of about 24 hours under storage conditions. In some embodiments, the droplets of the aerosol composition are characterized by a geometric standard deviation (GSD) of about 3 or less.

[0251] In some embodiments, provided herein are (e.g., pharmaceutical) compositions comprising components that enable improved efficacy or results based on the delivery of a polynucleotide. The compositions described elsewhere herein may be more effective in delivery to a particular cell, cell type, organ, or body region compared to a reference composition or compound. The compositions described elsewhere herein may be more effective in increasing expression of a polypeptide corresponding to the delivered polynucleotide. The compositions described elsewhere herein may be more effective in generating a greater number of cells expressing a polypeptide corresponding to the delivered polynucleotide. The compositions described elsewhere herein may result in increased uptake of the polynucleotide compared to a reference polynucleotide. The increased uptake may be the result of improved stability of the polynucleotide or improved targeting of the composition to a particular cell type or organ. In some embodiments, the SORT lipid results in greater expression or activity of a polynucleotide (or the polynucleotide's corresponding polypeptide) in a cell compared to the expression or activity achieved using a reference lipid composition comprising 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 1.1-fold greater expression or activity of a polynucleotide (or the polynucleotide's corresponding polypeptide) in a cell compared to the expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 2-fold greater expression or activity of a polynucleotide (or the polynucleotide's corresponding polypeptide) in a cell compared to the expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids.In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 5-fold greater expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a cell compared to the expression or activity achieved using a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG lipid. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 10-fold greater expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a cell compared to the expression or activity achieved using a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG lipid.

[0252] In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a greater plurality of cells compared to expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a greater plurality of cells than expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a greater plurality of cells than expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the polynucleotide's corresponding polypeptide) in a greater plurality of cells than expression or activity achieved using a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 10-fold greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a plurality of cells compared to the expression or activity achieved using a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.

[0253] In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in a greater number of cells uptake of the polynucleotide compared to that achieved with a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in a greater number of cells uptake of the polynucleotide compared to that achieved with a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG lipids.

[0254] method In some embodiments, provided herein are methods for nebulized delivery to lung cells of a subject, the method comprising administering to the subject a (e.g., pharmaceutical) composition (e.g., as described herein) comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid (e.g., as described herein) and (ii) a selective organ targeting (SORT) lipid (e.g., as described herein) other than the ionizable cationic lipid, thereby delivering the therapeutic agent to the lung cells in the lungs of the subject. In some embodiments, the method provides a (e.g., therapeutically) effective amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the lung epithelial cells of the subject. In some embodiments, the method provides a (e.g., therapeutically) effective amount or activity of the therapeutic agent in at least about 2%, 5%, or 10% of the lung ciliated cells of the subject. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung secretory cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung club cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung goblet cells. In some embodiments, the method provides an effective (e.g., therapeutically) amount or activity of the therapeutic agent in at least about 5%, 10%, 15%, or 20% of the subject's lung basal cells. In some embodiments, the lipid composition comprises a phospholipid. In some embodiments, the (e.g., pharmaceutical) composition comprising the therapeutic agent in combination with the lipid composition is an aerosol composition (such as those described herein).

[0255] In some embodiments, provided herein are methods for potent delivery to cells of a subject, the method comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) in combination with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0256] In some embodiments of the method, the cells are lung cells. In some embodiments, the lung cells are lung airway cells. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0257] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG-lipid.

[0258] In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 20-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 5-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic efficacy than the therapeutic efficacy achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater than the therapeutic effect achieved with a reference lipid composition.

[0259] In some embodiments of the method, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof, that is about 1.1-fold to about 20-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof, that is about 1.1-fold to about 10-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof, that is about 1.1-fold to about 5-fold greater than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipids achieve about a 10- to about 20-fold greater therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof, compared to the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipids achieve at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater therapeutic effect in cells selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof, compared to the therapeutic effect achieved with a reference lipid composition.

[0260] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in multiple cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG-lipid.

[0261] In some embodiments of the method, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 10-fold more cells than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 5-fold more cells than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in about 10-fold to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in at least about 1.1-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold more cells than the therapeutic effect achieved with a reference lipid composition.

[0262] In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 1.1-fold to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 1.1-fold to about 10-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 5-fold to about 10-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet cells and club cells, ciliated cells, and any combination thereof. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 10- to about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold more cells than the therapeutic effect achieved with a reference lipid composition, the cells being selected from basal cells, secretory cells, e.g., goblet and club cells, ciliated cells, and any combination thereof.

[0263] In some embodiments of the methods, the pharmaceutical compositions of the present application may be administered via any suitable route, including, for example, oral, rectal, vaginal, transmucosal, intratracheal or pulmonary, including inhalation, or enteral administration; intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0264] In some embodiments of the method, the pharmaceutical composition of the present application may be administered locally rather than systemically, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways depending on the tissue being targeted.

[0265] In some embodiments of the method, an aerosol containing the composition of the present application may be inhaled (in the case of nasal, tracheal, or bronchial delivery). In some embodiments, the composition of the present application may be injected, for example, at the site of injury, disease symptoms, or pain. In some embodiments, the composition of the present application may be provided in a lozenge for oral, tracheal, or esophageal application. In some embodiments, the composition of the present application may be provided in the form of a liquid, tablet, or capsule for administration to the stomach or intestine. In some embodiments, the composition of the present application may be provided in the form of a suppository for rectal or vaginal application. In some embodiments, the composition of the present application may also be delivered to the eye by use of an emulsion, eye drops, or injection.

[0266] In another aspect, provided herein is a method for targeted delivery to cells of a subject, comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) combined with a lipid composition as described herein, wherein the lipid composition comprises (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0267] In some embodiments of the method, the SORT lipid results in delivery of the therapeutic agent to a greater proportion of cell types compared to that achieved with a reference lipid composition. In some embodiments, the reference lipid composition does not include a SORT lipid. In some embodiments, the reference lipid composition does not include an amount of SORT lipid. In some embodiments, the reference lipid includes 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG-lipid.

[0268] In some embodiments of the method, the cells are lung cells. In some embodiments, the lung cells are lung airway cells. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0269] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a therapeutic effect in a first plurality of cells of a first cell type and a greater therapeutic effect in a second plurality of cells of a second cell type, in some embodiments, the first cell type is different from the second cell type.

[0270] In some embodiments of the method, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0271] In some embodiments of this method, the second cell type is lung cell.In some embodiments, the second cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0272] In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 20-fold larger than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 10-fold larger than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 5-fold larger than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 10-fold to about 20-fold larger than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than the first plurality of cells of the first cell type.

[0273] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type, in some embodiments, the first cell type is different from the second cell type.

[0274] In some embodiments of the method, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0275] In some embodiments of this method, the second cell type is lung cell.In some embodiments, the second cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0276] In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 20-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic efficacy in a first cell of a first cell type compared to the therapeutic efficacy achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold therapeutic effect in a first cell of a first cell type compared to the therapeutic effect achieved in a second cell of a second cell type.

[0277] In some embodiments, delivery of a therapeutic agent to a cell may result in changes to the genome, transcriptome, or expression level. The cell may be expanded or allowed to proliferate, and the changes may be inherited by cells generated from the cell to which the therapeutic agent was delivered. In this way, the therapeutic effect can be propagated to a larger number of cells. Alterations to the genome, transcriptome, or expression level may also persist within a given cell.

[0278] basal cell Basal cells originate from the undifferentiated columnar epithelium of the developing airways. They are located at the base of the columnar epithelium and are characterized by the presence of hemidesmosomes (characterized by α6β4 integrin), cytokeratins 5 and 14, and the nuclear protein p63. The distribution of basal cells varies depending on the level of the airway and the animal species. Airways with larger diameters contain more basal cells than those with smaller diameters. The number of basal cells decreases as the diameter of the airways decreases, and they are completely absent in the terminal bronchioles.

[0279] In another aspect, provided herein is a method for delivery to basal cells of a subject, the method comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) in combination with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid. In some embodiments, the basal cells are lung basal cells.

[0280] In some embodiments of the method, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect in which basal cells are detectable in the organ or tissue of the subject. In some embodiments, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect in which at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of basal cells are detectable in the organ or tissue of the subject.

[0281] In some embodiments of the method, the organ is a lung. In some embodiments, the tissue is lung tissue. In some embodiments, the tissue is lung airway tissue. In some embodiments, the method delivers a therapeutic agent to the subject's lung to produce a therapeutic effect detectable in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the basal cells in the subject's lung.

[0282] In some embodiments of the method, the pharmaceutical composition is administered to the subject via any suitable delivery method. In some embodiments, the pharmaceutical composition is administered to the subject by inhalation. In some embodiments, the pharmaceutical composition is administered to the subject by systemic administration, such as intravenous administration.

[0283] Ciliated cells Ciliated cells are cells that have ciliated structures on their cell surface. Examples of ciliated cells include, but are not limited to, respiratory ciliated cells, fallopian tube ciliated cells, endometrial ciliated cells, rete testis ciliated cells, vas deferens ciliated cells, and / or ciliated ependymal cells. Human airway ciliated cells have 200–300 cilia on their surface. Cilia are elongated, motile, cylindrical projections from the cell membrane, approximately 0.25 mm in diameter, containing microtubules and cytoplasm continuous with that of the cell. Human tracheal cilia are 5–8 mm long, becoming shorter in more distal airways.

[0284] Cilia are complex in structure, consisting of an axoneme anchored to the cell by a basal body and a filament bundle, with several small claw-like structures at the tip. The orientation of the basal body determines the orientation of the cilium and the direction of its effective beating. The axoneme contains nine pairs of microtubules surrounding a central pair, as well as radial spokes and peripheral nexin links, which largely maintain the cilium's wheel-like arrangement. The inner and outer arms are attached to the microtubules. The main structural protein of the doublet is tubulin. The inner and outer arms contain dynein, a protein classified as an ATPase. Dynein generates the force that drives the sliding motion of the microtubules responsible for ciliary movement. It is generally believed that the outer dynein arm is primarily responsible for the beating frequency, while the inner dynein arm, together with the radial spokes and nexin links, contributes to the beating waveform. Alterations in the structural integrity of the axoneme can result in a variety of abnormal beating states, ranging from quiescence to hyperactive states with abnormal patterns.

[0285] In another aspect, provided herein are methods for delivery to ciliated cells of a subject, the method comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) in combination with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid. In some embodiments, the ciliated cells are pulmonary ciliated cells.

[0286] In some embodiments of the method, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect detectable by ciliated cells in the organ or tissue of the subject, hi some embodiments, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect detectable by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of ciliated cells in the organ or tissue of the subject.

[0287] In some embodiments, the organ is a lung. In some embodiments, the tissue is lung tissue. In some embodiments, the tissue is lung airway tissue. In some embodiments, the method delivers a therapeutic agent to a subject's lungs to produce a therapeutic effect detectable in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of ciliated cells in the subject's lungs.

[0288] In some embodiments of the method, the pharmaceutical composition is administered to the subject via any suitable delivery method. In some embodiments, the pharmaceutical composition is administered to the subject by inhalation. In some embodiments, the pharmaceutical composition is administered to the subject by systemic administration, such as intravenous administration.

[0289] secretory cells "Secretory cells" refer to cells specialized for secretion. These cells are usually of epithelial origin and have a well-developed characteristic rough endoplasmic reticulum, or, in the case of cells secreting lipids or lipid-derived products, a well-developed smooth endoplasmic reticulum. Examples of secretory cells include salivary gland cells, mammary gland cells, lacrimal gland cells, auditory canal gland cells, eccrine sweat gland cells, apocrine sweat gland cells, sebaceous gland cells, Bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin's gland cells, Littré gland cells, endometrial cells, goblet cells of the respiratory and digestive tracts, mucous cells of the stomach, zymogenic cells of the gastric glands, acid-secreting cells of the gastric glands, acinar cells of the pancreas, Paneth cells of the small intestine, type II pneumocytes of the lungs, and club cells of the lungs. These include cells of the pituitary gland, anterior pituitary cells, intermediate pituitary cells, posterior pituitary cells, intestinal and respiratory cells, thyroid cells, parathyroid cells, adrenal cells, testicular cells, ovarian cells, cells of the juxtaglomerular apparatus of the kidney, cells that secrete extracellular matrix (e.g., epithelial cells, non-epithelial cells (fibroblasts, chondrocytes, osteoblasts / osteocytes, bone-forming cells, etc.)), and secretory cells of the immune system (e.g., Ig-producing B cells, cytokine-producing T cells, etc.).

[0290] In another aspect, provided herein is a method for delivery to secretory cells of a subject, the method comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) in combination with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid. In some embodiments, the secretory cells are pulmonary secretory cells.

[0291] In some embodiments of the method, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect detectable by secretory cells in the organ or tissue of the subject, hi some embodiments, the method delivers a therapeutic agent to an organ or tissue of a subject to produce a therapeutic effect detectable by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of secretory cells in the organ or tissue of the subject.

[0292] In some embodiments of the method, the organ is a lung. In some embodiments, the tissue is lung tissue. In some embodiments, the tissue is lung airway tissue. In some embodiments, the method delivers a therapeutic agent to the lung of a subject to produce a therapeutic effect detectable in at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of secretory cells in the lung of the subject.

[0293] In some embodiments of the method, the pharmaceutical composition is administered to the subject via any suitable delivery method. In some embodiments, the pharmaceutical composition is administered to the subject by inhalation. In some embodiments, the pharmaceutical composition is administered to the subject by systemic administration, such as intravenous administration.

[0294] Dosage Level In another aspect, a high-potency dosage form of a therapeutic (or prophylactic) agent formulated with selective organ targeting (SORT) lipids is provided, the dosage form comprising a therapeutic (or prophylactic) agent in combination with a lipid composition as described herein. In some embodiments, the lipid composition comprises (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0295] In some embodiments of the high-potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a lower dose of therapeutic agent than that required in a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG-lipid.

[0296] In some embodiments of the high-potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of therapeutic agent that is about 1.1 to about 20-fold lower than that required for a reference lipid composition. In some embodiments of the high-potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of therapeutic agent that is about 1.1 to about 10-fold lower than that required for a reference lipid composition. In some embodiments of the high-potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of therapeutic agent that is about 1.1 to about 5-fold lower than that required for a reference lipid composition. In some embodiments of the high-potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of therapeutic agent that is about 10 to about 20-fold lower than that required for a reference lipid composition. In some embodiments of the high potency dosage forms of the present application, the SORT lipid is present in the dosage form in an amount sufficient to achieve a therapeutic effect at a dose of therapeutic agent that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold lower than the amount required in a reference lipid composition.

[0297] In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 2.0, 1.5, 1.0, 0.5, 0.2, or 0.1 milligrams per kilogram of body weight (mg / kg, or mpk), or a dose range between and including any two of the foregoing values.

[0298] In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 2 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 1 milligram per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.5 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.2 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.1 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5 milligrams per milliliter (mg / mL) or less.

[0299] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligrams per milliliter (mg / mL), or a concentration range between and including any two of the foregoing values.

[0300] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 2 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 1 milligram per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 0.5 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 0.1 milligrams per milliliter (mg / mL) or less.

[0301] Any suitable dosage form can be prepared for delivery via, for example, oral, rectal, vaginal, transmucosal, intratracheal or pulmonary, including inhalation, or enteral administration; intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0302] In some embodiments, the dosage form may be administered locally rather than systemically, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways depending on the tissue being targeted.

[0303] In some embodiments, the dosage form is an inhalation aerosol containing the composition of the present invention for nasal, tracheal, or bronchial delivery. In some embodiments, the dosage form can be provided in a lozenge for oral, tracheal, or esophageal application. In some embodiments, the dosage form can be provided in the form of a liquid, tablet, or capsule for administration to the stomach or intestines. In some embodiments, the dosage form can be provided in the form of a suppository for rectal or vaginal application. In some embodiments, the dosage form can also be delivered to the eye by using an emulsion, eye drops, or injection.

[0304] In some embodiments, the administration of a dose of a therapeutic agent may be repeated.

[0305] subject Any subject in need of treatment can be treated using the methods of the present application. In some embodiments, the subject has been determined to be likely to respond to a therapeutic agent. For example, the subject may have, is suffering from, or is suspected of having a disease or condition. For example, the disease or disorder may be selected from the group consisting of genetic respiratory diseases, chronic inflammatory lung diseases, pulmonary fibrosis, central nervous system (CNS) disorders, immunodeficiencies, autoimmune diseases, cancer, infectious diseases, liver fibrosis, liver cirrhosis, metabolic disorders, muscular dystrophy, and viral infections. Therapeutic or prophylactic agents described elsewhere herein may be effective in providing a therapeutic benefit to a subject through various mechanisms, for example, gene therapy (e.g., requiring repeated administration), alteration (e.g., increase) of protein production, (e.g., in vivo) chimeric antigen receptor (CAR) T-cell generation, immuno-oncology, vaccine-based approaches, tumor suppressor reactivation, or other mechanisms.

[0306] In some embodiments, the subject has been determined to have a mutation (e.g., a missense or nonsense) in the target gene. In some embodiments, the mutation in the target gene is associated with a genetic disease or disorder. In some embodiments, the target gene encodes a protein selected from the group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1.

[0307] In some embodiments, the subject is determined to exhibit abnormal expression or activity of a protein or polynucleotide corresponding to the target gene. In some embodiments, the abnormal expression or activity of the protein or polynucleotide is associated with a genetic disease or disorder. In some embodiments, the protein is selected from the group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1. In some embodiments, the polynucleotide encodes a protein selected from the group consisting of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, and FBN1.

[0308] In some embodiments, the subject is selected from the group consisting of a mouse, a rat, a monkey, and a human. In some embodiments, the subject is a human.

[0309] In another aspect, provided herein is a method for potent delivery of a therapeutic agent (or prophylactic agent) to a cell, the method comprising contacting the cell with a pharmaceutical composition of the present application. In some embodiments of the method, the pharmaceutical composition comprises the therapeutic agent (or prophylactic agent) in combination with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0310] In some embodiments of the method, the cells are isolated from a subject. In some embodiments of the method, the cells are a cell line. In some embodiments of the method, the cells are lung cells. In some embodiments, the lung cells are lung airway cells. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0311] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells characterized by a greater therapeutic effect compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments, the reference lipid composition does not contain a SORT lipid. In some embodiments, the reference lipid composition does not contain an amount of SORT lipid. In some embodiments, the reference lipid comprises 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), a phospholipid, cholesterol, and a PEG-lipid.

[0312] In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 20-fold greater therapeutic efficacy than that achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic efficacy than that achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 5-fold greater therapeutic efficacy than that achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic efficacy than that achieved with a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater than the therapeutic effect achieved with a reference lipid composition.

[0313] In some embodiments of the method, the SORT lipids result in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in a plurality of cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 1.1-fold to about 20-fold more cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 1.1-fold to about 10-fold more cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 5-fold to about 10-fold more cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments of the method, the SORT lipids achieve a therapeutic effect in about 10-fold to about 20-fold more cells compared to the therapeutic effect achieved using a reference lipid composition. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in cells that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold greater than the therapeutic effect achieved with a reference lipid composition.

[0314] In another aspect, provided herein is a method for targeted delivery of a therapeutic agent (or prophylactic agent) to a cell type, comprising contacting the cell with a pharmaceutical composition of the present application. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) combined with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0315] In some embodiments of the method, the cells are isolated from a subject. In some embodiments of the method, the cells are a cell line. In some embodiments of the method, the cells are lung cells. In some embodiments, the lung cells are lung airway cells. Examples of lung airway cells that can be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells such as goblet cells and club cells, ciliated cells, and any combination thereof.

[0316] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a therapeutic effect in a first plurality of cells of a first cell type and a greater therapeutic effect in a second plurality of cells of a second cell type. In some embodiments, the first cell type is different from the second cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 20-fold greater than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 10-fold greater than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 1.1-fold to about 5-fold greater than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is about 10- to about 20-fold larger than the first plurality of cells of the first cell type. In some embodiments of the method, the SORT lipid achieves a therapeutic effect in a second plurality of cells of a second cell type that is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, or at least about 20-fold larger than the first plurality of cells of the first cell type.

[0317] In some embodiments of the method, the SORT lipid results in delivery of a therapeutic agent to cells of a subject characterized by a greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type. In some embodiments, the second cell type is different from the first cell type. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 20-fold greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves about a 10-fold to about a 20-fold therapeutic effect in a first cell of a first cell type compared to the therapeutic effect achieved in a second cell of a second cell type. In some embodiments of the method, the SORT lipid achieves at least about a 1.1-fold, at least about a 1.5-fold, at least about a 2-fold, at least about a 3-fold, at least about a 4-fold, at least about a 5-fold, at least about a 6-fold, at least about a 7-fold, at least about a 8-fold, at least about a 9-fold, at least about a 10-fold, at least about a 11-fold, at least about a 12-fold, at least about a 13-fold, at least about a 14-fold, at least about a 15-fold, at least about a 16-fold, at least about a 17-fold, at least about a 18-fold, at least about a 19-fold, or at least about a 20-fold therapeutic effect in a first cell of a first cell type compared to the therapeutic effect achieved in a second cell of a second cell type.

[0318] In some embodiments of the method, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0319] In some embodiments of this method, the second cell type is lung cell.In some embodiments, the second cell type is lung airway cell.The example of lung airway cell that can be targeted by delivery of the present application includes but is not limited to basal cell, secretory cell, such as goblet cell and club cell, ciliated cell and any combination thereof.

[0320] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting comprises administering to the subject a composition comprising a therapeutic agent in combination with a lipid composition.

[0321] Below are examples of compositions and composition ratings of the present disclosure: Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0322] List of Embodiments The following list of embodiments of the invention should be considered as disclosing various features of the invention, which features may be considered unique to the particular embodiment in which they are discussed or may be combinable with various other features as listed in other embodiments. Thus, simply because a feature is discussed in a particular embodiment does not necessarily mean that the use of that feature is limited to that embodiment.

[0323] Embodiment 1. A method for potent delivery to lung cells of a subject, comprising administering to the subject an aerosol composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to the cells of the subject characterized by a greater therapeutic effect (e.g., about 1.1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold) compared to the therapeutic effect achieved with a reference lipid composition (e.g., not including the amount of the SORT lipid), and optionally, the SORT lipid is selected from those set forth in Table 6, or a pharmaceutically acceptable salt thereof, or a subset of lipids and pharmaceutically acceptable salts thereof.

[0324] Embodiment 2. A method for potent delivery to lung cells of a subject, comprising administering to the subject an aerosol composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to cells of the subject characterized by a greater therapeutic effect in a plurality of lung cells (e.g., about 1.1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold) compared to the therapeutic effect achieved with a reference lipid composition (e.g., not including the amount of the SORT lipid), and optionally, the SORT lipid is selected from those set forth in Table 6, or a pharmaceutically acceptable salt thereof, or a subset of lipids and pharmaceutically acceptable salts thereof.

[0325] Embodiment 3. A method for targeted delivery to lung cells of a subject, comprising administering to the subject an aerosol composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) results in delivery of the therapeutic agent to a greater proportion of cell types compared to that achieved with a reference lipid composition, and optionally, the SORT lipid is selected from those set forth in Table 6, or a pharmaceutically acceptable salt thereof, or a subset of lipids and pharmaceutically acceptable salts thereof.

[0326] Embodiment 4. A method for targeted delivery to lung cells of a subject, comprising administering to the subject an aerosol composition comprising a therapeutic agent combined with a lipid composition comprising: (i) an ionizable cationic lipid; and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) effects delivery of the therapeutic agent to cells of the subject characterized by a therapeutic effect in a first plurality of lung cells of a first cell type and a second plurality of lung cells of a second cell type that is greater (e.g., about 1.1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold)

[0327] Embodiment 5. A method for targeted delivery to lung cells of a subject, comprising administering to the subject an aerosol composition comprising a therapeutic agent combined with a lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid, wherein the SORT lipid (e.g., an amount of the SORT lipid) effects delivery of the therapeutic agent to cells of the subject characterized by a greater therapeutic effect (e.g., about 1.1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold) in first lung cells of a first cell type of the subject compared to the therapeutic effect in second lung cells of a second cell type of the subject, wherein the first cell type is different from the second cell type, and optionally the SORT lipid is selected from those set forth in Table 6, or a pharmaceutically acceptable salt thereof, or a subset of lipids and pharmaceutically acceptable salts thereof.

[0328] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the lipid composition further comprises (iii) a phospholipid.

[0329] Embodiment 7. The method of any one of embodiments 1 to 6, wherein said administering is not intravenous.

[0330] Embodiment 8 The method of embodiment 7, wherein said administering comprises administering by inhalation.

[0331] Embodiment 9. The method of any one of embodiments 1 to 8, wherein said lipid composition comprises said SORT lipid at a molar percentage of about 20% to about 65%.

[0332] Embodiment 10. The method of any one of embodiments 1 to 9, wherein said lipid composition comprises said ionizable cationic lipid at a molar percentage of about 5% to about 30%.

[0333] Embodiment 11. The method of any one of embodiments 1 to 10, wherein said lipid composition comprises said phospholipid at a molar percentage of about 8% to about 23%.

[0334] Embodiment 12 The method of any one of embodiments 1 to 11, wherein said phospholipid is not ethylphosphocholine.

[0335] Embodiment 13. The method of any one of embodiments 1 to 12, wherein said lipid composition further comprises a steroid or steroid derivative.

[0336] Embodiment 14. The method of embodiment 13, wherein said lipid composition comprises said steroid or steroid derivative at a molar percentage of about 15% to about 46%.

[0337] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the lipid composition further comprises a polymer-conjugated lipid (e.g., a poly(ethylene glycol) (PEG)-conjugated lipid).

[0338] Embodiment 16. The method of embodiment 15, wherein said lipid composition comprises about 0.5% to about 10% molar percentage of said polymer-conjugated lipid.

[0339] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the therapeutic agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 20:1 or less.

[0340] Embodiment 18. The method of embodiment 17, wherein said N / P ratio is from about 5:1 to about 20:1.

[0341] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the molar ratio of said therapeutic agent to total lipids in said lipid composition is no greater than about 1:1, 1:10, 1:50, or 1:100.

[0342] Embodiment 20 The method of any one of embodiments 1 to 19, wherein at least about 85% of the therapeutic agent is encapsulated in particles of the lipid composition.

[0343] Embodiment 21. The method of any one of embodiments 1 to 20, wherein the lipid composition comprises a plurality of particles characterized by one or more of the following properties: (1) a (e.g., average) size of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of about 0.2 or less; and (3) a zeta potential of -10 millivolts (mV) to 10 mV.

[0344] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7.

[0345] Embodiment 23 The method of embodiment 22, wherein said apparent pKa of said lipid composition is about 8 or greater.

[0346] Embodiment 24. The method of embodiment 23, wherein said apparent pKa of said lipid composition is from about 8 to abo...

Claims

1. An aerosol composition for delivering a polynucleotide to lung cells, comprising aerosol droplets, the aerosol droplets comprising a polynucleotide in combination with a lipid composition; The lipid composition comprises: (i) an ionizable cationic lipid; and (ii) a phospholipid; and (iii) cholesterol, (iv) polyethylene glycol conjugated lipids (PEG lipids); Including, the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 microns (μm) to about 10 μm; Aerosol compositions.

2. The aerosol composition of claim 1, wherein the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 1.7 microns (μm) to about 2.3 μm.

3. The aerosol composition of claim 2, wherein the polynucleotide is messenger RNA (mRNA).

4. The aerosol composition of claim 3, wherein the mRNA encodes a therapeutic agent.

5. The aerosol composition of claim 3, wherein the mRNA encodes a CFTR protein.

6. The aerosol composition of claim 3, wherein the mRNA encodes DNAI1 protein.

7. The aerosol composition of claim 1, wherein the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

8. The aerosol composition of claim 1, wherein the lipid composition comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

9. The aerosol composition of claim 1, wherein the lipid composition contains DODAP at a molar percentage of about 20% to about 30%.

10. The aerosol composition of claim 1, wherein the ionizable lipid is an ionizable cationic lipid.

11. The aerosol composition of claim 1, wherein the lipid composition comprises (v) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid.

12. The aerosol composition of claim 1, wherein the lipid composition comprises (v) a selective organ targeting (SORT) lipid other than the ionizable cationic lipid in a molar percentage of about 20% to about 30%.

13. The aerosol composition of claim 11, wherein the lipid composition comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC).

14. The aerosol composition of claim 11, wherein the lipid composition comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP).

15. The aerosol composition of claim 11, wherein the lipid composition comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and the ionizable cationic lipid is 4A3-SC7.

16. The aerosol composition of claim 11, wherein the lipid composition comprises 1,2-dioleoyl-3-trimethylammonium-propane (14:0 TAP).

17. The aerosol composition of claim 1, wherein the ionizable cationic lipid is 4A3-SC7.

18. The aerosol composition of claim 1, wherein the lipid composition comprises ionizable cationic lipids at a molar percentage of about 15% to about 30%.

19. The aerosol composition of claim 1, wherein the lipid composition contains 4A3-SC7 at a molar percentage of about 15% to about 30%.

20. The aerosol composition of claim 1, wherein the lipid composition contains cholesterol at a molar percentage of about 15% to about 46%.

21. 2. The aerosol composition of claim 1, wherein the ionizable cationic lipid is selected from the ionizable cationic lipids listed in Table 4.

22. The lipid composition, (i) 14:0 EPC in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid in a molar percentage of about 1.5 to about 4.0; 14. The aerosol composition of claim 13, comprising:

23. The lipid composition, (i) 14:0 EPC in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid at a molar percentage of about 1.5 to about 4.0; (v) 4A3-SC7 in a molar percentage of 15% to 30% 14. The aerosol composition of claim 13, comprising:

24. The lipid composition, (i) 14:0 TAP in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid in a molar percentage of about 1.5 to about 4.0; 17. The aerosol composition of claim 16, comprising:

25. The lipid composition, (i) 14:0 TAP in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid at a molar percentage of about 1.5 to about 4.0; (v) 4A3-SC7 in a molar percentage of 15% to 30% 17. The aerosol composition of claim 16, comprising:

26. The lipid composition, (i) DODAP in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid in a molar percentage of about 1.5 to about 4.0; 15. The aerosol composition of claim 14, comprising:

27. The lipid composition, (i) DODAP in a molar percentage of about 20% to about 30%; (ii) a phospholipid; and (iii) cholesterol in a molar percentage of about 15% to about 46%; (iv) a PEG lipid at a molar percentage of about 1.5 to about 4.0; (v) 4A3-SC7 in a molar percentage of 15% to 30% 15. The aerosol composition of claim 14, comprising:

28. 1. A method for delivery of a polynucleotide to a lung cell of a subject, comprising: administering an aerosol composition comprising the aerosol droplets to lung cells of a subject; the aerosol droplets comprise a polynucleotide in combination with a lipid composition; The lipid composition comprises: (i) an ionizable cationic lipid; and (ii) a phospholipid; and (iii) cholesterol, (iv) polyethylene glycol conjugated lipids (PEG lipids); Including, the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 microns (μm) to about 10 μm; A method of delivering an effective amount of a polynucleotide to a lung cell.

29. 30. The method of claim 28, comprising administering the aerosol composition by inhalation.

30. Use of a lipid composition for the manufacture of a medicament for treating a pulmonary disease, comprising: delivering an aerosol composition comprising aerosol droplets; the aerosol droplets comprise a polynucleotide in combination with a lipid composition; The lipid composition comprises: (i) an ionizable cationic lipid; and (ii) a phospholipid; and (iii) cholesterol, (iv) polyethylene glycol conjugated lipids (PEG lipids); Including, the aerosol droplets have a mass median aerodynamic diameter (MMAD) of about 0.5 microns (μm) to about 10 μm; The polynucleotide is delivered to the lung in an amount effective to treat a pulmonary disease.

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