Compositions for delivering payload molecules to airway epithelia - Patent application

Abstract

The present disclosure provides LNPs that contain payload molecules, such as mRNA therapeutics, for the treatment of diseases or disorders that would benefit from delivery of the payload molecule to airway cells.

Description

[Background technology]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 278,973, filed November 12, 2021, the entire contents of which are incorporated herein by reference. (Background technology) Respiratory epithelial cells line the airways. Their primary function is to humidify the respiratory tract, protect the airways from potential pathogens, infections, and tissue damage, and / or facilitate gas exchange. Dysfunction of respiratory epithelial cells can lead to many disorders, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Delivery of payloads to respiratory epithelial cells can be used to induce immunity against antigens of interest and to modulate the function of airway epithelial cells, for example, to replace missing or mutant proteins or to increase or decrease the functionality of such cells.

[0002] For example, cystic fibrosis ("CF") is an autosomal recessive disease characterized by the abnormal accumulation of sticky, thick mucus in patients. CF is also known as cystic fibrosis of the pancreas, fibrocystic disease of the pancreas, or pancreatic fibrosis. Mucus is an important bodily fluid that lubricates and protects the lungs, reproductive system, digestive system, and other organs. However, CF patients produce thick, sticky mucus, which reduces the size of their airways, resulting in chronic coughing, wheezing, inflammation, bacterial infections, fibrosis, and lung cysts. Furthermore, in most CF patients, mucus blocks ducts in the pancreas, which prevents the release of insulin and digestive enzymes, resulting in diarrhea, malnutrition, poor growth, and weight loss (Gershman AJ et al., Cleve Clin J Med. 73:1065-1074 (2006)). The estimated incidence of CF is 1 in 2,500–3,500 births in Caucasians, but it is much rarer in other populations (Ratjen F. et al., Lancet 361:681–689 (2003)). Current treatments for CF only manage symptoms, not cure the disease. Specifically, antibiotics, anti-inflammatory drugs, bronchodilators, decongestants, a high-protein and high-fat diet, and vitamin supplements are prescribed to control symptoms. In advanced lung disease, lung transplantation has also been performed to provide patients with intact lungs. However, these treatments do not completely or reliably control the disease. New treatments have emerged that target the underlying cause of CF. These treatments modulate CFTR in patients with the Phe508del cystic fibrosis transmembrane conductance regulator (CFTR) mutation (Middleton PJ et al., N Engl J Med. 381:1809–1819 (2019)). However, approximately 1 in 100 CF patients do not have the Phe508del CFTR mutation and are therefore excluded from this treatment.

[0003] Thus, there is a need for improved therapies for treating disorders associated with airway epithelial cell dysfunction (e.g., CF), for targeting such airway epithelial cells for prophylactic treatment (e.g., immunization), or for treating other disorders that would benefit from therapeutic delivery of nucleic acid molecules or other payload molecules to airway epithelial cells. Summary of the Invention

[0004] The present disclosure provides LNP molecules for delivering nucleic acid molecules, e.g., mRNA therapeutics, to airway epithelial cells for the treatment of disorders associated with airway epithelial dysfunction or for the prophylactic benefit of patients. In one embodiment, the subject LNP molecules can be used to treat disorders associated with epithelial cell dysfunction, such as cystic fibrosis (CF), COPD, or asthma, as well as to administer vaccine payloads. The present disclosure provides LNPs that, when administered to cells (e.g., in vitro and in vivo), have improved properties, e.g., improved payload delivery to epithelial cells (e.g., as measured by cellular accumulation of the LNP, expression of a desired protein, and / or expression of mRNA).

[0005] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent primarily disposed on the outer surface of the core; Provided herein are nanoparticles comprising:

[0006] In one aspect, (a) Below: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid nanoparticle core comprising PEG-lipids; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising:

[0007] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in epithelial cells and about 5% or greater expression in epithelial cells.

[0008] In one aspect, provided herein is a process for preparing nanoparticles, comprising contacting lipid nanoparticles with a cationic agent, wherein the lipid nanoparticles comprise: (a) Below: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid nanoparticle core comprising PEG-lipids; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell.

[0009] In one aspect, provided herein are nanoparticles prepared by the processes described herein.

[0010] In one aspect, provided herein is a method of delivering a polynucleotide or polypeptide payload to a cell, the method comprising contacting the cell with a nanoparticle described herein.

[0011] In one aspect, provided herein is a method of treating or preventing a disease in a patient, the method comprising administering to the patient nanoparticles comprising a payload for treating or preventing a disease as described herein.

[0012] Each limitation of the present invention may encompass various embodiments of the present invention. Accordingly, each limitation of the present invention involving any one element or combination of elements is contemplated as being included in each aspect of the present invention. The present invention is not limited in its application to the details of construction or the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram of an exemplary first generation post-loading (PHL) process for preparing LNPs.

[0014] [Figure 2] FIG. 1 is a diagram of an exemplary second generation PHL process (comprehensive) for preparing LNPs.

[0015] [Figure 3] FIG. 1 is a diagram of an exemplary second generation PHL process (specific) for preparing LNPs.

[0016] [Figure 4] FIG. 1 is a diagram of an exemplary process for the preparation of a hollow lipid nanoparticle prototype ("neutral assembly"), in which hollow LNPs are mixed at pH 8.0, with the final formulation at pH 5.0.

[0017] [Figure 5] FIG. 1 is a diagram of an exemplary process for preparing LNPs using sterol amines.

[0018] [Figure 6] Small-angle X-ray scattering (SAXS) analysis of LNP-1 and LNP-1a.

[0019] [Figure 7]1 is a graph showing the general polarity Laurdan (GLP) of LNP-1 and LNP-1a. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides LNP molecules for delivering nucleic acid molecules or payload molecules to airway epithelial cells. For example, such LNP molecules can be used to deliver payload molecules, such as mRNA therapeutics for the treatment of cystic fibrosis (CF), to airway epithelial cells. Cystic fibrosis (CF) is a progressive genetic disease that causes persistent lung infections and limits breathing over time. This disease is characterized by mutations in both copies of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Without CFTR, which is involved in the production of sweat, digestive juices, and mucus, normally thin secretions become thick. mRNA therapeutics are particularly suitable for the treatment of CF because this technology delivers mRNA encoding CFTR into cells, resulting in the de novo synthesis of functional CFTR protein within the target cells. After delivery of the mRNA to the target cells, the desired CFTR protein is expressed by the cell's own translational machinery, thereby replacing the defective or missing protein with a fully functional CFTR protein. In another embodiment, such LNPs can be used to deliver nucleic acid molecules for gene editing, small molecules, or other payloads for alleviating epithelial cell dysfunction.In another embodiment, such LNPs can be used to deliver antigens to airway cells.In one embodiment, the antigen is in the form of an mRNA construct present in the LNP, which causes the expression of polypeptides or peptides that generate an immune response against the antigen. Lipid nanoparticles (LNPs) are ideal platforms for safely and effectively delivering payload molecules, such as mRNA, to target cells. LNPs have the unique ability to deliver nucleic acids through mechanisms including cellular uptake, intracellular trafficking, and endosomal release or escape. Some embodiments provided herein feature LNPs with improved properties. In some embodiments, the LNPs provided herein comprise a lipid nanoparticle core, a polynucleotide or polypeptide payload encapsulated within the core for delivery to cells, and a cationic agent primarily disposed on the outer surface of the nanoparticle. Without being bound by theory, LNPs with a cationic agent primarily disposed on the outer surface of the core can improve the accumulation of LNPs in cells, such as human bronchial epithelium (HBE), and can also improve the function of the payload molecule, as measured, for example, by mRNA expression in cells (e.g., airway epithelial cells).

[0021] In some embodiments, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent primarily disposed on the outer surface of the core; Provided herein are nanoparticles comprising:

[0022] In some embodiments, (a) Below: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid nanoparticle core comprising PEG-lipids; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent.

[0023] In some embodiments, (a) Below: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid nanoparticle core comprising PEG-lipids; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent located primarily on the outer surface of the core.

[0024] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit cellular accumulation in at least about 20% of cells and expression in about 5% or more of the cells. In some embodiments, the nanoparticles exhibit cellular accumulation in about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of the cells. In some embodiments, the nanoparticles exhibit expression in about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of the cells.

[0025] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent primarily disposed on the outer surface of the core; Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit cellular accumulation in at least about 20% of cells and expression in about 5% or more of the cells. In some embodiments, the nanoparticles exhibit cellular accumulation in about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of the cells. In some embodiments, the nanoparticles exhibit expression in about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of the cells.

[0026] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent primarily disposed on the outer surface of the core; Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit about 0.5% to 50% protein expression in cells. In some embodiments, the nanoparticles exhibit about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% protein expression in cells.

[0027] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit about 0.5% to 50% protein expression in cells. In some embodiments, the nanoparticles exhibit about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% protein expression in cells.

[0028] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in epithelial cells and about 5% or greater expression in epithelial cells. In some embodiments, the nanoparticles exhibit about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in epithelial cells. In some embodiments, the nanoparticles exhibit about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in epithelial cells. In some embodiments, the epithelial cells are HBE cells.

[0029] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of epithelial cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of epithelial cells.

[0030] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of lung cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of lung cells.

[0031] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of nasal cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of nasal cells.

[0032] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of alveolar epithelial cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of alveolar epithelial cells.

[0033] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in respiratory epithelial cells and about 5% or greater expression in respiratory epithelial cells. In some embodiments, the nanoparticles exhibit about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in respiratory epithelial cells. In some embodiments, the nanoparticles exhibit about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in respiratory epithelial cells.

[0034] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of respiratory epithelial cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of respiratory epithelial cells.

[0035] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of macrophages. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of macrophages.

[0036] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of HeLa cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of HeLa cells.

[0037] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in bronchial epithelial cells and about 5% or greater expression in bronchial epithelial cells. In some embodiments, the nanoparticles exhibit about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in respiratory epithelial cells. In some embodiments, the nanoparticles exhibit about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in bronchial epithelial cells.

[0038] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit protein expression in about 0.5% to about 50% of bronchial epithelial cells. In some embodiments, the nanoparticles exhibit protein expression in about 0.1% to about 60%, about 0.5% to about 40%, about 1% to about 30%, or about 1% to about 20% of bronchial epithelial cells.

[0039] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in HBE cells and about 5% or greater expression in HBE cells. In some embodiments, the nanoparticles exhibit about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in HBE cells. In some embodiments, the nanoparticles exhibit about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in HBE cells.

[0040] In one aspect, (a) a lipid nanoparticle core; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent; and Provided herein are nanoparticles comprising: wherein the nanoparticles exhibit at least about 20% cellular accumulation in healthy HBE cells in vitro and about 5% or greater expression in healthy HBE cells in vitro. In some embodiments, the nanoparticles exhibit about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% cellular accumulation in healthy HBE cells in vitro. In some embodiments, the nanoparticles exhibit about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% expression in healthy HBE cells in vitro.

[0041] In some embodiments, the cells referred to herein above and throughout the specification can be in vitro or in vivo cells. In some embodiments, the cells are in vitro cells. In some embodiments, the cells are in vivo cells.

[0042] In some embodiments, the nanoparticles of the present invention exhibit increased cell accumulation (e.g., in airway epithelial cells such as HBEs) compared to nanoparticles prepared of substantially the same composition but without the post-addition of a cationic agent (e.g., layering or contacting a preformed lipid nanoparticle with a cationic agent). In some embodiments, the nanoparticles of the present invention exhibit increased cell expression (e.g., in airway epithelial cells such as HBEs) compared to nanoparticles prepared of substantially the same composition but without the post-addition of a cationic agent (e.g., layering or contacting a preformed lipid nanoparticle with a cationic agent).

[0043] In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 0.1:1 to about 15:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 0.2:1 to about 10:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 10:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 8:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 7:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 6:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 5:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 4:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1.25:1 to about 3.75:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 1.25:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 2.5:1. In some embodiments, the weight ratio of cationic agent to polynucleotide payload is about 3.75:1.

[0044] In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 0.1:1 to about 20:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 10:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 9:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 8:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 7:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 6:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1 to about 5:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 1.5:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 2:1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 3: 1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 4: 1. In some embodiments, the molar ratio of cationic agent to polynucleotide payload is about 5: 1.

[0045] In some embodiments, the nanoparticles of the present invention have a zeta potential of about 5 mV to about 20 mV. In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 15 mV. In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 10 mV.

[0046] Zeta potential measures the surface charge of a colloidal dispersion. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between similarly charged adjacent particles in the dispersion. Zeta potential can be measured with a Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential using the principle of "massively parallel phase analysis light scattering" or MP-PALS. This measurement is more sensitive and induces less stress than ISO Method 13099-1:2012, which uses only one detection angle and requires higher voltages for operation. In some embodiments, the zeta potential of lipids in the hollow lipid nanoparticle compositions described herein is measured using an instrument that utilizes the principle of MP-PALS. Zeta potential can be measured with a Malvern Zetasizer (Nano ZS).

[0047] In some embodiments, the lipid nanoparticle core has a neutral charge at neutral pH.

[0048] In some embodiments, greater than about 80% of the cationic agent is on the surface of the nanoparticle. In some embodiments, greater than about 90% of the cationic agent is on the surface of the nanoparticle. In some embodiments, greater than about 95% of the cationic agent is on the surface of the nanoparticle.

[0049] In some embodiments, at least about 50% of the polynucleotide or polypeptide payload is encapsulated within the core. In some embodiments, at least about 75% of the polynucleotide or polypeptide payload is encapsulated within the core. In some embodiments, at least about 90% of the polynucleotide or polypeptide payload is encapsulated within the core. In some embodiments, at least about 95% of the polynucleotide or polypeptide payload is encapsulated within the core.

[0050] In some embodiments, the nanoparticles have a polydispersity value of less than about 0.4. In some embodiments, the nanoparticles have a polydispersity value of less than about 0.3. In some embodiments, the nanoparticles have a polydispersity value of less than about 0.2.

[0051] In some embodiments, the nanoparticles have an average diameter of about 40 nm to about 150 nm. In some embodiments, the nanoparticles have an average diameter of about 50 nm to about 100 nm. In some embodiments, the nanoparticles have an average diameter of about 60 nm to about 120 nm. In some embodiments, the nanoparticles have an average diameter of about 60 nm to about 100 nm. In some embodiments, the nanoparticles have an average diameter of about 60 nm to about 80 nm.

[0052] In some embodiments, the nanoparticles have a Laurdan general polarization of about 0.6 or greater. In some embodiments, the nanoparticles have a d-spacing of greater than about 6 nm. In some embodiments, the nanoparticles have a d-spacing of greater than about 7 nm.

[0053] In some embodiments, at least 50% of the nanoparticles have a surface fluidity value greater than the threshold polarization level. In some embodiments, at least 75% of the nanoparticles have a surface fluidity value greater than the threshold polarization level. In some embodiments, at least 90% of the nanoparticles have a surface fluidity value greater than the threshold polarization level. In some embodiments, at least 95% of the nanoparticles have a surface fluidity value greater than the threshold polarization level.

[0054] In some embodiments, when nanoparticles are contacted with a cell population, about 10% or more of the cell population accumulate nanoparticles. In some embodiments, when nanoparticles are contacted with a cell population, about 15% or more of the cell population accumulate nanoparticles. In some embodiments, when nanoparticles are contacted with a cell population, about 20% or more of the cell population accumulate nanoparticles. In some embodiments, when nanoparticles are contacted with a cell population, about 5% or more of the cells express the polynucleotide or polypeptide. In some embodiments, when nanoparticles are contacted with a cell population, about 10% or more of the cells express the polynucleotide or polypeptide. In some embodiments, the cell population is an epithelial cell population. In some embodiments, the cell population is a respiratory epithelial cell population. In some embodiments, the respiratory epithelial cell population is a pulmonary cell population. In some embodiments, the respiratory epithelial cell population is a nasal cell population. In some embodiments, the respiratory epithelial cell population is an alveolar epithelial cell population. In some embodiments, the respiratory epithelial cell population is a bronchial epithelial cell population. In some embodiments, the respiratory epithelial cell population is an HBE population. In some embodiments, the cell population is a pulmonary cell population. In some embodiments, the cell population is a nasal cell population. In some embodiments, the cell population is an alveolar epithelial cell population. In some embodiments, the cell population is a bronchial epithelial cell population. In some embodiments, the cell population is an HBE population. In some embodiments, the cell population is a HeLa population.

[0055] Cationic Agents Cationic agents can include any water-soluble molecule or substance that has a net positive charge and can attach to the surface of the lipid nanoparticle core. Such agents can be lipid-soluble but also soluble in aqueous solutions. Cationic agents can be charged at physiological pH. Physiological pH is the pH level normally observed in the human body. Physiological pH can be about 7.30-7.45 or about 7.35-7.45. Physiological pH can be about 7.40. Generally speaking, cationic agents are characterized by a net positive charge at physiological pH because they contain one or more basic functional groups that are protonated at physiological pH in aqueous media. For example, cationic agents can contain one or more amine groups, e.g., primary, secondary, or tertiary amines, each with a pKa of 8.0 or greater. The pKa can be greater than about 9.

[0056] In some embodiments, the cationic agent can be a cationic lipid, which is a water-soluble amphipathic molecule, with one portion of the molecule being hydrophobic, e.g., comprising a lipid portion, and the other portion of the molecule being hydrophilic and comprising one or more functional groups that are typically charged at physiological pH. The hydrophobic portion, including the lipid portion, can serve to anchor the cationic agent to the lipid nanoparticle core. The hydrophilic portion can serve to increase the surface charge of the lipid nanoparticle core. For example, the cationic agent can have a solubility of greater than about 1 mg / mL in alcohol. The solubility in alcohol can be greater than about 5 mg / mL. The solubility in alcohol can be greater than about 10 mg / mL. The solubility in alcohol can be greater than about 20 mg / mL in alcohol. The alcohol can be a C 1000 saturate, such as ethanol. 1~6 It may be alcohol.

[0057] The lipid portion of the molecule can be, for example, a structural lipid, a fatty acid, or similar hydrocarbyl group.

[0058] The structured lipid may be selected from, but is not limited to, steroids, diterpenoids, triterpenoids, cholestane, ursolic acid, or derivatives thereof.

[0059] In some embodiments, the structured lipid is a steroid selected from, but not limited to, cholesterol or phystosterol. In some embodiments, the structured lipid is a cholesterol analog. In some embodiments, the structured lipid is sitosterol, campesterol, or stigmasterol. In some embodiments, the structured lipid is a sitosterol, campesterol, or stigmasterol analog. In some embodiments, the structured lipid is β-sitosterol.

[0060] Fatty acids are those with 1 to 4 C 6~20 It contains a hydrocarbon chain. Fatty acids can be fully saturated or contain one to seven double bonds. Fatty acids can contain one to five heteroatoms along or pendant from the backbone.

[0061] In some embodiments, the fatty acid has two C 10~18 In some embodiments, the fatty acid comprises two C 10~18 In some embodiments, the fatty acid comprises two C 16 In some embodiments, the fatty acid comprises two C 14 In some embodiments, the fatty acid comprises a saturated hydrocarbon chain. 10~18 In some embodiments, the fatty acid comprises two C hydroxyl groups each having one double bond. 16~18 In some embodiments, the fatty acid comprises three C 8~18 Contains saturated hydrocarbon chains.

[0062] The hydrocarbyl group is 1 to 4 C 6~20 It consists of an alkyl chain, an alkenyl chain, an alkynyl chain, or a 3- to 10-membered cycloalkyl group, a cycloalkenyl group, or a cycloalkynyl group.

[0063] In some embodiments, the hydrocarbyl chain is C 8~10In some embodiments, the hydrocarbyl chain is C 8~10 It is alkenyl.

[0064] The hydrophilic portion may contain one to five functional groups that are charged at physiological pH of 7.3 to 7.4. The hydrophilic groups may contain basic functional groups that are protonated and positively charged at physiological pH, with at least one of the basic functional groups having a pKa of 8 or greater.

[0065] In some embodiments, the hydrophilic moiety comprises an amine group. The amine group may comprise one to four primary, secondary, or tertiary amines and mixtures thereof. The primary, secondary, or tertiary amine may be part of a larger amine containing a functional group selected from, but not limited to, -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N-. The amine may be contained in a 3- to 8-membered heteroalkyl or heteroaryl ring.

[0066] In some embodiments, the amine group comprises one or two terminal primary amines. In some embodiments, the amine group comprises one or two terminal primary amines and one internal secondary amine. In some embodiments, the amine group comprises one or two tertiary amines. In some embodiments, the tertiary amine is (CH3)2N-. In some embodiments, the amine group comprises one to two terminal (CH3)2N-.

[0067] The hydrophilic portion may comprise a phosphonium group, the counterion of which consists of a monovalent anion.

[0068] In some embodiments, three of the phosphonium substituents are isopropyl groups. In some embodiments, the counterion is a halo, hydrogen sulfate, nitrite, chloride, or bicarbonate. In some embodiments, the counterion is bromide.

[0069] In some embodiments, the cationic agent is a cationic lipid that is a sterol amine. A sterol amine has a sterol in its hydrophobic portion and an amine group in its hydrophilic portion. The sterol group can be selected from, but is not limited to, cholesterol, sitosterol, campesterol, stigmasterol, or derivatives thereof. The amine group can contain one to five primary, secondary, or tertiary amines, or a mixture thereof. At least one of the amines has a pKa of 8 or greater and is charged at physiological pH. The primary, secondary, or tertiary amine can be part of a larger amine containing a functional group selected from, but not limited to, -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N-. The amine can be contained in a 3- to 8-membered heteroalkyl or heteroaryl ring.

[0070] In some embodiments, the amine group of the sterol amine comprises one or two terminal primary amines. In some embodiments, the amine group comprises one or two terminal primary amines and one internal secondary amine. In some embodiments, the amine group comprises one or two tertiary amines. In some embodiments, the tertiary amine is (CH3)2N-. In some embodiments, the amine group comprises one to two terminal (CH3)2N-.

[0071] Sterol amines useful in the nanoparticles of the present invention include those of formula (A1): ALB(A1) or a salt thereof, wherein A is an amine group, L is an optional linker, and B is a sterol.

[0072] In some embodiments, the amine group is an alkyl (e.g., C 1~14 Alkyl, C 1~12 Alkyl, C 1~10 alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof, where the alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 and NH(5-6 membered heteroaryl), each optionally substituted with one, two, three, or four substituents selected from: -C(=O), -C(=O)N, -OC(=O)N, -CH-NH-C(O)-, -C(O)O-, -OC(O)-CH-CH-C(=O)N-, -SS-CH, or -SS-CH-CH-C(O)N-. In some embodiments, the sterol group is cholesterol, sitosterol, campesterol, stigmasterol, or a derivative thereof.

[0073] In some embodiments, the sterol amine has the formula A2a: [ka] or a salt thereof, wherein [ka] is a single or double bond, R 1 is C 1~14 Alkyl or C 1~14 is alkenyl; L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] is the basis of; Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2.

[0074] In some embodiments, the sterol amine has the formula A2a: [ka] or a salt thereof, wherein [ka] is a single or double bond, R 1 is C 1~14 Alkyl or C 1~14 is alkenyl; L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] is the basis of; Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2 (However, the compound of formula A2a is [ka] [ka] [ka] other than).

[0075] In some embodiments, [ka] is a double bond. In some embodiments, [ka] is a single bond.

[0076] In some embodiments, L a is -OC(=O), -OC(=O)N-, or -OC(=O)-CH2-CH2-C(=O)N-.

[0077] In some embodiments, n is 1. In some embodiments, n is 2.

[0078] In some embodiments, R 1 is C 1~14 In some embodiments, R 1 is C 1~14 In some embodiments, R is alkenyl. 1 teeth, [ka] is.

[0079] In some embodiments, Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, -C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or -C 1~6 alkyl-(5-6 membered heteroaryl), Here, the C 1~10 Alkyl, 3-8 membered heterocycloalkyl, -C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and -C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; Here, the C 1~10 Alkyl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, OH, -C 1~6 Each is optionally substituted with alkyl-OH, or NH2.

[0080] In some embodiments, the sterol amine has formula A2: [ka] or a salt thereof, wherein: [ka] is a single or double bond, R 1 is C 1~14 Alkyl or C 1~14 is alkenyl; L is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, or -SS-CH2-CH2-C(O)N-; Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2.

[0081] In some embodiments, the sterol amine has the formula A3a: [ka] or a salt thereof, wherein [ka] is a single or double bond; R 2 is H or C 1~6 is alkyl; L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] is the basis of; Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2.

[0082] In some embodiments, the sterol amine has the formula A3a: [ka] or a salt thereof, wherein [ka] is a single or double bond; R 2 is H or C 1~6 is alkyl; L a is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, -SS-CH2-CH2-C(O)N-, or a group represented by the formula (a): [ka] is the basis of; Y 1 is C 1~10Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2 (However, the compound of formula A2a is [ka] [ka] [ka] other than).

[0083] In some embodiments, [ka] is a double bond. In some embodiments, [ka] is a single bond.

[0084] In some embodiments, L a is -OC(=O), -OC(=O)N-, or -OC(=O)-CH2-CH2-C(=O)N-.

[0085] In some embodiments, n is 1. In some embodiments, n is 2.

[0086] In some embodiments, R 2 is H. In some embodiments, R 2 is ethyl.

[0087] In some embodiments, Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, -C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or -C 1~6 alkyl-(5-6 membered heteroaryl), Here, the C 1~10 Alkyl, 3-8 membered heterocycloalkyl, -C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and -C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; Here, the C 1~10 Alkyl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, OH, -C 1~6 Each is optionally substituted with alkyl-OH, or NH2.

[0088] In some embodiments, the sterol amine has formula A3: [ka] or a salt thereof, wherein: [ka] is a single or double bond; R 2 is H or C 1~6 is alkyl; L is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, or -SS-CH2-CH2-C(O)N-; Y 1 is C 1~10 Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2.

[0089] In some embodiments, Y 1 teeth, [ka] [ka] be selected.

[0090] In some embodiments, Y 1 teeth, [ka] [ka] is selected from.

[0091] In some embodiments, the sterol amine has formula A4: [ka] or a salt thereof, wherein: Z 1 is OH or C 3~6 is alkyl; L is absent, -O-, -SS-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -SS-CH2, or -SS-CH2-CH2-C(O)N-; Y 1 is C 1~10Alkyl, 3-8 membered heterocycloalkyl, 5-6 membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), or C 1~6 alkyl-(5-6 membered heteroaryl), wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 alkyl-(5-6 membered heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or a combination thereof; wherein the alkyl, 3- to 8-membered heterocycloalkyl, 5- to 6-membered heteroaryl, C 1~6 Alkyl-(3- to 8-membered heterocycloalkyl), and C 1~6 Alkyl-(5-6 membered heteroaryl) is C 1~6 Alkyl, halo, OH, O(C 1~6 alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 alkyl), N(C 1~6 alkyl) 2, 3- to 8-membered heterocycloalkyl (C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~14 each optionally substituted with one, two, three, or four substituents selected from NH(3- to 8-membered heterocycloalkyl), NH(5- to 6-membered heteroaryl), NH(3- to 8-membered heterocycloalkyl), and NH(5- to 6-membered heteroaryl); and n=1 or 2.

[0092] In some embodiments, Z 1 is OH. In some embodiments, Z 1 is C 3~6 It is alkyl.

[0093] In some embodiments, L is -C(=O)N-, -CH2-NH-C(=O)-, or -C(=O)O-.

[0094] In some embodiments, Y 1C containing 1 to 5 primary, secondary, or tertiary amines, or a combination thereof 1~10 In some embodiments, Y is alkyl. 1 teeth, [ka] is.

[0095] In some embodiments, n is 1. In some embodiments, n is 2.

[0096] In some embodiments, the sterol amine has formula A5: [ka] or a salt thereof, wherein: Z 2 is OH or isopropyl; L 3 is —CH—NH—C(O)—, —C(O)NH—, or —C(O)O—.

[0097] In some embodiments, the sterol amine is [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] or a salt thereof.

[0098] In some embodiments, the sterol amine is [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] is selected from.

[0099] In some embodiments, the sterol amine is [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] or a salt thereof.

[0100] In some embodiments, the sterol amine is [Table 4] or a salt thereof.

[0101] In some embodiments, the sterol amine is SA3: [ka] or a salt thereof, which is also referred to as GL-67. SA3 or GL-67 may be prepared according to processes known in the art or purchased from commercial suppliers such as Avanti® Polar Lipids, Inc. (SKU 890893).

[0102] In some embodiments, the cationic lipid is a modified amino acid, such as modified arginine, in which an amino acid residue having an amine-containing side chain is attached to a hydrophobic group, such as a sterol (e.g., cholesterol or its derivative), a fatty acid, or a similar hydrocarbyl group. At least one amine of the modified amino acid moiety has a pKa of 8.0 or higher. At least one amine of the modified amino acid moiety is positively charged at physiological pH. The amino acid residue may include, but is not limited to, arginine, histidine, lysine, tryptophan, ornithine, and 5-hydroxylysine. The amino acid is linked to the hydrophobic group via a linker.

[0103] In some embodiments, the modified amino acid is a modified arginine.

[0104] In some embodiments, the cationic agent is a non-lipid cationic agent. Examples of non-lipid cationic agents include, for example, benzalkonium chloride, cetylpyridium chloride, L-lysine monohydrate, or tromethamine.

[0105] In some embodiments, the lipid nanoparticles are 2-15%, 3-10%, 4-10%, 5-10%, 6-10%, 2-3%, 2-4%, 2-5%, 2-6%, 2-7%, 2-8%, 3-4%, 3-5%, 3-6%, 3-7%, 3-8%, 4-5%, 4-6%, 4-7%, 4-8%, 5-6%, 5-7%, 5-8%, 6-7%, 6-8%, 2%, 2.5%, 3%, 3.5%, 4%, 4%, 4. The lipid nanoparticles comprise a molar ratio of 0.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10% of a cationic agent (e.g., sterol amine). In some embodiments, the lipid nanoparticles comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, 0.5-15% PEG-modified lipid, and 2-10% of a cationic agent (e.g., sterol amine). In some embodiments, the lipid nanoparticles comprise a molar ratio of 40-60% ionizable cationic lipid, 5-15% non-cationic lipid, 30-50% sterol, 0.5-10% PEG-modified lipid, and 3-7% cationic agent. In some embodiments, the lipid nanoparticles comprise a molar ratio of 45-55% ionizable cationic lipid, 7.5-12.5% ​​non-cationic lipid, 35-45% sterol, 0.5-5% PEG-modified lipid, and 4.5-6% cationic agent. In some embodiments, the cationic agent is GL-67 or a salt thereof.

[0106] Other exemplary embodiments include (compounds that, when used in the tables, refer to ionized amino lipids). [Table 5] [Table 6] [Table 7] [Table 8]

[0107] In some embodiments, the weight ratio of cationic agent to polynucleotide is about 0.1:1 to about 15:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 0.2:1 to about 10:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 10:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 8:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 7:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 6:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 5:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1:1 to about 4:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1.25:1 to about 3.75:1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 1.25: 1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 2.5: 1. In some embodiments, the weight ratio of cationic agent to polynucleotide is about 3.75: 1.

[0108] In some embodiments, the molar ratio of cationic agent to polynucleotide is about 0.1:1 to about 20:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 10:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 9:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 8:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 7:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 6:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1 to about 5:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 1.5:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 2:1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 3: 1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 4: 1. In some embodiments, the molar ratio of cationic agent to polynucleotide is about 5: 1.

[0109] In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 20 mV. In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 20 mV. In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 15 mV. In some embodiments, the nanoparticles have a zeta potential of about 5 mV to about 10 mV.

[0110] In some embodiments, the lipid nanoparticle core has a neutral charge at neutral pH.

[0111] In some embodiments, greater than about 80% of the cationic agent is on the surface of the nanoparticle. In some embodiments, greater than about 90% of the cationic agent is on the surface of the nanoparticle. In some embodiments, greater than about 95% of the cationic agent is on the surface of the nanoparticle.

[0112] The term "lipid," as generally defined herein, refers to a small molecule having hydrophobic or amphiphilic properties. Lipids can be natural or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some cases, the amphiphilic properties of some lipids lead to the formation of liposomes, vesicles, or membranes in aqueous media.

[0113] Ionized lipids As used herein, the term "ionizable lipid" has its ordinary meaning in the art and may refer to a lipid containing one or more charged moieties. In some embodiments, an ionizable lipid may be positively or negatively charged. For example, an ionizable lipid may be positively charged at a relatively low pH, in which case it may be referred to as a "cationic lipid." In certain embodiments, an ionizable lipid molecule may contain an amine group and may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety that carries a formal charge of electrons, such as monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. A charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium groups, guanidine groups, and imidazolium groups. In certain embodiments, the charged moiety comprises an amine group. Examples of negatively charged groups or precursors thereof include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of a charged moiety may, in some cases, be altered by environmental conditions; for example, a change in pH may change the charge of the moiety and / or cause the moiety to become charged or uncharged. In general, the charge density of a molecule may be selected as desired.

[0114] It should be understood that the term "charge" or "charged moiety" does not refer to a "partial negative charge" or a "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meaning in the art. A "partial negative charge" can result when a functional group contains a bond that is polarized such that electron density is attracted to one atom of the bond, resulting in a partial negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this way.

[0115] In some embodiments, the ionizable lipid is an ionizable amino lipid. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0116] In some embodiments, the nanoparticles described herein comprise between about 30 mol% and about 60 mol% ionized lipids, hi some embodiments, the nanoparticles comprise between about 40 mol% and about 50 mol% ionized lipids.

[0117] The lipid nanoparticle compositions of the present invention may include one or more ionizable (e.g., ionizable amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH). Ionizable lipids include 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane, and the like. (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N ,N-Dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), The ionizable lipid may be selected from the non-limiting group consisting of (2Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)), and (2S)2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition, the ionizable lipid may also be a lipid containing a cyclic amine group.

[0118] The ionizable lipid may also be a compound disclosed in International Publication No. WO2017 / 075531A1 (incorporated herein by reference in its entirety). For example, the ionizable amino lipid may be: [ka] and any combination thereof, but are not limited to these.

[0119] The ionizable lipid may also be a compound disclosed in International Publication No. WO2015 / 199952A1 (incorporated herein by reference in its entirety). For example, the ionizable amino lipid may be: [ka] [ka] and any combination thereof, but are not limited to these.

[0120] In one embodiment, the ionizable lipids are those described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, and WO2013086354, U.S. Pat. The ionizable lipid may be selected from, but is not limited to, the ionizable lipids described in U.S. Patent Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, and US20130225836 (the contents of each of which are incorporated herein by reference in their entirety).

[0121] In another embodiment, the ionizable lipid may be selected from, but is not limited to, Formula A as set forth in International Publication No. WO2013116126 or US20130225836 (the contents of each of which are incorporated herein by reference in their entirety). In yet another embodiment, the ionizable lipid may be selected from, but is not limited to, Formula CLI-CLXXIX in International Publication No. WO2008103276, Formula CLI-CLXXIX in U.S. Patent No. 7,893,302, Formula CLI-CLXXXXII in U.S. Patent No. 7,404,969, and Formulas I-VI in U.S. Patent Publication No. US20100036115, and Formula I in U.S. Patent Publication No. US20130123338 (each of which is incorporated herein by reference in its entirety).

[0122] Non-limiting examples of cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylheneicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17 -dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien- 8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z) -N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosane-20-en-10-amine, (15Z)-N,N-dimethyleptacosane-15-en-10-amine, (14Z)-N,N-dimethylnonacosane-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacontriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheneicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16 -dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentyl cyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2- Decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien- 1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine 1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE, (2S)-1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(PENTYLOXY)PROPAN-2-AMINE, (2S)-1-(HEXYLOXY)-3-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE, 1-[(11Z,14Z)-ICOSA-11,14-DIEN-1-YLOXY]-N,N-DIMETHYL-3-(OCTYLOXY)PROPAN-2-AMINE (i) propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2 -amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0123] Additional examples of ionizable lipids include: [ka]

[0124] In one embodiment, the lipid may be a cleavable lipid, such as those described in International Publication No. WO2012170889 (incorporated herein by reference in its entirety). In one embodiment, the lipid may be synthesized by methods known in the art and / or the methods described in International Publication No. WO2013086354 (the contents of each of which are incorporated herein by reference in their entirety). In another embodiment, the lipid may be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids, as well as methods for making and using trialkyl cationic lipids, are described in International Patent Publication No. WO2013126803, the contents of which are incorporated herein by reference in their entirety.

[0125] In some embodiments, the ionizable lipid has formula (I): [ka] or a salt or isomer thereof, wherein: R1 is H, C 5~30 Alkyl, C 5~30 selected from the group consisting of alkenyl, -R*YR", -YR", -(CH2)n(NR4)R"M'R', and -R"M'R'; R2 and R3 are H, C 1~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atom to which they are attached form a heterocycle or carbocycle, which carbocycle is optionally substituted with C6 cycloalkyl or C5 alkyl; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R), -CH(CHQ), and unsubstituted C 1~6 alkyl, wherein the C 3~6 the carbocycle is optionally substituted with -OH or -OMe; Each Q is a carbocycle, a heterocycle, -OR, or -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n OR, -(CH2) nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N independently selected from (OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(O)OR; Or Q is [ka] Selected from; each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; R8 is C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is C 1~3 Alkyl, C 2~3alkenyl, and H, wherein C 1~3 alkyl is optionally substituted with -OH, -C(O)OH, -OMe, -O-benzyl; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR", -YR", and H, wherein C 1~18 The alkyl is optionally substituted with -OMe; Each R" is H, C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0126] In some embodiments, the ionizable lipid has formula (I): [ka] or a salt or isomer thereof, wherein: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are H, C 1~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) nCHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, where Q is a carbocycle, a heterocycle, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N( R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2) n -OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)N(R), -C(O)N(R)OR, and -C(R)N(R)C(O)OR, wherein each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are -C(O)O-, -OC(O)-, -C(O)N(R')-, independently selected from —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —SS—, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; R8 is C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0127] In some embodiments, a subset of compounds of formula (I) includes compounds where R4 is -(CH2) n Q, -(CH2) n When it is CHQR, -CHQR, or -CQ(R), (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R), or (ii) when n is 1 or 2, Q is not a 5-, 6-, or 7-membered heterocycloalkyl.

[0128] In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are H, C 1~14 Alkyl, C 2~14independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, wherein Q is selected from the group consisting of C 3~6 Carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R )S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, --O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and one or more heteroatoms selected from N, O, and S, and oxo (=O), OH, amino, monoalkylamino, or dialkylamino, and C 1~3 5-14 membered heterocycloalkyl substituted with one or more substituents selected from alkyl, where each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; R8, C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof. In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are H, C 1~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, wherein Q is selected from the group consisting of C 3~6 Carbocycle, 5-14 membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N( R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2) n and (i) R4 is -(CH2)N(R)OR, -N(R)C(=NR9)N(R), -N(R)C(=CHR9)N(R), -OC(O)N(R), -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)R, -N(OR)C(O)OR, -N(OR)C(O)N(R), -N(OR)C(S)N(R), -N(OR)C(=NR9)N(R), -N(OR)C(=CHR9)N(R), -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R), where each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5- to 14-membered heterocycle, and (i) R4 is -(CH2) n Q (n is 1 or 2), or (ii) R4 is -(CH2) n or (iii) when R4 is -CHQR and -CQ(R)2, Q is either a 5- to 14-membered heteroaryl or an 8- to 14-membered heterocycloalkyl; Each R5 is C 1~3 Alkyl, C 2~3independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; R8, C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0129] In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are H, C 1~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is C 3~6 Carbocyclic ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 alkyl, wherein Q is selected from the group consisting of C 3~6 Carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R )S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, --O(CH2) n -OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, wherein each n is independently selected from 1, 2, 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; R8, C 3~6 selected from the group consisting of carbocycles and heterocycles; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenyl, C 3~6 selected from the group consisting of carbocycles and heterocycles; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0130] In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are H, C 2~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is -(CH2) n Q or -(CH2) n CHQR, where Q is -N(R)2 and n is selected from 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3 selected from the group consisting of alkenyl, and H; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C1~12 Alkyl and C 1~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0131] In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20 selected from the group consisting of alkenyl, -R*YR", -YR", and -R"M'R'; R2 and R3 are C 1~14 Alkyl, C 2~14 independently selected from the group consisting of alkenyl, -R*YR", -YR", and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocycle or carbocycle; R4 is -(CH2) n Q, -(CH2) n -CHQR, -CHQR, and -CQ(R), where Q is -N(R) and n is selected from 1, 2, 3, 4, and 5; Each R5 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R6 is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, an aryl group, and a heteroaryl group; R7 is C 1~3 Alkyl, C 2~3selected from the group consisting of alkenyl, and H; Each R is C 1~3 Alkyl, C 2~3 independently selected from the group consisting of alkenyl, and H; Each R' is C 1~18 Alkyl, C 2~18 independently selected from the group consisting of alkenyl, -R*YR″, -YR″, and H; Each R” is C 3~14 Alkyl and C 3~14 alkenyl; Each R* is C 1~12 Alkyl and C 1~12 alkenyl; Each Y is independently C 3~6 It is a carbocyclic ring; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; or a salt or isomer thereof.

[0132] In some embodiments, a subset of compounds of formula (I) includes compounds of formula (IA): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M is a bond or M'; R is an unsubstituted C 1~3 Alkyl, or -(CH2) nQ, where Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; R and R are H, C 1~14 Alkyl, and C 2~14 alkenyl.

[0133] In some embodiments, a subset of compounds of formula (I) includes compounds of formula (II): [ka] or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M is a bond or M'; R is an unsubstituted C 1~3 Alkyl, or -(CH2) n Q, where n is 2, 3, or 4, and Q is OH, —NHC(S)N(R), —NHC(O)N(R), —N(R)C(O)R, —N(R)S(O)R, —N(R)R, —NHC(═NR)N(R), —NHC(═CHR)N(R), —OC(O)N(R), —N(R)C(O)OR, heteroaryl, or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —SS—, an aryl group, and a heteroaryl group; R and R are H, C 1~14 Alkyl, and C 2~14 alkenyl.

[0134] In some embodiments, a subset of compounds of formula (I) includes compounds of formula (IIa), (IIb), (IIc), or (IIe): [ka] or a salt or isomer thereof, wherein R4 is as described herein.

[0135] In some embodiments, a subset of compounds of formula (I) includes compounds of formula (IId): [ka] or a salt or isomer thereof, wherein n is 2, 3, or 4; m, R′, R″, and R2 through R6 are as described herein. For example, each of R2 and R3 is C 5~14 Alkyl and C 5~14 alkenyl.

[0136] In some embodiments, the compound of Formula (I) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0137] In a further embodiment, the compound of formula (I) is selected from the group consisting of: [ka]

[0138] In some embodiments, the compound of formula (I) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and salts and isomers thereof.

[0139] In some embodiments, the ionizable lipid is compound 429: [ka] or a salt thereof.

[0140] In some embodiments, the ionizable lipid is compound 18: [ka] or a salt thereof.

[0141] In some embodiments, the lipid nanoparticle composition comprises a lipid component comprising a compound described herein (e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId), or (IIe)).

[0142] In some embodiments, LNPs can be composed of an ionizable lipid containing a central piperazine moiety. Such LNPs can advantageously be composed of an ionizable lipid, a phospholipid, and a PEG-lipid, and can optionally include or lack a structured lipid. In some embodiments, the phospholipid is DSPC or DOP.

[0143] The ionizable lipids comprising a central piperazine moiety described herein can be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and / or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, the lipid compounds disclosed herein have lower immunogenicity compared to reference lipids (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index compared to a corresponding formulation comprising a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.

[0144] The lipid has the formula (III) [ka] or a salt or isomer thereof, wherein Ring A is [ka] and; t is 1 or 2; A1 and A2 are each independently selected from CH or N; Z is CH2 or absent, where when Z is CH2, dashed lines (1) and (2) each represent a single bond; when Z is absent, dashed lines (1) and (2) are both absent; R1, R2, R3, R4, and R5 are C 5~20 Alkyl, C 5~20 independently selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; X 1 , X 2 and X 3 are independently selected from the group consisting of a bond, —CH—, —(CH)—, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH—, —CH—C(O)—, —C(O)O—CH—, —OC(O)—CH—, —CH—C(O)O—, —CH—OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—; Each Y is independently C 3~6 It is a carbocyclic ring; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each R is C 1~3 Alkyl and C 3~6 independently selected from the group consisting of: carbocycle; Each R' is C 1~12 Alkyl, C2~12 independently selected from the group consisting of alkenyl, and H; Each R” is C 3~12 Alkyl and C 3~12 alkenyl; In the formula, ring A is [ka] If i)X 1 , X 2 , and X 3 at least one of is not -CH2-, and / or ii) At least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0145] In some embodiments, the compound is of any of formulas (IIIa1)-(IIIa6): [ka]

[0146] Compounds of any of formula (III) or (IIIa1)-(IIIa6), where applicable, include one or more of the following features:

[0147] In some embodiments, ring A is [ka] is.

[0148] In some embodiments, ring A is [ka] is.

[0149] In some embodiments, ring A is [ka] is.

[0150] In some embodiments, ring A is [ka] is.

[0151] In some embodiments, ring A is [ka] is.

[0152] In some embodiments, ring A is [ka] and the N atom in the ring is X 2 is connected to.

[0153] In some embodiments, Z is CH2.

[0154] In some embodiments, Z is absent.

[0155] In some embodiments, at least one of A1 and A2 is N.

[0156] In some embodiments, each of A1 and A2 is N.

[0157] In some embodiments, each of A1 and A2 is CH.

[0158] In some embodiments, A1 is N and A2 is CH.

[0159] In some embodiments, A1 is CH and A2 is N.

[0160] In some embodiments, X 1 , X 2 , and X 3 At least one of X is not -CH-. For example, in certain embodiments,1 is not -CH-. In some embodiments, X 1 , X 2 , and X 3 At least one of the is —C(O)—.

[0161] In some embodiments, X 2 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, or -CH2-OC(O)-.

[0162] In some embodiments, X 3 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, or -CH2-OC(O)-. In other embodiments, X 3 is -CH2-.

[0163] In some embodiments, X 3 is a bond or -(CH2)2-.

[0164] In some embodiments, R1 and R2 are the same. In certain embodiments, R1, R2, and R3 are the same. In some embodiments, R4 and R5 are the same. In certain embodiments, R1, R2, R3, R4, and R5 are the same.

[0165] In some embodiments, at least one of R, R, R, R, and R is -R"MR'. In some embodiments, R 1、R2、R3、R4、及びR5at most one of R is -R"MR'. For example, at least one of R, R, and R can be -R"MR', and / or at least one of R and R is -R"MR'. In certain embodiments, at least one M is -C(O)O-. In some embodiments, each M is -C(O)O-. In some embodiments, at least one M is -OC(O)-. In some embodiments, each M is -OC(O)-. In some embodiments, at least one M is -OC(O)O-. In some embodiments, each M is -OC(O)O-. In some embodiments, at least one R is C alkyl. In certain embodiments, each R is C alkyl. In some embodiments, at least one R is C alkyl. In certain embodiments, each R is C alkyl. In some embodiments, at least one R is C alkyl. In certain embodiments, each R" is a C6 alkyl. In some embodiments, at least one R" is a C7 alkyl. In certain embodiments, each R" is a C7 alkyl. In some embodiments, at least one R" is a C5 alkyl. In certain embodiments, each R' is a C5 alkyl. In other embodiments, at least one R is a C1 alkyl. In certain embodiments, each R' is a C1 alkyl. In some embodiments, at least one R' is a C2 alkyl. In certain embodiments, each R' is a C2 alkyl.

[0166] In some embodiments, at least one of R1, R2, R3, R4, and R5 is C 12 In certain embodiments, R1, R2, R3, R4, and R5 are each C 12 It is alkyl.

[0167] In certain embodiments, the compound is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0168] In other embodiments, the lipid has the formula (IV): [ka] or a salt or isomer thereof, wherein: A1 and A2 are each independently selected from CH or N, and at least one of A1 and A2 is N; Z is CH2 or absent, where when Z is CH2, dashed lines (1) and (2) each represent a single bond; when Z is absent, dashed lines (1) and (2) are both absent; R1, R2, R3, R4, and R5 are C 6~20 Alkyl and C 6~20 alkenyl; In the formula, ring A is [ka] If i) R1, R2, R3, R4, and R5 are the same (where R1 is C 12 Alkyl, C 18 Alkyl, or C 18 not alkenyl); ii)R 1、R2、R3、R4、及びR5 Only one of the 6~20 selected from alkenyl; iii) at least one of R1, R2, R3, R4, and R5 has a different number of carbon atoms than at least one of the other R1, R2, R3, R4, and R5; iv) R1, R2, and R3 are C 6~20 alkenyl, and R4 and R5 are selected from C 6~20 alkyl; or v) R1, R2, and R3 are C 6~20 alkyl, and R and R are selected from C 6~20 alkenyl.

[0169] In some embodiments, the compound has formula (IVa): [ka] It is of the type.

[0170] The compounds of formula (IV) or (IVa), where applicable, include one or more of the following features:

[0171] In some embodiments, Z is CH2.

[0172] In some embodiments, Z is absent.

[0173] In some embodiments, at least one of A1 and A2 is N.

[0174] In some embodiments, each of A1 and A2 is N.

[0175] In some embodiments, each of A1 and A2 is CH.

[0176] In some embodiments, A1 is N and A2 is CH.

[0177] In some embodiments, A1 is CH and A2 is N.

[0178] In some embodiments, R1, R2, R3, R4, and R5 are the same, and C 12 Alkyl, C 18 Alkyl, or C 18 In some embodiments, R1, R2, R3, R4, and R5 are the same and are not C9 alkyl or C 14 It is alkyl.

[0179] In some embodiments, R 1、R2、R3、R4、及びR5 Only one of the 6~20 In certain such embodiments, R, R, R, R, and R have the same number of carbon atoms. In some embodiments, R is selected from C 5~20 For example, R4 is selected from C 12 Alkenyl or C 18 It may be alkenyl.

[0180] In some embodiments, at least one of R1, R2, R3, R4, and R5 has a different number of carbon atoms than at least one other of R1, R2, R3, R4, and R5.

[0181] In certain embodiments, R1, R2, and R3 are C 6~20 alkenyl, and R and R are selected from C 6~20 In other embodiments, R1, R2, and R3 are selected from C alkyl. 6~20 alkyl, and R and R are selected from C 6~20 In some embodiments, R1, R2, and R3 have the same number of carbon atoms, and / or R4 and R5 have the same number of carbon atoms. For example, R1, R2, and R3, or R4 and R5, can have 6, 8, 9, 12, 14, or 18 carbon atoms. In some embodiments, R1, R2, and R3, or R4 and R5, can be selected from C 18 alkenyl (e.g., linoleyl). In some embodiments, R1, R2, and R3, or R4 and R5, are alkyl groups containing 6, 8, 9, 12, or 14 carbon atoms.

[0182] In some embodiments, R1 has a different number of carbon atoms than R2, R3, R4, and R5. In other embodiments, R3 has a different number of carbon atoms than R1, R2, R4, and R5. In further embodiments, R4 has a different number of carbon atoms than R1, R2, R3, and R5.

[0183] In some embodiments, the compound is selected from the group consisting of: [ka] [ka] [ka]

[0184] In other embodiments, the compound has formula (V): [ka] or a salt or isomer thereof, wherein: A3 is CH or N; A4 is CH2 or NH; at least one of A3 and A4 is N or NH; Z is CH2 or absent, where when Z is CH2, dashed lines (1) and (2) each represent a single bond; when Z is absent, dashed lines (1) and (2) are both absent; R1, R2, and R3 are C 5~20 Alkyl, C 5~20 independently selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, an aryl group, and a heteroaryl group; X 1 and X 2 are independently selected from the group consisting of -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, -CH2-OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-; Each Y is independently C 3~6 It is a carbocyclic ring; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each R is C 1~3 Alkyl and C 3~6 independently selected from the group consisting of: carbocycle; Each R' is C 1~12 Alkyl, C 2~12 independently selected from the group consisting of alkenyl, and H; Each R” is C 3~12 Alkyl and C 3~12 alkenyl.

[0185] In some embodiments, the compound has the formula (Va): [ka] It is of the type.

[0186] The compounds of formula (V) or (Va), where applicable, include one or more of the following features:

[0187] In some embodiments, Z is CH2.

[0188] In some embodiments, Z is absent.

[0189] In some embodiments, at least one of A3 and A4 is N or NH.

[0190] In some embodiments, A3 is N and A4 is NH.

[0191] In some embodiments, A3 is N and A4 is CH2.

[0192] In some embodiments, A3 is CH and A4 is NH.

[0193] In some embodiments, X 1 and X 2 At least one of X is not -CH-. For example, in certain embodiments, 1 is not -CH-. In some embodiments, X 1 and X 2 At least one of the is —C(O)—.

[0194] In some embodiments, X 2 is -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, or -CH2-OC(O)-.

[0195] In some embodiments, R1, R2, and R3 are C 5~20 Alkyl and C 5~20 In some embodiments, R1, R2, and R3 are the same. In certain embodiments, R1, R2, and R3 are independently selected from the group consisting of C6, C9, C 12 , or C 14 In other embodiments, R1, R2, and R3 are C 18 For example, R1, R2, and R3 can be linoleyl.

[0196] In some embodiments, the compound is selected from the group consisting of: [ka]

[0197] In another aspect, the present disclosure provides a compound of formula (VI): [ka] or a salt or isomer thereof, wherein: A6 and A7 are each independently selected from CH or N, wherein at least one of A6 and A7 is N; Z is CH2 or absent, where when Z is CH2, dashed lines (1) and (2) each represent a single bond; when Z is absent, dashed lines (1) and (2) are both absent; X 4 and X 5 are independently selected from the group consisting of -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH2-, -CH2-C(O)-, -C(O)O-CH2-, -OC(O)-CH2-, -CH2-C(O)O-, -CH2-OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-; R1, R2, R3, R4, and R5 are C 5~20Alkyl, C 5~20 independently selected from the group consisting of alkenyl, -R"MR', -R*YR", -YR", and -R*OR"; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)-, an aryl group, and a heteroaryl group; Each Y is independently C 3~6 It is a carbocyclic ring; Each R* is C 1~12 Alkyl and C 2~12 alkenyl; Each R is C 1~3 Alkyl and C 3~6 independently selected from the group consisting of: carbocycle; Each R' is C 1~12 Alkyl, C 2~12 independently selected from the group consisting of alkenyl, and H; Each R” is C 3~12 Alkyl and C 3~12 alkenyl.

[0198] In some embodiments, R1, R2, R3, R4, and R5 are each C 6~20 Alkyl and C 6~20 alkenyl.

[0199] In some embodiments, R1 and R2 are the same. In certain embodiments, R1, R2, and R3 are the same. In some embodiments, R4 and R5 are the same. In certain embodiments, R1, R2, R3, R4, and R5 are the same.

[0200] In some embodiments, at least one of R1, R2, R3, R4, and R5 is C 9~12 In certain embodiments, R1, R2, R3, R4, and R5 are each independently selected from the group consisting of C9, C 12 , or C14 In certain embodiments, R, R, R, R, and R are each C alkyl.

[0201] In some embodiments, A6 is N and A7 is N. In some embodiments, A6 is CH and A7 is N.

[0202] In some embodiments, X4 is -CH2- and X5 is -C(O)-. In some embodiments, X4 and X5 are -C(O)-.

[0203] In some embodiments, when A6 is N and A7 is N, at least one of X4 and X5 is not -CH2-, e.g., at least one of X4 and X5 is -C(O)-. In some embodiments, when A6 is N and A7 is N, at least one of R1, R2, R3, R4, and R5 is -R"MR'.

[0204] In some embodiments, at least one of R1, R2, R3, R4, and R5 is not -R"MR'.

[0205] In some embodiments, the compound is [ka] is.

[0206] In one embodiment, the compound has the formula: [ka]

[0207] PEG and PEG-modified lipids Generally, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein can be synthesized as described in International Patent Application No. PCT / US2016 / 000129, filed December 10, 2016, entitled "Compositions and Methods for Delivery of Therapeutic Agents," which is incorporated by reference in its entirety.

[0208] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, e.g., PEG, or a PEG-modified lipid. Such species may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. The PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid. In some embodiments, the PEG lipid is DMG-PEG 2k or compound 428.

[0209] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. PEG-DMG has the following structure: [ka]

[0210] In some embodiments, the nanoparticles described herein comprise between about 1 mol% and about 5 mol% PEG-lipid. In some embodiments, the nanoparticles comprise between about 1 mol% and about 2.5 mol% PEG-lipid.

[0211] In one embodiment, the PEG lipid useful in the present invention may be a PEGylated lipid described in International Publication No. WO2012099755 (the contents of which are incorporated herein by reference in their entirety). Any of these exemplary PEG lipids described herein may be modified to include hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a "PEG-OH lipid" (also referred to herein as a "hydroxy-PEGylated lipid") is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid contains one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG-OH lipid, i.e., the hydroxy-PEGylated lipid, contains an -OH group at the end of its PEG chain. Each possibility represents a separate embodiment of the present invention.

[0212] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VII): [ka] or a salt thereof, wherein: R 3 -OR O and; R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; L 1 is an optionally substituted C 1~10 alkylene, wherein the optionally substituted C 1~10 At least one methylene of the alkylene is independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NRN C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- has been replaced by; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] is of; L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6 One methylene unit of the alkylene is optionally —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- has been replaced by; R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl; optionally, where R 2 one or more methylene units in the formula (I) are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N)-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by; R N each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and p is 1 or 2.

[0213] In certain embodiments, the compound of formula (VII) is a PEG-OH lipid (i.e., R 3 HA-OR O and R O is hydrogen). In certain embodiments, the compound of formula (VII) has the formula (VII-OH): [ka] or a salt thereof.

[0214] In certain embodiments, D is a moiety obtained by click chemistry (e.g., a triazole). In certain embodiments, the compound of formula (VII) has formula (VII-a-1) or formula (VII-a-2): [ka] or a salt thereof.

[0215] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof, wherein s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0216] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0217] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0218] In certain embodiments, the compound of formula (VII) has the following formula: [ka] (wherein r is 1 to 100), or a salt thereof.

[0219] In certain embodiments, D is a moiety that is cleavable under physiological conditions (e.g., an ester, an amide, a carbonate, a carbamate, a urea). In certain embodiments, the compound of formula (VII) has formula (VII-b-1) or (VII-b-2): [ka] or a salt thereof.

[0220] In certain embodiments, the compound of formula (VII) has formula (VII-b-1-OH) or (VII-b-2-OH): [ka] or a salt thereof.

[0221] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0222] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0223] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0224] In certain embodiments, the compound of formula (VII) has the following formula: [ka] or a salt thereof.

[0225] In certain embodiments, the PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VIII). As used herein, the formula (VIII): [ka] or a salt thereof, wherein: R 3 -OR O and; R O is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive; R 5 is an optionally substituted C 10~40 Alkyl, optionally substituted C 10~40 alkenyl, or optionally substituted C 10~40 alkynyl; optionally, R 5 one or more methylene groups in the formula (I) are optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR NC(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-; and R N Each instance of is independently hydrogen, an optionally substituted alkyl, or a nitrogen protecting group.

[0226] In certain embodiments, the compound of formula (VIII) has the formula (VIII-OH): [ka] or a salt thereof.

[0227] In certain embodiments, the compound of formula (VIII) has the following formula: [ka] In some embodiments, r is 45, or a salt thereof.

[0228] In certain embodiments, the compound of formula (VIII) has the following formula: [ka] In some embodiments, r is 45, or a salt thereof.

[0229] In yet another embodiment, the compound of formula (VIII) is [ka] or a salt thereof.

[0230] In some embodiments, the compound of formula (VIII) is [ka] is.

[0231] In certain embodiments, the PEG lipid has the following formula: [ka] In some embodiments, r is 45, or a salt thereof.

[0232] phospholipids As defined herein, a phospholipid is any lipid containing a phosphate group. Phospholipids are a subset of non-cationic lipids. The lipid component of a lipid nanoparticle composition may comprise one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be organized into one or more lipid bilayers. Generally, a phospholipid may comprise a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-naturally occurring species, including naturally occurring species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids can be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, the alkyne group can undergo copper-catalyzed cycloaddition upon exposure to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cellular recognition, or for conjugating nanoparticle compositions to useful components such as targeting or imaging moieties (e.g., dyes).

[0233] In some embodiments, the nanoparticles described herein comprise between about 5 mol% and about 15 mol% phospholipids. In some embodiments, the nanoparticles comprise between about 8 mol% and about 13 mol% phospholipids. In some embodiments, the nanoparticles comprise between about 10 mol% and about 12 mol% phospholipids.

[0234] Phospholipids that are useful or potentially useful in the present compositions and methods include: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and The glycerol may be selected from the non-limiting group consisting of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment of the present invention.

[0235] In some embodiments, the lipid nanoparticle composition comprises DSPC. In certain embodiments, the lipid nanoparticle composition comprises DOPE. In some embodiments, the lipid nanoparticle composition comprises both DSPC and DOPE. In some embodiments, the lipid nanoparticles 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (4ME 16:0 PE) [ka] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC) [ka] 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), [ka] 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS) [ka] or a mixture thereof.

[0236] Examples of phospholipids include, but are not limited to: [ka] [ka]

[0237] In certain embodiments, phospholipids useful or potentially useful in the present invention are analogs or variants of DSPC.

[0238] In certain embodiments, phospholipids useful or potentially useful in the present invention have the formula (IX): [ka] or a salt thereof, wherein: Each R 1 are independently H or optionally substituted alkyl; or optionally, two R 1 are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] is of; L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6One methylene unit of the alkylene is optionally —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- has been replaced by; R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl; optionally, where R 2 one or more methylene units in the formula (I) are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by; R N each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and p is 1 or 2 (However, the compound has the formula: [ka] (In the formula, R 2 Each instance of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl).

[0239] In certain embodiments, phospholipids useful or potentially useful in the present invention have the formula (IX): [ka] or a salt thereof, wherein: Each R 1 are independently optionally substituted alkyl; or optionally, two R 1are joined together with the intervening atoms to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 are joined together with the intervening atoms to form an optionally substituted bicyclic carbocyclyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is a group having the formula: [ka] It is of L 2 Each instance of is independently a bond or an optionally substituted C 1~6 alkylene, wherein the optionally substituted C 1~6 One methylene unit of the alkylene is optionally —O—, —N(R N )-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, or -NR N C(O)N(R N )- has been replaced by; R 2 Each instance of is independently an optionally substituted C 1~30 Alkyl, optionally substituted C 1~30 alkenyl, or optionally substituted C 1~30 alkynyl; optionally, where R 2 one or more methylene units in the formula (I) are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N)-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by; R N each instance of is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and p is 1 or 2 (However, the compound has the formula: [ka] (In the formula, R 2 Each instance of is independently an unsubstituted alkyl, an unsubstituted alkenyl, or an unsubstituted alkynyl).

[0240] Modification of phospholipid head groups In certain embodiments, phospholipids useful or potentially useful in the present invention contain a modified phospholipid head group (e.g., a modified choline group). In certain embodiments, the phospholipid with a modified head group is DSPC or an analog thereof with a modified quaternary amine. For example, in embodiments of formula (IX), R 1 At least one of R is not methyl. 1 In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof, wherein each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each v is independently 1, 2, or 3. In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof.

[0241] In certain embodiments, the compound of formula (IX) has the following formula: [ka] [ka] or a salt thereof.

[0242] In certain embodiments, the compound of formula (IX) has formula (IX-a): [ka] or a salt thereof.

[0243] In certain embodiments, the phospholipid that is useful or potentially useful in the present invention comprises modified core.In certain embodiments, the phospholipid that has modified core described herein is DSPC or its analog with modified core structure.For example, in certain embodiments of formula (IX-a), group A is the following formula: [ka] It is not one of them.

[0244] In certain embodiments, the compound of formula (IX-a) has the following formula: [ka] or a salt thereof.

[0245] In certain embodiments, the compound of formula (IX) has the following formula: [ka] or a salt thereof.

[0246] In certain embodiments, the phospholipid that is useful or potentially useful in the present invention comprises a cyclic moiety instead of a glyceride moiety.In certain embodiments, the phospholipid that is useful in the present invention is DSPC or its analogue, which has a cyclic moiety instead of a glyceride moiety.In certain embodiments, the compound of formula (IX) is represented by formula (IX-b): [ka] or a salt thereof.

[0247] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-1): [ka] or a salt thereof, wherein: w is 0, 1, 2, or 3.

[0248] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-2): [ka] or a salt thereof.

[0249] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-3): [ka] or a salt thereof.

[0250] In certain embodiments, the compound of formula (IX-b) has the formula (IX-b-4): [ka] or a salt thereof.

[0251] In certain embodiments, the compound of formula (IX-b) has the following formula: [ka] or a salt thereof.

[0252] Modification of phospholipid tails In certain embodiments, the phospholipids useful or potentially useful in the present invention comprise modified tails.In certain embodiments, the phospholipids useful or potentially useful in the present invention are DSPC or its analogs with modified tails.As described herein, "modified tails" can be tails with relatively short or long aliphatic chains, branched aliphatic chains, substituted aliphatic chains, aliphatic chains in which one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof.For example, in certain embodiments, the compound of (IX) is of formula (IX-a) or a salt thereof, wherein R 2 In at least one example of R 2 Each example of 1~30 alkyl, where R 2 one or more methylene units in the formula (I) are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NR N )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N)-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O- is replaced by

[0253] In certain embodiments, the compound of formula (IX) has formula (IX-c): [ka] or a salt thereof, wherein: each x is independently an integer between 0 and 30, inclusive; In each example, G is an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, -N(R N )-, -O-, -S-, -C(O)-, -C(O)N(R N )-, -NR N C(O)-, -NR N C(O)N(R N )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R N )-, -NR N C(O)O-, -C(O)S-, -SC(O)-, -C(=NR N )-, -C(=NR N )N(R N )-, -NR N C(=NR N )-, -NR N C(=NRN )N(R N )-, -C(S)-, -C(S)N(R N )-, -NR N C(S)-, -NR N C(S)N(R N )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R N )S(O)-, -S(O)N(R N )-, -N(R N )S(O)N(R N )-, -OS(O)N(R N )-, -N(R N )S(O)O-, -S(O)2-, -N(R N )S(O)2-, -S(O)2N(R N )-, -N(R N )S(O)2N(R N )-, -OS(O)2N(R N )-, or -N(R N )S(O)2O-. Each possibility represents a separate embodiment of the present invention.

[0254] In certain embodiments, the compound of formula (IX-c) has the formula (IX-c-1): [ka] or a salt thereof, wherein: Each instance of v is independently 1, 2, or 3.

[0255] In certain embodiments, the compound of formula (IX-c) has the formula (IX-c-2): [ka] or a salt thereof.

[0256] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0257] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0258] In certain embodiments, the compound of formula (IX-c) has formula (IX-c-3): [ka] or a salt thereof.

[0259] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0260] In certain embodiments, the compound of formula (IX-c) has the following formula: [ka] or a salt thereof.

[0261] In certain embodiments, phospholipids useful or potentially useful in the present invention comprise a modified phosphocholine moiety, wherein the alkyl chain connecting the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Thus, in certain embodiments, phospholipids useful or potentially useful in the present invention are compounds of formula (IX), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, compounds of formula (IX) have the following formula: [ka] or a salt thereof.

[0262] In certain embodiments, the compound of formula (IX) has the following formula: [ka] [ka] or a salt thereof.

[0263] alternative lipids In certain embodiments, alternative lipids are used in place of the phospholipids of the present invention. Non-limiting examples of such alternative lipids include those having the following formula: [ka] [ka] Examples include:

[0264] structural lipids The lipid component of the lipid nanoparticle composition may include one or more structured lipids. Introducing a structured lipid into the lipid nanoparticle may help reduce aggregation of other lipids within the particle. The structured lipid may be selected from the group including, but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structured lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structured lipid is a steroid. In certain embodiments, the structured lipid is cholesterol. In certain embodiments, the structured lipid is a cholesterol analog. In certain embodiments, the structured lipid is alpha-tocopherol. Examples of structured lipids include, but are not limited to: [ka]

[0265] In some embodiments, the nanoparticles described herein may comprise about 20 mol% to about 60 mol% structured lipid. In some embodiments, the nanoparticles comprise about 30 mol% to about 50 mol% structured lipid. In some embodiments, the nanoparticles comprise about 35 mol% structured lipid. In some embodiments, the nanoparticles comprise about 40 mol% structured lipid. In some embodiments, the structured lipid is cholesterol or the following structure: [ka] It is a compound having the formula:

[0266] Molar ratio of lipid nanoparticle components In some embodiments, the payload is formulated with a delivery agent including, for example, a compound having Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; a compound having Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428; or a compound having Formula A1, A2, A3, A4, or A5, e.g., any one of SA1-SA41, or any combination thereof. In some embodiments, the delivery agent includes Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6±25:9.5±8:36.6±20:1.4±1.25:4.9±2.5. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6±12.5:9.5±4:36.6±10:1.4±0.75:4.9±1.25. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 47.6±6.25:9.5±2:36.6±5:1.4±0.375:4.9±0.625. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6:9.5:36.6:1.4:4.9. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6±25:9.5±8:36.6±20:1.4±1.25:4.9±2.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6±12.5:9.5±4:36.6±10:1.4±0.75:4.9±1.25.In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 47.6±6.25:9.5±2:36.6±5:1.4±0.375:4.9±0.625. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.6:9.5:36.6:1.4:4.9. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3±25:9.5±8:36.4±20:1.4±1.25:5.5±2.5. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3±12.5:9.5±4:36.4±10:1.4±0.75:5.5±1.25. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 47.3±6.25:9.5±2:36.4±5:1.4±0.375:5.5±0.625. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3:9.5:36.4:1.4:5.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3±25:9.5±8:36.4±20:1.4±1.25:5.5±2.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3±12.5:9.5±4:36.4±10:1.4±0.75:5.5±1.25.In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 47.3±6.25:9.5±2:36.4±5:1.4±0.375:5.5±0.625. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 47.3:9.5:36.4:1.4:5.5. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8±25:10.5±8:36.8±20:1.4±1.25:5.5±2.5. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8±12.5:10.5±4:36.8±10:1.4±0.75:5.5±1.25. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 45.8±6.25:10.5±2:36.8±5:1.4±0.375:5.5±0.625. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8:10.5:36.8:1.4:5.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8±25:10.5±8:36.8±20:1.4±1.25:5.5±2.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8±12.5:10.5±4:36.8±10:1.4±0.75:5.5±1.25.In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 45.8±6.25:10.5±2:36.8±5:1.4±0.375:5.5±0.625. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, e.g., in a molar ratio of about 45.8:10.5:36.8:1.4:5.5. In some embodiments, the delivery agent comprises Compound 18 or 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, in a concentration of, for example, about 30 to about 60 mol % Compound 18 or 236 (or a related suitable amino lipid) (e.g., 30 to 40, 40 to 45, 45 to 50, 50 to 55, or 55 to 60 mol % Compound 18 or 236 (or a related suitable amino lipid)), about 5 to about 20 mol % phospholipid (or a related suitable phospholipid or "helper lipid") (e.g., 5 to 10, 10 to 15, or 15 to 20 mol % phospholipid) (or a related suitable phospholipid or "helper lipid"), about 20 to about 50 mol % cholesterol (or related sterol or "non-cationic" lipid) (e.g., about 20-30, 30-35, 35-40, 40-45, or 45-50 mol % cholesterol) (or related sterol or "non-cationic" lipid)), about 0.05 to about 10 mol % PEG lipid (or other suitable PEG lipid) (e.g., 0.05-1, 1-2, 2-3, 3-4, 4-5, 5-7, or 7-10 mol % PEG lipid) (or other suitable PEG lipid)), and about 1 to about 10 mol % GL-67 or a salt thereof (e.g., 1-3, 3-5, 5-7, 7-10, 3-8, 3.5-6.5 mol % GL-67 or a salt thereof). Exemplary delivery agents may include, for example, molar ratios of 47.6:9.5:36.6:1.4:4.9, 47.3:9.5:36.4:1.4:5.5, or 45.8:10.5:36.8:1.4:5.5.In certain examples, exemplary delivery agents may include, for example, molar ratios of 48:9.5:35.5:1.5:5.5, 47:10:36:1.5:5.5, 46:10.5:36.5:1.5:5.5, 45:10.5:37.5:1.5:5.5, 48:9.5:36:1.5:5, 47:10:36.5:1.5:5, 46:10.5:37:1.5:5, or 45:10.5:38:1.5:5. In some embodiments, the delivery agent includes Compound 18 or 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, for example, in a molar ratio of about 47.6:9.5:36.6:1.4:4.9. In some embodiments, the delivery agent comprises Compound 18 or 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, for example, in a molar ratio of about 47.3:9.5:36.4:1.4:5.5. In some embodiments, the delivery agent comprises Compound 18 or 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, for example, in a molar ratio of about 45.8:10.5:36.8:1.4:5.5.

[0267] In some embodiments, a payload disclosed herein is formulated with a delivery agent comprising, for example, a compound having Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a compound having Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any combination thereof. In some embodiments, the delivery agent comprises Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 49.5±3:10.5±2:39±3:1±0.75. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 49.5±3:10.5±2:39±3:1±0.75. In some embodiments, the delivery agent comprises about 48-52 mol % Compound 18 or 236 (or a related suitable amino lipid) (e.g., 48-51, 48-50, 49-52, or 49-51 mol % Compound 18 or 236 (or a related suitable amino lipid)), about 9-12 mol % phospholipid (or a related suitable phospholipid or "helper lipid") (e.g., 9-11, 9-10, 10-12, 10-11.5, 10-11 mol % phospholipid (or a related suitable phospholipid or "helper lipid"). % cholesterol (or related sterol or "non-cationic" lipid) (e.g., about 36-41, 36-40, 37-40, or 38-40 mol % cholesterol (or related sterol or "non-cationic" lipid)), and about 0.25-2.5 mol % PEG lipid (or other suitable PEG lipid) (e.g., 0.25-2, 0.25-1.5, 0.25-2, or 0.5-1.5 mol % PEG lipid (or other suitable PEG lipid)).

[0268] In some embodiments, a payload disclosed herein is formulated with a delivery agent including, for example, a compound having Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; a compound having Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428; or a compound having Formula A1, A2, A3, A4, or A5, e.g., any one of SA1-SA41, or any combination thereof. In some embodiments, the delivery agent includes Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG in a molar ratio of, for example, about 46.5±3:10±2:36±3:1.25±0.75:4.5±1.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, for example, in a molar ratio of about 46.5±3:10±2:36±3:1.25±0.75:4.5±1.5.In some embodiments, the delivery agent comprises about 43-49 mol% Compound 18 or 236 (or a related suitable amino lipid) (e.g., 43-48, 44-48, 45-48, or 45.5-48 mol% Compound 18 or 236 (or a related suitable amino lipid)), about 8-12 mol% phospholipid (or a related suitable phospholipid or "helper lipid") (e.g., 8-11, 8-10, 9-12, 9-11, 9.5-10.5 mol% phospholipid (or a related suitable phospholipid or "helper lipid")), about 33-39 mol% cholesterol (or a related suitable cholesterol). % cholesterol (or related sterol or "non-cationic" lipid)) (e.g., about 33-38, 34-38, 35-38, or 36-37 mol % cholesterol (or related sterol or "non-cationic" lipid)), about 0.5-2 mol % PEG lipid (or other suitable PEG lipid) (e.g., 0.5-1.5, 0.75-1.5, or 1-1.5 mol % PEG lipid (or other suitable PEG lipid)), and about 3-6 mol % cationic agent (e.g., sterol amine) (e.g., 3-5, 3-4.5, 4-6, or 5-6 mol % cationic agent (e.g., sterol amine)).

[0269] In some embodiments, a payload disclosed herein is formulated with a delivery agent including, for example, a compound having Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; a compound having Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428; or a compound having Formula A1, A2, A3, A4, or A5, e.g., any one of SA1-SA41, or any combination thereof. In some embodiments, the delivery agent includes Compound 18, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 47±3:10±2:36±3:1.25±0.75:4.5±1.5. In some embodiments, the delivery agent comprises Compound 236, DSPC, cholesterol, and Compound 428 or PEG-DMG, e.g., in a molar ratio of about 46.5±3:10±2:36±3:1.25±0.75:4.5±1.5. In some embodiments, the delivery agent comprises about 43-49 mol% Compound 18 or 236 (or a related suitable amino lipid) (e.g., 43-48, 44-48, 45-48, or 45.5-48 mol% Compound 18 or 236 (or a related suitable amino lipid)), about 8-12 mol% phospholipid (or a related suitable phospholipid or "helper lipid") (e.g., 8-11, 8-10, 9-12, 9-11, 9.5-10.5 mol% phospholipid (or a related suitable phospholipid or "helper lipid")), about 33-39 mol% cholesterol (or a related sterol). % cholesterol (or related sterol or "non-cationic" lipid)) (e.g., about 33-38, 34-38, 35-38, or 36-37 mol % cholesterol (or related sterol or "non-cationic" lipid)), about 0.5-2 mol % PEG lipid (or other suitable PEG lipid) (e.g., 0.5-1.5, 0.75-1.5, or 1-1.5 mol % PEG lipid (or other suitable PEG lipid)), and about 3-6 mol % cationic agent (e.g., sterol amine) (e.g., 3-5, 3-4.5, 4-6, or 5-6 mol % cationic agent (e.g., sterol amine)).In some embodiments, the delivery agent comprises compound 18, DSPC, cholesterol, DMG-PEG-2k, and GL-67. In further embodiments, the delivery agent comprises about 45-48 mol% compound 18, about 9-11 mol% DSPC, about 35-38 mol% cholesterol, about 1-3 mol% DMG-PEG-2k, and about 4-6 mol% GL-67. In further embodiments, the delivery agent comprises about 45-48 mol% compound 18, about 9-11 mol% DSPC, about 35-38 mol% cholesterol, about 1-3 mol% DMG-PEG-2k, and about 4-6 mol% GL-67. In a further embodiment, the delivery agent comprises about 45.8-47.6 mol% Compound 18, about 9.5-10.5 mol% DSPC, about 36.4-36.8 mol% cholesterol, about 1.4 mol% DMG-PEG-2k, and about 4.9-5.5 mol% GL-67.

[0270] Unless otherwise specified, the molar ratios / percentages described herein refer to the composition for delivery and not to the cargo (e.g., nucleic acid therapeutic, e.g., polynucleotide, e.g., mRNA).

[0271] Payload molecules The compositions of the present disclosure can be used to deliver a wide variety of different agents to airway cells. Airway cells can be, for example, cells lining the respiratory passages in the oral cavity, nose, pharynx, or lungs. Therapeutic agents can mediate a therapeutic effect in such airway cells (e.g., directly or via a bystander effect). Typically, the therapeutic agents delivered by the compositions are nucleic acids, although non-nucleic acid agents, such as small molecules, chemotherapeutic agents, peptides, polypeptides, and other biomolecules, are also encompassed by the present disclosure. Nucleic acids that can be delivered include DNA-based molecules (i.e., containing deoxyribonucleotides) and RNA-based molecules (i.e., containing ribonucleotides). Furthermore, the nucleic acids can be naturally occurring forms of the molecules or chemically modified forms of the molecules (e.g., containing one or more modified nucleotides).

[0272] Agents for enhancing protein expression In one embodiment, the therapeutic agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to increase protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors).

[0273] In one embodiment, the therapeutic agent is a DNA therapeutic agent.The DNA molecule can be double-stranded DNA, single-stranded DNA (ssDNA), or a partially double-stranded DNA molecule, i.e., a molecule having a double-stranded portion and a single-stranded portion.In some cases, the DNA molecule is triple-stranded or partially triple-stranded, i.e., has a triple-stranded portion and a double-stranded portion.The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

[0274] A DNA therapeutic agent can be a DNA molecule capable of introducing a gene into a cell, e.g., a DNA molecule capable of encoding a transcript and allowing its expression. For example, a DNA therapeutic agent can encode a protein of interest, thereby increasing the expression of the protein of interest in the airway upon delivery by LNP. In some embodiments, the DNA molecule can be naturally derived, e.g., isolated from a natural source. In other embodiments, the DNA molecule is a synthetic molecule, e.g., a synthetic DNA molecule generated in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutic agents include plasmid expression vectors and viral expression vectors.

[0275] The DNA therapeutic agents described herein, for example, DNA vectors, may have a variety of different characteristics. The DNA therapeutic agents described herein, for example, DNA vectors, may contain non-coding DNA sequences. For example, the DNA sequence may contain at least one regulatory element of a gene, such as a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, etc. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, the DNA sequence described herein may have a non-coding DNA sequence operably linked to a transcriptionally active gene. In other embodiments, the DNA sequence described herein may have a non-coding DNA sequence that is not linked to a gene. That is, the non-coding DNA does not control the gene on the DNA sequence.

[0276] In some embodiments, the payload comprises at least one component of a gene regulator, i.e., a system that modifies the nucleic acid sequence of a DNA molecule, e.g., by altering a nucleic acid base, e.g., by introducing an insertion, deletion, mutation (e.g., a missense, silent, or nonsense mutation), duplication, or inversion, or any combination thereof. In some embodiments, the gene regulator comprises a DNA base editor, a CRISPR / Cas gene editing system, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, a meganuclease system, or a transposase system, or any combination thereof.

[0277] In some embodiments, the gene regulatory element comprises a template DNA. In some embodiments, the gene regulatory element does not comprise a template DNA. In some embodiments, the gene regulatory element comprises a template RNA. In some embodiments, the gene regulatory element does not comprise a template RNA.

[0278] In some embodiments, the gene regulator is a CRISPR / Cas gene editing system. In some embodiments, the CRISPR / Cas gene editing system includes a guide RNA (gRNA) molecule comprising a target sequence specific to the sequence of the target gene, and a peptide having nuclease activity (e.g., endonuclease activity) (e.g., a Cas protein or a fragment (e.g., a biologically active fragment) or variant thereof, such as a Cas9 protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas3 protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas12a protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas12e protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas13 protein, a fragment (e.g., a biologically active fragment) or variant thereof; or a Cas14 protein, a fragment (e.g., a biologically active fragment) or variant thereof).

[0279] In some embodiments, the CRISPR / Cas gene editing system comprises a gRNA molecule comprising a target sequence specific to the sequence of a target gene and a nucleic acid encoding a peptide having nuclease activity (e.g., endonuclease activity) (e.g., a Cas protein, or a fragment (e.g., a biologically active fragment) or variant thereof, such as a Cas9 protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas3 protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas12a protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas12e protein, a fragment (e.g., a biologically active fragment) or variant thereof; a Cas13 protein, a fragment (e.g., a biologically active fragment) or variant thereof; or a Cas14 protein, a fragment (e.g., a biologically active fragment) or variant thereof).

[0280] In some embodiments, the CRISPR / Cas gene editing system includes a nucleic acid encoding a gRNA molecule comprising a target sequence specific to the sequence of a target gene, and a Cas9 protein, a fragment (e.g., a biologically active fragment), or a variant thereof.

[0281] In some embodiments, the CRISPR / Cas gene editing system includes a nucleic acid encoding a gRNA molecule comprising a target sequence specific to the sequence of the target gene, and a nucleic acid encoding a Cas9 protein, a fragment (e.g., a biologically active fragment), or a variant thereof.

[0282] In some embodiments, the CRISPR / Cas gene editing system further comprises a DNA template. In some embodiments, the CRISPR / Cas gene editing system further comprises an RNA template. In some embodiments, the CRISPR / Cas gene editing system further comprises a reverse transcriptase.

[0283] In some embodiments of any of the methods, compositions, or cells disclosed herein, the gene regulator is a zinc finger nuclease (ZFN) system. In some embodiments, the ZFN system comprises a peptide having a zinc finger DNA-binding domain, a fragment (e.g., a biologically active fragment) or variant thereof; and / or having nuclease activity, e.g., endonuclease activity. In some embodiments, the ZFN system comprises a peptide having a zinc finger DNA-binding domain. In some embodiments, the zinc finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, or more zinc fingers. In some embodiments, the ZFN system comprises a peptide having nuclease activity, e.g., endonuclease activity. In some embodiments, the peptide having nuclease activity is a type II restriction 1-like endonuclease, e.g., FokI endonuclease. In some embodiments, the ZFN system comprises a nucleic acid encoding a peptide having a zinc finger DNA-binding domain, a fragment (e.g., a biologically active fragment) or variant thereof; and / or having nuclease activity, e.g., endonuclease activity.

[0284] In some embodiments, the ZFN system comprises a nucleic acid encoding a peptide having a zinc finger DNA binding domain. In some embodiments, the zinc finger binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, or more zinc fingers. In some embodiments, the ZFN system comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In some embodiments, the peptide having nuclease activity is a type II restriction 1-like endonuclease, e.g., FokI endonuclease.

[0285] In some embodiments, the system further comprises a template, eg, template DNA.

[0286] In some embodiments of any of the methods, compositions, or cells disclosed herein, the gene regulatory element is a transcription activator-like effector nuclease (TALEN) system. In some embodiments, the system comprises a peptide having a transcription activator-like (TAL) effector DNA-binding domain, a fragment (e.g., a biologically active fragment) or a variant thereof; and / or having nuclease activity, e.g., endonuclease activity. In some embodiments, the system comprises a peptide having a TAL effector DNA-binding domain, a fragment (e.g., a biologically active fragment) or a variant thereof. In some embodiments, the system comprises a peptide having nuclease activity, e.g., endonuclease activity. In some embodiments, the peptide having nuclease activity is a type II restriction 1-like endonuclease, e.g., FokI endonuclease.

[0287] In some embodiments, the system comprises a nucleic acid encoding a peptide having a transcription activator-like (TAL) effector DNA-binding domain, a fragment (e.g., a biologically active fragment) or variant thereof; and / or having nuclease activity, e.g., endonuclease activity. In some embodiments, the system comprises a nucleic acid encoding a peptide having a transcription activator-like (TAL) effector DNA-binding domain, a fragment (e.g., a biologically active fragment) or variant thereof. In some embodiments, the system comprises a nucleic acid encoding a peptide having nuclease activity, e.g., endonuclease activity. In some embodiments, the peptide having nuclease activity is a Type II restriction 1-like endonuclease, e.g., FokI endonuclease.

[0288] In some embodiments, the system further comprises a template, eg, template DNA.

[0289] In some embodiments of any of the methods, compositions, or cells disclosed herein, the gene regulator is a meganuclease system. In some embodiments, the meganuclease system comprises a peptide having a DNA binding domain and nuclease activity, such as a homing endonuclease. In some embodiments, the homing endonuclease comprises a LAGLIDADG endonuclease, a GIY-YIG endonuclease, a HNH endonuclease, a His-Cys box endonuclease, or a PD-(D / E)XK endonuclease, or a fragment (e.g., a biologically active fragment) or variant thereof, as described, for example, in Silva G. et al., (2011) Curr Gene Therapy 11(1):11-27.

[0290] In some embodiments, the meganuclease system comprises a nucleic acid encoding a peptide having a DNA-binding domain and nuclease activity (e.g., a homing endonuclease). In some embodiments, the homing endonuclease comprises a LAGLIDADG endonuclease, a GIY-YIG endonuclease, a HNH endonuclease, a His-Cys box endonuclease, or a PD-(D / E)XK endonuclease, or a fragment (e.g., a biologically active fragment) or variant thereof, e.g., as described in Silva G. et al., (2011) Curr Gene Therapy 11(1):11-27.

[0291] In some embodiments, the system further comprises a template, eg, template DNA.

[0292] In some embodiments of any of the methods, compositions, or cells disclosed herein, the gene regulatory element is a transposase system. In some embodiments, the transposase system comprises a nucleic acid sequence (e.g., a retrotransposon, e.g., an LTR retrotransposon or a non-LTR retrotransposon) encoding a peptide with reverse transcriptase activity and / or nuclease activity. In some embodiments, the transposase system includes a template, for example, an RNA template.

[0293] In one embodiment, the therapeutic agent is an RNA therapeutic agent.The RNA molecule can be single-stranded RNA, double-stranded RNA (dsRNA), or a partially double-stranded RNA molecule, that is, a molecule that has a double-stranded portion and a single-stranded portion.The RNA molecule can be a circular RNA molecule or a linear RNA molecule.

[0294] The RNA therapeutic agent can be an RNA therapeutic agent capable of introducing a gene into cells, for example, encoding a protein of interest, thereby increasing the expression of the protein of interest in airway cells. In some embodiments, the RNA molecule can be naturally derived, for example, isolated from a natural source. In other embodiments, the RNA molecule is a synthetic molecule, for example, a synthetic RNA molecule produced in vitro.

[0295] Non-limiting examples of RNA therapeutics include messenger RNA (mRNA) (e.g., encoding a protein of interest), modified mRNA (mmRNA), mRNA incorporating microRNA-binding site(s) (miR-binding site(s)), modified RNA containing functional RNA elements, microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers and Dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acids (LNA), and those encoding components of CRISPR / Cas9 technology, each of which is further described in the following subsections. In some embodiments, the RNA regulatory element comprises an RNA base editing system. In some embodiments, the RNA base editing system comprises a deaminase, e.g., RNA-specific adenosine deaminase (ADAR); a Cas protein, a fragment (e.g., a biologically active fragment) or variant thereof; and / or a guide RNA. In some embodiments, the RNA base editing system further comprises a template, e.g., a DNA template or an RNA template.

[0296] mRNA may be naturally occurring or non-naturally occurring mRNA. As described below, mRNA may contain one or more modified nucleic acid bases, modified nucleosides, or modified nucleotides, in which case mRNA may be referred to as "modified mRNA" or "mmRNA". As described herein, "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or its derivative and an organic base (e.g., purine or pyrimidine) or its derivative (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside containing a phosphate group.

[0297] An mRNA can include a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). An mRNA can include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900), or thousands (e.g., 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000) of base pairs. Any number (e.g., all, some, or none) of the nucleic acid bases, nucleosides, or nucleotides can be substituted, modified, or otherwise non-naturally occurring analogs of standard species. In certain embodiments, all of a particular nucleobase type may be modified.

[0298] In some embodiments, the mRNAs described herein may include a 5' cap structure, chain-terminating nucleotides, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a polyA sequence, and / or a polyadenylation signal.

[0299] A 5' cap structure or cap species is a compound containing two nucleoside moieties connected by a linker and can be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). The cap species can include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7th position connected by a triphosphate bond at the 5' position, such as m7G(5')ppp(5')G, commonly written as m7GpppG. The cap species can also be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, and m27,O2'GppppG.

[0300] The mRNA may alternatively or additionally comprise chain-terminating nucleosides. For example, chain-terminating nucleosides may include nucleosides deoxygenated at the 2' and / or 3' positions of the sugar group. Such species may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, incorporating chain-terminating nucleotides into the mRNA, e.g., at the 3' end, can stabilize the mRNA, as described, for example, in International Patent Publication WO 2013 / 103659.

[0301] Alternatively, or in addition, the mRNA may contain a stem loop, e.g., a histone stem loop. The stem loop may contain 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, the stem loop may contain 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located within, before, or after an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or a polyA sequence or tail. In some embodiments, the stem loop may affect one or more functions of the mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0302] Alternatively, or in addition, the mRNA may contain a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or primarily of adenine nucleotides or their analogs or derivatives. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the mRNA. In some embodiments, the polyA sequence may affect the nuclear export, translation, and / or stability of the mRNA.

[0303] The mRNA may alternatively or additionally contain a microRNA binding site.

[0304] In some embodiments, the mRNA is a bicistronic mRNA containing a first coding region and a second coding region, with an intervening sequence containing an internal ribosome entry site (IRES) sequence that allows internal translation initiation between the first and second coding regions, or an intervening sequence that encodes a self-cleaving peptide, such as a 2A peptide. IRES sequences and 2A peptides are typically used to enhance expression of multiple proteins from the same vector. For example, various IRES sequences, including the encephalomyocarditis virus IRES, are known and available in the art and can be used.

[0305] In some embodiments, mRNA of the present disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, modified mRNA may have useful properties compared to a reference unmodified mRNA, including enhanced stability, intracellular retention, enhanced translation, and / or lack of substantial induction of the innate immune response of a cell into which the mRNA is introduced. Thus, the use of modified mRNA may not only increase the efficiency of protein production, intracellular retention of nucleic acids, but may also reduce immunogenicity.

[0306] In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, or 4) different modified nucleobases, modified nucleosides, or modified nucleotides. In some embodiments, the mRNA comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, modified nucleosides, or modified nucleotides. In some embodiments, the modified mRNA may have reduced degradation in cells into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0307] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-Methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2- Thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-meth oxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (mcm5Um), Uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0308] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydro-cytidine (5-hydro-cytidine), 5-hydroxy-cytidine (5-hydroxy ... Hoxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl These include 2'-F-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0309] In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having modified adenines include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, Aminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonine Onylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thiamine 2'-O-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0310] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OhyW), undermodified hydroxywyosine (OhyW*), 7-deaza-guanosine, queusine (Q), epoxywyosine (Q), hydroxywyosine (OHyW* ... Queuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2 2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0311] In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0312] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases). In one embodiment, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the mRNA of the present disclosure is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) represents 75 to 100% of the uracils contained in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) represents 100% of the uracils contained in the mRNA.

[0313] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine. In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0314] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0315] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine. In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of two, three, or four of the aforementioned modified nucleobases).

[0316] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, the mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (e.g., a combination of two, three, or four of the foregoing modified nucleobases).

[0317] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyl-uridine. In some embodiments, the mRNA comprises 2'-O-methyl-uridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0318] In certain embodiments, the mRNA of the present disclosure is uniformly modified for a particular modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), which means that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, the mRNA of the present disclosure can be uniformly modified by replacing any type of nucleoside residue present in the sequence with a modified residue such as those described above.

[0319] In some embodiments, mRNAs of the present disclosure may be modified within the coding region (e.g., the open reading frame encoding a polypeptide). In other embodiments, mRNAs may be modified in regions other than the coding region. For example, in some embodiments, a 5'-UTR and / or a 3'-UTR are provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present within the coding region.

[0320] Examples of nucleoside modifications and combinations thereof that may be present in the mmRNAs of the present disclosure include, but are not limited to, those described in PCT Patent Application Publications WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0321] The mmRNAs of this disclosure may contain combinations of modifications to the sugar, nucleobase, and / or internucleoside linkages, which may include any one or more of the modifications described herein.

[0322] When a single modification is listed, the listed nucleoside or nucleotide represents 100% of the modified A, U, G, or C nucleotides or nucleosides. When percentages are listed, they represent the percentage of that particular A, U, G, or C nucleobase triphosphate out of the total amount of A, U, G, or C triphosphates present. For example, the combination 25% 5-aminoallyl-CTP + 75% CTP / 25% 5-methoxy-UTP + 75% UTP refers to a polynucleotide in which 25% of the cytosine triphosphates are 5-aminoallyl-CTP and 75% of the cytosines are CTP, while 25% of the uracils are 5-methoxy-UTP and 75% of the uracils are UTP. When no modified UTP is listed, naturally occurring ATP, UTP, GTP, and / or CTP are used at 100% of these nucleotide positions found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unmodified.

[0323] The mRNA of the present disclosure can be produced by means available in the art, including, but not limited to, in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, hybrid synthesis, small-area synthesis, and ligation methods can be used. In one embodiment, the mRNA is produced using IVT enzymatic synthesis. Methods for producing polynucleotides by IVT are known in the art and are described in International Application No. PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety. Thus, the present disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that can be used to in vitro transcribe the mRNA described herein.

[0324] Non-natural modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications can be on the internucleoside linkage, the purine or pyrimidine base, or the sugar. In certain embodiments, the modifications can be introduced by chemical synthesis or by polymerase enzymes at the end of the polynucleotide chain or elsewhere in the polynucleotide chain. Examples of modified nucleic acids and their synthesis are disclosed in PCT Application No. PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0325] Using either enzymatic or chemical ligation methods, polynucleotides or regions thereof can be conjugated to a variety of functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).

[0326] Therapeutic Agents for Reducing Protein Expression In one embodiment, the therapeutic agent reduces (i.e., decreases, inhibits, downregulates) protein expression. In one embodiment, the therapeutic agent reduces protein expression in target airway cells. Non-limiting examples of types of therapeutic agents that can be used to reduce protein expression include mRNA with introduced microRNA binding site(s) (miR binding sites), microRNA (miRNA), antagomir, small interfering RNA (siRNA) (including shortmers and Dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and CRISPR / Cas9 technology.

[0327] Peptide / Polypeptide Therapeutics In one embodiment, the therapeutic agent is a peptide therapeutic agent. In one embodiment, the therapeutic agent is a polypeptide therapeutic agent.

[0328] In some embodiments, the therapeutic or prophylactic payload comprises an mRNA encoding a secreted, membrane-bound, or intracellular protein, or a peptide, polypeptide, or biologically active fragment thereof.

[0329] In some embodiments, the therapeutic or prophylactic payload comprises an mRNA encoding a secreted protein, a peptide, a polypeptide, or a biologically active fragment thereof. In some embodiments, the therapeutic or prophylactic payload comprises an mRNA encoding a membrane-bound protein, a peptide, a polypeptide, or a biologically active fragment thereof. In some embodiments, the therapeutic or prophylactic payload comprises an mRNA encoding an intracellular protein, a peptide, a polypeptide, or a biologically active fragment thereof. In some embodiments, the therapeutic or prophylactic payload comprises a protein, a polypeptide, or a peptide.

[0330] In some embodiments, the peptide or polypeptide is naturally occurring, e.g., isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, e.g., a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In one embodiment, the peptide or polypeptide therapeutic of the composition is a naturally occurring peptide or polypeptide. In one embodiment, the peptide or polypeptide therapeutic of the composition is a modified version of a naturally occurring peptide or polypeptide (e.g., containing fewer than 3, fewer than 5, fewer than 10, fewer than 15, fewer than 20, or fewer than 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0331] LNPs containing cationic agents The LNPs of the invention comprise an LNP core and a cationic agent located primarily on the outer surface of the core. Such LNPs have a zeta potential greater than neutral at physiological pH.

[0332] Core lipid nanoparticles typically comprise one or more of the following components: lipids (which may include ionized amino lipids, phospholipids, helper lipids (which may be neutral lipids, zwitterionic lipids, anionic lipids, etc.)), structural lipids (e.g., cholesterol or cholesterol analogs), fatty acids, polymers, stabilizers, salts, buffers, solvents, etc.

[0333] Certain LNP cores provided herein comprise an ionizable lipid, such as an ionizable lipid, e.g., an ionizable amino lipid, a phospholipid, a structural lipid, and optionally a stabilizing agent (e.g., a molecule comprising polyethylene glycol), which may or may not be provided conjugated to another lipid.

[0334] The structured lipid may be, but is not limited to, a sterol, such as cholesterol. The structured lipid may be β-sitosterol.

[0335] The helper lipid is a non-cationic lipid. The helper lipid may comprise at least one fatty acid chain of at least 8C and at least one polar head group moiety.

[0336] When a molecule containing polyethylene glycol (i.e., PEG) is used, the molecule can be used as a stabilizer. In some embodiments, the molecule containing polyethylene glycol can be polyethylene glycol conjugated to a lipid, and thus can be provided as, for example, PEG-c-DOMG or PEG-DMG. Certain LNPs provided herein contain no PEGylated lipids or low levels of PEGylated lipids (including no alkyl-PEGylated lipids or low levels of alkyl-PEGylated lipids) and may be referred to herein as PEG-free or PEGylated lipid-free. Thus, some LNPs contain less than 0.5 mol% PEGylated lipids. In some cases, PEG can be an alkyl-PEG, such as methoxy-PEG. Still other LNPs contain non-alkyl-PEGs, such as hydroxy-PEGs, and / or non-alkyl-PEGylated lipids, such as hydroxy-PEGylated lipids. Certain LNPs provided herein contain high levels of PEGylated lipids. Some LNPs contain 0.5 mol% PEGylated lipids. Some LNPs contain more than 0.5 mol% PEGylated lipids. In some embodiments, the LNPs comprise 1.5 mol% PEGylated lipids. In some embodiments, the LNPs comprise 3.0 mol% PEGylated lipids. In some embodiments, the LNPs comprise 0.1 mol% to 3.0 mol% PEGylated lipids, 0.5 mol% to 2.0 mol% PEGylated lipids, or 1.0 mol% to 1.5 mol% PEGylated lipids.

[0337] In some embodiments, the core nanoparticle composition may have a molar ratio of Compound 18:phospholipid:cholesterol:N-lauroyl-D-erythro-sphinganylphosphorylcholine of 50:10:38.5:1.5, hi some embodiments, the core nanoparticle composition may have a molar ratio of Compound 18:DSPC:cholesterol:Compound 428 of 50:10:38.5:1.5.

[0338] Compound 428: [ka]

[0339] The nanoparticles of the present disclosure comprise at least one compound according to Formula (I). For example, the nanoparticle composition may comprise one or more of Compounds 1-147. The nanoparticles may also comprise various other components. For example, the nanoparticle composition may comprise, in addition to a lipid according to Formula (I) or (II), one or more other lipids, such as (i) at least one phospholipid, (ii) at least one structured lipid, (iii) at least one PEG-lipid, or (iv) any combination thereof.

[0340] In some embodiments, the nanoparticle composition comprises a compound of Formula (I) (e.g., compound 18, 25, 26, or 48). In some embodiments, the nanoparticle composition comprises a compound of Formula (I) (e.g., compound 18, 25, 26, or 48) and a phospholipid (e.g., DSPC, DOPE, or MSPC). In some embodiments, the nanoparticle composition comprises a compound of Formula (I) (e.g., compound 18, 25, 26, or 48) and a phospholipid (e.g., DSPC, DPPC, DOPE, or MSPC).

[0341] The present disclosure also provides a process for preparing nanoparticles comprising contacting lipid nanoparticles with a cationic agent, wherein the lipid nanoparticles are (a) Below: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) a lipid nanoparticle core comprising PEG-lipids; (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell.

[0342] In some embodiments, contacting the lipid nanoparticles with a cationic agent comprises dissolving the cationic agent in a non-ionic additive. In some embodiments, the non-ionic additive is selected from macrogol 15 hydroxystearate (HS 15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), Compound 428, polyoxyethylene sorbitan monooleate [TWEEN® 80], and d-α-tocopherol polyethylene glycol succinate (TPGS). In some embodiments, the non-ionic additive is macrogol 15 hydroxystearate (HS 15). In some embodiments, contacting the lipid nanoparticles with a cationic agent comprises dissolving the cationic agent in a buffer solution. In some embodiments, the buffer solution is phosphate buffered saline (PBS). In some embodiments, the buffer solution is a Tris-based buffer.

[0343] The processes described herein provide nanoparticles prepared, for example, by contacting lipid nanoparticles with a cationic agent. In some embodiments, the cationic agent can be a sterol amine such as GL-67. In some embodiments, the lipid nanoparticle core of the lipid nanoparticles optionally comprises a PEG-lipid. In some embodiments, the lipid nanoparticle core that forms the lipid nanoparticles that are contacted with a cationic agent is substantially free of PEG-lipid. In some embodiments, the PEG-lipid is added to the lipid nanoparticles together with the cationic agent before or after contact with the cationic agent.

[0344] In one embodiment, the LNPs of the invention can be made using conventional mixing techniques in which a nucleic acid payload is mixed with core LNP components to create a core LNP+payload. Once this loaded core LNP is prepared, the cationic agent is contacted with the loaded core LNP.

[0345] In another embodiment, the LNPs of the invention can be made using hollow LNPs as a starting point. For example, as shown in Figure 1, hollow LNPs are made before loading with a nucleic acid payload. Once the nucleic acid payload has been contacted with the LNP, a cationic agent can be added to form the LNPs of the invention.

[0346] For example, in one embodiment, in the post-loading (PHL) method, empty LNPs are first formulated by nanoprecipitation, and the buffer is exchanged into a low pH buffer (i.e., pH 5). Then, these empty LNPs are introduced into mRNA through a mixing event (which is also acidified at low pH). After the mixing step, the pH is neutralized using a pH adjustment method. Finally, PEG lipids, such as DMG-PEG-2k, are added to stabilize the particles. Then, these particles are concentrated to a target concentration and filtered. A cationic agent, such as GL67, is added.

[0347] A variation on the starting point for hollow LNPs is illustrated in Figure 2. Figure 2 shows the formation of hollow LNPs using lipids from the LNPs, excluding the PEG lipids. A nucleic acid solution is then contacted with the hollow LNP to form a loaded LNP. As illustrated by the dotted box in Figure 2, the PEG lipids can be added at one or two points during further processing of the loaded LNP, and the cationic agent can be added at any point during that further processing. Figure 3 is a more specific version of the process of Figure 2, where again the cationic agent can be added at any point during that further processing of the loaded LNP.

[0348] In some embodiments, the LNPs of the present invention can be prepared using nanoprecipitation, a unit operation that allows LNPs to self-assemble from their individual lipid components through dynamic mixing and subsequent maturation and serial dilution. This unit operation involves three separate steps: mixing of aqueous and organic inputs, maturation of the LNPs, and dilution after a controlled residence time. These steps are considered a single unit operation due to their continuous nature. The unit operation involves the continuous in-line combination of three liquid streams and one in-line maturation step: mixing of an aqueous buffer with a lipid stock solution, maturation through a controlled residence time, and dilution of the nanoparticles. The nanoprecipitation itself occurs in a scalable mixer designed to enable continuous, high-energy mixing of an aqueous solution with a lipid stock solution dissolved in ethanol. Both the aqueous solution and the lipid stock solution flow simultaneously into the mixing device continuously throughout the operation. The ethanol content, which maintains the lipids in solution, is rapidly reduced, causing all the lipids to precipitate from each other. Thus, the particles self-assemble within the mixing chamber.

[0349] One of the goals of the unit operation is to exchange the solution into a completely aqueous buffer solution that is free of ethanol to achieve the target concentration of LNPs, which can be achieved by first reaching a target processing concentration, then diafiltration, followed by a final concentration step (if necessary) once the ethanol has been completely removed.

[0350] In some embodiments, the LNPs of the present invention can be prepared using nanoprecipitation, a unit operation that allows LNPs to self-assemble from their individual lipid components through dynamic mixing and subsequent maturation and serial dilution. This unit operation involves three separate steps: mixing of aqueous and organic inputs, maturation of the LNPs, and dilution after a controlled residence time. These steps are considered a single unit operation due to their continuous nature. The unit operation involves the continuous in-line combination of three liquid streams and one in-line maturation step: mixing of an aqueous buffer with a lipid stock solution, maturation through a controlled residence time, and dilution of the nanoparticles. The nanoprecipitation itself occurs in a scalable mixer designed to enable continuous, high-energy mixing of an aqueous solution with a lipid stock solution dissolved in ethanol. Both the aqueous solution and the lipid stock solution flow simultaneously into the mixing device continuously throughout the operation. The ethanol content, which maintains the lipids in solution, is rapidly reduced, causing all the lipids to precipitate out of each other. Thus, the particles self-assemble within the mixing chamber.

[0351] One of the goals of the unit operation is to exchange the solution into a completely aqueous buffer solution that is free of ethanol to achieve the target concentration of LNPs, which can be achieved by first reaching a target processing concentration, then diafiltration, followed by a final concentration step (if necessary) once the ethanol has been completely removed.

[0352] In some aspects, the present disclosure provides a method of preparing a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow lipid nanoparticles (hollow LNPs), the method comprising: i) Below: ia) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) comprising hollow nanoparticle intermediates (hollow LNP intermediates); ib) maintaining the hollow LNP intermediate solution for a residence time; ic) adding the dilute solution to the hollow LNP intermediate solution, thereby forming an empty LNP solution comprising the hollow LNPs.

[0353] In some aspects, the present disclosure provides a method of preparing a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow lipid nanoparticles (hollow LNPs), the method comprising: i) Below: ia) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) comprising hollow nanoparticle intermediates (hollow LNP intermediates); ib) maintaining the hollow LNP intermediate solution for a residence time; ic) adding the dilute solution to the hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; and ii) processing the hollow LNP solution.

[0354] In some aspects, the present disclosure provides a method of preparing a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow lipid nanoparticles (hollow LNPs), the method comprising: ii) treating a solution of empty LNPs containing empty LNPs.

[0355] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: i) Below: ia) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) comprising hollow nanoparticle intermediates (hollow LNP intermediates); ib) maintaining the hollow LNP intermediate solution for a residence time; ic) adding the dilute solution to the hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; and ii) processing the hollow LNP solution; iii) a loading step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution, thereby forming a loaded LNP solution containing loaded lipid nanoparticles (loaded LNPs).

[0356] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: i) Below: ia) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) comprising hollow nanoparticle intermediates (hollow LNP intermediates); ib) maintaining the hollow LNP intermediate solution for a residence time; ic) adding the dilute solution to the hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; and ii) processing the hollow LNP solution; iii) a loading step, comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution, thereby forming a loaded LNP solution containing loaded lipid nanoparticles (loaded LNPs); iv) processing the loaded LNP solution, thereby forming a loaded LNP formulation.

[0357] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: i) Below: ia) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) comprising hollow nanoparticle intermediates (hollow LNP intermediates); ib) maintaining the hollow LNP intermediate solution for a residence time; ic) adding the dilute solution to the hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; and ii) processing the hollow LNP solution; iii) a loading step, comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution, thereby forming a loaded LNP solution containing loaded lipid nanoparticles (loaded LNPs); iv) processing the loaded LNP solution, thereby forming a loaded LNP formulation; v) adding a cationic agent.

[0358] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: iii) a loading step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution containing hollow LNPs, thereby forming a loaded nanoparticle solution (loaded LNP solution) containing loaded lipid nanoparticles (loaded LNPs).

[0359] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: iii) a loading step comprising mixing a nucleic acid solution comprising nucleic acids with a hollow LNP solution comprising hollow LNPs, thereby forming a loaded nanoparticle solution (loaded LNP solution) comprising loaded lipid nanoparticles (loaded LNPs); iv) processing the loaded LNP solution, thereby forming a loaded LNP formulation.

[0360] In some aspects, the present disclosure provides a method of preparing a lipid nanoparticle formulation (LNP formulation), comprising: iii) a loading step comprising mixing a nucleic acid solution comprising nucleic acids with a hollow LNP solution comprising hollow LNPs, thereby forming a loaded nanoparticle solution (loaded LNP solution) comprising loaded lipid nanoparticles (loaded LNPs); iv) processing the loaded LNP solution, thereby forming a loaded LNP formulation; v) adding a cationic agent.

[0361] In some embodiments, steps ia) to ic) are performed in separate operational units (eg, separate reaction devices).

[0362] In some embodiments, steps ia)-ic) are performed in a single operational unit. In some embodiments, steps ia)-ic) are performed in a continuous flow device such that step ic) is downstream of step ib), which is downstream of step ia).

[0363] In some embodiments, step ic) involves a single addition of the diluted solution.

[0364] In some embodiments, the diluted solution is added sequentially in step ic).

[0365] In some aspects, the present disclosure provides methods for producing hollow lipid nanoparticles (hollow LNPs), the methods comprising: i) a mixing step comprising mixing an ionizable lipid with a first buffer, thereby forming a hollow LNP, the hollow LNP comprising about 0.1 mol% to about 0.5 mol% of a polymeric lipid (e.g., a PEG-lipid).

[0366] In some aspects, the present disclosure provides a method of preparing a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow lipid nanoparticles (hollow LNPs), the method comprising: i) mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG-lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow LNPs.

[0367] In some aspects, the present disclosure provides a method of preparing a hollow lipid nanoparticle solution (hollow LNP solution) comprising hollow lipid nanoparticles (hollow LNPs), the method comprising: i) a mixing step comprising mixing a lipid solution comprising ionizable lipids, structured lipids, phospholipids, and PEG-lipids with an aqueous buffer solution comprising a first buffer, thereby forming a hollow lipid nanoparticle solution comprising hollow LNPs (hollow LNP solution); ii) processing the hollow LNP solution.

[0368] In some embodiments, the mixing step comprises mixing a lipid solution comprising ionized lipids with an aqueous buffer solution comprising a first buffering agent, thereby forming a hollow lipid nanoparticle solution comprising hollow LNPs (hollow LNP solution).

[0369] In some aspects, the present disclosure provides methods for preparing loaded lipid nanoparticles (loaded LNPs) associated with nucleic acids, the method comprising a loading step comprising ii) mixing nucleic acid with hollow LNPs, followed by adding a cationic agent, thereby forming loaded LNPs.

[0370] In some embodiments, the loading step comprises mixing a nucleic acid solution containing nucleic acids with an empty LNP solution, followed by adding a cationic agent, thereby forming a loaded lipid nanoparticle solution (loaded LNP solution) containing loaded LNPs.

[0371] In some embodiments, the empty LNPs or empty LNP solutions are subjected to the loading step without being held or stored.

[0372] In some embodiments, the empty LNPs or empty LNP solution are held for a period of time before being subjected to a loading step.

[0373] In some embodiments, the empty LNP or empty LNP solution is held for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, or about 24 hours before being subjected to the loading step.

[0374] In some embodiments, the empty LNPs or empty LNP solutions are stored for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years before being subjected to the loading step.

[0375] In some embodiments, the empty LNPs or empty LNP solutions are subjected to the loading step without being stored or held for any period of time after formation.

[0376] In some embodiments, the present disclosure provides methods that further include ii) treating the hollow LNP solution.

[0377] In some embodiments, the present disclosure provides methods further comprising iv) treating the loaded LNP solution, thereby forming a lipid nanoparticle formulation (LNP formulation).

[0378] In contrast to other production techniques (e.g., thin film rehydration / extrusion), ethanol dropwise precipitation has become the industry standard for producing nucleic acid-lipid nanoparticles. Precipitation reactions are advantageous due to their continuous nature, scalability, and ease of adoption. These processes typically use high-energy mixers (e.g., T-junctions, confined impinging jets, microfluidic mixers, vortex mixers) to controllably introduce lipids (in ethanol) into a suitable antisolvent (i.e., water) to drive liquid supersaturation and spontaneous precipitation into lipid particles. In some embodiments, the vortex mixer used is one described in U.S. Patent Application Nos. 62 / 799,636 and 62 / 886,592 (incorporated herein by reference in their entireties). In some embodiments, the microfluidic mixer used is one described in PCT Application No. WO / 2014 / 172045 (incorporated herein by reference in their entireties).

[0379] In some embodiments, the mixing step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.

[0380] In some embodiments, the loading step is performed using a T-junction, a confined impinging jet, a microfluidic mixer, or a vortex mixer.

[0381] In some embodiments, the mixing step is carried out at a temperature below about 30°C, below about 28°C, below about 26°C, below about 24°C, below about 22°C, below about 20°C, or below about ambient temperature.

[0382] In some embodiments, the loading step is carried out at a temperature below about 30°C, below about 28°C, below about 26°C, below about 24°C, below about 22°C, below about 20°C, or below about ambient temperature.

[0383] In some embodiments, the step of treating the hollow LNP solution or the loaded LNP solution comprises a first adding step that comprises adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs or loaded LNPs.

[0384] In some embodiments, the step of treating the hollow LNP solution comprises a first adding step that comprises adding polyethylene glycol lipids (PEG lipids) to the hollow LNP solution.

[0385] In some embodiments, the step of treating the hollow LNP solution comprises a first adding step that comprises adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs.

[0386] In some embodiments, the step of treating the loaded LNP solution comprises a first adding step that comprises adding polyethylene glycol lipids (PEG lipids) to the loaded LNP solution.

[0387] In some embodiments, the step of treating the loaded LNP solution comprises a first adding step that comprises adding polyethylene glycol lipids (PEG lipids) to the loaded LNPs.

[0388] In some embodiments, the first adding step comprises adding a polyethylene glycol solution containing PEG lipids (PEG solution) to the empty LNP solution or the loaded LNP solution.

[0389] In some embodiments, the step of treating the hollow LNP solution or the loaded LNP solution includes a second adding step that includes adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs or loaded LNPs.

[0390] In some embodiments, the step of treating the hollow LNP solution includes a second adding step that includes adding polyethylene glycol lipids (PEG lipids) to the hollow LNP solution.

[0391] In some embodiments, the step of treating the hollow LNP solution includes a second addition step that includes adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs.

[0392] In some embodiments, the step of treating the loaded LNP solution includes a second adding step that includes adding polyethylene glycol lipids (PEG lipids) to the loaded LNP solution.

[0393] In some embodiments, the step of treating the loaded LNP solution includes a second adding step that includes adding polyethylene glycol lipids (PEG lipids) to the loaded LNPs.

[0394] In some embodiments, the second adding step comprises adding a polyethylene glycol solution containing PEG lipids (PEG solution) to the empty LNP solution or the loaded LNP solution.

[0395] In some embodiments, the first addition step comprises adding to the hollow LNP or loaded LNP about 0.1 mol% to about 3.0 mol% PEG, about 0.2 mol% to about 2.5 mol% PEG, about 0.5 mol% to about 2.0 mol% PEG, about 0.75 mol% to about 1.5 mol% PEG, or about 1.0 mol% to about 1.25 mol% PEG.

[0396] In some embodiments, the first addition step comprises adding to the hollow LNP or loaded LNP about 0.1 mol% to about 3.0 mol% PEG, about 0.2 mol% to about 2.5 mol% PEG, about 0.5 mol% to about 2.0 mol% PEG, about 0.75 mol% to about 1.5 mol% PEG, or about 1.0 mol% to about 1.25 mol% PEG. In some embodiments, the first addition step comprises about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, or about 3.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0397] In some embodiments, the first addition step comprises about 1.75±0.5 mol%, about 1.75±0.4 mol%, about 1.75±0.3 mol%, about 1.75±0.2 mol%, or about 1.75±0.1 mol% (e.g., about 1.75 mol%) of PEG lipid (e.g., PEG 2k -DMG).

[0398] In some embodiments, after the first addition step, the empty LNP solution (e.g., empty LNPs) has a concentration of about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, or about 2.8 mol% , about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, about 4.0 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0399] In some embodiments, after the first addition step, the loaded LNP solution (e.g., loaded LNP) is about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol% , about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, about 4.0 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0400] In some embodiments, the second addition step comprises adding to the hollow LNP or loaded LNP about 0.1 mol% to about 3.0 mol% PEG, about 0.2 mol% to about 2.5 mol% PEG, about 0.5 mol% to about 2.0 mol% PEG, about 0.75 mol% to about 1.5 mol% PEG, or about 1.0 mol% to about 1.25 mol% PEG.

[0401] In some embodiments, the second addition step comprises adding to the hollow LNP or loaded LNP about 0.1 mol% to about 3.0 mol% PEG, about 0.2 mol% to about 2.5 mol% PEG, about 0.5 mol% to about 2.0 mol% PEG, about 0.75 mol% to about 1.5 mol% PEG, or about 1.0 mol% to about 1.25 mol% PEG.

[0402] In some embodiments, the second addition step comprises about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, or about 3.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0403] In some embodiments, the second addition step comprises about 1.0±0.5 mol%, about 1.0±0.4 mol%, about 1.0±0.3 mol%, about 1.0±0.2 mol%, or about 1.0±0.1 mol% (e.g., about 1.0 mol%) of a PEG lipid (e.g., PEG 2k -DMG).

[0404] In some embodiments, the second addition step comprises adding about 1.0 mol% PEG lipid to the empty or loaded LNP.

[0405] In some embodiments, after the second addition step, the empty LNP solution (e.g., empty LNPs) has a concentration of about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, or about 2.8 mol% , about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, about 4.0 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0406] In some embodiments, after the second addition step, the loaded LNP solution (e.g., loaded LNP) is about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol% , about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, about 4.0 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5.0 mol% PEG lipid (e.g., PEG 2k -DMG).

[0407] In some embodiments, the first addition step is carried out at a temperature below about 30°C, below about 28°C, below about 26°C, below about 24°C, below about 22°C, below about 20°C, or below about ambient temperature.

[0408] In some embodiments, the second addition step is carried out at a temperature below about 30°C, below about 28°C, below about 26°C, below about 24°C, below about 22°C, below about 20°C, or below about ambient temperature.

[0409] In some embodiments, the step of processing the empty LNP solution or the loaded LNP solution further comprises at least one step selected from filtration, pH adjustment, buffer exchange, dilution, dialysis, concentration, freezing, lyophilization, storage, and packaging.

[0410] In some embodiments, the step of treating the empty or loaded LNP solution further comprises pH adjustment.

[0411] In some embodiments, adjusting the pH comprises adding a second buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, and Tris buffer.

[0412] In some embodiments, the first addition step is performed before the pH adjustment.

[0413] In some embodiments, the first addition step is performed after the pH adjustment.

[0414] In some embodiments, the second addition step is performed before the pH adjustment.

[0415] In some embodiments, the second addition step is performed after the pH adjustment.

[0416] In some embodiments, the step of processing the empty or loaded LNP solution further comprises filtration.

[0417] In some embodiments, the filtration is tangential flow filtration (TFF).

[0418] In some embodiments, filtration removes organic solvents (e.g., alcohol or ethanol) from the LNP solution. In some embodiments, once the organic solvent (e.g., alcohol or ethanol) has been removed, the LNP solution is converted to a solution buffered at a neutral pH, pH 6.5 to 7.8, pH 6.8 to 7.5, preferably pH 7.0 to 7.2 (e.g., phosphate buffer or HEPES buffer). In some embodiments, the LNP solution is converted to a solution buffered at about pH 7.0 to about pH 7.2. In some embodiments, the resulting LNP solution is sterilized, for example, by filtration (e.g., through a 0.1-0.5 μm filter) prior to storage or use.

[0419] In some embodiments, the step of treating the empty LNP solution or the loaded LNP solution further comprises buffer exchange.

[0420] In some embodiments, the buffer exchange comprises the addition of an aqueous buffer solution comprising a third buffering agent.

[0421] In some embodiments, the first addition step is performed before buffer exchange.

[0422] In some embodiments, the first addition step is performed after buffer exchange.

[0423] In some embodiments, the second addition is performed before buffer exchange.

[0424] In some embodiments, the second addition step is performed after buffer exchange.

[0425] In some embodiments, the step of treating the empty or loaded LNP solution further comprises dilution.

[0426] In some embodiments, the step of treating the hollow or loaded LNP solution further comprises dialysis.

[0427] In some embodiments, the step of processing the empty or loaded LNP solution further comprises concentration.

[0428] In some embodiments, the step of treating the empty or loaded LNP solution further comprises freezing.

[0429] In some embodiments, the step of processing the empty or loaded LNP solution further comprises lyophilization.

[0430] In some embodiments, lyophilization involves freezing the loaded LNP solution at a temperature of about -100°C to about 0°C, about -80°C to about -10°C, about -60°C to about -20°C, about -50°C to about -25°C, or about -40°C to about -30°C.

[0431] In some embodiments, lyophilization further comprises drying the frozen loaded LNP solution to form lyophilized hollow LNPs or lyophilized loaded LNPs.

[0432] In some embodiments, drying is carried out at a vacuum ranging from about 50 mTorr to about 150 mTorr.

[0433] In some embodiments, drying is carried out at about -35°C to about -15°C.

[0434] In some embodiments, drying is carried out at about room temperature to about 25°C.

[0435] In some embodiments, the step of processing the empty LNP solution or the loaded LNP solution further comprises storing.

[0436] In some embodiments, storage comprises storing the empty or loaded LNP at a temperature of about -80°C, about -78°C, about -76°C, about -74°C, about -72°C, about -70°C, about -65°C, about -60°C, about -55°C, about -50°C, about -45°C, about -40°C, about -35°C, or about -30°C for at least 1 day, at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, or at least 1 year.

[0437] In some embodiments, storage comprises storing the empty or loaded LNP at a temperature of about -40°C, about -35°C, about -30°C, about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C for at least 1 day, at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, or at least 1 year.

[0438] In some embodiments, storage includes storing the hollow or loaded LNP at a temperature of about -40°C to about 0°C, about -35°C to about -5°C, about -30°C to about -10°C, about -25°C to about -15°C, about -22°C to about -18°C, or about -21°C to about -19°C for at least 1 day, at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, or at least 1 year.

[0439] In some embodiments, storage comprises storing the empty or loaded LNP at a temperature of about -20°C for at least 1 day, at least 2 days, at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 8 months, or at least 1 year.

[0440] In some embodiments, the step of processing the empty or loaded LNP solution further comprises packing.

[0441] As used herein, "packing" can refer to storing a pharmaceutical product in its final state or storing an empty LNP, loaded LNP, or LNP formulation during processing prior to placement in final packaging. Storage and / or packaging methods include, but are not limited to, refrigeration in sterile bags, refrigerated or frozen formulations in vials, lyophilized formulations in vials and syringes, etc.

[0442] In some embodiments, the step of treating the empty LNP solution or the loaded LNP solution comprises i ia) adding a cryoprotectant to the empty LNP solution or the loaded LNP solution.

[0443] In some embodiments, the step of treating the empty or loaded LNP solution comprises iib) filtering the empty or loaded LNP solution.

[0444] In some embodiments, the step of treating the empty LNP solution or the loaded LNP solution comprises: iia) adding a cryoprotectant to the hollow LNP solution or the loaded LNP solution; iic) filtering the empty or loaded LNP solution.

[0445] In some embodiments, the step of processing the empty LNP solution or the loaded LNP solution comprises the following steps: iib) adding cryoprotectants to hollow LNP solutions or loaded LNP solutions; iic) lyophilizing the hollow LNP solution or the loaded LNP solution, thereby forming a lyophilized LNP composition; iid) storing the hollow LNP solution or loaded LNP solution of the lyophilized LNP composition; and iie) adding a buffer solution to the empty LNP solution, the loaded LNP solution, or the lyophilized LNP composition, thereby forming the LNP formulation.

[0446] In some embodiments, the step of treating the empty LNP solution comprises iia) adding a cryoprotectant to the empty LNP solution.

[0447] In some embodiments, the step of treating the empty LNP solution comprises iib) filtering the empty LNP solution.

[0448] In some embodiments, treating the hollow LNP solution comprises: iia) adding a cryoprotectant to the hollow LNP solution; iic) filtering the hollow LNP solution.

[0449] In some embodiments, a cryoprotectant is added to the empty or loaded LNP solution prior to lyophilization. In some embodiments, the cryoprotectant comprises one or more cryoprotectants, each of which is independently selected from the group consisting of a polyol (e.g., a diol or triol, such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a non-surfactant sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG 1800, PEG 1900, PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600, PEG 2700, PEG 2800, PEG 2900, PEG 3000, PEG 3100, PEG 3200, PEG 3300, PEG 3400, PEG 3500, PEG 3600, PEG 3700, PEG 3800 2k-DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or a hydrate of any of these), or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or additive comprises sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or additive comprises sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[0450] In some embodiments, the cryoprotectant comprises a cryoprotectant present in a concentration of about 10 g / L to about 1000 g / L, about 25 g / L to about 950 g / L, about 50 g / L to about 900 g / L, about 75 g / L to about 850 g / L, about 100 g / L to about 800 g / L, about 150 g / L to about 750 g / L, about 200 g / L to about 700 g / L, about 250 g / L to about 650 g / L, about 300 g / L to about 600 g / L, about 350 g / L to about 550 g / L, about 400 g / L to about 500 g / L, and about 450 g / L to about 500 g / L. In some embodiments, the cryoprotectant comprises a cryoprotectant present at a concentration of about 10 g / L to about 500 g / L, about 50 g / L to about 450 g / L, about 100 g / L to about 400 g / L, about 150 g / L to about 350 g / L, about 200 g / L to about 300 g / L, and about 200 g / L to about 250 g / L. In some embodiments, the cryoprotectant comprises a cryoprotectant present at a concentration of about 10 g / L, about 25 g / L, about 50 g / L, about 75 g / L, about 100 g / L, about 150 g / L, about 200 g / L, about 250 g / L, about 300 g / L, about 300 g / L, about 350 g / L, about 400 g / L, about 450 g / L, about 500 g / L, about 550 g / L, about 600 g / L, about 650 g / L, about 700 g / L, about 750 g / L, about 800 g / L, about 850 g / L, about 900 g / L, about 950 g / L, and about 1000 g / L.

[0451] In some embodiments, the cryoprotectant comprises a cryoprotectant present at a concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 25 mM, about 8 mM to about 20 mM, about 9 mM to about 15 mM, and about 10 mM to about 15 mM. In some embodiments, the cryoprotectant comprises a cryoprotectant present at a concentration of about 0.1 mM to about 10 mM, about 0.5 mM to about 9 mM, about 1 mM to about 8 mM, about 2 mM to about 7 mM, about 3 mM to about 6 mM, and about 4 mM to about 5 mM. In some embodiments, the cryoprotectant comprises a cryoprotectant present at a concentration of about 0.1 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, and about 100 mM.

[0452] In some embodiments, the cryoprotectant comprises sucrose.

[0453] In some embodiments, the cryoprotectant comprises an aqueous solution comprising sucrose.

[0454] In some embodiments, the cryoprotectant comprises an aqueous solution comprising about 700±300 g / L, 700±200 g / L, 700±100 g / L, 700±90 g / L, 700±80 g / L, 700±70 g / L, 700±60 g / L, 700±50 g / L, 700±40 g / L, 700±30 g / L, 700±20 g / L, 700±10 g / L, 700±9 g / L, 700±8 g / L, 700±7 g / L, 700±6 g / L, 700±5 g / L, 700±4 g / L, 700±3 g / L, 700±2 g / L, or 700±1 g / L of sucrose.

[0455] In some embodiments, the cryoprotectant comprises an aqueous solution comprising sodium acetate and sucrose.

[0456] In some embodiments, the cryoprotectant is (a) about 5±1 mM, about 5±0.9 mM, about 5±0.8 mM, about 5±0.5 mM, about 5±0.6 mM, about 5±0.5 mM, about 5±0.4 mM, about 5±0.3 mM, about 5±0.2 mM, or about 5±0.1 mM sodium acetate; (b) about 700±300 g / L, 700±200 g / L, 700±100 g / L, 700±90 g / L, 700±80 g / L, 700±70 g / L, 700±60 g / L, 700±50 g / L, 700±40 g / L, 700±30 g / L, 700±20 g / L, 700±10 g / L, 700±9 g / L, 700±8 g / L, 700±7 g / L, 700±6 g / L, 700±5 g / L, 700±4 g / L, 700±3 g / L, 700±2 g / L, 700±5 g / L, 700±4 g / L, 700±3 g / L, 700±2 g / L, or 700±1 g / L of sucrose.

[0457] In some embodiments, the cryoprotectant comprises an aqueous solution comprising sodium acetate and sucrose, wherein the aqueous solution has a pH value of 5.0±2.0, 5.0±1.5, 5.0±1.0, 5.0±0.9, 5.0±0.8, 5.0±0.7, 5.0±0.6, 5.0±0.5, 5.0±0.4, 5.0±0.3, 5.0±0.2, or 5.0±0.1.

[0458] In some embodiments, the cryoprotectant is (a) about 5±1 mM, about 5±0.9 mM, about 5±0.8 mM, about 5±0.5 mM, about 5±0.6 mM, about 5±0.5 mM, about 5±0.4 mM, about 5±0.3 mM, about 5±0.2 mM, or about 5±0.1 mM sodium acetate; (b) an aqueous solution comprising about 700±300 g / L, 700±200 g / L, 700±100 g / L, 700±90 g / L, 700±80 g / L, 700±70 g / L, 700±60 g / L, 700±50 g / L, 700±40 g / L, 700±30 g / L, 700±20 g / L, 700±10 g / L, 700±9 g / L, 700±8 g / L, 700±7 g / L, 700±6 g / L, 700±5 g / L, 700±4 g / L, 700±3 g / L, 700±2 g / L, or 700±1 g / L of sucrose; The aqueous solution has a pH value of 5.0±2.0, 5.0±1.5, 5.0±1.0, 5.0±0.9, 5.0±0.8, 5.0±0.7, 5.0±0.6, 5.0±0.5, 5.0±0.4, 5.0±0.3, 5.0±0.2, or 5.0±0.1.

[0459] In some embodiments, lyophilization is performed in a suitable glass receptacle (e.g., a 10 mL cylindrical glass vial). In some embodiments, the glass receptacle is capable of withstanding extreme temperature changes from below -40°C to above room temperature over a short period of time and / or is cut to a uniform shape. In some embodiments, the lyophilization step comprises freezing the LNP solution at a temperature greater than about -40°C, thereby forming a frozen LNP solution; and drying the frozen LNP solution to form a lyophilized LNP composition. In some embodiments, the lyophilization step comprises freezing the LNP solution at a temperature greater than about -40°C and less than about -30°C. The freezing step linearly ramps from 20°C to -40°C over about 6 minutes, preferably at about 1°C per minute, to the final temperature. In some embodiments, the freezing step linearly ramps from 20°C to -40°C over about 6 minutes, at about 1°C per minute, to the final temperature. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out at a vacuum ranging from about 50 mTorr to about 150 mTorr. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out at a vacuum ranging from about 50 mTorr to about 150 mTorr, first at a low temperature ranging from about -35°C to about -15°C, and then at a high temperature ranging from room temperature to about 25°C. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out at a vacuum ranging from about 50 mTorr to about 150 mTorr, and the drying step is completed in 3-7 days. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out at a vacuum ranging from about 50 mTorr to about 150 mTorr, first at a low temperature ranging from about -35°C to about -15°C, and then at a high temperature ranging from room temperature to about 25°C, and the drying step is completed in 3-7 days. In some embodiments, the drying step is performed at a vacuum ranging from about 50 mTorr to about 100 mTorr. In some embodiments, the drying step is performed at a vacuum ranging from about 50 mTorr to about 100 mTorr, first at a lower temperature ranging from about -15°C to about 0°C, and then at an elevated temperature.

[0460] In some embodiments, the empty LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a pH of about 3.5 to about 8.0, about 4.0 to about 7.5, about 4.5 to about 7.0, about 5.0 to about 6.5, and about 5.5 to about 6.0. In some embodiments, the empty LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a pH of about 3.5, about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 4.5, about 5.5, about 6.5, about 7.0, about 7.5, and about 8.0.

[0461] In some embodiments, the LNP solution, loaded LNP solution, or lyophilized LNP composition is stored in a cryoprotectant comprising sucrose and sodium acetate. In some embodiments, the LNP solution, loaded LNP solution, or lyophilized LNP composition is stored in a cryoprotectant comprising about 150 g / L to about 350 g / L sucrose and about 3 mM to about 6 mM sodium acetate at a pH of about 4.5 to about 7.0. In some embodiments, the LNP solution, loaded LNP solution, or lyophilized LNP composition is stored in a cryoprotectant comprising about 200 g / L sucrose and 5 mM sodium acetate at a pH of about 5.0.

[0462] In some embodiments, the empty LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a temperature of about -80°C, about -78°C, about -76°C, about -74°C, about -72°C, about -70°C, about -65°C, about -60°C, about -55°C, about -50°C, about -45°C, about -40°C, about -35°C, or about -30°C prior to adding the buffer solution.

[0463] In some embodiments, the empty LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a temperature of about -40°C, about -35°C, about -30°C, about -25°C, about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C prior to adding the buffer solution.

[0464] In some embodiments, the hollow LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a temperature ranging from about −40° C. to about 0° C., about −35° C. to about −5° C., about −30° C. to about −10° C., about −25° C. to about −15° C., about −22° C. to about −18° C., or about −21° C. to about −19° C. prior to addition of the buffer solution.

[0465] In some embodiments, the empty LNP solution, loaded LNP solution, or lyophilized LNP composition is stored at a temperature of about -20°C prior to adding the buffer solution.

[0466] Certain aspects of this method are described in PCT Application No. WO / 2020 / 160397, which is incorporated herein by reference in its entirety.

[0467] Also described herein are cells containing nanoparticles. The cells can be epithelial cells. For example, the cells can be lung cells. The cells can be respiratory epithelial cells. For example, the cells can be lung cells, nasal cells, alveolar epithelial cells, or bronchial epithelial cells. The cells can be human bronchial epithelial (HBE) cells. The cells can be HeLa cells. Such cells can be contacted with LNPs in vitro or in vivo.

[0468] Pharmaceutical Compositions and Formulations The present disclosure provides pharmaceutical compositions and formulations comprising any of the nanoparticles described herein.

[0469] Pharmaceutical compositions or formulations may optionally contain one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions or formulations of the present disclosure may be sterile and / or pyrogen-free. General information on the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21 sted., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to nanoparticles containing a polynucleotide or polypeptide payload delivered as described herein.

[0470] The formulations and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such methods of preparation include bringing the nanoparticles into association with an excipient and / or one or more other accessory ingredients, and then, as necessary and / or desired, dividing, shaping, and / or packaging the product into desired single or multiple dose units.

[0471] Pharmaceutical compositions or formulations according to the present disclosure may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dose, e.g., one-half or one-third of such a dose.

[0472] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0473] Although the description of pharmaceutical compositions and formulations provided herein is directed primarily to pharmaceutical compositions and formulations suitable for administration to humans, one of skill in the art will understand that such compositions are generally suitable for administration to any other animal, e.g., non-human animals (e.g., non-human mammals).

[0474] As used herein, pharmaceutically acceptable excipients include, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersing or suspending aids, diluents, granulating and / or dispersing agents, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, binders, lubricants or oils, coloring agents, sweeteners or flavoring agents, stabilizers, antioxidants, antibacterial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelating agents, cryoprotectants, and / or bulking agents, as appropriate for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, the entire contents of which are incorporated herein by reference)).

[0475] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, and the like and / or combinations thereof.

[0476] Exemplary granulating and / or dispersing agents include, but are not limited to, starch, pregelatinized starch, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone), (providone), cross-linked poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, and the like, and / or combinations thereof.

[0477] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether) [BRIJ® 30]), PLUORINC® F 68, POLOXAMER® 188, and the like, and / or combinations thereof.

[0478] Exemplary binders include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, and the like, and combinations thereof.

[0479] Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations.To prevent oxidation, antioxidants can be added to the formulation.Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium metabisulfite or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.

[0480] Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, fumaric acid, malic acid, phosphoric acid, edetate sodium, tartaric acid, edetate trisodium, and the like, and combinations thereof.

[0481] Exemplary antibacterial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof.

[0482] Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), and the like, and combinations thereof.

[0483] In some embodiments, the pH of the polynucleotide solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers for controlling pH can include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and / or combinations thereof.

[0484] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, and the like, and combinations thereof.

[0485] The pharmaceutical compositions described herein can include a cryoprotectant to stabilize the polynucleotides described herein during freezing. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof.

[0486] The pharmaceutical compositions described herein can include bulking agents in the lyophilized polynucleotide formulation to produce a "pharmaceutical refined" cake and stabilize the lyophilized polynucleotide during long-term (e.g., 36 months) storage. Exemplary bulking agents of the present disclosure can include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

[0487] The composition may be in liquid or solid form. In some embodiments, the composition or formulation is in liquid form. In some embodiments, the composition is suitable for inhalation. The composition may be administered to the pulmonary tract. Aerosolized pharmaceutical formulations may be delivered to the lungs, preferably using a number of commercially available devices.

[0488] The compositions can be administered to the respiratory tract by any suitable method, such as intranasal instillation, intratracheal instillation, and intratracheal injection. In some embodiments, the compositions or nanoparticles are administered intranasally, intrabronchially, or via pulmonary administration. For example, the compositions and nanoparticles are administered via a nebulizer or inhaler.

[0489] In some embodiments, the composition is delivered to the lungs by inhalation of an aerosolized pharmaceutical formulation. Inhalation can occur through the subject's nose and / or mouth. Administration can occur by self-administering the formulation during inhalation or by administering the formulation to a subject wearing a respirator via the respirator. Exemplary devices for delivering formulations to the lungs include, but are not limited to, dry powder inhalers, pressurized metered-dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

[0490] Liquid formulations can be administered to a patient's lungs using a pressurized metered dose inhaler (pMDI). pMDIs generally include at least two components: a canister in which the liquid formulation is combined with one or more propellants and held under pressure, and a receptacle used to hold and actuate the canister. The canister may contain single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, that can expel the contents of the canister. Aerosolized medication is delivered from the pMDI by applying force to the canister, forcing it into the receptacle, opening the valve and transporting medication particles through the valve and through the receptacle outlet. Upon expulsion from the canister, the liquid formulation atomizes, forming an aerosol. pMDIs typically use one or more propellants to pressurize the contents of the canister and propel the liquid formulation out the receptacle outlet, forming an aerosol. Any suitable propellant can be utilized. The propellant can take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas.

[0491] Liquid formulations can also be administered using a nebulizer. A nebulizer is a liquid aerosol generator that converts a liquid formulation into a mist or cloud of fine droplets, preferably with an aerodynamic mass median diameter of less than 5 microns, which can be inhaled into the lower respiratory tract. This process is called atomization. When the aerosol cloud is inhaled, the droplets carry one or more active agents to the nose, upper respiratory tract, or deep lung. Any type of nebulizer can be used to administer the formulation to a patient, including, but not limited to, pneumatic (jet) nebulizers and electromechanical nebulizers. Pneumatic (jet) nebulizers use a supply of pressurized gas as the driving force for atomizing the liquid formulation. The compressed gas is delivered through a nozzle or nozzle, creating a low-pressure field that entrains the surrounding liquid formulation and shears it into a thin film or filament. The film or filament becomes unstable and breaks into fine droplets that are carried into the inhaled air by the compressed gas flow. A baffle inserted into the droplet plume selects out larger droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers atomize liquid formulations using electrically generated mechanical forces. The electromechanical driving force can be applied, for example, by vibrating the liquid formulation at ultrasonic frequencies or by forcing the bulk liquid through small holes in a thin film. This force generates a thin film or filament of liquid that breaks into microdroplets, forming a slow-moving aerosol stream that can be entrained in the inspiratory airflow. Liquid formulations can also be administered using electrohydrodynamic (EHD) aerosol devices. EHD aerosol devices use electrical energy to aerosolize a solution or suspension of a liquid formulation.

[0492] Dry powder inhalers (DPIs) typically use a mechanism such as a burst of gas to create a cloud of dry powder within a container, which can then be inhaled by the subject. In DPIs, the administered dose is stored in the form of an unpressurized dry powder, and upon actuation of the inhaler, the subject inhales the powder particles. In some cases, the powder can be delivered using a compressed gas (i.e., a propellant), similar to pressurized metered-dose inhalers (pMDIs). In some cases, DPIs can be breath-activated, meaning that the aerosol is created in precise response to inspiration. Dry powder inhalers typically deliver doses of less than a few tens of milligrams per inhalation to avoid inducing coughing. Examples of DPIs include the Turbohaler® inhaler (Astrazeneca, Wilmington, Del.), Clickhaler® inhaler (Innovata, Ruddington, Nottingham, UKL), Diskus® inhaler (Glaxo, Greenford, Middlesex, UK), EasyHaler® (Orion, Expoo, FI), Exubera® inhaler (Pfizer, New York, NY), Qdose® inhaler (Microdose, Monmouth Junction, NJ), and Spiros® inhaler (Dura, San Diego, Calif.).

[0493] The pharmaceutical compositions of the present invention are administered in an effective amount to produce a desired biological effect, e.g., a therapeutic or prophylactic effect (e.g., through expression of a normal gene product to replenish or replace a defective protein or to reduce undesired protein expression, as measured in some embodiments by the alleviation of one or more symptoms). The formulations can be administered in an effective amount to deliver a payload, e.g., to deliver LNPs to the apical membrane of respiratory and non-respiratory epithelial cells. In some embodiments, the pharmaceutical compositions are administered in an effective amount to induce missing CFTR activity in patients with CF or to increase existing levels of residual CFTR activity in patients with CF.

[0494] The presence of a desired biological activity, e.g., residual CFTR activity at the epithelial surface, can be readily detected using methods known in the art, including standard electrophysiological, biochemical, and / or histochemical techniques, whereby CFTR activity is identified and / or quantified using in vivo or ex vivo electrophysiological techniques, measurement of CT concentrations in sweat or saliva, or ex vivo biochemical or histochemical techniques that monitor CFTR cell surface density.

[0495] How to use Described herein are methods for treating or preventing a disease in a patient, wherein the disease is associated with dysfunction of airway cells. The method comprises administering to the patient nanoparticles or compositions comprising a nucleic acid payload as described herein for the treatment or prevention of the disease. For example, in one embodiment, the payload is a nucleic acid molecule, such as an mRNA molecule, and the disease is alleviated by the expression of a protein or polypeptide in airway epithelial cells. In some embodiments, the disease is cystic fibrosis.

[0496] In some embodiments, the nanoparticles described herein are used in a method for reducing cellular sodium levels in a subject in need thereof.

[0497] In some embodiments, the nanoparticles described herein are used to reduce levels of a metabolite (e.g., a substrate or product) associated with CF, and the method comprises administering to a subject an effective amount of a polynucleotide encoding a CFTR polypeptide.

[0498] In some embodiments, administration of an effective amount of a nanoparticle described herein reduces the level of a biomarker for CF, e.g., intracellular sodium levels. In some embodiments, administration of a nanoparticle described herein reduces the level of one or more biomarkers for CF, e.g., intracellular sodium levels, within a short period of time after administration of a nanoparticle described herein.

[0499] In some embodiments, administration of nanoparticles described herein to a subject reduces intracellular sodium levels in cells to a level that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% lower than the level observed before administration of the composition or formulation.

[0500] In some embodiments, provided herein is a method for delivering a polynucleotide payload or a polypeptide payload to a cell, comprising contacting the cell with a nanoparticle described herein. In some embodiments, administering the nanoparticle described herein causes CFTR expression in the cells of a subject. In some embodiments, administering the nanoparticle described herein increases the CFTR enzyme activity of the subject. For example, this method can increase the CFTR enzyme activity in at least some cells of the subject.

[0501] In some embodiments, administration of a nanoparticle described herein comprising mRNA encoding a CFTR polypeptide to a subject increases CFTR enzymatic activity in the subject's cells to a level that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% or more of the level of activity expected in a normal subject, e.g., a human not affected by CF.

[0502] In some embodiments, administration of the nanoparticles described herein results in expression of CFTR protein in at least a portion of the subject's cells, which persists for a period of time sufficient to result in significant chloride channel activity.

[0503] In some embodiments, expression of the encoded polypeptide is increased. In some embodiments, the polynucleotide, when introduced into a cell, increases the CFTR expression level of the cell by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% relative to the CFTR expression level of the cell before the polypeptide was introduced into the cell.

[0504] As will be appreciated by those skilled in the art, the sterol amines disclosed herein have additional uses. For example, the sterol amines can be used to treat inflammatory diseases. The sterol amines can also be used as antibacterial agents.

[0505] Kits and Devices The present disclosure provides various kits for conveniently and / or effectively using the claimed nanoparticles of the present disclosure. Typically, the kits include components in sufficient quantities and / or numbers to allow a user to perform multiple treatments of a subject(s) and / or to perform multiple experiments.

[0506] In one aspect, the present disclosure provides a kit comprising the nanoparticles of the present disclosure.

[0507] The kit may further include packaging and instructions and / or a delivery agent for forming the formulation. The delivery agent may include saline, a buffer solution, a lipidoid, or any delivery agent disclosed herein. In one embodiment, such a kit further includes an administration device such as a nebulizer or inhaler.

[0508] Pulmonary function tests and other tests to improve respiratory symptoms In some embodiments, nanoparticles or pharmaceutical compositions contain mRNA containing an open reading frame (ORF) encoding a polypeptide or protein. Such polypeptides or proteins can be tested for improvement of respiratory function or symptoms. For example, in one embodiment, a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide, when administered to a subject in need thereof, is sufficient to improve at least one measured respiratory volume by at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration, compared to at least one reference respiratory volume measured in a subject with untreated cystic fibrosis. Respiratory volume is the amount of air inhaled, exhaled, and stored in the lungs at any given time. Non-limiting examples of various respiratory volumes that can be measured are provided below.

[0509] Total lung capacity (TLC) is the volume of the lungs at maximum expansion and is the sum of the VC and RV. The average total lung capacity is 6000 ml, but this varies with age, height, sex, and health status.

[0510] Tidal volume (TV) is the volume of air that enters and leaves the lungs during quiet breathing. (TV denotes the lung fraction. When tidal volume is measured precisely, as in gas exchange calculations, the symbol TV or VT is used.) The average tidal volume is 500 ml.

[0511] Residual volume (RV) is the volume of air remaining in the lungs after maximal expiration. Residual volume (RV / TLC%) is expressed as a percentage of TLC.

[0512] Expiratory reserve volume (ERV) is the maximum volume of air that can be exhaled (above the tidal volume) during forced expiration.

[0513] Inspiratory reserve volume (IRV) is the maximum volume that can be inhaled from end-inspiratory position.

[0514] The maximum intake quantity (IC) is the sum of IRV and TV.

[0515] Inspiratory vital capacity (IVC) is the maximum volume of air that can be inspired from the point of maximum expiration.

[0516] Vital capacity (VC) is the volume of air exhaled after the deepest inspiration.

[0517] Functional residual capacity (FRC) is the volume of the lung at end-expiration.

[0518] Forced vital capacity (FVC) is the determination of vital capacity from a maximal forced expiratory effort.

[0519] Forced expiratory volume (hours) (FEV t FEV1 is the volume of air exhaled at the end of the first second of forced expiration. FEF xis the forced expiratory flow associated with the portion of the FVC curve. The modifier refers to the amount of FVC already exhaled. FEF max is the maximum instantaneous flow rate achieved during the FVC maneuver.

[0520] Forced inspiratory flow (FIF) is a specific measurement of the forced inspiratory curve and is expressed in terms similar to the forced expiratory curve. For example, the maximum inspiratory flow is expressed as FIF max Unless otherwise specified, the volume qualifier indicates the volume inspired from the RV at the time of measurement.

[0521] Peak expiratory flow (PEF) is the maximal forced expiratory flow measured with a peak flow meter.

[0522] Maximal ventilation (MVV) is the volume of air exhaled within a specific period during repeated maximal efforts.

[0523] synthesis As will be appreciated by those skilled in the art, the compounds provided herein (including salts and stereoisomers thereof) can be prepared using known organic synthesis techniques, or can be synthesized according to any of a number of possible synthetic routes, such as those shown in the following schemes.

[0524] The reactions for preparing the compounds described herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be one that does not substantially react with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out (for example, a temperature that can range from the freezing temperature of the solvent to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of multiple solvents. Depending on the specific reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0525] As used herein, the expressions "ambient temperature" or "room temperature" or "rt" are understood in the art and generally refer to a temperature near the temperature of the room in which the reaction is carried out (e.g., a temperature of about 20°C to about 30°C), e.g., the reaction temperature.

[0526] Preparation of the compounds described herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Protecting group chemistry is described, for example, in T.W. Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0527] The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) and normal phase silica chromatography.

[0528] Compounds of formula A2a can be prepared, for example, using the process illustrated in the following scheme. [ka]

[0529] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 1. A suitable reaction between cholesteryl chloroformate and an amine can be carried out under appropriate conditions to produce compounds of formula A2a. [ka]

[0530] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 2. A suitable reaction between cholesterol or a cholesterol derivative (such as stigmasterol) and 4-nitrophenyl chloroformate can be carried out under suitable conditions (such as using triethylamine and 4-dimethylaminopyridine). The product of the above reaction can be reacted with an amine under suitable conditions (such as using triethylamine) to give compounds of formula A2a. [ka]

[0531] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 3. A suitable reaction between cholesterol or a cholesterol derivative (such as stigmasterol) and a carboxylic acid in the presence of an activating reagent (such as EDC-HCl, DMAP, DCC, or pivalic anhydride) can be carried out under suitable conditions to provide compounds of formula A2a. [ka]

[0532] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 4. A suitable reaction between cholesterol hemisuccinate or a cholesterol hemisuccinate derivative and an activating agent can be carried out under suitable conditions. The product of the above reaction can be reacted with an amine under suitable conditions to give compounds of formula A2a. [ka]

[0533] The compound of formula A2a can be prepared via the synthetic route outlined in Scheme 5. The appropriate reaction between cholesteryl chloroformate and ethane-1,2-diamine can be carried out under appropriate conditions to give SA22. SA22 can be reacted with 2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide under appropriate conditions to give the compound of formula A2a. SA22 can also be reacted with dimethyl squarate under appropriate conditions, and the product of the reaction can be further reacted with a secondary amine under appropriate conditions to give the compound of formula A2a. [ka]

[0534] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 6. The appropriate reaction between an aminoalkyl carbamate and a guanidinylating agent can be carried out under appropriate conditions. The product of the above reaction can be reacted with HCl under appropriate conditions to give compounds of formula A2a. [ka]

[0535] Precursors to compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 7. A suitable reaction between cholesterol or a cholesterol derivative (such as stigmasterol) can be carried out under suitable conditions (such as using triethylamine and 4-dimethylaminopyridine). The product of the above reaction can be reacted with an amine under suitable conditions (such as using triethylamine) to provide precursors to compounds of formula A2a. [ka]

[0536] Precursors to compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 8. A suitable reaction between cholesterol or a cholesterol derivative (such as stigmasterol) and a boc-hemiester can be carried out under appropriate conditions. The product of the above reaction can be reacted under appropriate conditions to give precursors to compounds of formula A2a. [ka]

[0537] Intermediates for synthesizing compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 9. The appropriate reaction between spermidine or spermine and (E)-N-((tert-butoxycarbonyl)oxy)benzimidoyl cyanide (BOC-ON) can be carried out under appropriate conditions to provide intermediates for synthesizing compounds of formula A2a.

[0538] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly indicated herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0539] The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one member of the group, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0540] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more," and "at least one," may be used interchangeably herein. In certain embodiments, the terms "a" or "an" mean "single." In other embodiments, the terms "a" or "an" include "two or more" or "plurality."

[0541] Furthermore, as used herein, "and / or" refers to the specific disclosure of each of two particular features or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0542] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0543] Whenever an embodiment is described herein using the word "comprising," other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0544] Units, prefixes, and symbols are indicated in their International System of Units (SI) recognized format. Numerical ranges are intended to be inclusive of the numbers defining the range. When a range of values ​​is listed, it is to be understood that each intervening integer and fractional value between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included in or excluded from the range, and each range in which either, neither, or both limits are included is also encompassed within the scope of the disclosure. When values ​​are explicitly listed, it is understood that values ​​that are approximately the same quantity or amount as the recited value are also within the scope of the disclosure. When combinations are disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, when different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. When any element of the disclosure is disclosed as having multiple alternatives, examples of the disclosure in which each alternative is excluded alone or in any combination with the other alternatives are also disclosed herein. Multiple elements of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0545] About: The term "about" as used throughout this specification and claims in connection with numerical values ​​indicates an interval of accuracy well known and accepted by those skilled in the art. Such interval of accuracy is ±10%.

[0546] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can be considered to be up to one-tenth of the unit of the lower limit of that range for any particular value or subrange within the stated range in different embodiments of the present disclosure, unless the context clearly dictates otherwise.

[0547] Administered in Combination: As used herein, the term "administered in combination" or "combined administration" means that two or more agents are administered to a subject simultaneously or within an interval such that the effects of each agent on the patient can overlap. In some embodiments, they are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of each other. In some embodiments, the administration of the agents is spaced sufficiently close together so that a combined (e.g., synergistic) effect is achieved.

[0548] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and insects. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0549] Approximately: As used herein, the term "approximately" when applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (above or below) (except where such number would exceed 100% of the possible values).

[0550] Compound: As used herein, the term "compound" is meant to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" refers to any geometric isomer (e.g., cis and trans isomers), enantiomer, or diastereomer of a compound. The present disclosure includes all stereoisomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), enantiomeric mixtures, and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds, as well as means for resolving them into their component enantiomers or stereoisomers, are well known. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Additionally, compounds, salts, or complexes of the present disclosure may be prepared in combination with solvents or water molecules to form solvates and hydrates by routine methods.

[0551] Contacting: As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle are made to share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a nanoparticle composition and a mammalian cell placed within a mammal can be accomplished by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can include various amounts of the nanoparticle composition. Furthermore, multiple mammalian cells can be contacted with the nanoparticle composition. Another example of contacting is between a nanoparticle and a cationic agent. Contacting a nanoparticle with a cationic agent can refer to bringing the surface of the nanoparticle into physical contact with the cationic agent, allowing the cationic agent to form a non-bonded interaction with the nanoparticle. In some embodiments, contacting a nanoparticle with a cationic agent causes the cationic agent to intercalate into the nanoparticle, for example, starting from the surface of the nanoparticle. In some embodiments, the terms "layering," "coating," and "post-addition" and "addition" can be used to mean "contacting" with respect to contacting nanoparticles with a cationic agent.

[0552] Deliver: As used herein, the term "deliver" means providing an entity to a destination. For example, delivering a polynucleotide to a subject can include administering a nanoparticle composition comprising the polynucleotide to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cells can include contacting one or more cells with the nanoparticle composition.

[0553] Delivery agent: As used herein, "delivery agent" refers to any substance that at least partially facilitates the in vivo, in vitro, or ex vivo delivery of a polynucleotide to a target cell.

[0554] Diastereomers: As used herein, the term "diastereomers" means stereoisomers that are not mirror images of each other and are not superimposable with respect to one another.

[0555] Disposed: As used herein, the term "disposed" means that the molecule has formed a non-bonded interaction with the nanoparticle after the molecule and nanoparticle have come into contact with each other.

[0556] Dosing regimen: As used herein, a "dosing regimen" or "administration regimen" is a schedule or physician-determined regimen for administering treatment, prevention, or palliative care.

[0557] Effective amount: As used herein, the term "effective amount" of a drug refers to an amount sufficient to produce a beneficial or desired result, e.g., a clinical result, and therefore the "effective amount" depends on the context in which it is applied. For example, in the context of administering a drug to treat a protein defect (e.g., a CFTR defect), the effective amount of the drug is the amount of CFTR-expressing mRNA sufficient to alleviate, reduce, eliminate, or prevent the signs and symptoms associated with the CFTR defect, for example, compared to the severity of the symptoms observed without administration of the drug. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0558] Enantiomer: As used herein, the term "enantiomer" refers to each individual optically active form of a compound of the present disclosure having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), at least 90%, or at least 98%.

[0559] Encapsulate: As used herein, the term "encapsulate" means to close off, surround, or enclose.

[0560] Encapsulation efficiency: As used herein, "encapsulation efficiency" refers to the amount of polynucleotide that becomes part of a nanoparticle composition compared to the initial total amount of polynucleotide used to prepare the nanoparticle composition. For example, if 97 mg of polynucleotide is encapsulated in the nanoparticle composition out of a total of 100 mg of polynucleotide initially provided in the composition, the encapsulation efficiency may be 97%. As used herein, "encapsulation" may refer to completely, substantially, or partially enclosing, enclosing, surrounding, or enveloping.

[0561] Epithelial cell: As used herein, "epithelial cell" includes cells of epithelial origin. Examples of epithelial cells are respiratory epithelial cells, nasal epithelial cells, alveolar epithelial cells, pulmonary epithelial cells, or bronchial epithelial cells. In some embodiments, the epithelial cells are human bronchial epithelial (HBE) cells. In some embodiments, the epithelial cells are in vitro cells. In some embodiments, the epithelial cells are in vivo cells.

[0562] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) the production of an mRNA template from a DNA sequence (e.g., by transcription); (2) the processing of the mRNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) translation of the mRNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0563] Ex vivo: As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event may occur in an environment that is minimally altered from the natural (e.g., in vivo) environment.

[0564] Helper lipid: As used herein, the term "helper lipid" refers to a compound or molecule that contains a lipid portion (for insertion into a lipid layer, e.g., a lipid bilayer) and a polar portion (for interaction with the physiological solution at the surface of the lipid layer). Typically, helper lipids are phospholipids. The function of helper lipids is to "complement" amino lipids to increase the membrane fusogenicity of the bilayer and / or to help promote endosomal escape (e.g., of nucleic acids delivered to cells). Helper lipids are also believed to be important structural components of the surface of LNPs.

[0565] In vitro: As used herein, the term "in vitro" refers to events that take place not inside a living organism (e.g., an animal, plant, and / or microorganism) but in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.

[0566] In vivo: As used herein, the term "in vivo" refers to events that take place inside an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0567] Ionized amino lipid: The term "ionized amino lipid" includes lipids having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titrative amino head group (e.g., alkylamino or dialkylamino head group). Ionized amino lipids are typically protonated (i.e., positively charged) at pH below the pKa of the amino head group, and are substantially uncharged at pH above the pKa. Such ionized amino lipids include, but are not limited to, DLin-MC3-DMA (MC3) and (13Z,165Z)-N,N-dimethyl-3-nonidocosa-13-16-dien-1-amine (L608).

[0568] Isomer: As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any of the compounds of the present disclosure. It is recognized that the compounds of the present disclosure may have one or more chiral centers and / or double bonds and thus may exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present disclosure, the chemical structures depicted herein, and thus the compounds of the present disclosure, encompass all of the corresponding stereoisomers, i.e., both stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures (e.g., racemates). Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallizing the compounds as chiral salt complexes, or crystallizing the compounds in chiral solvents. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0569] Lipid nanoparticle core: As used herein, a lipid nanoparticle core is a lipid nanoparticle to which a subsequent layer of additional components, such as a cationic agent and / or a PEG-lipid or other lipid, can be added. In some embodiments, the lipid nanoparticle core comprises (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) optionally a PEG-lipid. In further embodiments, the lipid nanoparticle core comprises (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid.

[0570] Linker: As used herein, "linker" refers to a group of atoms, e.g., 10 to 1,000 atoms, which may be composed of atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be attached at one end to a modified nucleoside or nucleotide on a nucleobase or sugar moiety and at the second end to a payload, e.g., a detectable or therapeutic agent. The linker may be of sufficient length so as not to interfere with incorporation into a nucleic acid sequence. Linkers can be used for any useful purpose, such as to form polynucleotide multimers (e.g., via attachment of two or more chimeric polynucleotide molecules or IVT polynucleotides) or to form polynucleotide conjugates, and to administer a payload, as described herein. Examples of chemical groups that can be incorporated into linkers include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl (each of which may be optionally substituted as described herein). Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker that can be cleaved using reducing agents or photolysis, such as disulfide bonds (-SS-) or azo bonds (-N=N-). Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and / or photolysis, and ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0571] Pulmonary cell: As used herein, "pulmonary cell" includes cells derived from the lung. Pulmonary cells can be, for example, pulmonary epithelial cells, airway basal cells, bronchiolar exocrine cells, pulmonary neuroendocrine cells, alveolar cells, or airway epithelial cells. In some embodiments, the pulmonary cells are in vitro cells. In some embodiments, the pulmonary cells are in vivo cells.

[0572] Method of administration: As used herein, "method of administration" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The method of administration can be selected to target (e.g., specifically deliver) delivery to a particular area or system of the body.

[0573] The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2′-amino-LNA with a 2′-amino functionalization, and 2′-amino-α-LNA with a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0574] Patient: As used herein, "patient" refers to a subject who may be seeking or may need treatment, who needs treatment, who is undergoing treatment, who will be undergoing treatment, or who is receiving medical care from a trained professional for a particular disease or condition.

[0575] CFTR-related disease: As used herein, the term " CFTR-related disease " or " CFTR-related disorder " refers to the disease or disorder caused by the abnormal activity of CFTR (for example, decreased activity or increased activity), respectively.As a non-limiting example, cystic fibrosis is a CFTR-related disease.Many clinical variants of cystic fibrosis are known in the art.For example, see www.omim.org / entry / 219700.

[0576] The terms "CFTR enzymatic activity," "CFTR activity," and "cystic fibrosis transmembrane conductance regulator activity" are used interchangeably in this disclosure and refer to the ability of CFTR to transport chloride ions across a cell membrane. Thus, a fragment or variant that retains or has CFTR enzymatic activity or CFTR activity refers to a fragment or variant that undergoes measurable chloride transport across a cell membrane.

[0577] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to refer 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 of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0578] Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound), which has substantially non-toxic and non-inflammatory properties in patients. Examples of excipients include anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, excipients (diluents), film-forming or coating agents, flavors, fragrances, flow agents (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0579] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is modified by converting an existing acid or base moiety into its salt form (for example, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethoxybenzoate, and 2-hydroxy-ethoxybenzoate. Examples of suitable salts include toluenesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate hydrochloride, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or in a mixture of the two (generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used). Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0580] As used herein, the term "solvate" refers to a compound of the present disclosure in which molecules of a suitable solvent have been incorporated into the crystal lattice. A suitable solvent is one that is physiologically tolerable at the administered dosage. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents include ethanol, water (e.g., monohydrate, dihydrate, and trihydrate), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate."

[0581] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). Modified forms of polynucleotides, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides are also included. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including, for example, tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced, any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing non-nucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided the polymer contains nucleobases in an arrangement that allows base pairing and base stacking, such as those found in DNA and RNA. In certain embodiments, a polynucleotide includes an mRNA. In other embodiments, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be substituted with unnatural nucleobases, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of synthetic DNA, or A, C, G, and U (uridine) in the case of synthetic RNA.

[0582] Those skilled in the art will understand that the T base in the codon maps disclosed herein is present in DNA, and that in the corresponding RNA, the T base is replaced by a U base. For example, codon-nucleotide sequences disclosed herein in DNA form, such as vectors or in vitro translation (IVT) templates, have a T base that is transcribed as a U based on its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (including T) and their corresponding mRNA sequences (including U) are considered codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also understand that equivalent codon maps can be created by replacing one or more bases with unnatural bases. Thus, for example, the TTC codon (DNA map) corresponds to the UUC codon (RNA map), which in turn corresponds to the ΨΨC codon (RNA map in which U is replaced with pseudouridine).

[0583] Canonical AT and GC base pairs occur under conditions that allow hydrogen bonds to form between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy, N3, and C4-NH2 of cytidine and the C2-NH2, N'-H, and C6-oxy of guanosine, respectively. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications result in a nucleoside base that no longer effectively forms canonical base pairs with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) results in a modified nucleotide that does not base pair efficiently with guanosine but does base pair with isoguanosine (Collins et al., U.S. Pat. No. 5,681,702). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and the references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and the references cited therein; isoguanine nucleotides can be prepared using the methods described by Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 to Collins et al.Other unnatural base pairs can be synthesized by the method described by Piccirilli et al., 1990, Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement, 1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotide units that form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.

[0584] Polypeptide: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers can contain modified amino acids. These terms also encompass amino...

Claims

1. (a) Below: (i) ionizable cationic lipids, (ii) phospholipids, (iii) sterols, and (iv) PEG-modified lipid a lipid nanoparticle core comprising: (b) a polynucleotide or polypeptide payload encapsulated within the core for delivery to a cell; and (c) a cationic agent primarily disposed on the outer surface of the core; A nanoparticle comprising: the nanoparticles have a zeta potential greater than neutral at physiological pH; the cationic agent is a cationic lipid, and The nanoparticles comprise, in molar ratio, 20-60% ionized cationic lipid, 5-25% phospholipid, 25-55% sterol, 0.5-15% PEG-modified lipid, and 2-10% cationic agent; Nanoparticles.

2. The nanoparticles are, in a molar ratio: (a) 40-60% ionizable cationic lipid, 5-15% non-cationic lipid, 30-50% sterol, 0.5-10% PEG-modified lipid, and 3-7% cationic agent; or (b) 45-55% ionized cationic lipid, 7.5-12.5% ​​phospholipid, 35-45% sterol, 0.5-5% PEG-modified lipid, and 4.5-6% cationic agent.

2. The nanoparticle of claim 1, comprising:

3. Nanoparticles described in claim 1 or 2, wherein the cationic agent is GL-67 or a salt thereof. (a) a weight ratio of cationic agent to polynucleotide of about 0.1:1 to about 15:1; and / or (b) the molar ratio of cationic agent to polynucleotide is from about 0.1:1 to about 20:1; 3. Nanoparticles according to claim 1 or 2.

5. The nanoparticles described in claim 1 or 2, wherein the nanoparticles comprise compound 18 or 236, DSPC, cholesterol, compound 428 or PEG-DMG, and GL-67 or a salt thereof.

6. The nanoparticles are, in a molar ratio: (a) about 48-52 mol % of Compound 18 or 236, about 9-12 mol % of a phospholipid, about 36-42 mol % of cholesterol, and about 0.25-2.5 mol % of a PEG-modified lipid; or (b) about 43 to 49 mol % of compound 18 or 236, about 8 to 12 mol % of a phospholipid, about 33 to 39 mol % of cholesterol, about 0.5 to 2 mol % of a PEG-modified lipid, and about 3 to 6 mol % of a cationic agent 3. The nanoparticle of claim 1 or 2, comprising:

7. Nanoparticles described in claim 1 or 2, wherein the nanoparticles comprise compound 18, DSPC, cholesterol, and compound 428 or PEG-DMG, optionally in a molar ratio of about 49.5±3:10.5±2:39±3:1±0.

75.

8. Nanoparticles described in claim 1 or 2, wherein the nanoparticles comprise compound 236, DSPC, cholesterol, and compound 428 or PEG-DMG, optionally in a molar ratio of about 49.5±3:10.5±2:39±3:1±0.

75.

9. The nanoparticles comprising Compound 18, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, optionally in a molar ratio of: (a) about 47.6±25:9.5±8:36.6±20:1.4±1.25:4.9±2.5; or (b) about 47.3±25:9.5±8:36.4±20:1.4±1.25:5.5±2.5; or (c) approximately 45.8 ± 25: 10.5 ± 8: 36.8 ± 20: 1.4 ± 1.25: 5.5 ± 2.5; or (d) about 47±3:10±2:36±3:1.25±0.75:4.5±1.5; or (e) Approx. 46.5±3: 10±2: 36±3: 1.25±0.75: 4.5±1.5 3. The nanoparticle of claim 1 or 2, wherein:

10. The nanoparticles comprising Compound 236, DSPC, cholesterol, Compound 428 or PEG-DMG, and GL-67 or a salt thereof, optionally in a molar ratio of: (a) about 47.6±25:9.5±8:36.6±20:1.4±1.25:4.9±2.5; or (b) about 47.3±25:9.5±8:36.4±20:1.4±1.25:5.5±2.5; or (c) approximately 45.8 ± 25: 10.5 ± 8: 36.8 ± 20: 1.4 ± 1.25: 5.5 ± 2.5; or (d) Approx. 46.5±3: 10±2: 36±3: 1.25±0.75: 4.5±1.5 3. The nanoparticle of claim 1 or 2, wherein:

11. The nanoparticles of claim 1 or 2, wherein the nanoparticles comprise compound 18, DSPC, cholesterol, DMG-PEG-2k, and GL-67.

12. The nanoparticles described in claim 11, comprising, in molar ratios, about 45 to 48 mol% of compound 18, about 9 to 11 mol% of DSPC, about 35 to 38 mol% of cholesterol, about 1 to 3 mol% of DMG-PEG-2k, and about 4 to 6 mol% of GL-67.

13. The nanoparticles of claim 1 or 2, wherein the nanoparticles exhibit at least about 20% cellular accumulation in epithelial cells and about 5% or more expression in epithelial cells.

14. The nanoparticles of claim 1 or 2, wherein the nanoparticles exhibit approximately 0.5% to 50% protein expression in cells, and the cells are in vivo.

15. 3. The nanoparticle of claim 1 or 2, wherein the weight ratio of cationic agent to polynucleotide payload is about 1:1 to about 4:1, about 1.25:1 to about 3.75:1, about 1.25:1, about 2.5:1, or about 3.75:

1.

16. 3. The nanoparticles of claim 1 or 2, wherein the nanoparticles have a zeta potential of about 5 mV to about 20 mV, about 5 mV to about 20 mV, about 5 mV to about 15 mV, or about 5 mV to about 10 mV.

17. 3. The nanoparticle of claim 1 or 2, wherein the lipid nanoparticle core has a neutral charge at neutral pH.

18. 3. The nanoparticle of claim 1 or 2, wherein greater than about 80%, greater than 90%, greater than 95%, or greater than 95% of the cationic agent is on the surface of the nanoparticle.

19. 3. The nanoparticle of claim 1 or 2, wherein at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the polynucleotide or polypeptide payload is encapsulated within the core.

20. 3. The nanoparticles of claim 1 or 2, wherein the nanoparticles have a polydispersity value of less than about 0.4, less than about 0.3, or less than about 0.

2.

21. 3. The nanoparticles of claim 1 or 2, wherein the nanoparticles have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 100 nm, or about 60 nm to about 80 nm.

22. 3. The nanoparticles of claim 1 or 2, wherein the nanoparticles have a Laurdan general polarization (GPL) of about 0.6 or greater.

23. 3. The nanoparticle of claim 1 or 2, wherein the nanoparticle has a d-spacing of greater than about 6 nm or greater than about 7 nm.

24. 3. The nanoparticles of claim 1 or 2, wherein at least 50%, at least 75%, at least 90%, or at least 95% of the nanoparticles have a surface fluidity value greater than the threshold polarization level.

25. 3. The nanoparticle of claim 1 or 2, wherein when the nanoparticle contacts a cell population, about 10% or more, about 15% or more, or about 20% or more of the cell population accumulates the nanoparticle.

26. 3. The nanoparticle of claim 1 or 2, wherein when the nanoparticle is contacted with a population of cells, about 5% or more or about 10% or more of the cells express the polynucleotide or polypeptide.

27. The cell population (a) Epithelial cell population; (b) respiratory epithelial cell population; (c) nasal cell population; (d) alveolar epithelial cell population; (e) lung cell population; (f) a bronchial epithelial cell population; or (g) HBE group 3. The nanoparticle of claim 1 or 2, wherein:

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