Composition for delivering payload molecules to airway epithelium

Abstract

The present disclosure provides LNPs comprising payload molecules, eg, 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] Respiratory epithelial cells line the respiratory tract. Their primary functions include humidifying the airway, protecting it from potential pathogens, infections, and tissue damage, and / or facilitating gas exchange. Dysfunction of airway epithelial cells can lead to numerous disorders, including, for example, asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Delivery of payloads to respiratory epithelial cells can be used to induce immunity against a target antigen, and the function of airway epithelial cells can be modulated, for example, to replace missing or mutated proteins, or to increase or decrease the functionality of such cells.

[0002] For example, cystic fibrosis ("CF") is an autosomal recessive disorder characterized by the abnormal accumulation of thick, sticky mucus in patients. CF is also known as pancreatic cystic fibrosis, 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, in patients with CF, thick, sticky mucus is produced, which reduces the size of the airways, leading to chronic cough, wheezing, inflammation, bacterial infections, fibrosis, and pulmonary cysts. Furthermore, in most CF patients, the mucus obstructs the ducts within the pancreas, preventing the release of insulin and digestive enzymes, resulting in diarrhea, malnutrition, poor growth, and weight loss (Gershman AJet 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 far rarer in other populations (Ratjen F. et al., Lancet 361:681-689 (2003)). The latest treatments for CF only control symptoms, not cure the disease. Specifically, antibiotics, anti-inflammatory drugs, bronchodilators, decongestants, a high-protein, high-fat diet, and vitamin supplements are prescribed to control symptoms. In progressive lung disease, lung transplants are also performed to provide patients with undamaged lungs. However, these treatments do not completely or reliably control the disease. New treatments have emerged that focus on the root cause of CF. These treatments modulate CFTR in patients with mutations in the Phe508del cystic fibrosis membrane conductance regulator (CFTR) (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] Therefore, improved treatments are needed to address disorders associated with airway epithelial cell dysfunction (e.g., CF), to target such airway epithelial cells for prophylactic treatment (e.g., immunization), or to address other disorders that would benefit from the therapeutic delivery of nucleic acid molecules or other payload molecules to airway epithelial cells. [Overview of the project]

[0004] This disclosure provides LNP molecules for delivering nucleic acid molecules, such as mRNA therapeutics, to airway epithelial cells for the treatment of disorders associated with airway epithelial dysfunction or for the preventive 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 for administering vaccine payloads. This disclosure provides LNPs that, when administered to cells (e.g., in vitro and in vivo), have improved properties, such as improved payload delivery to epithelial cells (e.g., measured by cellular accumulation of LNPs, expression of desired proteins, and / or mRNA expression).

[0005] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) A cationic agent mainly arranged on the outer surface of the core, Nanoparticles containing the above are provided herein, which have a zeta potential above neutral at physiological pH.

[0006] In one embodiment, (a) Below: (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids A lipid nanoparticle core containing, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the following are provided herein.

[0007] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which exhibit at least about 20% cellular accumulation in epithelial cells and at least 5% expression in epithelial cells.

[0008] In one embodiment, a process for preparing nanoparticles is provided herein, comprising contacting lipid nanoparticles with a cationic agent, wherein the lipid nanoparticles are (a) Below: (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids A lipid nanoparticle core containing, (b) comprising a polynucleotide payload or polypeptide payload encapsulated within a core for delivery to cells.

[0009] In one embodiment, nanoparticles prepared by a process described herein are provided herein.

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

[0011] In one embodiment, a method for treating or preventing a patient's disease is provided herein, comprising administering to the patient nanoparticles comprising a payload for treating or preventing the disease described herein.

[0012] Each limitation of the present invention may encompass various embodiments of the present invention. Therefore, each limitation of the present invention involving any one element or combination of elements is expected to be included in each aspect of the present invention. In its application, the present invention is not limited to the configuration details or arrangement of components described in the following description or illustrated in the drawings. Other embodiments of the present invention are possible and can be practiced or carried out in various ways. [Brief explanation of the drawing]

[0013] [Figure 1] This is a diagram illustrating an exemplary first-generation post-loading (PHL) process for preparing LNPs. [Figure 2] This is a diagram illustrating an exemplary second-generation PHL process (comprehensive) for preparing LNPs. [Figure 3] This is a diagram illustrating an exemplary second-generation PHL process (specific) for preparing LNPs. [Figure 4] This is a diagram illustrating an exemplary process for preparing a prototype of hollow lipid nanoparticles ("neutral assembly"), where the hollow LNPs are mixed at pH 8.0, and the final formulation is pH 5.0. [Figure 5] This is a diagram illustrating an exemplary process for preparing LNPs using sterolamines. [Figure 6] This is a small-angle X-ray scattering (SAXS) analysis of LNP-1 and LNP-1a. [Figure 7] This graph shows the general polarity (GLP) of LNP-1 and LNP-1a. [Modes for carrying out the invention]

[0014] This 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. For example, cystic fibrosis (CF) is a progressive genetic disease that causes persistent lung infections and limits respiratory function over time. The disease is characterized by mutations in both copies of the gene for the cystic fibrosis membrane conductance regulator (CFTR) protein. Without CFTR, which is involved in the production of sweat, digestive fluids, and mucus, normally thin secretions become thick. mRNA therapeutics are particularly suitable for the treatment of CF because this technique delivers mRNA encoding CFTR into cells, followed by de novo synthesis of a functional CFTR protein in the target cells. After delivery of mRNA to the target cells, the desired CFTR protein is expressed by the cell's own translational mechanisms, so that the complete functional CFTR protein is replaced by a deficient or missing 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 yet 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 that results in the expression of a polypeptide or peptide that elicits an immune response to the antigen.

[0015] Lipid nanoparticles (LNPs) are an ideal platform for the safe and effective delivery of payload molecules, such as mRNA, to target cells. LNPs have a unique ability to deliver nucleic acids by mechanisms including cellular uptake, intracellular transport, and endosomal release or escape. Several 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 positioned on the outer surface of the nanoparticle. While not bound by any particular theory, LNPs having a cationic agent primarily positioned on the outer surface of the core may improve the accumulation of LNPs in cells such as human bronchial epithelium (HBE), and may also improve the function of payload molecules as measured by mRNA expression in cells (e.g., airway epithelial cells).

[0016] In some embodiments, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) A cationic agent mainly arranged on the outer surface of the core, Nanoparticles containing the above are provided herein, which have a zeta potential above neutral at physiological pH.

[0017] In some embodiments, (a) Below: (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids A lipid nanoparticle core containing, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the following are provided herein.

[0018] In some embodiments, (a) Below: (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids A lipid nanoparticle core containing, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) A cationic agent mainly arranged on the outer surface of the core, Nanoparticles containing the following are provided herein.

[0019] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing are provided herein, which exhibit cellular accumulation of at least about 20% of cells and expression of about 5% or more in cells. In some embodiments, the nanoparticles exhibit cellular accumulation of about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in cells. In some embodiments, the nanoparticles exhibit expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in cells.

[0020] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) A cationic agent mainly arranged on the outer surface of the core, Nanoparticles containing are provided herein, which exhibit cellular accumulation of at least about 20% of cells and expression of about 5% or more in cells. In some embodiments, the nanoparticles exhibit cellular accumulation of about 1% to about 75%, 5% to about 50%, about 10% to about 40%, or about 15% to about 25% in cells. In some embodiments, the nanoparticles exhibit expression of about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% in cells.

[0021] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) A cationic agent mainly arranged on the outer surface of the core, Nanoparticles containing the above are provided herein, which exhibit about 0.5% to 50% protein expression in cells. In some embodiments, the nanoparticles exhibit about 0.1% to 60%, about 0.5% to 40%, about 1% to 30%, or about 1% to 20% protein expression in cells.

[0022] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which exhibit about 0.5% to 50% protein expression in cells. In some embodiments, the nanoparticles exhibit about 0.1% to 60%, about 0.5% to 40%, about 1% to 30%, or about 1% to 20% protein expression in cells.

[0023] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles comprising the above are provided herein, which exhibit at least about 20% cellular accumulation in epithelial cells and about 5% or more 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.

[0024] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which exhibit protein expression of approximately 0.5% to 50% in epithelial cells. In some embodiments, the nanoparticles exhibit protein expression of approximately 0.1% to 60%, 0.5% to 40%, 1% to 30%, or 1% to 20% in epithelial cells.

[0025] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0026] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0027] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0028] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles comprising the above are provided herein, which exhibit at least about 20% cellular accumulation in respiratory epithelial cells and expression of about 5% or more 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.

[0029] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0030] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0031] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0032] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles comprising the above are provided herein, which exhibit at least about 20% cellular accumulation in bronchial epithelial cells and expression of about 5% or more 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.

[0033] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles containing the above are provided herein, which 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.

[0034] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles comprising the above are provided herein, which exhibit at least about 20% cellular accumulation in HBE cells and about 5% or more 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.

[0035] In one embodiment, (a) Lipid nanoparticle core, (b) A polynucleotide payload or polypeptide payload encapsulated within the core for delivery to cells, (c) Cationic agents and, Nanoparticles comprising the above are provided herein, which exhibit at least about 20% cellular accumulation in healthy HBE cells in vitro and expression of about 5% or more 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.

[0036] In some embodiments, the cells referred to above and throughout this specification may be in vitro cells or in vivo cells. In some embodiments, the cells are in vitro cells. In some embodiments, the cells are in vivo cells.

[0037] In some embodiments, the nanoparticles of the present invention exhibit increased cellular accumulation (e.g., in airway epithelial cells such as HBE) compared to nanoparticles of substantially the same composition but without the post-addition of a cationic agent (e.g., layering or contacting a cationic agent with pre-formed lipid nanoparticles). In some embodiments, the nanoparticles of the present invention exhibit increased cellular expression (e.g., in airway epithelial cells such as HBE) compared to nanoparticles of substantially the same composition but without the post-addition of a cationic agent (e.g., layering or contacting a cationic agent with pre-formed lipid nanoparticles).

[0038] In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 0.1:1 to about 15:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 0.2:1 to about 10:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 10:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 8:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 7:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 6:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 5:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is about 1:1 to about 4:1. In some embodiments, the weight ratio of the cationic agent to the polynucleotide payload is approximately 1.25:1 to approximately 3.75:1.

[0039] In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 0.1:1 to about 20:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 10:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 9:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 8:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 7:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 6:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1 to about 5:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 1.5:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is about 2:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is approximately 3:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is approximately 4:1. In some embodiments, the molar ratio of the cationic agent to the polynucleotide payload is approximately 5:1.

[0040] 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.

[0041] 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 the Wyatt Technologies Mobius Zeta Potential instrument. This instrument characterizes mobility and zeta potential by the principle of "ultraparallel phase-analytical 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 a high voltage 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 the Malvern Zetasizer (Nano ZS).

[0042] In some embodiments, the lipid nanoparticle core has a neutral charge at a neutral pH.

[0043] In some embodiments, more than 80% of the cationic agent is on the surface of the nanoparticles. In some embodiments, more than 90% of the cationic agent is on the surface of the nanoparticles. In some embodiments, more than 95% of the cationic agent is on the surface of the nanoparticles.

[0044] In some embodiments, at least about 50% of the polynucleotide payload or polypeptide payload is encapsulated within the core. In some embodiments, at least about 75% of the polynucleotide payload or polypeptide payload is encapsulated within the core. In some embodiments, at least about 90% of the polynucleotide payload or polypeptide payload is encapsulated within the core. In some embodiments, at least about 95% of the polynucleotide payload or polypeptide payload is encapsulated within the core.

[0045] 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.

[0046] 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.

[0047] In some embodiments, the general polarization of the nanoparticles is about 0.6 or greater. In some embodiments, the nanoparticles have a d-spacing greater than about 6 nm. In some embodiments, the nanoparticles have a d-spacing greater than about 7 nm.

[0048] 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.

[0049] In some embodiments, when nanoparticles come into contact with a cell population, more than 10% of the cell population accumulates the nanoparticles. In some embodiments, when nanoparticles come into contact with a cell population, more than 15% of the cell population accumulates the nanoparticles. In some embodiments, when nanoparticles come into contact with a cell population, more than 20% of the cell population accumulates the nanoparticles. In some embodiments, when nanoparticles come into contact with a cell population, more than 5% of the cells express polynucleotides or polypeptides. In some embodiments, when nanoparticles come into contact with a cell population, more than 10% of the cells express polynucleotides or polypeptides. 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 lung 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 lung 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.

[0050] Cationic agents Cationic agents may contain any water-soluble molecules or substances that have a net positive charge and can adhere to the surface of lipid nanoparticle cores. Such agents may also be lipid-soluble but are soluble in aqueous solutions. Cationic agents may be charged at physiological pH. Physiological pH is the pH level normally observed in the human body. Physiological pH may be about 7.30–7.45 or about 7.35–7.45. Physiological pH may 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 an aqueous medium. For example, cationic agents may contain one or more amine groups, e.g., primary, secondary, or tertiary amines, each having a pKa of 8.0 or greater. The pKa may be greater than about 9.

[0051] In some embodiments, the cationic agent may be a cationic lipid, which is a water-soluble amphiphilic molecule, and this molecule is partially hydrophobic, for example, containing a lipid moiety, while the other part of this molecule is hydrophilic and contains one or more functional groups that are normally charged at physiological pH. The hydrophobic moiety containing the lipid moiety may play a role in immobilizing the cationic agent on the lipid nanoparticle core. The hydrophilic moiety may play a role in increasing the charge on the surface of the lipid nanoparticle core. For example, the cationic agent may have a solubility of more than about 1 mg / mL in alcohol. The solubility in alcohol may be more than about 5 mg / mL. The solubility in alcohol may be more than about 10 mg / mL. The solubility in alcohol may be more than about 20 mg / mL in alcohol. The alcohol is C, such as ethanol. 1~6 It could be alcohol.

[0052] The lipid portion of the molecule may be, for example, a structural lipid, a fatty acid, or a similar hydrocarbyl group.

[0053] Structural lipids may be selected from, but are not limited to, steroids, diterpenoids, triterpenoids, cholestanes, ursolic acid, or derivatives thereof.

[0054] In some embodiments, the structural lipid is a steroid selected from, but not limited to, cholesterol or phystosterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is sitosterol, campesterol, or stigmasterol. In some embodiments, the structural lipid is an analog of sitosterol, campesterol, or stigmasterol. In some embodiments, the structural lipid is β-sitosterol.

[0055] The fatty acid contains 1 to 4 C 6~20 hydrocarbon chains. The fatty acid may be fully saturated or may contain 1 to 7 double bonds. The fatty acid may contain 1 to 5 heteroatoms along or pendant to the main chain.

[0056] In some embodiments, the fatty acid contains 2 C 10~18 hydrocarbon chains. In some embodiments, the fatty acid contains 2 C 10~18 saturated hydrocarbon chains. In some embodiments, the fatty acid contains 2 C 16 saturated hydrocarbon chains. In some embodiments, the fatty acid contains 2 C 14 saturated hydrocarbon chains. In some embodiments, the fatty acid contains 2 unsaturated C 10~18 hydrocarbon chains. In some embodiments, the fatty acid contains 2 C hydrocarbon chains each having 1 double bond. 16~18 In some embodiments, the fatty acid contains 3 C 8~18 saturated hydrocarbon chains.

[0057] The hydrocarbyl group consists of 1 to 4 C 6~20 alkyl chain, alkenyl chain, or alkynyl chain, or a 3- to 10-membered cycloalkyl group, cycloalkenyl group, or cycloalkynyl group.

[0058] In some embodiments, the hydrocarbyl chain is C 8~10It is alkyl. In some embodiments, the hydrocarbyl chain is C 8~10 It is Alkenil.

[0059] The hydrophilic portion may contain 1 to 5 functional groups that are charged at a physiological pH of 7.3 to 7.4. The hydrophilic group may contain a basic functional group that is protonated and positively charged at physiological pH. At least one of the basic functional groups has a pKa of 8 or more.

[0060] In some embodiments, the hydrophilic portion includes an amine group. The amine group may include 1 to 4 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.

[0061] 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-.

[0062] The hydrophilic portion may contain a phosphonium group. The counterion of the phosphonium ion consists of a monovalent anion.

[0063] In some embodiments, three of the substituents on the phosphonium are isopropyl groups. In some embodiments, the counterion is a halo, bisulfite, nitrite, chloric acid, or bicarbonate. In some embodiments, the counterion is a bromide.

[0064] In some embodiments, the cationic agent is a cationic lipid, which is a sterolamine. A sterolamine has a sterol on its hydrophobic portion and an amine group on its hydrophilic portion. The sterol group is selected from, but is not limited to, cholesterol, sitosterol, campesterol, stigmasterol, or derivatives thereof. The amine group may consist of 1 to 5 primary, secondary, or tertiary amines, or mixtures 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 amines may be part of a larger amine containing a functional group selected from -C(=N-)-N-, -C=CN-, -C=N-, or -NC(=N-)-N- (but not limited to these). The amine may be contained in a 3 to 8-membered heteroalkyl or heteroaryl ring.

[0065] In some embodiments, the amine group of the sterolamine contains one or two terminal primary amines. In some embodiments, the amine group contains one or two terminal primary amines and one internal secondary amine. In some embodiments, the amine group contains one or two tertiary amines. In some embodiments, the tertiary amine is (CH3)2N-. In some embodiments, the amine group contains one or two terminal (CH3)2N-.

[0066] A sterolamine useful for the nanoparticles of the present invention is given by formula (A1): ALB(A1) The formula includes molecules or salts thereof having: A is an amine group, L is an optional linker, and B is a sterol.

[0067] In some embodiments, the amine group is alkyl (e.g., C 1~14 Alkyl, C 1~12 Alkyl, C 1~10 Alkyl, etc.), 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C1~6 Alkyl-(5-6 member heteroaryl), where this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) includes 1-5 primary, secondary, or tertiary amines, or combinations thereof, where this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 The groups are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups). In some embodiments, the linker is absent or is -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-. In some embodiments, the sterol group is cholesterol, sitosterol, campesterol, stigmasterol, or a derivative thereof.

[0068] In some embodiments, the sterolamine is derived from formula A2a: [ka] or having a salt thereof, in the formula: [ka] These are single or double bonds, R 1 C 1~14 Alkyl or C 1~14 It is an alkenil; L a It does not exist, or -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 formula (a): [ka] It is the basis of; Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n = 1 or 2.

[0069] In some embodiments, the sterolamine is derived from formula A2a: [ka] or having a salt thereof, in the formula: [ka] These are single or double bonds, R 1 C 1~14 Alkyl or C 1~14 It is an alkenil; L a It does not exist, or -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 formula (a): [ka] It is the basis of; Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n=1 or 2 (However, the compound of formula A2a is, [ka] [ka] [ka] (This is unexpected.)

[0070] In some embodiments, [ka] It is a double bond. In some embodiments, [ka] It is a single bond.

[0071] In some embodiments, L a These are -OC(=O), -OC(=O)N-, or -OC(=O)-CH2-CH2-C(=O)N-.

[0072] In some embodiments, n is 1. In some embodiments, n is 2.

[0073] In some embodiments, R 1 C 1~14 It is alkyl. In some embodiments, R 1 C 1~14 It is an alkenyl. In some embodiments, R 1 teeth, [ka] That is the case.

[0074] In some embodiments, Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, -C 1~6 Alkyl-(3-8 member heterocycloalkyl), or -C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this C 1~10 Alkyl, 3-8 member heterocycloalkyl, -C 1~6 Alkyl-(3-8 member heterocycloalkyl), and -C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or combinations thereof; Here, this C 1~10 Alkyl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) is C 1~6 alkyl, OH, -C 1~6 It is optionally substituted with either an alkyl-OH group or an NH2 group.

[0075] In some embodiments, the sterolamine is derived from formula A2: [ka] or having a salt thereof, in the formula: [ka] is a single bond or a double bond, R 1 is C 1~14 alkyl or C 1~14 alkenyl; L is absent or is -O-, -S-S-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH2-NH-C(O)-, -C(O)O-, -OC(O)-CH2-CH2-C(=O)N-, -S-S-CH2, or -SS-CH2-CH2-C(O)N-; Y 1 is C 1~10 alkyl, a 3- to 8-member heterocycloalkyl, a 5- to 6-member heteroaryl, C 1~6 alkyl-(3- to 8-member heterocycloalkyl), or C 1~6 alkyl-(5- to 6-member heteroaryl), wherein this alkyl, 3- to 8-member heterocycloalkyl, 5- to 6-member heteroaryl, C 1~6 alkyl-(3- to 8-member heterocycloalkyl), and C 1~6 alkyl-(5- to 6-member heteroaryl) contains 1 to 5 primary, secondary, or tertiary amines, or combinations thereof, wherein this alkyl, 3- to 8-member heterocycloalkyl, 5- to 6-member heteroaryl, C 1~6 alkyl-(3- to 8-member heterocycloalkyl), and C 1~6 alkyl-(5- to 6-member 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, a 3- to 8-member heterocycloalkyl (optionally substituted with 1 to 5 primary, secondary, or tertiary amines, or combinations thereof and wherein the C 1~14 alkyl is optionally substituted), a 5- to 6-member heteroaryl, NH(3- to 8-member heterocycloalkyl), and NH(5- to 6-member heteroaryl), each optionally substituted with 1, 2, 3, or 4 substituents selected from; n = 1 or 2.

[0076] In some embodiments, the sterolamine is of formula A3a: [ka] or having a salt thereof, in the formula: [ka] It is either a single bond or a double bond; R 2 is H or C 1~6 It is alkyl; L a It does not exist, or -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 formula (a): [ka] It is the basis of; Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) is C 1~6 alkyl, halo, OH, O(C 1~6Alkyl), C 1~6 Alkyl-OH, NH2, NH(C 1~6 Alkyl), N(C 1~6 Alkyl) 2, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n = 1 or 2.

[0077] In some embodiments, the sterolamine is of formula A3a: [ka] or having a salt thereof, in the formula: [ka] It is either a single bond or a double bond; R 2 is H or C 1~6 It is alkyl; L a It does not exist, or -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 formula (a): [ka] It is the basis of; Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n=1 or 2 (However, the compound of formula A2a is, [ka] [ka] [ka] (This is unexpected.)

[0078] In some embodiments, [ka] It is a double bond. In some embodiments, [ka] It is a single bond.

[0079] In some embodiments, L a These are -OC(=O), -OC(=O)N-, or -OC(=O)-CH2-CH2-C(=O)N-.

[0080] In some embodiments, n is 1. In some embodiments, n is 2.

[0081] In some embodiments, R 2 is H. In some embodiments, R 2 It is ethyl.

[0082] In some embodiments, Y 1 C 1~10 Alkyl, 3-8 member heterocycloalkyl, -C 1~6 Alkyl-(3-8 member heterocycloalkyl), or -C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this C 1~10 Alkyl, 3-8 member heterocycloalkyl, -C 1~6 Alkyl-(3-8 member heterocycloalkyl), and -C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or combinations thereof; Here, this C 1~10 Alkyl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) is C 1~6 alkyl, OH, -C 1~6 It is optionally substituted with either an alkyl-OH group or an NH2 group.

[0083] In some embodiments, the sterolamine is derived from formula A3: [ka] or having a salt thereof, in the formula: [ka] It is either a single bond or a double bond; R 2 is H or C 1~6 It is alkyl; L is either nonexistent, -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 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n = 1 or 2.

[0084] In some embodiments, Y 1 teeth, [ka] [ka] Selected from.

[0085] In some embodiments, Y 1 teeth, [ka] [ka] (28) Selected from N(CH3)2.

[0086] In some embodiments, the sterolamine is derived from formula A4: [ka] or having a salt thereof, in the formula: Z 1 is OH or C 3~6 It is alkyl; L is either nonexistent, -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 C 1~10 Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), or C 1~6 It is an alkyl-(5-6 member heteroaryl) group, Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6Alkyl-(5-6 member heteroaryl) contains 1-5 primary, secondary, or tertiary amines, or a combination thereof. Here, this alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, C 1~6 Alkyl-(3-8 member heterocycloalkyl), and C 1~6 Alkyl-(5-6 member 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, 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) 1~14 They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, NH(3-8 member heterocycloalkyl groups), and NH(5-6 member heteroaryl groups); n = 1 or 2.

[0087] In some embodiments, Z 1 is OH. In some embodiments, Z 1 is C 3~6 It is alkyl.

[0088] In some embodiments, L is -C(=O)N-, -CH2-NH-C(=O)-, or -C(=O)O-.

[0089] In some embodiments, Y 1 C contains 1 to 5 primary, secondary, or tertiary amines, or combinations thereof. 1~10 It is alkyl. In some embodiments, Y 1 teeth, [ka] That is the case.

[0090] In some embodiments, n is 1. In some embodiments, n is 2.

[0091] In some embodiments, the sterolamine is derived from formula A5: [ka] or having a salt thereof, in the formula: Z 2 is OH or isopropyl; L 3 These are -CH2-NH-C(O)-, -C(O)NH-, or -C(O)O-.

[0092] In some embodiments, sterolamines are [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] or selected from its salts.

[0093] In some embodiments, sterolamines are [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0094] In some embodiments, sterolamines are [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] or selected from its salts.

[0095] In some embodiments, sterolamines are [Table 4] or selected from its salts.

[0096] In some embodiments, sterolamine is SA3: [ka] or a salt thereof, also known as GL-67. SA3 or GL-67 can be prepared according to processes known in the art, or can be purchased from commercial vendors such as Avanti® Polar Lipids, Inc. (SKU 890893).

[0097] 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 derivatives), a fatty acid, or a similar hydrocarbyl group. At least one amine in the modified amino acid moiety has a pKa of 8.0 or greater. At least one amine in 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 attached to the hydrophobic group via a linker.

[0098] In some embodiments, the modified amino acid is modified arginine.

[0099] 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.

[0100] Where used herein, the term “lipid” refers to small molecules having hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of the class of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenolipids. In some cases, the amphiphilic properties of certain lipids lead to the formation of liposomes, vesicles, or membranes in aqueous media.

[0101] Ionized lipids As used herein, the term “ionized lipid” has its usual meaning in the art and may refer to a lipid containing one or more charged moieties. In some embodiments, ionized lipids may be positively charged or negatively charged. For example, an ionized lipid may be positively charged at relatively low pH, in which case it may be referred to as a “cationic lipid.” In certain embodiments, an ionized lipid molecule may contain an amine group and may be referred to as an ionized aminolipid. As used herein, a “charged moiety” is a chemical moiety that possesses a formal charge of electrons, e.g., 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 imidizolium groups. In certain embodiments, the charged moiety includes an amine group. Examples of negatively charged groups or their precursors include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, and hydroxyl groups. The charge of the charged moiety may, in some cases, vary depending on environmental conditions; for example, changes in pH may alter the charge of this moiety and / or cause it to become charged or uncharged. Generally, the charge density of the molecule may be selected as needed.

[0102] It should be understood that the terms “charge” or “charged portion” do not refer to “partially negative charge” or “partially positive charge” on a molecule. The terms “partially negative charge” and “partially positive charge” are given their usual meanings in the art. “Partially 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 partially negative charge on that atom. Those skilled in the art will generally recognize bonds that can be polarized in this way.

[0103] In some embodiments, the ionized lipid is an ionized aminolipid. In one embodiment, the ionized aminolipid may have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

[0104] In some embodiments, the nanoparticles described herein contain about 30 mol% to about 60 mol% of ionized lipids. In some embodiments, the nanoparticles contain about 40 mol% to about 50 mol% of ionized lipids.

[0105] The lipid nanoparticle composition of the present invention may contain one or more ionized (e.g., ionized amino) lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH). The ionized lipids are 3-(didodecylamino)-N1,N1,4-tridedecyl-1-piperazineethaneamine (KL10), N1-[2-(didodecylamino)ethyl]N1,N4,N4-tridedecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), and 1,2-dilinoleyloxy-N,N-dimethylaminopropane (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,1 The following can be selected from a non-limiting group consisting of (2Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)) and (2S)2-({8-[(3β)-cholest-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2S)). In addition to these, ionized lipids may also be lipids containing cyclic amine groups.

[0106] Ionized lipids may also be compounds disclosed in International Publication No. WO2017 / 075531A1 (which is incorporated herein by reference in its entirety). For example, ionized aminolipids include: [ka] Examples include, but are not limited to, any combination thereof.

[0107] Ionized lipids may also be compounds disclosed in International Publication No. WO2015 / 199952A1 (which is incorporated herein by reference in its entirety). For example, ionized aminolipids include: [ka] [ka] Examples include, but are not limited to, any combination thereof.

[0108] In one embodiment, the ionized lipid is described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373, and WO2013086354, U.S. Ionized lipids may be selected from, but are not limited to, those described in U.S. Patent Publications 7,893,302, 7,404,969, 8,283,333, and 8,466,122, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, and S20130225836 (the contents of each of these are incorporated herein by reference in their entirety).

[0109] In another embodiment, the ionized lipid may be selected from, but is not limited to, formula A as described in International Publication WO2013116126 or US20130225836 (the contents of each of these are incorporated herein by reference in their entirety). In yet another embodiment, the ionized lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of International Publication WO2008103276, formula CLI-CLXXIX of U.S. Patent No. 7,893,302, formula CLI-CLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Publication US20100036115 and formula I of U.S. Patent Publication US20130123338 (each of these are incorporated herein by reference in their entirety).

[0110] As non-limiting examples, cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-diene-4-amine, and (14Z,17Z)-N,N-dimethyltricosa-14,17 -Diene-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene- 8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-diene-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-dimethylheptacosan-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-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethyleptacosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltritriaconta-24-en-10-amine, (20Z)-N,N-dimethylnonacosa-20-en-10-amine, (22Z)-N,N-dimethylhentriaconta-22-en-10-amine, (16Z)-N,N-dimethylpentacosa-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16 -Diene-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentyl [Cyclopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2- Decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-[(5Z)-octa-5-ene-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene- 1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-o [Cutadeca-6,9,12-triene-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy (C)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-ene-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-ene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadeca-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 -amines, 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-triene-10-amine or their pharmaceutically acceptable salts or stereoisomers may be selected.

[0111] Examples of the addition of ionized lipids include the following: [ka]

[0112] In one embodiment, the lipid may be a cleavable lipid, for example, one described in International Publication WO2012170889 (which is incorporated herein by reference in its entirety). In one embodiment, the lipid may be synthesized by methods known in the art and / or by methods described in International Publication WO2013086354 (each of which is incorporated herein by reference in its entirety). In another embodiment, the lipid may be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids, as well as methods for preparing and using trialkyl cationic lipids, are described in International Patent Publication WO2013126803, which is incorporated herein by reference in its entirety.

[0113] In some embodiments, the ionized lipid is expressed by formula (I): [ka] It may be a compound or salt or isomer of the compound, in which formula: 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 R2 and R3 are independently selected from the group consisting of alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 form a heterocycle or carbocycle with the atom to which they are bonded, and this carbocycle is optionally substituted with a C6 cycloalkyl or C5 alkyl group; R4 is C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, -CH(CH2Q)2, and unsubstituted C 1~6 Selected from the group consisting of alkyl groups, where this C 3~6 The carbon ring is optionally substituted with -OH or -OMe; Each Q is a carbocyclic ring, heterocyclic ring, -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 alkenyls and H; Each R6 is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; R8 is C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; Each R is C 1~3 Alkyl, C 2~3Independently selected from the group consisting of alkenyls and H, where C 1~3 Alkyl groups are optionally substituted with -OH, -C(O)OH, -OMe, or -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, where C 1~18 Alkyl groups are optionally substituted with -OMe; Each R is H, C 3~14 Alkyl and C 3~14 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each Y is independent of C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0114] In some embodiments, the ionized lipid is expressed by formula (I): [ka] It may be a compound or salt or isomer of the compound, in which formula: 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 R2 and R3 are independently selected from the group consisting of alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 form a heterocycle or carbocycle together with the atom to which they are bonded; R4 is C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted 1~6Selected from the group consisting of alkyl groups, where Q is a carbocyclic, heterocyclic, -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 Selected from 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 -C(R)N(R)2C(O)OR, 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 alkenyls and H; Each R6 is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; R8 is C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6Selected from the group consisting of carbocyclic and heterocyclic rings; Each R is C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each Y is independent of C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0115] In some embodiments, a subset of compounds of formula (I) has R4 -(CH2) n Q, -(CH2) n If it is CHQR, -CHQR, or -CQ(R)2, then (i) if n is 1, 2, 3, 4, or 5, Q is not -N(R)2, or (ii) if n is 1 or 2, Q is not a 5, 6, or 7-membered heterocycloalkyl.

[0116] 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 alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 together with the atoms to which they are bonded form a heterocycle or a carbon ring; R4 is C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 Selected from the group consisting of alkyl groups, where Q is C 3~6 5-14 member heteroaryl, -OR, -O(CH2) having one or more heteroatoms selected from a carbocyclic ring, N, O, and S. 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 (oxo(=O), OH, amino, monoalkylamino or dialkylamino, and C 1~3 Selected from 5- to 14-membered heterocycloalkyl groups having one or more substituents selected from alkyl groups, where each n is independently selected from 1, 2, 3, 4, and 5; Each R5, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls and H; Each R6, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; R8 is C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; Each R, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each Y independently, C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. This includes salts or isomers thereof.

[0117] In some embodiments, another subset of compounds of formula (I) includes: R1 is C 5~30 Alkyl, C 5~20Selected 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 alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 together with the atoms to which they are bonded form a heterocycle or a carbon ring; R4 is C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 Selected from the group consisting of alkyl groups, where Q is C 3~6 A carbon ring, a 5- to 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 Selected from 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, where each n is independently selected from 1, 2, 3, 4, and 5; Q is a 5- to 14-membered complex ring, and (i) R4 is -(CH2) n Q(n is 1 or 2) or (ii) R4 is -(CH2) n (iii) If R4 is CHQR (n is 1), or if R4 is -CHQR and -CQ(R)2, Q is either a 5-14 member heteroaryl or an 8-14 member heterocycloalkyl; Each R5, C 1~3Alkyl, C 2~3 Independently selected from the group consisting of alkenyls and H; Each R6, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; R8 is C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; Each R, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each Y independently, C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. This includes salts or isomers thereof.

[0118] 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 alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 together with the atoms to which they are bonded form a heterocycle or a carbon ring; R4 is C 3~6 Carbon ring, -(CH2) n Q, -(CH2) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C 1~6 Selected from the group consisting of alkyl groups, where Q is C 3~6 5-14 member heteroaryl, -OR, -O(CH2) having one or more heteroatoms selected from a carbocyclic ring, N, O, and S. 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)R 8、 -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 are selected, and each n is independently selected from 1, 2, 3, 4, and 5; Each R5, C 1~3 Alkyl, C 2~3Independently selected from the group consisting of alkenyls and H; Each R6, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; R8 is C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; R9 is H, CN, NO2, C 1~6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2~6 Alkenil, C 3~6 Selected from the group consisting of carbocyclic and heterocyclic rings; Each R, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each Y independently, C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. This includes salts or isomers thereof.

[0119] 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 alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 together with the atoms to which they are bonded form a heterocycle or a carbon ring; 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, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls and H; Each R6, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C 2~3 Selected from the group consisting of alkenyls and H; Each R, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 1~12 Independently selected from the group consisting of alkenils; Each Y independently, C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. This includes salts or isomers thereof.

[0120] 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 alkenyls, -R*YR'', -YR'', and -R*OR'', or R2 and R3 together with the atoms to which they are bonded form a heterocycle or a carbon ring; R4 is -(CH2) n Q, -(CH2) n A group consisting of CHQR, -CHQR, and -CQ(R)2 is selected, where Q is -N(R)2 and n is selected from 1, 2, 3, 4, and 5; Each R5, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls and H; Each R6, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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-, aryl groups, and heteroaryl groups; R7 is C 1~3 Alkyl, C2~3 Selected from the group consisting of alkenyls and H; Each R, C 1~3 Alkyl, C 2~3 Independently selected from the group consisting of alkenyls 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 Independently selected from the group consisting of alkenils; Each R* is C 1~12 Alkyl and C 1~12 Independently selected from the group consisting of alkenils; Each Y independently, C 3~6 It is a carbon ring; Each X is independently selected from the group consisting of F, Cl, Br, and I; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. This includes salts or isomers thereof.

[0121] In some embodiments, a subset of compounds of formula (I) includes formula (IA): [ka] This includes C, or its salts or isomers, where l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9, M1 is a bond or M'; and R4 is an unsubstituted C. 1~3 Alkyl, or -(CH2) nQ is a heteroaryl or heterocycloalkyl group, where Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -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-, aryl group, and heteroaryl group; R2 and R3 are H, C 1~14 Alkyl and C 2~14 It is independently selected from the group consisting of alkenils.

[0122] In some embodiments, a subset of compounds of formula (I) includes formula (II): [ka] This includes C, or its salts or isomers, where l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M'; R4 is an unsubstituted C 1~3 Alkyl, or -(CH2) n Q is a heteroaryl or heterocycloalkyl group, where n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -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-, aryl group, and heteroaryl group; R2 and R3 are H, C 1~14 Alkyl and C 2~14 It is independently selected from the group consisting of alkenils.

[0123] In some embodiments, a subset of compounds of formula (I) includes formulas (IIa), (IIb), (IIc), or (IIe): [ka] This includes the same as, or its salts or isomers, where R4 is as described herein.

[0124] In some embodiments, a subset of compounds of formula (I) includes formula (IId): [ka] This includes the compound or its salts or isomers, where n is 2, 3, or 4; m, R', R'', and R2-R6 are as described herein. For example, each of R2 and R3 is C 5~14 Alkyl and C 5~14 They can be independently selected from the group consisting of alkenils.

[0125] In some embodiments, compounds of formula (I) are selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0126] In further embodiments, the compound of formula (I) is selected from the group consisting of: [ka]

[0127] In some embodiments, the compounds of formula (I) are comprised of the following groups: [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] Selected from its salts and isomers.

[0128] In some embodiments, the ionized lipid is compound 429: [ka] or its salt.

[0129] In some embodiments, the ionized lipid is compound 18: [ka] or its salt.

[0130] In some embodiments, the lipid nanoparticle composition comprises a lipid component containing a compound described herein (for example, a compound according to formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId), or (IIe)).

[0131] In some embodiments, the LNP may be composed of an ionized lipid containing a central piperazine moiety. Such an LNP may advantageously consist of an ionized lipid, a phospholipid, and a PEG lipid, and may optionally contain a structural lipid or be lacking in structural lipids. In some embodiments, the phospholipid is DSPC or DOP.

[0132] Ionized lipids containing a central piperazine moiety as described herein may 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 to no immunogenicity. For example, the lipid compounds disclosed herein have lower immunogenicity compared to reference lipids (e.g., MC3, KC2, or DLinDMA). For example, formulations containing the lipids disclosed herein and a therapeutic or prophylactic agent have an increased therapeutic index compared to corresponding formulations containing the same therapeutic or prophylactic agent and a reference lipid (e.g., MC3, KC2, or DLinDMA).

[0133] Lipids are given by formula (III) [ka] It may be a compound of, or a salt or isomer thereof, in which, Ring A is, [ka] and; t is either 1 or 2; A1 and A2 are selected independently from CH or N; Z is either CH2 or absent, where if Z is CH2, the dashed lines (1) and (2) represent single bonds, respectively; if Z is absent, neither the dashed lines (1) nor (2) exist; R1, R2, R3, R4, and R5 are C 5~20 Alkyl, C 5~20 Independently selected from the group consisting of alkenyls, -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)2-, aryl groups, and heteroaryl groups; X 1 , X 2 , and X 3 These are independently selected from the group consisting of bonds, -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 independent of C 3~6 It is a carbon ring; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each R is C 1~3 Alkyl and C 3~6 Independently selected from the group consisting of carbon rings; Each R' is C 1~12 Alkyl, C 2~12 Independently selected from the group consisting of alkenyls and H; Each R is C 3~12Alkyl and C 3~12 Independently selected from the group consisting of alkenils, In the equation, ring A [ka] If that is the case, i)X 1 , X 2 , and X 3 At least one of them is not -CH2-; and / or ii) At least one of R1, R2, R3, R4, and R5 is -R”MR’.

[0134] In some embodiments, the compound is one of the formulas (IIIa1) to (IIIa6): [ka]

[0135] Any compound of formula (III) or (IIIa1) to (IIIa6) contains, where applicable, one or more of the following characteristics:

[0136] In some embodiments, ring A is [ka] That is the case.

[0137] In some embodiments, ring A is [ka] That is the case.

[0138] In some embodiments, ring A is [ka] That is the case.

[0139] In some embodiments, ring A is [ka] That is the case.

[0140] In some embodiments, ring A is [ka] That is the case.

[0141] In some embodiments, ring A is [ka] And the N atom in the ring is X 2 It is connected to this.

[0142] In some embodiments, Z is CH2.

[0143] In some embodiments, Z does not exist.

[0144] In some embodiments, at least one of A1 and A2 is N.

[0145] In some embodiments, each of A1 and A2 is N.

[0146] In some embodiments, each of A1 and A2 is CH.

[0147] In some embodiments, A1 is N and A2 is CH.

[0148] In some embodiments, A1 is CH and A2 is N.

[0149] In some embodiments, X 1 , X 2 , and X 3 At least one of them is not -CH2-. For example, in a particular embodiment, X 1 is not -CH2-. In some embodiments, X 1 , X 2 , and X3 At least one of them is -C(O)-.

[0150] In some embodiments, X 2 These are -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)-.

[0151] 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 It is -CH2-.

[0152] In some embodiments, X 3 This is a bond or -(CH2)2-.

[0153] 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.

[0154] In some embodiments, at least one of R1, R2, R3, R4, and R5 is -R"MR'. In some embodiments, at most one of R1, R2, R3, R4, and R5 is -R"MR'. For example, at least one of R1, R2, and R3 may be -R"MR', and / or at least one of R4 and R5 may be -R"MR'. In some particular 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)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 a C3 alkyl. In some particular embodiments, each R'' is a C3 alkyl. In some embodiments, at least one R'' is a C5 alkyl. In certain embodiments, each R'' is a C5 alkyl. In some embodiments, at least one R'' is a C6 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.

[0155] In some embodiments, at least one of R1, R2, R3, R4, and R5 is C 12 It is alkyl. In a particular embodiment, each of R1, R2, R3, R4, and R5 is C 12 It is alkyl.

[0156] In a particular embodiment, the compound is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0157] In other embodiments, lipids are given by formula (IV) [ka] or having a salt or isomer thereof, in the formula, A1 and A2 are independently selected from CH or N, and at least one of A1 and A2 is N; Z is either CH2 or absent, where if Z is CH2, the dashed lines (1) and (2) represent single bonds, respectively; if Z is absent, neither the dashed lines (1) nor (2) exist; R1, R2, R3, R4, and R5 are C 6~20 Alkyl, C 6~20 Independently selected from the group consisting of alkenils; In the equation, ring A [ka] If that is the case, i) Are R1, R2, R3, R4, and R5 the same (where R1 is C)? 12 Alkyl, C 18 Alkyl, or C 18 Not Alkenil); ii) Only one of R1, R2, R3, R4, and R5 is C 6~20 Selected from Alkenil; iii) At least one of R1, R2, R3, R4, and R5 has a different number of carbon atoms than the other at least one of R1, R2, R3, R4, and R5; iv) R1, R2, and R3 are C 6~20 Selected from alkenyl, R4 and R5 are C 6~20 Selected from alkyl groups; or v) R1, R2, and R3 are C 6~20 Selected from alkyl, R4 and R5 are C 6~20 Selected from Alkenil.

[0158] In some embodiments, the compound is of formula (IVa): [ka] It belongs to them.

[0159] Compounds of formula (IV) or (IVa) include, where applicable, one or more of the following characteristics:

[0160] In some embodiments, Z is CH2.

[0161] In some embodiments, Z does not exist.

[0162] In some embodiments, at least one of A1 and A2 is N.

[0163] In some embodiments, each of A1 and A2 is N.

[0164] In some embodiments, each of A1 and A2 is CH.

[0165] In some embodiments, A1 is N and A2 is CH.

[0166] In some embodiments, A1 is CH and A2 is N.

[0167] In some embodiments, R1, R2, R3, R4, and R5 are the same, C 12 Alkyl, C 18 Alkyl, or C 18 It is not an alkenyl. In some embodiments, R1, R2, R3, R4, and R5 are the same, and C9 alkyl or C 14 It is alkyl.

[0168] In some embodiments, only one of R1, R2, R3, R4, and R5 is C 6~20 Selected from alkenyls. In certain such embodiments, R1, R2, R3, R4, and R5 have the same number of carbon atoms. In some embodiments, R4 is C 5~20 Selected from alkenyls. For example, R4 is C 12 Alkenyl or C 18 It could be Alkenil.

[0169] In some embodiments, at least one of R1, R2, R3, R4, and R5 has a different number of carbon atoms than the other at least one of R1, R2, R3, R4, and R5.

[0170] In a particular embodiment, R1, R2, and R3 are C 6~20 Selected from alkenyl, R4 and R5 are C 6~20 Selected from alkyl. In other embodiments, R1, R2, and R3 are C 6~20 Selected from alkyl, R4 and R5 are C 6~20 Selected from alkenyls. 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 may have 6, 8, 9, 12, 14, or 18 carbon atoms. In some embodiments, R1, R2, and R3, or R4 and R5 are C 18 The alkenyl is (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.

[0171] 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.

[0172] In some embodiments, the compound is selected from the group consisting of: [ka] [ka] [ka]

[0173] In other embodiments, the compound is of formula (V) [ka] or having a salt or isomer thereof, in the formula, A3 is either CH or N; A4 is CH2 or NH; at least one of A3 and A4 is N or NH; Z is either CH2 or absent, where if Z is CH2, the dashed lines (1) and (2) represent single bonds, respectively; if Z is absent, neither the dashed lines (1) nor (2) exist; R1, R2, and R3 are C 5~20 Alkyl, C 5~20 Independently selected from the group consisting of alkenyls, -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)2-, aryl groups, and heteroaryl groups; X 1 and X 2 These 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 independent of C 3~6 It is a carbon ring; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each R is C 1~3 Alkyl and C 3~6 Independently selected from the group consisting of carbon rings; Each R' is C 1~12 Alkyl, C 2~12Independently selected from the group consisting of alkenyls and H; Each R is C 3~12 Alkyl and C 3~12 It is independently selected from the group consisting of alkenils.

[0174] In some embodiments, the compound is given by formula (Va): [ka] It belongs to them.

[0175] Compounds of formula (V) or (Va) include, where applicable, one or more of the following characteristics:

[0176] In some embodiments, Z is CH2.

[0177] In some embodiments, Z does not exist.

[0178] In some embodiments, at least one of A3 and A4 is N or NH.

[0179] In some embodiments, A3 is N and A4 is NH.

[0180] In some embodiments, A3 is N and A4 is CH2.

[0181] In some embodiments, A3 is CH and A4 is NH.

[0182] In some embodiments, X 1 and X 2 At least one of them is not -CH2-. For example, in a particular embodiment, X 1 is not -CH2-. In some embodiments, X 1 and X 2 At least one of them is -C(O)-.

[0183] In some embodiments, X 2These are -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)-.

[0184] In some embodiments, R1, R2, and R3 are C 5~20 Alkyl and C 5~20 They are independently selected from the group consisting of alkenyls. In some embodiments, R1, R2, and R3 are the same. In a particular embodiment, R1, R2, and R3 are C6, C9, C 12 , or C 14 It is alkyl. In other embodiments, R1, R2, and R3 are C 18 These are alkenils. For example, R1, R2, and R3 could be rhinorails.

[0185] In some embodiments, the compound is selected from the group consisting of: [ka]

[0186] In another aspect, this disclosure is related to formula (VI): [ka] The present invention provides a compound or salt or isomer thereof, in which, A6 and A7 are independently selected from CH or N, where at least one of A6 and A7 is N; Z is either CH2 or absent, where if Z is CH2, the dashed lines (1) and (2) represent single bonds, respectively; if Z is absent, neither the dashed lines (1) nor (2) exist; X 4 and X 5These 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 5~20 Alkyl, C 5~20 Independently selected from the group consisting of alkenyls, -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)2-, aryl groups, and heteroaryl groups; Each Y is independent of C 3~6 It is a carbon ring; Each R* is C 1~12 Alkyl and C 2~12 Independently selected from the group consisting of alkenils; Each R is C 1~3 Alkyl and C 3~6 Independently selected from the group consisting of carbon rings; Each R' is C 1~12 Alkyl, C 2~12 Independently selected from the group consisting of alkenyls and H; Each R is C 3~12 Alkyl and C 3~12 It is independently selected from the group consisting of alkenils.

[0187] In some embodiments, R1, R2, R3, R4, and R5 are C 6~20 Alkyl and C 6~20 It is independently selected from the group consisting of alkenils.

[0188] 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.

[0189] In some embodiments, at least one of R1, R2, R3, R4, and R5 is C 9~12 It is alkyl. In a particular embodiment, each of R1, R2, R3, R4, and R5 is independently C9, C 12 , or C 14 It is alkyl. In a particular embodiment, each of R1, R2, R3, R4, and R5 is a C9 alkyl.

[0190] In some embodiments, A6 is N and A7 is N. In some embodiments, A6 is CH and A7 is N.

[0191] In some embodiments, X4 is -CH2- and X5 is -C(O)-. In some embodiments, X4 and X5 are -C(O)-.

[0192] In some embodiments, when A6 is N and A7 is N, at least one of X4 and X5 is not -CH2-, but for example, 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'.

[0193] In some embodiments, at least one of R1, R2, R3, R4, and R5 is not -R"MR'.

[0194] In some embodiments, the compound is [ka] That is the case.

[0195] In one embodiment, the compound has the following formula: [ka]

[0196] PEG and PEG-modified lipids In general, some of the other lipid components (e.g., PEG lipids) of the various formulas described herein may be synthesized as described in International Patent Application PCT / US2016 / 000129, filed on 10 December 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents” (which is incorporated in its entirety by reference).

[0197] The lipid component of the lipid nanoparticle composition may include one or more molecules containing polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternatively referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids may be selected from a 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, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. In some embodiments, the PEG lipid is DMG-PEG 2k or compound 428.

[0198] In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG. PEG-DMG has the following structure: [ka]

[0199] In some embodiments, the nanoparticles described herein contain about 1 mol% to about 5 mol% of PEG-lipids. In some embodiments, the nanoparticles contain about 1 mol% to about 2.5 mol% of PEG-lipids.

[0200] In one embodiment, a 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 a hydroxyl group 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 or hydroxy-PEGylated lipid contains an -OH group at the end of the PEG chain. Each possibility represents a separate embodiment of the present invention.

[0201] In certain embodiments, the PEG lipid useful in the present invention is a compound of formula (VII). In this specification, formula (VII): [ka] A compound or salt thereof is provided, in the formula: R 3 is -OR O and; R O is a hydrogen atom, an optionally substituted alkyl group, or an oxygen protecting group; r is an integer between 1 and 100 (including both ends); L 1 C is a C that is optionally substituted. 1~10 It is an alkylene, and here this optionally substituted C 1~10At least one methylene group of the alkylene is independently and optionally substituted with a carbocyclylene, optionally substituted with a heterocyclylene, optionally substituted with an arylene, optionally substituted with a heteroarylene, -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 )- is replaced by; D is a portion obtained by click chemistry or a portion that can be cleaved under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is the formula: [ka] It belongs to; L 2 Each of these cases is independently, combined, or optionally substituted C 1~6 It is an alkylene, and here this optionally substituted C 1~6 One methylene unit of an alkylene can be optionally -O- or -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 )- is replaced by; R 2 Each of these cases is independently and arbitrarily substituted C 1~30 Alkyl, optionally substituted C 1~30 C is an alkenyl or optionally substituted C. 1~30It is an alkinyl; optionally, here R 2 One or more methylene units are independently and optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with 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 It is replaced by S(O)2O-; R NEach of these cases is independently a hydrogen, optionally substituted alkyl, or nitrogen protecting group; Ring B is an arbitrarily substituted carbocyclyl, an arbitrarily substituted heterocyclyl, an arbitrarily substituted aryl, or an arbitrarily substituted heteroaryl; p is either 1 or 2.

[0202] In certain embodiments, the compound of formula (VII) is a PEG-OH lipid (i.e., R 3 は-OR O And R O ( is hydrogen). In certain embodiments, the compound of formula (VII) is formula (VII-OH): [ka] It is either a substance or its salt.

[0203] In certain embodiments, D is a moiety obtained by click chemistry (e.g., triazole). In certain embodiments, the compound of formula (VII) is formula (VII-a-1) or (VII-a-2): [ka] It is either a substance or its salt.

[0204] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of the following, or a salt thereof, in the formula, s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0205] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0206] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0207] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of the following (where r is between 1 and 100 in the formula), or a salt thereof.

[0208] In certain embodiments, D is a portion that can be cleaved under physiological conditions (e.g., ester, amide, carbonate, carbamate, urea). In certain embodiments, the compound of formula (VII) is formula (VII-b-1) or (VII-b-2): [ka] It is either a substance or its salt.

[0209] In certain embodiments, the compound of formula (VII) is (VII-b-1-OH) or (VII-b-2-OH): [ka] It is either a substance or its salt.

[0210] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0211] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0212] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0213] In a particular embodiment, the compound of formula (VII) is: [ka] It is one of them, or a salt thereof.

[0214] 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). In this specification, formula (VIII): [ka] A compound or salt thereof is provided, in the formula: R 3 is -OR O and; R O is a hydrogen, optionally substituted alkyl, or oxygen protecting group; r is an integer between 1 and 100 (including both ends); R 5 C is a C that is optionally substituted. 10~40 Alkyl, optionally substituted C 10~40 C is an alkenyl or optionally substituted C. 10~40 It is alkinyl; optional, R 5One or more methylene groups are optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with 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 It is replaced by S(O)2O-; R N Each of these examples is, independently, hydrogen, an optionally substituted alkyl group, or a nitrogen protecting group.

[0215] In a particular embodiment, the compound of formula (VIII) is of formula (VIII-OH): [ka] It is either a substance or its salt.

[0216] In a particular embodiment, the compound of formula (VIII) is: [ka] It is one of the following, or a salt thereof. In some embodiments, r is 45.

[0217] In a particular embodiment, the compound of formula (VIII) is: [ka] It is one of the following, or a salt thereof. In some embodiments, r is 45.

[0218] In yet another embodiment, the compound of formula (VIII) is [ka] or its salt.

[0219] In some embodiments, the compound of formula (VIII) is [ka] That is the case.

[0220] In a particular embodiment, the PEG lipid is given by the following formula: [ka] It is one of the following, or a salt thereof. In some embodiments, r is 45.

[0221] Phospholipids As defined herein, phospholipids are any lipids containing a phosphate group. Phospholipids are a subset of noncationic lipids. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, for example, one or more (poly)unsaturated lipids. Phospholipids may be assembled into one or more lipid bilayers. Generally, phospholipids may include 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 an unrestricted 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-natural species, including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes, are also considered. For example, phospholipids may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkyne groups may undergo copper-catalyzed cycloaddition upon exposure to azides. Such reactions may be useful for functionalizing the lipid bilayer of nanoparticle compositions to promote membrane permeability or cell recognition, or for conjugating nanoparticle compositions with useful components such as targeting or imaging moieties (e.g., dyes).

[0222] In some embodiments, the nanoparticles described herein contain about 5 mol% to about 15 mol% of phospholipids. In some embodiments, the nanoparticles contain about 8 mol% to about 13 mol% of phospholipids. In some embodiments, the nanoparticles contain about 10 mol% to about 12 mol% of phospholipids.

[0223] Phospholipids that are useful or potentially useful in this composition and method are: 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-Dimiristoyl-sn-glycerophosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Diundecanoyl-sn-glycerophosphocholine (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-Dydocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-Difitanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-Difitanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4 ME 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-Dydocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and The following can be selected from a 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.

[0224] In some embodiments, the lipid nanoparticle composition includes DSPC. In certain embodiments, the lipid nanoparticle composition includes DOPE. In some embodiments, the lipid nanoparticle composition includes both DSPC and DOPE. In some embodiments, the lipid nanoparticles are 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (4 ME 16:0 PE) [ka] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC) [ka] 1,2-Difitanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(sodium salt)(4ME 16:0 PG), or [ka] 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4 ME 16:0 PS) [ka] or a mixture thereof.

[0225] Examples of phospholipids, though not limited to them, include the following: [ka] [ka]

[0226] In certain embodiments, the phospholipids that are useful or potentially useful in the present invention are analogs or variants of DSPCs.

[0227] In certain embodiments, the phospholipid that is useful or potentially useful in the present invention is formula (IX): [ka] A compound or salt thereof, in the formula: Each R 1 is independently an alkyl group that is H or optionally substituted; or optionally two R groups 1 However, it is linked together with the intervening atom to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 However, when connected together with intervening atoms, they form optionally substituted bicyclic carbocyclines or optionally substituted bicyclic heterocyclines; 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 the formula: [ka] It belongs to; L 2 Each of these cases is independently, combined, or optionally substituted C 1~6 It is an alkylene, and here this optionally substituted C 1~6One methylene unit of an alkylene can be optionally -O- or -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 )- is replaced by; R 2 Each of these cases is independently and arbitrarily substituted C 1~30 Alkyl, optionally substituted C 1~30 C is an alkenyl or optionally substituted C. 1~30 It is an alkinyl; optionally, here R 2 One or more methylene units are independently and optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with 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 It is replaced by S(O)2O-; R N Each of these cases is independently a hydrogen, optionally substituted alkyl, or nitrogen protecting group; Ring B is an arbitrarily substituted carbocyclyl, an arbitrarily substituted heterocyclyl, an arbitrarily substituted aryl, or an arbitrarily substituted heteroaryl; p is either 1 or 2. (However, this compound has the formula: [ka] (In the formula, R 2 Each of these examples is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl (not).

[0228] In certain embodiments, the phospholipid that is useful or potentially useful in the present invention is formula (IX): [ka] A compound or salt thereof, in the formula: Each R 1 is an alkyl group that is independently and optionally substituted; or optionally, two R 1However, it is linked together with the intervening atom to form an optionally substituted monocyclic carbocyclyl or an optionally substituted monocyclic heterocyclyl, or optionally, three R 1 However, when connected together with intervening atoms, they form optionally substituted bicyclic carbocyclines or optionally substituted bicyclic heterocyclines; 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 the formula: [ka] It belongs to; L 2 Each of these cases is independently, combined, or optionally substituted C 1~6 It is an alkylene, and here this optionally substituted C 1~6 One methylene unit of an alkylene can be optionally -O- or -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 )- is replaced by; R 2 Each of these cases is independently and arbitrarily substituted C 1~30 Alkyl, optionally substituted C 1~30 C is an alkenyl or optionally substituted C. 1~30 It is an alkinyl; optionally, here R 2 One or more methylene units are independently and optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with 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 It is replaced by S(O)2O-; R N Each of these cases is independently a hydrogen, optionally substituted alkyl, or nitrogen protecting group; Ring B is an arbitrarily substituted carbocyclyl, an arbitrarily substituted heterocyclyl, an arbitrarily substituted aryl, or an arbitrarily substituted heteroaryl; p is either 1 or 2. (However, this compound has the formula: [ka] (In the formula, R 2 Each of these examples is independently an unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl (not).

[0229] Modification of phospholipid heads In certain embodiments, the phospholipids that are useful or potentially useful in the present invention include a modified phospholipid head (e.g., a modified choline group). In certain embodiments, the phospholipid having a modified head is a DSPC or analog thereof having a modified quaternary amine. For example, in the embodiment of formula (IX), R 1 At least one of them is not methyl. In a particular embodiment, R 1 At least one of them is not hydrogen or methyl. In certain embodiments, the compound of formula (IX) is the following: [ka] One of the following, or a salt thereof, in the formula: 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.

[0230] In a particular embodiment, the compound of formula (IX) is: [ka] It is one of them, or a salt thereof.

[0231] In a particular embodiment, the compound of formula (IX) is: [ka] [ka] It is one of them, or a salt thereof.

[0232] In a particular embodiment, the compound of formula (IX) is formula (IX-a): [ka] It is either a substance or its salt.

[0233] In certain embodiments, phospholipids useful or potentially useful in the present invention include a modified core. In certain embodiments, the phospholipids having a modified core described herein are DSPCs or analogs thereof having a modified core structure. For example, in a certain embodiment of formula (IX-a), group A is of the following formula: [ka] It is not.

[0234] In a particular embodiment, the compound of formula (IX-a) is the following: [ka] It is one of them, or a salt thereof.

[0235] In a particular embodiment, the compound of formula (IX) is: [ka] It is one of them, or a salt thereof.

[0236] In certain embodiments, phospholipids useful or potentially useful in the present invention include a cyclic moiety instead of a glyceride moiety. In certain embodiments, phospholipids useful in the present invention are DSPCs or analogs thereof having a cyclic moiety instead of a glyceride moiety. In certain embodiments, a compound of formula (IX) is formula (IX-b): [ka] It is either a substance or its salt.

[0237] In a particular embodiment, the compound of formula (IX-b) is formula (IX-b-1): [ka] It is a substance or a salt thereof, in the formula: w is 0, 1, 2, or 3.

[0238] In a particular embodiment, the compound of formula (IX-b) is formula (IX-b-2): [ka] It is either a substance or its salt.

[0239] In a particular embodiment, the compound of formula (IX-b) is formula (IX-b-3): [ka] It is either a substance or its salt.

[0240] In a particular embodiment, the compound of formula (IX-b) is formula (IX-b-4): [ka] It is either a substance or its salt.

[0241] In a particular embodiment, the compound of formula (IX-b) is the following: [ka] It is one of them, or a salt thereof.

[0242] Modification of phospholipid tails In certain embodiments, phospholipids that are useful or potentially useful in the present invention include a modified tail. In certain embodiments, phospholipids that are useful or potentially useful in the present invention are DSPCs or analogs thereof having a modified tail. As used herein, a “modified tail” can be a tail having a relatively short or long aliphatic chain, a branched aliphatic chain, a substituted aliphatic chain, an aliphatic chain in which one or more methylene groups are replaced by cyclic or heteroatomic groups, or any combination thereof. For example, in certain embodiments, the compound of formula (IX) is the compound of formula (IX-a) or a salt thereof, where R 2 In at least one case, R 2 Each of these cases is a C that has been arbitrarily substituted. 1~30 It is alkyl, and here, R 2 One or more methylene units are independently and optionally substituted with carbocyclylene, optionally substituted with heterocyclylene, optionally substituted with arylene, optionally substituted with 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 It is replaced by S(O)2O-.

[0243] In a particular embodiment, the compound of formula (IX) is formula (IX-c): [ka] It is a substance or a salt thereof, in the formula: Each x is an independent integer between 0 and 30 (including both ends); In each case, G is a randomly substituted carbocyclylene, a randomly substituted heterocyclylene, a randomly substituted arylene, a randomly 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 It is independently selected from the group consisting of )S(O)2O-. Each possibility represents a separate embodiment of the present invention.

[0244] In a particular embodiment, the compound of formula (IX-c) is formula (IX-c-1): [ka] It is a substance or a salt thereof, in the formula: Each case of v is independently 1, 2, or 3.

[0245] In a particular embodiment, the compound of formula (IX-c) is formula (IX-c-2): [ka] It is either a substance or its salt.

[0246] In a particular embodiment, the compound of formula (IX-c) is of the following formula: [ka] It is either a substance or its salt.

[0247] In a particular embodiment, the compound of formula (IX-c) is as follows: [ka] or its salt.

[0248] In a particular embodiment, the compound of formula (IX-c) is formula (IX-c-3): [ka] It is either a substance or its salt.

[0249] In a particular embodiment, the compound of formula (IX-c) is of the following formula: [ka] It is either a substance or its salt.

[0250] In a particular embodiment, the compound of formula (IX-c) is as follows: [ka] or its salt.

[0251] In certain embodiments, the phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, where the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, the phospholipid useful or potentially useful in the present invention is a compound of formula (IX), where n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, the compound of formula (IX) is: [ka] It is one of them, or a salt thereof.

[0252] In a particular embodiment, the compound of formula (IX) is: [ka] [ka] It is one of them, or a salt thereof.

[0253] alternative lipids In certain embodiments, alternative lipids are used instead of the phospholipids of the present invention. Non-limiting examples of such alternative lipids include: [ka] [ka] These are some examples.

[0254] structural lipids The lipid components of a lipid nanoparticle composition may include one or more structural lipids. Introducing structural lipids into lipid nanoparticles may help reduce the aggregation of other lipids within the particles. Structural lipids 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 structural lipid is a sterol. As defined herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. Examples of structural lipids, but not limited to, include: [ka]

[0255] In some embodiments, the nanoparticles described herein may contain about 20 mol% to about 60 mol% of structural lipids. In some embodiments, the nanoparticles contain about 30 mol% to about 50 mol% of structural lipids. In some embodiments, the nanoparticles contain about 35 mol% of structural lipids. In some embodiments, the nanoparticles contain about 40 mol% of structural lipids. In some embodiments, the structural lipids may be cholesterol or the following structures: [ka] It is a compound that has [a certain characteristic].

[0256] Payload molecule The compositions of this disclosure can be used to deliver a wide variety of different agents to airway cells. Airway cells may be, for example, cells lining the respiratory tract in the oral cavity, nose, pharynx, or lungs. Therapeutic agents can mediate therapeutic effects in such airway cells (e.g., directly or via bystander effects). Typically, the therapeutic agents delivered by the compositions are nucleic acids, but non-nucleic acid agents such as small molecules, chemotherapeutic agents, peptides, polypeptides, and other biomolecules are also covered by this disclosure. Nucleic acids that can be delivered include DNA-based molecules (i.e., including deoxyribonucleotides) and RNA-based molecules (i.e., including ribonucleotides). Furthermore, nucleic acids may be in naturally occurring forms of molecules or in chemically modified forms of molecules (e.g., including one or more modified nucleotides).

[0257] Drugs to enhance protein expression In one embodiment, the therapeutic agent is a drug that enhances (i.e., increases, stimulates, or upregulates) protein expression. Non-limiting examples of types of therapeutic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR / Cas9 technology, ssDNA, and DNA (e.g., expression vectors).

[0258] In one embodiment, the therapeutic agent is a DNA therapeutic agent. The DNA molecule may be double-stranded DNA, single-stranded DNA (ssDNA), or a molecule that is partially double-stranded DNA, 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., having a triple-stranded portion and a double-stranded portion. The DNA molecule may be a circular DNA molecule or a linear DNA molecule.

[0259] A DNA therapeutic agent may be a DNA molecule capable of introducing a gene into a cell, for example, a DNA molecule that can encode a transcript and express it. For example, a DNA therapeutic agent may encode a protein of interest, thereby increasing the expression of that protein in the airways upon delivery by LNPs. In some embodiments, the DNA molecule may be of natural origin, for example, isolated from a natural source. In other embodiments, the DNA molecule is a synthetic molecule, for example, a synthetic DNA molecule produced 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.

[0260] The DNA therapeutic agents described herein, such as DNA vectors, may have a variety of different characteristics. The DNA therapeutic agents described herein, such as DNA vectors, may include non-coding DNA sequences. For example, the DNA sequence may include 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 a gene on the DNA sequence.

[0261] In some embodiments, the payload includes at least one component of a gene regulator, i.e., a system that modifies the nucleic acid sequence of a DNA molecule by altering nucleic acid bases, for example, by introducing insertions, deletions, mutations (e.g., missense mutations, silent mutations, or nonsense mutations), duplications, or inversions, or any combination thereof. In some embodiments, the gene regulator includes DNA base editing factors, CRISPR / Cas gene editing systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease systems, or transposase systems, or any combination thereof.

[0262] In some embodiments, the gene regulator includes template DNA. In some embodiments, the gene regulator does not include template DNA. In some embodiments, the gene regulator includes template RNA. In some embodiments, the gene regulator does not include template RNA.

[0263] In some embodiments, the gene regulator is a CRISPR / Cas gene editing system. In some embodiments, the CRISPR / Cas gene editing system comprises a guide RNA (gRNA) molecule containing a target sequence specific to the sequence of the target gene, and a peptide having nuclease activity (e.g., endonuclease activity) (e.g., Cas protein or its fragments (e.g., biologically active fragments) or variants, e.g., Cas9 protein, its fragments (e.g., biologically active fragments) or variants; Cas3 protein, its fragments (e.g., biologically active fragments) or variants; Cas12a protein, its fragments (e.g., biologically active fragments) or variants; Cas12e protein, its fragments (e.g., biologically active fragments) or variants; Cas13 protein, its fragments (e.g., biologically active fragments) or variants; or Cas14 protein, its fragments (e.g., biologically active fragments) or variants).

[0264] In some embodiments, the CRISPR / Cas gene editing system comprises a gRNA molecule containing 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., Cas protein or its fragments (e.g., biologically active fragments) or variants, e.g., Cas9 protein, its fragments (e.g., biologically active fragments) or variants; Cas3 protein, its fragments (e.g., biologically active fragments) or variants; Cas12a protein, its fragments (e.g., biologically active fragments) or variants; Cas12e protein, its fragments (e.g., biologically active fragments) or variants; Cas13 protein, its fragments (e.g., biologically active fragments) or variants; or Cas14 protein, its fragments (e.g., biologically active fragments) or variants).

[0265] In some embodiments, the CRISPR / Cas gene editing system comprises a nucleic acid encoding a gRNA molecule containing a target sequence specific to the sequence of the target gene, and a Cas9 protein, a fragment thereof (e.g., a biologically active fragment), or a variant thereof.

[0266] In some embodiments, the CRISPR / Cas gene editing system comprises a nucleic acid encoding a gRNA molecule containing a target sequence specific to the sequence of the target gene, and a nucleic acid encoding the Cas9 protein, a fragment thereof (e.g., a biologically active fragment), or a variant thereof.

[0267] In some embodiments, the CRISPR / Cas gene editing system further comprises template DNA. In some embodiments, the CRISPR / Cas gene editing system further comprises template RNA. In some embodiments, the CRISPR / Cas gene editing system further comprises reverse transcriptase.

[0268] 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 thereof (e.g., a biologically active fragment) or a variant; and / or having nuclease activity, e.g., endonuclease activity. In some embodiments, the ZFN system comprises a peptide having a Zn finger DNA-binding domain. In some embodiments, the Zn finger-binding domain comprises one, two, three, four, five, six, seven, eight, 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 I-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 thereof (e.g., a biologically active fragment) or a variant; and / or nuclease activity, e.g., endonuclease activity.

[0269] In some embodiments, the ZFN system comprises a nucleic acid encoding a peptide having a Zn finger DNA-binding domain. In some embodiments, the Zn finger-binding domain comprises one, two, three, four, five, six, seven, eight, or more zinc fingers. In some embodiments, the ZFN system comprises a nucleic acid encoding a peptide having nuclease activity, such as endonuclease activity. In some embodiments, the peptide having nuclease activity is a type II restriction I-like endonuclease, such as FokI endonuclease.

[0270] In some embodiments, the system further includes a template, such as template DNA.

[0271] In some embodiments of any of the methods, compositions, or cells disclosed herein, the gene regulator 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 thereof (e.g., a biologically active fragment), or a variant; 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 thereof (e.g., a biologically active fragment), or a variant. 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.

[0272] In some embodiments, the system comprises a nucleic acid encoding a peptide having a transcription activator-like (TAL) effector DNA-binding domain, a fragment thereof (e.g., a biologically active fragment), or a variant; and / or nuclease activity, such as 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 thereof (e.g., a biologically active fragment), or a variant. In some embodiments, the system comprises a nucleic acid encoding a peptide having nuclease activity, such as endonuclease activity. In some embodiments, the peptide having nuclease activity is a type II restriction 1-like endonuclease, such as FokI endonuclease.

[0273] In some embodiments, the system further includes a template, such as template DNA.

[0274] In some embodiments 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, for example, a LAGLIDADG endonuclease, GIY-YIG endonuclease, HNH endonuclease, His-Cys box endonuclease, or PD-(D / E)XK endonuclease, or a fragment (e.g., a biologically active fragment) or variant thereof, as described in Silva G. et al, (2011) Curr Gene Therapy 11(1):11-27.

[0275] In some embodiments, the meganuclease system comprises a nucleic acid encoding a peptide (e.g., a homing endonuclease) having a DNA-binding domain and nuclease activity. In some embodiments, the homing endonuclease includes, for example, LAGLIDADG endonuclease, GIY-YIG endonuclease, HNH endonuclease, His-Cys box endonuclease, or PD-(D / E)XK endonuclease, or a fragment (e.g., a biologically active fragment) or variant thereof, as described in Silva G. et al, (2011) Curr Gene Therapy 11(1):11-27.

[0276] In some embodiments, the system further includes a template, such as template DNA.

[0277] In some embodiments of the methods, compositions, or cells disclosed herein, the gene regulator is a transposase system. In some embodiments, the transposase system comprises a nucleic acid sequence encoding a peptide having reverse transcriptase activity and / or nuclease activity (e.g., a retrotransposon, e.g., an LTR retrotransposon or a non-LTR retrotransposon). In some embodiments, the transposase system comprises a template, e.g., an RNA template.

[0278] In one embodiment, the therapeutic agent is an RNA therapeutic agent. The RNA molecule may be single-stranded RNA, double-stranded RNA (dsRNA), or a molecule that is partially double-stranded RNA, i.e., a molecule having a double-stranded portion and a single-stranded portion. The RNA molecule may be a circular RNA molecule or a linear RNA molecule.

[0279] RNA therapeutics can introduce genes into cells, for example, by encoding a target protein to increase the expression of a target protein in airway cells. In some embodiments, the RNA molecule may be of natural origin, 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.

[0280] Non-limiting examples of RNA therapeutics include messenger RNA (mRNA) (e.g., those encoding the protein of interest), modified mRNA (mmRNA), mRNA with introduced microRNA binding sites (or miR binding sites), modified RNA containing functional RNA elements, microRNA (miRNA), antagomyl, small (short) interfering RNA (siRNA) (including shortmer and dicer substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA), and those encoding components of CRISPR / Cas9 technology, each of which is further described in the following subsections. In some embodiments, the RNA regulator includes an RNA base editing system. In some embodiments, the RNA base editing system includes a deaminase, e.g., RNA-specific adenosine deaminase (ADAR); a Cas protein, its fragment (e.g., a biologically active fragment) or variant; and / or guide RNA. In some embodiments, the RNA base editing system further includes a template, such as a DNA template or an RNA template.

[0281] mRNA may be naturally occurring mRNA or mRNA that does not exist in nature. mRNA may contain one or more modified nucleic acid bases, modified nucleosides, or modified nucleotides, as described below, in which case mRNA may be referred to as “modified mRNA” or “mmRNA”. As used herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleic acid base”). As used herein, “nucleotide” is defined as a nucleoside containing a phosphate group.

[0282] mRNA may contain a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and / or a coding region (e.g., an open reading frame). mRNA may contain any appropriate 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., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) base pairs. Any number (e.g., all, some, or zero) nucleic acid bases, nucleosides, or nucleotides may be substituted, modified, or otherwise non-naturally occurring analogs of the canonical species. In certain embodiments, all of a particular nucleic acid base type may be modified.

[0283] In some embodiments, the mRNA described herein may include a 5' cap structure, a strand termination nucleotide, and optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem-loop, a poly-A sequence, and / or a polyadenylation signal.

[0284] A 5' cap structure or cap species is a compound containing two nucleoside moieties linked by a linker, and may be selected from naturally occurring caps, non-naturally occurring caps or cap analogs, or anti-reverse caps (ARCAs). A cap species may contain one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may contain a guanine nucleotide and a guanine (G) nucleotide methylated at position 7, linked by a triphosphate bond at position 5', such as m7G(5')ppp(5')G, commonly described as m7GpppG. A cap species may also be an anti-reverse cap analog. An unrestricted list of possible cap types 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.

[0285] mRNA may contain, in addition to or instead, a chain termination nucleoside. For example, the chain termination nucleoside may include a nucleoside 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, e.g., 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine. In some embodiments, the introduction of a chain termination nucleotide into mRNA, for example at the 3' end, may result in mRNA stabilization, as described, for example, in International Patent Publication WO2013 / 103659.

[0286] mRNA may contain, or may contain additional, stem loops, such as histone stem loops. Stem loops may contain two, three, four, five, six, seven, eight, or more nucleotide base pairs. For example, a stem loop may contain four, five, six, seven, or eight nucleotide bases. Stem loops may be located in any region of mRNA. For example, a stem loop may be located before, or after, an untranslated region (5' or 3' untranslated region), a coding region, or within a polyA sequence or tail. In some embodiments, stem loops may affect one or more functions of mRNA, such as translation initiation, translation efficiency, and / or transcription termination.

[0287] mRNA may contain, instead or additionally, a poly(A) sequence and / or a polyadenylation signal. The poly(A) sequence may consist entirely or primarily of adenine nucleotides or their analogs or derivatives. The poly(A) sequence may be a tail located adjacent to the 3' untranslated region of mRNA. In some embodiments, the poly(A) sequence may affect mRNA nuclear export, translation, and / or stability.

[0288] mRNA may contain microRNA binding sites, either in place of or in addition to them.

[0289] In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region (having an intervening sequence containing an internal ribosome entry site (IRES) sequence that enables internal translation initiation between the first and second coding regions, or an intervening sequence encoding a self-cleaving peptide such as a 2A peptide). IRES sequences and 2A peptides are typically used to enhance the expression of multiple proteins from the same vector. For example, various IRES sequences, including encephalomyocarditis virus IRESs, are known and available in the art and can be used.

[0290] In some embodiments, the mRNA of this disclosure comprises one or more modified nucleic acid bases, modified nucleosides, or modified nucleotides (referred to as “modified mRNA” or “mmRNA”). In some embodiments, the modified mRNA may have useful properties compared to the reference unmodified mRNA, including enhanced stability, intracellular retention, enhanced translation, and / or substantial absence of induction of the innate immune response in the cell into which the mRNA is introduced. Thus, the use of modified mRNA may not only increase protein production efficiency and intracellular retention of nucleic acids, but also reduce immunogenicity.

[0291] In some embodiments, the mRNA contains one or more (e.g., 1, 2, 3, or 4) different modified nucleic acid bases, modified nucleosides, or modified nucleotides. In some embodiments, the mRNA contains 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 nucleic acid bases, modified nucleosides, or modified nucleotides. In some embodiments, the modified mRNA may reduce degradation in the cell into which the mRNA is introduced compared to the corresponding unmodified mRNA.

[0292] In some embodiments, the modified nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onyribonucleoside, 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine (m3U), 5-Methoxyuridine (mo5U), Uridine 5-oxyacetic acid (cmo5U), Uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (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-pseuduridine, 5-taurinomethyl-uridine (τm 5U), 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseuduridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseuduridine (m1s4ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m3ψ), 2-thio-1-methyl-pseuduridine,1-Methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine (D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-pseuduridine, 4-methoxy-2-thiopseuduridine, N1-methylpseuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseuduridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2 '-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methyl-pseudridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine Examples include din (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-ala-uridine, 2'-F-uridine, 2'-OH-ala-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0293] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), and 5-hydr Roxymethylcytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl Examples include thyl-cytidine (m5Cm), N4-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-ala-cytidine, 2'-F-cytidine, and 2'-OH-ala-cytidine.

[0294] In some embodiments, the modified nucleic acid base is a modified adenine.Exemplary nucleic acid bases and nucleosides having modified adenine 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-di Aminopurines, 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-thre 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-methyl Examples include 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-ala-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0295] In some embodiments, the modified nucleic acid base is modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanosine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), yosine (imG), methylyosine (mimG), 4-demethylyosine (imG-14), isoyosine (imG2), wibutosine (yW), peroxywibutosine (o2yW), hydroxywibutosine (OhyW), undermodified hydroxywibutosine (OhyW*), 7-deaza-guanosine, quosine (Q), and epixine. Siquosin (oQ), galactosyl-quiosin (galQ), mannosyl-quiosin (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), alkaeosin (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-guano N2-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 Examples include '-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-ala-guanosine, and 2'-F-guanosine.

[0296] In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (for example, two, three, or four combinations of the aforementioned modified nucleic acid bases).

[0297] In some embodiments, the modified nucleic acid bases are pseudouridine (ψ), N1-methylpseudridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudridine, 2-thio-1-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the mRNA of the Disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases). In one embodiment, the modified nucleic acid base is N1-methylpseudridine (m1ψ), and the mRNA of this disclosure is completely modified with N1-methylpseudridine (m1ψ). In some embodiments, N1-methylpseudridine (m1ψ) corresponds to 75-100% of the uracil in the mRNA. In some embodiments, N1-methylpseudridine (m1ψ) corresponds to 100% of the uracil in the mRNA.

[0298] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include N4-acetylcytidine (ac4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methylpseudoisocytidine, 2-thiocytidine (s2C), and 2-thio-5-methylcytidine. In some embodiments, the mRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0299] In some embodiments, the modified nucleic acid base is a modified adenine. Exemplary nucleic acid bases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenosine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, the mRNA of this disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (for example, combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0300] In some embodiments, the modified nucleic acid base is a modified guanine. Exemplary nucleic acid bases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), waiosine (imG), methylwaiosine (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 this disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (for example, combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0301] In some embodiments, the modified nucleic acid bases are 1-methyl-pseudridine (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 Disclosure comprises one or more combinations of the aforementioned modified nucleic acid bases (e.g., combinations of two, three, or four of the aforementioned modified nucleic acid bases).

[0302] In some embodiments, the mRNA contains pseudouridine (ψ). In some embodiments, the mRNA contains pseudouridine (ψ) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 1-methylpseudridine (m1ψ). In some embodiments, the mRNA contains 1-methylpseudridine (m1ψ) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 2-thiouridine (s2U). In some embodiments, the mRNA contains 2-thiouridine and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 5-methoxyuridine (mo5U). In some embodiments, the mRNA contains 5-methoxyuridine (mo5U) and 5-methylcytidine (m5C). In some embodiments, the mRNA contains 2'-O-methyluridine. In some embodiments, the mRNA comprises 2'-O-methyluridine and 5-methylcytidine (m5C). In some embodiments, the mRNA comprises N6-methyladenosine (m6A). In some embodiments, the mRNA comprises N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

[0303] In certain embodiments, the mRNA of the Disclosure may be uniformly modified for a specific modification (i.e., completely modified, modified throughout the entire sequence). For example, the mRNA may be uniformly modified with N1-methylpseudridine (m1ψ) or 5-methylcytidine (m5C), meaning that all uridine or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudridine (m1ψ) or 5-methylcytidine (m5C). Similarly, any type of nucleoside residue present in the sequence may be uniformly modified in the mRNA of the Disclosure by replacement with modification residues such as those described above.

[0304] In some embodiments, the mRNA of this disclosure may be modified within the coding region (e.g., the open reading frame encoding the polypeptide). In other embodiments, the mRNA may be modified in regions other than the coding region. For example, in some embodiments, 5'-UTR and / or 3'-UTR are provided, which may independently contain one or both of the following nucleoside modifications: In such embodiments, the nucleoside modifications may also be present within the coding region.

[0305] Examples of nucleoside modifications and their combinations that may be present in the mMRNAs of this disclosure include, but are not limited to, those described in PCT patent application publications WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

[0306] The mMRNAs of this disclosure may include combinations of modifications to sugars, nucleic acid bases, and / or nucleoside-to-nucleoside bonds. These combinations may include any one or more of the modifications described herein.

[0307] Where a single modification is listed, the listed nucleoside or nucleotide represents 100% of that modified A, U, G, or C nucleotide or nucleoside. Where a percentage is listed, these represent the percentage of that particular A, U, G, or C nucleic acid base 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 triphosphate is 5-aminoallyl-CTP and 75% of the cytosine is CTP, while 25% of the uracil is 5-methoxy-UTP and 75% of the uracil is UTP. Where a modified UTP is not listed, naturally occurring ATP, UTP, GTP, and / or CTP are used for 100% of the sites of these nucleotides found in the polynucleotide. In this example, all GTP and ATP nucleotides remain unmodified.

[0308] The mRNAs of this disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthesis methods. Enzymatic (IVT), solid-phase, liquid-phase, complex synthesis, small-region synthesis, and ligation methods may be utilized. In one embodiment, the mRNA is produced using an IVT enzymatic synthesis method. Methods for producing polynucleotides by IVT are publicly known in the art and are described in international application PCT / US2013 / 30062, the contents of which are incorporated herein by reference in their entirety. Accordingly, this disclosure also includes polynucleotides, such as DNA, constructs, and vectors, that may be used to transcribe the mRNAs described herein in vitro.

[0309] Unnaturally modified nucleic acid bases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In certain embodiments, the modifications may be on nucleoside bonds, purine or pyrimidine bases, or sugars. In certain embodiments, the modifications may be introduced by chemical synthesis or by polymerase enzymes to the ends of the polynucleotide chain or anywhere else on the polynucleotide chain. Examples of modified nucleic acids and their synthesis are disclosed in PCT application PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

[0310] Polynucleotides or their regions can be conjugated with various functional parts, such as targeting agents or delivery agents, fluorescent labels, liquids, or nanoparticles, using either enzymatic or chemical ligation methods. Conjugation and modification of polynucleotides are outlined in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

[0311] Therapeutic agents for reducing protein expression In one embodiment, the therapeutic agent is one that reduces (i.e., decreases, inhibits, or 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 microRNA binding sites (miR binding sites), microRNAs (miRNAs), antagomyl, small (short) interfering RNAs (siRNAs) (including shortmer and dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs), and CRISPR / Cas9 technology.

[0312] Peptide / polypeptide therapeutic agents In one embodiment, the therapeutic agent is a peptide therapeutic agent. In another embodiment, the therapeutic agent is a polypeptide therapeutic agent.

[0313] In some embodiments, the therapeutic or prophylactic payload includes a secreted protein, a membrane-bound protein, or an intercellular protein, or a peptide, polypeptide, or mRNA encoding a biologically active fragment thereof.

[0314] In some embodiments, the therapeutic or prophylactic payload includes a secreted protein, its peptide, polypeptide, or mRNA encoding a biologically active fragment. In some embodiments, the therapeutic or prophylactic payload includes a membrane-bound protein, its peptide, polypeptide, or mRNA encoding a biologically active fragment. In some embodiments, the therapeutic or prophylactic payload includes an intracellular protein, its peptide, polypeptide, or mRNA encoding a biologically active fragment. In some embodiments, the therapeutic or prophylactic payload includes a protein, polypeptide, or peptide.

[0315] In some embodiments, the peptide or polypeptide is of natural origin, for example, isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, for example, 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 agent of the composition is a naturally occurring peptide or polypeptide. In one embodiment, the peptide or polypeptide therapeutic agent of the composition is a modified version of a naturally occurring peptide or polypeptide (for example, containing fewer than 3, 5, 10, 15, 20, or 25 amino substitutions, deletions, or additions compared to its wild-type, naturally occurring peptide or polypeptide counterpart).

[0316] LNP containing cationic agents The LNP of the present invention comprises an LNP core and a cationic agent mainly disposed on the outer surface of the core. Such an LNP has a zeta potential that is above neutral at physiological pH.

[0317] Core lipid nanoparticles typically contain 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.

[0318] Certain LNP cores provided herein include ionized lipids, such as ionized aminolipids, phospholipids, structural lipids, and optionally, stabilizers (e.g., molecules containing polyethylene glycol) which may or may not be provided conjugated to another lipid.

[0319] The structural lipid may be, but is not limited to, a sterol (e.g., cholesterol). The structural lipid may be β-sitosterol.

[0320] Helper lipids are noncationic lipids. Helper lipids may contain at least one fatty acid chain of at least 8C and at least one polar head portion.

[0321] When a molecule containing polyethylene glycol (i.e., PEG) is used, that molecule can be used as a stabilizer. In some embodiments, the molecule containing polyethylene glycol may be polyethylene glycol conjugated to a lipid, and therefore may be provided, for example, as PEG-c-DOMG or PEG-DMG. Certain LNPs provided herein do not contain PEGylated lipids or contain low levels of PEGylated lipids (including not containing alkyl-PEGylated lipids or containing low levels of alkyl-PEGylated lipids), and may be referred to herein as PEG-free or PEGylated lipid-free. Therefore, some LNPs contain less than 0.5 mol% PEGylated lipids. In some cases, PEG may be alkyl-PEG, such as methoxy-PEG. Further LNPs contain non-alkyl-PEG, such as hydroxy-PEG, 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 LNP contains 1.5 mol% of PEGylated lipids. In some embodiments, the LNP contains 3.0 mol% of PEGylated lipids. In some embodiments, the LNP contains 0.1 mol% to 3.0 mol% of PEGylated lipids, 0.5 mol% to 2.0 mol% of PEGylated lipids, or 1.0 mol% to 1.5 mol% of PEGylated lipids.

[0322] In some embodiments, the core nanoparticle composition may have a formulation of compound 18:phospholipid:cholesterol:N-lauroyl-D-erythro-sphinganylphosphorylcholine in a molar ratio of 50:10:38.5:1.5. In some embodiments, the nanoparticle core composition may have a formulation of compound 18:DSPC:cholesterol:compound 428 in a molar ratio of 50:10:38.5:1.5.

[0323] Compound 428: [ka]

[0324] The nanoparticles of this disclosure comprise at least one compound according to formula (I). For example, a nanoparticle composition may comprise one or more compounds from 1 to 147. The nanoparticles may also comprise a variety of other components. For example, a nanoparticle composition may comprise one or more other lipids in addition to the lipid according to formula (I) or (II), such as (i) at least one phospholipid, (ii) at least one structural lipid, (iii) at least one PEG-lipid, or (iv) any combination thereof.

[0325] In some embodiments, the nanoparticle composition comprises a compound of formula (I) (e.g., compounds 18, 25, 26, or 48). In some embodiments, the nanoparticle composition comprises a compound of formula (I) (e.g., compounds 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., compounds 18, 25, 26, or 48) and a phospholipid (e.g., DSPC, DPPC, DOPE, or MSPC).

[0326] This disclosure also provides a process for preparing nanoparticles, which includes contacting lipid nanoparticles with a cationic agent, and these lipid nanoparticles (a) Below: (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids A lipid nanoparticle core containing, (b) comprising a polynucleotide payload or polypeptide payload encapsulated within a core for delivery to cells.

[0327] In some embodiments, contacting lipid nanoparticles with a cationic agent involves dissolving the cationic agent in a nonionic excipient. In some embodiments, the nonionic excipient is selected from macrogol 15 hydroxystearate (HS 15), 1,2-dimiristoyl-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 nonionic excipient is macrogol 15 hydroxystearate (HS 15). In some embodiments, contacting lipid nanoparticles with a cationic agent involves 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.

[0328] The processes described herein provide nanoparticles prepared, for example, by contacting lipid nanoparticles with a cationic agent. In some embodiments, the cationic agent may be a sterolamine such as GL-67. In some embodiments, the lipid nanoparticle core of the lipid nanoparticles optionally contains PEG-lipids. In some embodiments, the lipid nanoparticle core forming the lipid nanoparticles that come into contact with the cationic agent is substantially free of PEG-lipids. In some embodiments, the PEG-lipids are added to the lipid nanoparticles together with the cationic agent, either before or after contact with the cationic agent.

[0329] In one embodiment, the LNP of the present invention can be prepared using a conventional mixing technique to create a core LNP+payload by mixing a nucleic acid payload with a core LNP component. After preparing this mounted core LNP, a cationic agent is brought into contact with the mounted core LNP.

[0330] In another embodiment, the LNPs of the present invention can be fabricated using hollow LNPs as a starting point. For example, as shown in Figure 1, the hollow LNPs are fabricated before being loaded onto a nucleic acid payload. After the nucleic acid payload is brought into contact with the LNPs, a cationic agent can be added to form the LNPs of the present invention.

[0331] For example, in one embodiment, in a post-loading (PHL) method, first, hollow LNPs are formulated in a nanoprecipitation step, and the buffer is replaced with a low pH buffer (i.e., pH 5). Next, these hollow LNPs are introduced into mRNA through a mixing event (similarly acidified at a low pH). After the mixing step, the pH is neutralized using a pH adjustment method. Finally, a PEG lipid, such as DMG-PEG-2k, is added to stabilize the particles. Next, these particles are concentrated to the target concentration and filtered. A cationic agent, such as GL67, is added.

[0332] Figure 2 illustrates a modified form of the starting point for hollow LNPs. Figure 2 shows the formation of hollow LNPs using lipids from the LNPs, excluding PEG lipids. Next, the nucleic acid solution is brought into contact with the hollow LNPs to form loaded LNPs. As illustrated by the dotted box in Figure 2, PEG lipids can be added at one or two points during further processing of the loaded LNPs, and cationic agents can be added at any point during that further processing. Figure 3 is a more specific version of the process in Figure 2, where, as before, cationic agents can be added at any point during that further processing of the loaded LNPs.

[0333] In some embodiments, the LNPs of the present invention can be prepared using nanoprecipitation, which is a unit operation that causes the LNPs to self-assemble from their individual lipid components through dynamic mixing and subsequent maturation and serial dilution. This unit operation includes three individual 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 sequential nature. The unit operation involves a sequential inline combination of three liquid flows and one inline maturation step, namely, mixing of aqueous buffer and lipid stock, maturation through a controlled residence time, and dilution of nanoparticles. The nanoprecipitation itself occurs in a scale-appropriate mixer designed to allow continuous high-energy mixing of the aqueous solution and the lipid stock dissolved in ethanol. Both the aqueous solution and the lipid stock flow simultaneously into the mixing apparatus in a continuous manner throughout this operation. The ethanol content, which maintains the solubility of the lipids, is rapidly reduced, and all the lipids precipitate from one another. In this way, the particles self-assemble within the mixing chamber.

[0334] One of the objectives of the unit operation is to replace the solution with a complete aqueous buffer that does not contain ethanol and to reach the target concentration of LNP. This can be achieved by first reaching the target treatment concentration, then diafiltration, and then, once the ethanol has been completely removed, (if necessary) a final concentration step.

[0335] In some embodiments, the LNPs of the present invention can be prepared using nanoprecipitation, which is a unit operation that causes the LNPs to self-assemble from their individual lipid components through dynamic mixing and subsequent maturation and serial dilution. This unit operation includes three individual 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 sequential nature. The unit operation involves a sequential inline combination of three liquid flows and one inline maturation step, namely, mixing of aqueous buffer and lipid stock, maturation through a controlled residence time, and dilution of nanoparticles. The nanoprecipitation itself occurs in a scale-appropriate mixer designed to allow continuous high-energy mixing of the aqueous solution and the lipid stock dissolved in ethanol. Both the aqueous solution and the lipid stock flow simultaneously into the mixing apparatus in a continuous manner throughout this operation. The ethanol content, which maintains the solubility of the lipids, is rapidly reduced, and all the lipids precipitate from one another. In this way, the particles self-assemble within the mixing chamber.

[0336] One of the objectives of the unit operation is to replace the solution with a complete aqueous buffer that does not contain ethanol and to reach the target concentration of LNP. This can be achieved by first reaching the target treatment concentration, then diafiltration, and then, once the ethanol has been completely removed, (if necessary) a final concentration step.

[0337] In some embodiments, the present disclosure relates to a method for preparing a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow lipid nanoparticles (hollow LNPs), i) Below: ia) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) containing hollow nanoparticle intermediates (hollow LNP intermediates); ib) To retain the hollow LNP intermediate solution for the duration of the residence time; ic) Adding the diluted solution to the hollow LNP intermediate solution thereby forming a hollow LNP solution containing hollow LNPs, The present invention provides a method comprising a nanoprecipitation step including the following:

[0338] In some embodiments, the present disclosure relates to a method for preparing a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow lipid nanoparticles (hollow LNPs), i) Below: ia) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) containing hollow nanoparticle intermediates (hollow LNP intermediates); ib) To retain the hollow LNP intermediate solution for the duration of the residence time; ic) Adding a diluted solution to a hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; A nanoprecipitation step including; ii) A method comprising processing a hollow LNP solution is provided.

[0339] In some embodiments, the present disclosure relates to a method for preparing a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow lipid nanoparticles (hollow LNPs), ii) A method is provided which includes processing a hollow LNP solution containing hollow LNPs.

[0340] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), i) Below: ia) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) containing hollow nanoparticle intermediates (hollow LNP intermediates); ib) To retain the hollow LNP intermediate solution for the duration of the residence time; ic) Adding a diluted solution to a hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; A nanoprecipitation step including; ii) Processing the hollow LNP solution; iii) A method is provided which includes a mounting step of mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution to form a mounted LNP solution containing mounted lipid nanoparticles (mounted LNPs).

[0341] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), i) Below: ia) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) containing hollow nanoparticle intermediates (hollow LNP intermediates); ib) To retain the hollow LNP intermediate solution for the duration of the residence time; ic) Adding a diluted solution to a hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; A nanoprecipitation step including; ii) Processing the hollow LNP solution; iii) an onboarding step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution to form an onboard LNP solution containing onboard lipid nanoparticles (onboard LNPs); iv) A method is provided which includes processing a mounted LNP solution to form a mounted LNP formulation.

[0342] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), i) Below: ia) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle intermediate solution (hollow LNP intermediate solution) containing hollow nanoparticle intermediates (hollow LNP intermediates); ib) To retain the hollow LNP intermediate solution for the duration of the residence time; ic) Adding a diluted solution to a hollow LNP intermediate solution, thereby forming a hollow LNP solution containing hollow LNPs; A nanoprecipitation step including; ii) Processing the hollow LNP solution; iii) an onboarding step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution to form an onboard LNP solution containing onboard lipid nanoparticles (onboard LNPs); iv) Processing the mounted LNP solution to form a mounted LNP formulation; v) A method comprising adding a cationic agent is provided.

[0343] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), iii) A method is provided which includes a mounting step of mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution containing hollow LNPs, thereby forming a mounted nanoparticle solution (mounted LNP solution) containing mounted lipid nanoparticles (mounted LNPs).

[0344] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), iii) A mounting step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution containing hollow LNPs, thereby forming a mounted nanoparticle solution (mounted LNP solution) containing mounted lipid nanoparticles (mounted LNPs); iv) A method is provided which includes processing a mounted LNP solution to form a mounted LNP formulation.

[0345] In some embodiments, the present disclosure relates to a method for preparing lipid nanoparticle formulations (LNP formulations), iii) A mounting step comprising mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution containing hollow LNPs, thereby forming a mounted nanoparticle solution (mounted LNP solution) containing mounted lipid nanoparticles (mounted LNPs); iv) Processing the mounted LNP solution to form a mounted LNP formulation; v) A method comprising adding a cationic agent is provided.

[0346] In some embodiments, steps ia) to ic) are carried out in separate operating units (e.g., separate reaction devices).

[0347] In some embodiments, steps ia) to ic) are performed in a single operating unit. In some embodiments, steps ia) to ic) are performed in a continuous flow device such that step ic) is downstream of step ia) and step ib).

[0348] In some embodiments, the diluted solution is added once in step ic).

[0349] In some embodiments, the diluted solution is added continuously in step ic).

[0350] In some embodiments, the Disclosure provides a method for producing hollow lipid nanoparticles (hollow LNPs), the method comprising a mixing step of i) mixing an ionized lipid with a first buffer to form hollow LNPs, the hollow LNPs comprising about 0.1 mol% to about 0.5 mol% of polymer lipids (e.g., PEG lipids).

[0351] In some embodiments, the present disclosure relates to a method for preparing a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow lipid nanoparticles (hollow LNPs), i) A method is provided comprising a mixing step, which includes mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow LNPs.

[0352] In some embodiments, the present disclosure relates to a method for preparing a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow lipid nanoparticles (hollow LNPs), i) A mixing step comprising mixing a lipid solution containing ionized lipids, structural lipids, phospholipids, and PEG lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow LNPs; ii) A method comprising processing a hollow LNP solution is provided.

[0353] In some embodiments, the mixing step includes mixing a lipid solution containing ionized lipids with an aqueous buffer solution containing a first buffer, thereby forming a hollow lipid nanoparticle solution (hollow LNP solution) containing hollow LNPs.

[0354] In some embodiments, the Disclosure provides a method for preparing a loaded lipid nanoparticle (loaded LNP) associated with a nucleic acid, comprising the loading step of: ii) mixing a nucleic acid with a hollow LNP, followed by the addition of a cationic agent to thereby form the loaded LNP.

[0355] In some embodiments, the loading step includes mixing a nucleic acid solution containing nucleic acids with a hollow LNP solution, followed by the addition of a cationic agent to form a loaded lipid nanoparticle solution (loaded LNP solution) containing loaded LNPs.

[0356] In some embodiments, hollow LNPs or hollow LNP solutions are subjected to the mounting step without being held or stored.

[0357] In some embodiments, the hollow LNP or hollow LNP solution is held for a certain period of time before being subjected to the mounting step.

[0358] In some embodiments, the hollow LNP or hollow 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 mounting step.

[0359] In some embodiments, the hollow LNP or hollow LNP solution is subjected to the loading step after being stored for approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years.

[0360] In some embodiments, the hollow LNP or hollow LNP solution is subjected to the mounting step without being stored or held for a certain period of time after formation.

[0361] In some embodiments, the present disclosure provides a method that further includes ii) processing a hollow LNP solution.

[0362] In some embodiments, the present disclosure further provides a method comprising iv) processing a loaded LNP solution to form a lipid nanoparticle formulation (LNP formulation).

[0363] In contrast to other production techniques (e.g., thin-film rehydration / extrusion), ethanol dropwise precipitation has become the industry standard for the production of nucleic acid lipid nanoparticles. Precipitation reactions are advantageous due to their continuity, scalability, and ease of adoption. These processes typically utilize high-energy mixers (e.g., T-junctions, constrained impingement jets, microfluidic mixers, vortex mixers) to introduce lipids (in ethanol) into a suitable poor solvent (i.e., water) in a controllable manner, thereby promoting liquid supersaturation and spontaneous precipitation into lipid particles. In some embodiments, the vortex mixers used are those described in U.S. Patent Applications 62 / 799,636 and 62 / 886,592 (both incorporated herein by reference). In some embodiments, the microfluidic mixers used are those described in PCT Application WO / 2014 / 172045 (both incorporated herein by reference).

[0364] In some embodiments, the mixing step is carried out using a T-joint, a constrained impingement jet, a microfluidic mixer, or a vortex mixer.

[0365] In some embodiments, the mounting step is carried out using a T-joint, a constrained impingement jet, a microfluidic mixer, or a vortex mixer.

[0366] In some embodiments, the mixing step is carried out at a temperature below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 24°C, below approximately 22°C, below approximately 20°C, or near ambient temperature.

[0367] In some embodiments, the mounting step is performed at a temperature below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 24°C, below approximately 22°C, below approximately 20°C, or near ambient temperature.

[0368] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution includes a first addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNP or mounted LNP.

[0369] In some embodiments, the step of processing the hollow LNP solution includes a first addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNP solution.

[0370] In some embodiments, the step of processing the hollow LNP solution includes a first addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs.

[0371] In some embodiments, the step of processing the mounted LNP solution includes a first addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the mounted LNP solution.

[0372] In some embodiments, the step of processing the mounted LNP solution includes a first addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the mounted LNPs.

[0373] In some embodiments, the first addition step includes adding a polyethylene glycol solution containing PEG lipids (PEG solution) to a hollow LNP solution or a mounted LNP solution.

[0374] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution includes a second addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNP or mounted LNP.

[0375] In some embodiments, the step of processing the hollow LNP solution includes a second addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNP solution.

[0376] In some embodiments, the step of processing the hollow LNP solution includes a second addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the hollow LNPs.

[0377] In some embodiments, the step of processing the mounted LNP solution includes a second addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the mounted LNP solution.

[0378] In some embodiments, the step of processing the mounted LNP solution includes a second addition step, which involves adding polyethylene glycol lipids (PEG lipids) to the mounted LNPs.

[0379] In some embodiments, the second addition step includes adding a polyethylene glycol solution containing PEG lipids (PEG solution) to the hollow LNP solution or the mounted LNP solution.

[0380] In some embodiments, the first addition step includes adding about 0.1 mol% to about 3.0 mol% of PEG, about 0.2 mol% to about 2.5 mol% of PEG, about 0.5 mol% to about 2.0 mol% of PEG, about 0.75 mol% to about 1.5 mol% of PEG, and about 1.0 mol% to about 1.25 mol% of PEG to the hollow LNP or mounted LNP.

[0381] In some embodiments, the first addition step includes adding about 0.1 mol% to about 3.0 mol% of PEG, about 0.2 mol% to about 2.5 mol% of PEG, about 0.5 mol% to about 2.0 mol% of PEG, about 0.75 mol% to about 1.5 mol% of PEG, and about 1.0 mol% to about 1.25 mol% of PEG to the hollow LNP or mounted LNP. In some embodiments, the first addition step is to add 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% of PEG lipids (e.g., PEG 2k This includes adding -DMG.

[0382] In some embodiments, the first addition step involves adding 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 lipids (e.g., PEG 2k This includes adding -DMG.

[0383] In some embodiments, after the first addition step, the hollow LNP solution (e.g., hollow LNP) is divided into approximately 1.0 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, approximately 2.0 mol%, approximately 2.1 mol%, approximately 2.2 mol%, approximately 2.3 mol%, approximately 2.4 mol%, approximately 2.5 mol%, approximately 2.6 mol%, approximately 2.7 mol%, and approximately 2.8 mol%. Approximately 2.9 mol%, approximately 3.0 mol%, approximately 3.1 mol%, approximately 3.2 mol%, approximately 3.3 mol%, approximately 3.4 mol%, approximately 3.5 mol%, approximately 3.6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, or approximately 5.0 mol% of PEG lipids (e.g., PEG 2k (-DMG)

[0384] In some embodiments, after the first addition step, the mounted LNP solution (e.g., mounted LNP) is composed of approximately 1.0 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, approximately 2.0 mol%, approximately 2.1 mol%, approximately 2.2 mol%, approximately 2.3 mol%, approximately 2.4 mol%, approximately 2.5 mol%, approximately 2.6 mol%, approximately 2.7 mol%, and approximately 2.8 mol%. Approximately 2.9 mol%, approximately 3.0 mol%, approximately 3.1 mol%, approximately 3.2 mol%, approximately 3.3 mol%, approximately 3.4 mol%, approximately 3.5 mol%, approximately 3.6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, or approximately 5.0 mol% of PEG lipids (e.g., PEG 2k (-DMG)

[0385] In some embodiments, the second addition step includes adding about 0.1 mol% to about 3.0 mol% of PEG, about 0.2 mol% to about 2.5 mol% of PEG, about 0.5 mol% to about 2.0 mol% of PEG, about 0.75 mol% to about 1.5 mol% of PEG, and about 1.0 mol% to about 1.25 mol% of PEG to the hollow LNP or mounted LNP.

[0386] In some embodiments, the second addition step includes adding about 0.1 mol% to about 3.0 mol% of PEG, about 0.2 mol% to about 2.5 mol% of PEG, about 0.5 mol% to about 2.0 mol% of PEG, about 0.75 mol% to about 1.5 mol% of PEG, and about 1.0 mol% to about 1.25 mol% of PEG to the hollow LNP or mounted LNP.

[0387] In some embodiments, the second addition step is to add 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% of PEG lipids (e.g., PEG 2k This includes adding -DMG.

[0388] In some embodiments, the second addition step involves adding 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 PEG lipids (e.g., PEG 2k This includes adding -DMG.

[0389] In some embodiments, the second addition step includes adding about 1.0 mol% of PEG lipid to the hollow LNP or mounted LNP.

[0390] In some embodiments, after the second addition step, the hollow LNP solution (e.g., hollow LNP) is divided into approximately 1.0 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, approximately 2.0 mol%, approximately 2.1 mol%, approximately 2.2 mol%, approximately 2.3 mol%, approximately 2.4 mol%, approximately 2.5 mol%, approximately 2.6 mol%, approximately 2.7 mol%, and approximately 2.8 mol%. Approximately 2.9 mol%, approximately 3.0 mol%, approximately 3.1 mol%, approximately 3.2 mol%, approximately 3.3 mol%, approximately 3.4 mol%, approximately 3.5 mol%, approximately 3.6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, or approximately 5.0 mol% of PEG lipids (e.g., PEG 2k (-DMG)

[0391] In some embodiments, after the second addition step, the mounted LNP solution (e.g., mounted LNP) is composed of approximately 1.0 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, approximately 2.0 mol%, approximately 2.1 mol%, approximately 2.2 mol%, approximately 2.3 mol%, approximately 2.4 mol%, approximately 2.5 mol%, approximately 2.6 mol%, approximately 2.7 mol%, and approximately 2.8 mol%. Approximately 2.9 mol%, approximately 3.0 mol%, approximately 3.1 mol%, approximately 3.2 mol%, approximately 3.3 mol%, approximately 3.4 mol%, approximately 3.5 mol%, approximately 3.6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, or approximately 5.0 mol% of PEG lipids (e.g., PEG 2k (-DMG)

[0392] In some embodiments, the first addition step is carried out at a temperature below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 24°C, below approximately 22°C, below approximately 20°C, or near ambient temperature.

[0393] In some embodiments, the second addition step is carried out at a temperature below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 24°C, below approximately 22°C, below approximately 20°C, or near ambient temperature.

[0394] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes at least one step selected from filtration, pH adjustment, buffer exchange, dilution, dialysis, concentration, freezing, lyophilization, storage, and packing.

[0395] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes pH adjustment.

[0396] In some embodiments, pH adjustment involves adding a second buffer selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, and Tris buffer.

[0397] In some embodiments, the first addition step is performed before pH adjustment.

[0398] In some embodiments, the first addition step is performed after pH adjustment.

[0399] In some embodiments, the second addition step is performed before pH adjustment.

[0400] In some embodiments, the second addition step is performed after pH adjustment.

[0401] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes filtration.

[0402] In some embodiments, filtration is tangential flow filtration (TFF).

[0403] In some embodiments, filtration removes organic solvents (e.g., alcohol or ethanol) from the LNP solution. In some embodiments, after removing the organic solvents (e.g., alcohol or ethanol), the LNP solution is converted to a buffered solution (e.g., phosphate buffer or HEPES buffer) at a neutral pH, pH 6.5–7.8, pH 6.8–7.5, preferably pH 7.0–7.2. In some embodiments, the LNP solution is converted to a buffered solution at approximately pH 7.0–7.2. In some embodiments, the resulting LNP solution is sterilized before storage or use, for example, by filtration (e.g., through a 0.1–0.5 μm filter).

[0404] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes buffer exchange.

[0405] In some embodiments, buffer exchange involves adding an aqueous buffer solution containing a third buffering agent.

[0406] In some embodiments, the first addition step is performed before the buffer exchange.

[0407] In some embodiments, the first addition step is performed after the buffer exchange.

[0408] In some embodiments, the second addition is performed before the buffer exchange.

[0409] In some embodiments, the second addition step is performed after the buffer exchange.

[0410] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes dilution.

[0411] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes dialysis.

[0412] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes concentration.

[0413] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes freezing.

[0414] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes lyophilization.

[0415] In some embodiments, freeze-drying includes freezing the mounted LNP solution at temperatures of approximately -100°C to approximately 0°C, approximately -80°C to approximately -10°C, approximately -60°C to approximately -20°C, approximately -50°C to approximately -25°C, or approximately -40°C to approximately -30°C.

[0416] In some embodiments, freeze-drying further includes drying a frozen mounted LNP solution to form freeze-dried hollow LNPs or freeze-dried mounted LNPs.

[0417] In some embodiments, drying is carried out under a vacuum ranging from approximately 50 mTorr to approximately 150 mTorr.

[0418] In some embodiments, drying is carried out at a temperature of approximately -35°C to approximately -15°C.

[0419] In some embodiments, drying is carried out at around room temperature to about 25°C.

[0420] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes storage.

[0421] In some embodiments, storage includes storing the hollow LNP or mounted 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.

[0422] In some embodiments, storage includes storing the hollow LNP or mounted LNP at a temperature of approximately -40°C, approximately -35°C, approximately -30°C, approximately -25°C, approximately -20°C, approximately -15°C, approximately -10°C, approximately -5°C, approximately 0°C, approximately 5°C, approximately 10°C, approximately 15°C, approximately 20°C, or approximately 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.

[0423] In some embodiments, storage includes storing the hollow LNP or mounted LNP at a temperature of approximately -40°C to approximately 0°C, approximately -35°C to approximately -5°C, approximately -30°C to approximately -10°C, approximately -25°C to approximately -15°C, approximately -22°C to approximately -18°C, or approximately -21°C to approximately -19°C for at least one day, at least two days, at least one week, at least two weeks, at least four weeks, at least one month, at least two months, at least three months, at least six months, at least eight months, or at least one year.

[0424] In some embodiments, storage includes storing the hollow LNP or mounted 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.

[0425] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution further includes packing.

[0426] As used herein, “packing” may refer to the storage of a pharmaceutical product in its final state, or the storage of hollow LNPs, mounted LNPs, or LNP formulations during processing before being placed in final packaging. Storage and / or packing methods include, but are not limited to, refrigeration in sterile bags, refrigerated or frozen formulations in vials, and lyophilized formulations in vials and syringes.

[0427] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution includes iia) adding an antifreeze to the hollow LNP solution or the mounted LNP solution.

[0428] In some embodiments, the step of processing the hollow LNP solution or mounted LNP solution includes (i) filtering the hollow LNP solution or mounted LNP solution.

[0429] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution is: iia) Adding an antifreeze to the hollow LNP solution or the mounted LNP solution; iic) Includes filtering a hollow LNP solution or an mounted LNP solution.

[0430] In some embodiments, the step of processing a hollow LNP solution or a mounted LNP solution is as follows: iib) Add an antifreeze to the hollow LNP solution or the mounted LNP solution; iic) Freeze-drying a hollow LNP solution or a mounted LNP solution to form a freeze-dried LNP composition; iid) To store the hollow LNP solution or mounted LNP solution of the freeze-dried LNP composition; and iie) Adding a buffer solution to a hollow LNP solution, a mounted LNP solution, or a lyophilized LNP composition to form an LNP formulation, comprising one or more of the above.

[0431] In some embodiments, the step of processing the hollow LNP solution includes iia) adding an antifreeze to the hollow LNP solution.

[0432] In some embodiments, the step of processing the hollow LNP solution includes filtering the hollow LNP solution (i.b).

[0433] In some embodiments, the step of processing the hollow LNP solution is: iia) Adding an antifreeze to the hollow LNP solution; iic) Includes filtering the hollow LNP solution.

[0434] In some embodiments, an antifreeze is added to the hollow LNP solution or mounted LNP solution before freeze-drying. In some embodiments, the antifreeze comprises one or more cryoprotective agents, each of which is independently a polyol (e.g., diol or triol, e.g., 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 osmolite (e.g., L-proline or trimethylamine N-oxide dihydrate), or a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 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), organic solvents (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugars (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 salts (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 any hydrate thereof), or any combination thereof. In some embodiments, the antifreeze contains sucrose. In some embodiments, the antifreeze and / or excipient is sucrose. In some embodiments, the antifreeze contains sodium acetate. In some embodiments, the antifreeze and / or excipient is sodium acetate. In some embodiments, the antifreeze comprises sucrose and sodium acetate.

[0435] In some embodiments, the antifreeze agent includes a cryoprotectant present at concentrations of approximately 10 g / L to 1000 g / L, approximately 25 g / L to 950 g / L, approximately 50 g / L to 900 g / L, approximately 75 g / L to 850 g / L, approximately 100 g / L to 800 g / L, approximately 150 g / L to 750 g / L, approximately 200 g / L to 700 g / L, approximately 250 g / L to 650 g / L, approximately 300 g / L to 600 g / L, approximately 350 g / L to 550 g / L, approximately 400 g / L to 500 g / L, and approximately 450 g / L to 500 g / L. In some embodiments, the antifreeze includes cryoprotectants present at concentrations of approximately 10 g / L to approximately 500 g / L, approximately 50 g / L to approximately 450 g / L, approximately 100 g / L to approximately 400 g / L, approximately 150 g / L to approximately 350 g / L, approximately 200 g / L to approximately 300 g / L, and approximately 200 g / L to approximately 250 g / L. In some embodiments, the antifreeze agent includes a cryoprotectant present at concentrations of approximately 10 g / L, approximately 25 g / L, approximately 50 g / L, approximately 75 g / L, approximately 100 g / L, approximately 150 g / L, approximately 200 g / L, approximately 250 g / L, approximately 300 g / L, approximately 300 g / L, approximately 350 g / L, approximately 400 g / L, approximately 450 g / L, approximately 500 g / L, approximately 550 g / L, approximately 600 g / L, approximately 650 g / L, approximately 700 g / L, approximately 750 g / L, approximately 800 g / L, approximately 850 g / L, approximately 900 g / L, approximately 950 g / L, and approximately 1000 g / L.

[0436] In some embodiments, the antifreeze includes cryoprotective agents present at concentrations of approximately 0.1 mM to approximately 100 mM, approximately 0.5 mM to approximately 90 mM, approximately 1 mM to approximately 80 mM, approximately 2 mM to approximately 70 mM, approximately 3 mM to approximately 60 mM, approximately 4 mM to approximately 50 mM, approximately 5 mM to approximately 40 mM, approximately 6 mM to approximately 30 mM, approximately 7 mM to approximately 25 mM, approximately 8 mM to approximately 20 mM, approximately 9 mM to approximately 15 mM, and approximately 10 mM to approximately 15 mM. In some embodiments, the antifreeze includes cryoprotective agents present at concentrations of approximately 0.1 mM to approximately 10 mM, approximately 0.5 mM to approximately 9 mM, approximately 1 mM to approximately 8 mM, approximately 2 mM to approximately 7 mM, approximately 3 mM to approximately 6 mM, and approximately 4 mM to approximately 5 mM. In some embodiments, the antifreeze includes frost protection agents present at concentrations of approximately 0.1 mM, approximately 0.5 mM, approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM, approximately 85 mM, approximately 90 mM, approximately 95 mM, and approximately 100 mM.

[0437] In some embodiments, the antifreeze includes sucrose.

[0438] In some embodiments, the antifreeze includes an aqueous solution containing sucrose.

[0439] In some embodiments, the antifreeze agent comprises an aqueous solution containing approximately 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.

[0440] In some embodiments, the antifreeze includes an aqueous solution containing sodium acetate and sucrose.

[0441] In some embodiments, the antifreeze agent is (a) with sodium acetate in concentrations of approximately 5±1 mM, 5±0.9 mM, 5±0.8 mM, 5±0.5 mM, 5±0.6 mM, 5±0.5 mM, 5±0.4 mM, 5±0.3 mM, 5±0.2 mM, or 5±0.1 mM; (b) an aqueous solution containing sucrose in concentrations of approximately 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.

[0442] In some embodiments, the antifreeze comprises an aqueous solution containing sodium acetate and sucrose, the aqueous solution having 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.

[0443] In some embodiments, the antifreeze agent is (a) with sodium acetate in concentrations of approximately 5±1 mM, 5±0.9 mM, 5±0.8 mM, 5±0.5 mM, 5±0.6 mM, 5±0.5 mM, 5±0.4 mM, 5±0.3 mM, 5±0.2 mM, or 5±0.1 mM; (b) an aqueous solution containing approximately 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, This aqueous solution has pH values ​​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.

[0444] In some embodiments, lyophilization is carried out in a suitable glass receptacle (e.g., a 10 mL cylindrical glass vial). In some embodiments, the glass receptacle is resistant to extreme temperature changes from below -40°C to above room temperature over a short period of time and / or is cut into a uniform shape. In some embodiments, the lyophilization step includes freezing the LNP solution at a temperature above about -40°C to form a frozen LNP solution; and drying the frozen LNP solution to form a lyophilized LNP composition. In some embodiments, the lyophilization step includes freezing the LNP solution at a temperature above about -40°C and below about -30°C. The freezing step causes the temperature to drop linearly from 20°C to -40°C to the final temperature, preferably at about 1°C per minute, over about 6 minutes. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out under a vacuum in the range of approximately 50 mTorr to approximately 150 mTorr. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out under a vacuum in the range of approximately 50 mTorr to approximately 150 mTorr, first at a low temperature in the range of approximately -35°C to approximately -15°C, and then at a high temperature in the range of room temperature to approximately 25°C. In some embodiments, 12-15% sucrose can be used, and the drying step is carried out under a vacuum in the range of approximately 50 mTorr to approximately 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 under a vacuum in the range of approximately 50 mTorr to approximately 150 mTorr, first at a low temperature in the range of approximately -35°C to approximately -15°C, and then at a high temperature in the range of room temperature to approximately 25°C, and the drying step is completed in 3-7 days. In some embodiments, the drying step is carried out under a vacuum in the range of about 50 mTorr to about 100 mTorr. In some embodiments, the drying step is carried out under a vacuum in the range of about 50 mTorr to about 100 mTorr, first at a low temperature in the range of about -15°C to about 0°C, and then at a high temperature.

[0445] In some embodiments, hollow LNP solutions, mounted LNP solutions, or lyophilized LNP compositions are stored at pH levels of approximately 3.5 to approximately 8.0, approximately 4.0 to approximately 7.5, approximately 4.5 to approximately 7.0, approximately 5.0 to approximately 6.5, and approximately 5.5 to approximately 6.0. In some embodiments, hollow LNP solutions, mounted LNP solutions, or lyophilized LNP compositions are stored at pH levels of approximately 3.5, approximately 4.0, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 4.5, approximately 5.5, approximately 6.5, approximately 7.0, approximately 7.5, and approximately 8.0.

[0446] In some embodiments, the LNP solution, onboard LNP solution, or lyophilized LNP composition is stored in an antifreeze containing sucrose and sodium acetate. In some embodiments, the LNP solution, onboard LNP solution, or lyophilized LNP composition is stored in an antifreeze containing about 150 g / L to about 350 g of sucrose and about 3 mM to about 6 mM of sodium acetate at a pH of about 4.5 to about 7.0. In some embodiments, the LNP solution, onboard LNP solution, or lyophilized LNP composition is stored in an antifreeze containing about 200 g / L of sucrose and 5 mM of sodium acetate at a pH of about 5.0.

[0447] In some embodiments, the hollow LNP solution, mounted 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 before the addition of a buffer solution.

[0448] In some embodiments, the hollow LNP solution, mounted 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 before the addition of the buffer solution.

[0449] In some embodiments, the hollow LNP solution, mounted LNP solution, or lyophilized LNP composition is stored at temperatures in the range 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 before the addition of the buffer solution.

[0450] In some embodiments, the hollow LNP solution, mounted LNP solution, or lyophilized LNP composition is stored at a temperature of approximately -20°C before the addition of a buffer solution.

[0451] Certain aspects of this method are described in PCT application WO / 2020 / 160397, which is incorporated herein by reference in its entirety.

[0452] This specification also describes cells containing nanoparticles. The cells may be epithelial cells. For example, the cells may be lung cells. The cells may be respiratory epithelial cells. For example, the cells may be lung cells, nasal cells, alveolar epithelial cells, or bronchial epithelial cells. The cells may be human bronchial epithelial (HBE) cells. The cells may be HeLa cells. Such cells can be brought into contact with LNPs in vitro or in vivo.

[0453] Pharmaceutical compositions and preparations This disclosure provides pharmaceutical compositions and formulations comprising any of the nanoparticles described herein.

[0454] A pharmaceutical composition or formulation may optionally contain one or more additional active substances, such as therapeutically and / or prophylactically active substances. The pharmaceutical compositions or formulations of this disclosure may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents are, for example, Remington: The Science and Practice of Pharmacy 21. stThis can be found in Lippincott Williams & Wilkins, ed., 2005 (which is incorporated herein by reference in its entirety). In some embodiments, the composition is administered to a human, a human patient, or a human subject. For the purposes of this disclosure, the term “active ingredient” generally refers to nanoparticles comprising a polynucleotide or polypeptide payload delivered as described herein.

[0455] The formulations and pharmaceutical compositions described herein may be prepared by any method known or to be developed in the field of pharmacology. Generally, such preparation methods include the steps of associating nanoparticles with excipients and / or one or more other auxiliary components, and then, if necessary and / or desired, dividing, shaping, and / or packaging the product into desired single or multi-dose units.

[0456] The pharmaceutical compositions or formulations described herein may be prepared, packaged, and / or sold in bulk as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” refers to an individual amount of a pharmaceutical composition containing a specified amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject and / or a favorable proportion of such dose, for example, half or one-third of such dose.

[0457] The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition according to this disclosure may vary depending on the specificity, size, and / or condition of the object being treated, and further depending on the route through which the composition is administered.

[0458] The descriptions of pharmaceutical compositions and formulations provided herein primarily concern pharmaceutical compositions and formulations suitable for administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals (e.g., non-human mammals).

[0459] When used herein, pharmaceutically acceptable excipients include, but are not limited to, all solvents, dispersions, or other liquid vehicles, dispersing or suspending aids, diluents, granulators and / or dispersants, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, binders, lubricants or oils, colorants, sweeteners or flavorings, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic regulators, pH adjusters, buffers, chelating agents, antifreeze, and / or bulking agents. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) (the whole is incorporated herein by reference)).

[0460] Examples of diluents include, but are not limited to, calcium carbonate or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, and / or combinations thereof.

[0461] Examples of granulating and / or dispersing agents include, but are not limited to, starch, pregelatinized starch, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinylpyrrolidone), (providone), cross-linked poly(vinylpyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, and / or combinations thereof.

[0462] 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 gum, pectin, gelatin, egg yolk, casein, lanolin, 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 / or combinations thereof.

[0463] Examples of 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., gum arabic, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and combinations thereof.

[0464] Oxidation is a potential degradation pathway for mRNA, particularly liquid mRNA formulations. Antioxidants can be added to formulations to prevent oxidation. 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, and combinations thereof.

[0465] Examples of chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, and combinations thereof.

[0466] Examples of antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate or sodium benzoate, potassium sorbate or sodium sorbate, sodium propionate, sorbic acid, and combinations thereof.

[0467] Examples of 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 combinations thereof.

[0468] 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 include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and / or combinations thereof.

[0469] Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, or magnesium lauryl sulfate, and combinations thereof.

[0470] The pharmaceutical compositions described herein may include cryoprotectants for stabilizing the polynucleotides described herein during freezing. Examples of cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and combinations thereof.

[0471] The pharmaceutical compositions described herein can incorporate a bulking agent into a lyophilized polynucleotide preparation to produce a "pharmaceutically refined" cake, thereby stabilizing the lyophilized polynucleotide during long-term storage (e.g., 36 months). Exemplary bulking agents of this disclosure include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

[0472] 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 into a pulmonary duct. Aerosolized pharmaceutical formulations may be delivered to the lungs, preferably using a number of commercially available devices.

[0473] The composition may be administered into the respiratory tract by appropriate methods such as intranasal infusion, intratracheal infusion, and intratracheal injection. In some embodiments, the composition or nanoparticles are administered by intranasal administration, intrabronchial administration, or transpulmonary administration. For example, the composition and nanoparticles are administered by nebulizer or inhaler.

[0474] In some embodiments, the composition is delivered to the lungs by inhalation of an aerosolized pharmaceutical formulation. Inhalation may be performed through the nasal and / or oral cavity of the subject. Administration may be performed by self-administration of the formulation at the time of inhalation, or by administration of the formulation to a subject fitted with a respirator via a respirator. Exemplary devices for delivering the formulation to the lungs include, but are not limited to, dry powder inhalers, pressurized metered-dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

[0475] Liquid formulations may be administered to a patient's lungs using a pressurized metered-dose inhaler (pMDI). A pMDI typically comprises at least two components: a canister in which the liquid formulation is held under pressure in combination with one or more propellants, and a receptacle used to hold and operate the canister. The canister may contain a single or multiple dose of the formulation. The canister may include a valve, usually a throttling valve, that can dispense the contents of the canister. The aerosolizing agent is supplied from the pMDI by applying force to the canister, pushing it into the receptacle, which opens the valve and allows drug particles to be transported from the valve through the receptacle outlet. Upon dispensing from the canister, the liquid formulation atomizes and forms an aerosol. A pMDI typically uses one or more propellants to pressurize the contents of the canister and propel the liquid formulation out of the receptacle outlet to form an aerosol. Any suitable propellant can be used. Propellant can take various forms. For example, the propellant may be a compressed gas or a liquefied gas.

[0476] Liquid formulations can also be administered using a nebulizer. A nebulizer is a liquid aerosol generator that converts a liquid formulation into mist- or cloud-like microdroplets, preferably with an aerodynamic median diameter of less than 5 microns in diameter that can be inhaled into the lower respiratory tract. This process is called atomization. When the cloud-like aerosol is inhaled, the droplets carry one or more active ingredients into the nose, upper respiratory tract, or deep into the lungs. Formulations can be administered to patients using any type of nebulizer, including but not limited to air (jet) nebulizers and electromechanical nebulizers. Air (jet) nebulizers use the supply of pressurized gas as the driving force for atomization of the liquid formulation. The compressed gas is delivered through a nozzle or outlet, creating a low-pressure field that entrains the surrounding liquid formulation and shears it into a thin membrane or filament. The membrane or filament is unstable and breaks into microdroplets that are carried into the inhaled air by the compressed gas flow. A baffle inserted into the droplet plume filters out larger droplets and returns them to the bulk liquid reservoir. Electromechanical nebulizers atomize liquid formulations using electrically generated mechanical force. Electromechanical driving force can be applied, for example, by vibrating the liquid formulation at an ultrasonic frequency, or by passing the bulk liquid through a thin film with small pores. This force generates a liquid thin film or filament flow, which splits into microdroplets, forming a slowly moving aerosol flow that can be carried along by the inhalation flow. 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.

[0477] Dry powder inhalers (DPIs) typically use a mechanism, such as a gas burst, to create a cloud of dry powder inside a container, which the subject can then inhale. In DPIs, the dose to be administered is stored in the form of unpressurized dry powder, and the subject inhales the powder particles when the inhaler is activated. In some cases, compressed gas (i.e., propellant) can be used to deliver the powder, similar to pressurized metered-dose inhalers (pMDIs). In some cases, DPIs can be activated by respiration, meaning that an aerosol is created in response precisely to inhalation. Dry powder inhalers typically administer doses of less than tens of milligrams per inhalation to avoid inducing coughing. Examples of DPIs include the Turbohaler® inhaler (Astrazeneca, Wilmington, Del.), the Clickhaler® inhaler (Innovata, Ruddington, Nottingham, UKL), the Diskus® inhaler (Glaxo, Greenford, Middlesex, UK), the EasyHaler® inhaler (Orion, Expoo, FI), the Exubera® inhaler (Pfizer, New York, NY), the Qdose® inhaler (Microdose, Monmouth Junction, NJ), and the Spiros® inhaler (Dura, San Diego, Calif.).

[0478] The pharmaceutical composition of the present invention is administered in an effective amount to produce a desired biological effect, such as a therapeutic or prophylactic effect (for example, by the expression of normal gene products to replenish or replace a deficient protein or to reduce undesirable protein expression, measured, for example, by the alleviation of one or more symptoms in some embodiments). The formulation may be administered in an effective amount to deliver a payload, for example, to deliver LNPs to the apical membranes of respiratory and non-respiratory epithelial cells. In some embodiments, the pharmaceutical composition is administered in an effective amount to induce deficiency of CFTR activity in patients suffering from CF, or to increase the existing level of residual CFTR activity in patients suffering from CF.

[0479] The presence of desired biological activity, such as residual CFTR activity on the epithelial surface, can be readily detected using methods known in the art, including standard electrophysiological, biochemical, and / or histochemical techniques. CFTR activity can be identified and / or quantified using in vivo or ex vivo electrophysiological techniques, measurement of CT concentration in sweat or saliva, or ex vivo biochemical or histochemical techniques monitoring CFTR cell surface density.

[0480] How to use This specification describes methods for treating or preventing a patient's disease in which the disease is associated with dysfunction of airway cells. The methods include administering to a patient nanoparticles or compositions containing 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.

[0481] In some embodiments, the nanoparticles described herein are used in methods for reducing cellular sodium levels in subjects where such reduction is required.

[0482] In some embodiments, the nanoparticles described herein are used to reduce the levels of CF-related metabolites (e.g., substrates or products), and the method involves administering an effective amount of a polynucleotide encoding a CFTR polypeptide to the target.

[0483] In some embodiments, administration of an effective amount of the nanoparticles described herein reduces the level of a biomarker of CF, such as intracellular sodium levels. In some embodiments, administration of the nanoparticles described herein reduces the level of one or more biomarkers of CF, such as intracellular sodium levels, within a short period following administration of the nanoparticles described herein.

[0484] In some embodiments, administration of the nanoparticles described herein to a target reduces intracellular sodium levels in cells to a level 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.

[0485] In some embodiments, methods for delivering a polynucleotide payload or polypeptide payload to cells are provided herein, comprising contacting the cells with nanoparticles described herein. In some embodiments, administration of the nanoparticles described herein results in the expression of CFTR in target cells. In some embodiments, administration of the nanoparticles described herein increases the CFTR enzyme activity of target cells. For example, this method can increase CFTR enzyme activity in at least some of the target cells.

[0486] In some embodiments, administration of the nanoparticles described herein, which contain mRNA encoding a CFTR polypeptide, to a subject increases the CFTR enzyme activity in the target cells to 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 higher levels of activity expected in a normal subject, e.g., a human not suffering from CF.

[0487] In some embodiments, administration of the nanoparticles described herein induces the expression of CFTR protein in at least a subset of target cells, which persists for a period sufficient to produce significant chloride channel activity.

[0488] In some embodiments, the expression of the encoded polypeptide is increased. In some embodiments, when the polynucleotide is introduced into a cell, it increases the CFTR expression level of that cell to, for example, 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% compared to the CFTR expression level of the cell before the polypeptide was introduced into the cell.

[0489] As those skilled in the art will understand, the sterolamines disclosed herein have further applications. For example, sterolamines can be used in the treatment of inflammatory diseases. Sterolamines can also be used as antimicrobial agents.

[0490] Kits and devices This disclosure provides a variety of kits for the convenient and / or effective use of the claimed nanoparticles of this disclosure. Typically, the kits contain sufficient quantities and / or numbers of components to allow the user to perform multiple treatments on and / or experiments on the subject(s)(s).

[0491] In one embodiment, the Disclosure provides a kit comprising the nanoparticles of the Disclosure.

[0492] The kit may further include packaging and instructions and / or a delivery agent for forming the formulation composition. The delivery agent may include saline, a buffer solution, a lipidoid, or any delivery agent disclosed herein. In one embodiment, such a kit may further include an administration device such as a nebulizer or inhaler.

[0493] Pulmonary function tests and other tests for the improvement of respiratory symptoms In some embodiments, nanoparticles or pharmaceutical compositions comprising 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 membrane conductance regulator (CFTR) polypeptide, when administered to a subject in need, is sufficient to improve at least one respiratory volume measurement by at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% compared to at least one reference respiratory volume measured in a subject with untreated cystic fibrosis, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. Respiratory volume is the amount of air inhaled, exhaled, and stored in the lungs at any given time. A non-limiting example of various respiratory volumes that can be measured is given below.

[0494] Total lung volume (TLC) is the volume of the lung at maximum expansion and is the sum of VC and RV. The average total lung volume is 6000 ml, but this varies depending on age, height, sex, and health condition.

[0495] Tidal volume (TV) is the volume of air that enters and leaves the lungs during quiet breathing (TV indicates a lung fraction. When tidal volume is measured precisely, such as in gas exchange calculations, the symbol TV or VT is used). The average tidal volume is 500 ml.

[0496] Residual volume (RV) is the volume of air remaining in the lungs after maximum exhalation. Residual volume (RV / TLC%) is expressed as a percentage of TLC.

[0497] Expiratory reserve volume (ERV) is the maximum volume of air that can be exhaled (exceeding the tidal volume) when forcibly exhaling.

[0498] Inspiratory reserve volume (IRV) is the maximum volume that can be inhaled from the end-spiratory position.

[0499] The maximum intake volume (IC) is the sum of IRV and TV.

[0500] Inspiratory vital capacity (IVC) is the maximum volume of air inhaled from the point of maximum exhalation.

[0501] Lung capacity (VC) is the volume of air exhaled after the deepest inhalation.

[0502] Functional residual capacity (FRC) is the volume of the lungs at the end of expiratory position.

[0503] Forced vital capacity (FVC) is determined from the maximum forced expiratory effort.

[0504] Forced Expiratory Volume (FEV1) t FEV1 is a general term that represents the volume of air exhaled under forced exhalation during the first t seconds. FEF is the volume exhaled at the end of the first second of forced exhalation. xFEF is the forced expiratory flow rate associated with a portion of the FVC curve. The modifier refers to the amount of FVC already exhaled. max This is the maximum instantaneous flow rate achieved during the FVC procedure.

[0505] Forced inspiratory flow rate (FIF) is a specific measurement of the forced inspiratory curve and is expressed using terminology similar to that of the forced expiratory curve. For example, maximum inspiratory flow rate is expressed as FIF. max This is expressed as follows. Unless otherwise specified, the volume limiter indicates the volume drawn in from the RV at the time of measurement.

[0506] Peak expiratory flow (PEF) is the maximum forced expiratory flow rate measured by a peak flow meter.

[0507] Maximum ventilation (MVV) is the volume of air exhaled during a specific period between repeated maximal effort breaths.

[0508] synthesis As will be understood by those skilled in the art, the compounds provided herein (including their salts and stereoisomers) can be prepared using known organic synthesis techniques and can be synthesized according to any of a number of possible synthetic routes, such as those shown in the following scheme.

[0509] The reactions for preparing the compounds described herein can be carried out in suitable solvents readily available to those skilled in the art of organic synthesis. Suitable solvents may be those that are substantially inactive with the starting materials (reactants), intermediates, or products at the reaction temperature (for example, a temperature ranging from the freezing point to the boiling point of the solvent). A given reaction can be carried out in one solvent or a mixture of several solvents. Depending on the specific reaction step, a solvent suitable for that particular step can be selected by those skilled in the art.

[0510] As used herein, the terms “ambient temperature,” “room temperature,” or “rt” are understood in the art and generally refer to a temperature close to the temperature of the room in which the reaction takes place (e.g., a temperature of about 20°C to about 30°C), such as the reaction temperature.

[0511] The preparation of the compounds described herein may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemical action of protecting groups is described, for example, in TW Greene and PGMWuts, Protective Groups in Organic Synthesis, 3. rd This can be found in Ed., Wiley & Sons, Inc., New York (1999).

[0512] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means, e.g., nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) The compounds can be monitored by infrared spectroscopy, spectrophotometric methods (e.g., ultraviolet-visible), mass spectrometry, or chromatography, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin-layer chromatography (TLC). The compounds can be purified by those skilled in the art by various methods, including high-performance liquid chromatography (HPLC) and normal-phase silica chromatography.

[0513] Compounds of formula A2a can be prepared, for example, using the process illustrated in the following scheme.

[0514] [ka] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 1. Compounds of formula A2a can be produced by carrying out appropriate reactions between cholesteryl chloroformate and amines under appropriate conditions.

[0515] [ka] 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 (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the above reaction can then be reacted with an amine under suitable conditions (e.g., using triethylamine) to obtain compounds of formula A2a.

[0516] [ka] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 3. Compounds of formula A2a can be obtained by carrying out a suitable reaction between cholesterol or a cholesterol derivative (such as stigmasterol) and a carboxylic acid under appropriate conditions in the presence of an activating reagent (e.g., EDC-HCl, DMAP, DCC, or pivalic anhydride).

[0517] [ka] Compounds of formula A2a can be prepared via the synthetic route outlined in Scheme 4. Appropriate reactions between hemisuccinate cholesterol or a hemisuccinate cholesterol derivative and an activator can be carried out under appropriate conditions. The product of the above reaction can be reacted with an amine under appropriate conditions to obtain compounds of formula A2a.

[0518] [ka] The compound of formula A2a can be prepared via the synthetic route outlined in Scheme 5. SA22 can be obtained by carrying out a suitable reaction between cholesteryl chloroformate and ethane-1,2-diamine under suitable conditions. SA22 can be reacted with 2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide under suitable conditions to obtain the compound of formula A2a. SA22 can also be reacted with dimethyl squalate under suitable conditions, and the product of the reaction can be further reacted with a secondary amine under suitable conditions to obtain the compound of formula A2a.

[0519] [ka] The compound of formula A2a can be prepared via the synthetic route outlined in Scheme 6. A suitable reaction between an aminoalkyl carbamate and a guanidinylating agent can be carried out under suitable conditions. The product of the above reaction can be reacted with HCl under suitable conditions to obtain the compound of formula A2a.

[0520] [ka] Precursors to the compound of formula A2a can be prepared via the synthetic route outlined in Scheme 7. Appropriate reactions between cholesterol or cholesterol derivatives (such as stigmasterol) can be carried out under appropriate conditions (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the above reaction can then be reacted with an amine under appropriate conditions (e.g., using triethylamine) to obtain a precursor to the compound of formula A2a.

[0521] [ka] Precursors for the compound 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 suitable conditions. The product of the above reaction can then be reacted under suitable conditions to obtain a precursor for the compound of formula A2a.

[0522] [ka] Intermediates for synthesizing the compound of formula A2a can be prepared via the synthetic route outlined in Scheme 9. An intermediate for synthesizing the compound of formula A2a can be obtained by carrying out a suitable reaction between spermidine or spermine and (E)-N-((tert-butoxycarbonyl)oxy)benzimidoylcyanide (BOC-ON) under suitable conditions.

[0523] definition To make this disclosure more easily understandable, certain terms are defined first. Where used in this application, unless otherwise expressly indicated herein, each of the following terms shall have the meanings set forth below. Further definitions are provided throughout this application.

[0524] This disclosure includes embodiments in which exactly one element of the group is present, used, or otherwise related in a given product or process. This disclosure includes embodiments in which multiple or all of the group elements are present, used, or otherwise related in a given product or process.

[0525] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless otherwise clearly indicated by the context. The terms “a” (or “an”), and the terms “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 “plural.”

[0526] Furthermore, as used herein, “and / or” should be considered a specific disclosure of each of the two expressed characteristics or components, with or without the other. Accordingly, 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, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: 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).

[0527] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which this disclosure relates. 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 many common dictionaries of the terms used herein.

[0528] Other similar embodiments are also provided, which are described in this specification with the terms "comprising" and / or "essentially consisting of."

[0529] Units, prefixes, and symbols are expressed in the form recognized by their respective International System of Units (SI). Numerical ranges include the numbers that define the range. Where ranges of values ​​are enumerated, it should be understood that each integer value and each fraction thereof that lie between the enumerated upper and lower limits of that range are also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included in or excluded from that range, and each range that includes either, neither, or both of the limit values ​​is also included within this disclosure. Where values ​​are explicitly enumerated, it should be understood that values ​​that are substantially the same quantity or amount as the enumerated values ​​are also within the scope of this disclosure. Where combinations are disclosed, each of the subcombinations of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of this disclosure is disclosed as having multiple substitutes, examples of this disclosure in which each substitute is excluded, either individually or in any combination with other substitutes, are also disclosed herein. Multiple elements of this disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0530] About: The term “about” as used in relation to numbers throughout this specification and the claims indicates an acceptable interval of precision, which is well known to those skilled in the art. Such an interval of precision is ±10%.

[0531] Where a range is given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, any value expressed as a range may be considered to be any particular value or subrange within the range described in different embodiments of this disclosure up to one-tenth of the lower limit of that range, unless otherwise clearly indicated in the context.

[0532] Combined administration: As used herein, the terms “combined administration” or “combined administration” mean that two or more drugs are administered to a subject simultaneously or at intervals such that the effects of each drug on the patient may overlap. In some embodiments, the drugs are administered to each other within approximately 60, 30, 15, 10, 5, or 1 minute. In some embodiments, the drugs are administered at intervals close enough to each other to obtain a combined (e.g., synergistic) effect.

[0533] Animals: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any developmental stage. In some embodiments, “animal” refers to a non-human animal at any developmental stage. In certain embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and insects. In some embodiments, animals are transgenic animals, genetically modified animals, or clones.

[0534] Approximately: As used herein, the term “approximately” applied to the value of one or more subjects means a value similar to the stated reference value. In certain embodiments, unless otherwise stated or evident from the context, the term “approximately” means a range of values ​​that fall 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) (unless such a number exceeds 100% of the possible values).

[0535] Compounds: As used herein, the term “compound” means all stereoisomers and isotopes of the described structure. As used herein, the term “stereoisomer” means any geometric isomer (e.g., cis and trans isomers), enantiomer, or diastereomer of a compound. This disclosure encompasses all stereoisomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomer mixtures and stereoisomer mixtures, such as racemates. Enantiomer mixtures and stereoisomer mixtures of compounds, and means of dividing them into their enantiomers or stereoisomers are well known. “Isotopes” refer to atoms that have the same atomic number but different mass numbers due to a difference in the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of this disclosure can be prepared by conventional methods in combination with a solvent or water molecule to form solvates and hydrates.

[0536] Contact: As used herein, the term “contact” means to establish a physical connection between two or more entities. For example, contacting mammalian cells with a nanoparticle composition means to cause the mammalian cells and nanoparticles to share a physical connection. Methods for contacting cells with external entities both in vivo and ex vivo are well known in the field of biology. For example, contacting a nanoparticle composition with mammalian cells placed within a mammal can be carried out by various administration routes (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varying amounts of the nanoparticle composition. Furthermore, multiple mammalian cells can be contacted with the nanoparticle composition. A further example of contact is between nanoparticles and a cationic agent. Contacting nanoparticles with a cationic agent may mean causing the surface of the nanoparticles to be physically connected to the cationic agent so that the cationic agent can form an unbound interaction with the nanoparticles. In some embodiments, contacting nanoparticles with a cationic agent causes the cationic agent to be intercalated into the nanoparticles, for example, starting from the surface of the nanoparticles. In some embodiments, the terms “layering,” “coating,” and “post-addition” and “addition” may be used to mean “contacting” in relation to bringing nanoparticles into contact with a cationic agent.

[0537] To deliver: As used herein, the term “to deliver” means to provide an entity to a destination. For example, delivering a polynucleotide to a destination may include administering a nanoparticle composition containing a polynucleotide to a destination (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cells may include bringing one or more cells into contact with the nanoparticle composition.

[0538] Delivery agent: As used herein, “delivery agent” means any substance that at least partially facilitates the in vivo, in vitro, or ex vivo delivery of polynucleotides to target cells.

[0539] Diastereomer: As used herein, the term “diastereomer” means stereoisomers that are not mirror images of each other and cannot be superimposed on each other.

[0540] Arranged: As used herein, the term “arranged” means that after a molecule and a nanoparticle come into contact with each other, the molecule has formed a non-bonding interaction with the nanoparticle.

[0541] Dosage regimen: As used herein, “dosage regimen” or “administration regimen” refers to a schedule of treatment, prevention, or palliative care or a regimen determined by a physician.

[0542] Effective dose: As used herein, the term “effective dose” of a drug is the amount sufficient to produce a beneficial or desired outcome, such as a clinical outcome, and therefore the “effective dose” depends on the context in which it is applied. For example, in the context of administering a drug to treat a protein deficiency (e.g., CFTR deficiency), the effective dose of the drug is, for example, the amount of CFTR-expressing mRNA sufficient to alleviate, reduce, eliminate, or prevent the signs and symptoms associated with CFTR deficiency, compared to the severity of symptoms observed without administration of the drug. The term “effective dose” may be used interchangeably with “effective dose,” “therapeutic effective dose,” or “therapeutic effective dose.”

[0543] Enantiomers: As used herein, the term “enantiomer” means each individual optically active form of the compound herein having an optical purity or enantiomer excess of at least 80% (i.e., at least 90% of one enantiomer and up to 10% of the other enantiomer), at least 90%, or at least 98% (determined by standard methods of the art).

[0544] To seal: As used herein, the term “to seal” means to seal, enclose, or wrap.

[0545] Encapsulation efficiency: As used herein, “encapsulation efficiency” refers to the amount of polynucleotides that become part of the nanoparticle composition relative to the initial total amount of polynucleotides used to prepare the nanoparticle composition. For example, if 97 mg of polynucleotides out of a total of 100 mg of polynucleotides initially supplied to a nanoparticle composition are encapsulated in that composition, the encapsulation efficiency can be expressed as 97%. As used herein, “encapsulation” may mean completely, substantially, or partially sealing, enclosing, surrounding, or wrapping.

[0546] Epithelial cells: As used herein, “epithelial cells” include cells of epithelial origin. Examples of epithelial cells are respiratory epithelial cells, nasal epithelial cells, alveolar epithelial cells, lung epithelial cells, or bronchial epithelial cells. In some embodiments, epithelial cells are human bronchial epithelial (HBE) cells. In some embodiments, epithelial cells are in vitro cells. In some embodiments, epithelial cells are in vivo cells.

[0547] Expression: As used herein, “expression” of a nucleic acid sequence means one or more of the following: (1) the generation of an mRNA template from a DNA sequence (e.g., by transcription); (2) the processing of an mRNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) the translation of mRNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0548] ex vivo: As used herein, the term “ex vivo” refers to an event occurring outside of an organism (e.g., an animal, plant, or microorganism, or its cells or tissues). Ex vivo events may occur in environments minimally altered from their natural (e.g., in vivo) environment.

[0549] Helper Lipids: As used herein, the term “helper lipid” refers to a compound or molecule comprising a lipid portion (for insertion into lipid layers, e.g., lipid bilayers) and a polar portion (for interaction with physiological solutions on the surface of lipid layers). Typically, helper lipids are phospholipids. The function of helper lipids is to “complement” aminolipids to increase bilayer membrane fusion and / or to assist in facilitating endosomal extrusion (e.g., nucleic acids delivered to cells). Helper lipids are also considered to be important structural components of the surface of LNPs.

[0550] in vitro: As used herein, the term “in vitro” refers to events occurring in an artificial environment, such as a test tube or reaction vessel, a cell culture, or a petri dish, rather than inside a living organism (e.g., an animal, plant, and / or microorganism).

[0551] in vivo: As used herein, the term “in vivo” refers to events occurring inside an organism (for example, an animal, plant, or microorganism, or its cells or tissues).

[0552] Ionized aminolipids: The term "ionized aminolipids" includes lipids having one, two, three, or more fatty acid or fatty alkyl chains and pH titrable amino head groups (e.g., alkylamino or dialkylamino head groups). Ionized aminolipids are typically protonated (i.e., positively charged) at pH below the pKa of the amino head group and substantially uncharged at pH above the pKa. Examples of such ionized aminolipids include, but are not limited to, DLin-MC3-DMA(MC3) and (13Z,165Z)-N,N-dimethyl-3-nonidocosa-13-16-diene-1-amine (L608).

[0553] Isomers: As used herein, the term “isomer” means any tautomer, stereoisomer, enantiomer, or diastereomer of any of the compounds of this disclosure. The compounds of this disclosure may have one or more chiral centers and / or double bonds and are therefore recognized to exist as stereoisomers, e.g., double bond isomers (i.e., geometric E / Z isomers) or diastereomers (i.e., enantiomers (i.e., (+) or (-)) or cis / trans isomers). According to this disclosure, the chemical structures described herein and therefore the compounds of this disclosure encompass all of the corresponding stereoisomers, i.e., stereoisomerically pure forms (i.e., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomer mixtures and stereoisomer mixtures (i.e., racemates). Enantiomer and stereoisomer mixtures of the compounds disclosed herein can typically be separated into their enantiomers or stereoisomers by known methods such as chiral phase gas chromatography, chiral phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents, and catalysts by known asymmetric synthesis methods.

[0554] Lipid nanoparticle core: As used herein, a lipid nanoparticle core is a lipid nanoparticle to which a post-addition layer of additional components such as a cationic agent and / or PEG-lipid or other lipid may be added. In some embodiments, the lipid nanoparticle core comprises (i) an ionized lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) optionally a PEG-lipid. In further embodiments, the lipid nanoparticle core comprises (i) an ionized lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid.

[0555] Linker: As used herein, “linker” refers to, but is not limited to, a group of atoms, such as 10 to 1,000 atoms, and may consist of atoms or groups such as carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. A linker can be bound at a first end to a modified nucleoside or modified nucleotide on a nucleic acid base or sugar moiety, and at a second end to a payload, such as a detectable drug or therapeutic agent. A linker may be of sufficient length so as not to interfere with introduction into a nucleic acid sequence. A linker can be used for any useful purpose, such as forming a polynucleotide polymer (e.g., via the linkage of two or more chimeric polynucleotide molecules or IVT polynucleotides) or a polynucleotide conjugate, and for administering a payload, as described herein. Examples of chemical groups that can be introduced into the linker 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, as well as their derivatives. Other examples include, but are not limited to, cleavable moieties within the linker that can be cleaved using a reducing agent 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 by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and / or photolysis, and ester bonds, which can be cleaved by acidic or basic hydrolysis, for example.

[0556] Lung cells: As used herein, “lung cells” include cells of lung origin. Lung cells may be, for example, lung epithelial cells, airway basal cells, bronchiolar exocrine cells, pulmonary neuroendocrine cells, alveolar cells, or airway epithelial cells. In some embodiments, lung cells are in vitro cells. In some embodiments, lung cells are in vivo cells.

[0557] Method of administration: As used herein, “method of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. The method of administration may be selected to target delivery to a specific area or system of the body (e.g., specific delivery).

[0558] The term "nucleic acid" in its broadest sense includes any compound and / or substance containing polymers of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of this 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 having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0559] Patient: As used herein, “patient” means a person who may be seeking treatment, may need treatment, needs treatment, is receiving treatment, will receive treatment, or is receiving medical care from a specialist skilled in a particular disease or condition.

[0560] CFTR-related disorders: As used herein, the terms “CFTR-related disorders” or “CFTR-related conditions” refer, respectively, to diseases or disorders resulting from abnormal activity of CFTR (e.g., decreased or increased activity). As a non-limiting example, cystic fibrosis is a CFTR-related disorder. Numerous clinical variants of cystic fibrosis are known in the art. See, for example, www.omim.org / entry / 219700.

[0561] The terms “CFTR enzyme activity,” “CFTR activity,” and “cystic fibrosis membrane conductance regulatory factor activity” are used interchangeably in this disclosure and refer to the ability of CFTR to transport chloride ions across the cell membrane. Accordingly, CFTR enzyme activity or a fragment or variant possessing CFTR activity refers to a fragment or variant that performs measurable chloride transport across the cell membrane.

[0562] Pharmaceutically acceptable: The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, and / or dosage form that is suitable, within reasonable medical judgment, for use in contact with human and animal tissues in a reasonable benefit-to-risk ratio without excessive toxicity, irritation, allergic reaction, or other problems or complications.

[0563] Pharmacopoeia-acceptable excipients: As used herein, the term "pharmacopoeia-acceptable excipients" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is substantially non-toxic and non-inflammatory in the patient. Examples of excipients include anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, pigments (colorants), softeners, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, fluidizers (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersants, sweeteners, and hydration water. Examples of excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, 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.

[0564] pharmaceutically acceptable salts: This disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salt” means a derivative of a compound of the disclosed compound in which the parent compound is modified by converting an existing acidic or base moiety to 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 salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Typical acid addition salts include acetate, acetic acid, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptoneate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethane. Examples include sulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and valersates. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations (but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc.). The pharmaceutically acceptable salts of this disclosure include, for example, conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized by conventional chemical methods from a parent compound containing a basic or acidic moiety.Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two (generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used). A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17. th See also, Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, PHStahl and CGWermuth (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.

[0565] As used herein, the term “solvate” means a compound of the disclosure in which a molecule of a suitable solvent is introduced into the crystal lattice. A suitable solvent is one that is physiologically tolerable at the administered dose. For example, solvates may 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), NN'-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, and benzyl benzoate. When water is the solvent, the solvate is called a "hydrate."

[0566] Polynucleotide: As used herein, the term “polynucleotide” refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, this term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). This term also includes modified forms of polynucleotides (e.g., by alkylation and / or capping), as well as unmodified forms. More specifically, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) (including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or not), any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a non-nucleotide backbone, such as polyamides (e.g., peptide nucleic acids “PNA”) and polymorpholinopolymers, and other synthetic sequence-specific nucleic acid polymers (limited to those polymers containing nucleic acid bases in a configuration that allows for base pairing and base stacking as found in DNA and RNA). In certain embodiments, polynucleotides include mRNA. In other embodiments, mRNA is synthetic mRNA. In some embodiments, synthetic mRNA includes at least one non-natural nucleic acid base. In some embodiments, all nucleic acid bases of a particular class are replaced with non-natural nucleic acid bases (for example, all uridines in the polynucleotides disclosed herein may be replaced with non-natural nucleic acid bases, such as 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., synthetic RNA or synthetic DNA) comprises only natural nucleic acid bases, 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.

[0567] Those skilled in the art will understand that while T bases in the codon maps disclosed herein are present in DNA, in the corresponding RNA, T bases are replaced by U bases. For example, codon-nucleotide sequences disclosed herein in DNA form, such as vectors or in-vitro translation (IVT) templates, have T bases that are transcribed as U based on their corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (containing T) and their corresponding mRNA sequences (containing U) are considered codon-optimized nucleotide sequences of this disclosure. Those skilled in the art will also understand that equivalent codon maps can be created by replacing one or more bases with non-native bases. Thus, for example, a TTC codon (DNA map) corresponds to a UUC codon (RNA map), which then corresponds to a ΨΨC codon (RNA map in which U is replaced with pseudouridine).

[0568] Standard AT and GC base pairs are formed under conditions that allow for hydrogen bond formation 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. Therefore, 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 modification results in a nucleoside base that no longer effectively forms standard base pairs with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-aminopyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxypyrimidine) results in a modified nucleotide that does not effectively form a base pair with guanosine but does form a base pair with isoguanosine (Collins et al., U.S. Patent No. 5,681,702). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described in Switzer et al. (1993) Biochemistry 32:10489-10496 and the references cited therein; 2'-deoxy-5-methylisocytidine can be prepared by the method described in 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 in Switzer et al., 1993, cited above and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Patent No. 5,780,610 of Collins et al.Other non-natural base pairs can be synthesized by the method described in 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., cited above.

[0569] Polypeptides: The terms “polypeptide,” “peptide,” and “protein” are used herein interchangeably to refer to polymers of amino acids of any length. Polymers may include modified amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, e.g., conjugation with a labeling component. Polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art, are also included in this definition.

[0570] As used herein, this term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogs thereof. Polypeptides may be monomers or multimolecular complexes such as dimers, trimers, or tetramers. Polypeptides may also include single-chain or multi-chain polypeptides. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a “peptide” may be 50 amino acid lengths or less, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths.

[0571] Preventing: As used herein, the term “preventing” means partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, characteristics, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more signs and symptoms, characteristics, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition; and / or reducing the risk of developing a condition associated with an infection, disease, disorder, and / or condition.

[0572] Prophylactic: As used herein, “prophylactic” refers to a therapeutic or set of actions taken to prevent the spread of disease.

[0573] Prophylaxis: As used herein, “prophylaxis” refers to measures taken to maintain health and prevent the spread of disease. “Immunoprevention” refers to measures taken to produce.

[0574] Salts: In some embodiments, the pharmaceutical compositions disclosed herein contain salts of some of their lipid components. The term “salt” includes any anionic and cationic complexes. Non-limiting examples of anions include inorganic and organic anions, e.g., fluorides, chlorides, bromides, iodides, oxalic acid (e.g., hemioxalic acid), phosphoric acid, phosphonic acid, hydrogen phosphate, dihydrogen phosphate, oxides, carbonic acid, bicarbonate, nitric acid, nitrite, nitride, bisulfite, sulfide, bisulfuric acid, sulfuric acid, thiosulfate, bisulfuric acid, boric acid, formic acid, acetic acid, benzoic acid, citric acid, tartaric acid, lactic acid, acrylic acid, polyacrylic acid, fumaric acid, maleic acid Examples include ic acid, itaconic acid, glycolic acid, gluconic acid, malic acid, mandelic acid, tigric acid, ascorbic acid, salicylic acid, polymethacrylic acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, bromate, hypobromous acid, iodic acid, alkyl sulfonic acid, aryl sulfonic acid, arsenic acid, arsenous acid, chromic acid, dichromate, cyanide, cyanic acid, thiocyanic acid, hydroxide, peroxide, permanganic acid, and mixtures thereof.

[0575] Sample: As used herein, the terms “sample” or “biological sample” refer to a subset of its tissues, cells, or components (including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen). A sample may further include homogenates, lysates, or extracts prepared from a whole organism or a subset of its tissues, cells, or components, or fractions or parts thereof (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, external sections (of skin, respiratory, intestinal, and genitourinary tracts), tears, saliva, milk, blood cells, tumors, and organs). A sample may further refer to a medium such as a nutrient broth or gel that may contain cellular components such as proteins or nucleic acid molecules.

[0576] Single unit dose: As used herein, “single unit dose” is the dose of any therapeutic agent administered in one dose / in one instance / through a single route / at a single point of contact, i.e., in a single administration event.

[0577] Divided dose: As used herein, “divided dose” refers to a single unit dose or a total daily dose divided into two or more doses.

[0578] Stereoisomers: As used herein, the term “stereoisomer” means all possible different isomers and stereostructural forms that a compound may have (e.g., any compound of any of the formulas described herein), in particular all possible stereochemical and stereostructural isomers of the basic molecular structure, all diastereomers, enantiomers, and / or conformational isomers. Some of the compounds in this disclosure may exist in different tautomers, all of which are included within the scope of this disclosure.

[0579] Subject: "Subject," "individual," "animal," "patient," or "mammal" means any subject, especially mammalian subjects, for which diagnosis, prognosis prediction, or treatment is desired. Mammalian subjects include, but are not limited to, humans, domesticated animals, livestock, zoo animals, sports animals, and companion animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and cows); primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felines such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals (e.g., cows, pigs, and sheep); ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters, and guinea pigs. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In certain embodiments, the subject is a human patient requiring treatment.

[0580] Substantially: As used herein, the term “substantially” refers to a qualitative state indicating all or nearly all range or degree of the feature or characteristic in question. Those skilled in the art of biology will understand that it is rare, if any, for biological and chemical features to become complete, and / or progress toward completeness, or to achieve or avoid absolute results. Therefore, the term “substantially” is used herein to capture the potential lack of completeness inherent in many biological and chemical features.

[0581] Affected: An individual “affected” by a disease, disorder, and / or condition is diagnosed with that disease, disorder, and / or condition, or exhibits one or more signs and symptoms thereof.

[0582] Susceptible to disease: Individuals “suspicious” to a disease, disorder, and / or condition are those who have not been diagnosed with the disease, disorder, and / or condition and / or cannot exhibit the signs and symptoms of the disease, disorder, and / or condition, but who have a tendency to develop the disease or its signs and symptoms. In some embodiments, individuals susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) gene mutations associated with the development of the disease, disorder, and / or condition; (2) gene polymorphisms associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; (5) family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with microorganisms associated with the development of the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition develop that disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not develop that disease, disorder, and / or condition.

[0583] Synthesis: The term "synthetic" means artificially produced, prepared, and / or manufactured. The synthesis of the polynucleotides or other molecules of this disclosure may be chemical or enzymatic.

[0584] Therapeutic agent: The term “therapeutic agent” refers to a drug that, when administered to a subject, has therapeutic, diagnostic, and / or prophylactic effects, and / or elicits a desired biological and / or pharmacological effect. For example, in some embodiments, mRNA encoding a CFTR polypeptide may be a therapeutic agent.

[0585] Therapeutic dose: As used herein, the term “therapeutic dose” means the amount of a delivered agent (e.g., nucleic acids, drugs, therapeutic agents, diagnostic agents, prophylactic agents, etc.) that, when administered to a subject who is afflicted with or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, improve, diagnose, prevent, and / or delay the onset of such infection, disease, disorder, and / or condition.

[0586] Therapeutic outcome: As used herein, the term “therapeutic outcome” means an outcome that is sufficient to treat, improve, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject that is affected or susceptible to such infection, disease, disorder, and / or condition.

[0587] Total daily dose: As used herein, “total daily dose” refers to the amount given or prescribed within a 24-hour period. The total daily dose may be administered as a single unit dose or in divided doses.

[0588] Treatment, Therapy: As used herein, the terms “treatment” or “therapy” mean to partially or completely reduce, alleviate, improve, mitigate, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of, one or more signs, symptoms or characteristics of a disease, such as cystic fibrosis. For example, “treatment” of cystic fibrosis could mean reducing the signs and symptoms associated with the disease, extending the patient’s lifespan (increasing survival rate), reducing the severity of the disease, or preventing or delaying the onset of the disease. Treatment may be applied to subjects who do not show signs of a disease, disorder, and / or condition, and / or subjects who show only the initial signs of a disease, disorder, and / or condition, for the purpose of reducing the risk of developing a condition associated with the disease, disorder, and / or condition.

[0589] As used herein, the terms "alkyl" or "alkyl group" mean a linear or branched saturated hydrocarbon comprising one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms).

[0590] "C 1~14 The term "alkyl" refers to a linear or branched saturated hydrocarbon containing 1 to 14 carbon atoms. Alkyl alkyl groups may be optionally substituted.

[0591] As used herein, the terms “alkenyl” or “alkenyl group” mean a linear or branched hydrocarbon comprising two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more carbon atoms) and at least one double bond.

[0592] "C 2~14The term "alkenyl" refers to a linear or branched hydrocarbon containing 2 to 14 carbon atoms and at least one double bond. An alkenyl group may contain 1, 2, 3, 4, or more double bonds. Alkenyl groups may be optionally substituted.

[0593] As used herein, the terms “carbocyclic” or “carbocyclic group” mean a monocyclic or polycyclic system containing one or more rings of carbon atoms. The rings may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-membered rings.

[0594] "C 3~6 The term "carbocyclic ring" refers to a monocyclic ring...

Claims

1. (a) Lipid nanoparticle core, (b) A polynucleotide payload enclosed within the core for delivery to cells, (c) Cationic agents and Nanoparticles containing, The cationic agent is mainly disposed on the outer surface of the core, and more than 90% of the cationic agent is on the surface of the nanoparticles. The aforementioned nanoparticles have a zeta potential exceeding neutrality, approximately 5 mV to approximately 15 mV at a physiological pH. The lipid nanoparticle core, (i) Ionized lipids, (ii) Phospholipids, (iii) Structural lipids, and (iv) PEG-lipids The nanoparticles comprising the above.

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

3. The nanoparticles according to claim 1, wherein the nanoparticles exhibit about 0.5% to 50% protein expression in the cells, and the cells are in vivo.

4. The nanoparticle according to any one of claims 1 to 3, wherein the weight ratio of the cationic agent to the 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.

5. The nanoparticles according to any one of claims 1 to 4, wherein the nanoparticles have a zeta potential of about 5 mV to about 10 mV.

6. The nanoparticle according to any one of claims 1 to 5, wherein the lipid nanoparticle core has a neutral charge at a neutral pH.

7. The nanoparticle according to any one of claims 1 to 6, wherein more than 95% of the cationic agent is located on the surface of the nanoparticle.

8. The nanoparticles according to any one of claims 1 to 7, wherein at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the polynucleotide payload are encapsulated within the core.

9. The nanoparticles according to any one of claims 1 to 8, wherein the nanoparticles have a polydispersity value of less than about 0.4, less than about 0.3, or less than about 0.

2.

10. The nanoparticles according to any one of claims 1 to 9, 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.

11. The nanoparticle according to any one of claims 1 to 10, wherein the general polarity (GPL) of the Raurdan of the nanoparticle is about 0.6 or more.

12. The nanoparticles according to any one of claims 1 to 11, wherein the nanoparticles have a d-spacing greater than about 6 nm or greater than about 7 nm.

13. The nanoparticles according to any one of claims 1 to 12, 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.

14. The nanoparticles according to any one of claims 1 to 13, wherein when the nanoparticles come into contact with a cell population, about 10% or more, about 15% or more, or about 20% or more of the cell population accumulate the nanoparticles.

15. The nanoparticles according to any one of claims 1 to 14, wherein when the nanoparticles come into contact with a cell population, about 5% or more of the cells or about 10% or more of the cells express the polynucleotide or polypeptide.

16. The nanoparticle according to any one of claims 1 to 15, wherein the cell population is an epithelial cell population, a respiratory epithelial cell population, a nasal cell population, an alveolar epithelial cell population, a lung cell population, a bronchial epithelial cell population, or an HBE population.

17. Whether the cationic agent is a cationic lipid; The cationic agent is a cationic lipid, and the cationic lipid is a sterolamine comprising a hydrophobic portion and a hydrophilic portion; or The cationic agent is a cationic lipid, the cationic lipid is a sterolamine, and the sterolamine is, the sterolamine is, formula (A1): A-L-B (A1) A compound or salt thereof, in the formula: The nanoparticle according to any one of claims 1 to 16, wherein A is an amine group, L is an optionally selected linker, and B is a sterol.

18. The sterolamine is of formula A2a: 【Chemistry 1】 or a salt thereof, in the formula: 【Chemistry 2】 These are single or double bonds, R １ C １～１４ Alkyl or C １～１４ It is an alkenil; L ａ is absent, or -O-, -S-S-, -OC(=O), -C(=O)N-, -OC(=O)N-, CH ２ -NH-C(O)-, -C(=O)O-, -OC(=O)-CH ２ -CH ２ -C(=O)N-, -S-S-CH ２ , -SS-CH ２ -CH ２ -C(=O)N-, or formula (a): 【Transformation 3】 It is the basis of; Y １ C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), or -C １～６ It is alkyl-(5-6 member heteroaryl), Said C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), and -C １～６ Alkyl-(5-6 member heteroaryl) comprises 1-5 primary, secondary, or tertiary amines, or combinations thereof; Said C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), and -C １～６ Alkyl- (5-6 member heteroaryl) １～６ Alkyl, Halo, OH, -O(C) １～６ Alkyl), -C １～６ alkyl-OH, NH ２ ,-NH(C １～６ Alkyl), N (C １～６ Alkyl) ２ , 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) １～１４ They are optionally substituted with one, two, three, or four substituents independently selected from alkyl groups, 5-6 member heteroaryl groups, -NH- (3-8 member heterocycloalkyl groups), and -NH (5-6 member heteroaryl groups); n is either 1 or 2. The nanoparticles according to claim 17.

19. L ａ However, -OC(=O), -OC(=O)N-, or -OC(=O)-CH ２ -CH ２ The nanoparticle according to claim 18, wherein it is -C(=O)N-.

20. R １ but, 【Chemistry 4】 The nanoparticle according to claim 18 or 19.

21. Y １ However, C １～１０ Alkyl, 3-8 member heterocycloalkyl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), or -C １～６ It is alkyl-(5-6 member heteroaryl), Said C １～１０ Alkyl, 3-8 member heterocycloalkyl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), and -C １～６ Alkyl-(5-6 member heteroaryl) comprises 1-5 primary, secondary, or tertiary amines, or combinations thereof; Said C １～１０ Alkyl, C １～６ Alkyl-(3-8 member heterocycloalkyl), and C １～６ Alkyl- (5-6 member heteroaryl) １～６ Alkyl, OH, -C １～６ Alkyl-OH, or NH ２ Each is optionally replaced, Nanoparticles according to any one of claims 18 to 20.

22. Y １ but, 【Chemistry 5-1】 【Chemistry 5-2】 Nanoparticles according to any one of claims 18 to 21, selected from the above.

23. The sterolamine is, Formula A4: 【Transformation 6】 or a salt thereof, in the formula: Z １ is OH or C ３～６ It is alkyl; L either does not exist, or is -O-, -S-S-, -OC(=O), -C(=O)N-, -OC(=O)N-, -CH ２ -NH-C(=O)-, -C(=O)O-, -OC(=O)-CH ２ -CH ２ -C(=O)N-, -S-S-CH ２ , or -SS-CH ２ -CH ２ -C(O)N-; Y １ C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), or -C １～６ It is alkyl-(5-6 member heteroaryl), Said C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), and -C １～６ Alkyl-(5-6 member heteroaryl) comprises 1-5 primary, secondary, or tertiary amines, or combinations thereof; Said C １～１０ Alkyl, 3-8 member heterocycloalkyl, 5-6 member heteroaryl, -C １～６ Alkyl-(3-8 member heterocycloalkyl), and -C １～６ Alkyl- (5-6 member heteroaryl) １～６ Alkyl, Halo, OH, -O(C) １～６ Alkyl), -C １～６ alkyl-OH, NH ２ ,-NH(C １～６ Alkyl), -N(C １～６ Alkyl) ２ , 3-8 member heterocycloalkyl (containing 1-5 primary, secondary, or tertiary amines, or combinations thereof) １～１４ They are optionally substituted with one, two, three, or four substituents selected from alkyl groups, 5-6 member heteroaryl groups, -NH(3-8 member heterocycloalkyl groups), and -NH(5-6 member heteroaryl groups); n is either 1 or 2. Nanoparticles according to any one of claims 18 to 22.

24. Z １ However, the nanoparticle according to claim 23 is OH.

25. Z １ However, C ３～６ The nanoparticle according to claim 23, wherein it is alkyl.

26. L is -C(=O)N-, -CH ２ The nanoparticle according to any one of claims 23 to 25, wherein it is -NH-C(=O)- or -C(=O)O-.

27. Y １ However, C contains 1 to 5 primary, secondary, or tertiary amines, or combinations thereof. １～１０ A nanoparticle according to any one of claims 23 to 26, wherein the nanoparticle is alkyl.

28. Y １ but, 【Transformation 7】 The nanoparticle according to any one of claims 23 to 26.

29. The sterolamine mentioned above, Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 The nanoparticles according to claim 18, selected from or from a salt thereof.

30. The nanoparticles according to any one of claims 1 to 29, wherein the nanoparticles contain about 30 mol% to about 60 mol% or about 40 mol% to about 50 mol% of ionized lipids.

31. Ionized lipids, compound 18: 【Transformation 8】 The nanoparticles according to any one of claims 1 to 30, or a salt thereof.

32. The nanoparticles according to any one of claims 1 to 31, wherein the nanoparticles contain about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10 mol% to about 12 mol% of phospholipids.

33. The nanoparticles according to any one of claims 1 to 32, wherein the nanoparticles contain about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol%, about 35 mol%, or about 40 mol% of structural lipids.

34. The nanoparticles according to any one of claims 1 to 33, wherein the nanoparticles contain about 1 mol% to about 5 mol% of PEG-lipid, or about 1 mol% to about 2.5 mol% of PEG-lipid.

35. A cell comprising nanoparticles according to any one of claims 1 to 34.

36. A pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 34.

37. The pharmaceutical composition according to claim 36, wherein the composition is suitable for inhalation.

38. A pharmaceutical for treating or preventing a patient's disease, comprising a payload for treating or preventing the disease, comprising nanoparticles according to any one of claims 1 to 34 or a composition according to any one of claims 36 or 37.

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