Compositions and methods for targeted systemic delivery to cells - Patents.com

Abstract

Described herein are compositions, kits, and methods for potent systemic delivery to cells of a subject. Also described herein are pharmaceutical compositions comprising therapeutic or prophylactic agents combined with lipid compositions. The lipid compositions can comprise ionizable cationic lipids, phospholipids, and selective organ targeting lipids. Additionally, described herein are high potency dosage forms of therapeutic or prophylactic agents formulated with lipid compositions.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 164,534, filed March 23, 2021, and U.S. Provisional Application No. 63 / 305,652, filed February 1, 2022, the entireties of which are incorporated by reference herein for all purposes.

[0002] 2. Background of the Invention Therapeutic approaches such as CRISPR / Cas (clustered regularly interspaced short palindromic repeat / CRISPR-associated protein (Cas)) technology often require precise and potent delivery of therapeutic agents to target organs or cells, sometimes in a sequence-dependent manner. To date, there remains a clear need to achieve therapeutically safe and effective lipid-based carriers to achieve clinical outcomes in the context of genetic diseases and many other applications.

[0003] Summary of the Invention In certain aspects, the present application provides compositions for potent delivery of therapeutic agents to cells of a subject. In some embodiments, the compositions can be formulated for systemic (e.g., intravenous) administration, the compositions comprising: (i) an ionizable cationic lipid; (ii) a polymer-conjugated lipid; and (iii) a selective organ targeting (SORT) lipid, or a pharma- ceutically acceptable salt, stereoisomer, or tautomer thereof; a therapeutic agent in combination with a lipid composition comprising The selective organ targeting (SORT) lipid has the formula (IA): [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24)or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0004] In some embodiments, the compositions may be formulated for systemic (e.g., intravenous) administration, the compositions comprising: (i) an ionizable cationic lipid; (ii) a polymer-conjugated lipid; and (iii) a selective organ targeting (SORT) lipid, or a pharma- ceutically acceptable salt, stereoisomer, or tautomer thereof; a therapeutic agent in combination with a lipid composition comprising The selective organ targeting (SORT) lipid has the formula (S-III): [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0005] In certain aspects, the present application provides a method for the potent delivery of therapeutic agents to cells of a subject.In some embodiments, the method described herein can be for targeted delivery of therapeutic agents to spleen cells, and the method comprises administering (e.g., systemically) the composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the spleen cells of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the lung cells of the subject.

[0006] In some embodiments, the methods described herein may be for targeted delivery of a therapeutic agent to lung cells, the methods comprising administering (e.g., systemically) a composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the lung cells of the subject that is at least 1.1-fold greater than the corresponding amount or activity of the therapeutic agent achieved in the spleen cells of the subject.

[0007] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0008] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one color drawing. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures"), in which: [Figure 1] FIG. 1 shows exemplary structures of SORT lipids. [Diagram 2] FIG. 1 shows example structures of ionizable cationic lipid dendrimers or dendrons. [Diagram 3] FIG. 1 shows the stability and general properties of various LNP compositions. [Figure 4A]FIG. 1 shows IVIS organ imaging of the spleen, liver and lungs of a female dog following administration of pulmonary SORT. [Figure 4B] FIG. 1 shows IVIS organ imaging of the spleen, liver and lungs of male dogs following administration of pulmonary SORT. [Figure 5A] FIG. 13 shows IVIS organ imaging of spleen, liver and lungs of cynomolgus NHPs following lung SORT administration. [Figure 5B] FIG. 13 shows IVIS organ imaging of spleen, liver and lungs of cynomolgus NHPs following lung SORT administration. [Figure 6A] FIG. 13 shows IVIS organ imaging of mouse spleen, liver, kidneys and lungs following administration of luciferase mRNA formulated in 14:0 TAP SORT 5 hours later. [Figure 6B] FIG. 6B is a quantitative representation of the signal obtained in the lungs, spleen, and liver of the mice in FIG. 6A. [Figure 7A] FIG. 1 shows TR intensity expressed in hBEs treated with two different SORT LNPs. [Figure 7B] FIG. 1 shows % LDH released from hBE treated with two different SORT LNPs. [Figure 8A] FIG. 13 shows IVIS organ imaging of the spleen, liver and lungs of two beagles following intravenous bolus administration of luciferase mRNA formulated in 14:0 TAP SORT. [Figure 8B] FIG. 13 shows IVIS organ imaging of the spleen, liver and lungs of two beagles following intravenous administration of luciferase mRNA formulated in 14:0 TAP SORT with premedication. [Figure 9] FIG. 1 shows a compiled panel of IVIS organ imaging of dog and NHP spleen, liver, and lungs. [Figure 10A] FIG. 13 shows ex vivo imaging of bioluminescence in the spleen, lung, liver and kidney following intravenous administration of Luc mRNA / LNP using multiple LNP compositions. [Figure 10B]FIG. 10B is a quantitative graph of the bioluminescence data of FIG. 10A. [Figure 11A] FIG. 13 shows ex vivo imaging of bioluminescence in the spleen, lung, liver and kidney following intravenous administration of Luc mRNA / LNP using multiple LNP compositions. [Figure 11B] FIG. 11B is a quantitative graph of the bioluminescence data of FIG. 11A.

[0009] Detailed Description Before embodiments of the present disclosure are described, it is understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the invention.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein can be used to practice or test the present invention, but suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, shall prevail. Moreover, the materials, methods, and examples are merely illustrative and are not intended to be limiting. Numerous variations, modifications, and substitutions will readily occur to those skilled in the art without departing from the present invention.

[0011] In the context of this application, the following terms have the meanings ascribed to them unless specified otherwise.

[0012] As used throughout this specification and claims, the terms "a," "an," and "the" are generally used in the sense of meaning "at least one," "at least the first," "one or more," or "multiple" of the referenced component or step, unless an upper limit is specifically stated thereafter. For example, "cleavage sequence" as used herein means "at least the first cleavage sequence," but includes multiple cleavage sequences. As with the amount of any single agent, the operable limits and parameters of the combination will be known to those of skill in the art in light of this application.

[0013] The terms "polypeptide", "peptide" and "protein" are generally used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0014] As used herein in reference to a polypeptide structure, the "N-terminus" (or "amino terminus") and the "C-terminus" (or "carboxyl terminus") generally refer to the extreme amino and carboxyl termini, respectively, of a polypeptide.

[0015] The term "N-terminal sequence" as used herein with respect to a polypeptide or polynucleotide sequence of interest generally means that there are no other amino acid or nucleotide residues at the N-terminus prior to the N-terminal sequence in the polypeptide or polynucleotide sequence of interest. The term "C-terminal sequence" as used herein with respect to a polypeptide or polynucleotide sequence of interest generally means that there are no other amino acid or nucleotide residues at the C-terminus following the C-terminal sequence in the polypeptide or polynucleotide sequence of interest.

[0016] The terms "non-naturally occurring" and "non-naturally occurring" are used interchangeably herein. The term "non-naturally occurring" or "non-natural" as used herein with respect to a therapeutic or prophylactic agent generally means that the agent is not biologically derived in a mammal (including, but not limited to, a human). The terms "non-naturally occurring" and "non-naturally occurring" as applied to a sequence and used herein refer to a polypeptide or polynucleotide sequence that is not paired, complementary, or highly homologous to a wild-type or naturally occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, or less amino acid sequence identity with a natural sequence when properly aligned.

[0017] "Physiological conditions" refers to a set of conditions in a living host and in vitro conditions that mimic those of a living subject, including temperature, salt concentration, and pH. A large number of physiologically relevant conditions have been established for use in in vitro assays. Generally, physiological buffers contain physiological concentrations of salt and are adjusted to a neutral pH ranging from about 6.5 to about 7.8, preferably from about 7.0 to about 7.5. Various physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperatures range from about 25°C to about 38°C, preferably from about 35°C to about 37°C.

[0018] As used herein, the terms "treatment" or "treating" or "palliating" or "ameliorating" are used interchangeably herein. These terms generally refer to an approach to obtain a beneficial or desired result, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disease being treated. Also, therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disease, or improvement of one or more clinical parameters, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disease. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even if the disease may not have been diagnosed.

[0019] As used herein, "therapeutic effect" or "therapeutic benefit" generally refers to a physiological effect resulting from administration of a polypeptide of the present disclosure other than the ability to induce the production of antibodies against an antigenic epitope possessed by the biologically active protein, including, but not limited to, the alleviation, amelioration, or prevention of disease in a human or other animal, or the improvement of one or more clinical parameters associated with an underlying disease, or otherwise enhancing the physical or mental health of a human or animal. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, a symptom or symptom of a disease, or a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0020] The terms "therapeutically effective amount" and "therapeutically effective dose" as used herein generally refer to an amount of a drug or biologically active protein, alone or as part of a polypeptide composition, that can have any detectable beneficial effect on the symptoms, aspects, measured parameters or characteristics of a disease state or condition when administered to a subject in one or multiple doses. Such an effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0021] The term "equimolar dose" generally means that the amount of a material administered to a subject has an equivalent molar amount, based on the molecular weight of the material used in the dose.

[0022] The term "therapeutically effective and non-toxic dose" as used herein generally refers to a tolerable dose of a composition as defined herein that is high enough to cause tumor or cancer cell depletion, tumor removal, tumor shrinkage, or disease stabilization without or essentially without significant toxic effects in a subject.Such a therapeutically effective and non-toxic dose can be determined by dose escalation studies as described in the art, and should be below the dose that induces severe side effects.

[0023] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0024] When used in the context of a chemical group, "hydrogen" means -H, "hydroxy" means -OH, "oxo" means =O, "carbonyl" means -C(=O)-, "carboxy" means -C(=O)OH (also written as -COOH or -COH), "halo" means, independently, -F, -Cl, -Br or -I, "amino" means -NH, "hydroxyamino" means -NHOH, "nitro" means -NO, imino means =NH, "cyano" means -CN, and "isocyanato" means -C(=O)-. , -N=C=O, "azide" means -N3, in the monovalent context "phosphate" means -OP(O)(OH)2 or a deprotonated form thereof, and in the divalent context "phosphate" means -OP(O)(OH)O- or a deprotonated form thereof, "mercapto" means -SH, "thio" means =S, "sulfonyl" means -S(O)2-, "hydroxysulfonyl" means -S(O)2OH, "sulfonamide" means -S(O)2NH2, and "sulfinyl" means -S(O)-.

[0025] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. [ka] represents an optional bond, which, if present, is either a single bond or a double bond. [ka] represents a single or double bond. Thus, for example, the formula [ka] teeth, [ka] and it is understood that no such ring atom forms part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate a preferred stereochemistry when linking one or two stereoatoms. Instead, it covers all stereoisomers and mixtures thereof. The symbol [ka] is a bond (e.g., for methyl [ka] When drawn vertically across a group, it indicates the point of attachment of the group. Note that points of attachment are typically only identified in this way for larger groups in order to allow the reader to clearly identify the point of attachment. [ka] denotes a single bond in which the group attached to the thick end of the wedge is "off the page". [ka] denotes a single bond in which the group attached to the thick end of the wedge is "into the page". [ka] means a single bond with no defined geometry around the double bond (e.g., either E or Z). Thus, both options and combinations thereof are contemplated. Undefined valences on atoms of structures shown in this application implicitly represent a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is pointing out of the plane of the paper.

[0026] The group "R" may, for example, be of the formula [ka] When depicted as a "floating group" on a ring system, R may replace any hydrogen atom attached to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is formed. [ka] When depicted as a "floating group" on a fused ring system, such as in R may replace any hydrogen bonded to any ring atom of the fused ring, unless otherwise specified. Replaceable hydrogens include depicted hydrogens (e.g., hydrogen bonded to the nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and any hydrogens present depending on the identity of the ring atom (e.g., hydrogen bonded to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present in either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the bracketed group "R" represents a numerical variable. Unless otherwise specified, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms in the ring or ring system.

[0027] In chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that may be present in the group / class, the minimum number being as small as possible for that group / class, e.g., the group "alkenyl (C≦8) " or class "Alkenes (C≦8) " is understood to be 2. "Alkoxy" designates an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn~n'" defines both the minimum number (n) and the maximum number (n') of carbon atoms in the group. Thus, "alkyl (C2~10)" designates an alkyl group having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class they modify, and may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "Olefin C5 " are all synonymous.

[0028] When the term "saturated" is used to modify a compound or chemical group, it means that the compound or chemical group has no carbon-carbon double bonds or carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substituents of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. If such bonds are present, they do not exclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism. When the term "saturated" is used to modify a solution of a substance, it means that the substance cannot be dissolved any more in the solution.

[0029] The term "aliphatic", when used without the "substituted" modifier, indicates that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) joined by carbon-carbon single bonds, or unsaturated with one or more carbon-carbon double bonds (alkene / alkenyl), or unsaturated with one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0030] The term "aromatic," when used to modify an atom of a compound or chemical group, means that the compound or chemical group contains a planar unsaturated ring of atoms stabilized by the interaction of bonds that form the ring.

[0031] The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), -CH(CH3)2(i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl", when used without the "substituted" modifier, refers to a divalent saturated aliphatic group with one or two saturated carbon atoms as points of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl, as that term is defined above. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl where the replacement of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or -I) and no other atoms are present except carbon, hydrogen, and halogen. The group -CH2Cl is a non-limiting example of haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl where the replacement of hydrogen atoms is limited to fluoro and no other atoms are present except carbon, hydrogen, and fluorine. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0032] The term "cycloalkyl", when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl", when used without the "substituted" modifier, refers to a divalent saturated aliphatic group with two carbon atoms as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group [ka] is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula HR, where R is cycloalkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0033] The term "alkenyl", when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, no at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl", when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group having two carbon atoms as the point of attachment, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It should be noted that although alkenediyl groups are aliphatic, it is not excluded that the group forms part of an aromatic structure when attached at both ends. The terms "alkene" and "olefin" are synonymous and refer to the class of compounds having the formula HR, where R is alkenyl, as that term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to alkenes that have only one carbon-carbon double bond, which is part of a vinyl group at the end of the molecule. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0034] The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to the class of compounds having the formula HR, where R is alkynyl. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0035] The term "aryl", when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structures, all of the ring atoms being carbon, and the group being composed of no atoms other than carbon and hydrogen. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitations permitted) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -CHCHCH(ethylphenyl), naphthyl, and biphenyl. The term "arenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structures, all of the ring atoms being carbon, and the monovalent group being composed of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitations permitted) attached to the first aromatic ring or any additional aromatic rings present. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitations permitted). Non-limiting examples of arenediyl groups include: [ka] Examples include:

[0036] "Arene" refers to a class of compounds having the formula HR, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0037] The term "aralkyl," when used without the "substituted" modifier, refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used consistent with the definitions provided above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl groups are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

[0038] The term "heteroaryl", when used without the "substituted" modifier, refers to a monovalent aromatic group with an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heteroaryl group is composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. The heteroaryl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number limitations permitted) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group with a nitrogen atom as the point of attachment. The term "heteroarenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group being composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitations permitted). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number limitations permitted) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyl groups include: [ka] Examples include:

[0039] "Heteroarene" refers to a class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0040] The term "heterocycloalkyl", when used without the "substituted" modifier, refers to a monovalent non-aromatic group with a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heterocycloalkyl group is composed of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitations permitted) attached to a ring or ring system. Additionally, the term does not preclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl", when used without the "substituted" modifier, refers to a divalent cyclic group with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group is composed of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked through one or more of a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limit permitted). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limit permitted) attached to a ring or ring system.The term also does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: [ka] When these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0041] The term "acyl", when used without the "substituted" modifier, refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as these terms are defined above. The groups -CHO, -C(O)CH3(acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3), -C(O)CH(CH2), -C(O)CH6H5, -C(O)CH4CH3, -C(O)CH2C6H5, -C(O)(imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined in an analogous manner, except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom -C(S)R. The term "aldehyde" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms (including any hydrogen atoms directly bonded to the carbon atom of a carbonyl or thiocarbonyl group) are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups, -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0042] The term "alkoxy", when used without the "substituted" modifier, refers to the group -OR, where R is alkyl, as that term is defined above. Non-limiting examples include -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2), -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio", when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0043] The term "alkylamino," when used without the "substituted" modifier, refers to the group --NHR, where R is alkyl, as that term is defined above. Non-limiting examples include --NHCH and --NHCH.

[0044] The term "dialkylamino", when used without the "substituted" modifier, refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' taken together can represent an alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino", and "alkylsulfonylamino", when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH3. The term "alkylimino", when used without the "substituted" modifier, refers to the divalent group =NR, where R is alkyl, as that term is defined above. When any of these terms are used in conjunction with the "substituted" modifier, one or more hydrogen atoms bonded to a carbon atom are independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.The groups -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amide groups.

[0045] Unless otherwise indicated, all numerical values ​​expressing amounts of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in all cases by the term "about". Thus, unless otherwise indicated, the numerical parameters described in the specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present application. In general, the term "about" used in this specification when referring to a measurable value such as weight, time, dose, and the like, is meant to encompass variations from the stated amount of ±20% or ±10%, in one example, ±5%, in another example, ±3%, in another example, ±1%, and in yet another example, ±0.1%, where such variations are appropriate for carrying out the disclosed method.

[0046] As used in this application, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have a different degree of polymerization and therefore a different molar mass. Average molecular weight can be used to represent the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number average molar mass, weight (mass) average molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, average molecular weight represents either the number average molar mass or the weight average molar mass of the formula. In some embodiments, the average molecular weight is the number average molar mass. In some embodiments, average molecular weight can be used to represent the PEG components present in lipids.

[0047] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs is also open-ended, such as "comprises," "comprising," "has," "having," "includes," "including," etc. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other steps that are not listed.

[0048] The term "effective" as used herein and / or in the claims means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmacologically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to effectively effect such treatment for the disease.

[0049] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit half of a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0050] An "isomer" of a first compound is a distinct chemical compound in which each molecule contains the same constituent atoms as the first compound, but differs in the arrangement of those atoms in three dimensions.

[0051] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., a non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0052] The terms "assemble" or "assembled" as used herein in connection with delivery of a payload to a target cell generally refer to a covalent or non-covalent interaction or association, e.g., where a therapeutic or prophylactic agent is complexed with or encapsulated in a lipid composition.

[0053] As used herein, the term "lipid composition" generally refers to a composition that includes lipid compounds, including, but not limited to, lipoplexes, liposomes, lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

[0054] As used herein, the term "detectable" refers to the occurrence or change of a signal that is directly or indirectly detectable by either observation or instrumentation. Typically, the detectable response is the occurrence of a signal where the fluorophore is intrinsically fluorescent and does not undergo a signal change upon binding with a metal ion or biological compound. Alternatively, the detectable response is an optical response that results in a change in the wavelength distribution pattern or intensity of absorbance or fluorescence, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0055] The term "potent" or "potency" as used herein with respect to the delivery of therapeutic agents generally refers to a higher ability of a delivery system (e.g., lipid composition) to achieve or cause a desired amount, activity, or effect of a therapeutic or prophylactic agent in a cell (e.g., target cell) to any measurable extent, e.g., compared to a reference delivery system, of a desired level of translation, transcription, production, expression, or activity of a protein or gene. For example, a lipid composition with higher potency may achieve a desired therapeutic effect in a greater number of relevant cell populations, within a shorter response time, or for a longer duration.

[0056] As generally used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0057] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present application that is pharma- ceutically acceptable as defined above and has the desired pharmacological activity. Such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, and the like. Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as sucrose, fumaric, glucoheptonic, gluconic, glutamic, glycolic, heptanoic, hexanoic, hydroxynaphthoic, lactic, lauryl sulfuric, maleic, malic, malonic, mandelic, methanesulfonic, muconic, o-(4-hydroxybenzoyl)benzoic, oxalic, p-chlorobenzenesulfonic, phenyl-substituted alkanoic, propionic, p-toluenesulfonic, pyruvic, salicylic, stearic, succinic, tartaric, tert-butyl acetate, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present can react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharma- ceutically acceptable.Additional examples of pharma- ceutically acceptable salts and their methods of preparation and use are described in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0058] As used herein, the term "pharmaceutical acceptable carrier" means a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in carrying or transporting a chemical agent.

[0059] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptoms of the disease, and / or (2) delaying the onset of a disease symptom or symptom in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the symptoms or symptoms of the disease.

[0060] A "repeating unit" is the simplest structural entity of a material, e.g., a framework and / or polymer, whether organic, inorganic, or organometallic. In a polymer chain, the repeating units are linked together in succession along the chain like beads on a necklace. For example, the -[-CH2CH2-] of polyethylene nIn -, the repeat unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e. the number of repeat units connected to each other. If the value of "n" is left undefined or is absent, it simply indicates the repetition of the formula in brackets and the polymeric nature of the material. The concept of repeat units applies equally well when the connectivity between repeat units extends in three dimensions, such as in metal-organic frameworks, modified polymers, and thermosetting polymers. Within the context of dendrimers or dendrons, the repeat units may also be described as branch units, internal layers, or generations. Similarly, terminal groups may also be described as surface groups.

[0061] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms, but differ in the three-dimensional arrangement of those atoms. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also called a stereogenic center or stereoisomeric center, which is any point in a molecule that has groups such that any two groups, if swapped, would result in a stereoisomer, but not necessarily an atom. In organic compounds, the chiral center is typically a carbon, phosphorus, or sulfur atom, but in organic and inorganic compounds, other atoms can be stereogenic centers. A molecule can have multiple stereocenters, giving many stereoisomers. In compounds where stereoisomerism is due to tetrahedral stereocenters (e.g., tetrahedral carbons), the total number of hypothetical possible stereoisomers is 2. nn is the number of tetrahedral stereocenters. The number of stereoisomers of a molecule with symmetry is often less than the maximum possible number. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be separated or resolved using techniques known in the art. For any stereocenter or axis of chirality where the stereochemistry is not defined, it is contemplated that the stereoisomer or axis of chirality can exist as its R-, S-, or mixtures of R- and S-forms, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer.

[0062] "Treatment" or "treating" includes (1) inhibiting the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease (e.g., preventing further progression of the symptoms and / or symptoms), (2) ameliorating the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease (e.g., reversing the symptoms and / or symptoms), and / or (3) causing any measurable decrease in the disease in a subject or patient experiencing or exhibiting symptoms or symptoms of the disease.

[0063] The above definitions take precedence over any conflicting definitions in any references incorporated herein by reference. However, the fact that a particular term is defined should not be taken as indicating that the undefined term is unclear. Rather, all terms used are believed to describe the present disclosure in terms that allow a person skilled in the art to understand and practice the scope of the present application.

[0064] composition lipid composition In one aspect, provided herein is a lipid composition comprising (i) an ionizable cationic lipid and (iii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0065] Ionizable Cationic Lipids In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid contains one or more groups that are protonated at physiological pH, but may be deprotonated and uncharged at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group may contain one or more protonatable amines that can form cationic groups at physiological pH. Cationic ionizable lipid compounds may also include C6-C 24 They may further comprise one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These lipid groups may be linked via ester bonds or further attached via Michael addition to sulfur atoms. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0066] In some embodiments of the lipid compositions of the present application, ionizable cationic lipids refer to lipids and lipid-like molecules that have a nitrogen atom capable of acquiring an electric charge (pKa). These lipids are sometimes known in the literature as cationic lipids. These molecules that have amino groups typically have 2-6 hydrophobic chains, often C6-C7. 24The cationic ionizable lipids may have at least one or more than six tails, but may have alkyl or alkenyl, such as alkyl or alkenyl groups. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers that exhibit regular dendritic branching, formed by the sequential or generational addition of branches to or from a core, and are characterized by a core, at least one internal branch layer, and a surface branch layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994.) In other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular structure having an internal core, an internal layer (or "generation") of repeat units regularly attached to this initiator core, and an external surface of terminal groups attached to the outermost generation. A "dendron" is a dendrimer species having branches emanating from a center that can be tethered or tethered to a core, either directly or via a linking moiety, to form a larger dendrimer. In some embodiments, the dendrimer structure has repeating groups radiating from a central core that doubles with each repeat unit on each branch. In some embodiments, the dendrimers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms may be used to describe the compounds described herein that have a dendron-like appearance (e.g., molecules radiating from a single center).

[0067] Although dendrimers are polymers, they may be considered preferable to conventional polymers because they have a controllable structure, a single molecular weight, a large number of controllable surface functional groups, and traditionally assume a globular conformation after reaching a certain generation. Dendrimers can be prepared by sequentially reacting each repeat unit, creating monodisperse, tree-like and / or generationally structured polymer structures. Individual dendrimers consist of a central core molecule with dendritic wedges attached to one or more functional sites on the central core. The dendrimer surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups, depending on the assembly monomers used during preparation.

[0068] The functional groups and / or chemical properties of the core, repeat units, and surface or terminal groups can be modified to adjust their physical properties. Some properties that can be changed include, but are not limited to, solubility, toxicity, immunogenicity, and bioattachment capability. Dendrimers are often described by the generation and number of repeat units in the branches. A dendrimer consisting only of a core molecule is called generation 0, and each successive repeat unit along all branches is generation 1, generation 2, and so on, up to the terminal or surface groups. In some embodiments, half generations are possible that result only from a first condensation reaction with an amine and not from a second condensation reaction with a thiol.

[0069] The preparation of dendrimers requires a level of synthetic control that is achieved through a series of stepwise reactions involving building up the dendrimer with each successive group. Dendrimer synthesis can be convergent or divergent. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process that connects one generation to the previous generation and changes the functional groups for the next step of reaction. Functional group conversion is necessary to prevent uncontrolled polymerization. Such polymerization results in highly branched molecules that are not monodisperse, also known as hyperbranched polymers. Steric effects mean that continuing to react dendrimer repeat units will result in spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation and destroys the monodispersity of the molecule. Thus, in some embodiments, dendrimers of generations G1 to G10 are specifically contemplated. In some embodiments, the dendrimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeat units, or any range derivable therefrom. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by decreasing the spacing units in the branched polymer.

[0070] Furthermore, dendrimers have two main chemical environments: the environment created by the specific surface groups of the terminal generations, and the interior of the dendritic structure, which can be shielded from the bulk medium and the surface groups by the higher order structure. Because of these different chemical environments, dendrimers have found many different potential applications, including therapeutic applications.

[0071] In some embodiments of the lipid composition of the present application, dendrimers or dendrons are assembled using the difference in reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers or dendrons may contain secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylates with mercapto groups. In addition, the repeating units of the dendrimer or dendron may contain groups that are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows for dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches, each of which may contain one or more repeating units. Dendrimers or dendrons may be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the terminal groups are lipophilic groups such as long chain alkyl or alkenyl groups. In other embodiments, the terminal group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminal group is an aliphatic or aromatic group that contains an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In yet other embodiments, the terminal group is an aliphatic or aromatic group that contains one or more hydrogen bond donors such as a hydroxide group, an amide group, or an ester.

[0072] The cationic ionizable lipids of the present application may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic forms. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of the chemical formula are intended. The cationic ionizable lipids may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, single diastereomers are obtained. The chiral centers of the cationic ionizable lipids of the present application may have S or R configurations. Furthermore, it is contemplated that one or more of the cationic ionizable lipids may exist as structural isomers. In some embodiments, the compounds have the same formula but differ in connectivity to the nitrogen atom of the core. Without wishing to be bound by theory, it is believed that such cationic ionizable lipids exist because the starting monomer first reacts with a primary amine and then reacts with a statistically present secondary amine. Thus, a structural isomer may represent a mixture of fully reacted primary amines and subsequently reacted secondary amines.

[0073] The chemical formulas used to represent the cationic ionizable lipids of this application will typically only show one of several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given chemical formula, and regardless of which tautomer is most common, all tautomers of a given chemical formula are intended.

[0074] The cationic ionizable lipids of the present application, whether for use in the indications described herein or otherwise, may also have the advantage that they may be more effective, less toxic, longer acting, more potent, produce fewer side effects, be more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties, than compounds known in the prior art.

[0075] Additionally, the atoms constituting the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include tetrahydrofuran and tetrahydrofuran. 13 C and 14 Contains C.

[0076] It should be recognized that the specific anion or cation that forms part of any salt form of the cationic ionizable lipid provided herein is not critical, so long as the salt as a whole is pharma- ceutical acceptable.Additional examples of pharma- ceutical acceptable salts and their preparation and use are described in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0077] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C6-C 24 Contains an alkyl or alkenyl group.

[0078] Dendrimers or dendrons of formula (I) In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C8-C 24 In some embodiments, the ionizable cationic lipid comprises an alkyl group. Core-Repeat Unit-End Group (DI) or a pharma- ceutically acceptable salt thereof, wherein the core is linked to the repeat unit by removing one or more hydrogen atoms from the core and replacing said atoms with the repeat unit; The core has the formula: [ka] having During the ceremony X1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) , heterocycloalkyl (C≦12) , Heteroaryl (C≦12) or a substitution thereof, R1 is amino, hydroxy, or mercapto, or alkylamino; (C≦12) , dialkylamino (C≦12) or any of the substituted groups of these groups, a is 1, 2, 3, 4, 5, or 6; or The core has the formula: [ka] having During the ceremony, X2 is N(R5) y and R5 is hydrogen, alkyl (C≦18) or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino; (C≦12) , dialkylamino (C≦12) or any of the substituted groups of these groups, b is 1, 2, 3, 4, 5, or 6; z is 1, 2, 3, with the proviso that the sum of z and y is 3; or The core has the formula: [ka] having During the ceremony, X3 is -NR6-, where R6 is hydrogen, alkyl, (C≦8) or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , Heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or any of the substituted groups of these groups, R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) or any of the substituted groups of these groups; or a group of the formula: -N(R f ) f (CH2CH2N(R c )) e Rd , [ka] Based on During the ceremony, e and f are each independently 1, 2, or 3, with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) or substituted alkyl (C≦6) and c and d are each independently 1, 2, 3, 4, 5, or 6; or The core is an alkylamine (C≦18) , dialkylamine (C≦36) , heterocycloalkane (C≦12) or a substituted version of these groups, The repeat unit comprises a degradable diacyl and a linker, The degradable diacyl group has the formula: [ka] having During the ceremony, A1 and A2 each independently represent -O-, -S-, or -NR a - in which R a is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and Y3 is alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or a substitution of any of these groups; or the formula: [ka] Based on During the ceremony, X3 and X4 are alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil(C≦12) or any of the substituted groups of these groups, Y5 is a covalent bond, alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or any of the substituted groups of these groups, R9 is alkyl (C≦8) or substituted alkyl (C≦8) and The linker group has the formula: [ka] having During the ceremony, Y1 is alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or any of the substituted groups of these groups, When the repeat unit contains a linker group, the linker group contains separate degradable diacyl groups bonded to both the nitrogen and sulfur atoms of the linker group, when n is greater than 1, the first group in the repeat unit is a degradable diacyl group, and for each linker group, the next repeat unit contains two degradable diacyl groups bonded to nitrogen atoms of the linker group, and n is the number of linker groups present in the repeat unit; The terminal group is of the formula [ka] having During the ceremony, Y4 is alkanediyl (C≦18) , or alkanediyl (C≦18) Alkanediyl in which one or more of the above hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≦18) and R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12), N-heterocycloalkyl (C≦12) , -C(O)N(R 11 )-Alkanediyl (C≦6) -Heterocycloalkyl (C≦12) , -C(O)-alkylamino (C≦12) , -C(O)-dialkylamino (C≦12) , -C(O)-N-heterocycloalkyl (C≦12) where: R 11 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and The last degradable diacyl in the chain is attached to the terminal group, n is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the terminal group is of the formula [ka] is further defined in During the ceremony, Y4 is alkanediyl (C≦18) and R 10 In some embodiments, A1 and A2 are each independently -O- or -NR a -It is.

[0079] In some embodiments of a dendrimer or dendron of formula (DI), the core is of the formula [ka] is further defined in During the ceremony, X2 is N(R5) y and R5 is hydrogen or alkyl (C≦8) or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12)or a substitution of any of these groups, b is 1, 2, 3, 4, 5, or 6; z is 1, 2, or 3, with the proviso that the sum of z and y is 3.

[0080] In some embodiments of a dendrimer or dendron of formula (DI), the core is [ka] is further defined in During the ceremony, X3 is -NR6-, where R6 is hydrogen, alkyl, (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , Heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or a substitution of any of these groups, R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino. (C≦12) , dialkylamino (C≦12) or a substituent of any of these groups; or a group of the formula: -N(R f ) f (CH2CH2N(R c )) e R d , [ka] Based on During the ceremony, e and f are each independently 1, 2, or 3, with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) , or substituted alkyl (C≦6) and c and d are each independently 1, 2, 3, 4, 5, or 6.

[0081] In some embodiments of the dendrimer or dendron of formula (I), the terminal group is of the formula [ka] It is expressed as During the ceremony, Y4 is alkanediyl (C≦18) and R 10 is hydrogen.

[0082] In some embodiments of a dendrimer or dendron of formula (DI), the core is [ka] It is further defined as:

[0083] In some embodiments of the dendrimer or dendron of formula (DI), the degradable diacyl is [ka] It is further defined as:

[0084] In some embodiments of a dendrimer or dendron of formula (DI), the linker is [ka] is further defined as In the formula, Y1 is an alkanediyl (C≦8) or substituted alkanediyl (C≦8) It is.

[0085] In some embodiments of the dendrimer or dendron of formula (DI), the dendrimer or dendron is [ka] [ka] [ka] and pharma- ceutically acceptable salts thereof.

[0086] A dendrimer or dendron of formula (X) In some embodiments of the lipid composition, the ionizable cationic lipid has the formula: [ka] In some embodiments, the ionizable cationic lipid is a dendrimer or dendron of the formula [ka] The dendrimer or dendron is

[0087] In some embodiments of the lipid composition, the ionizable cationic lipid has the structural formula: [ka] A dendrimer or dendron of generation (g) having or a pharma- ceutically acceptable salt thereof, (a) The core has the structural formula (X コア ): [ka] Including, During the ceremony, Q, independently in each occurrence, is a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b - and R 2 is expressed independently for each occurrence as R 1g or -L 2 -NR 1e R 1f and R3a and R 3b is independently at each occurrence hydrogen or optionally substituted alkyl (e.g., C1-C6, such as C1-C3); R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) independently at each occurrence represents a connection point to a branch, a hydrogen, or an optionally substituted (e.g., C1-C 12 ) alkyl, L 0 , L 1 , and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; Alternatively, L 1 Part of R 1c and R 1d with one of the following to form a (e.g., C4-C6) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur); x 1 is 0, 1, 2, 3, 4, 5, or 6, (b) each branch of the plurality (N) of branches independently has the structural formula (X 分岐 ): [ka] Including, During the ceremony, * indicates the attachment point of the branch to the core, g is 1, 2, 3, or 4; Z=2 (g-1) and If g=1 then G=0; or if g≠1 then G=

number

[0088] X コア In some embodiments, Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b X コア In some embodiments, Q is, independently at each occurrence, a covalent bond. コア In some embodiments, Q is, independently at each occurrence, -O-. コア In some embodiments, Q is, independently at each occurrence, -S-. コア In some embodiments, Q is independently at each occurrence -NR 2 and R 2 is expressed independently for each occurrence as R 1g or -L 2 -NR 1e R 1f X コア In some embodiments, Q is independently at each occurrence -CR 3a R 3b R 3a and R 3a and R 3b is independently at each occurrence hydrogen or optionally substituted alkyl (eg, C1-C6, such as C1-C3).

[0089] X コアIn some embodiments of the present invention, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) is independently at each occurrence a connection point to a branch, hydrogen, or an optionally substituted alkyl. コア In some embodiments of the present invention, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) is independently at each occurrence a connection point to a branch, hydrogen. X コア In some embodiments of the present invention, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present), independently at each occurrence, the point of attachment to the branch, optionally substituted alkyl (e.g., C1-C 12 ).

[0090] X コア In some embodiments, L 0 , L 1 , and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or alternatively, L 1 Part of R 1c and R 1d and one of the following: X forms a heterocycloalkyl (e.g., C4-C6, containing one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur). コア In some embodiments, L 0 , L 1 , and L2 X may each independently at each occurrence be a covalent bond. コア In some embodiments, L 0 , L 1 , and L 2 X may each independently be hydrogen at each occurrence. コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents alkylene (e.g., C1-C 12 , C1 to C6 or C1 to C3, etc. コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents heteroalkylene (e.g., C1-C 12 , for example, C1 to C8 or C1 to C6). コア In some embodiments, L 0 , L 1 , and L 2 Each occurrence of X can be independently heteroalkylene (e.g., C2-C8 alkylene oxide, such as oligo(ethylene oxide)). コア In some embodiments, L 0 , L 1 , and L 2 may each independently at each occurrence be [alkylene]-[heterocycloalkyl]-[alkylene][(e.g., C1-C6) alkylene]-[(e.g., C4-C6) heterocycloalkyl]-[(e.g., C1-C6) alkylene]. X コア In some embodiments, L 0 , L 1 , and L 2 may each independently at each occurrence be [alkylene]-(arylene)-[alkylene][(e.g., C1-C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene]. X コア In some embodiments, L 0 , L 1 , and L 2may each independently at each occurrence be [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C1-C6) alkylene]-phenylene-[(e.g., C1-C6) alkylene]). X コア In some embodiments, L 0 , L 1 , and L 2 Each occurrence of X can independently be heterocycloalkyl (e.g., C4-C6 heterocycloalkyl). コア In some embodiments, L 0 , L 1 , and L 2 Each occurrence of X can independently be arylene (e.g., phenylene). コア In some embodiments, L 1 Part of R 1c and R 1d Forms a heterocycloalkyl with one of X コア In some embodiments, L 1 Part of R 1c and R 1d and one of the following to form a heterocycloalkyl (e.g., a C4-C6 heterocycloalkyl), which may contain one or two nitrogen atoms and, optionally, an additional heteroatom selected from oxygen and sulfur.

[0091] X コア In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C 12 (e.g., C2-C8) alkylene oxide (e.g., oligo(ethylene oxide, -(CH2CH2O) 1~4 -(CH2CH2)-, etc.), [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., [ka] ), and [(C1-C4) alkylene]-phenylene-[(C1-C4) alkylene] (e.g., [ka] ) is selected. コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents C1-C6 alkylene (e.g., C1-C3 alkylene), -(C1-C3 alkylene-O) 1~4 -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-, and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-. X コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence C1-C6 alkylene (e.g., C1-C3 alkylene). 0 , L 1 , and L 2 Each occurrence is independent of C2 to C 12 (e.g., C2-C8) alkylene oxide (e.g., -(C1-C3 alkylene-O) 1~4 -(C1-C3 alkylene)). X コア In some embodiments, L 0 , L 1 , and L 2 are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).

[0092] X コア In some embodiments, x 1is 0, 1, 2, 3, 4, 5, or 6. X コア In some embodiments, x 1 is 0. X コア In some embodiments, x 1 is 1. X コア In some embodiments, x 1 is 2. X コア In some embodiments, x 1 is 0, 3. X コア In some embodiments, x 1 is 4. X コア In some embodiments, x 1 is 5. X コア In some embodiments, x 1 is 6.

[0093] X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments, the core has the structural formula: [ka] wherein Q′ is —NR 2 -or-CR 3a R 3b - and q 1 and q 2 are each independently 1 or 2. コア In some embodiments, the core has the structural formula: [ka] Contains: X コア In some embodiments of the present invention, the core has the structure [ka] wherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C 12 and is a heteroaryl (C3 to C5, etc.). コア In some embodiments of the present invention, the core has the structure [ka] Includes.

[0094] X コア In some embodiments, the core comprises a structural formula set forth in Table 1, and pharma- ceutically acceptable salts thereof, where * indicates the point of attachment of the core to one of the multiple branches. In some embodiments, the exemplary cores in Table 1 are not limited to the listed stereoisomers (i.e., enantiomers, diastereomers).

[0095] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0096] X コア In some embodiments, the core comprises: [ka] [ka] [ka] and pharma- ceutically acceptable salts thereof, wherein * denotes the point of attachment of the core to one of the multiple branches or H. In some embodiments, * denotes the point of attachment of the core to one of the multiple branches.

[0097] X コア In some embodiments, the core has the structure [ka] where * indicates the point of attachment of the core to one of the branches or to H. In some embodiments, at least two branches are attached to the core. In some embodiments, at least three branches are attached to the core. In some embodiments, at least four branches are attached to the core.

[0098] X コア In some embodiments, the core has the structure [ka] where * indicates the point of attachment of the core to one of the branches or to H. In some embodiments, at least 4 branches are attached to the core. In some embodiments, at least 5 branches are attached to the core. In some embodiments, at least 6 branches are attached to the core.

[0099] In some embodiments, the plurality (N) of branches comprises at least three branches, at least four branches, at least five branches. In some embodiments, the plurality (N) of branches comprises at least three branches. In some embodiments, the plurality (N) of branches comprises at least four branches. In some embodiments, the plurality (N) of branches comprises at least five branches.

[0100] X 分岐 In some embodiments, g is 1, 2, 3, or 4. 分岐 In some embodiments, g is 1 and X 分岐 In some embodiments, g is 2 and X 分岐 In some embodiments, g is 3 and X 分岐 In some embodiments, g is 4.

[0101] X 分岐 In some embodiments, Z=2 (g-1) and if g=1 then G=0. X 分岐 In some embodiments, Z=2 (g-1) If g ≠ 1, then G =

number

[0102] X 分岐 In some embodiments of the invention, g=1, G=0, Z=1, and each branch of the plurality of branches has the structural formula [ka] Includes.

[0103] X 分岐 In some embodiments of the invention, g=2, G=1, Z=2, and each branch of the plurality of branches has the structure [ka] Includes.

[0104] X 分岐 In some embodiments of the invention, g=3, G=3, Z=4, and each branch of the plurality of branches is represented by the structural formula [ka] Includes.

[0105] X 分岐 In some embodiments of the invention, g=4, G=7, Z=8, and each branch of the plurality of branches is represented by the structural formula [ka] Includes.

[0106] In some embodiments, a dendrimer or dendron described herein of generation (g)=1 has the structure: [ka] has.

[0107] In some embodiments, a dendrimer or dendron described herein of generation (g)=1 has the structure: [ka] has.

[0108] Examples of formulations of dendrimers or dendrons described herein of generations 1-4 are shown in Table 2. The number of diacyl groups, linker groups, and terminal groups can be calculated based on g.

[0109] [Table 2]

[0110] In some embodiments, the diacyl group is independently represented by the structural formula [ka] where * indicates the point of attachment of the diacyl group at its proximal end and ** indicates the point of attachment of the diacyl group at its distal end.

[0111] X 分岐 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. 分岐 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkylene (e.g., C1-C 12 ) X 分岐 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C 12 ) X 分岐 In some embodiments of the diacyl group of Y 3 represents independently at each occurrence an optionally substituted allenylene (e.g., C1-C 12 ).

[0112] X 分岐 In some embodiments of the diacyl group of A 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -It is. X 分岐In some embodiments of the diacyl group of A 1 and A 2 is independently at each occurrence -O-. 分岐 In some embodiments of the diacyl group of A 1 and A 2 X is, independently at each occurrence, -S-. 分岐 In some embodiments of the diacyl group of A 1 and A 2 each occurrence independently represents -NR 4 - and R 4 is hydrogen or optionally substituted alkyl (e.g., C1-C6). X 分岐 In some embodiments of the diacyl group of m 1 and m 2 is independently at each occurrence 1, 2, or 3. In some embodiments of the X branched diacyl group, m 1 and m 2 X is independently 1 for each occurrence. 分岐 In some embodiments of the diacyl group of m 1 and m 2 X is, independently at each occurrence, 2. 分岐 In some embodiments of the diacyl group of m 1 and m 2 X is, independently at each occurrence, 3. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f X is independently at each occurrence hydrogen or optionally substituted alkyl. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f X is independently at each occurrence hydrogen. 分岐 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3fis independently at each occurrence an optionally substituted (eg, C1-C8) alkyl.

[0113] In some embodiments of the diacyl group, A 1 is -O- or -NH-. In some embodiments of the diacyl group, A 1 In some embodiments of the diacyl group, A 2 is -O- or -NH-. In some embodiments of the diacyl group, A 2 In some embodiments of the diacyl group, Y 3 is C1~C 12 (For example, C1 to C6, such as C1 to C3) alkylene.

[0114] In some embodiments of a diacyl group, the diacyl group can be independently represented at each occurrence by the structural formula [ka] and optionally, wherein R 3c , R 3d , R 3e , and R 3f is independently at each occurrence hydrogen or C1-C3 alkyl.

[0115] In some embodiments, the linker group is independently represented by the structural formula [ka] where ** indicates the point of attachment of the linker to the proximal diacyl group and *** indicates the point of attachment of the linker to the distal diacyl group.

[0116] X 分岐 In some embodiments where a linker group of the formula: 1 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. 分岐In some embodiments where a linker group of the formula: 1 is independently at each occurrence an optionally substituted alkylene (e.g., C1-C 12 ) X 分岐 In some embodiments where a linker group of the formula: 1 is independently at each occurrence an optionally substituted alkenylene (e.g., C1-C 12 ) X 分岐 In some embodiments where a linker group of the formula: 1 represents independently at each occurrence an optionally substituted allenylene (e.g., C1-C 12 ).

[0117] X 分岐 In some embodiments of the terminal group of X, each terminal group is independently selected from optionally substituted alkylthiol and optionally substituted alkenylthiol. 分岐 In some embodiments of the terminal group, each terminal group is an optionally substituted alkylthiol (e.g., C1-C 18 And C4~C 18 etc.) X 分岐 In some embodiments of the terminal group, each terminal group is an optionally substituted alkenyl thiol (e.g., C1-C 18 And C4~C 18 etc.).

[0118] X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 Alkenylthiol or C1-C 18 Alkylthiol, the alkyl or alkenyl part being halogen, C6-C 12 Aryl, C1-C 12 Alkylamino, C4-C6N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12alkylamino), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6N-heterocycloalkyl), -C(O)-(C1-C 12 -C(O)-(C4-C6N-heterocycloalkyl), and -C(O)-(C4-C6N-heterocycloalkyl), wherein the C4-C6N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0119] X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 (For example, C4~C 18 ) Alkenylthiol or C1-C 18 (For example, C4~C 18 ) alkylthiol, the alkyl or alkenyl portion being halogen, C6-C 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.), C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino) (e.g., [ka] ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocycloalkyl) (e.g., [ka] ), -C(O)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino) and -C(O)-(C4-C6N-heterocycloalkyl) (e.g., [ka] Optionally, each independently substituted with one or more substituents selected from the following: wherein the C4-C6 N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl. X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion being optionally substituted with one substituent -OH. X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion being C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (e.g., -NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.) and C4-C6 N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )) is optionally substituted with one substituent selected from X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 (For example, C4~C 18 ) Alkenylthiol or C1-C 18 (For example, C4~C 18 ) alkylthiol. X 分岐 In some embodiments of the terminal group, each terminal group is independently selected from C1 to C 18 (For example, C4~C 18 ) alkylthiol.

[0120] X 分岐 In some embodiments of the terminal groups, each terminal group is independently a structure set forth in Table 3. In some embodiments, the dendrimers or dendrons described herein may include a terminal group or a pharma- ceutically acceptable salt thereof selected in Table 3. In some embodiments, the exemplary terminal groups in Table 3 are not limited to the stereoisomers (i.e., enantiomers, diastereomers) listed.

[0121] [Table 3-1] [Table 3-2]

[0122] In some embodiments, the dendrimer or dendron of formula (X) is selected from those set forth in Table 4, and pharma- ceutically acceptable salts thereof.

[0123] [Table 4-1] [Table 4-2]

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

[0124] Other ionizable cationic lipids In some embodiments of the lipid composition, the cationic lipid has the structural formula (D-I'): [ka] Including, During the ceremony, a is 1 and b is 2, 3, or 4, or alternatively, b is 1 and a is 2, 3, or 4; m is 1 and n is 1, or alternatively, m is 2 and n is 0, or alternatively, m is 2 and n is 1; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H, -CHCH(OH)R 7 , -CH(R 7 )CH2OH, -CH2CH2C(=O)OR 7 , -CH2CH2C(=O)NHR 7 , and -CHR 7 wherein R 7 are independent, C3~C 18 Alkyl, C3-C with one C=C double bond 18selected from an alkenyl, an amino protecting group, -C(=NH)NH2, a poly(ethylene glycol) chain, and a receptor ligand; However, R 1 ~R 6 At least two moieties of are independently -CHCH(OH)R 7 , -CH(R 7 )CH2OH, -CH2CH2C(=O)OR 7 , -CH2CH2C(=O)NHR 7 , or -CH2R 7 wherein R 7 are independent, C3~C 18 Alkyl or C3-C with one C=C double bond 18 alkenyl, One or more of the nitrogen atoms in formula (D-I') may be protonated to provide a cationic lipid.

[0125] In some embodiments of the cationic lipid of formula (D-I'), a is 1. In some embodiments of the cationic lipid of formula (D-I'), b is 2. In some embodiments of the cationic lipid of formula (D-I'), m is 1. In some embodiments of the cationic lipid of formula (D-I'), n is 1. In some embodiments of the cationic lipid of formula (D-I'), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or -CHCH(OH)R 7 In some embodiments of the cationic lipid of formula (D-I′), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or [ka] In some embodiments of the cationic lipid of formula (D-I′), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or [ka] In some embodiments of the cationic lipid of formula (D-I′), R 7 is C3~C 18 Alkyl (e.g., C6-C 12 alkyl).

[0126] In some embodiments, the cationic lipid of formula (D-I') is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: [ka] It is.

[0127] In some embodiments, the cationic lipid of formula (D-I') is (11R,25R)-13,16,20-tris((R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: [ka] It is.

[0128] Additional cationic lipids that can be used in the compositions and methods of the present application include those described in J. McClellan, MC King, Cell 2010, 141, 210-217, and International Patent Publications WO2010 / 144740, WO2013 / 149140, WO2016 / 118725, WO2016 / 118724, WO2013 / 063468, WO2016 / 205691, WO2015 / 184256, WO2016 / 004202, WO2015 / 199952, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2017 / 049245, WO2017 / 173054, and WO2015 / 095340, which are incorporated herein by reference for all purposes. Examples of such ionizable cationic lipids include, but are not limited to, those shown in Table 5.

[0129] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14]

[0130] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in an amount of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is about 7.5 to about 20. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0131] In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 5% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 10% to about 25%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid at a molar percentage of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% molar percentage of ionizable cationic lipids. In some embodiments of the lipid composition of the present application, the lipid composition comprises at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, or at most about 30% molar percentage of ionizable cationic lipids.

[0132] Selective Organ Targeting (SORT) Lipids In some embodiments of the lipid compositions of the present application, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ. In some embodiments, the target organ is the lung, lung tissue, or lung cells. As used herein, the term "preferentially delivered" is used to refer to a composition that, upon delivery, is delivered to a target organ (e.g., lung), tissue, or cell at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the administered dose.

[0133] In some embodiments of the lipid composition, the lipid composition comprises one or more selective organ targeting (SORT) lipids that provide for selective delivery of the composition to a particular organ. In some embodiments, the SORT lipid is a C6-C 24 The alkyl or alkenyl group may have two or more alkyl or alkenyl chains.

[0134] In some embodiments of the lipid composition, the SORT lipid comprises a permanently positively charged moiety. The permanently positively charged moiety may be positively charged at physiological pH, so that the SORT lipid comprises a positive charge when the polynucleotide is delivered to the cell. In some embodiments, the positively charged moiety is a quaternary amine or a quaternary ammonium ion. In some embodiments, the SORT lipid comprises a counterion or otherwise complexes with or interacts with a counterion.

[0135] In some embodiments of the lipid composition, the SORT lipid is a permanent cationic lipid (i.e., comprises one or more hydrophobic moieties and a permanent cationic group). The permanent cationic lipid may contain a group that has a positive charge regardless of pH. One permanent cationic group that may be used in the permanent cationic lipid is a quaternary ammonium group. The permanent cationic lipid has the structural formula: [ka] wherein Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5, However, at least one of Y1, Y2, and Y3 is X2N + R3R4R5, R1 is C1~C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; X1 is O or NR a where R ais hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl; X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl; R3, R4, and R5 are each independently selected from C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 is a substituted alkenyl; A1 is X2N in the compound + It is an anion with a charge equal to the number of R3R4R5 groups.

[0136] In some embodiments of the SORT lipid, the permanent cationic SORT lipid has the structural formula: [ka] wherein R6 to R9 are each independently C1 to C 24 Alkyl, C1-C 24 Substituted alkyl, C1-C 24 Alkenyl, C1-C 24 substituted alkenyl, provided that at least one of R6 to R9 is C8 to C 24 Based on A2 is a monovalent anion.

[0137] In some embodiments of the lipid composition, the SORT lipid is an ionizable cationic lipid (i.e., comprises one or more hydrophobic components and an ionizable cationic group). The ionizable positively charged moiety may be positively charged at physiological pH. One ionizable cationic group that may be used in the ionizable cationic lipid is a tertiary amine group. In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl(C8~C24) or a substitution of any of these groups, R3 and R3' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) It is.

[0138] In some embodiments of the lipid composition, the SORT lipid comprises a head group of a particular structure. In some embodiments, the SORT lipid has the structural formula: [ka] wherein L is a linker and Z + is the positively charged moiety, and X - is a counter ion. In some embodiments, the linker is a biodegradable linker. The biodegradable linker may be degradable under physiological pH and temperature. The biodegradable linker may be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or a quaternary amine.

[0139] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R 1 and R 2 each independently represents an optionally substituted C 24 Alkyl or optionally substituted C6-C 24 It is alkenyl.

[0140] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] has.

[0141] In some embodiments of the lipid composition, the SORT lipid comprises a linker (L). In some embodiments, L is [ka] where: p and q are each independently 1, 2, or 3; R 4 is an optionally substituted C1-C6 alkyl.

[0142] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and R4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0143] In some embodiments of the lipid composition, the SORT lipid is a phosphatidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound is [ka] wherein: R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6)and X - is a monovalent anion.

[0144] In some embodiments of the lipid composition, the SORT lipid is a phosphocholine lipid. In some embodiments, the SORT lipid is an ethylphosphocholine. The ethylphosphocholine can be, for example and without limitation, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

[0145] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0146] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., the chloride salt).

[0147] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R4 and R4' are each independently an alkyl (C6~C24) , alkenyl (C6~C24) or a substitution of any of these groups, R4'' is an alkyl (C≦24) , alkenyl (C≦24) or a substitution of any of the groups R4''' is alkyl (C1~C8) , alkenyl (C2~C8) or a substitution of any of these groups, X2 is a monovalent anion.

[0148] By way of example, and not limitation, a SORT lipid of the formula in the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB) (e.g., the bromide salt).

[0149] In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0150] In some embodiments of the lipid composition, the SORT lipid is an anionic lipid. In some embodiments of the lipid composition, the SORT lipid has the structural formula: [ka] wherein R1 and R2 are each independently an alkyl(C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) or -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) It is.

[0151] In some embodiments of the lipid composition, the SORT lipid comprises one or more selected from the lipids set forth in Table 6.

[0152] [Table 6-1] [Table 6-2]

[0153] In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 20% to about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a SORT lipid at a molar percentage of about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises SORT lipids at a molar percentage of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises SORT lipids at a molar percentage of at most about 25%, at most about 30%, at most about 35%, at most about 40%, at least about 45%, at most about 50%, at most about 55%, at most about 60%, or at most about 65%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises SORT lipids in a molar percentage of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or a molar percentage in a range between (and including) any two of the foregoing values.

[0154] Additional lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises additional lipids, including, but not limited to, steroids or steroid derivatives, PEG lipids, and phospholipids.

[0155] Phospholipids or other zwitterionic lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises a phospholipid. In some embodiments, the phospholipid is a phospholipid having one or two long chains (e.g., C6-C 24 ) alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally an organic small molecule. The organic small molecule may be an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, which define lipids and lipid-like molecules that have both positive and negative charges.

[0156] In some embodiments of the lipid composition, the phospholipid is not ethylphosphocholine.

[0157] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of phospholipids relative to the total lipid composition of about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is about 7.5 to about 60. In some embodiments, the molar percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0158] In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 8% to about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 8% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 10% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a phospholipid at a molar percentage of about 12% to about 18%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at most about 8%, at most about 10%, at most about 12%, at most about 15%, at most about 18%, at most about 20%, or at most about 23%.

[0159] Steroids or steroid derivatives In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" refers to a class of compounds having a 4-ring, 17-carbon ring structure that may further comprise one or more substituents including an alkyl group, an alkoxy group, a hydroxy group, an oxo group, an acyl group, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid is represented by the formula [ka] In some embodiments, the steroid or steroid derivative comprises the above ring structure with one or more non-alkyl substitutions, as shown in the formula: [ka] In some embodiments of the present application, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is of the formula [ka] As described above, the cholestane derivatives include one or more non-alkyl substitutions of the ring system. In some embodiments, the cholestane or cholestane derivatives are cholestane or cholestane derivatives, or sterol or sterol derivatives. In other embodiments, the cholestane or cholestane derivatives are both cholestane and sterol or their derivatives.

[0160] In some embodiments of the lipid composition, the composition may further comprise a molar percentage of a steroid relative to the total lipid composition of about 40 to about 46. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45 to about 46, or any range derivable therein. In other embodiments, the molar percentage of a steroid relative to the total lipid composition is about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.

[0161] In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 15% to about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 25% to about 35%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 30% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative in a molar percentage of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative in a molar percentage of at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 46%.

[0162] Polymer-Conjugated Lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride that also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a diglyceride that also comprises one or more C6-C 24 Long chain alkyl or alkenyl groups or C6-C 24It is a compound containing a fatty acid group. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide complexes, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present application are taught by U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0163] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein R 12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or any of the substituted groups of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8)and x is 1 to 250. In some embodiments, R e is an alkyl group such as methyl. (C≦8) R 12 and R 13 are each independently an alkyl (C≦4~20) In some embodiments, x is 5 to 250. In some embodiments, x is 5 to 125, or x is 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0164] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein n1 is an integer from 1 to 100, and n2 and n3 are each independently selected from integers from 1 to 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0165] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of PEG lipid relative to the total lipid composition of about 4.0 to about 4.6. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range derivable therein. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0, or any range derivable therein.

[0166] In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer complex lipid at a molar percentage of about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer complex lipid at a molar percentage of about 1% to about 8%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer complex lipid at a molar percentage of about 2% to about 7%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer complex lipid at a molar percentage of about 3% to about 5%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer complex lipid at a molar percentage of about 5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10% polymer-conjugated lipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid in a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10%.

[0167] Pharmaceutical Compositions Therapeutic or prophylactic agents In another aspect, provided herein is a pharmaceutical composition comprising a therapeutic agent (or prophylactic agent) in combination with a lipid composition described herein.

[0168] In some embodiments of the pharmaceutical composition, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof. In some embodiments, the compound, polynucleotide, polypeptide, or a combination thereof is exogenous or heterologous to the cell or subject being treated by the pharmaceutical composition described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polynucleotide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polypeptide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof.

[0169] In some embodiments, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) for treating a pulmonary disease, such as asthma, COPD, or lung cancer. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a steroid, such as prednisone, hydrocortisone, prednisolone, methylprednisolone, or dexamethasone. In some embodiments, the therapeutic agent (or prophylactic agent) comprises Abraxane, afatinib dimaleate, Afinitor, Afinitor dispersible tablets (Afinitor Disperz), Alecensa, Alectinib, Alimta, Alumbrig, Atezolizumab, Avastin, Bevacizumab, Brigatinib, Capmatinib Hydrochloride, Carboplatin, Ceritinib, Crizotinib, Cyramza, Dabrafenib Mesylate, Dacomitinib, Docetaxel, Doxorubicin Hydrochloride, Durvalumab, Entrectinib, Erlotinib Hydrochloride, Everolimus, Gavreto, Gefitinib, Gilotrif, Gemcitabine, Ipilimumab, Iressa, Keytruda, Lorbrena, Mekinist, Methotrexate Sodium, Necitumumab, Nivolumab, Osimertinib Mesylate, Paclitaxel, These include pembrolizumab, pemetrexed disodium, pralsetinib, ramucirumab, LetVimo, selpercatinib, tabrecta, tafinlar, tagrisso, trametinib dimethylsulfoxide, vidinpro, vinorelbine tartrate, xalkori, yervoy, Zirabev, Zykadia, carboplatin, gemcitabine-cisplatin, afinitor, atezolizumab, durvalumab, etopofos, etoposide, hycamtin, imfinzi, keytruda, lurbinectedin, methotrexate sodium, nivolumab, opdivo, pembrolizumab, tecentriq, topotecan hydrochloride, trexol, or xepzelca. Other non-limiting examples of therapeutic (or prophylactic) agents that include compounds include 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acetretin, acrivastine, adenine, adenosine, alatrofloxacin, albendazole, albuterol, alclofenac,Aldesleukin, Alemtuzumab, Alfuzosin, Alitretinoin, Allobarbital, Allopurinol, All-trans-retinoic acid (ATRA), Aloxiprine, Alprazolam, Alprenolol, Altretamine, Amifostine, Amiloride, Aminoglutethimide, Aminopyrine, Amiodarone hydrochloride, Amitriptyline, Amlodipine, Amobarbital, Amodiaquine, Amoxapine, Amphetamine, Amphotericin, Amphotericin B, Ampicillin, Amphotericin Lenavir, amsacrine, amyl nitrate, amylobarbitone, anastrozole, amrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropine, azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbitone, BCG Live, beclamide, beclomethasone, bendroflumethiazide, benezepril, benidipine, benorylate, benperidol, bentazepam, benzamide, benzanthrazene, benzathine penicillin, benzahexol HCl, benznidazole, benzodiazepine, benzoic acid, bephenium hydroxynaphthoate, betamethasone, bevacizumab (Avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizola , budesonide, bumetanide, bupropion, busulfan, butalbital, butamben, butenafine HCl, butobarbitone, butobarbitone (butetal), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calciprotriene, calcitriol, calsterone, campbendazole, camphor, camptothecin, camptothecin analogues, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, carbofuran, carboplatin, carbromal, kallimazole, carmustine, cefamandole, cefazolin, cefixime,Ceftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, chlormethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil HCl, chlorpromazine, chlorpropamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol, chrysene, cilostazol, cimetidine, cinaridine, cinoxacin, ciprofibrate, Ciprofloxacin HCl, Cisapride, Cisplatin, Citalopram, Cladribine, Clarithromycin, Clemastine Fumarate, Clioquinol, Clobazam, Clofarabine, Clofazimine, Clofibrate, Clomiphene Citrate, Clomipramine, Clonazepam, Clopidogrel, Clotiazepam, Clotrimazole, Cloxacillin, Clozapine, Cocaine, Codeine, Colchicine, Colistin, Conjugated Estrogens, Corticosterone, Cortisone, Cortisone Acetate, Cyclizine, Cyclobarbital, Cyclohexasiloxane ... lobenzaprine, cyclobutane-spirobarbiturates, cycloethane-spirobarbiturates, cycloheptane-spirobarbiturates, cyclohexane-spirobarbiturates, cyclopentane-spirobarbiturates, cyclophosphamide, cyclopropane-spirobarbiturates, cycloserine, cyclosporine, cyproheptadine, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthron, dantrolene sodium, dapsone, darbepoetin alfa, dalodipine, daunorubicin, decoquinate , dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycorticosterone, desoxymetasone, dexamethasone, dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine, diatrizoic acid, diazepam, diazoxide, dichlorophene, dichloroprop, diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequiliin,Dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxanide furoate, dimenhydrinate, dimorpholamine, dinitramide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin, doxycycline, dromostanolone propionate, droperidol, dyphylline, echinocandin, econazo ethanol, econazole nitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone, epinephrine, epipodophyllotoxin derivatives, epirubicin, epoetin alfa, eposartan, equilenin, equilin, ergocalciferol, ergotamine tartrate, erlotinib, erythromycin, estradiol, estramustine, estriol, estrone, ethacrynic acid, ethambutol, etinamate, ethionamide, ethopropazine HCl, 4-aminobenzoic acid ethyl (benzocaine) ), ethylparaben, ethinyl estradiol, etodolac, etomidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecamyd acetate, floxacin, Lysine, Fludarabine, Fluconazole, Fluconazole, Flucytosine, Fludioxonil, Fludrocortisone, Fludrocortisone acetate, Flufenamic acid, Flunanisone, Flunarizine HCl, Flunisolide, Flunitrazepam, Fluocortolone, Fluometuron, Fluorene, Fluorouracil, Fluoxetine HCl, Fluoxymesterone, Flupentixol decanoate, Fluphenthixol decanoate, Flurazepam, Flurbiprofen, Fluticasone propionate, Fluvastatin, Folic acid, fosinopril, fosphenytoin sodium, frovatriptan, furosemide, fulvestrant, furazolidone, gabapentin, G-BHC (lindane), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glafenine, glibenclamide, gliclazide, glimepiride, glipizide, glutethimide, glyburide, glyceryl trinitrate (nitroglycerin), goserelin acetate, grepafloxacin, griseofulvin, guaifenesin, guanabenz acetate, guanine, halofantrine HCl, haloperidol, hydrazine lochlorothiazide, heptabarbital, heroin, hesperetin, hexachlorobenzene, hexetal, histrelin acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine, hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interferon alpha-2a, interferon alpha-2b, iodine dosamide, iopanoic acid, iprodione, irbesartan, irinotecan, isavuconazole, isocarboxazid, isoconazole, isoguanine, isoniazid, isopropyl barbiturate, isoproturon, isosorbide dinitrate, isosorbide mononitrate, izradipine, itraconazole, itraconazole, itraconazole (Itra), ivermectin, ketoconazole, ketoprofen, ketorolac, khellin, labetalol, lamivudine, lamotrigine, lanatoside C, lanosporazole, L-DOPA, leflunomide, lenalidomide, Letrozole, leucovorin, leuprolide acetate, levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin, lomustine, loperamide, loratadine, lorazepam, lorefloxacin, lormetazepam, losartan mesylate, lovastatin, lisuride maleate, maprotiline HCl, mazindol, mebendazole, meclizine HCl, meclofenamic acid, medazepam, medigoxin, medroxyprogesterone acetate, mefenamic acid, mefloquine HCl, megestrol acetate, melphalan,Mepenzolate bromide, meprobamate, meptazinol, mercaptopurine, mesalazine, mesna, mesoridazine, mestranol, methadone, methaqualone, methocarbamol, methoine, methotrexate, methoxsalen, mesuximide, methyclothiazide, methylphenidate, methylphenobarbitone, methyl p-hydroxybenzoate, methylprednisolone, methyltestosterone, methyprylon, methylsergide maleate, metoclopramide, metolazone, metoprolol, metronidazole, mianserin HCl, miconazole, mida Zolam, Mifepristone, Miglitol, Minocycline, Minoxidil, Mitomycin C, Mitotane, Mitoxantrone, Mofetil Mycophenolate, Molindone, Montelukast, Morphine, Moxifloxacin HCl, Nabumetone, Nadolol, Nalbuphine, Nalidixic Acid, Nandrolone, Naphthacene, Naphthalene, Naproxen, Naratriptan HCl, Natamycin, Nelarabine, Nelfinavir, Nevirapine, Nicardipine HCl, Nicotinamide, Nicotinic Acid, Nicoumarone, Nifedipine, Nilutamide, Nimodipine, Nimorazol , nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omoconazole, ondansetron HCl, oprelvequin, ornidazole, oxaliplatin, oxamniquine, oxantelembonate, oxaprozin, oxatomide, oxazepam, oxcarbazepine, oxyfendazole , oxiconazole, oxyprenolol, oxyphenbutazone, oxyphencyclimine HCl, paclitaxel, palifermin, pamidronate, p-aminosalicylic acid, pantoprazole, paramethadione, paroxetine HCl, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, penicillamine, pentaerythritol tetranitrate, pentazocine, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline,Perphenazine, Perphenazine pimozide, Perylene, Phenacemide, Phenacetin, Phenanthrene, Phenindione, Phenobarbital, Phenol barbitone, Phenolphthalein, Phenoxybenzamine, Phenoxybenzamine HCl, Phenoxymethylpenicillin, Phenoxymide, Phenylbutazone, Phenytoin, Pindolol, Pioglitazone, Pipobroman, Piroxicam, Pizotifen Maleate, Platinum Compounds, Plicamycin, Polyenes, Polymyxin B, Porfimer Sodium, Posaconazole (Posa), Pramipe Xol, prasterone, pravastatin, praziquantel, prazosin, prazosin HCl, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanil HCl, promethazine, propofol, propoxur, propranolol, propylparaben, propylthiouracil, prostaglandins, pseudoephedrine, pteridine-2-methylthiol, pteridine-2-thiol, pteridine-4-methylthiol, pteridine-4-thiol pteridine-7-methylthiol, pteridine-7-thiol, pyrantelembonate, pyrazinamide, pyrene, pyridostigmine, pyrimethamine, quetiapine, quinacrine, quinapril, quinidine, quinidine sulfate, quinine, quinine sulfate, rabeprazole sodium, ranitidine HCl, rasburicase, ravuconazole, repaglinide, reposal, reserpine, retinoids, rifabutin, rifampicin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatidine Liptan benzoate, rofecoxib, ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital, sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, stanolone, stanozolol, stavudine, stilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate, sulfacetamide,Sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide, sulfathiazole, sulindac, sulfabenzamide, sulfaacetamide, sulfadiazine, sulfadoxine, sulfafurazole, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfasalazine, sulfinpyrazone, sulpiride, sulthiame, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate , tamulosin, targretin, taxane, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole, terfenadine, testolactone, testosterone, tetracycline, tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine, theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, totoin (th otoin), thymine, tiagabine HCl, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone, triamterene, triazolam, triazole, triflupromazine, trimethoprim, trimipramine maleate, triphenylene, troglitazone, tromethamine, tropi and small molecules selected from: kamide, trovafloxacin, thibamate, ubidecarenone (coenzyme Q10), undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, barbicine, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirukast, zidovudine, zileton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, or zopiclone.

[0170] Polynucleotides In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) combined with the lipid composition comprises one or more polynucleotides. The present application is not limited in scope to any particular source, sequence, or type of polynucleotide. However, a person skilled in the art could easily identify related homologues in various other sources of polynucleotides, including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, monkey, baboon, cow, pig, horse, sheep, cat, and other species). It is contemplated that the polynucleotides used in the present application may include sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90%, and most preferably about 95% of nucleotides identical to the nucleotide sequence of the naturally occurring sequence. In another embodiment, the polynucleotide comprises a nucleic acid sequence that is complementary to a naturally occurring sequence, or 75%, 80%, 85%, 90%, 95% and 100% complementary to the sequence. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or more are contemplated herein.

[0171] In some embodiments, the polynucleotides used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the polynucleotide comprises complementary DNA (cDNA). Also contemplated are cDNAs with the addition of natural introns or introns from other genes, and such artificial molecules are sometimes referred to as "minigenes." At least these and other nucleic acids of the present application may be used, for example, as molecular weight standards in gel electrophoresis. The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA is that it contains primarily the coding sequence of the corresponding protein, as opposed to genomic DNA or DNA polymerized from unprocessed or partially processed genomic RNA templates. In some cases, complete or partial genomic sequences may be preferred, such as when non-coding regions are required for optimal expression or when targeting non-coding regions such as introns in antisense strategies.

[0172] In some embodiments, the polynucleotide comprises one or more segments comprising a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro ribonucleic acid (miRNA), a primary micro ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), or a double-stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one of the therapeutic agents (or prophylactic agents) described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide, such as a guide RNA (gRNA) or a guide DNA (gDNA), for forming a complex with a guide RNA-guided nuclease described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide-guided heterologous nuclease. The nuclease may be an endonuclease.Non-limiting examples of heterologous endonucleases guided by a guide polynucleotide include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and extremely halophilic archaea (Natronobacterium gregoryi) Argonaute (NgAgo); adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof; any derivative thereof; any variant thereof; and any fragment thereof.

[0173] Some embodiments of the therapeutic (or prophylactic) agents provided herein include a heterologous polypeptide that includes an actuator moiety. The actuator moiety may be configured to form a complex with a target polynucleotide corresponding to a target gene. In some embodiments, administration of the therapeutic (or prophylactic) agent results in altered expression or activity of the target gene. The therapeutic (or prophylactic) agent may include a heterologous polynucleotide that encodes an actuator moiety. The actuator moiety may be configured to form a complex with a target polynucleotide corresponding to a target gene. The heterologous polynucleotide may encode a guide polynucleotide configured to direct the actuator moiety to the target polynucleotide. The actuator moiety may include a heterologous endonuclease or a fragment thereof (e.g., directed by the guide polynucleotide to specifically bind to the target polynucleotide). The heterologous endonuclease may (1) be part of a ribonucleoprotein (RNP) or (2) form a complex with the guide polynucleotide. The heterologous endonuclease may be part of a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) protein complex. The heterologous endonuclease may be a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease. The heterologous endonuclease may comprise an inactivated endonuclease. The inactivated endonuclease may be fused to a regulatory moiety. The regulatory moiety may comprise a transcriptional activator, a transcriptional repressor, an epigenetic modifier, or a fragment thereof.

[0174] In some embodiments, the polynucleotide encodes at least one guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA))-guided heterologous endonuclease. In some embodiments, the polynucleotide encodes at least one guide polynucleotide and at least one heterologous endonuclease, and the guide polynucleotide can form a complex with the at least one heterologous endonuclease and guide it to cleave the locus of any one of the genes described herein. In some embodiments, the polynucleotide is a heterologous endonuclease guided by at least one guide polynucleotide, such as Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, Cas14, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, Casl encoding Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cul966; any derivative thereof; any variant thereof; or any fragment thereof. In some embodiments, Cas13 can include, but is not limited to, Cas13a, Cas13b, Cas13c, and Cas13d (e.g., CasRx).

[0175] In some embodiments, the heterologous endonuclease comprises an inactivating endonuclease, optionally fused to a regulatory moiety, such as an epigenetic modifier, for remodeling the epigenome to mediate expression of a selected gene of interest. In some cases, epigenetic modifiers can include methyltransferases, demethylases, dismutases, alkylating enzymes, depurinases, oxidases, photolyases, integrases, transposases, recombinases, polymerases, ligases, helicases, glycosylases, acetyltransferases, deacetylases, kinases, phosphatases, ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, ubiquitin ligases, deubiquitinating enzymes, adenylate-forming enzymes, AMPylases, deAMPylases, SUMOylases, deSUMOylases, ribosylases, derivosylases, N-myristoyltransferases, chromotin remodeling enzymes, proteases, oxidoreductases, transferases, hydrolases, lyases, isomerases, synthases, synthetases, or demyristoylating enzymes. In some embodiments, the epigenetic modifier may include one or more selected from the group consisting of p300, TET1, LSD1, HDAC1, HDAC8, HDAC4, HDAC11, HDT1, SIRT3, HST2, CobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV39H1, DIM5, KYP, SUVR4, Set4, Set1, SETD8, and TgSET8.

[0176] In some embodiments, the polynucleotide encodes a guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA)) that is at least partially complementary to a genomic region of a gene, such that when the guide polynucleotide binds to the gene, the guide polynucleotide recruits a nuclease guided by the guide polynucleotide to cleave the region and genetically modify it. Examples of genes that can be modified by nucleases guided by guide polynucleotides include CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0177] In some embodiments, the polynucleotide comprises or encodes at least one mRNA, the expression of which restores the function of a defective gene in a subject treated with a pharmaceutical composition described herein.

[0178] In some embodiments, the polynucleotide of the present application comprises at least one chemical modification of one or more nucleotides. In some embodiments, the chemical modification increases the specificity of the guide polynucleotide (such as a guide RNA (gRNA) or a guide DNA (gDNA)) binding to a complementary genomic locus (e.g., a genomic locus of any one of the genes described herein). In some embodiments, the at least one chemical modification increases resistance to nuclease digestion when the polynucleotide is administered to a subject in need of administration of the polynucleotide. In some embodiments, the at least one chemical modification reduces immunogenicity when the polynucleotide is administered to a subject in need of administration of the polynucleotide. In some embodiments, the at least one chemical modification stabilizes a scaffold, such as a tRNA scaffold. Such chemical modifications may have desirable properties, such as enhanced resistance to nuclease digestion or increased binding affinity with a target genomic locus relative to a polynucleotide that does not have at least one chemical modification.

[0179] In some embodiments, the at least one chemical modification comprises a modification to a sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety that comprises one or more non-bridging sugar substituents, including but not limited to, substituents at the 2' and / or 5' positions. Examples of suitable sugar substituents at the 2' position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2' position is allyl, amino, azido, thio, O-allyl, O-C1-C2-C3-amino, 1-methyl- ... 10 Alkyl, O--C1~C 10 Substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ), and O--CH2--C(=O)--N(R m )(R n ) and each R mand Rn are independently H or substituted or unsubstituted C1-C 10 and alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises two or more non-bridging sugar substituents, such as TF-5'-methyl sugar moieties.

[0180] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, a 2'-substituted nucleoside is a nucleoside that is substituted with halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S, or N(R m )-Alkynyl;O-Alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ) or O--CH2--C(=O)--N(R m )(R n ), each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0181] In some embodiments, the 2'-substituted nucleoside is selected from the group consisting of F, NH, N, OCF, O--CH, O(CH)NH, CH-CH=CH, O--CH-CH=CH, OCHCHOCH, O(CH)SCH, O--(CH)--O--N(R m )(R n), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamides (O--CH2--C(=O)--N(R m )(R n ), each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 It is an alkyl.

[0182] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent group selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0183] In some embodiments, a 2'-substituted nucleoside comprises a sugar moiety that includes a 2'-substituent group selected from F, O--CH3, and OCH2CH2OCH3.

[0184] Certain modified sugar moieties include a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4' to 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents include -[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O-- or --C(R a R b)--O--N(R)--; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2'(LNA); 4'-(CH2)--S-2'; 4'-(CH2)2--O-2'(ENA); 4'-CH(CH3)--O-2'(cEt) and 4'-CH(CHOCH3)--O-2' and analogs thereof; 4'-C(CH3)(CH3)--O-2' and analogs thereof; 4'-CH2--N(OCH3)-2' and analogs thereof; 4'-CH2--O--N(CH3)-2'; 4'-CH2--O--N(R)-2' and 4'-CH2--N(R)--O-2'-, where each R is independently H, a protecting group, or a C1-C 12 alkyl; 4'-CH2--N(R)--O-2', where R is H, C1-C 12 alkyl, or those which are protecting groups; 4'-CH2--C(H)(CH3)-2'; and 4'-CH2--C(=CH2)-2' and analogs thereof.

[0185] In some embodiments, such 4' to 2' bridges are independently -[C(R a )(R b )] n --, --C(R a )=C(R b )--, --C(R a )=N--, ​​--C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a )2--, --S(=O) x -- and --N(R a )—, x is 0, 1, or 2, n is 1, 2, 3, or 4, and each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12Alkynyl, Substituted C2-C 12 Alkynyl, C5-C 20 Aryl, Substituted C5-C 20 aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), where each J1 and J2 is independently H, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, Substituted C2-C 12 Alkynyl, C5-C 20 Aryl, Substituted C5-C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C 12 Aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0186] Nucleosides that contain a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include: (A) α-L-methyleneoxy (4'-CH2--O-2') BNAs, (B) β-D-methyleneoxy (4'-CH2--O-2') BNAs (also called locked nucleic acids or LNAs), (C) ethyleneoxy (4'-(CH2)2--O-2') BNAs, (D) aminooxy (4'-CH2--O--N(R)-2') BNAs, (E) oxyamino (4'-CH2--N(R)--O-2') BNAs, (F) methyl(methyleneoxy) (4'-CH(CH3)--O-2' ) BNAs (also known as constrained ethyl or cEt), (G) methylene-thio (4'-CH2--S-2') BNAs, (H) methyleneamino (4'-CH2-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH2--CH(CH3)-2') BNAs, (J) propylene carbocyclic (4'-(CH2)3-2') BNAs, and (K) methoxy(ethyleneoxy) (4'-CH(CH2OMe)-O-2') BNAs (also known as constrained MOE or cMOE).

[0187] In some embodiments, the bicyclic sugar moiety and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, nucleosides containing a 4'-2' methyleneoxy bridge may be in the α-L or β-D configuration. Previously, α-L-methyleneoxy (4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that exhibit antisense activity.

[0188] In some embodiments, the substituted sugar moiety includes one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted sugars and 4' to 2' bridged sugars, where LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0189] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described above. For example, some sugar surrogates include a 4'-sulfur atom and substituents at the 2' and / or 5' positions. As additional examples, carbocyclic bicyclic nucleosides with a 4' to 2' bridge are described.

[0190] In some embodiments, the sugar surrogate comprises a ring with more than 5 atoms. For example, in some embodiments, the sugar surrogate comprises a 6-membered tetrahydropyran. Such tetrahydropyran may be further modified or substituted. Nucleosides that include such modified tetrahydropyran include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA), and fluoro-HNA (F-HNA).

[0191] Many other bicyclo- and tricyclo-sugar surrogate ring systems are known in the art that can be used to modify nucleosides and incorporated into antisense compounds.

[0192] Combinations of modifications are also provided, such as, but not limited to, 2'-F-5'-methyl substituted nucleosides, replacement of the ribosyl ring oxygen atom with S, and further substitutions at the 2' position, alternatively 5'-substitution of bicyclic nucleic acids. In some embodiments, 4'-CH2--O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies are also described.

[0193] In some embodiments, the present application provides polynucleotides that include modified nucleosides. These modified nucleotides may include modified sugars, modified nucleobases, and / or modified linkages. The particular modifications are selected so that the resulting polynucleotide has desirable properties. In some embodiments, the polynucleotide includes one or more RNA-like nucleosides. In some embodiments, the polynucleotide includes one or more DNA-like nucleotides.

[0194] In some embodiments, the nucleosides of the present application comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present application comprise one or more modified nucleobases.

[0195] In some embodiments, the modified nucleobase is selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine. amine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0196] In some embodiments, the present application provides a polynucleotide comprising linked nucleosides. In such embodiments, the nucleosides can be linked using any internucleoside linkage. Two major classes of internucleoside linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linkage groups include, but are not limited to, methylenemethylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to native phosphodiester linkages. In some embodiments, internucleoside linkages with chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those of skill in the art.

[0197] The polynucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined in terms of absolute stereochemistry as (R) or (S), α or β, as in the case of sugar anomers, or (D) or (L), as in the case of amino acids, etc. The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0198] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (3'-CH2--N(CH3)--O-5'), amide-3 (3'-CH2--C(=O)--N(H)-5'), amide-4 (3'-CH2--N(H)--C(=O)-5'), formacetal (3'-O--CH2--O-5'), and thioformacetal (3'-S--CH2--O-5'). Further neutral internucleoside linkages include non-ionic linkages including siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include non-ionic linkages including mixed component parts of N, O, S, and CH2.

[0199] Additional modifications may be made to other positions of the oligonucleotide, particularly the 3' position of the sugar of the 3'-terminal nucleotide and the 5' position of the 5'-terminal nucleotide. For example, one additional modification of the ligand-conjugated polynucleotide of the present application involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate triethylammonium, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0200] In some embodiments, the polynucleotide described herein comprises or encodes at least one tRNA described herein. In some embodiments, the tRNA expressed from the polynucleotide restores the function of at least one defective tRNA in the subject treated by the pharmaceutical composition described herein. In some embodiments, the at least one tRNA expressed by the polynucleotide described herein can comprise a tRNA that encodes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylaniline, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the at least one tRNA expressed by the polynucleotide described herein can comprise a tRNA that encodes arginine, tryptophan, glutamic acid, glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine.

[0201] Polypeptides In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent combined with the lipid composition comprises one or more polypeptides. Some polypeptides may comprise an enzyme, such as any one of the nuclease enzymes described herein. For example, the nuclease enzyme can be a CRISPR-associated (Cas) protein or Cas nuclease, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and extremely halophilic archaea (Natronobacterium gregoryi Argonaute (NgAgo); Adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof; any derivative thereof; any variant thereof; and any fragment thereof. In some embodiments, the nuclease enzyme may be selected from Cas proteins, such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, These may include Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.In some embodiments, a Cas protein can complex with a guide polynucleotide described herein to form a CRISPR ribonucleoprotein (RNP).

[0202] The nuclease in the compositions described herein may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct the cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence, of any one of the genes described herein.

[0203] CRISPR enzyme may be mutated with respect to the corresponding wild type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence.For example, the aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cuts a single strand).In some embodiments, Cas9 nickase can be used in combination with a guide sequence, for example, two guide sequences that target the sense strand and antisense strand of a DNA target, respectively.This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0204] In some embodiments, the present application provides a polypeptide that contains one or more therapeutic proteins. Therapeutic proteins that can be included in the composition include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, clotting factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase α, tissue plasminogen activator (TPA) activase, glucocerebrosidase, interferon (IF) beta-1a, interferon beta-1b, interferon gamma, interferon α, TNF-α, IL-1 through IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle stimulating hormone (FSH), and Factor VIII.

[0205] In some embodiments, the polypeptide comprises a peptide sequence that is at least partially identical to any of the therapeutic (or prophylactic) agents that comprise the peptide sequence. For example, the polypeptide may comprise a peptide sequence that is at least partially identical to an antibody (e.g., a monoclonal antibody) for treating a disease such as cancer.

[0206] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject treated with a pharmaceutical composition described herein.

[0207] In some embodiments, the pharmaceutical composition of the present application includes multiple payloads combined with (e.g., encapsulated therein) a lipid composition. The multiple payloads combined with the lipid composition may be configured for gene editing or gene expression modification. The multiple payloads combined with the lipid composition may include a polynucleotide encoding an actuator portion (e.g., including a heterologous endonuclease such as Cas) or a polynucleotide encoding an actuator portion. The multiple payloads combined with the lipid composition may further include one or more (e.g., one or two) guide polynucleotides. The multiple payloads combined with the lipid composition may further include one or more donor or template polynucleotides. The multiple payloads combined with the lipid composition may include a ribonucleoprotein (RNP).

[0208] In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is (about) 20:1 or less, (about) 15:1 or less, (about) 10:1 or less, or (about) 5:1 or less. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is less than (about) 20:1, (about) 15:1, (about) 10:1, or (about) 5:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 10:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 15:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 5:1 to about 10:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 5:1 to about 15:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 15:1 to about 20:1.

[0209] In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 50:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:20. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 20:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 50:1 to about 1:70. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 70:1 to about 1:100. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipid of the lipid composition is (about) 1:1 or less, (about) 1:5 or less, (about) 1:10 or less, (about) 1:15 or less, (about) 1:20 or less, (about) 1:25 or less, (about) 1:30 or less, (about) 1:35 or less, (about) 1:40 or less, (about) 1:45 or less, (about) 1:50 or less, (about) 1:60 or less, (about) 1:70 or less, (about) 1:80 or less, (about) 1:90 or less, or (about) 1:100 or less. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipid of the lipid composition is less than (about) 1:1, less than (about) 1:5, less than (about) 1:10, less than (about) 1:15, less than (about) 1:20, less than (about) 1:25, less than (about) 1:30, less than (about) 1:35, less than (about) 1:40, less than (about) 1:45, less than (about) 1:50, less than (about) 1:60, less than (about) 1:70, less than (about) 1:80, less than (about) 1:90, or less than (about) 1:100.

[0210] In some embodiments of the pharmaceutical compositions of the present application, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% of the therapeutic agent is encapsulated in particles of the lipid composition.

[0211] In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles characterized by one or more of the following properties: (1) a (e.g., average) size of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of about 0.2 or less; and (3) a zeta potential of -10 millivolts (mV) to 10 mV. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 70 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a size (e.g., average) of about 60 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a size (e.g., average) of up to about 100 nanometers (nm), up to about 90 nanometers (nm), up to about 85 nanometers (nm), up to about 80 nanometers (nm), up to about 75 nanometers (nm), up to about 70 nanometers (nm), up to about 65 nanometers (nm), up to about 60 nanometers (nm), up to about 55 nanometers (nm), or up to about 50 nanometers (nm). In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of at least about 100 nanometers (nm), at least about 90 nanometers (nm), at least about 85 nanometers (nm), at least about 80 nanometers (nm), at least about 75 nanometers (nm), at least about 70 nanometers (nm), at least about 65 nanometers (nm), at least about 60 nanometers (nm), at least about 55 nanometers (nm), or at least about 50 nanometers (nm). (E.g., average) size can be determined by size exclusion chromatography (SEC).The (eg, average) size can be determined by spectroscopic or image-based methods, such as dynamic light scattering, static light scattering, multi-angle light scattering, laser light scattering, or dynamic image analysis, or a combination thereof.

[0212] In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.05 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.3. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.2 to about 0.5. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 or less, or about 0.05 or less.

[0213] In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -30 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 0 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 5 millivolts (mV) or less. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 10 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of 15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of 20 millivolts (mV) or less.

[0214] In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a negative zeta potential of -30 millivolts (mV) or more. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 0 millivolts (mV) or more. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 5 millivolts (mV) or more. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 10 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a zeta potential of 15 millivolts (mV) or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition comprises a plurality of particles having a zeta potential of 20 millivolts (mV) or more.

[0215] In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, or about 13 or more. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 13. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 10. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 9 to about 11. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 10 to about 13. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 8 to about 12. In some embodiments of the pharmaceutical composition of the present application, the lipid composition has an apparent pKa of about 10 to about 12.

[0216] In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is at least about 1.1 times greater than the therapeutic effect achieved in lung cells, in some embodiments, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is about 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 75 times greater, at least 100 times greater, at least 200 times greater, or at least 300 times greater. In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is at least about 1.1 times greater than that achieved in liver cells. In some embodiments, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is about 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than that achieved in liver cells. In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is at least about 1.1 times greater than that achieved in liver cells.In some embodiments, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is about 1.1 fold greater, at least 1.5 fold greater, at least 2 fold greater, at least 2.5 fold greater, at least 3 fold greater, at least 3.5 fold greater, at least 4 fold greater, at least 4.5 fold greater, at least 5 fold greater, at least 5.5 fold greater, at least 6 fold greater, at least 7 fold greater, at least 8 fold greater, at least 9 fold greater, at least 10 fold greater, at least 15 fold greater, at least 18 fold greater, or at least 20 fold greater than the therapeutic effect achieved in liver cells.

[0217] In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is at least about 1.1 times greater than that achieved in spleen cells. In some embodiments, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is about 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than that achieved in spleen cells. In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is at least about 1.1 times greater than that achieved in liver cells. In some embodiments, the SORT lipid in the composition achieves a therapeutic effect in spleen cells that is about 1.1 fold greater, at least 1.5 fold greater, at least 2 fold greater, at least 2.5 fold greater, at least 3 fold greater, at least 3.5 fold greater, at least 4 fold greater, at least 4.5 fold greater, at least 5 fold greater, at least 5.5 fold greater, at least 6 fold greater, at least 7 fold greater, at least 8 fold greater, at least 9 fold greater, at least 10 fold greater, at least 15 fold greater, at least 18 fold greater, or at least 20 fold greater in lung cells compared to the therapeutic effect achieved in liver cells. In some embodiments of the composition, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is at least about 1.1 fold greater than the therapeutic effect achieved in liver cells.In some embodiments, the SORT lipid in the composition achieves a therapeutic effect in lung cells that is about 1.1 fold greater, at least 1.5 fold greater, at least 2 fold greater, at least 2.5 fold greater, at least 3 fold greater, at least 3.5 fold greater, at least 4 fold greater, at least 4.5 fold greater, at least 5 fold greater, at least 5.5 fold greater, at least 6 fold greater, at least 7 fold greater, at least 8 fold greater, at least 9 fold greater, at least 10 fold greater, at least 15 fold greater, at least 18 fold greater, or at least 20 fold greater than the therapeutic effect achieved in liver cells.

[0218] method In some embodiments of the methods, the pharmaceutical compositions of the application may be administered via any suitable route, including, for example, oral, rectal, vaginal, mucosal, intratracheal or pulmonary, including inhalation, or enteral administration; intramuscular, subcutaneous, intramedullary injection, as well as parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0219] In some embodiments of the method, the pharmaceutical composition of the present application may be administered locally rather than systemically, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways depending on the tissue being targeted.

[0220] In some embodiments, the compositions of the present application may be injected, for example, at the site of injury, disease symptoms, or pain. In some embodiments, the compositions of the present application may be provided in a lozenge for oral, tracheal, or esophageal application. In some embodiments, the compositions of the present application may be provided in the form of a liquid, tablet, or capsule for administration to the stomach or intestines. In some embodiments, the compositions of the present application may be provided in the form of a suppository for rectal or vaginal application. In some embodiments, the compositions of the present application may also be delivered to the eye by use of an emulsion, eye drops, or injection.

[0221] In some embodiments, provided herein is a method for potent delivery to cells of a subject, comprising administering to a subject a pharmaceutical composition as described herein. In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) combined with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (iii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0222] In some embodiments of any of the methods described herein, a method of delivering a therapeutic agent to spleen cells comprises administering (e.g., systemically) a composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the spleen cells of the subject that is at least 1.1-fold greater than the corresponding amount or activity of the therapeutic agent achieved in the lung cells of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the spleen cells is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 75 times greater, at least 100 times greater, at least 200 times greater, or at least 300 times greater.

[0223] In some embodiments of the method of delivering a therapeutic agent to spleen cells, the method provides an effective amount or activity of the therapeutic agent in the spleen cells of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the spleen cells is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject.

[0224] In some embodiments of the method of delivering a therapeutic agent to spleen cells, the method provides an effective amount or activity of the therapeutic agent in the lung cells of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the lung cells is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject.

[0225] In some embodiments of any of the methods described herein, a method of delivering a therapeutic agent to a lung cell comprises administering (e.g., systemically) a composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the lung cells of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the spleen cells of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the lung cells is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in the spleen cells of the subject.

[0226] In some embodiments of the method of delivering a therapeutic agent to a lung cell, the method provides an effective amount or activity of the therapeutic agent in the lung cell of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cell of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the lung cell is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cell of the subject.

[0227] In some embodiments of the method of delivering a therapeutic agent to a lung cell, the method provides an effective amount or activity of the therapeutic agent in the spleen cells of the subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the spleen cells is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in the liver cells of the subject.

[0228] In some embodiments, delivery of a therapeutic agent to a cell may alter the genome, transcriptome, or expression level. The cell may be expanded or allowed to expand, and the alterations may be inherited by cells generated from the cell to which the therapeutic agent was delivered. In this way, the therapeutic effect may be propagated to a larger number of cells. Alterations in the genome, transcriptome, or expression level may also persist within a given cell.

[0229] Dosage Levels In another aspect, a high potency dosage form of a therapeutic (or prophylactic) agent formulated with selective organ targeting (SORT) lipids is provided, the dosage form comprising a therapeutic (or prophylactic) agent in combination with a lipid composition as described herein. In some embodiments, the lipid composition comprises (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0230] In some embodiments, the therapeutic agent is present in the dosage form at a dosage of about 2.0, 1.5, 1.0, 0.5, 0.2, or 0.1 milligrams per kilogram of body weight (mg / kg, or mpk), or a dosage range between and including any two of the foregoing values.

[0231] In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 2 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 1 milligram per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.5 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.2 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a dose of about 0.1 milligrams per kilogram of body weight (mg / kg, or mpk) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5 milligrams per milliliter (mg / mL) or less.

[0232] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligrams per milliliter (mg / mL), or a concentration range between and including any two of the foregoing values.

[0233] In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 5 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 2 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 1 milligram per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 0.5 milligrams per milliliter (mg / mL) or less. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of about 0.1 milligrams per milliliter (mg / mL) or less.

[0234] Any suitable dosage form may be prepared for delivery via, for example, oral, rectal, vaginal, mucosal, intratracheal or pulmonary, including inhalation, or enteral administration; intramuscular, subcutaneous, intramedullary injection, as well as parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0235] In some embodiments, the dosage form may be administered locally rather than systemically, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways depending on the tissue being targeted.

[0236] In some embodiments, the dosage form is an inhalation aerosol containing the composition of the present invention for nasal, tracheal, or bronchial delivery. In some embodiments, the dosage form can be provided in a lozenge for oral, tracheal, or esophageal application. In some embodiments, the dosage form can be provided in the form of a liquid, tablet, or capsule for administration to the stomach or intestines. In some embodiments, the dosage form can be provided in the form of a suppository for rectal or vaginal application. In some embodiments, the dosage form can also be delivered to the eye by using an emulsion, eye drops, or injection.

[0237] In some embodiments, administration of a dose of a therapeutic agent may be repeated.

[0238] subject Any subject in need of treatment can be treated by the methods of the present application. In some embodiments, the subject has been determined to be likely to respond to a therapeutic agent. For example, the subject may have, is suffering from, or is suspected of having a disease or condition. The therapeutic or prophylactic agents described elsewhere herein may be effective to provide a therapeutic effect to the subject by a variety of mechanisms, for example, via gene therapy (e.g., requiring repeated administration), alteration (e.g., increase) of protein production, chimeric antigen receptor (CAR) T cell generation (e.g., in vivo), immuno-oncology, vaccine-based approaches, reactivation of tumor suppressors, or other mechanisms.

[0239] In some embodiments, the subject has been determined to have a (e.g., missense or nonsense) mutation in the target gene. In some embodiments, the mutation in the target gene is associated with a genetic disease or disorder.

[0240] In some embodiments, the subject has been determined to exhibit aberrant expression or activity of a protein or polynucleotide corresponding to the target gene, in some embodiments, the aberrant expression or activity of the protein or polynucleotide is associated with a genetic disease or disorder.

[0241] In some embodiments, the subject is selected from the group consisting of a mouse, a rat, a monkey, and a human. In some embodiments, the subject is a human.

[0242] In another aspect, provided herein is a method for potent delivery of a therapeutic agent (or prophylactic agent) to a cell, comprising contacting the cell with a pharmaceutical composition of the present application.In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) combined with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (iii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid.The lipid composition may further comprise a phospholipid.

[0243] In some embodiments of this method, the cell is isolated from a subject. In some embodiments of this method, the cell is a cell line.

[0244] In another aspect, provided herein is a method for targeted delivery of a therapeutic agent (or prophylactic agent) to a cell type, comprising contacting the cell with a pharmaceutical composition of the present application.In some embodiments of the method, the pharmaceutical composition comprises a therapeutic agent (or prophylactic agent) combined with a lipid composition as described herein, the lipid composition comprising (i) an ionizable cationic lipid and (ii) a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid.The lipid composition may further comprise a phospholipid.

[0245] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting comprises administering to the subject a composition comprising a therapeutic agent in combination with a lipid composition.

[0246] Below are examples of compositions and composition evaluations of the present disclosure. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0247] LIST OF EMBODIMENTS The following listing of embodiments of the invention should be considered as disclosing various features of the invention, which features may be considered specific to the particular embodiment in which they are discussed or which may be combined with various other features as listed in other embodiments. Thus, simply because a feature is discussed in a particular embodiment, the use of that feature is not necessarily limited to that embodiment.

[0248] Embodiment 1. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising: (i) an ionizable cationic lipid; (ii) a polymer-conjugated lipid; and (iii) a selective organ targeting (SORT) lipid, or a pharma- ceutically acceptable salt, stereoisomer, or tautomer thereof; a therapeutic agent in combination with a lipid composition comprising The selective organ targeting (SORT) lipids described above have the structure of formula (IA): [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0249] Embodiment 2. A SORT lipid having the structure of formula (IA): [ka] and combinations thereof.

[0250] Embodiment 3. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising: (i) an ionizable cationic lipid; (ii) a polymer-conjugated lipid; and (iii) a selective organ targeting (SORT) lipid, or a pharma- ceutically acceptable salt, stereoisomer, or tautomer thereof; a therapeutic agent in combination with a lipid composition comprising The aforementioned selective organ targeting (SORT) lipid has the structure of formula (S-III): [ka] wherein R1 and R2 are each independently an alkyl (C8~C24) , alkenyl (C8~C24) or a substitution of any of these groups, R3, R3' and R3'' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0251] Embodiment 4. A SORT lipid having the structure of formula (S-III): [ka] 4. The composition of embodiment 3, wherein

[0252] Embodiment 5. The ionizable cationic lipid has the structural formula: [ka] A dendrimer or dendron of generation (g) having or a pharma- ceutically acceptable salt thereof, (a) The core has the structural formula (X コア ): [ka] Including, During the ceremony, Q, independently in each occurrence, is a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b - and R 2 is expressed independently for each occurrence as R 1g or -L 2 -NR 1e R 1f and R 3a and R 3b is independently at each occurrence hydrogen or optionally substituted alkyl (e.g., C1-C6, such as C1-C3); R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) independently at each occurrence represents a connection point to a branch, a hydrogen, or an optionally substituted (e.g., C1-C 12 ) alkyl, L 0 , L 1 , and L 2 represents independently at each occurrence a covalent bond (e.g., C1-C 12 alkylene (e.g., C1 to C6 or C1 to C3); 12 and selected from C1-C8 or C1-C6 (e.g., C2-C8 alkylene oxide, such as oligo(ethylene oxide)), [(e.g., C1-C6) alkylene]-[(e.g., C4-C6) heterocycloalkyl]-[(e.g., C1-C6) alkylene], [(e.g., C1-C6) alkylene]-(arylene)-[(e.g., C1-C6) alkylene] (e.g., [(e.g., C1-C6) alkylene]-phenylene-[(e.g., C1-C6) alkylene]), (e.g., C4-C6) heterocycloalkyl, and arylene (e.g., phenylene); or Alternatively, L 1 Part of R1c and R 1d with one of the following to form a (e.g., C4-C6) heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur); x 1 is 0, 1, 2, 3, 4, 5, or 6, (b) each branch of the plurality (N) of branches independently has the structural formula (X 分岐 ): [ka] Including, During the ceremony, * indicates the attachment point of the branch to the core, g is 1, 2, 3, or 4; Z=2 (g-1) and If g=1 then G=0; or if g≠1 then G=

number

[0253] Embodiment 6.x 1 The composition of embodiment 5, wherein is 0, 1, 2, or 3.

[0254] Embodiment 7.R 1a , R 1b , R 1c , R 1d , R1e , R 1f , and R 1g (when present) are each independently at each occurrence a connection point to a branch (e.g., indicated by *), hydrogen, or C1-C 12 alkyl (e.g., C1-C8 alkyl, such as C1-C6 alkyl or C1-C3 alkyl), the alkyl portion being -OH, C4-C8 (e.g., C4-C6) heterocycloalkyl (e.g., piperidinyl (e.g., [ka] ), N-(C1-C3 alkyl)-piperidinyl (e.g., [ka] ), piperazinyl (e.g. [ka] ), N-(C1-C3 alkyl)-piperazinyl (e.g., [ka] ), morpholinyl (e.g., [ka] ), N-pyrrolidinyl (e.g., [ka] ), pyrrolidinyl (e.g. [ka] ), or N-(C1-C3 alkyl)-pyrrolidinyl (e.g., [ka] )), (e.g., C6~C 10 ) aryl, and C3-C5 heteroaryl (e.g., imidazolyl (e.g., [ka] ) or pyridinyl (e.g. [ka] 7. The composition of embodiment 5 or 6, optionally substituted with one or more substituents each independently selected from:

[0255] Embodiment 8.R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) are each independently at each occurrence a connection point to a branch (e.g., indicated by *), hydrogen, or C1-C 12 The method of embodiment 7, wherein the alkyl moiety is optionally substituted with one substituent -OH.

[0256] Embodiment 9.R 3a and R 3b The composition of any one of embodiments 5 to 8, wherein each occurrence of is independently hydrogen.

[0257] Embodiment 10. The composition of any one of embodiments 5 to 9, wherein the plurality (N) of branches comprises at least three (e.g., at least four, or at least five) branches.

[0258] Embodiment 11. The composition of any one of embodiments 5 to 10, wherein g=1, G=0, and Z=1.

[0259] Embodiment 12. Each branch of the plurality of branches has the structure [ka] 12. The composition of embodiment 11, comprising:

[0260] Embodiment 13. The composition of any one of embodiments 5 to 10, wherein g=2, G=1, and Z=2.

[0261] Embodiment 14. Each branch of the plurality of branches has the structure [ka] 14. The composition of embodiment 13, comprising:

[0262] Embodiment 15. The core has the structural formula: [ka] 15. The composition of any one of embodiments 5 to 14, comprising:

[0263] Embodiment 16. The core has the structural formula: [ka] 15. The composition of any one of embodiments 5 to 14, comprising:

[0264] Embodiment 17. The core has the structural formula: [ka] 17. The composition of embodiment 16, comprising:

[0265] Embodiment 18. The core has the structural formula: [ka] 17. The composition of embodiment 16, comprising:

[0266] Embodiment 19. The core has the structural formula: [ka] Q' is -NR 2 -or-CR 3a R 3b - and q 1 and q 2The composition of any one of embodiments 5 to 14, wherein each is independently 1 or 2.

[0267] Embodiment 20. The core has the structural formula: [ka] 20. The composition of embodiment 19, comprising:

[0268] Embodiment 21. The core has the structure [ka] wherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C 12 The composition of any one of embodiments 5 to 14, wherein the heteroaryl is C3-C5).

[0269] Embodiment 22. The core has the structure [ka] 15. The composition of any one of embodiments 5 to 14, comprising:

[0270] Embodiment 23. The composition of any one of embodiments 5 to 14, wherein the core is selected from those set forth in Table 1, or a subset thereof.

[0271] Embodiment 24. The core is [ka] [ka] and pharma- ceutically acceptable salts thereof, wherein * indicates the point of attachment of the core to one branch of the plurality of branches.

[0272] Embodiment 25. The core is [ka] and pharma- ceutically acceptable salts thereof, wherein * indicates the point of attachment of the core to one branch of the plurality of branches.

[0273] 26. The core has the structure [ka] 15. The composition of any one of embodiments 5 to 14, having the formula:

[0274] Embodiment 27 The composition of embodiment 26, wherein at least two branches are attached to the core.

[0275] Embodiment 28 The composition of embodiment 26, wherein at least three branches are attached to the core.

[0276] Embodiment 29. The composition of embodiment 26, wherein at least four branches are attached to the core.

[0277] Embodiment 30. The core has a structure [ka] 15. The composition of any one of embodiments 5 to 14, having the formula:

[0278] Embodiment 31 The composition of embodiment 30, wherein at least four branches are attached to the core.

[0279] Embodiment 32 The composition of embodiment 30, wherein at least 5 branches are attached to the core.

[0280] Embodiment 33 The composition of embodiment 30, wherein at least six branches are attached to the core.

[0281] Embodiment 34.A 1 The composition of any one of embodiments 5 to 33, wherein is -O- or -NH-.

[0282] Embodiment 35.A 1 The composition of embodiment 34, wherein is -O-.

[0283] Embodiment 36.A 2 The composition of any one of embodiments 5 to 35, wherein is -O- or -NH-.

[0284] Embodiment 37.A 2 The composition of embodiment 36, wherein is -O-.

[0285] Embodiment 38.Y 3 But, C1~C 12 The composition of any one of embodiments 5 to 37, wherein the alkylene is C1-C6, such as C1-C3.

[0286] Embodiment 39. The diacyl group is represented independently at each occurrence by the formula [ka] and optionally, wherein R 3c , R 3d , R 3e , and R 3f The composition of any one of embodiments 5 to 38, wherein each occurrence of is independently hydrogen or C1-C3 alkyl.

[0287] Embodiment 40.L 0 , L 1 , and L 2 each occurrence independently represents a covalent bond, C1-C6 alkylene (e.g., C1-C3 alkylene), C2-C 12 (e.g., C2-C8) alkylene oxides (e.g., oligo(ethylene oxide), e.g., -(CH2CH2O) 1~4-(CH2CH2)-), [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., [ka] ), and [(C1-C4) alkylene]-phenylene-[(C1-C4) alkylene] (e.g., [ka] 40. The composition of any one of embodiments 5 to 39, wherein the composition is selected from:

[0288] Embodiment 41.L 0 , L 1 , and L 2 each occurrence independently represents C1-C6 alkylene (e.g., C1-C3 alkylene), -(C1-C3 alkylene-O) 1~4 The composition of embodiment 40, wherein the alkylene is selected from -(C1-C3 alkylene), -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-, and -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-.

[0289] Embodiment 42.L 0 , L 1 , and L 2 is independently at each occurrence C1-C6 alkylene (e.g., C1-C3 alkylene).

[0290] Embodiment 43.L 0 , L 1 , and L 2 However, each occurrence is independent, C2~C 12 (e.g., C2-C8) alkylene oxide (e.g., -(C1-C3 alkylene-O) 1~4 -(C1-C3 alkylene).

[0291] Embodiment 44.L 0 , L 1 , and L 2are each independently at each occurrence selected from [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-phenylene-(C1-C3 alkylene)-) and [(C1-C4) alkylene]-[(C4-C6) heterocycloalkyl]-[(C1-C4) alkylene] (e.g., -(C1-C3 alkylene)-piperazinyl-(C1-C3 alkylene)-).

[0292] Embodiment 45. Each terminal group is independently C1-C 18 (For example, C4~C 18 ) Alkenylthiol or C1-C 18 (For example, C4~C 18 ) alkylthiol, the alkyl or alkenyl portion being halogen, C6-C 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.), C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino) (e.g., [ka] ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocycloalkyl) (e.g., [ka] ), -C(O)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino) and -C(O)-(C4-C6N-heterocycloalkyl) (e.g., [ka] 45. The composition of any one of embodiments 5 to 44, wherein the C4-C6 N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0293] Embodiment 46. Each terminal group is independently C1-C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion being C6-C 12 Aryl (e.g., phenyl), C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.), C4-C6N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] )), -OH, -C(O)OH, -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C1-C 12 alkylamino (e.g., mono- or dialkylamino) (e.g., [ka] ), -C(O)N(C1-C3 alkyl)-(C1-C6 alkylene)-(C4-C6 N-heterocycloalkyl) (e.g., [ka] ), and -C(O)-(C4-C6N-heterocycloalkyl) (e.g., [ka] 46. ​​The composition of embodiment 45, wherein the C4-C6 N-heterocycloalkyl portion of any of the preceding substituents is optionally substituted with C1-C3 alkyl or C1-C3 hydroxyalkyl.

[0294] Embodiment 47. Each terminal group is independently C1-C 18 (For example, C4~C 18 47. The composition of embodiment 46, wherein the alkyl moiety is optionally substituted with one substituent -OH.

[0295] Embodiment 48. Each terminal group is independently C1-C 18 (For example, C4~C 18 ) alkylthiol, the alkyl portion being C1-C 12 (e.g., C1-C8) alkylamino (e.g., C1-C6 monoalkylamino (-NHCH2CH2CH2CH3, etc.) or C1-C8 dialkylamino (e.g., [ka] etc.) and C4-C6 N-heterocycloalkyl (e.g., N-pyrrolidinyl ( [ka] ), N-piperidinyl ( [ka] ), N-azepanyl ( [ka] 47. The composition of embodiment 46, optionally substituted with one substituent selected from:

[0296] Embodiment 49. Each terminal group is independently C1-C 18 (For example, C4~C 18 ) Alkenylthiol or C1-C 18 (For example, C4~C 18 ) alkylthiol.

[0297] Embodiment 50. Each terminal group is independently C1-C 18 (For example, C4~C 18 50. The composition of embodiment 47 or 49, wherein the thiol is an alkylthiol.

[0298] Embodiment 51. Each terminal group is: [ka] The composition of embodiment 50, wherein the compound is independently selected from the group consisting of:

[0299] Embodiment 52. The composition of any one of embodiments 5 to 44, wherein each terminal group is independently selected from those set forth in Table 3, or any subset thereof.

[0300] Embodiment 53. The composition of any one of embodiments 1 to 4, wherein the ionizable cationic lipid is selected from those set forth in Table 4, or a pharma- ceutically acceptable salt thereof, or a subset of the lipids and their pharma- ceutically acceptable salts.

[0301] Embodiment 54. The composition of any one of embodiments 1 to 4, wherein the ionizable cationic lipid is selected from those set forth in Table 4 or Table 5, or a pharma- ceutically acceptable salt thereof, or a subset of the lipids and their pharma- ceutically acceptable salts.

[0302] Embodiment 55. The composition of any one of embodiments 1 to 54, wherein the lipid composition further comprises a phospholipid.

[0303] Embodiment 56. The composition of embodiment 55, wherein the phospholipid is at a molar percentage of about 8% to about 23%.

[0304] Embodiment 57. The composition of any one of embodiments 1 to 56, wherein the phospholipid further comprises a steroid or a steroid derivative.

[0305] Embodiment 58. The composition of embodiment 57, wherein the steroid or steroid derivative is in a molar percentage of about 15% to about 46%.

[0306] Embodiment 59. The composition of any one of embodiments 1 to 58, wherein the ionizable cationic lipid is at a molar percentage of about 5% to about 30%.

[0307] Embodiment 60. The composition of any one of embodiments 1 to 59, wherein the polymer-conjugated lipid is at a molar percentage of about 0.5% to about 10%.

[0308] Embodiment 61. The composition of any one of embodiments 1 to 59, wherein the polymer-conjugated lipid is at a molar percentage of about 1% to about 10%.

[0309] Embodiment 62. The composition of any one of embodiments 1 to 59, wherein the polymer-conjugated lipid is at a molar percentage of about 2% to about 10%.

[0310] Embodiment 63. The composition of any one of embodiments 1 to 62, wherein the SORT lipid is at a molar percentage of about 20% to about 65%.

[0311] Embodiment 64. The composition of any one of embodiments 1 to 63, wherein the therapeutic agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 20:1 or less.

[0312] Embodiment 65. The composition of embodiment 64, wherein the N / P ratio is from about 5:1 to about 20:1.

[0313] Embodiment 66. The composition of any one of embodiments 1 to 65, wherein the molar ratio of therapeutic agent to total lipid of said lipid composition is less than about 1:1, 1:10, 1:50, or 1:100.

[0314] Embodiment 67. The composition of any one of embodiments 1 to 66, wherein at least about 85% of said therapeutic agent is encapsulated in particles of said lipid composition.

[0315] Embodiment 68. The lipid composition as described above, comprising: (1) A size (e.g., average) of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of about 0.2 or less; and (3) A negative zeta potential of -10 millivolts (mV) to 10 mV 68. The composition of any one of the preceding embodiments, comprising a plurality of particles characterized by one or more of the following properties:

[0316] Embodiment 69. The composition of any one of embodiments 1 to 68, wherein said lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7.

[0317] Embodiment 70. The composition of embodiment 69, wherein said apparent pKa of said lipid composition is about 7 or greater.

[0318] Embodiment 71. The composition of embodiment 69, wherein the apparent pKa of the lipid composition is about 8 or greater.

[0319] Embodiment 72. The composition of embodiment 69, wherein the apparent pKa of the lipid composition is from about 8 to about 13.

[0320] Embodiment 73. A method for targeted delivery of a therapeutic agent to spleen cells, the method comprising administering (e.g., systemically) a composition according to any one of embodiments 1 to 72, thereby providing an effective amount or activity of said therapeutic agent in said spleen cells of said subject that is at least 1.1-fold greater than the corresponding amount or activity of said therapeutic agent achieved in said subject's lung cells.

[0321] Embodiment 74. The method of embodiment 73, wherein the effective amount or activity of said therapeutic agent in said spleen cells of said subject is at least 1.1 times greater, at least 2.5 times greater, at least 3.5 times greater, at least 10 times greater, at least 5.5 times greater, at least 10 times greater, at least 15 times greater, at least 20 times greater, or at least 50 times greater than the corresponding amount or activity of said therapeutic agent achieved in said subject's lung cells.

[0322] Embodiment 75. A method for targeted delivery of a therapeutic agent to lung cells, comprising administering (e.g., systemically) a composition according to any one of embodiments 1 to 72, thereby providing an effective amount or activity of said therapeutic agent in said lung cells of said subject that is at least 1.1-fold greater than the corresponding amount or activity of said therapeutic agent achieved in said spleen cells of said subject.

[0323] Embodiment 76. The method of embodiment 75, wherein the effective amount or activity of the therapeutic agent in the lung cells of the subject is at least 1.1 times greater, at least 5 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, at least 20 times greater, or at least about 50 times greater than the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

[0324] Working Example Example 1. Preparation of lipid nanoparticles modified with DOTAP or DODAP Lipid nanoparticles (LNPs) are the most effective class of carriers for nucleic acid delivery in vivo. Historically, effective LNPs have been composed of four components: ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and the lipid poly(ethylene glycol) (PEG). However, these LNPs only result in general delivery of nucleic acids, not targeted delivery to organs or tissues. LNPs typically deliver RNA only to the liver. Therefore, new formulations of LNPs have been sought to provide targeted nucleic acid delivery.

[0325] Four standard lipids were mixed in a molar ratio of 15:15:30:3 with or without the addition of permanent cationic lipids. Briefly, LNPs were prepared by mixing dendrimer or dendron lipids (ionizable cationic), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cationic). Alternatively, DOTAP can be used in place of DODAP to generate LNPs containing DODAP. The structures of DODAP and DODAP are shown in Figure 1. The various dendrimer or dendron lipids that can be used are shown in Figure 2.

[0326] For the preparation of LNP formulations, dendrimer or dendron lipids, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at the desired molar ratio. mRNA was dissolved in citrate buffer (10 mM, pH 4.0). The mRNA was then diluted into the lipid solution, and a weight ratio of 40:1 (total lipid:mRNA) was achieved by rapidly mixing the mRNA into the lipid solution at a volume ratio of 3:1 (mRNA:lipid, v / v). The solution was then incubated at room temperature for 10 min. For the formation of DOTAP-modified LNP formulations, mRNA was dissolved in 1× PBS or citrate buffer (10 mM, pH 4.0) and rapidly mixed into ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP, and allowed to settle at a weight ratio of 40:1 (total lipid:mRNA), and a volume ratio of 3:1 (mRNA:lipid). Formulations are designated X%DOTAP Y (or X%DODAP Y), where X represents the molar percentage of DOTAP (or DODAP) in total lipids and Y represents the type of dendrimer or dendron lipid. Alternatively, formulations may be designated YX%DOTAP or YX%DODAP, where X represents the molar percentage of DOTAP (or DODAP) in total lipids and Y represents the type of dendrimer or dendron lipid.

[0327] Example 2. Stability of SORT LNPs The lipid compositions described herein were produced using either microfluidic or cross / tee mixing methods and tested for stability.The physical properties including size, polydispersity index (PDI) and zeta potential were characterized by dynamic light scattering (DLS) from various exemplary lipid (LNP) compositions (measured three times separately for each formulation) and are shown in Table 7.

[0328] Encapsulation efficiency was tested using the Ribogreen RNA assay (Zhao et al., 2016). Briefly, when mRNA was dissolved in acidic buffer (10 mM citrate, pH 4), it was encapsulated in LNPs with >95% efficiency. The properties of two types of LNPs (5A2-SC8 20% DODAP ("Liver-SORT") and 5A2-SC8 50% DOTAP ("Lung-SORT")) were observed over a period of 28 days. Figure 3 shows the change in physical properties of the lipid composition over a period of 28 days.

[0329] [Table 7]

[0330] Example 3: Pulmonary-SORT Intravenous Administration Study This Example summarizes several studies in which LUNG-SORT was administered intravenously and systemically to multiple animal species (e.g., mice, rats, dogs, and non-human primates (NHPs) (e.g., rhesus monkeys, cynomolgus monkeys)).

[0331] overview Most subjects received a single dose of luciferase mRNA encapsulated in Lung-SORT LNPs (e.g., 5A2-SC8 with 50% DOTAP). A portion of subjects received two doses. The lower doses administered to dogs and NHPs were translated from mouse studies. Whole animal imaging, organ imaging, blood chemistry, hematology, immunoreactivity, complement, and tolerability parameters were evaluated in the study. Lung-SORT LNPs demonstrated selectivity across species to target the lung and avoid other organs and tissues. Various DOTAP alternative LNPs (e.g., 14:0 TAP) were tested in vivo (e.g., mice) and in vitro (e.g., hBE) and showed improved tolerability, higher potency, and equivalent or better selectivity. A second lung-SORT ("RTX0031") using 14:0 TAP (SORT lipid) has also been tested in animal studies (e.g., dogs), and the formulation was highly potent and highly selective for lung targeting (similar results were obtained with IV bolus and IV infusion, with no signs of infusion-related reactions (IRR)).

[0332] The exemplary "Lung-SORT LNP" tested herein is a five-component lipid nanoparticle composition comprising about 11.9% 5A2-SC8 (an ionizable cationic lipid), about 50% DOTAP (a SORT lipid), about 11.9% DOPE lipid, about 23.8% cholesterol, and about 2.4% DMG-PEG (a PEG-conjugated lipid), with each lipid component defined as a molar % of the total lipid composition.

[0333] An exemplary DOTAP replacement LNP replaces DOTAP (SORT lipid) with 14:0 TAP as the SORT lipid (referred to as "RTX0031 LNP"). The RTX0031 LNP composition tested herein is a five-component lipid nanoparticle composition comprising about 14.3% 5A2-SC8 (an ionizable cationic lipid), about 40% 14:0 TAP (SORT lipid), about 14.3% DOPE, about 28.6% cholesterol, and about 2.8% DMG-PEG (a PEG-conjugated lipid), with each lipid component defined as a molar % of the total lipid composition.

[0334] NHP Intravenous Lung-SORT LNP Study Lung-SORT LNPs containing 0.1 mg / kg luciferase mRNA were delivered to NHPs by intravenous bolus over 5 minutes without premedication (e.g., steroids). Whole-body bioluminescence imaging was performed 4 hours after intravenous bolus administration. Results showed that the Lung-SORT LNP formulation was active in NHPs and highly potent, with mRNA delivery occurring outside the liver and significant signal observed in the lungs. Additionally, the tolerability of Lung-SORT LNPs was evaluated in NHPs. Without wishing to be bound by any theory, DOTAP and / or the method of administration may have contributed to tolerability.

[0335] Herein, RTX0031 LNP was tested. Additionally, treatment-related parameters were further tested (e.g., slow infusion with premedication if necessary).

[0336] [Table 8]

[0337] Figures 4A and 4B show IVIS organ imaging of the spleen, liver, and lungs of female and male dogs following administration of Lung-SORT LNPs (5A2-SC8 with 50% DOTAP). The high signal seen in the lungs indicated that Lung-SORT LNPs were selective for delivery to the lungs.

[0338] Both dogs showed signs consistent with a mild infusion-related reaction. The female dog (1) had very mild tachycardia compared to baseline. Blood chemistry in the female dog showed only minor changes with 2-fold increases in AST, CK, and LDH. Hematological evaluation showed no significant changes. The male dog had mucosal congestion and mild lethargy / weakness (ventral flexion of the neck) during the first 15 minutes after injection, which resolved within the first 30 minutes after injection and was not severe enough to require intervention. The male (2) dog had mild bradycardia compared to baseline, observed during the first hour after infusion.

[0339] [Table 9]

[0340] Figure 5A and Figure 5B show IVIS organ imaging of the spleen, liver, and lungs of both cynomolgus NHPs following administration of Lung-SORT LNPs (5A2-SC8 containing 50% DOTAP). The high signal seen in the lungs indicated that Lung-SORT LNPs were selective for delivery to the lungs.

[0341] No adverse events were observed during or after the first and second doses of the LUNG-SORT formulation. Blood chemistry tests in cynomolgus NHP subjects showed elevated AST, ALT, CK, and LDH after the first dose, and elevated LDH and CO2 after the second dose. Hematology tests in cynomolgus NHP subjects showed elevated neutrophils and decreased lymphocytes and eosinophils after the first and second doses.

[0342] Lung-SORT LNPs (5A2-SC8 containing 50% DOTAP) were delivered as an intravenous bolus to rats, dogs, rhesus monkeys, and cynomolgus monkeys. Lung-SORT LNP was well tolerated when delivered as an intravenous bolus without premedication, with no adverse events noted during two doses in cynomolgus monkeys and only mild signs of IRR after a single dose in dogs. Changes in AST, CK, LDH, and neutrophil / lymphocyte were blood markers of good tolerability across species tested.

[0343] Rats tested had differences in potency and toxicity compared to mice, dogs, and NHP subjects tested, showing signs of toxicity only at the 1 mg / mL dose, with no favorable results.

[0344] Mouse Study of RTX0031 LNP (DOTAP Alternative for Lung-SORT) Figure 6A shows IVIS organ imaging of the spleen, liver, kidneys and lungs of three mice after administration of luciferase mRNA formulated in RTX0031 LNPs for 5 hours. The higher signal seen in the lungs compared to the liver, spleen and kidneys indicates that RTX0031 LNPs were selective for delivery to the lungs. Figure 6B quantitatively shows the signal obtained in the lungs, spleen and liver of three mice. In vivo studies may provide information that RTX0031 LNPs have improved tolerability and potency.

[0345] Comparison of RTX0031 LNPs (14:0 TAP as SORT lipid) with lung-SORT LNPs (DOTAP as SORT lipid) in an hBE study RTX0031 LNPs and Lung-SORT LNPs were tested for tolerability and potency in hBEs. hBEs were administered 12 μg of Tomato red (TR) mRNA formulated in Lung-SORT and RTX0031 LNP formulations in the apical fluid. TR expression was assessed by fluorescence microscopy 24 hours after treatment. Figure 7A shows the TR intensity expressed in treated hBEs of the two LNP compositions tested. RTX0031 LNPs showed higher TR intensity compared to the Lung-SORT LNP (5A2-SC8 with 50% DOTAP) formulation. Additionally, cytotoxicity (LDH release) was assessed 48 hours after treatment. Figure 7B shows the % LDH released from treated hBEs of the two LNP compositions. The Lung-SORT LNP (5A2-SC8 with 50% DOTAP) formulation showed a higher LDH release rate than the RTX0031 LNP formulation. This in vitro study may provide information that the RTX0031 LNP formulation has improved tolerability and potency.

[0346] [Table 10]

[0347] Figure 8A shows IVIS organ imaging of the spleen, liver, and lungs of two beagles after an intravenous bolus administration of luciferase mRNA formulated in RTX0031 LNPs. The male dog showed some hypersalivation immediately after TA treatment, but it lasted less than 1-2 minutes. The female dog showed no signs of adverse reactions during or after TA treatment. Both dogs showed only minor changes in body temperature, heart rate, and respiratory rate, mainly related to the stress of handling. The female dog experienced seizures during luciferin administration. Blood chemistry showed an increase in ALT in the male dogs by approximately 3 years of age. Hematological changes were minimal, with decreases in lymphocytes and eosinophils, especially in the male dogs.

[0348] Figure 8B shows IVIS organ imaging of the spleen, liver, and lungs of two beagles after intravenous injection of luciferase mRNA formulated in RTX0031 LNPs along with premedication. None of the dogs showed any clinical signs or adverse reactions during or after TA administration. One dog had a seizure during luciferin administration. Blood chemistry showed increases in ALT (2-fold), CK (3-fold), and LDH (5-fold) in the female dog. Only minor hematological changes were noted, particularly decreases in lymphocytes and eosinophils.

[0349] FIG. 9 shows a compiled panel of IVIS organ imaging of dog and NHP spleen, liver, and lungs seen in FIGS. 4A, 4B, 5A, 5B, 8A, and 8B.

[0350] [Table 11]

[0351] Example 4: Organ-selective tropism of spleen SORT and lung SORT formulations Luc mRNA / LNPs containing 5A2-SC8 lipid and 40% SORT lipid as described in Table 12 were administered intravenously to mice at a dose of 0.05 mpk. Each SORT lipid discussed in Table 12 was administered to n=4 mice per group. Organs were harvested 5 hours after LNP administration and the kidneys, lungs, spleen, and liver were imaged by IVIS to determine the selective organ tropism of the different SORT lipids.

[0352] An exemplary LNP tested herein is a five-component lipid nanoparticle composition comprising about 14.3% 5A2-SC8 (an ionizable cationic lipid), about 40% SORT lipid from Table 12, about 14.3% DOPE, about 28.6% cholesterol, and about 2.8% DMG-PEG (a PEG-conjugated lipid), with each lipid component defined as a molar % of the total lipid composition.

[0353] [Table 12]

[0354] IVIS images in Figure 11A show varying organ selectivity of 12:0 EPC, 14:0 EPC, 14:1 EPC, 16:0 EPC, 18:0 EPC, 18:1 EPC, and 16:0-18:1 EPC SORT lipids in SORT LNPs delivered to mice. Higher intensities localized to the spleen and lungs compared to the liver indicate the organ targeting directional nature of SORT LNPs and the influence of SORT lipids. Figure 11B quantitatively shows IVIS data for mouse liver, spleen, and lungs with different SORT lipids tested. Each EPC lipid tested has higher selectivity for spleen and lungs compared to liver in all cases tested, as evidenced by higher emission intensities. Certain EPC lipids have higher levels of selectivity between lung and spleen, delivering payload more effectively.

[0355] The IVIS images in Figure 12A show the change in organ selectivity of 14:0 TAP, 16:0 TAP, 18:0 TAP, 18:1 TAP, 18:0 DDAB, and 18:1 DOTMA SORT lipids of SORT LNP administered to mice. The higher intensity localized to the lung compared to the liver and spleen indicates the organ targeting directional nature of SORT LNP and the influence of SORT lipids. Figure 12B quantitatively shows the IVIS data of the liver, spleen, and lung of mice with different SORT lipids tested. Most of the SORT lipids tested have higher selectivity for the lung compared to the liver and spleen as indicated by higher emission intensity. Certain SORT lipids have higher selectivity between the lung and spleen, delivering the payload more effectively.

[0356] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. It is therefore contemplated that the present invention will cover such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. A composition formulated for systemic administration, the composition comprising: (i) an ionizable cationic lipid; and (ii) a polymer-conjugated lipid; and (iii) Structural formula (SI'): 【Chemistry 1】 [In the formula, R 1 and R 2 are each independently alkyl (C8-C24), alkenyl (C8-C24), or a substitution of either group; R 3 , R 3 ' and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6); X − is a monovalent anion. Or structural formula (IA): 【Chemistry 2】 [In the formula, R １ and R ２ are each independently an alkyl （Ｃ８～Ｃ２４） , alkenyl （Ｃ８～Ｃ２４） or a substitution of any of these groups, R ３ , R ３ ' and R ３ Each " is independently an alkyl （Ｃ≦６） or substituted alkyl （Ｃ≦６） and X － is a monovalent anion. an additional ionizable lipid other than the ionizable cationic lipid, A composition comprising a therapeutic agent in combination with a lipid composition comprising:

2. The composition described in claim 1, wherein the lipid composition further comprises (iv) a phospholipid, and / or (v) a steroid or steroid derivative.

3. The lipid composition comprises: (A) the phospholipid in a molar percentage of 8% to 23%; and / or (B) said steroid or steroid derivative in a molar percentage between 15% and 46%; and / or (C) the ionizable cationic lipid in a molar percentage of 5% to 30%; and / or (D) the polymer-conjugated lipid in a molar percentage of 0.5% to 10%; and / or (E) an additional ionizable lipid other than the ionizable cationic lipid in a molar percentage of 20% to 65%. The composition of claim 2 comprising:

4. 4. The composition of any one of claims 1 to 3, wherein the therapeutic agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 20:1 or less, optionally the N / P ratio is between 5:1 and 20:

1.

5. The composition described in any one of claims 1 to 4, wherein the molar ratio of the therapeutic agent to the total lipids of the lipid composition is 1:1 or more, 1:5 or more, 1:10 or more, 1:15 or more, 1:20 or more, 1:25 or more, 1:30 or more, 1:35 or more, 1:40 or more, 1:45 or more, 1:50 or more, 1:60 or more, 1:70 or more, 1:80 or more, 1:90 or more, or 1:100 or more.

6. The lipid composition comprises: (1) a size (e.g., average) of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of 0.2 or less; and (3) Zeta potential of -10 millivolts (mV) to 10 mV The particle includes a plurality of particles characterized by one or more of the following properties: A composition according to any one of claims 1 to 5.

7. 7. The composition of any one of claims 1 to 6, wherein the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7, optionally the apparent pKa of the lipid composition is 8 or greater, or the apparent pKa of the lipid composition is between 8 and 13.

8. A composition described in any one of claims 2 to 7, wherein the lipid composition comprises the phospholipid in a molar percentage of about 8% to about 23%, or the steroid or steroid derivative in a molar percentage of about 15% to about 46%.

9. A composition described in any one of claims 1 to 8, wherein the lipid composition comprises the ionizable cationic lipid in a molar percentage of about 5% to about 30%, or the polymeric conjugate lipid in a molar percentage of about 0.5% to about 10%, or about 1% to about 10%.

10. A composition described in any one of claims 1 to 9, wherein the lipid composition comprises an additional ionizable lipid separate from the ionizable cationic lipid in a molar percentage of about 20% to about 65%.

11. The composition of any one of claims 1 to 10, wherein at least about 85% of the therapeutic agent is encapsulated in particles of the lipid composition.

12. The composition of any one of claims 1 to 11, wherein the ionizable cationic lipid is selected from those set forth in Tables 4 or 5, or a pharma- ceutically acceptable salt thereof, or a subset of lipids and pharma- ceutically acceptable salts thereof, optionally wherein the ionizable cationic lipid selected from those set forth in Tables 4 or 5 comprises 4A3SC4 or 5A2-SC8.

13. The method of claim 1, wherein the ionizable lipid has the structural formula: Core-Repeating Unit-Terminal Group (DI) or a pharma- ceutically acceptable salt thereof, said core is linked to 2 to 6 repeat units by removing 2 to 6 hydrogen atoms from the core and replacing the hydrogen atoms with the repeat units; The core has the formula: 【Chemistry 3】 [In formula (D-IV), X 3 is —NR 6 —, —O—, or optionally substituted alkylaminodiyl(C≦8) where R 6 is hydrogen, alkyl(C≦8) or substituted alkyl(C≦8); R 3 and R 4 are each independently amino, optionally substituted alkylamino (C≦12), or optionally substituted dialkylamino (C≦12); c and d are each independently 1, 2, 3, 4, 5, or 6. having The repeating unit has the formula: 【Chemistry 4】 [In formula (D-VII), A 1 and A 2 are each independently —O—; Y 3 is —CH 2 CH 2 —; R 9 is —CH 3 . and The terminal group has the formula: 【Chemistry 5】 [In formula (D-VIII), Y 4 is alkanediyl(C≦18) or alkenediyl(C≦18), R 10 is hydrogen. The composition of any one of claims 1 to 11, having the formula:

14. The method of claim 1, wherein the ionizable lipid has the structural formula: 【Chemistry 6】 [In the formula, N is 2 to 6; g=1. or a pharma- ceutically acceptable salt thereof, The core has the structural formula: 【Chemistry 7】 [In the formula, L 1 is C 1 -C 6 -alkylene; R 1a , R 1b , R 1c , R 1d , R 1e , and R 1f are each independently at each occurrence a C 1 -C 12 alkyl substituted with a branch attachment point or optionally one -OH. having Each diacyl group independently has the structural formula: 【Chemistry 8】 [In the formula, * indicates the point of attachment at the proximal end of the diacyl group; ** indicates the point of attachment of the diacyl group at its distal end; Y 3 is, independently at each occurrence, C 1 -C 12 alkylene; A 1 and A 2 are each —O—; m 1 and m 2 are each independently at each occurrence 1, 2, or 3; R 3c , R 3d , R 3e , and R 3f are each independently at each occurrence hydrogen or C 1 -C 8 alkyl. and The composition of any one of claims 1 to 11, wherein each terminal group is a C 4 -C 18 -alkenyl thiol. (A) the polymer-conjugated lipid is DMG-PEG, and / or (B) The composition according to any one of claims 1 to 13, wherein the phospholipid is DOPE.

16. The composition of any one of claims 1 to 15, wherein the additional ionizable lipid other than the ionizable cationic lipid comprises 14:0 TAP, 12:0 EPC, 14:0 EPC, 14:1 EPC, 16:0 EPC, 18:0 EPC, 18:1 EPC, 16:0-18:1 EPC, 16:0 TAP; 18:0 TAP, 18:1 TAP, 18:0 DDAB, or 18:1 DOTMA.

17. The method of claim 1, wherein an additional ionizable lipid, separate from the ionizable cationic lipid, has the following structure: 【Chemistry 9】 16. The composition of claim 1 , comprising a lipid having the formula:

18. The method of claim 17, further comprising: 【Chemistry 10】 The composition of claim 1 selected from the group consisting of:

19. The composition of any one of claims 1 to 7, wherein the lipid composition comprises 14.3% 5A2-SC8, 40% 14:0 TAP, 14.3% DOPE, 28.6% cholesterol, and 2.8% DMG-PEG, wherein each lipid component is defined as a mol % of the total lipid composition.

20. 20. The composition of any one of claims 1 to 19 for use in a method of delivering a therapeutic agent to spleen cells, the method comprising administering the composition, which, upon systemic administration, provides an effective amount or activity of the therapeutic agent in the spleen cells of a subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the lung cells of the subject.

21. 21. The composition for use of claim 20, wherein the effective amount or activity of the therapeutic agent in the spleen cells of the subject is at least 1.1 fold greater, at least 1.5 fold greater, at least 2 fold greater, at least 2.5 fold greater, at least 3 fold greater, at least 3.5 fold greater, at least 4 fold greater, at least 4.5 fold greater, at least 5 fold greater, at least 5.5 fold greater, at least 6 fold greater, at least 7 fold greater, at least 8 fold greater, at least 9 fold greater, at least 10 fold greater, at least 15 fold greater, at least 18 fold greater, at least 20 fold greater, at least 30 fold greater, at least 40 fold greater, at least 50 fold greater, at least 75 fold greater, at least 100 fold greater, at least 200 fold greater, or at least 300 fold greater.

22. 20. A composition according to any one of claims 1 to 19 for use in a method of delivering a therapeutic agent to lung cells, the method comprising administering the composition, which, upon systemic administration, provides an effective amount or activity of the therapeutic agent in the lung cells of a subject that is at least 1.1 times greater than the corresponding amount or activity of the therapeutic agent achieved in the splenocytes of the subject.

23. 23. The composition for use of claim 22, wherein the effective amount or activity of the therapeutic agent in the lung cells of the subject is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, or at least 20 times greater than the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

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