CN117750948A - Compositions and methods for targeted systemic delivery to cells - Google Patents

Abstract

Described herein are compositions, kits, and methods for effective systemic delivery to cells of a subject. Also described herein are pharmaceutical compositions comprising therapeutic or prophylactic agents assembled to lipid compositions. The lipid composition can comprise an ionizable cationic lipid, a phospholipid, and a selective organ-targeting lipid. Further described herein are highly potent dosage forms of therapeutic or prophylactic agents formulated with lipid compositions.

Description

Compositions and methods for targeted systemic delivery to cells

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application Ser. Nos. 63/164,534, filed 3/23/2021, and 63/305,652, filed 2/2022, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Background

Therapeutic approaches such as CRISPR/Cas (regularly spaced clustered short palindromic repeats/CRISPR-associated protein (Cas)) technology generally require precise and efficient delivery of one or more therapeutic agents to one or more target organs or target cells (sometimes in a sequence-dependent manner). To date, there remains a clear need to achieve therapeutically safe and effective lipid-based vectors to achieve clinical outcome in the context of genetic diseases and many other applications.

Disclosure of Invention

In certain aspects, the present application provides compositions for the effective delivery of therapeutic agents to cells of a subject. In some embodiments, the composition may be formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (IA):

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

In some embodiments, the composition may be formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (IA):

Wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

In certain aspects, the present application provides methods for the efficient delivery of therapeutic agents to cells of a subject. In some embodiments, the methods described herein can be used to target delivery of a therapeutic agent to spleen cells, the method comprising 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 times the corresponding amount or activity of the therapeutic agent achieved in lung cells of the subject.

In some embodiments, the methods described herein can be used to target delivery of a therapeutic agent to a lung cell, the method comprising administering (e.g., systemically) a composition described herein, thereby providing an effective amount or activity of the therapeutic agent in the lung cell of the subject that is at least 1.1 times the corresponding amount or activity of the therapeutic agent achieved in spleen cells of the subject.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference contradict the disclosure included in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Drawings

The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Upon request and payment of the necessary fee, the authority will provide a copy of the present patent or patent application publication with a colored drawing. 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"), of which:

fig. 1 shows an example of the structure of a SORT lipid.

Fig. 2 shows an exemplary structure of a dendrimer or dendron of an ionizable cationic lipid.

Figure 3 shows the stability and general characteristics of various LNP compositions.

Fig. 4A shows IVIS organs of spleen, liver and lung of female dogs imaged after administration of lung-SORT.

Fig. 4B shows IVIS organ imaging of spleen, liver and lung of male dogs after administration of lung-SORT.

Fig. 5A shows IVIS organ imaging of spleen, liver and lung of cynomolgus monkey NHP after administration of lung-SORT.

Fig. 5B shows IVIS organ imaging of spleen, liver and lung of cynomolgus monkey NHP after administration of lung-SORT.

FIG. 6A shows IVIS organ imaging of spleen, liver, kidney and lung of mice 5h after administration of luciferase mRNA formulated with 14:0TAP SORT.

Fig. 6B quantitatively shows the signals obtained at the lungs, spleen and liver of the mice in fig. 6A.

Fig. 7A shows TR intensities expressed in hBE treated with two different SORT LNPs.

Fig. 7B shows the% LDH released from hBE treated with two different SORT LNPs.

FIG. 8A shows IVIS organ imaging of spleen, liver and lung of two beagle dogs after intravenous bolus administration of luciferase mRNA formulated in 14:0TAP SORT.

FIG. 8B shows IVIS organ imaging of spleen, liver and lung of two beagle dogs after intravenous infusion of luciferase mRNA formulated in 14:0TAP SORT with the precursor drug.

Fig. 9 shows a compilation of IVIS organ images of spleen, liver and lung of dogs and NHPs.

Fig. 10A shows ex vivo imaging of bioluminescence of spleen, lung, liver and kidney after intravenous delivery of Luc mRNA/LNP using various compositions of LNP.

FIG. 10B shows a graph of the bioluminescence data of FIG. 10A quantitatively.

Fig. 11A shows ex vivo imaging of bioluminescence of spleen, lung, liver and kidney after intravenous delivery of Luc mRNA/LNP using various compositions of LNP.

FIG. 11B shows a graph of the bioluminescence data of FIG. 11A quantitatively.

Detailed Description

Before describing embodiments of the present disclosure, it is to be 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. Many modifications, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

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

In the context of the present application, the following terms have the meanings given to them unless otherwise indicated:

as used throughout the specification and claims, the terms "a" and "an" and "the" are used generally in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality/a plurality" of the referenced components or steps, unless the upper limit is specified below. For example, as used herein, "cleavage sequence" means "at least a first cleavage sequence", but includes a plurality of cleavage sequences. The operational limitations and parameters of the combination, as well as the amount of any single agent, will be known to those of ordinary skill in the art in light of this application.

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

As used herein in the context of polypeptide structure, "N-terminal" (or "amino-terminal") and "C-terminal" (or "carboxy-terminal") generally refer to the extreme amino-and carboxy-terminals, respectively, of a polypeptide.

As used herein, the term "N-terminal sequence" with respect to a polypeptide of interest 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 of the polypeptide of interest or polynucleotide sequence of interest. As used herein, the term "C-terminal sequence" with respect to a polypeptide of interest or polynucleotide sequence of interest generally means that there are no other amino acid or nucleotide residues at the C-terminus after the C-terminal sequence of the polypeptide of interest or polynucleotide sequence of interest.

The terms "non-naturally occurring" and "non-natural" are used interchangeably herein. As used herein, the term "non-naturally occurring" or "non-natural" in reference 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). As applied to sequences and as used herein, the term "non-naturally occurring" or "non-natural" means a polypeptide or polynucleotide sequence that does not have a counterpart, is not complementary, or does not have a high degree of homology to a wild-type or naturally occurring sequence found in a mammal. For example, when properly aligned, non-naturally occurring polypeptides or fragments may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to the native sequence.

"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, pH. A number of physiologically relevant conditions have been established for in vitro assays. Typically, the physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8 and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al (2001). The physiologically relevant temperature ranges from about 25 ℃ to about 38 ℃ and preferably from about 35 ℃ to about 37 ℃.

As used herein, the terms "treatment" or "moderating" or "improving" are used interchangeably herein. These terms generally refer to methods for achieving a beneficial or desired result, including but not limited to therapeutic benefit and/or prophylactic benefit. Therapeutic benefit means eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms or ameliorating one or more clinical parameters associated with a potential disorder such that an improvement is observed in the subject, although the subject may still have a potential disorder. For prophylactic benefit, the compositions may be administered to subjects at risk of developing a particular disease, or to subjects reporting one or more physiological symptoms of a disease, even though a diagnosis of such a disease may not have been made.

As used herein, "therapeutic effect" or "therapeutic benefit" generally refers to a physiological effect, including but not limited to, alleviation, amelioration, or prophylaxis of a disease or amelioration of one or more clinical parameters associated with a potential disorder in a human or other animal, or otherwise enhancing the physical or mental health of a human or animal, as a result of administration of a polypeptide of the present disclosure, rather than by the ability to induce production of antibodies to an epitope possessed by a biologically active protein. For prophylactic benefit, the compositions may be administered to subjects at risk of developing a particular disease, recurrence of a previous disease, a disorder or symptom of a disease, or to subjects reporting one or more physiological symptoms of a disease, even though such a disease may not have been diagnosed.

As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" generally refer to an amount of a drug or biologically active protein, alone or as part of a polypeptide composition, that is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or feature of a disease state or disorder when administered to a subject in one dose or repeated doses. Such effects need not be absolutely beneficial. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

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

As used herein, the term "therapeutically effective and nontoxic dose" generally refers to a tolerable dose of a composition as defined herein that is sufficiently high to result in the consumption of tumor or cancer cells, tumor elimination, tumor shrinkage, or disease stabilization without or substantially without major toxic effects in the subject. Such therapeutically effective and nontoxic dosages can be determined by dose escalation studies described in the art and should be below those that induce severe adverse side effects.

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

When used in the context of chemical groups: "hydrogen" means-H; "hydroxy" means-OH; "oxo" means =o; "carbonyl" means-C (=o) -; "carboxyl" means-C (=O) OH (also written as-COOH or-CO ₂ H) The method comprises the steps of carrying out a first treatment on the surface of the "halo" means independently-F, -Cl, -Br or-I; "amino" means-NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the "hydroxyamino" means-NHOH; "nitro" means-NO ₂ The method comprises the steps of carrying out a first treatment on the surface of the "imino" means =nh; "cyano" means-CN; "isocyanate" means-n=c=o; "azido" means-N ₃ The method comprises the steps of carrying out a first treatment on the surface of the In the monovalent context, "phosphoric acid" means-OP (O) (OH) ₂ Or a deprotonated form thereof; in the divalent context, "phosphoric acid" means-OP (O) (OH) O-or its deprotonated form; "mercapto" means-SH; and "thio" means = S; "sulfonyl" means-S (O) ₂ -; "hydroxysulfonyl" means-S (O) ₂ OH; "sulfonamide" means-S (O) ₂ NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And "sulfinyl" means-S (O) -.

In the context of the chemical formula, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. The symbol "- - -" represents an optional bond, which if present is a single bond or a double bond. Sign symbolRepresents a single bond or a double bond. Thus, for example, the formula->Comprises->And it should be understood that none of such ring atoms form part of more than one double bond. Furthermore, it should be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when one or both stereoisomerically derived atoms are attached. Instead, it covers all stereoisomers and mixtures thereof. When drawn perpendicular to the key (e.g. +. >Methyl) is represented, symbol->Indicating the attachment point of the group. It should be noted that the attachment point of the larger group is typically only identified in this way to help the reader to identify the attachment point explicitly. Sign->Meaning a single bond in which the group attached to the thick end of the wedge is "out of the page". Sign->Meaning a single bond in which the group attached to the thick end of the wedge "goes into the page". Sign symbolMeaning single bonds, wherein the geometry around the double bond (e.g., E or Z) is undefined. Thus, both options and combinations thereof are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. The thick dots on the carbon atoms indicate that the hydrogen attached to this carbon is out of the plane of the paper.

When the group "R" is depicted as a "floating group" on a ring system, for example, in the formula:

r may replace any hydrogen atom attached to any ring atom, including the depicted, implicit, or well-defined hydrogen, so long as a stable structure is formed. When the group "R" is depicted as a "floating group" on a fused ring system, for example in the formula:

r may replace any hydrogen attached to any ring atom of the fused ring unless otherwise indicated. Alternative hydrogens include those depicted (e.g., hydrogen attached to nitrogen in the formulas above), implicit hydrogens (e.g., hydrogen of the formulas above that are not shown but should be understood to be present), well-defined hydrogens, and their presence as an optional hydrogen depending on the identity of the ring atom (e.g., hydrogen attached to group X when X equals-CH), so long as a stable structure is formed. In the depicted example, R may reside on a 5-or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" immediately following the group "R" enclosed in brackets represents a numerical variable. Unless otherwise indicated, this variable may be 0, 1, 2 or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms of the ring or ring system.

For chemical conversionThe chemical groups and classes of compounds, the number of carbon atoms in a group or class being indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group/class. "C.ltoreq.n" defines the maximum number (n) of carbon atoms that can be in a group/class, the minimum number being as small as possible for the group/class in question, e.g., it will be understood that in the group "alkenyl _(C≤8) "or class" of olefins _(C≤8) The minimum number of carbon atoms in "is two. "alkoxy" with the designation alkoxy having 1 to 10 carbon atoms _(C≤10) "compare". "Cn-n '" defines the minimum number (n) and maximum number (n') of carbon atoms in the group. Thus, "alkyl group _(C2-10) "designate those alkyl groups having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class they modify, and it may or may not be enclosed in brackets, without any change in meaning. Thus, the terms "C5 olefins", "C5-olefins", "olefins _(C5) "and" olefins _C5 "all are synonymous.

When used to modify a compound or chemical group, the term "saturated" means having no carbon-carbon double bonds and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted forms of saturated groups, one or more carbonyl oxygen double bonds or carbon-nitrogen double bonds may be present. And when such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term "saturated" is used to modify a solution of a substance, it means that no more such substance can be dissolved in such solution.

When used without a "substituted" modifier, the term "aliphatic" means that the compound or chemical group so modified is acyclic or cyclic, rather than an aromatic hydrocarbon compound or group. In aliphatic compounds/groups, the carbon atoms may be linked together in a straight chain, branched or non-aromatic ring (alicyclic). The aliphatic compound/group may be saturated, i.e. linked by a single carbon-carbon bond (alkane/alkyl), or unsaturated, having one or more carbon-carbon double bonds (alkene/alkenyl) or having one or more carbon-carbon triple bonds (alkyne/alkynyl).

When used to modify a compound or a chemical group atom, the term "aromatic" means that the compound chemical group contains a ring of planar unsaturated atoms that are stabilized by interactions that form a ring bond.

When used without a "substituted" modifier, the term "alkyl" refers to a monovalent saturated aliphatic radical having a carbon atom as the point of attachment, a straight or branched chain acyclic structure, and no atoms other than carbon and hydrogen. group-CH ₃ (Me)、-CH ₂ CH ₃ (Et)、-CH ₂ CH ₂ CH ₃ (n-Pr or propyl), -CH (CH) ₃ ) ₂ (i-Pr、 ⁱ Pr or isopropyl) -CH ₂ CH ₂ CH ₂ CH ₃ (n-Bu)、-CH(CH ₃ )CH ₂ CH ₃ (sec-butyl) -CH ₂ CH(CH ₃ ) ₂ (isobutyl), -C (CH) ₃ ) ₃ (tert-butyl, t-Bu or ^t Bu) and-CH ₂ C(CH ₃ ) ₃ (neopentyl) is a non-limiting example of an alkyl group. The term "alkanediyl" when used without a "substituted" modifier refers to a divalent saturated aliphatic group having one or two saturated carbon atoms, a straight or branched chain acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen, as one or more attachment points. group-CH ₂ - (methylene) -CH ₂ CH ₂ -、-CH ₂ C(CH ₃ ) ₂ CH ₂ -and-CH ₂ CH ₂ CH ₂ Are non-limiting examples of alkanediyl groups. "alkane" refers to a class of compounds having the formula H-R, wherein R is the term alkyl as defined above. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this. The following groups are non-limiting examples of substituted alkyl groups: -CH ₂ OH、-CH ₂ Cl、-CF ₃ 、-CH ₂ CN、-CH ₂ C(O)OH、-CH ₂ C(O)OCH ₃ 、-CH ₂ C(O)NH ₂ 、-CH ₂ C(O)CH ₃ 、-CH ₂ OCH ₃ 、-CH ₂ OC(O)CH ₃ 、-CH ₂ NH ₂ 、-CH ₂ N(CH ₃ ) ₂ and-CH ₂ CH ₂ Cl. The term "haloalkyl" is a subset of substituted alkyl groups in which the substitution of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or-I) such that no atoms other than carbon, hydrogen, and halogen are present. group-CH ₂ Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which the substitution of hydrogen atoms is limited to fluorine, such that no atoms other than carbon, hydrogen, and fluorine are present. group-CH ₂ F、-CF ₃ and-CH ₂ CF ₃ Is a non-limiting example of a fluoroalkyl group.

When used without a "substituted" modifier, the term "cycloalkyl" refers to a monovalent saturated aliphatic radical having a carbon atom as the point of attachment (the carbon atom forms 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 (CH) ₂ ) ₂ (cyclopropyl), cyclobutyl, cyclopentyl or cyclohexyl (Cy). When used without a "substituted" modifier, the term "cycloalkanediyl" refers to a divalent saturated aliphatic group having two carbon atoms as attachment points, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Radicals (C)Is cycloalkanediylNon-limiting examples of (a) are provided. "cycloalkane" refers to a class of compounds having the formula H-R, wherein R is the term cycloalkyl as defined above. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

The term "alkenyl" when used without a "substituted" modifier refers to a monovalent unsaturated aliphatic radical having a carbon atom as the point of attachment, a straight or branched chain acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include: -ch=ch ₂ (vinyl), -ch=chch ₃ 、-CH＝CHCH ₂ CH ₃ 、-CH ₂ CH＝CH ₂ (allyl) -CH ₂ CH＝CHCH ₃ And-ch=chch=ch ₂ . When used without a "substituted" modifier, the term "alkenediyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as attachment points, a straight or branched chain 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 radicals-CH=CH-, -CH=C (CH ₃ )CH ₂ -、-CH＝CHCH ₂ -and-CH ₂ CH＝CHCH ₂ Are non-limiting examples of alkenediyl groups. It should be noted that while the alkenediyl group is aliphatic, once attached at both ends, this group is not excluded as forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to a class of compounds having the formula H-R, wherein R is the term alkenyl as defined above. Similarly, the terms "terminal olefin" and "alpha-olefin"synonymous and means an olefin having only one carbon-carbon double bond, wherein the bond is part of a vinyl group at the end of the molecule. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this. The groups-ch=chf, -ch=chcl and-ch=chbr are non-limiting examples of substituted alkenyl groups.

The term "alkynyl" when used without a "substituted" modifier refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a straight or branched chain 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 exclude the presence of one or more non-aromatic carbon-carbon double bonds. The radicals-C.ident.CH, -C.ident.CCH ₃ and-CH ₂ C≡CCH ₃ Is a non-limiting example of an alkynyl group. "alkyne" refers to a class of compounds having the formula H-R, wherein R is alkynyl. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

When used without "substituted" modifiersThe term "aryl" refers to a monovalent unsaturated aromatic radical having an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structures, wherein the ring atoms are all carbon, and wherein the atoms making up the radical are free of atoms other than carbon and hydrogen. If 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 or aralkyl groups (carbon number limitation allows) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl) phenyl, -C ₆ H ₄ CH ₂ CH ₃ (ethylphenyl), naphthyl, and monovalent radicals derived from biphenyl. When used without a "substituted" modifier, the term "arenediyl" refers to a divalent aromatic radical having two aromatic carbon atoms as attachment points that form part of one or more six-membered aromatic ring structures in which the ring atoms are both carbon, and in which the atoms making up the monovalent radical have no atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limitation allows) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. The unfused rings may be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number limitation allows). Non-limiting examples of aromatic diyl groups include:

"aromatic hydrocarbon" refers to a class of compounds having the formula H-R, wherein R is the term aryl as defined above. Benzene and toluene are non-limiting examples of aromatic hydrocarbons. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

When used without a "substituted" modifier, the term "aralkyl" refers to a monovalent group-alkanediyl-aryl, wherein the terms alkanediyl and aryl are each used in a manner consistent with the definition provided above. Non-limiting examples include: benzyl (benzyl, bn) and 2-phenyl-ethyl. When the term aralkyl is used with a "substituted" modifier, one or more hydrogen atoms from the alkanediyl and/or aryl groups are independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this. Non-limiting examples of substituted aralkyl groups: (3-chlorophenyl) -methyl and 2-chloro-2-phenyl-ethan-1-yl.

The term "heteroaryl" when used without a "substituted" modifier refers to a monovalent aromatic radical having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the atoms making up the heteroaryl group are free of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl rings may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. If 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 limitation allows) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include: furyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, picolyl, oxazolyl, phenylpyridyl, pyridyl (pyridinyl/pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as an attachment point. When without the "substituted" modifier, the term "heteroarenediyl" refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, said atoms forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the atoms making up the divalent group are free of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. The unfused rings may be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number limitation allows). As used herein, the term does not exclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation allows) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroarene diradicals include:

"heteroarenes" refers to a class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with a "substituted" modifier, one or more hydrogen atoms being independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

When used without a "substituted" modifier, the term "heterocycloalkyl" refers to a monovalent non-aromatic radical having a carbon or nitrogen atom as an attachment point that forms part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the atoms making up the heterocycloalkyl do not have atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. The heterocycloalkyl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen or sulfur. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number limitation allows) attached to a ring or ring system. In addition, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as an attachment point. N-pyrrolidinyl is an example of such a group. When used without a "substituted" modifier, the term "heterocycloalkyl diyl" refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two attachment points, said atoms forming part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein the atoms making up the divalent group are free of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. The unfused rings may be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl (carbon number limitation allows). As used herein, the term does not exclude the presence of one or more alkyl groups (carbon number limitation allows) attached to a ring or ring system. In addition, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyldiyl groups include:

When these terms are used with a "substituted" modifier, one or more hydrogen atoms being independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

The term "acyl" when used without a "substituted" modifier refers to the group-C (O) R, wherein R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl as those terms defined above. The radicals-CHO, -C (O) CH ₃ (acetyl, ac), -C (O) CH ₂ CH ₃ 、-C(O)CH ₂ CH ₂ CH ₃ 、-C(O)CH(CH ₃ ) ₂ 、-C(O)CH(CH ₂ ) ₂ 、-C(O)C ₆ H ₅ 、-C(O)C ₆ H ₄ CH ₃ 、-C(O)CH ₂ C ₆ H ₅ C (O) (imidazolyl) is a non-limiting example of an acyl group. "thioacyl" is defined in a similar manner except that the oxygen atom of the group-C (O) R has been replaced by a sulfur atom-C (S) R. The term "aldehyde" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by a —cho group. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms (including hydrogen atoms directly attached to a carbon atom of a carbonyl or thiocarbonyl group, e.g.If any) have been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this. group-C (O) CH ₂ CF ₃ 、-CO ₂ H (carboxyl) -CO ₂ CH ₃ (methylcarboxyl) -CO ₂ CH ₂ CH ₃ 、-C(O)NH ₂ (carbamoyl) and-CON (CH) ₃ ) ₂ Is a non-limiting example of a substituted acyl group.

The term "alkoxy" when used without a "substituted" modifier refers to the group-OR, where R is the term alkyl as defined above. Non-limiting examples include: -OCH ₃ (methoxy) -OCH ₂ CH ₃ (ethoxy) -OCH ₂ CH ₂ CH ₃ 、-OCH(CH ₃ ) ₂ (isopropoxy), -OC (CH) ₃ ) ₃ (tert-butoxy), -OCH (CH) ₂ ) ₂ -O-cyclopentyl and-O-cyclohexyl. When used without "substituted" modifiers, the terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy" and "acyloxy" refer to groups as defined by-OR, wherein each R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl and acyl. The term "Alkoxydiyl" refers to the divalent group-O-Alkyldiyl-, -O-alkanediyl-O-or-alkanediyl-O-alkanediyl-. When used without a "substituted" modifier, the terms "alkylthio" and "acyl" refer to the group-SR, where R is each alkyl and acyl. The term "alcohol" corresponds to an alkane as defined above wherein at least one hydrogen atom has been replaced by a hydroxyl group. The term "ether" corresponds to an alkane as defined above, which At least one hydrogen atom of which has been replaced by an alkoxy group. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms having been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this.

The term "alkylamino" when used without a "substituted" modifier refers to the group-NHR, where R is the term alkyl as defined above. Non-limiting examples include: -NHCH ₃ and-NHCH ₂ CH ₃ 。

When used without a "substituted" modifier, the term "dialkylamino" refers to the group-NRR ', where R and R ' may be the same or different alkyl groups, or R and R ' may together represent an alkanediyl group. Non-limiting examples of dialkylamino groups include: -N (CH) ₃ ) ₂ and-N (CH) ₃ )(CH ₂ CH ₃ ). When used without "substituted" modifiers, the terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino" and "alkylsulfonylamino" refer to groups as defined by-NHR, where R is each cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy and alkylsulfonyl. Non-limiting examples of arylamino groups are-NHC ₆ H ₅ . The term "alkylaminodiyl" refers to the divalent group-NH-alkanediyl-, -NH-alkanediyl-NH-or-alkanediyl-NH-alkanediyl-. The term "amido (acylamino)" when used without a "substituted" modifier refers to the group-NHR, where R is the term acyl as defined above. Acyl groupNon-limiting examples of amine groups are-NHC (O) CH ₃ . When used without a "substituted" modifier, the term "alkylimino" refers to a divalent group = NR, where R is the term alkyl as defined above. When any of these terms is used with a "substituted" modifier, one or more hydrogen atoms attached to a carbon atom have been independently replaced by-OH-F, -Cl, -Br, -I, -NH ₂ 、-NO ₂ 、-CO ₂ H、-CO ₂ CH ₃ 、-CN、-SH、-OCH ₃ 、-OCH ₂ CH ₃ 、-C(O)CH ₃ 、-NHCH ₃ 、-NHCH ₂ CH ₃ 、-N(CH ₃ ) ₂ 、-C(O)NH ₂ 、-C(O)NHCH ₃ 、-C(O)N(CH ₃ ) ₂ 、-OC(O)CH ₃ 、-NHC(O)CH ₃ 、-S(O) ₂ OH, or-S (O) ₂ NH ₂ Instead of this. group-NHC (O) OCH ₃ and-NHC (O) NHCH ₃ Are non-limiting examples of substituted amide groups.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present application. Generally, the term "about" as used herein, when referring to a measurable value (such as the amount of weight, time, dose, etc.), is meant to encompass a variation of ±20% or ±10% of the specified amount in one example, a variation of ±5% of the specified amount in another example, a variation of ±3% of the specified amount in another example, a variation of ±1% of the specified amount in another example, and a variation of ±0.1% of the specified amount in yet another example, and such a variation is suitable for performing the disclosed method.

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 polymerization level and thus a different molar mass. The average molecular weight may be used to represent the molecular weight of a plurality of polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weights: number average molar mass, weight (mass) average molar mass and Z average molar mass. In the context of the present application, unless otherwise indicated, the average molecular weight represents the number average molar mass or weight average molar mass of the formula. In some embodiments, the average molecular weight is a number average molar mass. In some embodiments, the average molecular weight may be used to describe the PEG component present in the lipid.

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

The term "effective" as that term is used in the specification and/or claims means sufficient to achieve the desired, intended or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the compound that, when administered to a subject or patient to treat a disease, is sufficient to effect such treatment of the disease.

As used herein, the term "IC ₅₀ "means the amount of inhibitor that is 50% of the maximum response obtained. Such quantitative measures indicate how much particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e., enzyme, cell receptor or microorganism) by half.

An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but the atoms differ in three-dimensional configuration.

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

As used herein, the term "assembled" or "assembled" in the context of delivering a payload to one or more target cells generally refers to one or more covalent or non-covalent interactions or associations, e.g., such that a therapeutic or prophylactic agent is complexed with or encapsulated in a lipid composition.

As used herein, the term "lipid composition" generally refers to a composition comprising one or more lipid compounds including, but not limited to, cationic liposome/nucleic acid complexes (lipoplex), liposomes, lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

As used herein, the term "detectable" refers to the occurrence or change in a signal that can be detected either directly or indirectly by observation or by instrumentation. Typically, the detectable response is the occurrence of a signal, wherein the fluorophore is intrinsic fluorescent and does not produce a signal change upon binding to the metal ion or biological compound. Alternatively, the detectable response is an optical response that results in a change in the wavelength profile or the intensity of absorbance or fluorescence or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above. Other detectable responses include, for example, chemiluminescence, phosphorescence, radioisotope radiation, magnetic attraction, and electron density.

As used herein, the term "potent" or "potency" in connection with the delivery of one or more therapeutic agents generally refers to the greater ability of a delivery system (e.g., a lipid composition) to achieve or bring about a desired amount, activity, or effect (such as a desired level of translation, transcription, production, expression, or activity of a protein or gene) of a therapeutic or prophylactic agent in a cell (e.g., a target cell) to any measurable extent (e.g., relative to a reference delivery system). For example, a lipid composition with higher potency may achieve a desired therapeutic effect in a larger population of related cells, within a shorter response time, or for a longer period of time.

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

By "pharmaceutically acceptable salt" is meant a salt of a pharmaceutically acceptable compound of the present application as defined above, and having the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or acid addition salts with organic acids such as 1, 2-ethyldisulfonic acid, 2-hydroxyethylsulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4' -methylenebis (3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo [2.2.2] oct-2-ene-1-carboxylic acid, acetic acid, aliphatic monocarboxylic and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethylsulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, hexadienedicarboxylic acid, o- (4-hydroxybenzoyl) benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, t-butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that may be formed when the acidic proton present is capable of reacting with an inorganic or organic base. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be appreciated that the particular anion or cation forming part of any salt of the present disclosure is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and methods of their preparation and Use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P.H.Stahl & C.G.Wermuth et al, verlag Helvetica Chimica Acta, 2002).

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

"prevention" or "prophylaxis" includes: (1) Inhibiting the onset of a disease in a subject or patient who may be at risk of and/or susceptible to the disease but who has not experienced or displayed any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of the disease in a subject or patient who may be at risk of and/or susceptible to the disease but who has not experienced or displayed any or all of the pathology or symptomatology of the disease.

"repeat units" are the simplest structural entities of certain materials (e.g., frames and/or polymers), whether organic, inorganic, or metal-organic. In the case of polymer chains, the repeating units are continuously linked together along the chain, like the beads of a necklace. For example, in polyethylene- [ -CH ₂ CH ₂ -] _n In which the repeating units are-CH ₂ CH ₂ -. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units linked together. When the value of "n" is undefined or when "n" is absent, it simply specifies the repetition of the formula in brackets as well as the polymeric nature of the material. The concept of repeating units is equally applicable in the case of a connective three-dimensional extension between repeating units, such as in metal organic frameworks, modified polymerizationsAnd thermosetting polymers, etc. In the context of dendrimers or dendrons, the repeating units may also be described as branching units, internal layers, or generations. Similarly, the terminating group may also be described as a surface group.

"stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but in which the atoms differ in three-dimensional configuration. "enantiomers" are stereoisomers of a given compound that mirror each other, as in the left and right hand. "diastereomers" are stereoisomers of the diastereomers of a given compound. Chiral molecules contain chiral centers, also known as stereocenters or stereogenic centers, which are any point in the molecule carrying a group, but not necessarily atoms, such that the interchange of any two groups results in stereoisomers. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, but other atoms may also be stereocenters in organic and inorganic compounds. The molecule may have multiple stereocenters, thereby giving rise to a number of stereoisomers. In compounds where the stereoisomerism is due to a tetrahedral stereogenic centre (e.g. tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2 ⁿ Where n is the number of tetrahedral stereocenters. Molecules with symmetry typically have less than the maximum possible number of stereoisomers. The 50:50 mixture of enantiomers is referred to as the racemic mixture. Alternatively, a mixture of enantiomers may be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers may be resolved or separated using techniques known in the art. It is contemplated that for any stereocenter or chiral axis for which stereochemistry has not been defined, such stereocenter or chiral axis may be present in its R form, S form or as a mixture 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% or less, more preferably 10% or less, even more preferably 5% or less, or most preferably 1% or less of one or more of the other speciesStereoisomers of the compounds.

"treating" or "treating" includes (1) inhibiting the disease (e.g., preventing further development of the pathology and/or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, (2) ameliorating the disease (e.g., reversing the pathology and/or symptoms) in a subject or patient experiencing or exhibiting the pathology or symptoms of the disease, and/or (3) experiencing or exhibiting any measurable reduction in the pathology or symptoms of the disease in a subject or patient.

The above definitions supersede any conflicting definition in any reference incorporated herein by reference. However, the fact that certain terms are defined should not be construed to indicate that any undefined term is undefined. Rather, all terms used are to be considered as descriptive of the present disclosure in terms such that one of ordinary skill can understand the scope and practice of the present application.

Composition and method for producing the same

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 isolated from an ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

Ionizable cationic lipids

In some embodiments of the lipid compositions of the present application, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the cationically ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated at a pH above 8, 9, 10, 11, or 12 and have no charge. The ionizable cationic groups may contain one or more protonatable amines capable of forming cationic groups at physiological pH. The cationically ionizable lipid compound may further comprise one or more lipid components, such as two or more having C ₆ -C ₂₄ Fatty acids of alkyl or alkenyl carbon groups. These lipid groups may be attached by ester linkages or may be further added to the sulfur atom by michael addition. In some embodiments, theseThe compound may be a dendrimer, dendron, polymer, or combination thereof.

In some embodiments of the lipid compositions herein, ionizable cationic lipids refer to lipids and lipid-like molecules having a nitrogen atom with an available charge (pKa). These lipids may be referred to in the literature as cationic lipids. These molecules with amino groups typically have between 2 and 6 hydrophobic chains, often alkyl or alkenyl groups (such as C ₆ -C ₂₄ Alkyl or alkenyl), but may have at least 1 or more than 6 tails. In some embodiments, these cationically ionizable lipids are dendrimers, which are polymers exhibiting regular dendritic branches, formed by sequentially or stepwise adding branching layers to or from a core and characterized by a core, at least one internal branching layer, and a surface branching layer. (see Petar r. Dvornic and Donald a. Tomalia in chem. In Britain,641-645, 8 th 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 repeating units regularly attached to the initiator core, and an external surface attached to the terminal groups of the outermost generation. "dendron" is a dendritic polymer having branches emanating from a focal point that is attached directly or through a linking moiety to a core or that can be attached to a core to form a larger dendritic polymer. In some embodiments, the dendrimer has repeating groups radiating from a central core that doubles with each repeating unit of 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 a compound described herein (e.g., a molecule radiating from a single focus) that has a dendron-like appearance.

While dendrimers are polymers, dendrimers may be more preferred than traditional polymers because they have a controlled structure, a single molecular weight, many and controllable surface functional groups, and traditionally take a spherical configuration after a specific generation is reached. Dendrimers can be prepared by sequential reactions of each repeating unit to produce monodisperse, tree-like and/or generation structured polymer structures. The individual dendrimers consist of a central core molecule with dendritic wedges attached to one or more functional sites on the central core. Depending on the assembly monomer used during preparation, the dendrimer surface layer may have various functional groups disposed thereon, including anionic, cationic, hydrophilic or lipophilic groups.

The physical properties of the core, repeat units and surface or terminating groups can be modulated by changing their functional and/or chemical properties. Some properties that may be altered include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesion. Dendrimers are generally described by the number of repeating units in their generations or branches. Dendrimers consisting of only core molecules are referred to as generation 0, while each successive repeat unit along all branches is generation 1, generation 2, etc., up to a termination or surface group. In some embodiments, half-generations may result from only a first condensation reaction with an amine, but not a second condensation reaction with a thiol.

The preparation of dendrimers requires a level of control of synthesis achieved by a series of stepwise reactions involving the building up of the dendrimer by each successive group. Dendrimer synthesis may be convergent or divergent. In the divergent dendrimer synthesis process, the molecules are assembled from the core to the periphery in a stepwise process that involves attaching one generation to the previous generation and then changing the functional groups for the next reaction stage. Functional group conversion is necessary to prevent uncontrolled polymerization. Such polymerization will result in highly branched molecules that are not monodisperse but are otherwise referred to as hyperbranched polymers. The continued reaction of the dendrimer repeat units results in a globular or spherical molecule due to steric effects until steric overcrowding prevents complete reaction in a particular generation and disrupts the monodispersity of the molecule. Thus, in some embodiments, G1-G10 generation dendrimers are specifically contemplated. In some embodiments, the dendrimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units or any range derivable therein. In some embodiments, the dendrimer used herein is G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) may be increased by decreasing the spacer units in the branched polymer.

In addition, dendrimers have two main chemical environments: the environment created by the specific surface groups on the termination generation; and the interior of the dendritic structure, which can be shielded from the bulk medium and surface groups due to the higher order structure. Because of these different chemical environments, dendrimers have found many different potential uses, including in therapeutic applications.

In some embodiments of the lipid compositions of the present application, the dendrimers or dendrons are assembled using differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers or dendrons may include secondary or tertiary amines and thioethers formed by reacting 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 repeat units may contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendritic polymerization to proceed in only one direction. In other embodiments, the core molecule is a polyamine having a plurality of different dendritic branches, each dendritic branch comprising one or more repeat units. Dendrimers or dendrons are formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms (such as nitrogen atoms). In some embodiments, the terminating group is a lipophilic group (such as a long chain alkyl or alkenyl group). In other embodiments, the terminating group is a long chain haloalkyl or haloalkenyl. In other embodiments, the terminating group is one containing an ionizable group (such as amine (-NH) ₂ ) Or carboxylic acid (-CO) ₂ H) Aliphatic or aromatic groups). In still other embodiments, the terminating group is an aliphatic or aromatic group containing one or more hydrogen bond donors (such as hydroxide groups, amide groups, or esters).

The cationically 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, all chiral, non-corresponding, racemic, epimeric and all geometric isomeric forms of a chemical formula are contemplated unless the specific stereochemistry or isomeric form is specifically indicated. The cationically ionizable lipids can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral center of the cationically ionizable lipid of the present application may have an S or R configuration. Furthermore, it is contemplated that one or more cationically ionizable lipids may exist as a structural isomer. In some embodiments, the compounds have the same formula but different connectivity to the nitrogen atom of the core. Without wishing to be bound by any theory, it is believed that such cationically ionizable lipids are present because the starting monomer reacts first with the primary amine and then statistically with any secondary amine present. Thus, the structural isomer may be present as a mixture of fully reacted primary amine, and then reacted secondary amine.

The chemical formula used to represent the cationically ionizable lipids of the present application will typically show only one of several different tautomers possible. For example, many types of keto 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 of a given formula is depicted and whichever is most common, all tautomers of a given formula are contemplated.

The cationically ionizable lipids of the present application may also have the following advantages: they may be more potent, less toxic, longer acting, more potent, produce fewer side effects, be more readily absorbed and/or have better pharmacokinetic characteristics (e.g., higher oral bioavailability and/or lower clearance) and/or have other useful pharmacological, physical or chemical properties than the compounds known in the art, whether for the indications described herein or otherwise.

In addition, the atoms comprising the cationically ionizable lipids of the present application are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. As a general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ¹³ C and C ¹⁴ C。

It should be appreciated that the particular anion or cation forming part of any salt form of the cationically ionizable lipid provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and methods of their preparation and Use are presented in Handbook of Pharmaceutical Salts:properties, and Use (2002), which are incorporated herein by reference.

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 groups are 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 C ₆ -C ₂₄ Alkyl or alkenyl.

Dendrimers or dendrons of formula (I)

In some embodiments of the lipid composition, the ionizable cationic lipid comprises at least two C ₈ -C ₂₄ An alkyl group. In some embodiments, the ionizable cationic lipid is a dendrimer further defined by the formulaSubstance or dendron:

core-repeating unit-terminating group (D-I)

Wherein the core repeat unit is attached to the repeat unit by removing one or more hydrogen atoms from the core and replacing the atoms with repeat units, and wherein:

the core has the formula:

wherein:

X ₁ is amino or alkylamino _(C≤12) Dialkylamino group _(C≤12) Heterocycloalkyl group _(C≤12) Heteroaryl group _(C≤12) Or a substituted form thereof;

R ₁ is amino, hydroxy or mercapto or alkylamino _(C≤12) Dialkylamino group _(C≤12) Or a substituted form of any of these groups; and is also provided with

a is 1, 2, 3, 4, 5 or 6; or (b)

The core has the formula:

wherein:

X ₂ is N (R) ₅ ) _y ；

R ₅ Is hydrogen or alkyl _(C≤18) Or substituted alkyl _(C≤18) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

y is 0, 1 or 2, provided that the sum of y and z is 3;

R ₂ is amino, hydroxy or mercapto or alkylamino _(C≤12) Dialkylamino group _(C≤12) Or a substituted form of any of these groups;

b is 1, 2, 3, 4, 5 or 6; and is also provided with

z is 1, 2, 3; provided that the sum of z and y is 3; or (b)

The core has the formula:

wherein:

X ₃ is-NR ₆ -, wherein R is ₆ Is hydrogen or alkyl _(C≤8) Or substituted alkyl _(C≤8) -O-or alkylamino-diyl _(C≤8) Alkoxy di-radicals _(C≤8) Aromatic hydrocarbon diradicals _(C≤8) Heteroarene diradicals _(C≤8) Heterocycloalkanediyl _(C≤8) Or a substituted form of any of these groups;

R ₃ and R is ₄ Each independently is amino, hydroxy or mercapto or alkylamino _(C≤12) Dialkylamino group _(C≤12) Or a substituted form of any of these groups; or a group of the formula: -N (R) _f ) _f (CH ₂ CH ₂ N(R _c )) _e R _d 、

Wherein:

e and f are each independently 1, 2 or 3; provided that the sum of e and f is 3;

R _c 、R _d and R is _f Each independently is hydrogen, alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

c and d are each independently 1, 2, 3, 4, 5 or 6; or (b)

The core is alkylamine _(C≤18) Dialkylamines _(C≤36) Heterocycloalkanes _(C≤12) Or a substituted form of any of these groups;

wherein the repeating unit comprises a degradable diacyl and a linker;

the degradable diacyl has the formula:

wherein:

A ₁ and A ₂ Each independently is-O-, -S-or-NR _a -, wherein:

R _a is hydrogen or alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

Y ₃ Is alkanediyl _(C≤12) Alkenediyl radicals _(C≤12) Aromatic hydrocarbon diradicals _(C≤12) Or a substituted form of any of these groups; or a group of the formula:

wherein:

X ₃ and X ₄ Is alkanediyl _(C≤12) Alkenediyl radicals _(C≤12) Aromatic hydrocarbon diradicals _(C≤12) Or a substituted form of any of these groups;

Y ₅ is a covalent bond or an alkanediyl group _(C≤12) Alkenediyl radicals _(C≤12) Aromatic hydrocarbon diradicals _(C≤12) Or a substituted form of any of these groups; and is also provided with

R ₉ Is an alkyl group _(C≤8) Or substituted alkyl _(C≤8) ；

The linker group has the formula:

wherein:

Y ₁ is alkanediyl _(C≤12) Alkenediyl radicals _(C≤12) Aromatic hydrocarbon diradicals _(C≤12) Or a substituted form of any of these groups; and is also provided with

Wherein when the repeating units comprise a linker group, then if n is greater than 1, the linker group comprises an independent degradable diacyl group attached to both the nitrogen and sulfur atoms of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, wherein for each linker group the next repeating unit comprises two degradable diacyl groups attached to the nitrogen atoms of the linker group; and wherein n is the number of linker groups present in the repeating unit; and is also provided with

The terminating group has the formula:

wherein:

Y ₄ is alkanediyl _(C≤18) Or alkanediyl radicals _(C≤18) Wherein the alkyl is a diyl group _(C≤18) One or more hydrogen atoms of which have been replaced by-OH-F, -Cl, -Br, -I, -SH, -OCH ₃ 、-OCH ₂ CH ₃ 、-SCH ₃ or-OC (O) CH ₃ Replacement;

R ₁₀ is hydrogen, carboxyl, hydroxyl, or

Aryl group _(C≤12) Alkylamino group _(C≤12) Dialkylamino group _(C≤12) N-heterocycloalkyl group _(C≤12) 、-C(O)N(R ₁₁ ) Alkyldiyl radicals _(C≤6) -heterocycloalkyl group _(C≤12) (C (O) -alkylamino _(C≤12) (C (O) -dialkylamino) _(C≤12) (C (O) -N-heterocycloalkyl) _(C≤12) Wherein:

R ₁₁ is hydrogen or alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

Wherein the final degradable diacyl group in the chain is attached to a terminating group;

n is 0, 1, 2, 3, 4, 5 or 6;

or a pharmaceutically acceptable salt thereof. In some embodiments, the terminating group is further defined by the formula:

wherein:

Y ₄ is alkanediyl _(C≤18) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₀ Is hydrogen. In some embodiments, a ₁ And A ₂ Each independently is-O-or-NR _a -。

In some embodiments of the dendrimer or dendron of formula (D-I), the core is further defined by the formula:

wherein:

X ₂ is N (R) ₅ ) _y ；

R ₅ Is hydrogen or alkyl _(C≤8) Or substituted alkyl _(C≤18) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

y is 0, 1 or 2, provided that the sum of y and z is 3;

R ₂ is amino, hydroxy or mercapto or alkylamino _(C≤12) Dialkylamino group _(C≤12) Or a substituted form of any of these groups;

b is 1, 2, 3, 4, 5 or 6; and is also provided with

z is 1, 2, 3; provided that the sum of z and y is 3.

In some embodiments of the dendrimer or dendron of formula (D-I), the core is further defined by the formula:

wherein:

X ₃ is-NR ₆ -, wherein R is ₆ Is hydrogen or alkyl _(C≤8) Or substituted alkyl _(C≤8) -O-or alkylamino-diyl _(C≤8) Alkoxy di-radicals _(C≤8) Aromatic hydrocarbon diradicals _(C≤8) Heteroarene diradicals _(C≤8) Heterocycloalkanediyl _(C≤8) Or a substituted form of any of these groups;

R ₃ and R is ₄ Each independently is amino, hydroxy or mercapto or alkylamino _(C≤12) Dialkylamino group _(C≤12) Or a substituted form of any of these groups; or a group of the formula: -N (R) _f ) _f (CH ₂ CH ₂ N(R _c )) _e R _d 、

Wherein:

e and f are each independently 1, 2 or 3; provided that the sum of e and f is 3;

R _c 、R _d and R is _f Each independently is hydrogen, alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

c and d are each independently 1, 2, 3, 4, 5 or 6.

In some embodiments of the dendrimer or dendron of formula (I), the terminating group is represented by the formula:

wherein:

Y ₄ is alkanediyl _(C≤18) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₁₀ Is hydrogen.

In some embodiments of the dendrimer or dendron of formula (D-I), the core is further defined as:

in some embodiments of the dendrimer or dendron of formula (D-I), the degradable diacyl is further defined as:

dendritic polymers of the formula (D-I)In some embodiments of the compound or dendron, the linker is further defined as

Wherein Y is ₁ Is alkanediyl _(C≤8) Or substituted alkanediyl _(C≤8) 。

In some embodiments of the dendrimer or dendron of formula (D-I), the dendrimer or dendron is selected from the group consisting of:

And pharmaceutically acceptable salts thereof.

Dendrimers or dendrons of formula (X)

In some embodiments of the lipid composition, the ionizable cationic lipid is of formula (la)Is a dendrimer or dendron of (i). In some embodiments, the ionizable cationic lipid is a dendrimer or dendron of the formula:

in some embodiments of the lipid composition, the ionizable cationic lipid is a dendrimer or dendron of generation (g) having the structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

(a) The core comprises a structural formula (X _Core(s) )：

Wherein:

q is independently at each occurrence a covalent bond, -O-, -S-, -NR ² -or-CR ^3a R ^3b -；

R ² At each occurrence independently R ^1g or-L ² -NR ^1e R ^1f ；

R ^3a And R is ^3b Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkyl group;

R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f and R is ^1g Each occurrence, if present, is independently at each occurrence a point of attachment to a branch, hydrogen, or optionally substituted (e.g., C ₁ -C ₁₂ ) An alkyl group;

L ⁰ 、L ¹ and L ² Each occurrence is independently selected from the group consisting of covalent bond, alkylene, heteroalkylene, [ alkylene ]]- [ heterocycloalkyl ]]- [ alkylene group ]][ alkylene group ]]- (arylene) - [ alkylene ] ]Heterocycloalkyl, and arylene; or (b)

Alternatively, L ¹ Part of (A) and R ^1c And R is ^1d One of which is formed (e.g., C ₄ -C ₆ ) Heterocycloalkyl (e.g., containing one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur); and is also provided with

x ¹ 0, 1, 2, 3, 4, 5 or 6; and is also provided with

(b) Each of the plurality of (N) branches independently comprises a structural formula (X) _Branching )：

Wherein:

* Indicating the point of attachment of the branch to the core;

g is 1, 2, 3 or 4;

Z＝2 ^(g-1) ；

when g=1, g=0; or when the g is not equal to 1,

(c) Each diacyl independently comprises a structural formulaWherein:

* Indicating the point of attachment of the diacyl group at its proximal end;

* Indicating the point of attachment of the diacyl group at its distal end;

Y ³ independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group;

A ¹ and A ² Each occurrence is independently of the others-O-, -S-or-NR ⁴ -, wherein:

R ⁴ is hydrogen or optionally substituted (e.g., C ₁ -C ₆ ) An alkyl group;

m ¹ and m ² Each occurrence is independently 1, 2, or 3; and is also provided with

R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₈ ) An alkyl group; and is also provided with

(d) Each linker group independently comprises a structural formula

Wherein:

* Indicating the point of attachment of the linker to the proximal diacyl group;

* Indicating the point of attachment of the linker to the distal diacyl group; and is also provided with

Y ₁ Independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group; and is also provided with

(e) Each terminating group is independently selected from optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkyl thiols and optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkenyl thiols.

At X _Core(s) In some embodiments of (2), Q is independently at each occurrence a covalent bond, -O-, -S-, -NR ² -or-CR ^3a R ^3b . At X _Core(s) Q is independently a covalent bond at each occurrence. At X _Core(s) In some embodiments of (2), Q is independently at each occurrence-O-. At X _Core(s) In some embodiments of (2), Q is independently at each occurrence-S-. At X _Core(s) In some embodiments of (2), Q is independently at each occurrence-NR ² And R is ² At each occurrence independently R ^1g or-L ² -NR ^1e R ^1f . At X _Core(s) In some embodiments of (2), Q is independently at each occurrence-CR ^3a R ^3b R ^3a And R is ^3a And R is ^3b Each occurrence is independently hydrogen or optionally substituted alkyl (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ )。

At X _Core(s) In some embodiments of (2), R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently at each occurrence a point of attachment to a branch, hydrogen, or optionally substituted alkyl. At X _Core(s) One of (2)In some embodiments, R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if any, is independently of the point of attachment to the branch, hydrogen. At X _Core(s) In some embodiments of (2), R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently at each occurrence a point of attachment to a branch, optionally substituted alkyl (e.g., C ₁ -C ₁₂ )。

At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence is independently selected from the group consisting of covalent bond, alkylene, heteroalkylene, [ alkylene ]]- [ heterocycloalkyl ]]- [ alkylene group ]][ alkylene group ]]- (arylene) - [ alkylene ]]Heterocycloalkyl and arylene; or alternatively, L ¹ Part of (A) and R ^1c And R is ^1d One of which forms a heterocycloalkyl group (e.g., C ₄ -C ₆ And contains one or two nitrogen atoms and optionally further heteroatoms selected from oxygen and sulfur). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each independently at each occurrence may be a covalent bond. At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each independently at each occurrence may be hydrogen. At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently can be an alkylene group (e.g., C ₁ -C ₁₂ Such as C ₁ -C ₆ Or C ₁ -C ₃ ). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently can be a heteroalkylene (e.g., C ₁ -C ₁₂ Such as C ₁ -C ₈ Or C ₁ -C ₆ ). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently can be a heteroalkylene (e.g., C ₂ -C ₈ Alkylene oxides, such as oligo (ethylene oxide)). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently may be]- [ heterocycloalkyl ]]- [ alkylene group ]][ (e.g. C ₁ -C ₆ ) Alkylene group]- [ (e.g. C) ₄ -C ₆ ) Heterocycloalkyl group]- [ (e.g. C) ₁ -C ₆ ) Alkylene group]. At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently may be]- (arylene) - [ alkylene ]][ (e.g. C ₁ -C ₆ ) Alkylene group]- (arylene) - [ (e.g. C) ₁ -C ₆ ) Alkylene group]. At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently may be]- (arylene) - [ alkylene ]](e.g., [ (e.g., C) ₁ -C ₆ ) Alkylene group]Phenylene- [ (e.g., C) ₁ -C ₆ ) Alkylene group]). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence independently can be a heterocycloalkyl (e.g., C ₄ -C ₆ Heterocycloalkyl). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each independently at each occurrence may be arylene (e.g., phenylene). At X _Core(s) In some embodiments of L ¹ Part of (A) and R ^1c And R is ^1d Forms a heterocycloalkyl group. At X _Core(s) In some embodiments of L ¹ Part of (A) and R ^1c And R is ^1d One of which forms a heterocycloalkyl group (e.g., C ₄ -C ₆ Heterocycloalkyl), and the heterocycloalkyl group may contain one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur.

At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from a covalent bond, C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ Alkylene group, C ₂ -C ₁₂ (e.g., C ₂ -C ₈ ) Alkylene oxides (e.g., oligo (ethylene oxide), such as- (CH) ₂ CH ₂ O) _1-4 -(CH ₂ CH ₂ )-)、[(C ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g.,) And [ (C) ₁ -C ₄ ) Alkylene group]-phenylene- [ (C) ₁ -C ₄ ) Alkylene group](e.g.)>). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ Alkylene) - (C) ₁ -C ₃ alkylene-O) _1-4 -(C ₁ -C ₃ Alkylene) - (C) ₁ -C ₃ Alkylene) -phenylene- (C ₁ -C ₃ Alkylene) -and- (C ₁ -C ₃ Alkylene) -piperazinyl- (C ₁ -C ₃ Alkylene group) -. At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each occurrence is independently C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ An alkylene group). In some embodiments, L ⁰ 、L ¹ And L ² Each occurrence is independently C ₂ -C ₁₂ (e.g., C ₂ -C ₈ ) Alkylene oxides (e.g., - (C) ₁ -C ₃ alkylene-O) _1-4 -(C ₁ -C ₃ Alkylene)). At X _Core(s) In some embodiments of L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from [ (C) ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g., - (C) ₁ -C ₃ Alkylene) -phenylene- (C ₁ -C ₃ Alkylene groupGroup) -) and [ (C) ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g., - (C) ₁ -C ₃ Alkylene) -piperazinyl- (C ₁ -C ₃ Alkylene) -.

At X _Core(s) In some embodiments of (2), x ¹ Is 0, 1, 2, 3, 4, 5 or 6. At X _Core(s) In some embodiments of (2), x ¹ Is 0. At X _Core(s) In some embodiments of (2), x ¹ Is 1. At X _Core(s) In some embodiments of (2), x ¹ Is 2. At X _Core(s) In some embodiments of (2), x ¹ Is 0, 3. At X _Core(s) In some embodiments of (2), x ¹ Is 4. At X _Core(s) In some embodiments of (2), x ¹ Is 5. At X _Core(s) In some embodiments of (2), x ¹ Is 6.

At X _Core(s) In some embodiments of (2), the core comprises the structural formula:(e.g.,). At X _Core(s) In some embodiments of (2), the core comprises the structural formula:At X _Core(s) In some embodiments of (2), the core comprises the structural formula:(e.g., ). At X _Core(s) In some embodiments of (2), the core comprises the structural formula:(e.g.)>). At X _Core(s) In some embodiments of (2), the core comprises the structural formula:At X _Core(s) In some embodiments of (2), the core comprises the structural formula:(e.g.)> ). At X _Core(s) In some embodiments of (2), the core comprises the structural formula:(e.g.)>Such as->). At X _Core(s) In some embodiments of (2), the core comprises the structural formula:wherein Q' is-NR ² -or-CR ^3a R ^3b -；q ¹ And q ² Each independently is 1 or 2. At X _Core(s) In some embodiments of (2), the core comprises the structural formula: (e.g., ). At X _Core(s) In some embodiments of (2), the core comprises the structural formula +.> (e.g., ) Wherein ring a is optionally substituted aryl or optionally substituted (e.g., C ₃ -C ₁₂ Such as C ₃ -C ₅ ) Heteroaryl groups. At X _Core(s) In some embodiments of (2), the core comprises the structural formula +.>

At X _Core(s) The core comprises the structural formula set forth in table 1 and pharmaceutically acceptable salts thereof, wherein the points of attachment of the core to one of the plurality of branches are indicated. In some embodiments, the exemplary cores of table 1 are not limited to the stereoisomers (i.e., enantiomers, diastereomers) listed.

TABLE 1 exemplary core Structure

At X _Core(s) In some embodiments of (2), the core comprises a structural formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein x indicates the point of attachment of the core to one of the plurality of branches or H. In some embodiments, wherein indicates the point of attachment of the core to one of the plurality of branches.

At X _Core(s) In some embodiments of (2), the core has a structureWhere indicates the attachment point or H of the core to one of the branches. In some embodiments, at least 2 branches are attached to the core. In some embodiments, at least 3 branches are attached to the core. In some embodiments, at least 4 branches are attached to the core.

At X _Core(s) In some embodiments of (2), the core has a structure Where indicates the attachment point or H of the core to one of the branches. 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.

In some embodiments, the plurality of (N) branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality of (N) branches comprises at least 3 branches. In some embodiments, the plurality of (N) branches comprises at least 4 branches. In some embodiments, the plurality of (N) branches comprises at least 5 branches.

At X _Branching In some embodiments of (2), g is 1, 2, 3, or 4. At X _Branching In some embodiments of (2), g is 1. At X _Branching In some embodiments of (2), g is 2. At X _Branching In some embodiments of (2), g is 3. At X _Branching In some embodiments of (2), g is 4.

At X _Branching In some embodiments of (2), z=2 ^(g-1) And when g=1, g=0. At X _Branching In some embodiments of (2), z=2 ^(g-1) And when g +.1,

at X _Branching In some embodiments of (2), g=1, g=0, z=1, and each of the plurality of branches comprises a structural formula

In some embodiments of the X branches, g=2, g=1, z=2, and each branch of the plurality of branches comprises a structural formula

In some embodiments of the X branches, g=3, z=4, and each branch of the plurality of branches comprises a structural formula

At X _Branching G=4, g=7, z=8, and each of the plurality of branches comprises a structural formula

In some embodiments, a dendrimer or dendron described herein having the generation (g) =1 has the following structure:

in some embodiments, a dendrimer or dendron described herein having (g) =1 generation has the following structure:

exemplary formulaic formulations of the dendrimers or dendrons described herein for generations 1-4 are shown in table 2. The number of diacyl groups, linker groups and termination groups can be calculated based on g.

TABLE 2 formulation of dendritic polymer or dendron groups based on generation (g)

	g＝1	g＝2	g＝3	g＝4
						Number of diacyl groups	1	1+2＝3	1+2+2 2 ＝7	1+2+2 2 +2 3 ＝15	1+2+…+2 g-1
Number of linker groups	0	1	1+2	1+2+2 2	1+2+…+2 g-2
						Number of terminating groups	1	2	2 2	2 3	2 (g-1)

In some embodiments, the diacyl groups independently comprise the structural formulaIndicating the point of attachment of the diacyl group at its proximal end and indicating the point of attachment of the diacyl group at its distal end.

At X _Branching In some embodiments of the diacyl group of (2), Y ³ Independently at each occurrence is an optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted arylene group. At X _Branching In some embodiments of the diacyl group of (2), Y ³ Independently at each occurrence is an optionally substituted alkylene (e.g., C ₁ -C ₁₂ ). At X _Branching In some embodiments of the diacyl group of (2), Y ³ Independently at each occurrence is an optionally substituted alkenylene group (e.g., C ₁ -C ₁₂ ). At X _Branching In some embodiments of the diacyl group of (2), Y ³ Independently at each occurrence an optionally substituted arylene group (e.g., C ₁ -C ₁₂ )。

At X _Branching In some embodiments of the diacyl group of A ¹ And A ² Each occurrence is independently of the others-O-, -S-or-NR ⁴ -. At X _Branching In some embodiments of the diacyl group of A ¹ And A ² Each occurrence is independently-O-. At X _Branching In some embodiments of the diacyl group of A ¹ And A ² Each occurrence is independently-S-. At X _Branching In some embodiments of the diacyl group of A ¹ And A ² Each occurrence is independently-NR ⁴ -, and R ⁴ Is hydrogen or optionally substituted alkyl (e.g., C ₁ -C ₆ ). At X _Branching In some embodiments of the diacyl group of (2), m ¹ And m ² Each occurrence is independently 1, 2 or 3. At X _Branching In some embodiments of the diacyl group of (2), m ¹ And m ² Each independently at each occurrence is 1. At X _Branching In some embodiments of the diacyl group of (2), m ¹ And m ² Each independently at each occurrence is 2. At X _Branching In some embodiments of the diacyl group of (2), m ¹ And m ² At each timeEach occurrence is independently 3. At X _Branching In some embodiments of the diacyl group of R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or optionally substituted alkyl. At X _Branching In some embodiments of the diacyl group of R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen. At X _Branching In some embodiments of the diacyl group of R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently an optionally substituted (e.g., C ₁ -C ₈ ) An alkyl group.

In some embodiments of the diacyl group, A ¹ is-O-or-NH-. In some embodiments of the diacyl group, A ¹ is-O-. In some embodiments of the diacyl group, A ² is-O-or-NH-. In some embodiments of the diacyl group, A ² is-O-. In some embodiments of the diacyl group, Y ³ Is C ₁ -C ₁₂ (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkylene group.

In some embodiments of the diacyl group, at each occurrence, independently comprises a structural formula(e.g.)>Such as->) And optionally R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or C ₁ -C ₃ An alkyl group.

In some embodiments, the linker groups independently comprise the structural formula* Indicating the joint and proximityAttachment points for pendant diacyl groups, and indicates attachment points of the linker to the distal diacyl group.

At X _Branching In some embodiments of the linker group (if present), Y ₁ Independently at each occurrence is an optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted arylene group. At X _Branching In some embodiments of the linker group (if present), Y ₁ Independently at each occurrence is an optionally substituted alkylene (e.g., C ₁ -C ₁₂ ). At X _Branching In some embodiments of the linker group (if present), Y ₁ Independently at each occurrence is an optionally substituted alkenylene group (e.g., C ₁ -C ₁₂ ). At X _Branching In some embodiments of the linker group (if present), Y ₁ Independently at each occurrence an optionally substituted arylene group (e.g., C ₁ -C ₁₂ )。

At X _Branching In some embodiments of the terminal groups of (a), each terminal group is independently selected from optionally substituted alkyl thiols and optionally substituted alkenyl thiols. At X _Branching In some embodiments of the terminating groups of (a) each terminating group is an optionally substituted alkyl thiol (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ). At X _Branching In some embodiments of the terminating groups of (a) each terminating group is an optionally substituted alkenyl thiol (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ )。

At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ Alkenyl thiols or C ₁ -C ₁₈ An alkyl thiol, and the alkyl or alkenyl moieties are optionally substituted with one or more moieties each independently selected from halogen, C ₆ -C ₁₂ Aryl, C ₁ -C ₁₂ Alkylamino, C ₄ -C ₆ N-heterocycloalkyl, -OH, -C (O) N (C) ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₁ -C ₁₂ Alkyl groupAmino), -C (O) N (C) ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₄ -C ₆ N-heterocycloalkyl), -C (O) - (C) ₁ -C ₁₂ Alkylamino) and-C (O) - (C) ₄ -C ₆ N-heterocycloalkyl), and C of any of the foregoing substituents ₄ -C ₆ The N-heterocycloalkyl moiety optionally being C ₁ -C ₃ Alkyl or C ₁ -C ₃ Hydroxyalkyl substitution.

At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkenyl thiols or C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more moieties each independently selected from halogen, C ₆ -C ₁₂ Aryl (e.g., phenyl), C ₁ -C ₁₂ (e.g., C ₁ -C ₈ ) Alkylamino (e.g., C ₁ -C ₆ Mono-alkylamino (such as-NHCH ₂ CH ₂ CH ₂ CH ₃ ) Or C ₁ -C ₈ Di-alkylamino groups (such as))、C ₄ -C ₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl->N-piperidinyl->N-azepanyl)、-OH、-C(O)OH、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₁ -C ₁₂ Alkylamino (e.g., mono-or di-alkylamino)) (e.g., (-)>)、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₄ -C ₆ N-heterocycloalkyl) (e.g.)>)、-C(O)-(C ₁ -C ₁₂ Alkylamino (e.g., mono-or di-alkylamino)), and-C (O) - (C) ₄ -C ₆ N-heterocycloalkyl) (e.g.)>) Wherein C is a substituent of any of the foregoing substituents ₄ -C ₆ The N-heterocycloalkyl moiety optionally being C ₁ -C ₃ Alkyl or C ₁ -C ₃ Hydroxyalkyl substitution. At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl moiety is optionally substituted with one substituent-OH. At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl moiety is optionally substituted with one selected from C ₁ -C ₁₂ (e.g., C ₁ -C ₈ ) Alkylamino (e.g., C ₁ -C ₆ Mono-alkylamino (such as-NHCH ₂ CH ₂ CH ₂ CH ₃ ) Or C ₁ -C ₈ Di-alkylamino groups (such as) And C) ₄ -C ₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl)N-piperidinyl->N-azepanyl->) Is substituted by a substituent of (a). At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkenyl thiols or C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkyl mercaptans. At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkyl mercaptans.

At X _Branching In some embodiments of the terminating groups of (2), each terminating group is independently a structure set forth in table 3. In some embodiments, a dendrimer or dendron described herein may comprise a terminating group selected from table 3 or a pharmaceutically acceptable salt thereof. In some embodiments, examples of the terminating groups of table 3 are not limited to the stereoisomers (i.e., enantiomers, diastereomers) listed.

TABLE 3 exemplary termination group/peripheral Structure

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

TABLE 4 exemplary ionizable cationic lipid-dendrimers or lipid-dendrons

Other ionizable cationic lipids

In some embodiments of the lipid composition, the cationic lipid comprises the structural formula (D-I'):

wherein:

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; and is also provided with

R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ And R is ⁶ Each independently selected from H, -CH ₂ CH(OH)R ⁷ 、-CH(R ⁷ )CH ₂ OH、-CH ₂ CH ₂ C(＝O)OR ⁷ 、-CH ₂ CH ₂ C(＝O)NHR ⁷ and-CH ₂ R ⁷ Wherein R is ⁷ Independently selected from C ₃ -C ₁₈ Alkyl, C having one C=C double bond ₃ -C ₁₈ Alkenyl, protecting group for amino, -C (=nh) NH ₂ Poly (ethylene glycol) chains and receptor ligands;

provided that R ¹ To R ⁶ At least two moieties of (a) are independently selected from-CH ₂ CH(OH)R ⁷ 、-CH(R ⁷ )CH ₂ OH、-CH ₂ CH ₂ C(＝O)OR ⁷ 、-CH ₂ CH ₂ C(＝O)NHR ⁷ or-CH ₂ R ⁷ Wherein R is ⁷ Independently selected from C ₃ -C ₁₈ Alkyl or C having one C=C double bond ₃ -C ₁₈ Alkenyl groups; and is also provided with

Wherein one or more of the nitrogen atoms indicated in formula (D-I') may be protonated to provide a cationic lipid.

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 is ⁶ Each independently is H or-CH ₂ CH(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 is ⁶ Each independently is H orIn some embodiments of the cationic lipid of formula (D-I'), R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ And R is ⁶ Each independently is H or->In some embodiments of the cationic lipid of formula (D-I'), R ⁷ Is C ₃ -C ₁₈ Alkyl (e.g., C ₆ -C ₁₂ Alkyl).

In some embodiments, the cationic lipid of formula (D-I') is 13,16,20-tris (2-hydroxydodecyl) -13,16,20,23-tetraazacyclopentadecane-11, 25-diol:

in some embodiments, the cationic lipid of formula (D-I') is (11R, 25R) -13,16,20-tris ((R) -2-hydroxydodecyl) -13,16,20,23-tetraazacyclopentadecane-11, 25-diol:

additional cationic lipids that may be used in the present compositions and methods include those described in J.McClellan, M.C.King, cell 2010,141,210-217 and international patent publications WO 2010/144740, WO 2013/14943, WO 2016/118725, WO 2016/118724, WO 2013/063268, WO 2016/205691, WO 2015/184356, WO 2016/004202, WO 2015/199952, WO 2017/004143, WO 2017/075531, WO 2017/117528, WO 2017/049245, WO 2017/173054 and WO 2015/095340, which are incorporated herein by reference for all purposes. Examples of those ionizable cationic lipids include, but are not limited to, those as shown in table 5.

Table 5: exemplary ionizable cationic lipids

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

In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of the ionizable cationic lipid from about 5% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises from about 10% to about 25% by mole of the ionizable cationic lipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of the ionizable cationic lipid from about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of the ionizable cationic lipid from about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of the ionizable cationic lipid from about 20% to about 30%. In some embodiments of the lipid compositions 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% by mole of the ionizable cationic lipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of 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% of the ionizable cationic lipid.

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 a lung, a lung tissue, or a lung cell. As used herein, the term "preferentially deliver" is used to refer to a composition that, when delivered, delivers at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of its amount administered to a target organ (e.g., lung), tissue, or cell.

In some embodiments of the lipid composition, the lipid composition comprises one or more selective organ targeting (SORT) lipids that result in selective delivery of the composition to a specific organ. In some embodiments, the SORT lipid may have two or more C ₆ -C ₂₄ Alkyl or alkenyl chains.

In some embodiments of the lipid composition, the SORT lipid comprises a permanently positively charged moiety. The permanently positively charged moiety may be positively charged at physiological pH such that the SORT lipid comprises a positive charge upon delivery of the polynucleotide 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 or is otherwise complexed with a counterion.

In some embodiments of the lipid composition, the SORT lipid is a permanently cationic lipid (i.e., comprises one or more hydrophobic components and one permanently cationic group). The permanently cationic lipid may contain groups that have a positive charge (irrespective of pH). One permanent cationic group that may be used in the permanent cationic lipid is a quaternary ammonium group. The permanently cationic lipid may comprise the structural formula:wherein:

Y ₁ 、Y ₂ or Y ₃ Each independently is X ₁ C(O)R ₁ Or X ₂ N ⁺ R ₃ R ₄ R ₅ ；

Provided that Y ₁ 、Y ₂ And Y ₃ At least one of them is X ₂ N ⁺ R ₃ R ₄ R ₅ ；

R ₁ Is C ₁ -C ₂₄ Alkyl, C ₁ -C ₂₄ Substituted alkyl, C ₁ -C ₂₄ Alkenyl, C ₁ -C ₂₄ Substituted alkenyl;

X ₁ is O or NR _a Wherein R is _a Is hydrogen, C ₁ -C ₄ Alkyl or C ₁ -C ₄ Substituted alkyl;

X ₂ is C ₁ -C ₆ Alkyldiyl or C ₁ -C ₆ Substituted alkanediyl;

R ₃ 、R ₄ and R is ₅ Each independently is C ₁ -C ₂₄ Alkyl, C ₁ -C ₂₄ Substituted alkyl, C ₁ -C ₂₄ Alkenyl, C ₁ -C ₂₄ Substituted alkenyl; and is also provided with

A ₁ Is of charge equal to X in the compound ₂ N ⁺ R ₃ R ₄ R ₅ Anions in the number of groups.

In some embodiments of the SORT lipid, the permanent cationic SORT lipid has the structural formula:wherein:

R ₆ -R ₉ each independently is C ₁ -C ₂₄ Alkyl, C ₁ -C ₂₄ Substituted alkyl, C ₁ -C ₂₄ Alkenyl, C ₁ -C ₂₄ Substituted alkenyl; provided that R ₆ -R ₉ At least one of which is C ₈ -C ₂₄ A group; and is also provided with

A ₂ ^- Is a monovalent anion.

In some embodiments of the lipid composition, the SORT lipid is an ionizable cationic lipid (i.e., comprises one or more hydrophobic components and one ionizable cationic group). The ionizable positively charged moiety may be positively charged at physiological pH. One ionizable cationic group that can 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:(S-I' a) wherein: />

R ₁ And R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group; and R is ₃ And R is ₃ ' each independently is an alkyl group _(C≤6) Or substituted alkyl _(C≤6) 。

In some embodiments of the lipid composition, the SORT lipid comprises a head group of a specific structure. In some embodiments, the SORT lipid comprises a headgroup having the following structural formula:wherein L is a linker; z is Z ⁺ Is a positively charged moiety and X ^- Is a counter ion. In some embodiments, the linker is a biodegradable linker. The biodegradable linker may be degradable at physiological pH and temperature. The biodegradable linker may be degraded by a protein or enzyme from the subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or a quaternary amine.

In some embodiments of the lipid composition, the SORT lipid has the structural formula:wherein R is ¹ And R is ² Each independently is optionally substituted C ₆ -C ₂₄ Alkyl or optionally substituted C ₆ -C ₂₄ Alkenyl groups.

In some embodiments of the lipid composition, the SORT lipid has the structural formula:

in some embodiments of the lipid composition, the SORT lipid comprises a linker (L). In some embodiments, L isWherein:

p and q are each independently 1, 2 or 3; and is also provided with

R ⁴ Is optionally substituted C ₁ -C ₆ An alkyl group.

In some embodiments of the lipid composition, the SORT lipid has the structural formula:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

R ₄ Is an alkyl group _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

In some embodiments of the lipid composition, the SORT lipid is phosphatidylcholine (e.g., 14:0 epc). In some embodiments, the phosphatidylcholine compound is further defined as:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

In some embodiments of the lipid composition, the SORT lipid is a phosphorylcholine lipid. In some embodiments, the SORT lipid is ethyl phosphorylcholine. For example, ethyl phosphorylcholine may be, but is not limited to, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-ethyl phosphorylcholine, 1, 2-distearoyl-sn-glycero-3-ethyl phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-ethyl phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine, 1, 2-dilauroyl-sn-glycero-3-ethyl phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethyl phosphorylcholine.

In some embodiments of the lipid composition, the SORT lipid has the structural formula:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) ；

X ^- Is a monovalent anion.

For example, but not limited thereto, the SORT lipid of the structural formula of the previous paragraph is 1, 2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., chloride salt).

In some embodiments of the lipid composition, the SORT lipid has the structural formula:(S-II'), wherein:

R ₄ and R is ₄ ' each independently is an alkyl group _(C6-C24) Alkenyl group _(C6-C24) Or a substituted form of either group;

R ₄ "is alkyl _(C≤24) Alkenyl group _(C≤24) Or a substituted form of either group;

R ₄ "' is alkyl _(C1-C8) Alkenyl group _(C2-C8) Or a substituted form of either group; and is also provided with

X ₂ ^- Is a monovalent anion.

For example, but not limited thereto, the SORT lipid of the structural formula of the previous paragraph is Dimethyl Dioctadecyl Ammonium (DDAB) (e.g., bromide salt).

In some embodiments of the lipid composition, the SORT lipid has the structural formula:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently isAlkyl group _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

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:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ is hydrogen or alkyl _(C≤6) Or substituted alkyl _(C≤6) or-Y ₁ -R ₄ Wherein:

Y ₁ is alkanediyl _(C≤6) Or substituted alkanediyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ₄ Is an acyloxy group _(C≤8-24) Or substituted acyloxy groups _(C≤8-24) 。

In some embodiments of the lipid composition, the SORT lipid comprises one or more lipids selected from the group set forth in table 6.

TABLE 6 exemplary SORT lipids

X ^- Is a counter ion (e.g. Cl ^- 、Br ^- Etc

In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid of about 20% to about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid of about 25% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid from about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid of about 20% to about 50%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid of about 30% to about 60%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of SORT lipid of about 25% to about 60%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a mole percent 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% of the SORT lipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent 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% of the SORT lipid. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a mole percent of SORT lipid of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% or a range between any two of the foregoing values (inclusive).

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.

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 may contain one or two long chains (e.g., C ₆ -C ₂₄ ) Alkyl or alkenyl, glycerol or sphingosine, one or two phosphate groups and optionally smallAnd (5) a molecule. The small organic molecule may be an amino acid, a sugar, or an amino substituted alkoxy group (such as choline or ethanolamine). In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein the zwitterionic lipids define lipids and lipid-like molecules having both positive and negative charges.

In some embodiments of the lipid composition, the phospholipid is not ethyl phosphorylcholine.

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

In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 8% to about 23% phospholipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 10% to about 20% phospholipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 15% to about 20% phospholipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 8% to about 15% phospholipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 10% to about 15% phospholipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 12% to about 18% phospholipid. In some embodiments of the lipid compositions of the present application, the lipid composition comprises 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% by mole of phospholipids. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of up to (about) 8%, up to (about) 10%, up to (about) 12%, up to (about) 15%, up to (about) 18%, up to (about) 20%, or up to (about) 23%.

Steroid or steroid derivative

In some embodiments of the lipid compositions of the present application, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, the term "steroid" is a class of compounds having a four-ring 17 carbocyclic ring structure that may further comprise one or more substitutions including alkyl, alkoxy, hydroxy, oxo, acyl, or double bonds between two or more carbon atoms. In one aspect, the ring structure of the steroid comprises three fused cyclohexyl rings and fused cyclopentyl rings, as shown in the formula:in some embodiments, the steroid derivative comprises the above-described ring structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:In some embodiments of the present application, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestanes, the ring structure is further defined by the formula: / >As described above, the cholestane derivative comprises one or more non-alkyl substitutions of the above-mentioned ring system. In some embodiments, the cholestane or cholestane derivative is cholestene or cholestene derivative orSterols or sterol derivatives. In other embodiments, the cholestane or cholestane derivative is both cholestene and sterol or a derivative thereof.

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

In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 15% to about 46% of a steroid or steroid derivative. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of about 20% to about 40% of a steroid or steroid derivative. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of about 25% to about 35% of a steroid or steroid derivative. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of about 30% to about 40% of a steroid or steroid derivative. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of about 20% to about 30% of a steroid or steroid derivative. In some embodiments of the lipid compositions of the present application, the lipid composition comprises 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% of a steroid or steroid derivative in mole percent. In some embodiments of the lipid composition of the present application, the lipid composition comprises a mole percent of up to (about) 15%, up to (about) 20%, up to (about) 25%, up to (about) 30%, up to (about) 35%, up to (about) 40%, up to (about) 45%, or up to (about) 46% of a steroid or steroid derivative.

Polymer conjugated lipids

In some embodiments of the lipid compositions of the present application, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride that also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is one containing one or more C attached to a linker group with a PEG chain ₆ -C ₂₄ Long chain alkyl or alkenyl or C ₆ -C ₂₄ Fatty acid group compounds. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, conjugated PEG ceramides, PEG-modified dialkylamines and PEG-modified 1, 2-diacyloxypropan-3-amines, PEG-modified diacylglycerols, and dialkylglycerols. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, 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 from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The PEG modified molecular weight 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 may be used in the present application are taught by U.S. patent 5,820,873, WO 2010/141069, or U.S. patent 8,450,298, which are incorporated herein by reference.

In some embodiments of the lipid composition of the present application, the PEG lipid has the structural formula:wherein: r is R ₁₂ And R is ₁₃ Each independently is an alkyl group _(C≤24) Alkenyl group _(C≤24) Or any of these groupsA substitution pattern; r is R _e Is hydrogen or alkyl _(C≤8) Or substituted alkyl _(C≤8) The method comprises the steps of carrying out a first treatment on the surface of the And x is 1-250. In some embodiments, R _e Is an alkyl group _(C≤8) Such as methyl. R is R ₁₂ And R is ₁₃ Each independently is an alkyl group _(C≤4-20) . In some embodiments, x is 5 to 250. In one embodiment, x is 5 to 125 or x is 100 to 250. In some embodiments, the PEG lipid is 1, 2-dimyristoyl-sn-glycerol, polyethylene glycol monomethyl ether.

In some embodiments of the lipid composition of the present application, the PEG lipid has the structural formula:wherein: n is n ₁ Is an integer between 1 and 100, and n ₂ And n ₃ Each independently selected from integers between 1 and 29. In some embodiments, n ₁ 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, n ₁ From about 30 to about 50. In some embodiments, n ₂ From 5 to 23. In some embodiments, n ₂ From 11 to about 17. In some embodiments, n ₃ From 5 to 23. In some embodiments, n ₃ From 11 to about 17.

In some embodiments of the lipid composition of the present application, the composition further comprises a molar percentage of PEG lipid of about 4.0 to about 4.6 of the total lipid composition. In some embodiments, the mole percent 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 mole percent is about 1.5 to about 4.0. In some embodiments, the mole percent 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.

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

Pharmaceutical composition

Therapeutic or prophylactic agent

In another aspect, provided herein are pharmaceutical compositions comprising a therapeutic (or prophylactic) agent assembled with a lipid composition as described herein.

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

In some embodiments, the pharmaceutical composition comprises a therapeutic (or prophylactic) agent for treating a lung disease (such as asthma, COPD or lung cancer). In some embodiments, the therapeutic (or prophylactic) agent comprises a steroid (such as prednisone, hydrocortisone, prednisolone, methylprednisolone, or dexamethasone). In some embodiments of the present invention, in some embodiments, the therapeutic agent (or prophylactic agent) comprises albumin-bound paclitaxel, afatinib dimaleate, ai Feini torr (Afinal), afinal Disperz, ai Leti ni (Alexena), aletinib, ainid, orange (Alunbig), alemtuzumab, avastin, bevacizumab, bragg inib, carbamazetinib hydrochloride (Capmatinib Hydrochloride), carboplatin, seritinib, crizotinib, lei Molu monoclonal antibody, darafenib mesylate, dactyltinib, docetaxel, doxorubicin hydrochloride, diminulinb, emtrictinib hydrochloride, erlotinib hydrochloride, everolimus, ganaxoto (Gavreto) gefitinib, ji Tairui (gilotril), gemcitabine, ipilimumab, iressa (Iressa), curettan (Keytruda), lafutilib (lorerena), mejining (Mekinist), methotrexate sodium, cetuximab (Necitumumab), nivolumab, octreotide mesylate, paclitaxel, pemetrexed disodium, platinib (Pralsetinib), ramucirumab (Ramucirumab), certeplatinib (Retevmo), cerpettinib (Selpercatinib), tabecta, tyloxapol (Tafinlar), tyloxapol (tagriso), qu Meiti nim dimethyl sulfoxide, polyose (Vizimpro), vinblastic tartrate Rebaudibin, racetam (Xalkori), yiwoy (Yervoy), zirabev, prandial (Zykadia), carboplatin, gemcitabine-cisplatin, ai Feini torr, alemtuzumab, dimaruzumab, etoposide phosphate (etoposis), etoposide (Etoposide), and mevinin (Hycamtin), inflifen (Imfinzi), cocoa, lu Bike-substituted (lurbinectein), methotrexate sodium, nivolumab, ohio (Opdivo), pemuzumab, termacy (tecentiq), topotecan hydrochloride, terrensu (Trexall), or Zepzelca. Other non-limiting examples of therapeutic (or prophylactic) agents comprising a compound include small molecules selected from the group consisting of: 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetophenylsulfonyl cyclohexaurea, acitretin, atorvastatin, adenine, adenosine, ala Qu Shaxing, albendazole, salbutamol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alretinate, dienbutamol, allopurinol, all-trans-retinoic acid (ATA), alopram, alprazolam, alprenolol, altretamine, amifostine, amiloride, amiluminol, amitriptolide, amiodarone HCl, amitriptyline, amlodipine, isobarbital, amolaquinine, amoxapine, amphetamine, amphotericin B, amitraz ampicillin, amprenavir, amsacrine, amyl nitrate, isopentobutyric, anastrozole, amirinone, anthracene, anthracyclines, aprepitant, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropinothioprine, auranofin, azacitidine, azapropine, azathioprine, azamidete, azithromycin, aztreonam, chlorpheniramine, barbital, live BCG, beclamide, beclomethasone, benfotiazine, benazepril (benazezepril), benidipine, benoride, benazepam, benzamide, benzanthracene, benzathine, benzosoxazole, benzonidazole, benzodiazepine Benzoic acid, benphenning hydroxynaphthoate,Betamethasone, bevacizumab (avastin), betasalbuta, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bleomycin, bortezomib, brinzolamide, diazepam, bromocriptine mesylate, bromoperidol, brotizolam, budesonide, bumetanide, bupropion, busulfan, butamol, ambroxol, butyl amino, butenafine HCl, butabarbital, butamol, butoconazole nitrate, butyl p-hydroxybenzoate, caffeine, calcitol, calcipotriol, calcitriol, kaptone, candidazole, camphor camptothecine, camptothecine analogues, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, carbofuran, carboplatin, carbobromourea, carbimazole, carmustine, cefamandole, cefazolin, ceftizoline, cefixime, cefuroxime axetil (cefuroxime axetil), celecoxib, cefradine, cerivastatin, cetirizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, chlormezothiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil HCl, chlorpromazine, chlorsulfoprourea, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol >Cilostazol, cimetidine, cinnarizine, cinnoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clomazone fumarate, clioquinol, clobazate, clofarabine, clofazimine, antomone citrate, chlorimipram, clonazepam, clopidogrel, chlorthiazepam, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estrogens, corticosterone, cortisone acetate, cyprodide, cyclohexabarbital, cyclobenzaprine, cyclobutane-spirobarbital salt, cyclohexane-spirobarbital salt, cyclophosphamide, cyclopropane-barbital salt, serine,Cyclosporin, cyproheptadine, cytarabine, cytosine, dacarbazine, actinomycin D, danazol, danthron, dantrolene sodium, dapsone, dapoxetine alpha, dapodipine, daunomycin, decoquinate, dehydroepiandrosterone, delavirdine, dimegycline, dilniinterleukin (denieukin), deoxycorticosterone, desoxymethasone (desoxymethyl), dexamethasone, dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexpropidium, dexpropoxyphene, heroin, diatrizoic acid, diazepam, dichlorophenone, 2, 4-D propionic acid, diclofenac, biscoumarin, desipramine, diflunisal, de Ji Tuoxin, digoxin, dihydrocodeine, dihydroequinestrone, dihydromethanesulfonate, diiodoquinoline, diltiazel, dichloroniter (diloxamide furoate), theanine, temozolomide, moxidec dinitolmide, diosgenin, diphenoxylate HCl, biphenyl, dipyridamole, dirithromycin, propidium, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin HCl, doxorubicin, doxycycline, droxiranolone propionate, norfloxacin, asthma, echinocandins, econazole nitrate, efavirenz, ellipticine, enalapril, enmomab, methimazole, epinephrine, epipodophyllotoxin derivatives, epirubicin, alfazotine, irbesartan (epothilan), equilin, mestrane, calciferol, ergotamine, erlotinib, erythromycin, estradiol, estramustine, estriol, estramustine, ethambutol, propargyl, ethionamine, ethionamide, prizepine, pri35 HCl, epothilone, ethionamide, and other drugs, ethyl-4-aminobenzoate (benzocaine), ethyl p-hydroxybenzoate, ethinyl estradiol, etodolac, etomidate, etoposide, itrate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, cyromazine, fenbuconazole, fenfluramine, fenofibrate, fenoldopam, fenoprofen calcium, fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine, fegrid, finasteride, flucarbamide acetate (flecamide acetate), floxuridine, fludarabine, fluconazole, flucytosine, fludioxonil, fludrocortisone Pine, fludrocortisone acetate, flufenamic acid, fluanidone (fluanidone), flunarizine HCl, flunisolide, flunitrazepam, flucorolone, flubenuron (fluometreron), fluorene, fluorouracil, fluxion HCl, fluoxytestosterone, trifluothioxanthecanoate (flupenthixol decanoate), flubentazone decanoate (fluphenthixol decanoate), fluazepam, flurbiprofen, fluticasone propionate, fluvastatin, folic acid, fosinopril (fosinopril), fosantoin sodium, frotriptan, furben-zene acid, fulvestrant, furazolidone, gabapentine, G-BHC (lindan), gefitinib, gemcitabine, gemfibrozil, gemtuzumab, glazin, glibenclamide, glimepiride, glipizide, gliflozin, glycon, glycerol trinitrate (nitroglycerin), goserelin, dapaglifloxacin acetate; griseofulvin, guaifenesin, guanabenz acetate, guanine, halofantrine HCl, haloperidol, hydrochlorothiazide, pimobarbital, heroin, hesperetin, hexachlorobenzene, hexabarbital, histrelin acetate, hydrocortisone, hydroflumothiazide, hydroxyurea, hyoscyamine, hypoxanthine, temozolomide, ibuprofen, idarubicin, idazophosamide, dihydroequilin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interferon alpha-2 a, interferon alpha-2 b, idamide, iofenamic acid, iprodione, irbesartan, irinotecan, epothilone, isocarboxazole, isocarboxazid, isoconazole, isoguanine, isoniazide, isoprobarbide, isoproturon, isosorbide dinitrate, isosorbide mononitrate, isradipine, itraconazole (Itra), ivermectin (ivermectin), ketoconazole, ketoprofen, ketorolac, khellin, labetalol, lamivudine, lamotrigine, digitalis C (lanatoside C), lansoprazole (lanosunazole), L-DOPA, leflunomide, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, levofloxacin, lidocaine, linuron, lisinopril, lomefloxacin, cyclohexanimide, loperamide, loratadine, lorazepam, lorefloxacin, lomefloxacin, lovastatin Statin, ergotoethylurea maleate, maprotiline HCl, mazindole (mazzinol), mebendazole, chlorphenazine HCl, meclofenamic acid, meddazepam, megigiosine, medroxyprogesterone acetate, mefenamic acid, mefloquine HCl, megestrol acetate, melphalan, bromomeprate (mepenzolate bromide), meprobamate, mepropanol, mercaptopurine, mesalazine, mesna, mebendazole ethinyl methyl ether, methadone, mequinone (methaqualone), methocarbamol, methoprene (methoin), methotrexate, methoxalin, methoxamine, mechlothiazide, methylphenidate, methylparaben, methylprednisolone, methyltestosterone, norethindrone, mecigergot maleate, metoclopramide, metolazone, metoprolol, metronidazole Mizilin HCl, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mycophenolate mofetil, molindone (monodone), montelukast, morphine, moxifloxacin HCl, nabumetone, nadolol, nanobuphine, nalidixic acid, nandrolone, naphthacene, naproxen, naratriptan HCl, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotinamide, nicotinic acid, neoanticoagulants (nicoumalone), nifedipine, nilutamide, nimodipine, nimorazole, nisoldipine, nitrozepam, nitrofurantoin, furfurazone, nifurazodone, nifedipine, norethimide, norfumerozolomab (norethidone), norethindrone, norgestrel, progesterone, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, omeprazole, omuconazole, ondansetron HCl, epleril, ornidazole, oxaliplatin, olo Sha Ni quine, oxepizin (oxantelbonate), oxaprozin, oxamide, norhydroxy-diazepine, oxcarbazepine, oxfendazole, oxicam, oxyphenbutazone, oxybenzylamine HCl, paclitaxel, palifemine, pamidronate, para-aminosalicylic acid, pantoprazole, methoprene, paroxetine HCl, pegasase, peginase, pefeglastim (peigpost), gfmetrexed disodium, penicillamine, quaternary season Pentatetrol tetranitrate, pentazocine, pentobarbital, pentobatin, pentobalamin perphenazine, perphenazine pimidamide, perylene, phenylacetyl urea, phenacetin, phenanthrene, phenylindene dione, phenobarbital, phenobarbitone, phenolphthalein, phenoxybenzylamine, phenoxazine, phenobarbital, phenoxazine phenoxybenzamine HCl, phenoxymethylpenicillin, benzoin, phenylbutazone, phenytoin, pindolol, pioglitazone, pipobromine, piroxicam, benzothiadiazine maleate, platinum compounds, plicamycin, polyenes, polymyxin B, porphin sodium (porfimerodium), posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel prazosin, prazosin HCl, prednisolone, prednisone, primidone, protuberant, probenecid, probucol, procarbazine, progesterone, proguanil HCl, promazine, propofol, propoxur, propranolol (propranolol), propyl p-hydroxybenzoate, propylthiouracil, prostaglandin, pseudoephedrine, pteridine-2-methyl-thiol, pteridine-2-thiol, pteridine-4-methyl-thiol, pteridine-4-thiol, pteridine-7-methyl-thiol, pteridine-7-thiol, thiopyrimidine pamoate, pyrazinamide, pyrene, pyrimidine, quetiapine, quinine, quinapril, quinidine sulfate, quinine sulfate, sodium rabeprazole, ranitidine HCl, labyrinase, raffmonazole, repaglinide, bicyclooctabarbital, reserpine, retinoids, rifabutin (rifabutin), rifampin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib, ropinirole HCl, rosiglitazone, saccharin, albuterol, salicylamide, salicylic acid, saquinavir, saxitin sec-bupivamidine, secobarbital, sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone, androsanolone, stavudine, diethylstilbestrol, streptozotocin, strychnine, sulconazole nitrate, sulfacetamide, sulfadiazine, sulfamethazine, sulfadimidine, and pharmaceutical compositions Sulfamethoxazole, sulfa, sulfathiazole, sulindac, sulfabenzoyl, sulfacetamide, sulfadiazine, sulfadoxine, sulfaisoxazole (sulphafurazole), sulfamethyidine (sulphamerazine), sulfamethoxazole (sulpha-methoxazole), sulfapyridine, sulfasalazine (sulphasalazine), benzenesulfonzolone, sulpiride, shu Saiqin, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, tabebitude, tamoxifen citrate, tamulosin (tamulosin), bexarotene, taxane, tazarotene, telmisartan, hydroxy diazine, temozoline, teniposide, tenoxicam, terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole, terfenadine, lactone (stonone), ketone, tetracycline, tetrahydrocannabinol, tetraoxyphenol thalidomide, thebaine, theobromine, theophylline, thiabendazole, thiamphenicol, thioguanine, thioridazine, thiotepa, ethylpental, thymine, tiagabine HCl, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine HCl, tolasulfurea, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, retinoic acid, triamcinolone, triazosin, triazolam, triazoline, trifluoperazine, trimethoprim maleate, benzophenanthrene, troglitazone, tromethamine, topiramate, temabane, ubiquinone (coenzyme Q10), undecanoic acid, uracil nitrogen mustard (uracil) uric acid, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin, pentobarbital (vinbarbital), vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, or zopiclone.

Polynucleotide

In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent assembled with the lipid composition comprises one or more polynucleotides. The scope of the present application is not limited to any particular polynucleotide source, sequence or type; however, one of ordinary skill in the art can readily identify related homologs of various other sources of polynucleotides, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, apes, chimpanzees, apes, baboons, cows, pigs, horses, sheep, cats, and other species). It is contemplated that polynucleotides as used herein may comprise sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, a sequence has at least about 50%, typically at least about 60%, more typically about 70%, most typically about 80%, preferably at least about 90% and most preferably about 95% nucleotides identical to the nucleotide sequence of a naturally occurring sequence. In another embodiment, the polynucleotide comprises a nucleic acid sequence that is complementary to a naturally occurring sequence or is complementary to 75%, 80%, 85%, 90%, 95% and 100% of a naturally occurring sequence. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer are contemplated herein.

In some embodiments, polynucleotides as used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. However, in a preferred embodiment, the polynucleotide will comprise complementary DNA (cDNA). cDNA plus a natural intron or an intron derived from another gene is also contemplated; such engineered molecules are sometimes referred to as "minigenes". At a minimum, these and other nucleic acids of the present application can be used as molecular weight standards in gel electrophoresis, for example. The term "cDNA" is intended to mean DNA prepared using messenger RNA (mRNA) as a template. In contrast to genomic DNA or DNA polymerized from genomic, unprocessed or partially processed RNA templates, the advantage of using cDNA is that cDNA mainly contains the coding sequence for the corresponding protein. It may sometimes be preferable to have complete or partial genomic sequences, such as where non-coding regions are required for optimal expression or where non-coding regions (such as introns) are targeted in an antisense strategy.

In some embodiments, the polynucleotide comprises one or more segments comprising small interfering ribonucleic acid (siRNA), short hairpin RNA (shRNA), microribonucleic acid (miRNA), primary microribonucleic acid (pri-miRNA), long non-coding RNA (lncRNA), messenger ribonucleic acid (mRNA), regularly spaced clustered short palindromic repeats (CRISPR) -associated nucleic acid, CRISPR-RNA (crRNA), single guide ribonucleic acid (sgRNA), transactivation CRISPR ribonucleic acid (tracrRNA), plasmid deoxyribonucleic acid (pDNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide (ASO), antisense ribonucleic acid (RNA), guide ribonucleic acid, deoxyribonucleic acid (DNA), double-stranded deoxyribonucleic acid (dsDNA), single-stranded deoxyribonucleic acid (ssDNA), single-stranded ribonucleic acid (ssRNA), or double-stranded ribonucleic acid (dsRNA). In some embodiments, the polynucleotide encodes at least one therapeutic (or prophylactic) agent described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide, such as guide RNA (gRNA) or guide DNA (gDNA), for complexing with a guide RNA-guided nuclease described herein. In some embodiments, the polynucleotide encodes at least one heterologous nuclease directed by the directing polynucleotide. The nuclease may be an endonuclease. Non-limiting examples of guide polynucleotide directed heterologous endonucleases can be selected from 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; a recombinase; a invertase; a transposase; argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), eukaryotic Argonaute (eAgo), and halophil griseum Argonaute (Natronobacterium gregoryi Argonaute) (NgAgo)); adenosine Deaminase (ADAR) acting on RNA; CIRT, PUF, homing endonuclease or any functional fragment thereof, any derivative thereof; any variant thereof; and any fragments thereof.

Some embodiments of the therapeutic (or prophylactic) agents provided herein include a heterologous polypeptide comprising an actuation moiety. The actuation portion may be configured to 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 comprise a heterologous polynucleotide encoding an actuating moiety. The actuation portion may be configured to complex with a target polynucleotide corresponding to a target gene. The heterologous polynucleotide may encode a guide polynucleotide configured to direct the actuation portion to the target polynucleotide. The actuating moiety may comprise a heterologous endonuclease or fragment thereof (e.g., directed by a guide polynucleotide to specifically bind a target polynucleotide). The heterologous endonuclease may be (1) part of a Ribonucleoprotein (RNP) and (2) complexed with a guide polynucleotide. The heterologous endonuclease may be part of a regularly-spaced clustered short palindromic repeats (CRISPR)/CRISPR associated (Cas) protein complex. The heterologous endonuclease may be a regularly spaced clustered short palindromic repeats (CRISPR) -associated (Cas) endonuclease. The heterologous endonuclease may comprise an inactivated endonuclease. The inactivated endonuclease may be fused to a regulatory portion. The regulatory portion may comprise a transcriptional activator, a transcriptional repressor, an epigenetic modifier, or a fragment thereof.

In some embodiments, the polynucleotide encodes at least one heterologous endonuclease directed by a guide polynucleotide, such as a guide RNA (gRNA) or a guide DNA (gDNA). In some embodiments, the polynucleotide encodes at least one guide polynucleotide and at least one heterologous endonuclease, wherein the guide polynucleotide can complex with the at least one heterologous endonuclease and direct the at least one heterologous endonuclease to cleave a genetic locus of any of the genes described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide-directed heterologous endonuclease, such as Cas9, cas12, cas13, cpf1 (or Cas12 a), C2C1, C2 (or Cas13 a), cas13b, cas13C, cas13d, cas14, C2C3, casl, caslB, cas2, cas3, cas4, cas5e (CasD), cas6e, cas6f, cas7, cas8a, cas8al, cas8a2, cas8b, cas8C, csnl, csxl2, cas10d, caslO, caslOd, casF, casG, casH, csyl, csy2, csy3, cas Csel (CasA), cse2 (CasB), cse3 (CasE), cse4 (CasC), cscl, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmrl, cmr3, cmr4, cmr5, cmr6, csbl, csb2, csb3, csxl7, csxl4, csxlO, csxl6, csaX, csx3, csxl5, csfl, csf2, csf3, csf4, or Cul966; any derivative thereof; any variant thereof; or any fragment thereof. In some embodiments, cas13 may include, but is not limited to, cas13a, cas13b, cas13c, and Cas13d (e.g., casRx).

In some embodiments, the heterologous endonuclease comprises an inactivated endonuclease, optionally fused to a regulatory portion, such as an epigenetic modifier for remodeling of the epigenetic genome that mediates expression of the selected gene of interest. In some cases, the epigenetic modifier may include a methyltransferase, a demethylase, a dismutase, an alkylating enzyme, a depurinase, an oxidase, a photocleavable enzyme, an integrase, a transposase, a recombinase, a polymerase, a ligase, a helicase, a glycosylase, an acetyltransferase, a deacetylase, a kinase, a phosphatase, a ubiquitin activating enzyme, a ubiquitin conjugating enzyme, a ubiquitin ligase, a desubiquitinase, an adenylate forming enzyme, an AMP-transferase (AMPylator), a desamp-transferase, a SUMO-transferase, a dessumo-transferase, a ribosyl enzyme, a N-myristoyl transferase, a chromatin-remodelling enzyme, a protease, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a synthase, a synthetase, or a desmyristoyl-transferase. In some cases, the epigenetic modifier can comprise one or more selected from the group consisting of: p300, TET1, LSD1, HDAC8, HDAC4, HDAC11, HDT1, SIRT3, HST2, cobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV H1, DIM5, KYP, SUVR4, set1, SETD8, and TgSET8.

In some embodiments, the polynucleotide encodes a guide polynucleotide that is at least partially complementary to a genomic region of a gene, such as a guide RNA (gRNA) or a guide DNA (gDNA), wherein upon binding of the guide polynucleotide to the gene, the guide polynucleotide recruits a nuclease that the guide polynucleotide directs to cleave and genetically modify the region. Examples of genes that can be modified by a nucleic acid guided by a guide polynucleotide include CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

In some embodiments, the polynucleotide comprises or encodes at least one mRNA that, upon expression, restores function of a defective gene in a subject treated by a pharmaceutical composition described herein.

In some embodiments, a 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 binding of a guide polynucleotide, such as guide RNA (gRNA) or guide DNA (gDNA), to a complementary genomic locus (e.g., a genomic locus of any of the genes described herein). In some embodiments, the at least one chemical modification increases resistance to nuclease digestion when the polynucleotide is subsequently administered to a subject in need thereof. In some embodiments, the at least one chemical modification reduces immunogenicity when the polynucleotide is subsequently administered to a subject in need thereof. In some embodiments, the at least one chemical modification stabilizes a scaffold (such as a tRNA scaffold). Such chemical modifications may have desirable properties, such as increased resistance to nuclease digestion or increased binding affinity to a target genomic locus relative to a polynucleotide without the at least one chemical modification.

In some embodiments, the at least one chemical modification comprises modification of a sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, including but not limited to substituents at the 2 'and/or 5' positions. Examples of suitable sugar substituents at the 2' -position include, but are not limited to: 2'-F, 2' -OCH ₃ ("OMe" or "O-methyl") and 2' -O (CH) ₂ ) ₂ OCH ₃ ("MOE"). In certain embodiments, the sugar substituent at the 2' position is selected from allyl, amino, azido, thio, O-allyl, O- -C ₁ -C ₁₀ Alkyl, O- -C ₁ -C ₁₀ Substituted alkyl;OCF ₃ 、O(CH ₂ ) ₂ SCH ₃ 、O(CH ₂ ) ₂ --O--N(R _m )(R _n ) And O- -CH ₂ --C(＝O)--N(R _m )(R _n ) Wherein each Rm and Rn is independently H or substituted or unsubstituted C ₁ -C ₁₀ An alkyl group. 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 saccharide comprises more than one non-bridging saccharide substituent, e.g., a T-F-5' -methyl saccharide moiety.

Nucleosides comprising 2 '-substituted sugar moieties are referred to as 2' -substituted nucleosides. In some embodiments, the 2 '-substituted nucleoside comprises a 2' -substituent selected from the group consisting of: halo, allyl, amino, azido, SH, CN, OCN, CF ₃ 、OCF ₃ O, S or N (R) _m ) -an alkyl group; o, S or N (R) _m ) -an alkenyl group; o, S or N (R) _m ) -alkynyl; O-alkylene-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH) ₂ ) ₂ SCH ₃ 、O(CH ₂ ) ₂ --O--N(R _m )(R _n ) Or O- -CH ₂ --C(＝O)--N(R _m )(R _n ) Wherein each R is _m And R is _n Independently H, an amino protecting group or a substituted or unsubstituted C ₁ -C ₁₀ An alkyl group. These 2' -substituents may be further substituted with one or more substituents independently selected from hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO ₂ ) Thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl substituents.

In some embodiments, the 2 '-substituted nucleoside comprises a 2' -substituent selected from the group consisting of: F. NH (NH) ₂ 、N ₃ 、OCF ₃ 、O--CH ₃ 、O(CH ₂ ) ₃ NH ₂ 、CH ₂ —CH＝CH ₂ 、O--CH ₂ —CH＝CH ₂ 、OCH ₂ CH ₂ OCH ₃ 、O(CH ₂ ) ₂ SCH ₃ 、O--(CH ₂ ) ₂ --O--N(R _m )(R _n )、O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ And N-substituted acetamides (O- -CH) ₂ --C(＝O)--N(R _m )(R _n ) Wherein each R is _m And R is _n Independently H, an amino protecting group or a substituted or unsubstituted C ₁ -C ₁₀ An alkyl group.

In some embodiments, the 2 '-substituted nucleoside comprises a sugar moiety comprising a 2' -substituent selected from the group consisting of: F. OCF (optical fiber) ₃ 、O--CH ₃ 、OCH ₂ CH ₂ OCH ₃ 、O(CH ₂ ) ₂ SCH ₃ 、O(CH ₂ ) ₂ --O--N(CH ₃ ) ₂ 、--O(CH ₂ ) ₂ O(CH ₂ ) ₂ N(CH ₃ ) ₂ And O- -CH ₂ --C(＝O)--N(H)CH ₃ 。

In some embodiments, the 2 '-substituted nucleoside comprises a sugar moiety comprising a 2' -substituent selected from the group consisting of: F. o- -CH ₃ And OCH ₂ CH ₂ OCH ₃ 。

Some modified sugar moieties contain bridging sugar substituents that form a second ring, thereby producing a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety comprises a bridge between the 4 'and 2' furanose ring atoms. Examples of such 4 'to 2' sugar substituents include, but are not limited to: - - [ 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'-CH ₂ -2'、4'-(CH ₂ ) ₂ -2'、4'-(CH ₂ )--O-2'(LNA)；4'-(CH ₂ )--S-2'；4'-(CH ₂ ) ₂ --O-2'(ENA)；4'-CH(CH ₃ ) - -O-2 '(cEt) and 4' -CH (CH) ₂ OCH ₃ ) -O-2' and analogs thereof; 4' -C (CH) ₃ )(CH ₃ ) -O-2' and analogs thereof; 4' -CH ₂ --N(OCH ₃ ) -2' and analogues thereof; 4' -CH ₂ --O--N(CH ₃ )-2'；4'-CH ₂ - -O- -N (R) -2 'and 4' -CH ₂ -N (R) -O-2' -, wherein each R is independently H, a protecting group or C ₁ -C ₁₂ An alkyl group; 4' -CH ₂ -N (R) -O-2' wherein R is H, C ₁ -C ₁₂ Alkyl or a protecting group; 4' -CH ₂ --C(H)(CH ₃ ) -2'; and 4' -CH ₂ --C(＝CH ₂ ) -2' and analogues thereof.

In some embodiments, such 4 'to 2' bridges independently comprise 1 to 4 linking groups independently selected from the group consisting of: - - [ 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 ) - - -; wherein: x is 0, 1 or 2; n is 1, 2, 3 or 4; each R _a And R is _b Independently H, a protecting group, hydroxy, C ₁ -C ₁₂ Alkyl, substituted C ₁ -C ₁₂ Alkyl, C ₂ -C ₁₂ Alkenyl, substituted C ₂ -C ₁₂ Alkenyl, C ₂ -C ₁₂ Alkynyl, substituted C ₂ -C ₁₂ Alkynyl, C ₅ -C ₂₀ Aryl, substituted C ₅ -C ₂₀ Aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C ₅ -C ₇ Alicyclic group, substituted C ₅ -C ₇ Alicyclic, halogen, OJ ₁ 、NJ ₁ J ₂ 、SJ ₁ 、N ₃ 、COOJ ₁ Acyl (C (=o) -H), substituted acyl, CN, sulfonyl (S (=o) ₂ -J ₁ ) Or synergistic sulfone (S (=O) -J) ₁ ) The method comprises the steps of carrying out a first treatment on the surface of the And each J ₁ And J ₂ H, C independently ₁ -C ₁₂ Alkyl, substituted C ₁ -C ₁₂ Alkyl, C ₂ -C ₁₂ Alkenyl, substituted C ₂ -C ₁₂ Alkenyl, C ₂ -C ₁₂ Alkynyl, substituted C ₂ -C ₁₂ Alkynyl, C ₅ -C ₂₀ Aryl, substituted C ₅ -C ₂₀ Aryl, acyl (C (=o) -H), substituted acyl, heterocyclyl, substituted heterocyclyl, C ₁ -C ₁₂ Aminoalkyl, substitutedC ₁ -C ₁₂ Aminoalkyl or a protecting group.

Nucleosides comprising a bicyclic sugar moiety are known as bicyclic nucleosides or BNA. Bicyclic nucleosides include, but are not limited to, (A) alpha-L-methyleneoxy (4' -CH) ₂ -O-2 ') BNA, (B) beta-D-methyleneoxy (4' -CH) ₂ - -O-2 ') BNA (also known as locked nucleic acid or LNA), (C) ethyleneoxy (4' - (CH) ₂ ) ₂ - -O-2 ') BNA, (D) aminooxy (4' -CH) ₂ -O- -N (R) -2 ') BNA, (E) oxyamino (4' -CH) ₂ -N (R) -O-2 ') BNA, (F) methyl (methyleneoxy) (4' -CH (CH) ₃ ) - -O-2 ') BNA (also known as constrained ethyl or cEt), (G) methylene-thio (4' -CH) ₂ - -S-2 ') BNA, (H) methylene-amino (4' -CH2-N (R) -2 ') BNA, (I) methyl carbocycle (4' -CH) ₂ --CH(CH ₃ ) -2 ') BNA, (J) propylene carbocycle (4' - (CH) ₂ ) ₃ -2 ') BNA, (K) methoxy (ethyleneoxy) (4' -CH (CH) ₂ OMe) -O-2') BNA (also known as constrained MOE or cMOE).

In some embodiments, the bicyclic sugar moiety and nucleosides incorporating such bicyclic sugar moiety are further defined by an isomeric configuration. For example, nucleosides comprising a 4'-2' methylene-oxy bridge can be in the α -L configuration or the β -D configuration. Previously, α -L-methyleneoxy (4' -CH ₂ - -O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that exhibit antisense activity.

In some embodiments, the substituted sugar moiety comprises one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5' -substituted sugar and 4' -2' -bridging sugar, wherein the LNA is substituted with, for example, 5' -methyl or 5' -vinyl).

In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of the naturally occurring sugar is substituted with, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moieties further comprise bridging and/or non-bridging substituents as described above. For example, certain sugar substitutes contain a 4' -sulfur atom and substituents at the 2' -and/or 5' -positions. As a further example, carbocyclic bicyclic nucleosides having 4'-2' bridges have been described.

In some embodiments, the sugar substitute comprises a ring having no 5 atoms. For example, in some embodiments, the sugar substitute comprises a six-membered tetrahydropyran. Such tetrahydropyran may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol Nucleic Acids (HNA), altritol Nucleic Acids (ANA), mannitol Nucleic Acids (MNA) and fluorohna (F-HNA).

Many other bicyclic and tricyclic sugar substitution ring systems are also known in the art, which can be used to modify nucleosides for incorporation into antisense compounds.

Combinations of modifications are also provided, not limited to, such as 2'-F-5' -methyl substituted nucleosides and substitution of ribosyl epoxy atoms with S, as well as further substitution at the 2 '-position or alternatively, 5' -substitution of the bicyclic nucleic acid. In some embodiments, 4' -CH ₂ - -O-2 'bicyclic nucleoside is further substituted at the 5' position with 5 '-methyl or 5' -vinyl). Also described are the synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies.

In some embodiments, the present application provides polynucleotides comprising modified nucleosides. Those modified nucleotides may comprise modified sugars, modified nucleobases and/or modified linkages. The particular modification is selected so that the resulting polynucleotide has the desired characteristics. In some embodiments, the polynucleotide comprises one or more RNA-like nucleosides. In some embodiments, the polynucleotide comprises one or more DNA-like nucleotides.

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.

In some embodiments, the modified nucleobase is selected from the group consisting of: 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-aminopropyl adenine, 5-propynyluracil as defined herein; 5-propynyl cytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine,6-methyl and other alkyl derivatives of 2-aminoadenine, 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-propynyl CH ₃ ) Uracil and cytosine; other alkynyl derivatives of pyrimidine bases; 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halogeno, in particular 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-deazapguanine and 7-deazapurine, 3-deazapguanine and 3-deazapurine, universal bases, hydrophobic bases, promiscuous bases, enlarged size bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([ 5, 4-b) ][1,4]Present benzoxazin-2 (3H) -ones, phenothiazine cytidine (1H-pyrimido [5, 4-b)][1,4]Benzothiazin-2 (3H) -one), G-clamp such as substituted phenothiazine cytidine (e.g., 9- (2-aminoethoxy) -H-pyrimido [5, 4-13)][1,4]The benzoxazine-2 (3H) -ketone and carbazole cytidine ² H-pyrimido [4,5-b]Indol-2-one), pyridoindole cytidine (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 (e.g., 7-deaza-adenine, 7-deaza-guanosine, 2-aminopyridine, and 2-pyridone).

In some embodiments, the present application provides polynucleotides comprising linked nucleosides. In such embodiments, nucleosides can be linked together using any internucleoside linkage. Two main classes of internucleoside linkages are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (p=o), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (p=s). Representative phosphorus-free internucleoside linkages include, but are not limited to, methylenemethylimino (- -CH) ₂ --N(CH ₃ )--O--CH ₂ - - - - -, a thiodiester (- -O- -C (O) - -S- - -, a thiocarbamate (- -O- -C (O) (NH) - -S- -; siloxane (- -O- -Si (H)) ₂ - -O- -; and N, N' -dimethylhydrazine (- -CH) ₂ --N(CH ₃ )--N(CH ₃ ) - -). Modified linkages can be used to alter (typically increase) nuclease resistance of the oligonucleotide compared to the native phosphodiester linkage. In some embodiments, the internucleoside linkages having chiral atoms can be prepared as a racemic mixture or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkyl phosphates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free internucleoside linkages are well known to those skilled in the art.

Polynucleotides described herein contain one or more asymmetric centers and thus produce enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined as (R) or (S), α or β (such as for a gluconeohead), or (D) or (L) (such as for an amino acid, etc.), depending on absolute stereochemistry. Antisense compounds provided herein include all such possible isomers and their racemic and optically pure forms.

Neutral internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMIs (3' -CH) ₂ --N(CH ₃ ) - -O-5 '), amide-3 (3' -CH) ₂ -C (=o) -N (H) -5 '), amide-4 (3' -CH) ₂ -N (H) -C (=o) -5 '), methylal (3' -O-CH ₂ - -O-5 ') and thiomethylal (3' -S- -CH) ₂ -O-5'). Other neutral internucleoside linkages include nonionic linkages comprising siloxanes (dialkylsiloxanes), carboxylates, formamides, sulfides, sulfonates and amides (see, e.g., carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, edit ACS Symposium Series 580; chapter 3 and 4, 40-65). Other neutral internucleoside linkages include N, O, S and CH which comprise mixtures ₂ Nonionic attachment of component parts.

Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3 'position of the sugar on the 3' terminal nucleotide and the 5 'position of the 5' terminal nucleotide. For example, one additional modification of the ligand-conjugated polynucleotides of the present application involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance 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, thioether (e.g., hexyl-5-tritylthiol), thiol cholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipid (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate), polyamine or polyethylene glycol chains or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties.

In some embodiments, a polynucleotide described herein comprises or encodes at least one tRNA described herein. In some embodiments, the function of at least one defective tRNA in a subject being treated by a pharmaceutical composition described herein is recovered from a tRNA expressed by a polynucleotide. In some embodiments, at least one tRNA expressed from a polynucleotide described herein can include a tRNA encoding alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, lysine, methionine, aniline, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, at least one tRNA expressed from a polynucleotide described herein can include a tRNA encoding arginine, tryptophan, glutamic acid, glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine.

Polypeptides

In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent assembled with the lipid composition comprises one or more polypeptides. Some polypeptides may include enzymes, such as any of the nucleases described herein. For example, the nuclease may comprise a CRISPR-associated (Cas) protein or Cas nuclease, including a type I CRISPR-associated (Cas) polypeptide, a type II CRISPR-associated (Cas) polypeptide, a type III CRISPR-associated (Cas) polypeptide, a type IV CRISPR-associated (Cas) polypeptide, a type V CRISPR-associated (Cas) polypeptide, and a type VI CRISPR-associated (Cas) polypeptide; zinc Finger Nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA Binding Proteins (RBPs); CRISPR-associated RNA binding proteins; a recombinase; a invertase; a transposase; argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaea Argonaute (aAgo), eukaryotic Argonaute (eAgo), and halophil griseum Argonaute (NgAgo)); adenosine Deaminase (ADAR) acting on RNA; CIRT, PUF, homing endonuclease or any functional fragment thereof, any derivative thereof; any variant thereof; and any fragments thereof. In some embodiments, the nuclease may comprise a Cas protein, such as Cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also referred to as Csn1 and Csx 12), cas10, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csfl, csf2, csf3, csf4, homologs thereof, or modified versions thereof. In some embodiments, the Cas protein can be complexed with a guide polynucleotide described herein to form a CRISPR Ribonucleoprotein (RNP).

The nuclease in the compositions described herein can be Cas9 (e.g., from streptococcus pyogenes(s) or streptococcus pneumoniae (s)). CRISPR enzymes can direct cleavage of one or both strands at a location of a target sequence, such as within the target sequence and/or within the complement of a target sequence of any of the genes described herein.

The CRISPR enzyme may be mutated relative to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or a chain strand of a target polynucleotide comprising a target sequence. For example, substitution of aspartic acid to alanine in the RuvC I catalytic domain of Cas9 from streptococcus pyogenes (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves single strand). In some embodiments, cas9 nickase may be used in combination with one or more guide sequences (e.g., two guide sequences that target the sense and antisense strands of a DNA target, respectively). This combination allows both chains to be nicked and used for NHEJ or HDR.

In some embodiments, the present application provides polypeptides comprising one or more therapeutic proteins. Therapeutic proteins that may be included in the compositions include a wide range of molecules such as cytokines, chemokines, interleukins, interferons, growth factors, clotting factors, anticoagulants, blood factors, bone morphogenic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include Erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), alpha-galactosidase A, alpha-L-iduronidase, thyroid stimulating hormone alpha, N-acetylgalactosamine-4-sulfatase (rhaSB), alfa-streptase, tissue Plasminogen Activator (TPA) activating enzyme, glucocerebrosidase, interferon (IF) beta-1 a, interferon beta-1 b, interferon gamma, interferon alpha, TNF-alpha, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin alpha, daptomtin alpha, follicle Stimulating Hormone (FSH), and factor VIII.

In some embodiments, the polypeptide comprises a peptide sequence that is at least partially identical to any therapeutic (or prophylactic) agent comprising the peptide sequence. For example, the polypeptide may comprise a peptide sequence that is at least partially identical to an antibody (e.g., monoclonal antibody) used to treat a disease, such as cancer.

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

In some embodiments, the pharmaceutical compositions of the present application comprise a plurality of payloads assembled with (e.g., encapsulated within) a lipid composition. The plurality of payloads assembled with the lipid composition may be configured for gene editing or gene expression modification. The plurality of payloads assembled with the lipid composition may comprise a polynucleotide encoding an actuation moiety (e.g., comprising a heterologous endonuclease such as Cas) or a polynucleotide encoding an actuation moiety. The plurality of payloads assembled with the lipid composition may further comprise one or more (e.g., one or two) guide polynucleotides. The plurality of payloads assembled with the lipid composition may further comprise one or more donor or template polynucleotides. The plurality of payloads assembled with the lipid composition may comprise Ribonucleoprotein (RNP).

In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is no more than (about) 20:1, no more than (about) 15:1, no more than (about) 10:1, or no more than (about) 5:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is not less than (about) 20:1, not less than (about) 15:1, not less than (about) 10:1, or not less than (about) 5:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 5:1 to about 20:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 10:1 to about 20:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 15:1 to about 20:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 5:1 to about 10:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 5:1 to about 15:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 5:1 to about 20:1. In some embodiments of the pharmaceutical compositions of the present application, the therapeutic (or prophylactic) agent is a polynucleotide and the molar ratio of nitrogen in the lipid composition to phosphorus in the polynucleotide (N/P ratio) is from about 15:1 to about 20:1.

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 from about 1:1 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 from about 1:1 to about 1:50. 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 from about 50:1 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 from about 1:1 to about 1:20. 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 20:1 to about 1:50. 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 from about 50:1 to about 1:70. 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 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 no more than (about) 1:1, no more than (about) 1:5, no more than (about) 1:10, no more than (about) 1:15, no more than (about) 1:20, no more than (about) 1:25, no more than (about) 1:30, no more than (about) 1:35, no more than (about) 1:40, no more than (about) 1:45, no more than (about) 1:50, no more than (about) 1:60, no more than (about) 1:70, no more than (about) 1:80, no more than (about) 1:90, or no more than (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 not less than (about) 1:1, not less than (about) 1:5, not less than (about) 1:10, not less than (about) 1:15, not less than (about) 1:20, not less than (about) 1:25, not less than (about) 1:30, not less than (about) 1:35, not less than (about) 1:40, not less than (about) 1:45, not less than (about) 1:50, not less than (about) 1:60, not less than (about) 1:70, not less than (about) 1:80, not less than (about) 1:90, or less than (about) 1:100.

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 the particles of the lipid composition.

In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles, the particles characterized by one or more of the following: (1) A dimension of 100 nanometers (nm) or less (e.g., average); (2) a polydispersity index (PDI) of no more than about 0.2; and (3) a zeta potential of from 10 millivolts (mV) to 10 mV.

In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 70 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 50 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of about 60 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of at most about 100 nanometers (nm), at most about 90 nanometers (nm), at most about 85 nanometers (nm), at most about 80 nanometers (nm), at most about 75 nanometers (nm), at most about 70 nanometers (nm), at most about 65 nanometers (nm), at most about 60 nanometers (nm), at most about 55 nanometers (nm), or at most about 50 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present application, the lipid composition comprises a plurality of particles having a (e.g., average) size of at least about 100 nanometers (nm), at least about 90 nanometers (nm), at least about 85 nanometers (nm), at least about 80 nanometers (nm), at least about 75 nanometers (nm), at least about 70 nanometers (nm), at least about 65 nanometers (nm), at least about 60 nanometers (nm), at least about 55 nanometers (nm), or at least about 50 nanometers (nm). The (e.g., average) size may be determined by Size Exclusion Chromatography (SEC). The (e.g., average) size may be determined by one or more spectroscopic methods or one or more image-based methods (e.g., dynamic light scattering, static light scattering, multi-angle light scattering, laser light scattering, or dynamic image analysis, or a combination thereof).

In some embodiments of the pharmaceutical compositions 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 compositions 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 compositions 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 compositions 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 compositions of the present application, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of no more than about 0.5, no more than about 0.4, no more than about 0.3, no more than about 0.2, no more than about 0.1, or no more than about 0.05.

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

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

In some embodiments of the pharmaceutical compositions 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 compositions 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 compositions of the present application, the lipid composition has an apparent pKa of about 8 to about 13. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition has an apparent pKa of about 8 to about 10. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition has an apparent pKa of about 9 to about 11. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition has an apparent pKa of about 10 to about 13. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition has an apparent pKa of about 8 to about 12. In some embodiments of the pharmaceutical compositions of the present application, the lipid composition has an apparent pKa of about 10 to about 12.

In some embodiments of the composition, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the lung cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, or at least 300-fold as compared to the therapeutic effect achieved in the lung cells. In some embodiments of the composition, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments of the composition, the SORT lipids in the lipid composition achieve a therapeutic effect in the lung cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in a lung cell of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold as compared to the therapeutic effect achieved in a liver cell.

In some embodiments of the composition, the SORT lipids in the composition achieve a therapeutic effect in the lung cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the spleen cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in the lung cells of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold as compared to the therapeutic effect achieved in the spleen cells. In some embodiments of the composition, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in the spleen cells of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments of the composition, the SORT lipids in the lipid composition achieve a therapeutic effect in the lung cells of about at least 1.1 fold as compared to the therapeutic effect achieved in the liver cells. In some embodiments, the SORT lipids in the composition achieve a therapeutic effect in a lung cell of about 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold as compared to the therapeutic effect achieved in a liver cell.

Method

In some embodiments of the methods, the pharmaceutical compositions of the present application may be administered by any suitable route, including, for example, oral, rectal, vaginal, transmucosal, pulmonary (including intratracheal or inhalation), or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

In some embodiments of the methods, the pharmaceutical compositions of the present application may be administered in a local rather than systemic manner, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in the form of a slow release formulation. Local delivery can be performed in a variety of ways, depending on the tissue to be targeted.

In some embodiments, the compositions of the present application may be injected into a site of injury, disease manifestations, or pain, for example. In some embodiments, the compositions of the present application may be provided in the form of lozenges for oral, tracheal or esophageal applications. In some embodiments, the compositions of the present application may be supplied in the form of a liquid, tablet or capsule for administration to the stomach or intestine. In some embodiments, the compositions of the present application may be supplied in the form of suppositories for rectal or vaginal application. In some embodiments, the compositions of the present application may even be delivered to the eye by using creams, drops or even injections.

In some embodiments, provided herein are methods for effectively delivering to cells of a subject, the methods comprising administering to the subject a pharmaceutical composition as described herein. In some embodiments of the methods, the pharmaceutical composition comprises a therapeutic (or prophylactic) agent assembled with a lipid composition as described herein, wherein the lipid composition comprises (i) an ionizable cationic lipid; and (iii) a selective organ-targeting (SORT) lipid isolated from an ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

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

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

In some embodiments of the method of delivering a therapeutic agent to a spleen cell, 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 the corresponding amount or activity of the therapeutic agent in 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-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold greater than the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

In some embodiments of any of the methods described herein, the 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 cell of the subject that is at least 1.1 times the corresponding amount or activity of the therapeutic agent achieved in 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-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold greater than the corresponding amount or activity of the therapeutic agent achieved in spleen cells of the subject.

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 the corresponding amount or activity of the therapeutic agent in a liver cell of the subject. In some embodiments, the effective amount or activity of the therapeutic agent in the lung cells is at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold greater than the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

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 cell of the subject that is at least 1.1 times the corresponding amount or activity of the therapeutic agent achieved in 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-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 18-fold, or at least 20-fold that of the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

In some embodiments, delivery of a therapeutic agent to a cell can alter the genome, transcriptome, or expression level. The cells may be allowed or capable of propagating and the changes may be transferred to cells produced by the cells to which the therapeutic agent is delivered. In this way, therapeutic effects can be transmitted to a greater number of cells. Changes in genome, transcriptome, or expression level may also persist in a given cell.

Level of administration

In another aspect, a highly potent dosage form of a therapeutic (or prophylactic) agent formulated with a selective organ-targeting (SORT) lipid is provided, the dosage form comprising the therapeutic (or prophylactic) agent assembled 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 isolated from an ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

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

In some embodiments, the therapeutic agent is present in the dosage form in a dose of no more than about 2 milligrams per kilogram (mg/kg or mpk) of body weight. In some embodiments, the therapeutic agent is present in the dosage form in a dosage of no more than about 1 milligram per kilogram (mg/kg or mpk) of body weight. In some embodiments, the therapeutic agent is present in the dosage form in a dose of no more than about 0.5 milligrams per kilogram (mg/kg or mpk) of body weight. In some embodiments, the therapeutic agent is present in the dosage form in a dose of no more than about 0.2 milligrams per kilogram (mg/kg or mpk) of body weight. In some embodiments, the therapeutic agent is present in the dosage form in a dose of no more than about 0.1 milligrams per kilogram (mg/kg or mpk) of body weight. In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 5 milligrams per milliliter (mg/mL).

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 range between any two of the foregoing values, inclusive.

In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 5 milligrams per milliliter (mg/mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 2 milligrams per milliliter (mg/mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 1 milligram per milliliter (mg/mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 0.5 milligrams per milliliter (mg/mL). In some embodiments, the therapeutic agent is present in the dosage form at a concentration of no more than about 0.1 milligrams per milliliter (mg/mL).

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

In some embodiments, the dosage form may be administered in a local rather than systemic manner, for example, via direct injection of the pharmaceutical composition into the target tissue, preferably in the form of a slow release formulation. Local delivery can be performed in a variety of ways, depending on the tissue to be targeted.

In some embodiments, the dosage form is an inhalation aerosol containing the composition of the invention for nasal, tracheal or bronchial delivery. In some embodiments, the dosage form may be provided in the form of a lozenge for oral, tracheal or esophageal use. In some embodiments, the dosage form may be supplied in the form of a liquid, tablet or capsule for administration to the stomach or intestine. In some embodiments, the dosage form may be supplied in the form of suppositories for rectal or vaginal use. In some embodiments, the dosage form may, even by use of a cream, drops or even an injection, be delivered to the eye.

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

A subject

Any subject in need thereof may be treated with the methods of the present application. In some embodiments, it has been determined that the subject is likely to respond to a therapeutic agent. For example, the subject may have, be suffering from, or suspected of having a disease or disorder. One or more therapeutic or prophylactic agents as described elsewhere herein can be effective to provide a therapeutic effect to a subject by a variety of mechanisms, e.g., via gene therapy (e.g., requiring repeated administration), altering (e.g., increasing) protein production, (e.g., in vivo) Chimeric Antigen Receptor (CAR) T cell production, immunooncology, vaccine-based methods, reactivation of tumor suppressors, or other mechanisms.

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.

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

In some embodiments, the 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.

In another aspect, provided herein is a method for the effective delivery of a therapeutic (or prophylactic) agent to a cell, the method comprising contacting the cell with a pharmaceutical composition of the present application. In some embodiments of the methods, the pharmaceutical composition comprises a therapeutic (or prophylactic) agent assembled with a lipid composition as described herein, wherein the lipid composition comprises (i) an ionizable cationic lipid; and (iii) a selective organ-targeting (SORT) lipid isolated from an ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

In some embodiments of the methods, the cells are isolated from the subject. In some embodiments of the method, the cell is a cell line.

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

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 assembled with a lipid composition.

The following are examples of compositions of the present disclosure and evaluations of the compositions. It should be understood that various other embodiments may be practiced in view of the general description provided above.

List of embodiments

The following list of embodiments of the invention is to be considered as disclosing various features of the invention which may be considered as specific to the particular embodiments discussed, or may be combined with various other features as set forth in other embodiments. Thus, the use of a feature is not necessarily limited to one embodiment, simply because such feature is discussed in the context of a particular embodiment.

Embodiment 1. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (IA):

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

Embodiment 2. The composition according to embodiment 1, wherein the SORT lipid having the structure of formula (IA) is selected from the group consisting of:

And combinations thereof.

Embodiment 3. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (S-III) or a pharmaceutically acceptable salt, stereoisomer, tautomer thereof:

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

Embodiment 4. The composition according to embodiment 3, wherein the SORT lipid having the structure of formula (S-III) is

Embodiment 5. The composition of any of embodiments 1-4, wherein the ionizable cationic lipid is a (g) generation dendrimer or dendron having the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

(a) The core comprises a structural formula (X _Core(s) )：

Wherein:

q at each timeAt the present time, independently is a covalent bond, -O-, -S-, -NR ² -or-CR ^3a R ^3b -；

R ² At each occurrence independently R ^1g or-L ² -NR ^1e R ^1f ；

R ^3a And R is ^3b Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkyl group;

R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f and R is ^1g Each occurrence, if present, is independently at each occurrence a point of attachment to a branch, hydrogen, or optionally substituted (e.g., C ₁ -C ₁₂ ) An alkyl group;

L ⁰ 、L ¹ and L ² Each independently at each occurrence selected from a covalent bond, (e.g., C) ₁ -C ₁₂ Such as C ₁ -C ₆ Or C ₁ -C ₃ ) Alkylene, (e.g. C) ₁ -C ₁₂ Such as C ₁ -C ₈ Or C ₁ -C ₆ ) Heteroalkylene (e.g., C ₂ -C ₈ Alkylene oxides, such as oligo (ethylene oxide)), [ (e.g., C) ₁ -C ₆ ) Alkylene group]- [ (e.g. C) ₄ -C ₆ ) Heterocycloalkyl group]- [ (e.g. C) ₁ -C ₆ ) Alkylene group](e.g., C) ₁ -C ₆ ) Alkylene group]- (arylene) - [ (e.g. C) ₁ -C ₆ ) Alkylene group](e.g., [ (e.g., C) ₁ -C ₆ ) Alkylene group]Phenylene- [ (e.g., C) ₁ -C ₆ ) Alkylene group]) (e.g., C ₄ -C ₆ ) Heterocycloalkyl and arylene (e.g., phenylene); or (b)

Alternatively, L ¹ Part of (A) and R ^1c And R is ^1d One of which is formed (e.g., C ₄ -C ₆ ) Heterocycloalkyl (e.g., containing one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur); and is also provided with

x ¹ 0, 1, 2, 3, 4, 5 or 6; and is also provided with

(b) By a means ofEach of the plurality of (N) branches independently comprises a structural formula (X) _Branching )：

Wherein:

* Indicating the point of attachment of the branch to the core;

g is 1, 2, 3 or 4;

Z＝2 ^(g-1) ；

when g=1, g=0; or when the g is not equal to 1,

(c) Each diacyl independently comprises a structural formulaWherein:

* Indicating the point of attachment of the diacyl group at its proximal end;

* Indicating the point of attachment of the diacyl group at its distal end;

Y ³ independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group;

A ¹ and A ² Each occurrence is independently of the others-O-, -S-or-NR ⁴ -, wherein:

R ⁴ is hydrogen or optionally substituted (e.g., C ₁ -C ₆ ) An alkyl group;

m ¹ and m ² Each occurrence is independently 1, 2, or 3; and is also provided with

R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₈ ) An alkyl group; and is also provided with

(d) Each linker group independently comprises a structural formula

Wherein:

* Indicating the point of attachment of the linker to the proximal diacyl group;

* Indicating the point of attachment of the linker to the distal diacyl group; and is also provided with

Y ₁ Independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group; and is also provided with

(e) Each terminating group is independently selected from optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkyl thiols and optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkenyl thiols.

Embodiment 6 the composition of embodiment 5 wherein x ¹ Is 0, 1, 2 or 3.

Embodiment 7 the composition of embodiment 5 or 6 wherein R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently a point of attachment to a branch (e.g., as indicated by a), hydrogen, or C ₁ -C ₁₂ Alkyl (e.g., C ₁ -C ₈ Alkyl groups, e.g. C ₁ -C ₆ Alkyl or C ₁ -C ₃ Alkyl), wherein the alkyl moieties are optionally substituted with one or more moieties each independently selected from-OH, C ₄ -C ₈ (e.g., C ₄ -C ₆ ) Heterocycloalkyl (e.g., piperidinyl (e.g.,)、N-(C ₁ -C ₃ alkyl) -piperidinyl (e.g.)>) Piperazinyl (e.g.)>)、N-(C ₁ -C ₃ Alkyl) -pipienyl (e.g.)>) Morpholinyl (e.g.)>) N-pyrrolidinyl (e.g.)>) A pyrrolidinyl group (e.g.,) Or N- (C) ₁ -C ₃ Alkyl) -pyrrolidinyl (e.g.)>) (e.g., C) ₆ -C ₁₀ ) Aryl, and C ₃ -C ₅ Heteroaryl (e.g., imidazolyl (e.g.,>) Or pyridyl (e.g.)>) Substituted with a substituent of (c).

Embodiment 8. The method according to embodiment 7, wherein R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently a point of attachment to a branch (e.g., as indicated by a), hydrogen, or C ₁ -C ₁₂ Alkyl (e.g., C ₁ -C ₈ Alkyl groups, e.g. C ₁ -C ₆ Alkyl or C ₁ -C ₃ Alkyl), wherein the alkyl moiety is optionally substituted with one substituent-OH.

Embodiment 9 according toThe composition of any of embodiments 5-8, wherein R ^3a And R is ^3b Each occurrence is independently hydrogen.

Embodiment 10. The composition of any of embodiments 5-9, wherein the plurality of (N) branches comprises at least 3 (e.g., at least 4 or at least 5) branches.

Embodiment 11 the composition of any one of embodiments 5-10, wherein g = 1; g=0; and z=1.

Embodiment 12. The composition of embodiment 11, wherein each of the plurality of branches comprises a structural formula

Embodiment 13 the composition of any one of embodiments 5-10, wherein g = 2; g=1; and z=2.

Embodiment 14. The composition of embodiment 13, wherein each of the plurality of branches comprises a structural formula

Embodiment 15 the composition of any of embodiments 5-14, wherein the core comprises the structural formula:(e.g.)>)。

Embodiment 16. The composition of any of embodiments 5-14, wherein the core comprises the structural formula:

Embodiment 17 the composition of embodiment 16 wherein the core comprises the structural formula:(e.g.)> )。

Embodiment 18. The composition of embodiment 16, wherein the core comprises the structural formula:(e.g.)>Such as->Or->)。

Embodiment 19 the composition of any of embodiments 5-14, wherein the core comprises the structural formula:wherein Q' is-NR ² -or-CR ^3a R ^3b -；q ¹ And q ² Each independently is 1 or 2.

Embodiment 20. The composition of embodiment 19, wherein the core comprises the structural formula:(e.g.)> )。

Embodiment 21 the composition of any of embodiments 5-14, wherein the core comprises a structural formula(e.g.)> ) Wherein ring a is optionally substituted aryl or optionally substituted (e.g., C ₃ -C ₁₂ Such as C ₃ -C ₅ ) Heteroaryl groups.

Embodiment 22 the composition of any of embodiments 5-14, wherein the core comprises a polymer having the formula

Embodiment 23. The composition of any of embodiments 5-14, wherein the core is selected from those set forth in table 1 or a subset thereof.

Embodiment 24. The composition of any of embodiments 5-14, wherein the core comprises a structural formula selected from the group consisting of:

And pharmaceutically acceptable salts thereof, wherein x indicates the point of attachment of the core to one of the plurality of branches.

Embodiment 25 the composition of any of embodiments 5-14, wherein the core comprises a structural formula selected from the group consisting of: and pharmaceutically acceptable salts thereof, wherein x indicates the point of attachment of the core to one of the plurality of branches.

Embodiment 26 the composition of any of embodiments 5-14, wherein the core has a structureWherein an attachment point or H of the core to one of the plurality of branches is indicated.

Embodiment 27. The composition of embodiment 26, wherein at least 2 branches are attached to the core.

Embodiment 28. The composition of embodiment 26, wherein at least 3 branches are attached to the core.

Embodiment 29. The composition of embodiment 26 wherein at least 4 branches are attached to the core.

Embodiment 30 the composition of any of embodiments 5-14, wherein the core has a structureWherein an attachment point or H of the core to one of the plurality of branches is indicated.

Embodiment 31 the composition of embodiment 30 wherein at least 4 branches are attached to the core.

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

Embodiment 33. The composition of embodiment 30, wherein at least 6 branches are attached to the core.

Embodiment 34 the composition of any of embodiments 5-33, wherein A ¹ is-O-or-NH-.

Embodiment 35 the composition of embodiment 34 wherein A ¹ is-O-.

Embodiment 36 the composition of any of embodiments 5-35, wherein A ² is-O-or-NH-.

Embodiment 37 the composition of any embodiment 36, wherein A ² is-O-.

Embodiment 38 the composition of any of embodiments 5-37, wherein Y ³ Is C ₁ -C ₁₂ (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkylene group.

Embodiment 39 the composition of any of embodiments 5-38 wherein the diacyl groups, at each occurrence, independently comprise a structural formula(e.g.)>Such as->) Optionally wherein R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or C ₁ -C ₃ An alkyl group.

Embodiment 40 the composition of any of embodiments 5-39, wherein L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from a covalent bond, C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ Alkylene group, C ₂ -C ₁₂ (e.g., C ₂ -C ₈ ) Alkylene oxides (e.g. oligomerisation [ ]Ethylene oxide), such as- (CH) ₂ CH ₂ O) _1-4 -(CH ₂ CH ₂ )-)、[(C ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g.,) And [ (C) ₁ -C ₄ ) Alkylene group]-phenylene- [ (C) ₁ -C ₄ ) Alkylene group](e.g.)>)。

Embodiment 41 the composition of embodiment 40, wherein L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ Alkylene) - (C) ₁ -C ₃ alkylene-O) _1-4 -(C ₁ -C ₃ Alkylene) - (C) ₁ -C ₃ Alkylene) -phenylene- (C ₁ -C ₃ Alkylene) -, and- (C) ₁ -C ₃ Alkylene) -piperazinyl- (C ₁ -C ₃ Alkylene group) -.

Embodiment 42 the composition of embodiment 40 wherein L ⁰ 、L ¹ And L ² Each occurrence is independently C ₁ -C ₆ Alkylene (e.g., C ₁ -C ₃ An alkylene group).

Embodiment 43 the composition of embodiment 40, wherein L ⁰ 、L ¹ And L ² Each occurrence is independently C ₂ -C ₁₂ (e.g., C ₂ -C ₈ ) Alkylene oxides (e.g., - (C) ₁ -C ₃ alkylene-O) _1-4 -(C ₁ -C ₃ Alkylene)).

Embodiment 44 the composition of embodiment 40 wherein L ⁰ 、L ¹ And L ² Each at each occurrence is independently selected from [ (C) ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g., - (C) ₁ -C ₃ Alkylene) -phenylene- (C ₁ -C ₃ Alkylene) -) and [ (C) ₁ -C ₄ ) Alkylene group]-[(C ₄ -C ₆ ) Heterocycloalkyl group]-[(C ₁ -C ₄ ) Alkylene group](e.g., - (C) ₁ -C ₃ Alkylene) -piperazinyl- (C ₁ -C ₃ Alkylene) -.

Embodiment 45 the composition of any of embodiments 5-44 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkenyl thiols or C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl or alkenyl moiety is optionally substituted with one or more moieties each independently selected from halogen, C ₆ -C ₁₂ Aryl (e.g., phenyl), C ₁ -C ₁₂ (e.g., C ₁ -C ₈ ) Alkylamino (e.g., C ₁ -C ₆ Mono-alkylamino (such as-NHCH ₂ CH ₂ CH ₂ CH ₃ ) Or C ₁ -C ₈ Di-alkylamino groups (such as))、C ₄ -C ₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl->N-piperidinyl->N-azepanyl->)、-OH、-C(O)OH、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₁ -C ₁₂ Alkylamino (e.g., mono-or di-alkylamino)) (e.g., (-)>)、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₄ -C ₆ N-heterocycloalkyl) (e.g.)>)、-C(O)-(C ₁ -C ₁₂ Alkylamino (e.g., mono-or di-alkylamino)), and-C (O) - (C) ₄ -C ₆ An N-heterocycloalkyl group) (e.g.,) Wherein C is a substituent of any of the foregoing substituents ₄ -C ₆ The N-heterocycloalkyl moiety optionally being C ₁ -C ₃ Alkyl or C ₁ -C ₃ Hydroxyalkyl substitution.

Embodiment 46 the composition of embodiment 45 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl moiety is optionally substituted with one or more (e.g., one) each independently selected from C ₆ -C ₁₂ Aryl (e.g., phenyl), C ₁ -C ₁₂ (e.g., C ₁ -C ₈ ) Alkylamino (e.g., C ₁ -C ₆ Mono-alkylamino (such as-NHCH ₂ CH ₂ CH ₂ CH ₃ ) Or C ₁ -C ₈ Di-alkylamino groups (such as ))、C ₄ -C ₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl->N-piperidinyl->N-azepanyl)、-OH、-C(O)OH、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₁ -C ₁₂ Alkylamino (e.g., mono-or di-alkylamino)) (e.g., (-)>)、-C(O)N(C ₁ -C ₃ Alkyl) - (C ₁ -C ₆ Alkylene) - (C ₄ -C ₆ N-heterocycloalkyl) (e.g.)>) and-C (O) - (C) ₄ -C ₆ N-heterocycloalkyl) (e.g.)>) Wherein C is a substituent of any of the foregoing substituents ₄ -C ₆ The N-heterocycloalkyl moiety optionally being C ₁ -C ₃ Alkyl or C ₁ -C ₃ Hydroxyalkyl substitution.

Embodiment 47 the composition of embodiment 46 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl moiety is optionally substituted with one substituent-OH.

Embodiment 48 the composition of embodiment 46 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) An alkyl thiol, wherein the alkyl moiety is optionally substituted with one selected from C ₁ -C ₁₂ (e.g., C ₁ -C ₈ ) Alkylamino (e.g., C ₁ -C ₆ Mono-alkylamino (such as-NHCH ₂ CH ₂ CH ₂ CH ₃ ) Or C ₁ -C ₈ Di-alkylamino groups (such as) And C) ₄ -C ₆ N-heterocycloalkyl (e.g., N-pyrrolidinyl->N-piperidinyl->N-azepanyl- >) Is substituted by a substituent of (a).

Embodiment 49 the composition of embodiment 45 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkenyl thiols or C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkyl mercaptans.

Embodiment 50. The composition of embodiments 47 or 49 wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkyl mercaptans.

Embodiment 51. The composition of embodiment 50 wherein each terminating group is independently selected from the group consisting of:

embodiment 52 the composition of any of embodiments 5-44 wherein each terminating group is independently selected from those set forth in table 3 or a subset thereof.

Embodiment 53 the composition of any of embodiments 1-4, wherein the ionizable cationic lipid is selected from those set forth in table 4 or a pharmaceutically acceptable salt thereof, or a subset of said lipids and pharmaceutically acceptable salts thereof.

Embodiment 54 the composition of any of embodiments 1-4, wherein the ionizable cationic lipid is selected from those set forth in table 4 or table 5 or a pharmaceutically acceptable salt thereof, or a subset of said lipid and a pharmaceutically acceptable salt thereof.

Embodiment 55 the composition of any one of embodiments 1-54, wherein the lipid composition further comprises a phospholipid.

Embodiment 56. The composition of embodiment 55, wherein the mole percent of the phospholipids is from about 8% to about 23%.

Embodiment 57 the composition of any of embodiments 1-56, wherein the lipid composition further comprises a steroid or steroid derivative.

Embodiment 58 the composition of embodiment 57 wherein the mole percent of the steroid or steroid derivative is about 15% to about 46%.

Embodiment 59 the composition of any one of embodiments 1-58, wherein the mole percent of the ionizable cationic lipid is from about 5% to about 30%.

Embodiment 60 the composition of any of embodiments 1-59, wherein the molar percentage of the polymer-conjugated lipid is about 0.5% to about 10%.

Embodiment 61 the composition of any of embodiments 1-59, wherein the mole percent of the polymer conjugated lipid is from about 1% to about 10%.

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

Embodiment 63 the composition of any of embodiments 1-62, wherein the mole percent of the SORT lipid is about 20% to about 65%.

Embodiment 64 the composition of any of embodiments 1-63, wherein the therapeutic agent is a polynucleotide; and wherein the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is no more than about 20:1.

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

Embodiment 66. The composition of any of embodiments 1-65, wherein the molar ratio of the therapeutic agent to the total lipids of the lipid composition is no more than about 1:1, 1:10, 1:50, or 1:100.

Embodiment 67 the composition of any of embodiments 1-66, wherein at least about 85% of said therapeutic agent is encapsulated in particles of said lipid composition.

Embodiment 68 the composition of any of embodiments 1-67, wherein the lipid composition comprises a plurality of particles characterized by one or more of the following:

(1) A dimension of 100 nanometers (nm) or less (e.g., average);

(2) A polydispersity index (PDI) of no more than about 0.2; and

(3) -a negative zeta potential of 10 millivolts (mV) to 10 mV.

Embodiment 69 the composition of any one of embodiments 1-68 wherein the lipid composition has an apparent ionization constant (pKa) outside of the range of 6 to 7.

Embodiment 70. The composition of embodiment 69, wherein the apparent pKa of the lipid composition is about 7 or greater.

Embodiment 71 the composition of embodiment 69, wherein said apparent pKa of said lipid composition is about 8 or greater.

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

Embodiment 73. A method for targeted delivery of a therapeutic agent to spleen cells, the method comprising administering (e.g., systemically) the composition of any one of embodiments 1-72, 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 the corresponding amount or activity of the therapeutic agent achieved in lung cells of the subject.

Embodiment 74. The method of embodiment 73, wherein the effective amount or activity of the therapeutic agent in the spleen cells of the subject is at least 1.1-fold, at least 2.5-fold, at least 3.5-fold, at least 10-fold, at least 5.5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or at least 50-fold that of the corresponding amount or activity of the therapeutic agent achieved in lung cells of the subject.

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

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-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 18-fold, at least 20-fold, or at least about 50-fold that of the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.

Examples

Example 1: preparation of DOTAP or DODAP modified lipid nanoparticles

Lipid Nanoparticles (LNP) are the most effective class of carriers for in vivo nucleic acid delivery. Historically, effective LNPs were composed of 4 components: ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and lipidic poly (ethylene glycol) (PEG). However, these LNPs result in only general delivery of nucleic acids, not organ or tissue targeted delivery. LNP typically delivers RNA only to the liver. Accordingly, efforts have sought to find new formulations to provide targeted nucleic acid delivery.

The four typical types of lipids were mixed in a molar ratio of 15:15:30:3 with or without the addition of permanent cationic lipids. Briefly, LNP is prepared by mixing dendrimer or dendron lipids (ionizable cations), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cations). Alternatively, DOTAP may be substituted for DOTAP to produce an LNP comprising DOTAP. The structure of the DODAP and DODAP is shown in FIG. 1. A variety of dendrimers or dendron lipids that may be used are shown in fig. 2.

To prepare the LNP formulation, the dendrimer or dendron lipid, DOPE, cholesterol and DMG-PEG are dissolved in ethanol in the desired molar ratio. mRNA was dissolved in citrate buffer (10 mM, pH 4.0). The mRNA was then diluted into the lipid solution by rapidly mixing the mRNA into the lipid solution in a 3:1 volume ratio (mRNA: lipid, v/v) to achieve a 40:1 weight ratio (total lipid: mRNA). The solution was then incubated at room temperature for 10min. To form DOTAP modified LNP formulations, mRNA was dissolved in 1 x PBS or citrate buffer (10 mm, ph 4.0) and rapidly mixed into ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP at a fixed weight ratio of 40:1 (total lipid: mRNA) and a volume ratio of 3:1 (mRNA: lipid). The formulation is named X% DOTAP Y (or X% DOTAP Y), where X represents the molar percentage of DOTAP (or DOTAP) in the total lipid and Y represents the type of dendrimer or dendron lipid. Alternatively, the formulation may be named Y X% DOTAP or Y X% DOTAP, wherein X represents the molar percentage of DOTAP (or DOTAP) in the total lipid and Y represents the type of dendrimer or dendron lipid.

Example 2: SORT LNP stability:

exemplary lipid compositions as described herein were produced using microfluidic mixing methods or cross/tee mixing methods and tested for stability. Physical characteristics including size, polydispersity index (PDI) and zeta potential were characterized by Dynamic Light Scattering (DLS) (3 measurements per formulation alone) from various exemplary Lipid (LNP) compositions and are illustrated in table 7.

Encapsulation efficiency was tested using a Ribogreen RNA assay (Zhao et al 2016). Briefly, mRNA was encapsulated in LNP with >95% efficiency when dissolved in acidic buffer (10 mM citrate, pH 4). Features of both types of lnp (5 A2-SC8 with 20% DOTAP ("liver-SORT") and 5A2-SC8 with 50% DOTAP ("lung-SORT")) were observed over 28 days. Fig. 3 illustrates the change in physical characteristics of the lipid composition over a 28 day duration.

Table 7: SORT LNP feature

Example 3: lung-SORT IV study

This example compiles several studies of intravenous and systemic administration of lung-SORT to multiple species (e.g., mice, rats, dogs, and non-human primate (NHP) (e.g., rhesus macaque), cynomolgus monkey (Cynomolgus macaques)).

SUMMARY

A single dose of luciferase mRNA encapsulated in lung-SORT LNP (e.g., 5A2-SC8 with 50% DOTAP) is administered to the majority of test subjects. Two doses were administered to some subjects. The low dose administered to dogs and NHP was translated from a mouse study. In this study, whole animal imaging, organ imaging, blood chemistry, hematology, immunoreactivity, complement, and tolerability parameters were evaluated. lung-SORT LNP shows selectivity across species to target the lung and avoid other organs and tissues. A variety of DOTAP-substituted LNPs (e.g., 14:0 tap) were tested in vivo (e.g., mice) and in vitro (e.g., in hBE) and exhibited improved tolerability, higher potency, and similar or better selectivity. Animal experiments also tested (e.g., in dogs) used a second lung-SORT ("RTX 0031") formulation with 14:0tap (SORT lipid), which was highly potent, very selective for targeting the lung (results of intravenous bolus and intravenous infusion are similar), with no signs of infusion-related reactions (IRR).

The exemplary "lung-SORT LNP" tested herein is a 5-component lipid nanoparticle composition comprising about 11.9%5a2-SC8 (ionizable cationic lipid), about 50% DOTAP (SORT lipid), about 11.9% DOPE lipid, about 23.8% cholesterol, and about 2.4% dmg-PEG (PEG conjugated lipid), wherein each lipid component is defined as mol% of the total lipid composition.

Exemplary DOTAP instead of LNP DOTAP (SORT lipid) was replaced with 14:0TAP as SORT lipid (also known as "RTX0031 LNP"). Such RTX0031 LNP compositions tested herein are 5-component lipid nanoparticle compositions comprising about 14.3%5a2-SC8 (ionizable cationic lipid), about 40%14:0tap (SORT lipid), about 14.3% DOPE, about 28.6% cholesterol, and about 2.8% DMG-PEG (PEG conjugated lipid), wherein each lipid component is defined as mol% of the total lipid composition.

NHP IV Lung-SORT LNP study

lung-SORT LNP containing 0.1mg/kg luciferase mRNA was delivered to NHP via intravenous bolus over 5min without any pre-drug (e.g., steroid). Systemic bioluminescence imaging was performed 4h after intravenous bolus administration. The present study showed that lung-SORT LNP formulations were active in NHP and highly potent, as mRNA delivery was extrahepatic and significant signaling was observed in the lung. In addition, the tolerance of lung-SORT LNP was assessed in NHP. Without wishing to be bound by any theory, DOTAP and/or the method of administration may play a role in tolerability.

RTX0031 LNP was tested herein. In addition, treatment-related parameters (e.g., slow infusion and on-demand precursor medication) were further tested.

TABLE 8 parameters of the beagle lung SORT LNP (5A 2-SC8 with 50% DOTAP) study

Figures 4A and 4B show IVIS organ imaging of spleen, liver and lung in female and male dogs after administration of lung-SORT LNP (5 A2-SC8 with 50% DOTAP). The higher signal seen in the lung indicates that lung-SORT LNP is selective for lung delivery.

Both dogs showed signs consistent with the response associated with mild infusion. Female (first) dogs had very mild tachycardia compared to their baseline. Female dogs showed only small changes in blood chemistry, with 2-fold increases in AST, CK and LDH. Hematological evaluation showed no significant change. Male dogs showed mucosal congestion and mild sleepiness/muscle weakness (abdominal flexion of the neck) 15min before injection, which resolved within the first half hour after infusion and were not severe enough to require intervention. Male (second) dogs had mild bradycardia compared to baseline and were observed within the first hour after infusion.

TABLE 9 cynomolgus monkey lung-SORT LNP (5A 2-SC8 with 50% DOTAP) study

Figures 5A and 5B show IVIS organ imaging of spleen, liver and lung of two cynomolgus monkey NHPs after administration of lung-SORT LNP (5A 2-SC8 with 50% DOTAP). The higher signal seen in the lung indicates that lung-SORT LNP is selective for lung delivery.

No adverse reactions were observed during or after the first or second administration of the lung-SORT formulation. The blood chemistry of cynomolgus monkey NHP test subjects showed AST, ALT, CK and LDH increase after the first dose and LDH and CO after the second dose ₂ And (3) increasing. The hematology of the cynomolgus monkey NHP test subjects showed neutrophil increase and lymphocyte and eosinophil decrease after the first and second doses.

In rats, dogs, rhesus and cynomolgus monkeys, lung-SORT LNP (5A 2-SC8 with 50% DOTAP) was delivered as an intravenous bolus

When delivered as intravenous bolus without any prodrugs, lung-SORT LNP was well tolerated and showed no adverse reactions during 2 administrations to cynomolgus monkeys and only mild signs of IRR in dogs after a single administration. AST, CK, LDH, and neutrophil/lymphocyte changes are good indicators of tolerogenic blood markers across the test species.

As an advantageous result, the rats tested did not provide differences in efficacy and toxicity compared to the mice, dogs and NHP subjects tested, showing signs of toxicity only at a dose of 1 mg/mL.

Studies of RTX0031 LNP (DOTAP instead of lung-SORT) in mice

FIG. 6A shows IVIS organ imaging of spleen, liver, kidney and lung of three mice 5h after administration of luciferase mRNA formulated with RTX0031 LNP. The higher signal seen in the lung compared to liver, spleen and kidney suggests that RTX0031 LNP is selective for pulmonary delivery. Figure 6B quantitatively shows the signals obtained at the lungs, spleen and liver of 3 mice. In vivo studies can provide information that RTX0031 LNP has improved tolerability and efficacy.

Studies of RTX0031 LNP (14:0 TAP as SORT lipid) and Lung-SORT LNP (DOTAP as SORT lipid) in hBE

The tolerance and efficacy of RTX0031 LNP and lung-SORT LNP were tested in hBE. Tip fluid (therapeutic liquid) with 12ug of formulated Tomato Red (TR) mRNA formulated in lung-SORT and RTX0031 LNP formulations was administered to hBE. TR expression was assessed 24h after treatment by fluorescence microscopy. Figure 7A shows TR intensity expressed in hBE treated with the two LNP compositions tested. RTX0031 LNP showed a higher TR strength compared to the lung-SORT LNP (5A 2-SC8 with 50% DOTAP) formulation. In addition, cytotoxicity (LDH release) was assessed 48h after treatment. Fig. 7B shows the% LDH released from hBE treated with two LNP compositions. lung-SORT LNP (5 A2-SC8 with 50% DOTAP) formulations showed higher% LDH release than RTX0031 LNP formulations. In vitro studies can provide information that RTX0031 LNP formulations have improved tolerability and efficacy.

TABLE 10 LNP study in beagle dogs

¹ Injection volume: intravenous bolus injection of 0.5mL and intravenous infusion of 10-15mL

² The precursor medicines for relieving IRR are as follows: dexamethasone 0.1mg/kg IV, acetaminophen 10mg/kg PO, diphenhydramine 1mg/kg IV, famotidine 0.5mg/kg IV for slow injection

In mice, RTX0031 LNP was about 2 times more potent than lung-SORT LNP (5 A2-SC8 with 50% DOTAP)

Figure 8A shows IVIS organ imaging of spleen, liver and lung of two beagle dogs after intravenous bolus administration of luciferase mRNA formulated in RTX0031 LNP. Shortly after TA treatment, male dogs had some salivation, which was observed in less than one to two minutes. Female dogs had no signs of adverse reactions during or after TA treatment. For both dogs, the temperature, heart rate and respiration rate showed only minor changes and were primarily related to the stress caused by the treatment. Female dogs underwent seizures during administration of luciferin. Blood chemistry showed an ALT increase in male dogs of about 3 years. Hematology showed only small changes, especially lymphopenia and eosinophilia in male dogs.

Figure 8B shows IVIS organs imaged of spleen, liver and lung of two beagle dogs after intravenous infusion of luciferase mRNA formulated in RTX0031 LNP with the precursor drug. Neither dog showed any signs of clinical observation or adverse reaction during or after TA administration. One of the dogs underwent seizures during administration of luciferin. Blood chemistry showed increases in ALT (2-fold), CK (3-fold) and LDH (5-fold) in female dogs. Hematology shows only small changes, in particular lymphopenia and eosinophilia.

Fig. 9 shows a compilation of IVIS organ imaging of spleen, liver and lung of dogs and NHPs as seen in fig. 4A, 4B, 5A, 5B, 8A and 8B.

TABLE 11 Assembly beagle and NHP Lung-SORT LNP and RTX0031 LNP study model

Example 4: organ selective tendencies of spleen SORT and lung ORT formulations

Luc mRNA/LNP comprising 5A2-SC8 lipid and 40% of the SORT lipids as described in table 12 was administered intravenously to mice at a dose of 0.05 mpk. Each SORT lipid studied in table 12 had n=4 mice/group. After 5h following LNP administration, organs were excised and IVIS imaged for kidneys, lungs, spleen and liver to determine the selective organ tropism for the different SORT lipids.

The exemplary LNP tested herein was a 5-component lipid nanoparticle composition comprising about 14.3%5a2-SC8 (ionizable cationic lipid), about 40% of the SORT lipid from table 12, about 14.3% DOPE, about 28.6% cholesterol, and about 2.8% DMG-PEG (PEG conjugated lipid), wherein each lipid component is defined as mol% of the total lipid composition.

Table 12: SORT LNP with different SORT lipids

SORT lipids	Size (nm)	PDI	Encapsulation (%)
				12:0EPC	45.3	0.16	97
14:0EPC	50.7	0.10	96
				14:1EPC	52.9	0.13	94
16:0EPC	60.9	0.16	93
				18:0EPC	80.3	0.08	95
18:1EPC	56.8	0.10	95
				16:0-18:1EPC	50.7	0.09	95
14:0TAP	104.4	0.15	96
				16:0TAP	133.9	0.12	97
18:0TAP	241.0	0.13	96
				18:1DOTAP	61.0	0.12	95
18:0DDAB	133.8	0.17	96
				18:1DOTMA	66.8	0.03	95

The IVIS image of fig. 11A shows the different organ selectivities of the 12:0epc, 14:0epc, 14:1epc, 16:0epc, 18:0epc, 18:1epc, and 16:0-18:1epc SORT lipids delivered to the SORT LNP of mice. The higher intensity of localization to spleen and lung compared to liver shows organ targeting tropism of SORT LNP and the effect of SORT lipids. Figure 11B shows quantitatively IVIS data of liver, spleen and lung of mice tested with different SORT lipids. In all cases tested, each EPC lipid tested had higher selectivity for spleen and lung than liver, as seen by higher luminescence intensity. Certain EPC lipids provide more efficient payload delivery and a level of selectivity between the lung and spleen.

The IVIS image of fig. 12A shows the different organ selectivities of 14:0tap, 16:0tap, 18:0tap, 18:1tap, 18:0ddab and 18:1dotma SORT lipids delivered to the SORT LNP of mice. The higher intensity of localization to the lung compared to liver and spleen shows the organ-targeting tropism of SORT LNP and the effect of SORT lipids. Figure 12B shows quantitatively IVIS data of liver, spleen and lung of mice tested with different SORT lipids. Most of the tested SORT lipids have higher selectivity compared to liver and spleen, as seen by higher luminescence intensity. Certain SORT lipids provide more efficient payload delivery and a level of selectivity between the lung and spleen.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The invention is not limited by the specific embodiments provided in the specification. While the invention has been described with reference to the foregoing specification, the description and illustration of embodiments herein are not intended to be construed in a limiting sense. Many modifications, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein in terms of various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the invention and their equivalents and methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims (56)

1. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (IA):

wherein:

R ₁ and R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

2. The composition of claim 1, wherein the SORT lipid having the structure of formula (IA) is selected from the group consisting of:

and combinations thereof.

3. A composition formulated for systemic (e.g., intravenous) administration, the composition comprising a therapeutic agent assembled with a lipid composition comprising:

(i) An ionizable cationic lipid;

(ii) A polymer conjugated lipid; and

(iii) A selective organ-targeting (SORT) lipid having the structure of formula (S-III) or a pharmaceutically acceptable salt, stereoisomer, tautomer thereof:

wherein:

R ₁ And R is ₂ Each independently is an alkyl group _(C8-C24) Alkenyl group _(C8-C24) Or a substituted form of either group;

R ₃ 、R ₃ ' and R ₃ "each independently is alkyl _(C≤6) Or substituted alkyl _(C≤6) The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ^- Is a monovalent anion.

4. A composition according to claim 3, wherein the SORT lipid having the structure of formula (S-III) is

5. The composition of claim 1 or 2, wherein the ionizable cationic lipid is a (g) generation dendrimer or dendron having the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein:

(a) The core comprises a structural formula (X _Core(s) )：

Wherein:

q is independently at each occurrence a covalent bond, -O-, -S-, -NR ² -or-CR ^3a R ^3b -；

R ² At each occurrence independently R ^1g or-L ² -NR ^1e R ^1f ；

R ^3a And R is ^3b Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkyl group;

R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f and R is ^1g Each occurrence, if present, is independently at each occurrence a point of attachment to a branch, hydrogen, or optionally substituted (e.g., C ₁ -C ₁₂ ) An alkyl group;

L ⁰ 、L ¹ and L ² Each independently at each occurrence selected from a covalent bond, (e.g., C) ₁ -C ₁₂ Such as C ₁ -C ₆ Or C ₁ -C ₃ ) Alkylene, (e.g. C) ₁ -C ₁₂ Such as C ₁ -C ₈ Or C ₁ -C ₆ ) Heteroalkylene (e.g., C ₂ -C ₈ Alkylene oxides, such as oligo (ethylene oxide)), [ (e.g., C) ₁ -C ₆ ) Alkylene group]- [ (e.g. C) ₄ -C ₆ ) Heterocycloalkyl group]- [ (e.g. C) ₁ -C ₆ ) Alkylene group](e.g., C) ₁ -C ₆ ) Alkylene group]- (arylene) - [ (e.g. C) ₁ -C ₆ ) Alkylene group](e.g., [ (e.g., C) ₁ -C ₆ ) Alkylene group]Phenylene- [ (e.g., C) ₁ -C ₆ ) Alkylene group]) (e.g., C ₄ -C ₆ ) Heterocycloalkyl and arylene (e.g., phenylene); or (b)

Alternatively, L ¹ Part of (A) and R ^1c And R is ^1d One of which is formed (e.g., C ₄ -C ₆ ) Heterocycloalkyl (e.g., containing one or two nitrogen atoms and optionally additional heteroatoms selected from oxygen and sulfur); and is also provided with

x ¹ 0, 1, 2, 3, 4, 5 or 6; and is also provided with

(b) Each of the plurality of (N) branches independently comprises a structural formula (X) _Branching )：

Wherein:

* Indicating the point of attachment of the branch to the core;

g is 1, 2, 3 or 4;

Z＝2 ^(g-1) ；

when g=1, g=0; or when the g is not equal to 1,

(c) Each diacyl independently comprises a structural formulaWherein:

* Indicating the point of attachment of the diacyl group at its proximal end;

* Indicating the point of attachment of the diacyl group at its distal end;

Y ³ independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group;

A ¹ and A ² Each occurrence is independently of the others-O-, -S-or-NR ⁴ -, wherein:

R ⁴ is hydrogen or optionally substituted (e.g., C ₁ -C ₆ ) An alkyl group;

m ¹ and m ² Each occurrence is independently 1, 2, or 3; and is also provided with

R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or optionally substituted (e.g., C ₁ -C ₈ ) An alkyl group; and is also provided with

(d) Each linker group independently comprises a structural formula

Wherein:

* Indicating the point of attachment of the linker to the proximal diacyl group;

* Indicating the point of attachment of the linker to the distal diacyl group; and is also provided with

Y ₁ Independently at each occurrence is optionally substituted (e.g., C ₁ -C ₁₂ ) Alkylene, optionally substituted (e.g., C ₁ -C ₁₂ ) Alkenylene or optionally substituted (e.g., C ₁ -C ₁₂ ) An arylene group; and is also provided with

(e) Each terminating group is independently selected from optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkyl thiols and optionally substituted (e.g., C ₁ -C ₁₈ Such as C ₄ -C ₁₈ ) Alkenyl thiols.

6. The composition of claim 5, wherein x ¹ Is 0, 1, 2 or 3.

7. According to the weightsThe composition of claim 5 wherein R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently a point of attachment to a branch (e.g., as indicated by a), hydrogen, or C ₁ -C ₁₂ Alkyl (e.g., C ₁ -C ₈ Alkyl groups, e.g. C ₁ -C ₆ Alkyl or C ₁ -C ₃ Alkyl), wherein the alkyl moieties are optionally substituted with one or more moieties each independently selected from-OH, C ₄ -C ₈ (e.g., C ₄ -C ₆ ) Heterocycloalkyl (e.g., piperidinyl (e.g., )、N-(C ₁ -C ₃ alkyl) -piperidinyl (e.g.)>) Piperazinyl (e.g.)>)、N-(C ₁ -C ₃ Alkyl) -pipienyl (e.g.)>) Morpholinyl (e.g.)>) N-pyrrolidinyl (e.g.)>) Pyrrolidinyl (e.g.)>) Or N- (C) ₁ -C ₃ Alkyl) -pyrrolidinyl (e.g.)>) (e.g., C) ₆ -C ₁₀ ) Aryl, and C ₃ -C ₅ Heteroaryl (e.g., imidazolyl (e.g.,>) Or pyridyl (e.g.)>) Substituted with a substituent of (c).

8. The method of claim 7, wherein R ^1a 、R ^1b 、R ^1c 、R ^1d 、R ^1e 、R ^1f And R is ^1g Each occurrence, if present, is independently a point of attachment to a branch (e.g., as indicated by a), hydrogen, or C ₁ -C ₁₂ Alkyl (e.g., C ₁ -C ₈ Alkyl groups, e.g. C ₁ -C ₆ Alkyl or C ₁ -C ₃ Alkyl), wherein the alkyl moiety is optionally substituted with one substituent-OH.

9. The composition of claim 5 wherein R ^3a And R is ^3b Each occurrence is independently hydrogen.

10. The composition of claim 5, wherein the plurality of (N) branches comprises at least 3 (e.g., at least 4 or at least 5) branches.

11. The composition of claim 5, wherein g = 1; g=0; and z=1.

12. The composition of claim 11, wherein each of the plurality of branches comprises a structural formula

13. The composition of claim 5, wherein g = 2; g=1; and z=2.

14. The composition of claim 13, wherein each of the plurality of branches comprises a structural formula

15. The composition of claim 5, wherein the core is selected from those set forth in table 1 or a subset thereof.

16. The composition of claim 5, wherein the core comprises a structural formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein x indicates the point of attachment of the core to one of the plurality of branches.

17. The composition of claim 5, wherein the core has a structureWherein an attachment point or H of the core to one of the plurality of branches is indicated.

18. The composition of claim 17, wherein at least 2 branches are attached to the core.

19. The composition of claim 17, wherein at least 3 branches are attached to the core.

20. The composition of claim 17, wherein at least 4 branches are attached to the core.

21. The composition of claim 5, wherein the core has a structureWherein an attachment point or H of the core to one of the plurality of branches is indicated.

22. The composition of claim 21, wherein at least 4 branches are attached to the core.

23. The composition of claim 21, wherein at least 5 branches are attached to the core.

24. The composition of claim 21, wherein at least 6 branches are attached to the core.

25. The composition of claim 5, wherein a ¹ is-O-or-NH-.

26. The composition of claim 25, wherein a ¹ is-O-.

27. The composition of claim 5, wherein a ² is-O-or-NH-.

28. The method of claim 27Compositions wherein A ² is-O-.

29. The composition of claim 5 wherein Y ³ Is C ₁ -C ₁₂ (e.g., C ₁ -C ₆ Such as C ₁ -C ₃ ) An alkylene group.

30. The composition of claim 5, wherein the diacyl groups, at each occurrence, independently comprise a structural formula(e.g.)>Such as->) Optionally wherein R ^3c 、R ^3d 、R ^3e And R is ^3f Each occurrence is independently hydrogen or C ₁ -C ₃ An alkyl group.

31. The composition of claim 5, wherein each terminating group is independently C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkenyl thiols or C ₁ -C ₁₈ (e.g., C ₄ -C ₁₈ ) Alkyl mercaptans.

32. The composition of claim 5, wherein each terminating group is independently selected from those set forth in table 3 or a subset thereof.

33. The composition of claim 1 or 2, wherein the ionizable cationic lipid is selected from those set forth in table 4 or a pharmaceutically acceptable salt thereof, or a subset of the lipids and pharmaceutically acceptable salts thereof.

34. The composition of claim 1 or 2, wherein the ionizable cationic lipid is selected from those set forth in table 4 or table 5 or a pharmaceutically acceptable salt thereof, or a subset of the lipids and pharmaceutically acceptable salts thereof.

35. The composition of claim 1 or 2, wherein the lipid composition further comprises a phospholipid.

36. The composition of claim 35, wherein the mole percent of phospholipids is from about 8% to about 23%.

37. The composition of claim 1 or 2, wherein the lipid composition further comprises a steroid or steroid derivative.

38. The composition of claim 37, wherein the mole percent of the steroid or steroid derivative is about 15% to about 46%.

39. The composition of claim 1 or 2, wherein the mole percent of the ionizable cationic lipid is from about 5% to about 30%.

40. The composition of claim 1 or 2, wherein the mole percent of polymer conjugated lipid is from about 0.5% to about 10%.

41. The composition of claim 1 or 2, wherein the mole percent of polymer conjugated lipid is from about 1% to about 10%.

42. The composition of claim 1 or 2, wherein the mole percent of polymer conjugated lipid is from about 2% to about 10%.

43. The composition of claim 1 or 2, wherein the mole percent of the SORT lipid is from about 20% to about 65%.

44. The composition of claim 1 or 2, wherein the therapeutic agent is a polynucleotide; and wherein the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N/P ratio) is no more than about 20:1.

45. The composition of claim 44, wherein the N/P ratio is from about 5:1 to about 20:1.

46. The composition of claim 1 or 2, wherein the molar ratio of the therapeutic agent to the total lipid of the lipid composition is no more than about 1:1, 1:10, 1:50, or 1:100.

47. The composition of claim 1 or 2, wherein at least about 85% of the therapeutic agent is encapsulated in particles of the lipid composition.

48. The composition of claim 1 or 2, wherein the lipid composition comprises a plurality of particles characterized by one or more of the following:

(1) A dimension of 100 nanometers (nm) or less (e.g., average);

(2) A polydispersity index (PDI) of no more than about 0.2; and

(3) -a negative zeta potential of 10 millivolts (mV) to 10 mV.

49. The composition of claim 1 or 2, wherein the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7.

50. The composition of claim 49, wherein the apparent pKa of the lipid composition is about 7 or greater.

51. The composition of claim 49, wherein the apparent pKa of the lipid composition is about 8 or greater.

52. The composition of claim 49, wherein the apparent pKa of the lipid composition is from about 8 to about 13.

53. A method for targeted delivery of a therapeutic agent to spleen cells, the method comprising administering (e.g., systemically) the composition of claim 1 or 2, 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 the corresponding amount or activity of the therapeutic agent achieved in lung cells of the subject.

54. The method of claim 53, wherein the effective amount or activity of the therapeutic agent in the spleen cells of the subject is at least 1.1-fold, at least 2.5-fold, at least 3.5-fold, at least 10-fold, at least 5.5-fold, at least 10-fold, at least 15-fold, at least 20-fold, or at least 50-fold that of the corresponding amount or activity of the therapeutic agent effected in lung cells of the subject.

55. A method for targeted delivery of a therapeutic agent to a lung cell, the method comprising administering (e.g., systemically) the composition of claim 1 or 2, thereby providing an effective amount or activity of the therapeutic agent in the lung cell of the subject that is at least 1.1 times the corresponding amount or activity of the therapeutic agent achieved in spleen cells of the subject.

56. The method of claim 55, wherein the effective amount or activity of the therapeutic agent in the lung cells of the subject is at least 1.1-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 18-fold, at least 20-fold, or at least about 50-fold greater than the corresponding amount or activity of the therapeutic agent achieved in liver cells of the subject.