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

Abstract

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

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 294,066, filed December 27, 2021, which is incorporated by reference in its entirety.

[0002] Statement regarding federally sponsored research This invention was made with Government support under Grant No. R01 EB025192-01A1 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] background Nucleic acids, which enable gene silencing, expression, and editing, have great potential for use as genetic medicines in multiple clinical settings, including cancer, inherited genetic disorders, and infectious diseases. Due to the unfavorable pharmacokinetic properties of nucleic acids, viral and non-viral delivery approaches are used to facilitate nucleic acid delivery to target cells. Lipid nanoparticles (LNPs) represent the most clinically mature non-viral platform for the safe and effective delivery of genetic medicines. Indeed, LNPs were the enabling technology for the 2018 US FDA approval of the first siRNA drug, Onpattro, and for the mRNA vaccines currently in circulation for immunization against the SARS-CoV-2 virus, the causative agent of the COVID-19 pandemic. Despite this advancement, intravenously (IV) administered LNPs typically accumulate in the liver and are internalized by liver hepatocytes, thereby greatly limiting the scope of their therapeutic applications. Summary of the Invention

[0004] overview The present disclosure provides, in some embodiments, a composition comprising a therapeutic agent assembled with a lipid composition. The lipid composition may comprise an ionizable cationic lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains each comprising about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and, for example, a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. The lipid composition may be characterized by an apparent ionization constant (pKa) of about 6 to about 6 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.

[0005] In some embodiments, the present disclosure provides compositions comprising a therapeutic agent assembled with a lipid composition. The lipid composition may comprise an ionizable cationic lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains each comprising about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and, for example, a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. The lipid composition may be characterized by an apparent ionization constant (pKa) outside the range of about 6 to about 7 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 6 or less as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 3 to about 6 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay.

[0006] In some embodiments, the present disclosure provides a composition comprising a therapeutic agent assembled with a lipid composition. The lipid composition may comprise an ionizable cationic lipid; a polymer-conjugated lipid comprising one or more hydrocarbon chains each comprising about 8 to about 20 (e.g., about 8 to about 18, about 8 to about 16, or about 8 to about 14) carbon atoms; and, for example, a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. The lipid composition may be characterized by an apparent ionization constant (pKa) of about 8 or greater as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 9 or greater as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 8 to about 13 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by an apparent ionization constant (pKa) of about 9 to about 13 as determined by a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) titration assay. In some embodiments, the lipid composition is characterized by a zeta (ζ) potential of the lipid composition of about -10 millivolts (mV) to about 10 mV as determined by dynamic light scattering (DLS). In some embodiments, the lipid composition is characterized by a zeta (ζ) potential of the lipid composition of about 0 millivolts (mV) to about 10 mV as determined by dynamic light scattering (DLS). In some embodiments, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. In some embodiments, the one or more hydrocarbon chains each contain from about 8 to about 18 carbon atoms. In other embodiments, the one or more hydrocarbon chains each contain from about 8 to about 16 carbon atoms. In yet other embodiments, the one or more hydrocarbon chains each contain from about 8 to about 14 carbon atoms.In some embodiments, one of the one or more hydrocarbon chains of the polymer-conjugated lipid contains 3 or less unsaturated carbon-carbon bonds. In other embodiments, one of the one or more hydrocarbon chains of the polymer-conjugated lipid contains 2 or less unsaturated carbon-carbon bonds. In some embodiments, the polymer-conjugated lipid comprises a polymer having a molecular weight of about 100 Daltons (Da) to about 100,000 Da. In other embodiments, the polymer-conjugated lipid comprises a polymer having a molecular weight of about 500 Da to about 100,000 Da. In some embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 20%. In other embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 15%. In still other embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 10%.

[0007] In some embodiments, the present disclosure provides cationic ionizable lipids (e.g., ionizable cationic lipids). In some embodiments, the cationic ionizable lipids comprise dendrons or dendrimers comprising one or more branches, each of which comprises two or more degradable functional groups. In some embodiments, the cationic ionizable lipids are dendrons or dendrimers comprising one or more diacyl groups. In some embodiments, the ionizable cationic lipids have the structural formula: In some embodiments, the core is a generation (g) dendrimer or dendron having the structural formula (X コア ): TIFF2025500547000002.tif13128, wherein Q is independently for each occurrence a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b - and R 2 is expressed independently for each occurrence as R1g or -L 2 -NR 1e R 1f and R 3a and R 3b are each independently hydrogen or substituted at each occurrence (e.g., C 1 -C 6 , for example C 1 -C 3 ) alkyl, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) are each independently at each occurrence an attachment point to a branch, a hydrogen, or an optionally substituted (e.g., C 1 -C 12 ) alkyl, and L 0 , L 1 , and L 2 represents, independently at each occurrence, a covalent bond (e.g., C 1 -C 12 , for example C 1 -C 6 or C 1 -C 3 ) alkylene, (e.g., C 1 -C 12 , for example 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]-[(e.g., C 4 -C 6 )heterocycloalkyl]-[(e.g., C 1 -C 6 ) alkylene], [(e.g., C 1 -C 6 ) alkylene]-(arylene)-[(e.g., C 1 -C 6 ) alkylene] (e.g., [(e.g., C 1 -C 6) alkylene]-phenylene-[(e.g., C 1 -C 6 ) alkylene]), (e.g., C 4 -C 6 ) heterocycloalkyl, and arylene (e.g., phenylene); or alternatively, L 1 Part of R 1c and R 1d One of the following (e.g., C 4 -C 6 ) heterocycloalkyl (e.g., containing 1 or 2 nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur), and x 1 is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, each branch of the plurality (N) of branches is independently represented by the structural formula (X 分枝 ): TIFF2025500547000003.tif11128, where * indicates the attachment point of the branch to the core, g is 1, 2, 3, or 4, and Z is 2 (g-1) and when g is 1, G is 0, or when g ≠ 1, G is In some embodiments, each diacyl group is independently represented by the structural formula TIFF2025500547000005.tif16128, in which * indicates the attachment point of a diacyl group at its proximal end, ** indicates the attachment point of a diacyl group at its distal end, and Y 3 is, independently at each occurrence, 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 ) arenylene, and A 1 and A 2 is, independently at each occurrence, -O-, -S-, or -NR 4 - where R 4may be hydrogen or substituted (e.g., C 1 -C 6 ) alkyl, m 1 and m 2 is independently at each occurrence 1, 2, or 3, and R 3c , R 3d , R 3e , and R 3f are each independently hydrogen or substituted at each occurrence (e.g., C 1 -C 8 In some embodiments, each linker group is independently represented by the structural formula TIFF2025500547000006.tif12128, where ** indicates the point of attachment of the linker to the proximal diacyl group, *** indicates the point of attachment of the linker to the distal diacyl group, and Y 1 is, independently at each occurrence, 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 In some embodiments, each terminal group is independently optionally substituted (e.g., C 1 -C 18 , for example C 4 -C 18 ) alkylthiols, and optionally substituted (e.g., C 1 -C 18 , for example C 4 -C 18 In some embodiments, x is selected from the group consisting of alkenyl thiols. 1 is 0, 1, 2, or 3. In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) each independently in each occurrence represents a connection point to a branch (e.g., as indicated by *), hydrogen, or C1 -C 12 Alkyl (e.g., C 1 -C 8 Alkyl, e.g. C 1 -C 6 Alkyl or C 1 -C 3 alkyl), the alkyl portion being -OH, C 4 -C 8 (For example, C 4 -C 6 ) heterocycloalkyl (e.g., piperidinyl (e.g., TIFF2025500547000007.tif15128), N-(C 1 -C 3 alkyl)-piperidinyl (e.g. TIFF2025500547000008.tif16128), piperazinyl (e.g., TIFF2025500547000009.tif13128), N-(C 1 -C 3 Alkyl)-piperadizinyl (e.g. TIFF2025500547000010.tif16128), morpholinyl (e.g., TIFF2025500547000011.tif13128), N-pyrrolidinyl (e.g., TIFF2025500547000012.tif13128), pyrrolidinyl (e.g., TIFF2025500547000013.tif13128), or N-(C 1 -C 3 alkyl)-pyrrolidinyl (e.g. TIFF2025500547000014.tif13128)), (e.g., C 6 -C 10 ) aryl, and C 3 -C 5 Heteroaryl (e.g., imidazolyl (e.g., TIFF2025500547000015.tif14128), or pyridinyl (e.g., In some embodiments, R is optionally substituted with one or more substituents each independently selected from the group consisting of: 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) each independently in each occurrence represents a connection point to a branch (e.g., as indicated by *), hydrogen, or C 1 -C 12 Alkyl (e.g., C 1 -C 8 Alkyl, e.g. C 1 -C 6 Alkyl or C 1 -C 3 In some embodiments, R is an alkyl group, the alkyl portion of which is optionally substituted with one substituent -OH. 3a and R 3b are each independently at each occurrence hydrogen. In some embodiments, the plurality of (N) branches comprises at least 3 (e.g., at least 4, or at least 5) branches. In some embodiments, g=1; G=0; and Z=1. In some embodiments, each branch of the plurality of branches is represented by the structural formula In some embodiments, g=2; G=1; and Z=2. In some embodiments, each branch of the plurality of branches is represented by the structural formula In some embodiments, g=3; G=3; and Z=4. In some embodiments, each branch of the plurality of branches is represented by the structural formula In some embodiments, g=4; G=7; and Z=8. In some embodiments, each branch of the plurality of branches is represented by the structural formula Contains TIFF2025500547000020.tif79154.

[0008] In some embodiments, the core has the structural formula: In some embodiments, the core has the structural formula: In some embodiments, the core has the structural formula: In other embodiments, the core comprises the structural formula: In yet other embodiments, the core comprises the structural formula: TIFF2025500547000025.tif13128, wherein Q′ is —NR 2 -OR-CR 3a R 3b -And;q 1 and q 2 is each independently 1 or 2. In some embodiments, the core has the structural formula: In some embodiments, the core comprises the structural formula TIFF2025500547000027.tif65149, wherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C 3 -C 12 , for example C 3 -C 5 ) heteroaryl. In some embodiments, the core is of the formula TIFF2025500547000028.tif20128. In some embodiments, the core has TIFF2025500547000029.tif236150, and pharma- ceutically acceptable salts thereof, wherein * indicates the attachment point of the core to one branch of the multiple branches. 1 In some embodiments, A is -O- or -NH-. 2 In some embodiments, Y is -O- or -NH-. 3 is C 1-C 12 (For example, C 1 -C 6 , for example C 1 -C 3 ) alkylene. In some embodiments, a diacyl group is represented independently at each occurrence by the structural formula TIFF2025500547000030.tif37128, optionally comprising: 3c , R 3d , R 3e , and R 3f is independently at each occurrence hydrogen or C 1 -C 3 In some embodiments, L is alkyl. 0 , L 1 , and L 2 represents, independently at each occurrence, a covalent bond; C 1 -C 6 Alkylene (e.g., C 1 -C 3 alkylene), C 2 -C 12 (For example, C 2 -C 8 ) alkylene oxides (e.g., oligo(ethylene oxide), e.g., -(CH 2 CH 2 O) 1-4 -(CH 2 CH 2 )-), [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., TIFF2025500547000031.tif16128), and [(C 1 -C 4 ) alkylene]-phenylene-[(C 1 -C 4 ) alkylene] (e.g., TIFF2025500547000032.tif16128). In some embodiments, L 0 , L 1 , and L 2each occurrence independently represents 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 In some embodiments, L is selected from 0 , L 1 , and L 2 each occurrence independently represents C 1 -C 6 Alkylene (e.g., C 1 -C 3 In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents C 2 -C 12 (For example, C 2 -C 8 ) alkylene oxides (e.g., -(C 1 -C 3 Alkylene-O) 1-4 -(C 1 -C 3 In some embodiments, L is 0 , L 1 , and L 2 are each independently, at each occurrence, 1 -C 4 ) alkylene]-[(C 4 -C 6 )Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 Alkylene)-phenylene-(C1 -C 3 alkylene)-), and [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 Alkylene)-piperazinyl-(C 1 -C 3 alkylene)-).

[0009] In some embodiments, each end group is independently C 1 -C 18 (For example, C 4 -C 18 ) alkenylthiol or C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl or alkenyl moiety being halogen, C 6 -C 12 Aryl (e.g., phenyl), C 1 -C 12 (For example, C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 Mono-alkylamino (e.g. -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 Di-alkylamino (e.g. TIFF2025500547000033.tif18128)), C 4 -C 6 N-heterocycloalkyl (e.g., N-pyrrolidinyl TIFF2025500547000034.tif15128, N-Piperidinyl TIFF2025500547000035.tif15128, N-Azepanil TIFF2025500547000036.tif15128), -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., TIFF2025500547000037.tif13128), -C(O)N(C 1 -C 3 Alkyl)-(C 1 -C 6 Alkylene)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000038.tif14128), -C(O)-(C 1 -C 12 alkylamino (e.g., mono- or di-alkylamino), and -C(O)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000039.tif18128), wherein C is any of the aforementioned substituents. 4 -C 6 The N-heterocycloalkyl moiety is 1 -C 3 Alkyl or C 1 -C 3 In some embodiments, each terminal group is independently substituted with C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl portion being C 6 -C 12 Aryl (e.g., phenyl), C 1 -C 12 (For example, C 1 -C 8 ) alkylamino (e.g., C 1 -C6 Mono-alkylamino (e.g. -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 Di-alkylamino (e.g. TIFF2025500547000040.tif17128)), C 4 -C 6 N-heterocycloalkyl (e.g., N-pyrrolidinyl TIFF2025500547000041.tif15128, N-Piperidinyl TIFF2025500547000042.tif15128, N-Azepanil TIFF2025500547000043.tif15128), -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., TIFF2025500547000044.tif13128), -C(O)N(C 1 -C 3 Alkyl)-(C 1 -C 6 Alkylene)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000045.tif14128), and -C(O)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000046.tif18128), wherein C of any of the foregoing substituents is optionally substituted with one or more (e.g., one) substituents each independently selected from 4 -C 6 The N-heterocycloalkyl moiety is 1 -C 3 Alkyl or C 1-C 3 In some embodiments, each terminal group is independently substituted with C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl moiety being optionally substituted with one substituent -OH. In some embodiments, each terminal group is independently selected from the group consisting of C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl portion being C 1 -C 12 (For example, C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 Mono-alkylamino (e.g. -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 Di-alkylamino (e.g. TIFF2025500547000047.tif18128)) and C 4 -C 6 N-heterocycloalkyl (e.g., N-pyrrolidinyl TIFF2025500547000048.tif15128, N-Piperidinyl TIFF2025500547000049.tif15128, N-Azepanil In some embodiments, each terminal group is independently substituted with one substituent selected from the group consisting of: 1 -C 18 (For example, C 4 -C 18 ) alkenylthiol or C 1 -C 18 (For example, C 4 -C 18 In some embodiments, each terminal group is independently C 1 -C 18(For example, C 4 -C 18 ) alkylthiol. In some embodiments, each terminal group is independently TIFF2025500547000051.tif61155.

[0010] In some embodiments, the dendrimer or dendron is TIFF2025500547000052.tif179156TIFF2025500547000053.tif190150TIFF2025500547000054.tif147150TIFF2025500547000055.tif89128, and pharma- ceutically acceptable salts thereof. In some embodiments, the lipid composition comprises about 5% to about 30% molar percentage of the ionizable cationic lipid. In some embodiments, the lipid composition further comprises a phospholipid. In some embodiments, the lipid composition comprises about 5% to about 30% molar percentage of the phospholipid. In other embodiments, the lipid composition comprises about 8% to about 23% molar percentage of the phospholipid. In some embodiments, the phospholipid is not ethylphosphocholine. In some embodiments, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of the steroid or steroid derivative of about 15% to about 46%. In some embodiments, the steroid or steroid derivative is cholesterol. In some embodiments, the SORT lipid is cationic. In some embodiments, the SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety).

[0011] In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000056.tif26128.

[0012] In some embodiments, L is a bond or a (e.g., biodegradable) linker. 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. In some embodiments, R', R'', and R''' are each independently alkyl. (C≦6) or substituted alkyl (C≦6) In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000057.tif23128.

[0013] In some embodiments, R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6) In some embodiments, the SORT lipid comprises a permanent cationic moiety (e.g., a quaternary ammonium ion). In some embodiments, the SORT lipid comprises a counterion to the permanent cationic moiety. In some embodiments, the SORT lipid is an alkylated phosphocholine (e.g., ethylphosphocholine). In some embodiments, the SORT lipid has the structural formula: TIFF2025500547000058.tif5128, where L is a bond or a (e.g., biodegradable) linker; Z + is a positively charged moiety (e.g., a quaternary ammonium ion); and X - is a counter ion. In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000059.tif26128.

[0014] In some embodiments, R 1 and R 2 each independently represents an optionally substituted C 6 -C 24 Alkyl or optionally substituted C 6 -C 24 In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000060.tif26128.

[0015] In some embodiments, R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. In some embodiments, R', R'', and R''' are each independently alkyl. (C≦6) or substituted alkyl (C≦6) In some embodiments, X - is a monovalent anion. In some embodiments, L is In some embodiments, p and q are each independently 1, 2, or 3. In some embodiments, R 4 is optionally substituted C 1 -C 6 In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000062.tif24128.

[0016] In some embodiments, R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6)In some embodiments, R 4 is an alkyl (C≦6) or substituted alkyl (C≦6) In some embodiments, X - is a monovalent anion. In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000063.tif24128.

[0017] In some embodiments, R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6) In some embodiments, X - is a monovalent anion. In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000064.tif15128.

[0018] In some embodiments, R 4 and R 4 ' are each independently an alkyl (C6-C24) , alkenyl (C6-C24) or a substituted version of either group. 4 '' is an alkyl (C≦24) , alkenyl (C≦24) or a substituted version of either group. 4 ''' is an alkyl (C1-C8) , alkenyl (C2-C8) or a substituted version of either group. 2is a monovalent anion. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid from about 20% to about 65%. In some embodiments, the SORT lipid is zwitterionic. In some embodiments, the SORT lipid comprises a hydrophobically modified phosphate, sulfonate, or carboxylate anion. In some embodiments, the SORT lipid is anionic. In some embodiments, the SORT lipid has the structural formula: I have TIFF2025500547000065.tif24128.

[0019] In some embodiments, R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group. 3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y 1 -R 4 In some embodiments, Y 1 is an alkanediyl (C≦6) or substituted alkanediyl (C≦6) In some embodiments, R 4 is acyloxy (C≦8-24) or substituted acyloxy (C≦8-24) It is.

[0020] In some embodiments, the lipid composition is characterized by an average diameter of about 200 nanometers (nm) or less as determined by dynamic light scattering (DLS). In other embodiments, the lipid composition is characterized by an average diameter of about 150 nanometers (nm) or less as determined by dynamic light scattering (DLS). In still other embodiments, the lipid composition is characterized by an average diameter of about 100 nanometers (nm) or less as determined by dynamic light scattering (DLS). In some embodiments, the lipid composition is characterized by a polydispersity index (PDI) of about 0.2 or less as determined by dynamic light scattering (DLS). In some embodiments, the lipid composition is characterized by a lipid fusion percentage of at least about 5%, 6%, 7%, 8%, 9%, or 10% as determined by a fluorescence resonance energy transfer (FRET)-based assay.

[0021] In some embodiments, the therapeutic agent comprises a compound, a polynucleotide, a polypeptide, a protein, or a combination thereof. In some embodiments, the therapeutic agent comprises a polypeptide or a protein. In some embodiments, the therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro ribonucleic acid (miRNA), a primary micro ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CLRs), a nucleotide sequence encoding a ... The therapeutic agent may be a CRISPR-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof. In some embodiments, the therapeutic agent comprises a polynucleotide; and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 20:1 or less. In some embodiments, the N / P ratio is about 5:1 to about 20:1. In some embodiments, the therapeutic agent comprises two or more polynucleotides comprising the polynucleotide. In some embodiments, the molar ratio of the therapeutic agent to total lipid of the lipid composition is about 1:1, 1:10, 1:50, or 1:100 or less. In some embodiments, at least about 85% of the therapeutic agent is encapsulated in particles of the lipid composition. In some embodiments, the SORT lipid is present in the composition in an amount sufficient to achieve a therapeutic effect at a dose of the therapeutic agent lower (e.g., at least about 1.1-fold or 10-fold) than that required in a reference lipid composition.In some embodiments, the therapeutic agent (e.g., heterologous polynucleotide) is present in the composition at a dose of about 2 milligrams per kilogram of body weight (mg / kg, or mpk) or less. The therapeutic agent (e.g., heterologous polynucleotide) is present in the intravenous composition at a dose of about 1.0, 0.5, 0.1, 0.05, or 0.01 mg / kg of body weight or less. In some embodiments, the therapeutic agent is present in the aerosol composition at a dose of about 1.0, 0.5, 0.1, 0.05, or 0.01 mg / kg of body weight or less. In some embodiments, the therapeutic agent (e.g., heterologous polynucleotide) is present in the intravenous dosage form at a concentration of about 5 or 2 milligrams per milliliter (mg / mL) or less.

[0022] The present disclosure also provides, in some embodiments, a method for targeted delivery of therapeutic agent to organ or cells therein in a subject in need thereof.The method can include administering to a subject a therapeutic agent assembled with a lipid composition comprising ionizable cationic lipid; polymer-conjugated lipid; and, for example, selective organ targeting (SORT) lipid, which is separate from the ionizable cationic lipid and the polymer-conjugated lipid, and when administered, the surface of the lipid composition binds to a plurality of target proteins, as determined by incubation assay, and the plurality of target proteins comprises a first target protein with a weight or mass ratio of about 20:1, 15:1, or 10:1 or less to a second target protein different from the first target protein, thereby delivering the therapeutic agent to a target organ or target cell in the subject.In some embodiments, the composition is according to any of the compositions described herein. In some embodiments, the method provides an amount, expression, or activity of the therapeutic agent in the organ of the subject or in the cells therein that is greater (e.g., at least about 2-fold) than that achieved with a corresponding reference lipid composition (e.g., without binding to the multiple target proteins). In some embodiments, the method provides an amount, expression, or activity of the therapeutic agent in the organ of the subject or in the cells therein that is greater (e.g., at least about 2-fold) than that achieved without the polymer-conjugated lipid. In some embodiments, the method provides an amount, expression, or activity of the therapeutic agent in the organ of the subject or in the cells therein that is greater (e.g., at least about 2-fold) than that achieved in a reference organ or reference cells.In some embodiments, the therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro ribonucleic acid (miRNA), a primary micro ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof.

[0023] In some embodiments, the present disclosure provides a method for targeted delivery of a therapeutic agent to an organ (e.g., liver) or cells therein (e.g., liver) in a subject in need thereof, comprising administering to the subject a therapeutic agent assembled with a lipid composition comprising an ionizable cationic lipid; a polymer-conjugated lipid; and, for example, a selective organ targeting (SORT) lipid other than the ionizable cationic lipid and the polymer-conjugated lipid, wherein upon administration, the surface of the lipid composition binds to a plurality of target proteins as determined by incubation assay, the plurality of target proteins comprising apolipoprotein E (Apo E) and serum albumin, thereby delivering the therapeutic agent to a target organ or target cells in the subject. In some embodiments, Apo E is present in the plurality of target proteins at a weight or mass ratio of about 6:1, 5:1, 4:1, or 3:1 or less to serum albumin as determined by incubation assay. In some embodiments, the plurality of target proteins further comprises complement C1q subcomponent subunit A, immunoglobulin heavy constant μ, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, or any combination thereof, as determined by incubation assay. In some embodiments, the SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety). In some embodiments, the SORT lipid is an ionizable cationic lipid. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid of about 5% to about 65%. In some embodiments, the lipid composition is according to any lipid composition provided herein.In some embodiments, the method provides an amount, expression, or activity of the therapeutic agent in the liver or liver cells in the subject that is greater (e.g., at least about 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold) than that achieved with a corresponding reference lipid composition (e.g., without the binding to the multiple target proteins).

[0024] In some embodiments, the disclosure provides a method for targeted delivery of a therapeutic agent to a non-liver organ or non-liver cells therein in a subject in need thereof, comprising administering to the subject a therapeutic agent assembled with a lipid composition comprising an ionizable cationic lipid; a polymer-conjugated lipid; and, e.g., a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein upon administration, the surface of the lipid composition interacts with Apolipoprotein E (Apo E) to a lesser extent than with an exogenous protein in the subject that is not Apo E, as determined by an incubation assay, wherein the endogenous protein that is not Apo E is selected from beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement C1q subcomponent subunit A, vitronectin, and serum paraoxonase / arylesterase 1, thereby delivering the therapeutic agent to the non-liver organ or non-liver cells in the subject. In some embodiments, the non-liver organ comprises a lung, spleen, bone marrow, or lymph node. In some embodiments, the non-liver cell comprises a lung cell, a spleen cell, or a macrophage. In some embodiments, apolipoprotein E (Apo E) is not the most abundant protein in the plurality of target proteins. In some embodiments, upon administration, the surface of the lipid composition interacts with apolipoprotein C (Apo C) to a lesser extent than with apolipoprotein E (Apo E) in the subject as determined by an incubation assay. In some embodiments, the method provides an amount or activity of the therapeutic agent in the liver or cells therein in the subject that is less than that achieved without the polymer-conjugated lipid. In some embodiments, the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid of about 5% to about 65%. In some embodiments, the lipid composition is according to any of the lipid compositions described herein.

[0025] In some embodiments, the present disclosure provides a method for targeted delivery of therapeutic agent to lung or lung cells in a subject in need thereof, comprising administering therapeutic agent assembled with lipid composition to the subject, said lipid composition comprises ionizable cationic lipid; polymer-conjugated lipid; and selective organ targeting (SORT) lipid, for example, other than said ionizable cationic lipid and said polymer-conjugated lipid, and when administered, the surface of lipid composition binds to a plurality of target proteins, as determined by incubation assay, said plurality of target proteins comprises vitronectin (Vtn) and clusterin, thereby delivering therapeutic agent to lung or lung cells in the subject.In some cases, vitronectin is present in the plurality of target proteins at a weight or mass ratio of about 6:1 or 5:1 or less to clusterin, as determined by incubation assay. In some embodiments, the multiple target proteins are selected from the group consisting of serum paraoxonase / arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 (β2-GP1), or apolipoprotein H (Apo E), as determined by an incubation assay. H), immunoglobulin (Ig) μ chain C region, α-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen α chain, vitamin K-dependent protein Z, α-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein AI, immunoglobulin heavy constant α, immunoglobulin (Ig) gamma-2A chain C region, β-globin, complement C1q subcomponent subunit C, protein Z-dependent protease inhibitor, or any combination thereof. In some embodiments, the SORT lipid is a cationic lipid. In some embodiments, the SORT lipid is a permanent cationic lipid.In some embodiments, the SORT lipid is an ionizable cationic lipid. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid of about 5% to about 65%. In some embodiments, the lipid composition is according to any lipid composition provided herein. In some embodiments, the method provides for an amount, expression, or activity of the therapeutic agent in the lung or lung cells in the subject that is greater (e.g., at least about 2-fold, 5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold) than that achieved with a corresponding reference lipid composition (e.g., no binding to the multiple target proteins). In some embodiments, the therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro ribonucleic acid (miRNA), a primary micro ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof.

[0026] In some embodiments, the present application provides a method for targeted delivery of a therapeutic agent to the spleen, bone marrow, or lymph nodes, or cells therein, in a subject in need thereof, comprising administering to the subject a therapeutic agent assembled with a lipid composition, the lipid composition comprising an ionizable cationic lipid; a polymer-conjugated lipid; and, for example, a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid, wherein upon administration, the surface of the lipid composition binds to a plurality of target proteins, as determined by an incubation assay, the plurality of target proteins comprising beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H) in a weight or mass ratio of about 20:1, 15:1, or 10:1 or less to a second target protein different from beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H), thereby delivering the therapeutic agent to the spleen, bone marrow, or lymph nodes, or cells in the subject. In some embodiments, the cells comprise spleen cells or macrophages. In some embodiments, the second target protein is selected from immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein CI, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) μ chain C region, serum albumin, serum paraoxonase / arylesterase 1, immunoglobulin heavy constant alpha, and immunoglobulin kappa variable 6-13. In some embodiments, the SORT lipid is a permanent cationic lipid or an anionic lipid. In some embodiments, the SORT lipid is a permanent cationic lipid. In some embodiments, the SORT lipid is an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid of about 5% to about 65%. In some embodiments, the lipid composition is according to any of the lipid compositions provided herein.In some embodiments, the method provides an amount, expression, or activity of the therapeutic agent in the lung or lung cells in the subject that is greater (e.g., at least about 2-fold) than that achieved with a corresponding reference lipid composition (e.g., without binding to the multiple target proteins).

[0027] In some embodiments, the present disclosure provides a method for targeted delivery of therapeutic agents to non-splenic organs or non-splenic cells in a subject in need thereof, comprising administering to the subject a therapeutic agent assembled with a lipid composition, the lipid composition comprising ionizable cationic lipids; polymer-conjugated lipids; and selective organ targeting (SORT) lipids, e.g., separate from the ionizable cationic lipids and the polymer-conjugated lipids, wherein upon administration, the surface of the lipid composition binds to a plurality of target proteins, as determined by incubation assay, and the plurality of target proteins comprises a first target protein to a second target protein different from the first target protein in a weight or mass ratio of about 20:1, 15:1, or 10:1 or less, thereby delivering the therapeutic agent to non-splenic organs or non-splenic cells in the subject. In some embodiments, the non-splenic organ is not spleen, bone marrow, or lymph node. In some embodiments, the non-splenic cell is not spleen cell or macrophage. In some embodiments, beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H) is not the most abundant protein in the plurality of target proteins. In some embodiments, the plurality of target proteins comprises clusterin. In some embodiments, the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of the SORT lipid of about 5% to about 65%. In some embodiments, the lipid composition is according to any lipid composition provided herein.In some embodiments, the therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a micro ribonucleic acid (miRNA), a primary micro ribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof.

[0028] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. In some embodiments, the one or more hydrocarbon chains each comprise about 8 to about 20 carbon atoms. In some embodiments, the one or more hydrocarbon chains each comprise about 8 to about 18 carbon atoms. In some embodiments, the one or more hydrocarbon chains each comprise about 8 to about 16 carbon atoms. In some embodiments, the one or more hydrocarbon chains each comprise about 8 to about 14 carbon atoms. In some embodiments, one of the one or more hydrocarbon chains of the polymer-conjugated lipid comprises 3 or less unsaturated carbon-carbon bonds. In some embodiments, one of the one or more hydrocarbon chains of the polymer-conjugated lipid comprises 2 or less unsaturated carbon-carbon bonds. In some embodiments, the polymer-conjugated lipid comprises a polymer having a molecular weight of about 100 Daltons (Da) to about 100,000 Da. In some embodiments, the polymer-conjugated lipid comprises a polymer having a molecular weight of about 500 Da to about 100,000 Da. In some embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 20%. In some embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 15%. In some embodiments, the lipid composition comprises a molar percentage of the polymer-conjugated lipid of about 0.5% to about 10%. In some embodiments, the administering step comprises intravenous administration. In some embodiments, the subject's bodily fluid (e.g., plasma or serum) comprises the plurality of target proteins. In some embodiments, the plurality of target proteins are a plurality of endogenous proteins of the subject.

[0029] Additional aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present application are shown and described. As will be understood, the present application is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0030] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to supersede and / or supersede any such conflicting material. [Brief description of the drawings]

[0031] The features of the present disclosure are set forth with particularity in the appended claims. 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 disclosure are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG.").

[0032] [Figure 1A]1A-1E show selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery, including their unique biodistribution and ionization behavior. As shown in FIG. 1A, by adding a supplemental (e.g., fifth) SORT molecule to a reference, e.g., four-component lipid composition (15:15:30:3 5A2-SC8:DOPE:cholesterol:C14-PEG2K mol:mol), the tissue-specific activity of the delivered mRNA changes based on the chemical structure of the included SORT molecule. For example, ionizable cationic lipids (DODAP) enhance liver-specific mRNA translation (Liver SORT- 5A2-SC8:DOPE:Cholesterol:C14-PEG2K:DODAP at 15:15:30:3:15.75), anionic lipids (18PA) result in spleen-specific mRNA translation (Spleen SORT- 5A2-SC8:DOPE:Cholesterol:C14-PEG2K:18PA at 15:15:30:3:27), and cationic quaternary ammonium lipids (DOTAP) result in lung-specific mRNA translation (Lung SORT- 5A2-SC8:DOPE:Cholesterol:C14-PEG2K:DOTAP at 15:15:30:3:63). [Figure 1B] Figures 1A-1E show selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery, including their unique biodistribution and ionization behavior. Figure 1B shows ex vivo fluorescence of Cy5-labeled mRNA in major organs extracted from C57BL / 6 mice intravenously injected with exemplary SORT LNPs (as described herein) incorporating increasing percentages of various SORT molecules (0.5 mg / kg mRNA / body weight, 6 hours). [Figure 1C]Figures 1A-1E show selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery, including their unique biodistribution and ionization behavior. Figure 1C shows the relative mean Cy5 fluorescence measured in liver, lung, and spleen as a function of percent inclusion of SORT molecules (0.5 mg / kg mRNA / body weight, n=2). SORT molecules promote biodistribution of mRNA to target organs. Data are shown as mean ± sem. [Figure 1D] Figures 1A-1E show selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery, including their unique biodistribution and ionization behavior. Figure 1D shows representative TNS assay curves for determining the apparent pKa of exemplary SORT LNPs (as described herein) incorporating increasing percentages of ionizable cationic lipid, anionic lipid, or cationic lipid SORT molecules. The apparent pKa was defined as the point at which 50% of the TNS fluorescence was achieved. [Figure 1E] Figures 1A-1E show selective organ targeting (SORT) lipid compositions (e.g., nanoparticles) for tissue-specific mRNA delivery, including their unique biodistribution and ionization behavior. As shown in Figure 1E, LNPs were assigned a tissue specificity score based on the tissue in which functional luciferase mRNA was detected (liver expression = 1.0, spleen expression = 2.0, lung expression = 3.0). For the 67 LNPs tested in the TNS assay, the apparent pKa of the LNP correlated with the specificity of tissue delivery of luciferase mRNA. [Diagram 2]Figures 2A-2H show, by way of example, that multiple steps are involved in the mechanism of SORT LNP tissue targeting, including the formation of a unique protein corona. Figure 2A shows, by way of example only, a proposed three-step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs, in which (1) PEG-lipid detachment allows (2) distinct serum proteins to bind to the SORT LNPs, resulting in (3) cellular internalization in the target tissue via receptor-mediated uptake. Figure 2B shows ex vivo bioluminescence of major organs isolated from C57BL / 6 mice IV-injected with exemplary liver, spleen, and lung SORT LNPs incorporating either sheddable PEG-lipids (C14-PEG2K) or less-sheddable PEG-lipids (C18-PEG2K) (0.1 mg FLuc mRNA / kg body weight, 6 hours). The total luminescence generated by each organ was reduced when using PEG-lipids that were difficult to shed, suggesting that PEG-lipid detachment is a key process for effective mRNA delivery by the SORT LNPs tested. Figure 2C shows quantification of the total luminescence generated by functional protein translated from FLuc mRNA in target organs of C57BL / 6 mice IV-injected with exemplary liver SORT LNPs, spleen SORT LNPs, and lung SORT LNPs incorporating either C14-PEG2K or C18-PEG2K (0.1 mg FLuc mRNA / kg body weight, 6 hours). Figure 2D shows ELISA quantification of serum hEPO in C57BL / 6 mice treated with exemplary liver SORT LNPs, spleen SORT LNPs, or lung SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA / kg body weight, 6 hours). The use of PEGs that are difficult to shed reduces the efficacy of SORT LNPs. 2E shows SDS-PAGE of serum proteins adsorbed to the surfaces of a reference mDLNP, an exemplary liver SORT LNP, an exemplary spleen SORT LNP, and an exemplary lung SORT LNP. LNPs with different organ targeting properties bind distinct serum proteins.FIG. 2F shows the average abundance of proteins with distinct biological functions in the protein corona of reference mDLNPs and exemplary liver SORT LNPs, spleen SORT LNPs, and lung SORT LNPs. The choice of SORT molecule results in large-scale differences in the functional population of serum proteins that bind to the LNPs. FIG. 2G shows the isoelectric distribution of the most enriched proteins, which constitute 80% of the protein corona of the LNPs. The head group structure of the SORT molecule affects the isoelectric distribution of the protein corona. FIG. 2H shows the top five most abundant serum proteins that bind to different exemplary SORT LNPs (n=3). The chemical structure of the SORT molecule influences the first-ranked serum proteins that are most highly enriched on the surface of the exemplary SORT LNPs. Data are shown as mean±sem. Statistical significance was determined using an unpaired two-tailed Student's t-test (*, p<0.05). [Diagram 3]Figures 3A-3D show that distinct serum proteins control the uptake and efficacy of exemplary SORT LNPs in vitro. As shown in Figure 3A, exemplary SORT LNPs were pre-incubated with either ApoE, β2-GPI, or Vtn prior to treating relevant cell lines to measure cellular uptake (Cy5-mRNA tracking) or functional mRNA delivery (bioluminescence). Figure 3B shows representative images of cellular uptake of uncoated and coated SORT LNPs (as examples) taken up by relevant cell types. Incubation of an exemplary SORT LNP with the protein to which it most tightly binds increases mRNA uptake in cell lines expressing the cognate receptor (250 ng mRNA per well, 1.5 h, scale bar = 50 μm). Figure 3C shows quantification of Cy5-mRNA fluorescence in cells treated with uncoated or coated SORT LNPs as examples (250 ng mRNA per well, 1.5 h, n = 10). Statistical significance was determined using unpaired two-tailed Student's t-test (****, p<0.0001; *, p<0.05). Figure 3D shows the activity of functional luciferase protein translated from mRNA delivered by exemplary SORT LNPs preincubated with the respective proteins in relevant cell lines (25 ng mRNA, 24 hours, n=4). Statistical significance was determined using one-way ANOVA with Brown-Forsythe test (****, p<0.0001; ***, p<0.001; *, p<0.05). Individual proteins bind exclusively to specific SORT LNPs and enhance mRNA delivery only to cell lines expressing their cognate receptors. Data are shown as mean ± sem. [Figure 4]Figures 4A-4C show that extrahepatic mRNA delivery occurs via an ApoE-independent mechanism. Figure 4A shows ex vivo bioluminescence generated by functional protein translated from FLuc mRNA in major organs isolated from wild-type C57BL / 6 mice IV-injected with reference mDLNP or exemplary liver SORT LNP, spleen SORT LNP, or lung SORT LNP (0.1 mg / kg FLuc mRNA, 6 hours). The role of ApoE on the efficacy of SORT LNP varies based on the chemical structure of the included SORT molecule. Figure 4B shows ex vivo bioluminescence generated by functional protein translated from FLuc mRNA in major organs isolated from ApoE- / - mice IV-injected with reference mDLNP or exemplary liver SORT LNP, spleen SORT LNP, or lung SORT LNP (0.1 mg / kg FLuc mRNA, 6 hours). FIG. 4C shows quantification of total bioluminescence produced by target organs isolated from wild-type and ApoE- / - mice treated with reference mDLNPs or exemplary liver SORT LNPs, spleen SORT LNPs, or lung SORT LNPs. (0.1 mg / kg FLuc mRNA, 6 hours, n=3). Data are shown as mean ± sem. Statistical significance was determined using unpaired two-tailed Student's t-tests (**, p<0.01; *, p<0.05; ns, p>0.05). Removal of ApoE from serum using gene knockout results in a significant reduction in hepatic mRNA delivery by reference mDLNPs and exemplary liver SORT LNPs. In contrast, exemplary spleen SORT LNPs enhance spleen targeting when ApoE is depleted from serum, while the efficacy of exemplary lung SORT LNPs is not affected by ApoE removal. [Diagram 5] Figure 5 shows the TNS fluorescence curves of exemplary SORT LNPs formulated with various mass fractions of cationic, anionic, ionizable, and zwitterionic lipids. The apparent pKa of the LNPs is calculated as the pH at which 50% of the TNS fluorescence is measured. [Figure 6]FIG. 6 shows apparent pKa values ​​calculated from exemplary SORT LNPs incorporating cationic, anionic, ionizable, and zwitterionic lipids at various mass fractions. [Figure 7] Figures 7A-7B show cellular uptake and functional mRNA delivery into low-density lipoprotein receptor (LDL-R)-expressing Hep G2 cells by exemplary SORT LNPs preincubated with ApoE. Figure 7A shows, as an example, quantification of Cy5-mRNA fluorescence in Hep G2 cells treated with either uncoated or ApoE-coated liver SORT LNPs (250 ng mRNA per well, 1.5 h, n=10). Statistical significance was determined using an unpaired two-tailed Student's t-test (****, p<0.0001). Figure 7B shows the activity of functional luciferase protein translated from mRNA delivered by exemplary SORT LNPs preincubated with increasing amounts of ApoE (25 ng mRNA, 24 h, n=4). Statistical significance was determined using one-way ANOVA with Brown-Forsythe test (ns, p>0.05). [Figure 8] Figures 8A-8B show cellular uptake and functional mRNA delivery into αvβ3-expressing U87-MG cells by exemplary SORT LNPs preincubated with Vtn. Figure 8A shows, as an example, quantification of Cy5-mRNA fluorescence in U87-MG cells treated with either uncoated or Vtn-coated lung SORT LNPs (250 ng mRNA per well, 1.5 h, n=10). Statistical significance was determined using an unpaired two-tailed Student's t-test (****, p<0.0001). Figure 8B shows the activity of functional luciferase protein translated from mRNA delivered by exemplary SORT LNPs preincubated with increasing amounts of Vtn (25 ng mRNA, 24 h, n=4). Statistical significance was determined using one-way ANOVA with Brown-Forsythe test (****, p<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Before describing aspects of the present disclosure, it is to be understood that such aspects are provided by way of example only, and that various alternatives to the aspects of the present disclosure described herein can be used in practicing the invention. Numerous modifications, changes, and substitutions will readily occur to those skilled in the art without departing from the invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein can be used in the practice or testing of this invention, but suitable methods and materials are described below. In case of conflict, this patent specification, including definitions, will control. In addition, the materials, methods, and examples are only illustrative and are not intended to be limiting. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from this invention.

[0035] In the context of this application, the following terms have the meanings ascribed to them unless specified otherwise.

[0036] As used throughout the specification and claims, the terms "a", "an" and "the" are generally used in the sense of meaning "at least one", "at least the first", "one or more", or "multiple" of the referenced component or step, unless an upper limit is specifically stated thereafter. For example, as used herein, "cleavage sequence" means "at least the first cleavage sequence", but includes multiple cleavage sequences. The operable limits and parameters of combinations, as well as the amounts of any single agent, will be known to those skilled in the art in light of this application.

[0037] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0038] As used herein in the context of a polypeptide structure, the "N-terminus" (or "amino terminus") and the "C-terminus" (or "carboxyl terminus") generally refer to the distal amino and carboxyl termini, respectively, of a polypeptide.

[0039] The term "N-terminal sequence", when used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that at the N-terminus, the N-terminal sequence in the polypeptide or polynucleotide sequence of interest is not preceded by any other amino acid or nucleotide residues. The term "C-terminal sequence", when used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that at the C-terminus, the C-terminal sequence in the polypeptide or polynucleotide sequence of interest is not followed by any other amino acid or nucleotide residues.

[0040] The terms "non-naturally occurring" and "non-natural" are used interchangeably herein. The term "non-naturally occurring" or "non-natural" when used herein in relation to a therapeutic or prophylactic agent generally means that the agent is not biologically derived in a mammal (including, but not limited to, a human). The term "non-naturally occurring" or "non-natural" when applied to a sequence and as used herein means a polypeptide or polynucleotide sequence that does not have a counterpart, is not complementary to, or has a high degree of homology with, a wild-type or naturally occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or less amino acid sequence identity with a natural sequence when aligned as appropriate.

[0041] "Physiological conditions" refers to in vitro conditions, including temperature, salt concentration, pH, that mimic a set of conditions in a living host and those conditions of a living subject. A large number of physiologically relevant conditions for use in in vitro assays have been established. Generally, physiological buffers contain physiological concentrations of salt and are adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. Various physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperatures range from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.

[0042] As used herein, the terms "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably herein. These terms generally refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. Therapeutic benefit refers to eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved with eradication or amelioration of one or more of the physiological symptoms, or improvement of one or more clinical parameters associated with the underlying disorder, such that improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder. For preventive benefit, the composition may be administered to a subject at risk of developing a particular disease, or to a subject who reports one or more of the physiological symptoms of the disease, even though the diagnosis of the disease may not have been made for the disease.

[0043] As used herein, "therapeutic effect" or "therapeutic benefit" generally refers to physiological effects resulting from administration of a polypeptide of the present disclosure other than the ability to induce the production of antibodies against an antigenic epitope possessed by the biologically active protein, including, but not limited to, the alleviation, amelioration, or prevention of disease in humans or other animals, or the improvement of one or more clinical parameters associated with an underlying disorder, or otherwise enhancing the physical or mental health of a human or animal. For prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, a disease state or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease may not have been diagnosed.

[0044] The terms "therapeutically effective amount" and "therapeutically effective dose" as used herein generally refer to an amount of a drug or biologically active protein, alone or as part of a polypeptide composition, that can have any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition when administered to a subject in one or repeated doses. Such an effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0045] The term "equivalent molar dose" generally means that the amount of a material administered to a subject has an equivalent molar amount based on the molecular weight of the material used in the dose.

[0046] The term "therapeutically effective and non-toxic dose" as used herein generally refers to a tolerated dose of a composition as defined herein that is large enough to cause tumor or cancer cell depletion, tumor removal, tumor shrinkage, or disease stabilization without or essentially without significant toxic effects in a subject.Such a therapeutically effective and non-toxic dose can be determined by dose escalation studies as described in the art, and should be below the dose that induces severe adverse side effects.

[0047] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0048] When used in the context of chemical groups: "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (-COOH or -CO 2 "halo" means, independently, -F, -Cl, -Br, or -I; "amino" means -NH 2 "hydroxyamino" means -NHOH; "nitro" means -NO2 "imino" means =NH; "cyano" means -CN; "isocyanato" means -N=C=O; "azido" means -N 3 in the monovalent context, "phosphate" means -OP(O)(OH) 2 or its deprotonated form; in the divalent context, "phosphate" 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 "Sulfonamide" means -S(O) 2 NH 2 and "sulfinyl" means --S(O)--.

[0049] In the context of chemical formulas, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. TIFF2025500547000066.tif4128 represents any bond, if present, which may be either a single bond or a double bond. TIFF2025500547000067.tif4128 represents a single or double bond. Thus, for example, the formula TIFF2025500547000068.tif10128 is TIFF2025500547000069.tif11128, and it is understood that no such ring atom forms part of more than one double bond. It is further noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when connecting one or two stereogenic atoms. Instead, it covers all stereoisomers and mixtures thereof. The symbol TIFF2025500547000070.tif4128 shows that when drawn vertically across a bond (e.g. for methyl TIFF2025500547000071.tif6128), indicating the point of attachment of the group. It is noted that attachment points are typically only identified in this fashion on larger groups to aid the reader in unambiguously identifying the attachment points. TIFF2025500547000072.tif4128 represents a single bond in which the group attached to the thick end of the wedge is "off the page". TIFF2025500547000073.tif4128 represents a single bond in which the group attached to the thick end of the wedge is "into the page". TIFF2025500547000074.tif4128 refers to a single bond with no defined geometry (e.g., either E or Z) around the double bond. Thus, both options and combinations thereof are contemplated. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.

[0050] The group "R" may, for example, be of the formula: When depicted as a "floating group" on a ring system in TIFF2025500547000075.tif14128, R may replace any hydrogen atom attached to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is formed. When depicted as a "floating group" on a fused ring system, as in TIFF2025500547000076.tif17128, R may replace any hydrogen attached to any of the ring atoms of either of the fused rings, unless otherwise specified. Replaceable hydrogens include depicted hydrogens (e.g., hydrogens attached to the nitrogen in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not shown but are understood to be present), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens attached to group X when X is equal to -CH-), so long as a stable structure is formed. In the depicted example, R may be present on either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the bracketed group "R" represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms on the ring or ring system.

[0051] For chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, the minimum number being as small as possible for the group / class in question, e.g., the group "alkenyl (C≦8) " or class "Alkenes (C≦8) The minimum number of carbon atoms in "alkoxy" is understood to be 2. "Alkoxy" designates an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Thus, "alkyl (C2-10) " designates an alkyl group having from 2 to 10 carbon atoms. These carbon number designations may precede or follow the chemical group or class it modifies, and may be enclosed in parentheses or not, without representing any change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "OlefinC5 " are all synonymous.

[0052] The term "saturated" when used to modify a compound or chemical group means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as described below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bonds. In the case of substituted versions of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. And, when such bonds are present, it does not preclude carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism. When the term "saturated" is used to modify a solution of a substance, it means that the substance is no longer soluble in the solution.

[0053] The term "aliphatic", when used without the "substituted" modifier, denotes that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms can be linked together in a straight chain, branched chain, or non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) linked by a single carbon-carbon bond, or unsaturated with one or more carbon-carbon double bonds (alkene / alkenyl), or unsaturated with one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0054] The term "aromatic," when used to modify an atom of a compound or chemical group, means that the compound or chemical group contains a planar, unsaturated ring of atoms stabilized by the interaction of the bonds that form the ring.

[0055] The term "alkyl" when used without the "substituted" modifier refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The 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-Butyl, t-Bu, or t Bu), and -CH 2 C(CH 3 ) 3 (neo-pentyl) is a non-limiting example of an alkyl group. The term "alkanediyl," when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as points of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group -CH 2 -(methylene), -CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 - and -CH 2 CH 2 CH 2- is a non-limiting example of an alkanediyl group. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -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 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 CH2 The term "haloalkyl" is a subset of substituted alkyl in which the replacement of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or -I) so that no other atoms other than carbon, hydrogen, and halogens are present. The group -CH 2 Cl is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl where the replacement of hydrogen atoms is limited to fluoro, such that no other atoms are present other than carbon, hydrogen, and fluorine. The group -CH 2 F, -CF 3 , and -CH 2 CF 3 are non-limiting examples of fluoroalkyl groups.

[0056] The term "cycloalkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, which carbon atom forms part of one or more non-aromatic ring structures, which has no carbon-carbon double or triple bonds, and which has no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH 2 ) 2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. TIFF2025500547000077.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula HR, where R is cycloalkyl as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 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 has been replaced by

[0057] The term "alkenyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, 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 The term "alkenediyl," when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, which is linear or branched, has a linear or branched acyclic structure, has at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and has no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH 3 )CH 2 -, -CH=CHCH 2 - and -CH 2CH=CHCH 2 - is a non-limiting example of an alkenediyl group. It is noted that although alkenediyl groups are aliphatic, once attached at both ends, the group is not precluded from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to a class of compounds having the formula HR, where R is alkenyl as that term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene with only one carbon-carbon double bond, which is part of a vinyl group at the end of the molecule. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms may be independently replaced with -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 The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0058] The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH 3 , and -CH 2 C≡CHH 3 is a non-limiting example of an alkynyl group. "Alkyne" refers to the class of compounds having the formula HR where R is alkynyl. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 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 has been replaced by

[0059] The term "aryl", when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more six-membered aromatic ring structures, where the ring atoms are all carbon and the group is composed of no atoms other than carbon and hydrogen. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (where the carbon number limit permits) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C 6 H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and biphenyl. The term "arenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structures, where the ring atoms are all carbon, and the monovalent group is composed of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limit permitting) attached to the first aromatic ring or any additional aromatic rings present. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be connected via one or more of the following: a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limit permitting). Non-limiting examples of arenediyl groups include: Contains TIFF2025500547000078.tif33128.

[0060] "Arene" refers to the class of compounds having the formula HR, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 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 has been replaced by

[0061] The term "aralkyl" when used without the "substituted" modifier refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl groups are independently substituted with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH2 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 Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

[0062] The term "heteroaryl", when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, where at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the heteroaryl group is composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. The heteroaryl ring may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where the carbon number limitation permits) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the point of attachment. The term "heteroarenediyl", when used without the "substituted" modifier, 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 the two points of attachment, the atoms forming part of one or more aromatic ring structures, where at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group is composed of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be connected through one or more of the following: a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitation permitting).As used herein, the term does not preclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where the carbon number limitation permits) attached to the aromatic ring or ring system. Non-limiting examples of heteroarenediyl groups include: Contains TIFF2025500547000079.tif15128.

[0063] "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 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 has been replaced by

[0064] The term "heterocycloalkyl", when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, which forms part of one or more non-aromatic ring structures, where at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the heterocycloalkyl group is composed of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. Heterocycloalkyl rings can contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups (where the carbon number limit permits) attached to a ring or ring system. Also, 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 the point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl", when used without the "substituted" modifier, refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structures, where at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group is composed of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When there is more than one ring, the rings may be fused or unfused. Non-fused rings may be connected through one or more of the following: a covalent bond, an alkanediyl group, or an alkenediyl group (carbon number limitation permitting).As used herein, the term does not preclude the presence of one or more alkyl groups (where the carbon number limitation permits) attached to the ring or ring system. Nor does the term preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: When these terms are used with the "substituted" modifier, one or more hydrogen atoms 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 has been replaced by

[0065] The term "acyl," when used without the "substituted" modifier, refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as those terms are defined above. The groups -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 is replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) are independently replaced with -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 The group -C(O)CH 2 CF 3 , -CO 2 H(carboxyl), -CO2 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.

[0066] The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, where R is alkyl, as that term is defined above. Non-limiting examples include: -OCH 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. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent groups -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio", when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" refers to an alkane, as defined above, in which at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms are used with the "substituted" modifier, one or more of the hydrogen atoms can be independently replaced with an alkoxy group, such as -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) 2OH, or -S(O) 2 NH 2 has been replaced by

[0067] The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, where R is alkyl, as that term is defined above. Non-limiting examples include -NHCH 3 and -NHCH 2 CH 3 Includes:

[0068] The term "dialkylamino," when used without the "substituted" modifier, refers to the group --NRR', where R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl. Non-limiting examples of dialkylamino groups include --N(CH 3 ) 2 and -N(CH 3 )(CH 2 CH 3 The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino", and "alkylsulfonylamino", when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -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 the "substituted" modifier, refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH 3The term "alkylimino," when used without the "substituted" modifier, refers to the divalent group =NR, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more of the hydrogen atoms attached to the carbon atom are independently selected from -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 The group -NHC(O)OCH 3 and -NHC(O)NHCH 3 is a non-limiting example of a substituted amide group.

[0069] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0070] 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 level of polymerization and therefore a different molar mass. Average molecular weight can be used to describe the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number average molar mass, weight (mass) average molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, average molecular weight refers to either the number average molar mass or the weight average molar mass of the formula. In some embodiments, the average molecular weight is number average molar mass. In some embodiments, average molecular weight can be used to describe the PEG components present in lipids.

[0071] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other unlisted steps.

[0072] The term "effective," as that term is used in the specification and / or claims, means adequate to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmacologically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to effect treatment for such disease.

[0073] As used herein, "IC50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximal response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is required to inhibit half of a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0074] An "isomer" of a first compound is a distinct chemical compound whose each molecule contains the same constituent atoms as the first compound, but differs in the arrangement of those atoms in three dimensions.

[0075] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., a non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0076] The terms "assemble" or "assembled" as used herein, in the context of delivery of a payload to a target cell, generally refer to a covalent or non-covalent interaction or association, such as, for example, a therapeutic or prophylactic agent being complexed with or encapsulated in a lipid composition.

[0077] As used herein, the term "lipid composition" generally refers to a composition that includes lipid compounds, including, but not limited to, lipoplexes, liposomes, lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

[0078] As used herein, the term "detectable" refers to the occurrence or change of a signal that is directly or indirectly detectable by either observation or instrumentation. Typically, the detectable response is the occurrence of a signal where the fluorophore is inherently fluorescent and does not cause a change in signal upon binding to a metal ion or biological compound. Alternatively, the detectable response is an optical response that results in a change in wavelength distribution pattern or absorbance or fluorescence intensity, or a change in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0079] The term "potent" or "potency", as used herein in relation to the delivery of a therapeutic agent, generally refers to a higher ability of a delivery system (e.g., a lipid composition) to achieve or produce a desired amount, activity, or effect (such as a desired level of translation, transcription, production, expression, or activity of a protein or gene) of a therapeutic or prophylactic agent in a cell (e.g., a target cell) to any measurable extent, for example, compared to a reference delivery system. For example, a lipid composition with higher potency may achieve a desired therapeutic effect in a larger population of relevant cells, within a shorter response time, or lasting for a longer period of time.

[0080] As generally used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are 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, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0081] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present application that are pharma- ceutically acceptable as defined above and have the desired pharmacological activity. Such salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or salts of 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethylenediaminetetraacetic ... Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharma- ceutically acceptable salts, and methods for their preparation and use, are provided in Handbook of Pharmaceutical Salts: Properties, and Use (PH Stahl & CG Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0082] As used herein, the term "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in the carrying or transport of a chemical agent.

[0083] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptomology of the disease, and / or (2) delaying the onset of disease pathology or symptomology in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptomology of the disease.

[0084] A "repeating unit" is the simplest structural entity of a particular material, e.g., a framework and / or polymer, whether organic, inorganic, or metal-organic. In a polymer chain, the repeating units are linked together sequentially along the chain, like beads on a necklace. For example, polyethylene-[-CH 2 CH 2 -] n -, the repeat unit is -CH 2 CH 2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units linked together. If the value of "n" is left undefined or is absent, it simply specifies the repetition of the formula within the brackets and the polymerizability of the material. The concept of repeat units applies equally when the connectivity between repeat units extends in three dimensions, for example, in metal-organic frameworks, modified polymers, thermosetting polymers, etc. Within the context of dendrimers or dendrons, the repeat units may also be described as branching units, internal layers, or generations. Similarly, the end groups may also be described as surface groups.

[0085] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the arrangement of those atoms in three dimensions. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain chiral centers, also called stereocenters or stereogenic centers, which are any points, but not necessarily atoms, in a molecule that bear groups such that the exchange of any two groups results in a stereoisomer. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, but it is also possible for other atoms to be stereocenters in organic and inorganic compounds. Molecules can have multiple stereocenters, giving rise to many stereoisomers. In compounds whose stereoisomerism is due to a tetrahedral asymmetric center (e.g., a regular tetrahedral carbon), the total number of hypothetical possible stereoisomers is 2 nn is the number of tetrahedral stereocenters. Molecules with symmetry often have less than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or axis of chirality where the stereochemistry is not defined, it is contemplated that the stereocenter or axis of chirality can exist 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%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer.

[0086] "Treatment" or "treating" includes: (1) inhibiting a disease (e.g., halting further progression of the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease; (2) ameliorating a disease (e.g., reversing the pathology and / or symptomology) in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease; and / or (3) causing any measurable reduction in a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease.

[0087] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that a particular term is defined should not be taken to indicate that any term not defined is unclear. Rather, all terms used are believed to describe the present disclosure in terms that allow one skilled in the art to appreciate the scope and to practice the present application.

[0088] composition lipid composition In some embodiments, provided herein is a lipid composition comprising an ionizable cationic lipid; a polymer-conjugated lipid; and, for example, a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid. The lipid composition may further comprise a phospholipid.

[0089] Ionizable Cationic Lipids In some embodiments of the lipid composition of the present application, the lipid composition comprises an ionizable cationic lipid. In some embodiments, the cationic 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 do not have a charge. The ionizable cationic group can contain one or more protonatable amines that can form a cationic group at physiological pH. The cationic ionizable lipid compound can also be C 6 -C 24 They may further comprise one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These lipid groups may be attached through ester bonds or further added through Michael addition to sulfur atoms. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0090] In some embodiments of the lipid compositions of the present application, ionizable cationic lipids refer to lipids and lipid-like molecules that have a nitrogen atom capable of acquiring an electric charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules that have an amino group typically have 2-6 hydrophobic chains, often C 6 -C 24The cationic ionizable lipids may have at least one or more, six tails, with alkyl or alkenyl groups such as alkyl or alkenyl groups. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers that exhibit regular dendritic branching formed by the successive or generational addition of layers of branches to or from a core, characterized by a core, at least one layer of internal branches, and a layer of surface branches. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994). In other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular configuration having an internal core, an internal layer (or "generation") of repeating units regularly attached to this initiator core, and an external surface of terminal groups attached to the outermost generation. A "dendron" is a type of dendrimer with branches emanating from a focal point that are or can be attached to a core, either directly or through a linking moiety, to form a larger dendrimer. In some embodiments, the dendrimer structure has repeating groups radiating from a central core that doubles at each repeat unit for each branch. In some embodiments, the dendrimers described herein can be described as small molecules, medium-sized molecules, lipids, or lipid-like substances. These terms can be used to describe the compounds described herein that have a dendron-like appearance (e.g., molecules that radiate from a single focal point).

[0091] Although dendrimers are polymers, they may be preferred over traditional polymers because they have a controllable structure, a single molecular weight, multiple and controllable surface functionalities, and traditionally adopt a globular conformation after reaching a certain generation. Dendrimers can be prepared by sequential reactions of each repeating unit to produce monodisperse, dendritic, and / or generationally structured polymer structures. Individual dendrimers consist of a central core molecule with dendritic wedges attached to one or more functional sites on the central core. The dendrimer surface layer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups, according to the assembly monomers used during preparation.

[0092] Their physical properties can be adjusted by modifying the functional groups and / or chemical properties of the core, repeating units, and surface or end groups. Some properties that can be changed include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesive ability. Dendrimers are often described by the number of repeating units in their generation or branch. A dendrimer consisting of only the core molecule is called generation 0, and each successive repeating unit along all branches is generation 1, generation 2, etc., up to the end or surface groups. In some embodiments, half-generations are possible that result only from the first condensation reaction with an amine and not from the second condensation reaction with a thiol.

[0093] The preparation of dendrimers requires a level of synthetic control, achieved through a series of stepwise reactions involving building up the dendrimer with each successive group. Dendrimer synthesis can be of convergent or divergent type. During divergent dendrimer synthesis, molecules are assembled from the core to the periphery in a stepwise process involving attaching one generation to the previous one and then changing the functional groups for the next step of reaction. The conversion of functional groups is necessary to prevent uncontrolled polymerization. Such polymerization would result in highly branched molecules that are not monodisperse, otherwise known as hyperbranched polymers. Due to steric effects, continuing to react dendrimer repeat units results in spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation, destroying the monodispersity of the molecule. Thus, in some embodiments, dendrimers of generations G1 to G10 are specifically contemplated. In some embodiments, dendrimers contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. In some embodiments, dendrimers used herein are G0, G1, G2, or G3. However, the number of possible generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by reducing the spacing units in the branched polymer.

[0094] Additionally, dendrimers have two main chemical environments: the environment created by the specific surface groups on the terminal generations, and the interior of the dendritic structure that can be shielded from the bulk medium and the surface groups due to the higher order structure. Because of these different chemical environments, dendrimers find many different potential uses, including in therapeutic applications.

[0095] In some embodiments of the lipid composition of the present disclosure, dendrimers or dendrons are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers or dendrons may contain secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylate with mercapto groups. Additionally, the repeating units of the dendrimer or dendron may contain groups that are degradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine with multiple different dendritic branches, each of which may contain one or more repeating units. Dendrimers or dendrons may be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the terminal group is a lipophilic group, such as a long chain alkyl or alkenyl group. In other embodiments, the terminal group is a long chain haloalkyl or haloalkenyl group. In other embodiments, the terminal group is an amine (-NH 2 ) or carboxylic acid (-CO 2 In yet other embodiments, the terminal group is an aliphatic or aromatic group containing an ionizable group such as a hydroxide group, an amide group, or an ester.

[0096] The cationic ionizable lipids of the present application may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic, epimeric, and all geometric isomeric forms of the chemical formula are intended unless a particular stereochemistry or isomeric form is specifically indicated. The cationic ionizable lipids may appear as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, single diastereomers are obtained. The chiral centers of the cationic ionizable lipids of the present application may have S or R configurations. Furthermore, it is contemplated that one or more of the cationic ionizable lipids may exist as structural isomers. In some embodiments, the compounds have the same formula but different connectivity to the nitrogen atom of the core. Without wishing to be bound by any theory, it is believed that such cationic ionizable lipids exist because the starting monomer reacts first with a primary amine and then statistically reacts with any secondary amine present. Thus, a structural isomer may represent a mixture of a fully reacted primary amine and a subsequently reacted secondary amine.

[0097] The chemical formula used to represent cationic ionizable lipids in this application typically only shows one of several different tautomers.For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups.Similarly, many types of imine groups exist in equilibrium with enamine groups.All tautomers of a given chemical formula are intended, regardless of which tautomer is depicted for a given formula and which one is most prevalent.

[0098] The cationic ionizable lipids of the present application may also have the advantage that they are more effective, less toxic, longer acting, more potent, produce fewer side effects, are more easily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties over compounds known in the prior art, whether for use in the indications described herein or otherwise.

[0099] In addition, the atoms constituting the cationic ionizable lipids of the present application are intended to include all isotopic forms of such atoms. As used herein, isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include: 13 C and 14 Contains C.

[0100] It should be recognized that the specific anion or cation that forms part of any salt form of the cationic ionizable lipid provided herein is not critical, so long as the salt as a whole is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts, as well as their preparation and use methods, are provided in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0101] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the ionizable cationic lipid is a dendrimer or dendron. In some embodiments, the ionizable cationic lipid comprises at least two C 6 -C 24 Contains an alkyl or alkenyl group.

[0102] Dendrimers of formula (I) In some embodiments of the lipid composition, the ionizable cationic lipid has at least two C 8 -C 24 In some embodiments, the ionizable cationic lipid comprises an alkyl group. Core-Repeat Unit-End Group (DI) or a pharma- ceutically acceptable salt thereof, wherein the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and replacing that atom with the repeating unit, and wherein The core has the formula: I have TIFF2025500547000081.tif10128, During the ceremony: X 1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) , heterocycloalkyl (C≦12) , Heteroaryl (C≦12) or a replacement version thereof; R 1 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version of any of these groups; and a is 1, 2, 3, 4, 5, or 6; or The core has the formula: has TIFF2025500547000082.tif12128, During the ceremony: X 2 is N(R 5 ) y and; R 5 is hydrogen, alkyl (C≦18) , or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R 2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3; provided that the sum of z and y is 3; or The core has the formula: TIFF2025500547000083.tif11128, During the ceremony: X 3 -NR 6 - where R 6 is hydrogen, alkyl (C≦8) Or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , Heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or a substituted version of any of these groups; R 3 and R 4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version of any of these groups; or a group of the formula: -N(Rf ) f (CH 2 CH 2 N(R c )) e R d , Based on TIFF2025500547000084.tif39128; Where: e and f are each independently 1, 2, or 3; with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) , or substituted alkyl (C≦6) and; c and d are each independently 1, 2, 3, 4, 5, or 6; or The core is an alkylamine (C≦18) , dialkylamine (C≦36) , heterocycloalkane (C≦12) or a substituted version of any of these groups; wherein the repeating unit comprises a degradable diacyl and a linker; The degradable diacyl group has the formula: I have TIFF2025500547000085.tif17128: During the ceremony: A 1 and A 2 are each independently -O-, -S-, or -NR a -where: R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; Y 3 is an alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or a substituted version of any of these groups; or a group of the formula: Based on TIFF2025500547000086.tif11128: Where: X3 and X 4 is an alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or a substituted version of any of these groups; Y 5 is a covalent bond, alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or a substituted version of any of these groups; and R 9 is an alkyl (C≦8) or substituted alkyl (C≦8) and; The linker group has the formula: I have TIFF2025500547000087.tif16128, During the ceremony: Y 1 is an alkanediyl (C≦12) , Alkene Diyl (C≦12) , Arendjiil (C≦12) or a substituted version of any of these groups; and wherein if the repeat unit contains a linker group, the linker group contains independent degradable diacyl groups attached to both the nitrogen and sulfur atoms of the linker group, if n is greater than 1, the first group in the repeat unit is a degradable diacyl group, and for each linker group, the next repeat unit contains two degradable diacyl groups attached to nitrogen atoms of the linker group; and n is the number of linker groups present in the repeat unit; and The terminal group has the formula: I have TIFF2025500547000088.tif12128: During the ceremony: Y 4 is an alkanediyl (C≦18) , or alkanediyl (C≦18) One or more of the hydrogen atoms above are -OH, -F, -Cl, -Br, -I, -SH, -OCH 3 , -OCH 2 CH 3 , -SCH 3, or -OC(O)CH 3 Alkanediyl replaced by (C≦18) and; R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , N-heterocycloalkyl (C≦12) , -C(O)N(R 11 )-Alkanediyl (C≦6) -Heterocycloalkyl (C≦12) , -C(O)-alkylamino (C≦12) , -C(O)-dialkylamino (C≦12) , -C(O)-N-heterocycloalkyl (C≦12) where: R 11 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and; where the last degradable diacyl in the chain is attached to the terminal group; n is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the terminal group has the formula: Further defined by TIFF2025500547000089.tif12128, During the ceremony: Y 4 is an alkanediyl (C≦18) and R 10 is hydrogen. 1 and A 2 each independently represents -O- or -NR a -It is.

[0103] In some embodiments of the dendrimer or dendron of formula (DI), the core has the formula: Further defined by TIFF2025500547000090.tif12128, During the ceremony: X 2 is N(R 5 ) y and; R5 is hydrogen or alkyl (C≦8) , or substituted alkyl (C≦18) and y is 0, 1, or 2, with the proviso that the sum of y and z is 3; R 2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, and 3; however, the sum of z and y is 3.

[0104] In some embodiments of the dendrimer or dendron of formula (DI), the core has the formula: Further defined by TIFF2025500547000091.tif11128, During the ceremony: X 3 -NR 6 - where R 6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiyl (C≦8) , Arendjiil (C≦8) , Heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) or a substituted version of any of these groups; R 3 and R 4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) or a substituted version 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 , Based on TIFF2025500547000092.tif19166; Where: e and f are each independently 1, 2, or 3; with the proviso that the sum of e and f is 3; R c , R d , and R f are each independently hydrogen, alkyl, (C≦6) , or substituted alkyl (C≦6) and; c and d are each independently 1, 2, 3, 4, 5, or 6.

[0105] In some embodiments of the dendrimer or dendron of formula (I), the terminal group has the formula: Represented by TIFF2025500547000093.tif12128, During the ceremony: Y 4 is an alkanediyl (C≦18) and R 10 is hydrogen.

[0106] In some embodiments of the dendrimer or dendron of formula (DI), the core is Further defined as TIFF2025500547000094.tif130148.

[0107] In some embodiments of the dendrimer or dendron of formula (DI), the degradable diacyl is Further defined as TIFF2025500547000095.tif15128.

[0108] In some embodiments of the dendrimer or dendron of formula (DI), the linker is TIFF2025500547000096.tif16128, where Y 1 is an alkanediyl (C≦8) or substituted alkanediyl (C≦8) It is.

[0109] In some embodiments of the dendrimer or dendron of formula (DI), the dendrimer or dendron is TIFF2025500547000097.tif188141TIFF2025500547000098.tif184140; and pharma- ceutically acceptable salts thereof.

[0110] A dendrimer or dendron of formula (X) In some embodiments of the lipid composition, the ionizable cationic lipid has the formula In some embodiments, the ionizable cationic lipid is a dendrimer or dendron of the formula: TIFF2025500547000100.tif22128 dendrimer or dendron.

[0111] In some embodiments of the lipid composition, the ionizable cationic lipid has the structural formula: TIFF2025500547000101.tif22128, or a pharma- ceutically acceptable salt thereof, wherein (a) The core has the structural formula (X コア ): TIFF2025500547000102.tif13128, During the ceremony: Q is, independently for each occurrence, a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b - and; R 2 is expressed independently for each occurrence as R 1g or -L 2 -NR 1e R 1f and; R 3a and R 3b are each independently hydrogen or substituted at each occurrence (e.g., C 1 -C 6 , for example C1 -C 3 ) alkyl; R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) are each independently at each occurrence an attachment point to a branch, a hydrogen, or an optionally substituted (e.g., C 1 -C 12 ) alkyl; L 0 , L 1 , and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or Alternatively, L 1 Part of R 1c and R 1d One of the following (e.g., C 4 -C 6 ) forming a heterocycloalkyl (e.g., containing one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur); and x 1 is 0, 1, 2, 3, 4, 5, or 6; and (b) each branch of the plurality (N) of branches independently has the structural formula (X 分枝 ): TIFF2025500547000103.tif11128, During the ceremony: * indicates the attachment point of the branch to the core; g is 1, 2, 3, or 4; Z=2 (g-1) ; When g=1, G=0; or when g≠1, TIFF2025500547000104.tif5128; (c) each diacyl group independently has the structural formula Including TIFF2025500547000105.tif16128, During the ceremony: * indicates the attachment point of the diacyl group at its proximal end; ** indicates the attachment point of the diacyl group at its distal end; Y 3 is, independently at each occurrence, 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 ) arenylene; A 1 and A 2 is, independently at each occurrence, -O-, -S-, or -NR 4 -where: R 4 may be hydrogen or substituted (e.g., C 1 -C 6 ) alkyl; m 1 and m 2 is, independently at each occurrence, 1, 2, or 3; and R 3c , R 3d , R 3e , and R 3f are each independently hydrogen or substituted at each occurrence (e.g., C 1 -C 8 ) alkyl; and (d) each linker group independently has the structural formula TIFF2025500547000106.tif12128, During the ceremony: ** indicates the point of attachment of the linker to the proximal diacyl group; *** indicates the point of attachment of the linker to the distal diacyl group; and Y 1 is, independently at each occurrence, 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 ) allenylene; and (e) each terminal group may independently be substituted (e.g., C 1 -C 18 , for example C 4 -C 18 ) alkylthiols, and optionally substituted (e.g., C 1 -C 18 , for example C 4 -C 18 ) alkenyl thiols.

[0112] X コア In some embodiments, Q is independently at each occurrence a covalent bond, -O-, -S-, -NR 2 - or -CR 3a R 3b X コア In some embodiments, Q is, independently at each occurrence, a covalent bond. コア In some embodiments, Q is, independently at each occurrence, -O-. コア In some embodiments, Q is, independently at each occurrence, -S-. コア In some embodiments, Q is independently at each occurrence -NR 2 and R 2 is expressed independently for each occurrence as R 1g or -L 2 -NR 1e R 1f X コア In some embodiments, Q is independently at each occurrence -CR 3a R 3b R 3a and R 3a and R 3b is independently at each occurrence hydrogen or optionally substituted alkyl (e.g., C 1 -C 6 , for example C 1 -C 3 ).

[0113] X コア In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) is independently at each occurrence an attachment point to a branch, hydrogen, or optionally substituted alkyl. コア In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) is independently at each occurrence a point of attachment to a branch, hydrogen. コア In some embodiments, R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present), independently at each occurrence, represent the attachment point to the branch, the optionally substituted alkyl (e.g., C 1 -C 12 ).

[0114] X コア In some embodiments, L 0 , L 1 , and L 2 is independently selected at each occurrence from a covalent bond, alkylene, heteroalkylene, [alkylene]-[heterocycloalkyl]-[alkylene], [alkylene]-(arylene)-[alkylene], heterocycloalkyl, and arylene; or alternatively, L 1 Part of R 1c and R 1d and heterocycloalkyl (e.g., C 4 -C 6and containing one or two nitrogen atoms, and optionally additional heteroatoms selected from oxygen and sulfur. X コア In some embodiments, L 0 , L 1 , and L 2 X may each independently at each occurrence be a covalent bond. コア In some embodiments, L 0 , L 1 , and L 2 X may be, independently at each occurrence, hydrogen. コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence an alkylene (e.g., C 1 -C 12 , for example C 1 -C 6 or C 1 -C 3 ) X コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence heteroalkylene (e.g., C 1 -C 12 , for example C 1 -C 8 or C 1 -C 6 ) X コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence heteroalkylene (e.g., C 2 -C 8 X may be an alkylene oxide, such as oligo(ethylene oxide). コア In some embodiments, L 0 , L 1 , and L 2 represents, independently at each occurrence, [alkylene]-[heterocycloalkyl]-[alkylene] [(e.g., C 1 -C 6 ) alkylene]-[(e.g., C4 -C 6 )heterocycloalkyl]-[(e.g., C 1 -C 6 ) alkylene. X コア In some embodiments, L 0 , L 1 , and L 2 represents, independently at each occurrence, [alkylene]-(arylene)-[alkylene] [(e.g., C 1 -C 6 ) alkylene]-(arylene)-[(e.g., C 1 -C 6 ) alkylene. X コア In some embodiments, L 0 , L 1 , and L 2 represents, independently at each occurrence, [alkylene]-(arylene)-[alkylene] (e.g., [(e.g., C 1 -C 6 ) alkylene]-phenylene-[(e.g., C 1 -C 6 ) alkylene]). X コア In some embodiments, L 0 , L 1 , and L 2 is independently at each occurrence heterocycloalkyl (e.g., C 4 -C 6 Heterocycloalkyl). X コア In some embodiments, L 0 , L 1 , and L 2 Each occurrence of X can be, independently, arylene (e.g., phenylene). コア In some embodiments, L 1 Part of R 1c and R 1d and one of X to form a heterocycloalkyl. コア In some embodiments, L 1 Part of R 1c and R 1d and heterocycloalkyl (e.g., C 4-C 6 heterocycloalkyl), which can contain one or two nitrogen atoms and, optionally, additional heteroatoms selected from oxygen and sulfur.

[0115] X コア In some embodiments, L 0 , L 1 , and L 2 represents, independently at each occurrence, a covalent bond; C 1 -C 6 Alkylene (e.g., C 1 -C 3 alkylene), C 2 -C 12 (For example, C 2 -C 8 ) alkylene oxides (e.g., oligo(ethylene oxide), e.g., -(CH 2 CH 2 O) 1-4 -(CH 2 CH 2 )-), [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., TIFF2025500547000107.tif16128), and [(C 1 -C 4 ) alkylene]-phenylene-[(C 1 -C 4 ) alkylene] (e.g., TIFF2025500547000108.tif16128). X コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents 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)-. X コア In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents C 1 -C 6 Alkylene (e.g., C 1 -C 3 In some embodiments, L 0 , L 1 , and L 2 each occurrence independently represents C 2 -C 12 (For example, C 2 -C 8 ) alkylene oxides (e.g., -(C 1 -C 3 Alkylene-O) 1-4 -(C 1 -C 3 X is an alkylene. コア In some embodiments, L 0 , L 1 , and L 2 are each independently, at each occurrence, 1 -C 4 ) alkylene]-[(C 4 -C 6 )Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 Alkylene)-phenylene-(C 1 -C 3 alkylene)-), and [(C 1 -C 4 ) alkylene]-[(C 4 -C 6)Heterocycloalkyl]-[(C 1 -C 4 ) alkylene] (e.g., -(C 1 -C 3 Alkylene)-piperazinyl-(C 1 -C 3 alkylene)-).

[0116] X コア In some embodiments, x 1 is 0, 1, 2, 3, 4, 5, or 6. X コア In some embodiments, x 1 is 0. X コア In some embodiments, x 1 is 1. X コア In some embodiments, x 1 is 2. X コア In some embodiments, x 1 is 0, 3. X コア In some embodiments, x 1 is 4. X コア In some embodiments, x 1 is 5. X コア In some embodiments, x 1 is 6.

[0117] X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000109.tif14128. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000110.tif15128. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000111.tif37128. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000112.tif14128. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000113.tif16128. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000114.tif56156. X コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000115.tif58147. X コア In some embodiments, the core has the structural formula: TIFF2025500547000116.tif13128, wherein Q′ is —NR 2 -OR-CR 3a R 3b -And;q 1 and q 2 are each independently 1 or 2. コア In some embodiments, the core has the structural formula: Contains TIFF2025500547000117.tif85162. X コア In some embodiments, the core has the structural formula TIFF2025500547000118.tif65150, wherein ring A is an optionally substituted aryl or an optionally substituted (e.g., C 3 -C 12 , for example C 3 -C 5 ) heteroaryl. X コア In some embodiments, the core has the structural formula I have TIFF2025500547000119.tif21128.

[0118] X コア In some embodiments, the core comprises the structural formula shown in Table 1, and pharma- ceutically acceptable salts thereof, where * indicates the attachment point of the core to one of the branches. In some embodiments, the exemplary cores in Table 1 are not limited to the listed stereoisomers (i.e., enantiomers, diastereomers).

[0119] Table 1: Exemplary core structures TIFF2025500547000120.tif64151TIFF2025500547000121.tif229151TIFF2025500547000122.tif223151TIFF2025500547000123.tif155151

[0120] X コア In some embodiments, the core comprises: TIFF2025500547000124.tif46150TIFF2025500547000125.tif226150, and pharma- ceutically acceptable salts thereof, wherein * indicates the attachment point of the core to one branch of the multiple branches.

[0121] In some embodiments, the plurality (N) of branches comprises at least 3 branches, at least 4 branches, at least 5 branches. In some embodiments, the plurality (N) of branches comprises at least 3 branches. In some embodiments, the plurality (N) of branches comprises at least 4 branches. In some embodiments, the plurality (N) of branches comprises at least 5 branches.

[0122] X 分枝 In some embodiments, g is 1, 2, 3, or 4. 分枝 In some embodiments, g is 1. 分枝 In some embodiments, g is 2. 分枝 In some embodiments, g is 3. 分枝 In some embodiments, g is 4.

[0123] X 分枝 In some embodiments, Z=2 (g-1) And when g=1, G=0. X 分枝 In some embodiments, Z=2 (g-1) and g ≠ 1, The file is TIFF2025500547000126.tif6128.

[0124] X 分枝 In some embodiments of the present invention, g=1, G=0, Z=1, and each branch of the plurality of branches comprises the structural formula: Contains TIFF2025500547000127.tif7128.

[0125] X 分枝 In some embodiments, g=2, G=1, Z=2, and each branch of the plurality of branches has the structural formula Contains TIFF2025500547000128.tif21128.

[0126] X 分枝 In some embodiments, g=3, G=3, Z=4, and each branch of the plurality of branches has the structural formula Includes TIFF2025500547000129.tif43128.

[0127] X 分枝 In some embodiments, g=4, G=7, Z=8, and each branch of the plurality of branches has the structural formula Contains TIFF2025500547000130.tif79157.

[0128] In some embodiments, the dendrimers or dendrons described herein having generation (g)=1 have the structure: I have TIFF2025500547000131.tif17128.

[0129] In some embodiments, the dendrimers or dendrons described herein having generation (g)=1 have the structure: I have TIFF2025500547000132.tif38128.

[0130] Exemplary formulations of dendrimers or dendrons described herein for generations 1-4 are shown in Table 2. The number of diacyl groups, linker groups, and terminal groups can be calculated based on g.

[0131] Table 2. Dendrimer or dendron group composition based on generation (g) TIFF2025500547000133.tif22151

[0132] In some embodiments, the diacyl group is independently represented by the structural formula TIFF2025500547000134.tif16128, where * indicates the attachment point of a diacyl group at the proximal end and ** indicates the attachment point of a diacyl group at the distal end.

[0133] X 分枝 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkylene, an optionally substituted alkenylene, or an optionally substituted allenylene. 分枝 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkylene (e.g., C 1 -C 12 ) X 分枝 In some embodiments of the diacyl group of Y 3 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ) X 分枝 In some embodiments of the diacyl group of Y 3 represents independently at each occurrence an optionally substituted allenylene (e.g., C 1 -C 12 ).

[0134] X 分枝 In some embodiments of the diacyl group of A 1 and A 2 is, independently at each occurrence, -O-, -S-, or -NR 4 -It is. X 分枝 In some embodiments of the diacyl group of A 1 and A 2 is independently at each occurrence -O-. 分枝In some embodiments of the diacyl group of A 1 and A 2 is, independently at each occurrence, -S-. 分枝 In some embodiments of the diacyl group of A 1 and A 2 each occurrence independently represents -NR 4 - and R 4 is hydrogen or optionally substituted alkyl (e.g., C 1 -C 6 ) X 分枝 In some embodiments of the diacyl group of m 1 and m 2 is independently 1, 2, or 3 at each occurrence. 分枝 In some embodiments of the diacyl group of m 1 and m 2 X is independently 1 for each occurrence. 分枝 In some embodiments of the diacyl group of m 1 and m 2 X is, independently at each occurrence, 2. 分枝 In some embodiments of the diacyl group of m 1 and m 2 X is, independently at each occurrence, 3. 分枝 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f X is independently at each occurrence hydrogen or optionally substituted alkyl. 分枝 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f is independently at each occurrence hydrogen. 分枝 In some embodiments of the diacyl group of R 3c , R 3d , R 3e , and R 3f are each independently optionally substituted at each occurrence (e.g., C 1 -C 8 ) alkyl.

[0135] In some embodiments of the diacyl group, A 1 In some embodiments of the diacyl group, A is -O- or -NH-. 1 In some embodiments of a diacyl group, A 2 In some embodiments of the diacyl group, A is -O- or -NH-. 2 In some embodiments of a diacyl group, Y 3 is C 1 -C 12 (For example, C 1 -C 6 , for example C 1 -C 3 ) alkylene.

[0136] In some embodiments of a diacyl group, the diacyl group can be independently represented at each occurrence by the structural formula TIFF2025500547000135.tif41128 and, optionally, R 3c , R 3d , R 3e , and R 3f is independently at each occurrence hydrogen or C 1 -C 3 It is an alkyl.

[0137] In some embodiments, the linker group is independently represented by the structural formula TIFF2025500547000136.tif12128, where ** indicates the point of attachment of the linker to the proximal diacyl group and *** indicates the point of attachment of the linker to the distal diacyl group.

[0138] X if present 分枝 In some embodiments of the linker group, Y 1 is independently at each occurrence optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted allenylene. 分枝 In some embodiments of the linker group, Y 1 is independently at each occurrence an optionally substituted alkylene (e.g., C1 -C 12 ) X if it exists 分枝 In some embodiments of the linker group, Y 1 is independently at each occurrence an optionally substituted alkenylene (e.g., C 1 -C 12 ) X if it exists 分枝 In some embodiments of the linker group, Y 1 represents independently at each occurrence an optionally substituted allenylene (e.g., C 1 -C 12 ).

[0139] X 分枝 In some embodiments of the terminal groups of X, each terminal group is independently selected from an optionally substituted alkylthiol and an optionally substituted alkenylthiol. 分枝 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkylthiol (e.g., C 1 -C 18 , for example C 4 -C 18 ) X 分枝 In some embodiments of the terminal groups, each terminal group is an optionally substituted alkenyl thiol (e.g., C 1 -C 18 , for example C 4 -C 18 ).

[0140] X 分枝 In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of 1 -C 18 Alkenylthiol or C 1 -C 18 The alkylthiol and the alkyl or alkenyl moiety is selected from the group consisting of halogen, C 6 -C 12 Aryl, C 1 -C 12 Alkylamino, C 4 -C 6 N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C 1 -C3 Alkyl)-(C 1 -C 6 Alkylene)-(C 1 -C 12 alkylamino), -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), any of the aforementioned substituents. 4 -C 6 The N-heterocycloalkyl moiety is 1 -C 3 Alkyl or C 1 -C 3 It may be substituted with hydroxyalkyl.

[0141] X 分枝 In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of 1 -C 18 (For example, C 4 -C 18 ) alkenylthiol or C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl or alkenyl moiety being halogen, C 6 -C 12 Aryl (e.g., phenyl), C 1 -C 12 (For example, C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 Mono-alkylamino (e.g. -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C8 Di-alkylamino (e.g. TIFF2025500547000137.tif18128)), C 4 -C 6 N-heterocycloalkyl (e.g., N-pyrrolidinyl TIFF2025500547000138.tif15128, N-Piperidinyl TIFF2025500547000139.tif15128, N-Azepanil TIFF2025500547000140.tif15128), -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., TIFF2025500547000141.tif14128), -C(O)N(C 1 -C 3 Alkyl)-(C 1 -C 6 Alkylene)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000142.tif14128), -C(O)-(C 1 -C 12 alkylamino (e.g., mono- or di-alkylamino), and -C(O)-(C 4 -C 6 N-heterocycloalkyl) (e.g., TIFF2025500547000143.tif18128), wherein C is any of the aforementioned substituents. 4 -C 6 The N-heterocycloalkyl moiety is 1 -C 3 Alkyl or C 1 -C 3 It may be substituted with hydroxyalkyl. 分枝In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl portion of which may be substituted with one substituent -OH. X 分枝 In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of 1 -C 18 (For example, C 4 -C 18 ) alkylthiol, the alkyl portion being C 1 -C 12 (For example, C 1 -C 8 ) alkylamino (e.g., C 1 -C 6 Mono-alkylamino (e.g. -NHCH 2 CH 2 CH 2 CH 3 ) or C 1 -C 8 Di-alkylamino (e.g. TIFF2025500547000144.tif18128)), and C 4 -C 6 N-heterocycloalkyl (e.g., N-pyrrolidinyl TIFF2025500547000145.tif15128, N-Piperidinyl TIFF2025500547000146.tif15128, N-Azepanil TIFF2025500547000147.tif15128). 分枝 In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of 1 -C 18 (For example, C 4 -C 18 ) alkenylthiol or C 1 -C 18 (For example, C 4 -C 18 ) alkylthiol. X 分枝 In some embodiments of the terminal groups, each terminal group is independently selected from the group consisting of1 -C 18 (For example, C 4 -C 18 ) alkylthiol.

[0142] X 分枝 In some embodiments of the end groups, each end group is independently a structure shown in Table 3. In some embodiments, the dendrimers or dendrons described herein can include a selected end group in Table 3 or a pharma- ceutically acceptable salt thereof. In some embodiments, the exemplary end groups in Table 3 are not limited to the stereoisomers (i.e., enantiomers, diastereomers) listed.

[0143] Table 3: Exemplary end groups / peripheral structures TIFF2025500547000148.tif207151TIFF2025500547000149.tif220151TIFF2025500547000150.tif76151

[0144] In some embodiments, the dendrimer or dendron of formula (X) is selected from those shown in Table 4 and pharma- ceutically acceptable salts thereof.

[0145] Table 4: Exemplary ionizable cationic lipo-dendrimers or lipo-dendrons TIFF2025500547000151.tif98162TIFF2025500547000152.tif174162TIFF2025500547000153.tif179162TIFF2025 500547000154.tif160162TIFF2025500547000155.tif172162TIFF2025500547000156.tif226162TIFF20255005470 00157.tif161162TIFF2025500547000158.tif164162TIFF2025500547000159.tif157162TIFF2025500547000160.t if217162TIFF2025500547000161.tif211162TIFF2025500547000162.tif174162TIFF2025500547000163.tif132162 TIFF2025500547000164.tif227162TIFF2025500547000165.tif182162TIFF2025500547000166.tif181162TIFF202 5500547000167.tif225162TIFF2025500547000168.tif224162TIFF2025500547000169.tif123162TIFF20255005470 00170.tif137162TIFF2025500547000171.tif201162TIFF2025500547000172.tif215162TIFF2025500547000173.t if199162TIFF2025500547000174.tif205162TIFF2025500547000175.tif229162TIFF2025500547000176.tif159162

[0146] Other ionizable cationic lipids In some embodiments of the lipid composition, the cationic lipid has the structural formula (D-I'): TIFF2025500547000177.tif19128 included, During the ceremony: a is 1 and b is 2, 3, or 4; or alternatively, b is 1 and a is 2, 3, or 4; m is 1 and n is 1; or alternatively, m is 2 and n is 0; or alternatively, m is 2 and n is 1; and R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently 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 7 is independently 3 -C 18 Alkyl, C with one C=C double bond 3 -C 18 Alkenyl, amino protecting group, -C(=NH)NH 2 , a poly(ethylene glycol) chain, and a receptor ligand; However, R 1 ~R 6 At least two moieties in are independently -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 where R 7 is independently 3 -C 18Alkyl or C with one C=C double bond 3 -C 18 alkenyl; and wherein one or more of the nitrogen atoms shown in formula (D-I') may be protonated to provide a cationic lipid.

[0147] In some embodiments of the cationic lipid of formula (D-I'), a is 1. In some embodiments of the cationic lipid of formula (D-I'), b is 2. In some embodiments of the cationic lipid of formula (D-I'), m is 1. In some embodiments of the cationic lipid of formula (D-I'), n is 1. In some embodiments of the cationic lipid of formula (D-I'), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or -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 6 are each independently H or In some embodiments of the cationic lipid of formula (D-I'), R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H or 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).

[0148] In some embodiments, the cationic lipid of formula (D-I') is 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: The file is TIFF2025500547000180.tif75128.

[0149] In some embodiments, the cationic lipid of formula (D-I') is (11R,25R)-13,16,20-tris((R)-2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol: The file is TIFF2025500547000181.tif75128.

[0150] Additional cationic lipids that can be used in the compositions and methods of the present application include those described in J. McClellan, MC King, Cell 2010, 141, 210-217, as well as International Patent Publications WO 2010 / 144740, WO 2013 / 149140, WO 2016 / 118725, WO 2016 / 118724, WO 2013 / 063468, WO 2016 / 205691, WO 2015 / 184256, WO 2016 / 004202, WO 2015 / 199952, WO 2017 / 004143, WO 2017 / 075531, WO 2017 / 117528, WO Examples of ionizable cationic lipids include those described in WO 2017 / 049245, WO 2017 / 173054, and WO 2015 / 095340, which are incorporated herein by reference for all purposes. Examples of such ionizable cationic lipids include, but are not limited to, those shown in Table 5.

[0151] Table 5. Exemplary ionizable cationic lipids TIFF2025500547000182.tif79151TIFF2025500547000183.tif227151TIFF2025500547000184.tif165151TIFF20255005470 00185.tif228151TIFF2025500547000186.tif224151TIFF2025500547000187.tif227151TIFF2025500547000188.tif190151 TIFF2025500547000189.tif207151TIFF2025500547000190.tif206151TIFF2025500547000191.tif207151TIFF20255005470 00192.tif227151TIFF2025500547000193.tif206151TIFF2025500547000194.tif216151TIFF2025500547000195.tif209151

[0152] In some embodiments of the lipid composition of the present application, the ionizable cationic lipid is present in an amount of about 20 to about 23. In some embodiments, the molar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 20. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0153] In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of ionizable cationic lipid of about 5% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of ionizable cationic lipid of about 10% to about 25%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of ionizable cationic lipid of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of ionizable cationic lipid of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of ionizable cationic lipid of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, or at least (about) 30% molar percentage of ionizable cationic lipid. In some embodiments of the lipid composition of the present application, the lipid composition comprises at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, or at most (about) 30% molar percentage of ionizable cationic lipid.

[0154] Selective Organ Targeting (SORT) Lipids In some embodiments of the lipid compositions of the present application, the lipid (e.g., nanoparticle) composition is preferentially delivered to a target organ. In some embodiments, the target organ is the lung, lung tissue, or lung cells. As used herein, the term "preferentially delivered" is used to refer to a composition that, when delivered, is delivered to a target organ (e.g., lung), tissue, or cell at least 25% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%) of the amount administered.

[0155] In some embodiments of the lipid composition, the lipid composition comprises one or more selective organ targeting (SORT) lipids that provide for selective delivery of the composition to a particular organ. In some embodiments, the SORT lipid comprises two or more C 6 -C 24 The alkyl or alkenyl chain may be of the formula:

[0156] In some embodiments of the lipid composition, the SORT lipid comprises a permanently positively charged portion. The permanently positively charged portion can be positively charged at physiological pH, so that the SORT lipid comprises a positive charge during delivery of polynucleotide to cells. In some embodiments, the positively charged portion is a quaternary amine or a quaternary ammonium ion. In some embodiments, the SORT lipid comprises a counterion or is otherwise complexed or interacted with a counterion.

[0157] In some embodiments of the lipid composition, the SORT lipid is a permanent cationic lipid (i.e., comprises one or more hydrophobic components and a permanent cationic group). The permanent cationic lipid may contain a group that has a positive charge regardless of pH. One permanent cationic group that may be used in the permanent cationic lipid is a quaternary ammonium group. The permanent cationic lipid has the structural formula: TIFF2025500547000196.tif11128, During the ceremony: Y 1 , Y 2 , or Y 3 are each independently 1 C(O)R 1 or X 2 N + R 3 R 4 R 5 and; However, Y 1 , Y 2 , and Y 3 At least one of the 2 N + R 3 R 4 R5 and; R 1 is C 1 -C 24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 is a substituted alkenyl; X 1 is O or NR a where R a is hydrogen, C 1 -C 4 Alkyl, or C 1 -C 4 is a substituted alkyl; X 2 is C 1 -C 6 Alkanediyl or C 1 -C 6 is a substituted alkanediyl; R 3 , R 4 , and R 5 are each independently 1 -C 24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 is a substituted alkenyl; and A 1 is the X in the compound 2 N + R 3 R 4 R 5 It is an anion with a charge equal to the number of groups.

[0158] In some embodiments of the SORT lipid, the permanent cationic SORT lipid has the structural formula: I have TIFF2025500547000197.tif13128, During the ceremony: R 6 ~R 9 are each independently 1 -C24 Alkyl, C 1 -C 24 Substituted alkyl, C 1 -C 24 Alkenyl, C 1 -C 24 substituted alkenyl; 6 ~R 9 At least one of them is C 8 -C 24 is a group of; and A 2 is a monovalent anion.

[0159] In some embodiments of the lipid composition, the SORT lipid is an ionizable cationic lipid (i.e., comprises one or more hydrophobic components and an ionizable cationic group). The ionizable positively charged moiety can be positively charged at physiological pH. One ionizable cationic group that can be used in the ionizable cationic lipid is a tertiary ammine group. In some embodiments of the lipid composition, the SORT lipid has the structural formula: TIFF2025500547000198.tif24128, During the ceremony: R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group; and R 3 and R 3 ' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) It is.

[0160] In some embodiments of the lipid composition, the SORT lipid comprises a head group of a particular structure. In some embodiments, the SORT lipid has the structural formula: TIFF2025500547000199.tif5128, where L is a linker; Z + is a positively charged moiety and X -is a counter ion. In some embodiments, the linker is a biodegradable linker. The biodegradable linker can be degradable under physiological pH and temperature. The biodegradable linker can be degraded by proteins or enzymes from the subject. In some embodiments, the positively charged moiety is a quaternary ammonium ion or a quaternary amine.

[0161] In some embodiments of the lipid composition, the SORT lipid has the structural formula: TIFF2025500547000200.tif26128, where R 1 and R 2 each independently represents an optionally substituted C 6 -C 24 Alkyl or optionally substituted C 6 -C 24 It is alkenyl.

[0162] In some embodiments of the lipid composition, the SORT lipid has the structural formula: I have TIFF2025500547000201.tif26128.

[0163] In some embodiments of the lipid composition, the SORT lipid comprises a linker (L). In some embodiments, L is TIFF2025500547000202.tif14128, During the ceremony: p and q are each independently 1, 2, or 3; and R 4 is optionally substituted C 1 -C 6 It is an alkyl.

[0164] In some embodiments of the lipid composition, the SORT lipid has the structural formula: has TIFF2025500547000203.tif24128, During the ceremony: R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl(C8-C24) or a substituted version of either group; R 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6) and; R 4 is an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0165] In some embodiments of the lipid composition, the SORT lipid is phosphotidylcholine (e.g., 14:0 EPC). In some embodiments, the phosphatidylcholine compound further comprises: Defined as TIFF2025500547000204.tif25128, During the ceremony: R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group; R 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0166] In some embodiments of the lipid composition, the SORT lipid is a phosphocholine lipid. In some embodiments, the SORT lipid is an ethylphosphocholine. The ethylphosphocholine can be, for example and without limitation, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine.

[0167] In some embodiments of the lipid composition, the SORT lipid has the structural formula: has TIFF2025500547000205.tif24128, During the ceremony: R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group; R 3 , R 3 ', and R 3 Each " is independently an alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion.

[0168] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is 1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP) (e.g., the chloride salt).

[0169] In some embodiments of the lipid composition, the SORT lipid has the structural formula: has TIFF2025500547000206.tif14128, During the ceremony: R 4 and R 4 ' are each independently an alkyl (C6-C24) , alkenyl (C6-C24) or a substituted version of either group; R 4 '' is an alkyl (C≦24) , alkenyl (C≦24) or a substituted version of either group; R 4 ''' is an alkyl (C1-C8) , alkenyl (C2-C8) or a substituted version of either group; and X 2 is a monovalent anion.

[0170] By way of example, and not limitation, a SORT lipid of the structural formula in the immediately preceding paragraph is dimethyldioctadecylammonium (DDAB) (e.g., the bromide salt).

[0171] In some embodiments of the lipid composition, the SORT lipid comprises one or more selected from the lipids shown in Table 6.

[0172] Table 6. Exemplary SORT lipids TIFF2025500547000207.tif97151X - is the counter ion (e.g. Cl - , Br - etc.).

[0173] In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage 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 molar percentage 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 molar percentage of SORT lipid of about 30% to about 55%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage 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 molar percentage 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 molar percentage 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 molar percentage of SORT lipid of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, or at least about 65%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of SORT lipid of at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, or at most about 65%. In some embodiments of the lipid compositions of the present application, the lipid composition comprises a molar percentage of SORT lipid of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65%, or a range between (inclusive) any two of the foregoing values.

[0174] 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.

[0175] 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 comprises one or two long chains (e.g., C 6 -C 24 ) alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and optionally a small organic molecule. The small organic molecule can be an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is a distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids define lipids and lipid-like molecules that have both positive and negative charges.

[0176] In some embodiments of the lipid composition, the phospholipid is not ethylphosphocholine.

[0177] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of phospholipids relative to the total lipid composition of about 20 to about 23. In some embodiments, the molar percentage is from about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 60. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0178] In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 8% to about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 10% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 15% to about 20%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 8% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 10% to about 15%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of about 12% to about 18%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 23%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of phospholipids of at most about 8%, at most about 10%, at most about 12%, at most about 15%, at most about 18%, at most about 20%, or at most about 23%.

[0179] Steroids or steroid derivatives In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure that may further comprise one or more substitutions, including an alkyl group, an alkoxy group, a hydroxy group, an oxo group, an acyl group, or a double bond between two or more carbon atoms. In some embodiments, the ring structure of the steroid is represented by the formula: As shown in TIFF2025500547000208.tif18128, the steroid derivative comprises three fused cyclohexyl rings and a fused cyclopentyl ring. In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative has the formula TIFF2025500547000209.tif19128. In some embodiments of the present application, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure has the formula: Further defined by TIFF2025500547000210.tif36128. As above, cholestane derivatives include one or more non-alkyl substitutions of the above ring system. In some embodiments, cholestane or cholestane derivatives are cholestene or cholestene derivatives, or sterol or sterol derivatives. In other embodiments, cholestane or cholestane derivatives are both cholestere and sterol or derivatives thereof.

[0180] In some embodiments of the lipid composition, the composition may further comprise a molar percentage of steroid to total lipid composition of about 40 to about 46. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45 to about 46, or any range derivable therein. In other embodiments, the molar percentage of steroid to total lipid composition is about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.

[0181] In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 15% to about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 20% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 25% to about 35%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 30% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of steroids or steroid derivatives of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of steroids or steroid derivatives of at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 46%.

[0182] Polymer-conjugated lipids In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride that also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid comprises one or more C glycerides attached to a linker group with a PEG chain. 6 -C 24 Long chain hydrocarbon groups (e.g., C 6 -C 24 Long chain alkyl or alkenyl groups, or C 6-C 24In some embodiments, the alkyl, alkenyl, or fatty acid group is from about 6 carbon atoms to about 24 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is at least about 6 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is at most about 24 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is from about 6 carbon atoms to about 8 carbon atoms, from about 6 carbon atoms to about 10 carbon atoms, from about 6 carbon atoms to about 12 carbon atoms, from about 6 carbon atoms to about 14 carbon atoms, from about 6 carbon atoms to about 16 carbon atoms, from about 6 carbon atoms to about 20 carbon atoms, from about 6 carbon atoms to about 22 carbon atoms, from about 6 carbon atoms to about 24 carbon atoms, from about 8 carbon atoms to about 10 carbon atoms, from about 8 carbon atoms to about 12 carbon atoms, from about 8 carbon atoms to about 14 carbon atoms, from about 8 carbon atoms to about 16 carbon atoms, from about 8 carbon atoms to about 20 carbon atoms, from about 8 carbon atoms to about 22 carbon atoms, from about 8 carbon atoms to about 24 carbon atoms, from about 10 carbon atoms to about 12 carbon atoms, from about 10 carbon atoms to about 14 carbon atoms, from about 10 carbon atoms to about 16 carbon atoms, , about 10 carbon atoms to about 20 carbon atoms, about 10 carbon atoms to about 22 carbon atoms, about 10 carbon atoms to about 24 carbon atoms, about 12 carbon atoms to about 14 carbon atoms, about 12 carbon atoms to about 16 carbon atoms, about 12 carbon atoms to about 20 carbon atoms, about 12 carbon atoms to about 22 carbon atoms, about 12 carbon atoms to about 24 carbon atoms, about 14 carbon atoms to about 16 carbon atoms, about 14 carbon atoms to about 20 carbon atoms, about 14 carbon atoms to about 22 carbon atoms, about 14 carbon atoms to about 24 carbon atoms, about 16 carbon atoms to about 20 carbon atoms, about 16 carbon atoms to about 22 carbon atoms, about 16 carbon atoms to about 24 carbon atoms, about 20 carbon atoms to about 22 carbon atoms, about 20 carbon atoms to about 24 carbon atoms, or about 22 carbon atoms to about 24 carbon atoms. In some embodiments, the alkyl, alkenyl, or fatty acid group is about 6 carbon atoms, about 8 carbon atoms, about 10 carbon atoms, about 12 carbon atoms, about 14 carbon atoms,It is about 16 carbon atoms, about 20 carbon atoms, about 22 carbon atoms, or about 24 carbon atoms. In some embodiments, the long chain hydrocarbon group(s) can contain one or more unsaturated carbon bonds (e.g., double or triple carbon-carbon bonds). In some embodiments, the long chain hydrocarbon group can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more unsaturated carbon bonds. In some embodiments, the hydrocarbon group can contain 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less unsaturated carbon bonds. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, it is a PEG-modified diasteroylphosphatidylethanolamine or a PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. In some embodiments, the polymer conjugated lipid has a molecular weight of about 500 to about 100,000 Daltons (Da). In some embodiments, the polymer-conjugated lipid has a molecular weight of about 100, 200, 300, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, or more Da.The polymer conjugated lipid has a molecular weight of about 100, 200, 300, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, or more. In some embodiments, the polymer conjugated lipid has a molecular weight of 100,000, 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, 500, 300, 200, 100 or less. In some embodiments, the polymer conjugated lipid has a molecular weight of about 100 Da to about 100,000 Da. In some embodiments, the polymer conjugated lipid has a molecular weight of at least about 100 Da. In some embodiments, the polymer conjugated lipid has a molecular weight of at most about 100,000 Da. In some embodiments, the polymer-conjugated lipid has a molecular weight of from about 100 Da to about 200 Da, from about 100 Da to about 300 Da, from about 100 Da to about 500 Da, from about 100 Da to about 1,000 Da, from about 100 Da to about 2,000 Da, from about 100 Da to about 5,000 Da, from about 100 Da to about 10,000 Da, from about 100 Da to about 20,000 Da, from about 100 Da to about 50,000 Da, from about 100 Da to about 100,000 Da, from about 200 Da to about 300 Da, from about 200 Da to about 500 Da, from about 200 Da to about 1,000 Da, from about 200 Da to about 2,000 Da, from about 200 Da to about 5,000 Da, from about 200 Da to about 10,000 Da, from about 200 Da to about 20,000 Da. Da, about 200 Da to about 50,000 Da, about 200 Da to about 100,000 Da, about 300 Da to about 500 Da, about 300 Da to about 1,000 Da, about 300 Da to about 2,000 Da, about 300 Da to about 5,000 Da, about 300 Da to about 10,000 Da, about 300 Da to about 20,000 Da, about 300 Da to about 50,000 Da, about 300 Da to about 100,000 Da, about 500 Da to about 1,000 Da, about 500 Da to about 2,000 Da, about 500 Da to about 5,000 Da, about 500 Da to about 10,000 Da, about 500 Da to about 20,000 Da, about 500 Da to about 50,000 Da, about 500 Da to about 100,000 Da,Approximately 1,000 Da to approximately 2,000 Da, approximately 1,000 Da to approximately 5,000 Da, approximately 1,000 Da to approximately 10,000 Da, approximately 1,000 Da to approximately 20,000 Da, approximately 1,000 Da to approximately 50,000 Da, approximately 1,000 Da to approximately 100,000 Da, approximately 2,000 Da to approximately 5,000 Da, about 2,000 Da to about 10,000 Da, about 2,000 Da to about 20,000 Da, about 2,000 Da to about 50,000 Da, about 2,000 Da to about 100,000 Da, about 5,000 Da to about 10,000 Da, about 5,000 Da to about 20,000 Da, about 5,000 Da ~ approx. 50,000 Da, approx. 5,000 The molecular weight is about 100,000 Da, about 10,000 Da to about 20,000 Da, about 10,000 Da to about 50,000 Da, about 10,000 Da to about 100,000 Da, about 20,000 Da to about 50,000 Da, about 20,000 Da to about 100,000 Da, or about 50,000 Da to about 100,000 Da. In some embodiments, the polymer-conjugated lipid has a molecular weight of about 100 Da, about 200 Da, about 300 Da, about 500 Da, about 1,000 Da, about 2,000 Da, about 5,000 Da, about 10,000 Da, about 20,000 Da, about 50,000 Da, or about 100,000 Da. Some non-limiting examples of lipids that can be used in the present application are taught by U.S. Pat. No. 5,820,873, WO 2010 / 141069, or U.S. Pat. No. 8,450,298, which are incorporated herein by reference.

[0183] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: TIFF2025500547000211.tif18128, where R 12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or a substituted version of any of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8)and x is 1 to 250. In some embodiments, R e is an alkyl (C≦8) , for example methyl. 12 and R 13 are each independently an alkyl (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, methoxypolyethylene glycol.

[0184] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: TIFF2025500547000212.tif40128, where n 1 is an integer from 1 to 100, and n 2 and n 3 are each independently selected from an integer of 1 to 29. 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. 1 is about 30 to about 50. In some embodiments, n 2 In some embodiments, n is 5 to 23. 2 In some embodiments, n is from 11 to about 17. 3 In some embodiments, n is 5 to 23. 3 is between 11 and about 17.

[0185] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of PEG lipid to total lipid composition of about 4.0 to about 4.6. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range derivable therein. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0, or any range derivable therein.

[0186] In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of about 1% to about 8%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of about 2% to about 7%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of about 3% to about 5%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of about 5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a molar percentage of polymer-conjugated lipid 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%.

[0187] Pharmaceutical Compositions Therapeutic or prophylactic agents In some embodiments, provided herein are pharmaceutical compositions comprising a therapeutic agent (or prophylactic agent) assembled with the lipid compositions described herein.

[0188] In some embodiments of the pharmaceutical composition, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof. In some embodiments, the compound, the polynucleotide, the polypeptide, or a combination thereof is exogenous or heterologous to the cell or subject being treated by the pharmaceutical composition described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polynucleotide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a polypeptide described herein. In some embodiments, the therapeutic agent (or prophylactic agent) comprises a compound, a polynucleotide, a polypeptide, or a combination thereof.

[0189] In some embodiments, the pharmaceutical composition comprises a therapeutic (or prophylactic) agent for treating a pulmonary 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, the therapeutic (or prophylactic) agent comprises Abraxane, afatinib dimaleate, Afinitor, Afinitor Disperz, Alecensa, Alectinib, Alimta, Alunbrig, Atezolizumab, Avastin, Bevacizumab, Brigatinib, Capmatinib hydrochloride, Carboplatin, Ceritinib, Crizotinib, Cyramza, Dabrafenib mesylate, Dacomitinib, Docetaxel, Doxorubicin hydrochloride, Durvalumab, Entrectinib, Erlotinib hydrochloride, Everolimus, Gavreto, Gefitinib, Gilotrif, Gemcitabine, Ipilimumab, Iressa, Keytruda, Lorbrena, Mekinist, Methotrexate sodium, Necitumumab, Nivolumab, Osimertinib mesylate, Paclitaxel, Pembrolizumab, Pemet These include: Rexed disodium, Pralsetinib, Ramucirumab, Retevmo, Selpercatinib, Tabrecta, Tafinlar, Tagrisso, Trametinib dimethyl sulfoxide, Vizimpro, Vinorelbine tartrate, Xalkori, Yervoy, Zirabev, Zykadia, Carboplatin, Gemcitabine-cisplatin, Afinitor, Atezolizumab, Durvalumab, Etopophos, Etoposide, Hycamtin, Imfinzi, Keytruda, Lurbinectedin, Methotrexate sodium, Nivolumab, Opdivo, Pembrolizumab, Tecentriq, Topotecan hydrochloride, Trexall, or Zepzelca. Other non-limiting examples of therapeutic (or prophylactic) agents including compounds include small molecules selected from the following: 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acetretin, acrivastine, adenine,Adenosine, alatrofloxacin, albendazole, albuterol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol, all-trans retinoic acid (ATRA), aloxiprine, alprazolam, alprenolol, altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone HCl, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphotericin, B, ampicillin, amprenavir, amsacrine, amyl nitrate, amylobarbitone, anastrozole, amrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropine azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbitone, BCG raw, beclamide, beclomethasone, bendroff Lumethiazide, benezepril, benidipine, benorylate, benperidol, bentazepam, benzamide, benzanthrazene, benzathine penicillin, benzhexol HCl, benznidazole, benzodiazepine, benzoic acid, bephenium hydroxynaphthoate, betamethasone, bevacizumab (avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate ... Muperidol, Brotizolam, Budesonide, Bumetanide, Bupropion, Busulfan, Butalbital, Butamben, Butenafine HCl, Butobarbitone, Butobarbitone (Butetal), Butoconazole, Butoconazole Nitrate, Butylparaben, Caffeine, Calcifediol, Calciprotriene, Calcitriol, Calsterone, Cambendazole, Camphor, Camptothecin, Camptothecin Analogs, Candesartan, Capecitabine, Capsaicin, Captopril, Carbamazepine, Carbimazole, Carbofuran, Carboplatin,Carbromal, Kalimazole, Carmustine, Cefamandole, Cefazolin, Cefixime, Ceftazidime, Cefuroxime axetil, Celecoxib, Cephradine, Cerivastatin, Cetrizine, Cetuximab, Chlorambucil, Chloramphenicol, Chlordiazepoxide, Chlormethiazole, Chloroquine, Chlorothiazide, Chlorpheniramine, Chlorproguanil HCl, Chlorpromazine, Chlorpropamide, Chlorprothixene, Chlorpyrifos, Chlortetracycline, Chlorthalidone, Chlorzoxazone, Cholecalciferol , chrysene, cilostazol, cimetidine, cinaridine, cinoxacin, ciprofibrate, ciprofloxacin HCl, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estradiol cyclosporine, corticosterone, cortisone, cortisone acetate, cyclizine, cyclobarbital, cyclobenzaprine, cyclobutane-spirobarbiturate, cycloethane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopropane-spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, danthron, dantrolene sodium , dapsone, darbepoetin alfa, dalodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycorticosterone, desoximetasone, dexamethasone, dexamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine, diatrizoic acid, diazepam, diazoxide, dichlorophene, dichlorprop, diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin,Dihydrocodeine, dihydroequilin, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxamide furoate, dimenhydrinate, dimorpholamine, dinitrumide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin, doxycycline, dromostanolone propionate, droperidol, dyphylline, echinocandin, Econazole, Econazole Nitrate, Efavirenz, Ellipticine, Enalapril, Enlimomab, Enoximone, Epinephrine, Epipodophyllotoxin Derivatives, Epirubicin, Epoetin Alfa, Eposartan, Equilenin, Equilin, Ergocalciferol, Ergotamine Tartrate, Erlotinib, Erythromycin, Estradiol, Estramustine, Estriol, Estrone, Ethacrynic Acid, Ethambutol, Ethinamate, Ethionamide, Ethopropazine HCl, Ethyl-4-aminobenzoate (Benzocaine), Ethylparaben Ben, ethinyl estradiol, etodolac, etomidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpiclonil, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecamid acetate, floxuridine, fludarabine, fluconazole, flu Conazole, Flucytosine, Fludioxonil, Fludrocortisone, Fludrocortisone Acetate, Flufenamic Acid, Flunanisone, Flunarizine HCl, Flunisolide, Flunitrazepam, Fluocortolone, Fluometuron, Fluorene, Fluorouracil, Fluoxetine HCl, Fluoxymesterone, Flupentixol Decanoate, Fluphenthixol Decanoate, Flurazepam, Flurbiprofen, Fluticasone Propionate, Fluvastatin, Folic Acid, Fosenopril, Fosphenytoin Sodium, Frovatriptan, Furosemide,Fulvestrant, Furazolidone, Gabapentin, G-BHC (Lindan), Gefitinib, Gemcitabine, Gemfibrozil, Gemtuzumab, Glafenine, Glibenclamide, Gliclazide, Glimepiride, Glipizide, Glutethimide, Glyburide, Glyceryl trinitrate (nitroglycerin), Goserelin acetate, Grepafloxacin, Griseofulvin, Guaifenesin, Guanabenz acetate, Guanine, Halofantrine HCl, Haloperidol, Hydrochlorothiazide, Heptabarbital, Heroin, Hesperetin, Hexachlorobenzate hexetal, histrelin acetate, hydrocortisone, hydroflumethiazide, hydroxyurea, hyoscyamine, hypoxanthine, ibritumomab, ibuprofen, idarubicin, idobutal, ifosfamide, ihydroequilenin, imatinib mesylate, imipenem, indapamide, indinavir, indomethacin, indoprofen, interferon alpha-2a, interferon alpha-2b, iodamide, iopanoic acid, iprodione, irbesartan, irinotecan, isavuconazole, isocalor Voxazid, Isoconazole, Isoguanine, Isoniazid, Isopropyl Barbiturate, Isoproturon, Isosorbide Dinitrate, Isosorbide Mononitrate, Izradipine, Itraconazole, Itraconazole (Itra), Ivermectin, Ketoconazole, Ketoprofen, Ketorolac, Kellin, Labetalol, Lamivudine, Lamotrigine, Lanatoside C, Lanosprazole, L-DOPA, Leflunomide, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Levofloxacin, Lido Cain, Linuron, Lisinopril, Lomefloxacin, Lomustine, Loperamide, Loratadine, Lorazepam, Lorefloxacin, Lormetazepam, Losartan Mesylate, Lovastatin, Lisuride Maleate, Maprotiline HCl, Mazindol, Mebendazole, Meclizine HCl, Meclofenamic Acid, Medazepam, Medigoxin, Medroxyprogesterone Acetate, Mefenamic Acid, Mefloquine HCl, Megestrol Acetate, Melphalan, Mepenzolate Bromide, Meprobamate, Meptazinol, Mercaptopurine, Mesalazine, Mesna, Mesoridazine,Mestranol, methadone, methaqualone, methocarbamol, methoine, methotrexate, methoxsalen, methsuximide, methyclothiazide, methylphenidate, methylphenobarbitone, methyl-p-hydroxybenzoate, methylprednisolone, methyltestosterone, methyprylon, methysergide maleate, metoclopramide, metolazone, metoprolol, metronidazole, mianserin HCl, miconazole, midazolam, mifepristone, miglitol, minocycline, minoxidil, mitomycin C, mitotane, mitoxantrone, mofetil mycophenolate, molindone, montelukast, morphine, moxifloxacin HCl, nabumetone, nadolol, na, Rubuphin, nalidixic acid, nandrolone, naphthacene, naphthalene, naproxen, naratriptan HCl, natamycin, nelarabine, nelfinavir, nevirapine, nicardipine HCl, nicotinamide, nicotinic acid, nicoumarin, nifedipine, nilutamide, nimodipine, nimorazole, nisoldipine, nitrazepam, nitrofurantoin, nitrofurazone, nizatidine, nofetumomab, norethisterone, norfloxacin, norgestrel, nortriptyline HCl, nystatin, estradiol, ofloxacin, olanzapine, Omeprazole, Omoconazole, Ondansetron HCl, Oprelvequin, Ornidazole, Oxaliplatin, Oxamniquine, Oxanterembonate, Oxaprozin, Oxatomide, Oxazepam, Oxcarbazepine, Oxfendazole, Oxiconazole, Oxprenol, Oxyphenbutazone, Oxyphencyclimine HCl, Paclitaxel, Palifermin, Pamidronate, p-Aminosalicylic Acid, Pantoprazole, Paramethadione, Paroxetine HCl, Pegasusamide, Pegaspargase, Pegfilgras Chim, pemetrexed disodium, penicillamine, pentaerythritol tetranitrate, pentazocine, pentazocine, pentobarbital, pentobarbitone, pentostatin, pentoxifylline, perphenazine, perphenazine pimozide, perylene, phenacemide, phenacetin, phenanthrene, phenindione, phenobarbital, phenol barbitone, phenolphthalein, phenoxybenzamine, phenoxybenzamine HCl, phenoxymethylpenicillin, phensuximide, phenylbutazone, phenytoin, pindo rol, pioglitazone, pipobroman, piroxicam, pizotifen maleate, platinum compounds, plicamycin, polyenes, polymyxin B, porfimer sodium, posaconazole (Posa), pramipexole, prasterone, pravastatin, praziquantel, prazosin, prazosin HCl, prednisolone, prednisone, primidone, probarbital, probenecid, probucol, procarbazine, prochlorperazine, progesterone, proguanil HCl, promethazine, propofol, propoxur, propranolol,Propylparaben, Propylthiouracil, Prostaglandins, Pseudoephedrine, Pteridine-2-methyl-thiol, Pteridine-2-thiol, Pteridine-4-methyl-thiol, Pteridine-4-thiol, Pteridine-7-methyl-thiol, Pteridine-7-thiol, Pyrantel embonate, Pyrazinamide, Pyrene, Pyridostigmine, Pyrimethamine, Quetiapine, Quinacrine, Quinapril, Quinidine, Quinidine Sulfate, Quinine, Quinine Sulfate, Rabeprazole Sodium, Ranitidine HCl, Rasburicase, Ravuconazole , repaglinide, reposar, reserpine, retinoids, rifabutin, rifampicin, rifapentine, rimexolone, risperidone, ritonavir, rituximab, rizatriptan benzoate, rofecoxib, ropinirole HCl, rosiglitazone, saccharin, salbutamol, salicylamide, salicylic acid, saquinavir, sargramostim, secbutabarbital, secobarbital, sertaconazole, sertindole, sertraline HCl, simvastatin, sirolimus, sorafenib, sparfloxacin, spiramycin, spironolactone methadone, stanolone, stanozolol, stavudine, stilbestrol, streptozocin, strychnine, sulconazole, sulconazole nitrate, sulfacetamide, sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide, sulfathiazole, sulindac, sulfabenzamide, sulfaacetamide, sulfadiazine, sulfadoxine, sulfafurazole, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfasalazine, sulfinpyrazone, sulpiride, sulpha lutiam, sumatriptan succinate, sunitinib maleate, tacrine, tacrolimus, talbutal, tamoxifen citrate, tamulosin, targretin, taxane, tazarotene, telmisartan, temazepam, temozolomide, teniposide, tenoxicam, terazosin, terazosin HCl, terbinafine HCl, terbutaline sulfate, terconazole, terfenadine, testolactone, testosterone, tetracycline, tetrahydrocannabinol, tetroxoprim, thalidomide, thebaine, theobromine, theophylline, thiabendazole,Thiamphenicol, thioguanine, thioridazine, thiotepa, thotoin, thymine, tiagabine HCl, tibolone, ticlopidine, tinidazole, tioconazole, tirofiban, tizanidine HCl, tolazamide, tolbutamide, tolcapone, topiramate, topotecan, toremifene, tositumomab, tramadol, trastuzumab, trazodone HCl, tretinoin, triamcinolone, triamterene, triazolam, triazole, triflupromazine, trimethoprim, trimipramine maleate, tri Phenylene, troglitazone, tromethamine, tropicamide, trovafloxacin, tibamate, ubidecarenone (coenzyme Q10), undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronic acid, zolmitriptan, zolpidem, or zopiclone.

[0190] Polynucleotides In some embodiments of the pharmaceutical composition of the present disclosure, the therapeutic agent (or prophylactic agent) assembled with the lipid composition comprises one or more polynucleotides. However, the present application is not limited in scope to any particular source, sequence, or type of polynucleotide, since those skilled in the art can easily identify related homologues in various other sources of polynucleotides, including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). It is contemplated that the polynucleotides used in the present application can include sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, the sequence has at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90%, and most preferably about 95% of the nucleotides are identical to the nucleotide sequence of the naturally occurring sequence. In another embodiment, the polynucleotide comprises a nucleic acid sequence that is complementary to a naturally occurring sequence or is 75%, 80%, 85%, 90%, 95%, and 100% complementary to the naturally occurring sequence. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or more are contemplated herein.

[0191] In some embodiments, the polynucleotides used herein may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the polynucleotides will include complementary DNA (cDNA). Also contemplated is cDNA plus natural introns or introns from another gene; such engineered molecules are sometimes referred to as "minigenes". At a minimum, these and other nucleic acids of the present application may be used, for example, as molecular weight standards in gel electrophoresis. The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA, as opposed to genomic DNA or DNA polymerized from genomic, unprocessed or partially processed RNA templates, is that cDNA primarily contains the coding sequence of the corresponding protein. There are times when a complete or partial genomic sequence is preferred, for example, when non-coding regions are required for optimal expression, or when non-coding regions such as introns should be targeted in antisense strategies.

[0192] 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), clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, CRISPR-RNA (crRNA), single guide ribonucleic acid (sgRNA), trans-activating 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 of the therapeutic agents (or prophylactic agents) described herein. In some embodiments, the polynucleotide encodes at least one guide polynucleotide, such as a guide RNA (gRNA) or a guide DNA (gDNA), for complexing with a guide RNA-guided nuclease described herein. In some embodiments, the polynucleotide encodes a heterologous nuclease guided by at least one guide polynucleotide. The nuclease can be an endonuclease.Non-limiting examples of heterologous endonucleases guided by a guide polynucleotide include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregorii (Natronobacterium gregoryi) Argonaute (NgAgo); adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease, or any functional fragment thereof, any derivative thereof; any variant thereof; and any fragment thereof.

[0193] In some embodiments, the therapeutic (or prophylactic) agent is a transfer ribonucleic acid (tRNA), which introduces an amino acid into the growing peptide chain of a protein of a target gene.

[0194] Some embodiments of the therapeutic (or prophylactic) agents provided herein include a heterologous polypeptide comprising an actuator portion. The actuator portion can be arranged 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 altered expression or activity of the target gene can be detectable, for example, in at least about 1% (e.g., at least about 2%, 5%, 10%, 15%, or 20%) of cells (e.g., lung cells, e.g., lung basal cells) of the subject. The therapeutic (or prophylactic) agent can include a heterologous polynucleotide encoding an actuator portion. The actuator portion can be arranged to complex with a target polynucleotide corresponding to the target gene. The heterologous polynucleotide can encode a guide polynucleotide arranged to direct the actuator portion to the target polynucleotide. The actuator portion can include a heterologous endonuclease or a fragment thereof (e.g., directed by the guide polynucleotide to specifically bind to the target polynucleotide). The heterologous endonuclease can be (1) a part of a ribonucleoprotein (RNP) and (2) complexed with a guide polynucleotide. The heterologous endonuclease can be a part of a clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) protein complex. The heterologous endonuclease can be a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease. The heterologous endonuclease can include an inactivating endonuclease. The inactivating endonuclease can be fused to a regulatory moiety. The regulatory moiety can include a transcriptional activator, a transcriptional repressor, an epigenetic modifier, or a fragment thereof.

[0195] In some embodiments, the polynucleotide encodes a heterologous endonuclease that is guided by at least one guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA)). In some embodiments, the polynucleotide encodes at least one guide polynucleotide and at least one heterologous endonuclease, where the guide polynucleotide can complex with and guide the at least one heterologous endonuclease to cleave a gene locus of any one of the genes described herein. In some embodiments, the polynucleotide is a heterologous endonuclease guided by at least one guide polynucleotide, such as Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, Cas14, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, , CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, or Cul966; any derivative thereof; any variant thereof; or any fragment thereof. In some embodiments, Cas13 can include, but is not limited to, Cas13a, Cas13b, Cas13c, and Cas13d (e.g., CasRx).

[0196] In some embodiments, the heterologous endonuclease comprises an inactivated endonuclease, optionally fused to a regulatory moiety, such as an epigenetic modifier for remodeling the epigenome to mediate expression of a selected gene of interest. In some cases, the epigenetic modifier can include a methyltransferase, a demethylase, a dismutase, an alkylating enzyme, a depurinase, an oxidase, a photolyase, 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 deubiquitinating enzyme, an adenylate-forming enzyme, an AMPylator, a de-AMPylator, a SUMOylating enzyme, a deSUMOylating enzyme, a ribosylase, a derivosylase, an N-myristoyltransferase, a chromotin remodeling enzyme, a protease, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a synthase, a synthetase, or a demyristoylating enzyme. In some examples, the epigenetic modifier can include one or more selected from the group consisting of p300, TET1, LSD1, HDAC1, HDAC8, HDAC4, HDAC11, HDT1, SIRT3, HST2, CobB, SIRT5, SIR2A, SIRT6, NUE, vSET, SUV39H1, DIM5, KYP, SUVR4, Set4, Set1, SETD8, and TgSET8.

[0197] In some embodiments, the polynucleotide encodes a guide polynucleotide (such as a guide RNA (gRNA) or guide DNA (gDNA)) that is at least partially complementary to a genomic region of a gene, where upon binding of the guide polynucleotide to the gene, the guide polynucleotide recruits a nuclease guided by the guide polynucleotide to cleave the region and genetically modify it. Examples of genes that can be modified by nucleases guided by guide polynucleotides include CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0198] In some embodiments, polynucleotide comprises or codes for at least one mRNA, which when expressed by the mRNA restores the function of defective gene in the subject treated by the pharmaceutical composition described herein.For example, polynucleotide comprises or codes for the mRNA expressing wild-type CFTR protein, which can be used to rescue the subject suffering from congenital mutation in CFTR protein. Other examples of mRNAs that can be expressed from the polynucleotides include mRNAs encoding DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1.

[0199] In some embodiments, the polynucleotide of the present application comprises at least one chemical modification of one or more nucleotides. In some embodiments, the chemical modification increases the specificity of the guide polynucleotide (such as a guide RNA (gRNA) or a guide DNA (gDNA)) binding to a complementary genomic locus (e.g., a genomic locus of any one of the genes described herein). In some embodiments, the at least one chemical modification increases resistance to nuclease digestion when the polynucleotide is administered to a subject in need thereof. In some embodiments, the at least one chemical modification reduces immunogenicity when the polynucleotide is administered to a subject in need thereof. In some embodiments, the at least one chemical modification stabilizes a scaffold, such as a tRNA scaffold. Such chemical modifications may have desirable properties, such as enhanced resistance to nuclease digestion or increased binding affinity to a target genomic locus, compared to a polynucleotide that does not have at least one chemical modification.

[0200] In some embodiments, at least one chemical modification comprises a modification to the sugar moiety. In some embodiments, the modified sugar moiety is a substituted sugar moiety that includes 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 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 an 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 )(Rn ), and O--CH 2 --C(=O)--N(R m )(R n wherein each R and R is independently selected from H, or substituted or unsubstituted C 1 -C 10 is alkyl. Examples of sugar substituents at the 5' position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, such as a TF-5'-methyl sugar moiety.

[0201] Nucleosides that include a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, 2'-substituted nucleosides include halo, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S, or N(R m )-Alkynyl;O-Alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(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 ), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C 1 -C 10 These 2'-substituent groups are hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO 2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0202] In some embodiments, the 2'-substituted nucleoside is selected from the group consisting of F, NH 2 , N 3 , OCF 3 , O--CH 3 , O(CH 2 ) 3 NH 2 , C.H. 2 -CH=CH 2 , O--CH 2 -CH=CH 2 , O.C.H. 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 ), where each R m and R n are independently H, an amino protecting group, or a substituted or unsubstituted C 1 -C 10 It is an alkyl.

[0203] In some embodiments, the 2'-substituted nucleoside is F, OCF 3 , O--CH 3 , O.C.H. 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 The sugar moiety comprises a 2'-substituent group selected from:

[0204] In some embodiments, the 2'-substituted nucleoside is F, O--CH 3 , and O.C.H. 2 CH 2 OCH 3 The sugar moiety comprises a 2'-substituent group selected from:

[0205] Certain modified sugar moieties include a bridged sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-2' sugar substituents include --[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O--, or --C(R a R b )--O--N(R)--;4'-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 its analogs; 4'-C(CH 3 )(CH 3 )--O-2' and its analogues; 4'-CH 2 --N(OCH 3 )-2' and its analogues; 4'-CH 2 --O--N(CH 3 )-2';4'-CH 2 --O--N(R)-2' and 4'-CH 2 --N(R)--O-2'-, where each R is independently H, a protecting group, or C 1 -C 12 alkyl);4'-CH 2 --N(R)--O-2' (where 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 analogs thereof.

[0206] In some embodiments, such a 4'-2' bridge is independently --[C(R a )(R b )] n --, --C(R a )=C(R b )--, --C(R a )=N--, ​​--C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a ) 2 --, --S(=O) x -- and --N(R a wherein x is 0, 1, or 2; n is 1, 2, 3, or 4; each R a and R b are independently H, a protecting group, a hydroxyl, C 1 -C 12 Alkyl, substituted C 1 -C 12 Alkyl, C2 -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, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C 5 -C 7 Alicyclic radicals, substituted C 5 -C 7 Alicyclic radicals, halogens, OJ 1 , N.J. 1 J 2 , S.J. 1 , N 3 , COOJ 1 , acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O) 2 -J 1 ), or sulfoxyl (S(=O)-J 1 ) and each J 1 and J. 2 are independently H, 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, acyl (C(=O)--H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C 1 -C 12 Aminoalkyl, substituted C 1 -C 12 aminoalkyl, or a protecting group.

[0207] Nucleosides that contain a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include: (A) α-L-methyleneoxy (4'-CH 2 --O-2')BNA, (B) β-D-methyleneoxy (4'-CH 2 --O-2') BNA (also called 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 called constrained ethyl or cEt), (G) methylene-thio (4'-CH 2 --S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH 2 --CH(CH 3 )-2') BNA, (J) propylene carbocyclic (4'-(CH 2 ) 3 -2') BNA, and (K) methoxy(ethyleneoxy) (4'-CH(CH 2 OMe)-O-2') BNA (also called constrained MOE or cMOE).

[0208] In some embodiments, the bicyclic sugar moiety and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, nucleosides containing a 4'-2' methylene-oxy bridge can be in the .alpha.-L or .beta.-D configuration. Previously, .alpha.-L-methyleneoxy (4'-CH 2 --O-2') bicyclic nucleosides have been incorporated into antisense polynucleotides that have demonstrated antisense activity.

[0209] 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 and 4'-2' bridged sugars, where LNA is substituted, e.g., with a 5'-methyl or 5'-vinyl group).

[0210] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of naturally occurring sugar is replaced with, for example, sulfur, carbon, or nitrogen atom. In some such embodiments, such modified sugar moiety also includes bridging and / or non-bridging substituents as described above. For example, certain sugar surrogates include a 4'-sulfur atom and substitutions at the 2'-position and / or 5'-position. As an additional example, carbocyclic bicyclic nucleosides with 4'-2' bridges are described.

[0211] In some embodiments, the sugar surrogate comprises a ring with more than 5 atoms. For example, in some embodiments, the sugar surrogate comprises a 6-membered tetrahydropyran. Such tetrahydropyrans can be further modified or substituted. Nucleosides that comprise such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA), and fluoro-HNA (F-HNA).

[0212] In some embodiments, a modified THP nucleoside of formula VII is provided, wherein q 1 , q 2 , q 3 , q 4 , q 5 , q 6 , and q 7 are each H. In certain embodiments, q 1 , q 2 , q 3 , q 4 , q 5 , q 6 , and q 7 At least one of q is other than H. 1 , q2 , q 3 , q 4 , q 5 , q 6 , and q 7 In some embodiments, a THP nucleoside of formula VII is provided, wherein R 1 and R 2 is F. In certain embodiments, R 1 is fluoro and R 2 is H and R 1 is methoxy and R 2 is H, and R 1 is methoxyethoxy, and R 2 is H.

[0213] Many other bicyclo- and tricyclosugar surrogate ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds.

[0214] Combinations of modifications are also provided, including but not limited to, 2'-F-5'-methyl substituted nucleosides, and replacement of the ribosyl ring oxygen atom with S, and further substitutions at the 2'-position of the bicyclic nucleic acid, or alternatively, 5'-substitutions. 2 The --O-2' bicyclic nucleosides are further substituted at the 5' position with a 5'-methyl or 5'-vinyl group. The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described along with their oligomerization and biochemical studies.

[0215] In some embodiments, the present application provides polynucleotides that include modified nucleosides. These modified nucleotides may include modified sugars, modified nucleobases, and / or modified linkages. The particular modifications are selected so that the resulting polynucleotide has desirable characteristics. In some embodiments, the polynucleotide includes one or more RNA-like nucleosides. In some embodiments, the polynucleotide includes one or more DNA-like nucleotides.

[0216] 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.

[0217] In some embodiments, the modified nucleobase is selected from: universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases, as defined herein. 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, as defined herein, such as 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH 3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2 H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.

[0218] In some embodiments, the application provides polynucleotides comprising linked nucleosides. In such embodiments, the nucleosides may be linked to one another using any internucleoside linkage. Two major classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linking groups include methylenemethylimino (--CH 2 --N(CH 3 )--O--CH 2 --), thiodiesters (--O--C(O)--S--), thionocarbamates (--O--C(O)(NH)--S--); siloxanes (--O--Si(H) 2 --O--); and N,N'-dimethylhydrazine (--CH 2 --N(CH 3 )--N(CH 3 Modified linkages include, but are not limited to, alkyl phosphonates and alkyl thiophosphates. Modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester linkages. In some embodiments, internucleoside linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods for the preparation of phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0219] The polynucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), e.g., α or β for sugar anomers, or (D) or (L) for, e.g., amino acids, etc. All such possible isomers, as well as their racemic and optionally optically pure forms, are included in the antisense compounds provided herein.

[0220] Neutral internucleoside linkages include, but are not limited to, phosphotriester, methylphosphonate, MMI (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'), formacetal (3'-O--CH 2 --O-5'), and thioform acetal (3'-S--CH 2 --O-5'). Additional neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates, and amides (see, e.g., Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include mixed N, O, S, and CH 2 Non-ionic linkages containing moieties are included.

[0221] Additional modifications can 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 polynucleotide of the present application includes chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0222] In some embodiments, the polynucleotide described herein comprises or encodes at least one tRNA described herein. In some embodiments, the tRNA expressed from the polynucleotide restores the function of at least one defective tRNA in a subject treated with the pharmaceutical composition described herein. In some embodiments, the at least one tRNA expressed by the polynucleotide described herein can comprise a tRNA encoding alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylaniline, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, the at least one tRNA expressed by the polynucleotide described herein can comprise a tRNA encoding arginine, tryptophan, glutamic acid, glutamine, serine, tyrosine, lysine, leucine, glycine, or cysteine. In some embodiments, the tRNA encoded by the polynucleotide described herein can restore the expression of any one of the genes described herein.In some embodiments, the tRNA encoded by the polynucleotide described herein can restore the expression of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM or FBN1.

[0223] Polypeptides In some embodiments of the pharmaceutical composition of the present disclosure, the therapeutic agent (or prophylactic agent) assembled with the lipid composition comprises one or more, one or more polypeptides. Some polypeptides may comprise an enzyme, such as any one of the nuclease enzymes described herein. For example, the nuclease enzymes include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins. (RBP); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregorii Argonaute (NgAgo)); adenosine deaminases acting on RNA (ADAR); CIRTs, PUFs, homing endonucleases, or any functional fragment thereof, any derivative thereof; any variant thereof; and any fragment thereof. In some embodiments, the nuclease enzyme includes a Cas protein, such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Cs These may include n2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.In some embodiments, a Cas protein can be complexed with a guide polynucleotide described herein to form a CRISPR ribonucleoprotein (RNP).

[0224] The nuclease in the compositions described herein can be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme can direct the cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence, of any one of the genes described herein. For example, the CRISPR enzyme can be directed to and cleave at the genomic locus of CFTR, DNAH5, DNAH11, BMPR2, FAH, PAH, IDUA, COL4A3, COL4A4, COL4A5, PKD1, PKD2, PKHD1, SLC3A1, SLC7A9, PAX9, MYO7A, CDH23, USH2A, CLRN1, GJB2, GJB6, RHO, DMPK, DMD, SCN1A, SCN1B, F8, F9, NGLY1, p53, PPT1, TPP1, hERG, PPT1, ATM, or FBN1,

[0225] CRISPR enzyme may be mutated against corresponding wild type enzyme, so that mutated CRISPR enzyme lacks the ability to cut one or both strands of target polynucleotide containing target sequence.For example, aspartic acid to alanine substitution (D10A) in RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cuts both strands to a nickase (cuts single strand).In some embodiments, Cas9 nickase can be used in combination with guide sequence, for example, two guide sequences that target the sense strand and antisense strand of DNA target, respectively.This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0226] In some embodiments, the present disclosure provides a polypeptide that contains one or more therapeutic proteins.The therapeutic proteins that can be included in the composition include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, clotting factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase α, tissue plasminogen activator (TPA) Activase, glucocerebrosidase, interferon (IF) beta-1a, interferon beta-1b, interferon gamma, interferon α, TNF-α, IL-1 through IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin α, follicle stimulating hormone (FSH), and Factor VIII.

[0227] In some embodiments, the polypeptide comprises a peptide sequence that is at least partially identical to any of the therapeutic (or prophylactic) agents that comprise the peptide sequence. For example, the polypeptide may comprise a peptide sequence that is at least partially identical to an antibody (e.g., a monoclonal antibody) for treating a lung disease, such as lung cancer.

[0228] In some embodiments, the polypeptide comprises a peptide or protein that restores the function of a defective protein in a subject treated with the pharmaceutical composition described herein. For example, the polynucleotide comprises a peptide or protein that restores the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which can be used to rescue a subject suffering from an inborn error that results in the expression of a mutated CFTR protein. Other examples of rescue include wild-type dynein axonemal heavy chain 5, dynein axonemal heavy chain 11, bone morphogenetic protein receptor type 2, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, α-L-iduronidase, type IV collagen α3 chain, type IV collagen α4 chain, type IV collagen α5 chain, polycystin 1, polycystin 2, fibrocystin (or polyductin), solute carrier family 3 member 1, solute carrier family 7 member 9, paired box (Paired Box), and other proteins that can be used to rescue a subject suffering from an inborn error that results in the expression of a mutated CFTR protein. box gene 9, myosin VIIA, cadherin-associated 23, Usherin, cularin 1, gap junction beta-2 protein, gap junction beta-6 protein, rhodopsin, myotonic dystrophy protein kinase, dystrophin, sodium voltage-dependent channel alpha subunit 1, sodium voltage-dependent channel beta subunit 1, coagulation factor VIII, coagulation factor IX, N-glycanase 1, tumor protein p53, palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, Kv11.1 (alpha subunit of potassium ion channel), palmitoyl protein thioesterase 1, ATM serine / threonine kinase, or fibrillin 1 peptide or protein to a subject in need thereof.

[0229] In some embodiments, the pharmaceutical composition of the present application comprises a plurality of payloads assembled with (e.g., encapsulated therein) a lipid composition. The plurality of payloads assembled with the lipid composition can be arranged for gene editing or gene expression modification. The plurality of payloads assembled with the lipid composition can comprise a polynucleotide encoding an actuator portion (e.g., including a heterologous endonuclease such as Cas) or a polynucleotide encoding an actuator portion. The plurality of payloads assembled with the lipid composition can further comprise one or more (e.g., one or two) guide polynucleotides. The plurality of payloads assembled with the lipid composition can further comprise one or more donor or template polynucleotides. The plurality of payloads assembled with the lipid composition can comprise a ribonucleoprotein (RNP).

[0230] In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is (about) 20:1 or less, (about) 15:1 or less, (about) 10:1 or less, or (about) 5:1 or less. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is (about) 20:1 or more, (about) 15:1 or more, (about) 10:1 or more, or (about) 5:1 or more. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 10:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 15:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 5:1 to about 10:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphoric acid in the polynucleotide (N / P ratio) is about 5:1 to about 15:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 5:1 to about 20:1. In some embodiments of the pharmaceutical composition of the present application, the therapeutic agent (or prophylactic agent) is a polynucleotide, and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 15:1 to about 20:1.

[0231] In some embodiments of the pharmaceutical composition of the present disclosure, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 50:1 to about 1:100. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 1:1 to about 1:20. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 20:1 to about 1:50. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 50:1 to about 1:70. In some embodiments of the pharmaceutical composition of the present application, the molar ratio of the therapeutic agent to the total lipids of the lipid composition is about 70:1 to about 1:100. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipid of the lipid composition is (about) 1:1 or less, (about) 1:5 or less, (about) 1:10 or less, (about) 1:15 or less, (about) 1:20 or less, (about) 1:25 or less, (about) 1:30 or less, (about) 1:35 or less, (about) 1:40 or less, (about) 1:45 or less, (about) 1:50 or less, (about) 1:60 or less, (about) 1:70 or less, (about) 1:80 or less, (about) 1:90 or less, or (about) 1:100 or less. In some embodiments of the pharmaceutical compositions of the present application, the molar ratio of therapeutic agent to total lipid of the lipid composition is (about) 1:1 or more, (about) 1:5 or more, (about) 1:10 or more, (about) 1:15 or more, (about) 1:20 or more, (about) 1:25 or more, (about) 1:30 or more, (about) 1:35 or more, (about) 1:40 or more, (about) 1:45 or more, (about) 1:50 or more, (about) 1:60 or more, (about) 1:70 or more, (about) 1:80 or more, (about) 1:90 or more, or (about) 1:100 or more.

[0232] In some embodiments of the pharmaceutical compositions of the present disclosure, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% of the therapeutic agent is encapsulated in particles of the lipid composition.

[0233] In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles characterized by one or more of the following characteristics: (1) a (e.g., average) size of 100 nanometers (nm) or less; (2) a polydispersity index (PDI) of about 0.2 or less; and (3) a zeta potential of -10 millivolts (mV) to 10 mV.

[0234] In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a size (e.g., average) 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 size (e.g., average) of about 70 nanometers (nm) to about 100 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a size (e.g., average) of about 50 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a size (e.g., average) of about 60 nanometers (nm) to about 80 nanometers (nm). In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a (e.g., average) size of at most about 100 nanometers (nm), at most about 90 nanometers (nm), at most about 85 nanometers (nm), at most about 80 nanometers (nm), at most about 75 nanometers (nm), at most about 70 nanometers (nm), at most about 65 nanometers (nm), at most about 60 nanometers (nm), at most about 55 nanometers (nm), or at most about 50 nanometers (nm). In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a size (e.g., average) 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 size (e.g., average) can be determined by spectroscopic or image-based methods, such as dynamic light scattering, static light scattering, multi-angle light scattering, laser light scattering, or dynamic image analysis, or a combination thereof.

[0235] In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.05 to about 0.5. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.5. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.1 to about 0.3. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.2 to about 0.5. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a polydispersity index (PDI) of about 0.5 or less, about 0.4 or less, about 0.3 or less, about 0.2 or less, about 0.1 or less, or about 0.05 or less.

[0236] In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or less. In some embodiments of the pharmaceutical composition of the present disclosure, 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 disclosure, 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 disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of 20 millivolts (mV) or less.

[0237] In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -5 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -10 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -15 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -20 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of -30 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 0 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 5 millivolts (mV) or greater. In some embodiments, the lipid composition comprises a plurality of particles having a zeta potential of 10 millivolts (mV) or greater. In some embodiments of the pharmaceutical compositions of the present disclosure, 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 disclosure, the lipid composition comprises a plurality of particles having a negative zeta potential of 20 millivolts (mV) or more.

[0238] In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent ionization constant (pKa) outside the range of 6 to 7. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 8 or more, about 9 or more, about 10 or more, about 11 or more, about 12 or more, or about 13 or more. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 8 to about 13. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 8 to about 10. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 9 to about 11. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 10 to about 13. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 8 to about 12. In some embodiments of the pharmaceutical composition of the present disclosure, the lipid composition has an apparent pKa of about 10 to about 12.

[0239] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent characterized by one or more of the following: (a) a therapeutic effect in a cell of the subject that is greater than that achieved with a reference lipid composition; (b) a therapeutic effect in a plurality of cells of the subject that is greater than that achieved with a reference lipid composition; (c) a therapeutic effect in a first plurality of cells of a first cell type and in a greater number of second plurality of cells of a second cell type; and (d) a therapeutic effect in a first cell of a first cell type of the subject that is greater than that in a second cell of a second cell type of the subject. In some embodiments, the first cell type is different from the second cell type.

[0240] In some embodiments of the pharmaceutical composition of the present disclosure, the cell is a lung cell. In some embodiments, the lung cell is a lung airway cell. Exemplary lung airway cells that can be targeted by the delivery of the present disclosure include, but are not limited to, basal cells.

[0241] In some embodiments of the pharmaceutical compositions of the present disclosure, the therapeutic effect is characterized by a therapeutically effective amount of the therapeutic agent, e.g., in the lungs, lung cells, lung cells, or lung cell types of the subject. In some embodiments, the therapeutic effect is characterized by the activity of the therapeutic agent, e.g., in the lungs, lung cells, lung cells, or lung cell types of the subject. In some embodiments, the therapeutic effect is characterized by the effect of the therapeutic agent, e.g., in the lungs, lung cells, lung cells, or lung cell types of the subject. In some embodiments, a greater therapeutic effect is characterized by a greater therapeutic amount of the therapeutic agent. In some embodiments, a greater therapeutic effect is characterized by a greater activity of the therapeutic agent. In some embodiments, a greater therapeutic effect is characterized by a greater effect of the therapeutic agent.

[0242] In some embodiments of the pharmaceutical composition of the present disclosure, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent to cells of a subject, characterized by a greater therapeutic effect compared to that achieved with a reference lipid composition. In some embodiments, the reference lipid composition does not include a SORT lipid. In some embodiments, the reference lipid composition does not include an amount of SORT lipid. In some embodiments, the reference lipid includes 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), phospholipid, cholesterol, and PEG-lipid.

[0243] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold therapeutic efficacy compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold therapeutic efficacy compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold therapeutic efficacy compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold therapeutic efficacy compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect that is at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than that achieved with a reference lipid composition.

[0244] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold therapeutic efficacy in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold therapeutic efficacy in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold therapeutic efficacy in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold therapeutic efficacy in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in basal cells that is at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than that achieved with a reference lipid composition.

[0245] In some embodiments of the pharmaceutical composition of the present disclosure, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent to cells of a subject, characterized by a therapeutic effect in a plurality of cells that is greater than that achieved with a reference lipid composition. In some embodiments, the reference lipid composition does not include SORT lipid. In some embodiments, the reference lipid composition does not include an amount of SORT lipid. In some embodiments, the reference lipid includes 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), phospholipid, cholesterol, and PEG-lipid.

[0246] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves a therapeutic effect in about 1.1-fold to about 20-fold more cells than that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 10-fold more cells than that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 5-fold to about 10-fold more cells than that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 10-fold to about 20-fold more cells than that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in a cell that is at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than that achieved with a reference lipid composition.

[0247] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves a therapeutic effect in about 1.1-fold to about 20-fold more cells in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 10-fold more cells in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 5-fold to about 10-fold more cells in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 10-fold to about 20-fold more cells in basal cells compared to that achieved with a reference lipid composition. In some embodiments, the SORT lipid achieves a therapeutic effect in about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold more cells than that achieved with a reference lipid composition in basal cells.

[0248] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent to cells of a subject characterized by a therapeutic effect in a first plurality of cells of a first cell type and a greater therapeutic effect in a second plurality of cells of a second cell type. In some embodiments, the first cell type is different from the second cell type.

[0249] In some embodiments of the pharmaceutical composition of the present disclosure, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The exemplary lung airway cell that can be targeted by the delivery of the present application includes but is not limited to basal cell.

[0250] In some embodiments of the pharmaceutical compositions of the present disclosure, the second cell type is a lung cell. In some embodiments, the second cell type is a lung airway cell.

[0251] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves a therapeutic effect in about 1.1-fold to about 20-fold more of the second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in about 1.1-fold to about 10-fold more of the second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in about 5-fold to about 10-fold more of the second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in about 10-fold to about 20-fold more of the second plurality of cells of the second cell type compared to the first plurality of cells of the first cell type. In some embodiments, the SORT lipid achieves a therapeutic effect in at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold more of a second plurality of cells of a second cell type compared to a first plurality of cells of a first cell type.

[0252] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition provides delivery of a therapeutic agent to a cell of a subject characterized by a greater therapeutic effect in a first cell of a first cell type compared to a second cell of a second cell type. In some embodiments, the first cell type is different from the second cell type.

[0253] In some embodiments of the pharmaceutical composition of the present disclosure, the first cell type is lung cell.In some embodiments, the first cell type is lung airway cell.The example of lung airway cell that can be targeted by the delivery of the present application includes but is not limited to basal cell.

[0254] In some embodiments of the pharmaceutical compositions of the present disclosure, the second cell type is a lung cell. In some embodiments, the second cell type is a lung airway cell.

[0255] In some embodiments of the pharmaceutical compositions of the present disclosure, the SORT lipid in the pharmaceutical composition achieves about 1.1-fold to about 20-fold greater therapeutic effect in a first cell of a first cell type compared to that achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 1.1-fold to about 10-fold greater therapeutic effect in a first cell of a first cell type compared to that achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 5-fold to about 10-fold greater therapeutic effect in a first cell of a first cell type compared to that achieved in a second cell of a second cell type. In some embodiments, the SORT lipid achieves about 10-fold to about 20-fold greater therapeutic effect in a first cell of a first cell type compared to that achieved in a second cell of a second cell type. In some embodiments of the methods, the SORT lipid achieves a therapeutic effect in a first cell of a first cell type that is at least about 1.1 fold, at least about 1.5 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 16 fold, at least about 17 fold, at least about 18 fold, at least about 19 fold, or at least about 20 fold greater than that achieved in a second cell of a second cell type.

[0256] In some embodiments, provided herein are (e.g., pharmaceutical) compositions that include components that allow for improved efficacy or outcome based on the delivery of polynucleotides. The compositions described elsewhere herein may be more effective in delivery to a particular cell, cell type, organ, or body region compared to a reference composition or compound. The compositions described elsewhere herein may be more effective in producing an increased expression of the polypeptide corresponding to the delivered polynucleotide. The compositions described elsewhere herein may be more effective in producing a greater number of cells that express the polypeptide corresponding to the delivered polynucleotide. The compositions described elsewhere herein may result in increased uptake of the polynucleotide compared to a reference polynucleotide. The increased uptake may be the result of improved stability of the polynucleotide or improved targeting of the composition to a particular cell type or organ. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising 13,16,20-tris(2-hydroxydodecyl)-13,16,20,23-tetraazapentatricontane-11,25-diol ("LF92"), phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids.In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 5-fold greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG-lipid. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in at least 10-fold greater expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a cell compared to that achieved with a reference lipid composition comprising LF92, phospholipid, cholesterol, and PEG-lipid.

[0257] In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in a greater number of cells than that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in at least 1.1-fold greater number of cells than that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in at least 2-fold greater number of cells than that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in at least 5-fold greater number of cells than that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in expression or activity of the polynucleotide (or the corresponding polypeptide of the polynucleotide) in at least a 10-fold greater number of cells compared to that achieved with a reference lipid composition comprising LF92, a phospholipid, cholesterol, and a PEG-lipid.

[0258] In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in increased uptake of the polynucleotide in a plurality of cells compared to that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids. In some embodiments, the SORT lipid is present in the lipid composition in an amount that results in increased uptake of the polynucleotide in a plurality of cells compared to that achieved with a reference lipid composition comprising LF92, phospholipids, cholesterol, and PEG-lipids.

[0259] Protein Corona Binding In some embodiments, upon administration, the surface of the pharmaceutical composition as described herein binds to one or more target proteins, including a protein corona. In some embodiments, the surface of the pharmaceutical composition can bind to the first target protein. In some embodiments, the surface of the pharmaceutical composition can bind to the first target protein and the second target protein. The surface of the pharmaceutical composition can bind to the first target protein and the second target protein in a weight or mass ratio. The weight or mass ratio can be determined, for example, by incubation assay.

[0260] The surface of the pharmaceutical composition as disclosed herein may include a protein corona. The protein corona may include one or more (e.g., serum or blood) proteins. The one or more proteins may include apolipoproteins, complement proteins, immune proteins, coagulation proteins, or any other proteins. In some embodiments, the one or more proteins include a target protein. The one or more target proteins may include apolipoproteins, complement proteins, immune proteins, coagulation proteins, or any other proteins. In some embodiments, the one or more target proteins are alpha-2-HS-glycoprotein, complement C1q subcomponent subunit C, alpha-1-antitrypsin 1-3, Ig alpha chain C region, Ig μ chain C region (fragment), serine protease inhibitor A3K, apolipoprotein CI, serum albumin, immunoglobulin heavy variable 1-34 (fragment), vitamin K-dependent protein Z, immunoglobulin kappa variable 6-13, Ig gamma-2 B chain C region, histone H, beta-2-glycoprotein 1, Ig heavy chain V region X44, protease inhibitor A3K ... Protein Z-dependent protease inhibitor, immunoglobulin heavy constant α (fragment), C-reactive protein, mannose-binding protein C, immunoglobulin kappa variable 1-110 (fragment), β-casein, immunoglobulin heavy constant μ, serum paraoxonase / arylesterase, glycosylphosphatidylinositol-specific phospholipase D1, inter-α-trypsin inhibitor, heavy chain, immunoglobulin heavy constant γ2C (fragment), complement C3, immunoglobulin kappa variable 17-127 (fragment), Ig heavy chain V region AC38 205.12, complement factor D, serotransferrin, β-globin, coagulation factor VII, Ig kappa chain V-III region 50S10.1, α-S1-casein, inter-α-trypsin inhibitor heavy chain H3, apolipoprotein A-IV, protein Igkv12-41 (fragment), α-S2-casein-like A, Ig heavy chain V region 6.96, clusterin, murinoglobulin-1, lactadherin, fibrinogen β chain, coagulation factor V, Ig κ chain V-II region 26-10, Ig γ-1 chain C region secretory type (fragment), immunoglobulin heavy constant γ3 (fragment), platelet factor 4, apolipoprotein AI, lipopolysaccharide binding protein, immunoglobulin heavy variable 5-9 (fragment), Ig κ chain VV region HP 124E, histidine-rich glycoprotein, Ig heavy chain V-III region J606, Ig κ chain V-III region PC 2880 / PC 1229, Ig κ chain VV region HP 124E1, band 3 anion transport protein, immunoglobulin kappa variable 17-121 (fragment), apolipoprotein N, plasminogen, immunoglobulin kappa chain variable 8-30 (fragment), complement C1s-A subcomponent, hemoglobin subunit beta-2, immunoglobulin kappa variable 1-135 (fragment), vitamin K-dependent protein C, H-2 class I histocompatibility antigen, Q10 alpha chain, alpha globin 1, thrombospondin-1, apolipoprotein D, coagulation factors, fibrinogen gamma chain, immunoglobulin heavy variable 7-1 (fragment), immunoglobulin kappa variable 4-57 (fragment), immunoglobulin heavy variable These include variable V1-5, Ig heavy chain V region 914, histone H2B, immunoglobulin heavy constant gamma 3 (fragment), apolipoprotein E, fibrinogen alpha chain, complement C1q subcomponent subunit A, immunoglobulin heavy variable 5-9 (fragment), immunoglobulin kappa constant, apolipoprotein C-III, immunoglobulin heavy constant gamma 2B (fragment), prothrombin, complement C1q subcomponent subunit, carboxypeptidase N catalytic chain, vitronectin, immunoglobulin kappa variable 12-46 (fragment), Ig gamma-2A chain C region, membrane-bound, or immunoglobulin kappa variable 12-44 (fragment).

[0261] In some embodiments, the pharmaceutical composition may bind to the first target protein and the second target protein at a certain weight or mass ratio. The weight or mass ratio may range from 1:1 to 20:1 or more. In some embodiments, the weight or mass ratio may be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 or more. In some embodiments, the weight or mass ratio can be 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, or less.

[0262] In some embodiments, the composition of the protein corona may determine the organ and / or cell type tropism (e.g., targeting) of the pharmaceutical composition as described herein. Organ and / or cell type targeting may be determined by the presence or absence of a particular target protein, the weight or mass ratio between one target protein and another target protein(s), or some combination thereof. The presence, absence, and / or weight or mass ratio between one protein or another or between groups of proteins may be determined by incubation assays. In some embodiments, the incubation assay may include incubating the lipid composition in a serum or plasma sample from an organism (e.g., a mouse). Proteins bound to the lipid composition may be isolated and / or purified and quantified by any suitable technique known in the art, such as Bradford assay or other colorimetric assays, UV-vis spectroscopy, biuret assay, and fluorescence assay. Proteins isolated from such samples may be further characterized or identified by mass spectrometry, gel electrophoresis (e.g., native gels, SDS PAGE gels), or other suitable techniques known in the art.

[0263] The targeting of a composition comprising a given protein corona can be determined by measuring (directly, indirectly, and / or relatively) the amount of the composition or a portion thereof (e.g., payload, therapeutic agent) delivered to one or more organs or cell types. For example, measuring the targeting of a composition comprising a protein corona can include measuring the amount, expression, or activity of a therapeutic payload contained in the composition in a target organ or cell type versus a reference organ or cell type. In some embodiments, the amount, expression, or activity of a therapeutic agent delivered by a composition comprising a protein corona is at least about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, or more. In some embodiments, the amount, expression, or activity of a therapeutic agent delivered by a composition comprising a protein corona is less than about 1 / 20, about 1 / 19, about 1 / 18, about 1 / 17, about 1 / 16, about 1 / 15, about 1 / 14, about 1 / 13, about 1 / 12, about 1 / 11, about 1 / 10, about 1 / 9, about 1 / 8, about 1 / 7, about 1 / 6, about 1 / 5, about 1 / 4, about 1 / 3, about 1 / 2, or even less.

[0264] Assays for determining targeting of compositions comprising a protein corona may include any suitable quantitative procedure or functional assay known in the art. As non-limiting examples, such assays may include quantification of the emission of a fluorescent payload or its transcription / translation products, immunofluorescence assays targeting the payload or its translation products, etc.

[0265] In some embodiments, the protein corona comprises apolipoprotein E (Apo E) and serum albumin. In such cases, the composition comprising the protein corona may target the liver or liver cells. In some embodiments, Apo E is present in a weight or mass ratio of about 6:1, about 5:1, about 4:1, or about 3:1 or less to serum albumin. In some embodiments, the protein corona further comprises complement C1q subcomponent subunit A, immunoglobulin heavy constant μ, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, clusterin, another protein, or any combination thereof, as determined by incubation assay. In some embodiments, the protein corona comprises at least one, at least two, or at least three proteins listed in Table 10 (e.g., not listed in Table 9 or listed differently from Table 9).

[0266] In some embodiments, the protein corona contains less Apo E than endogenous proteins that are not Apo E, as determined by incubation assay. In some embodiments, the endogenous proteins are beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement C1q subcomponent subunit A, vitronectin, and serum paraoxonase / arylesterase 1, clusterin, another protein, or a combination thereof. In some cases, the protein corona further contains apolipoprotein C (Apo C). In such cases, the protein corona may contain less Apo C than Apo E, as determined by incubation assay.

[0267] In some embodiments, the protein corona comprises vitronectin and clusterin. In such cases, the composition comprising the protein corona may target the lung or lung cells. In some embodiments, vitronectin is present in a weight or mass ratio of about 6:1 or about 5:1 or less to clusterin as determined by incubation assay. In some embodiments, the protein corona comprises serum paraoxonase / arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo E) as determined by incubation assay. H), immunoglobulin (Ig) μ chain C region, α-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen α chain, vitamin K-dependent protein Z, α-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein AI, immunoglobulin heavy constant alpha, immunoglobulin (Ig) gamma-2A chain C region, β-globin, complement C1q subcomponent subunit C, protein Z-dependent protease inhibitor, clusterin, another protein, or any combination thereof. In some embodiments, the protein corona comprises at least one, at least two, or at least three proteins listed in Table 12 (e.g., not listed in Table 9 or listed differently from Table 9).

[0268] In some embodiments, the protein corona comprises beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H) and a second target protein different from beta-2 glycoprotein 1 or Apo H. Beta-2 glycoprotein 1 or Apo H may be present in a weight or mass ratio of about 20:1, 15:1, 10:1 or less to the second target protein. In such cases, the pharmaceutical composition may target the spleen, bone marrow, or lymph nodes, or cells therein. In some embodiments, the cells are spleen cells or macrophages. In some embodiments, the second target protein comprises immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein CI, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) μ chain C region, serum albumin, serum paraoxonase / arylesterase 1, immunoglobulin heavy constant alpha, clusterin, and immunoglobulin kappa variable 6-13, another protein, or a combination thereof. In some embodiments, the protein corona comprises at least one, at least two, or at least three proteins listed in Table 11 (e.g., not listed in Table 9 or listed differently from Table 9). In some embodiments, the protein corona does not comprise beta-2 glycoprotein 1 or Apo H. In such cases, the composition may target an organ, or cells therein, that is not the spleen, bone marrow, or lymph node. The composition may not target spleen cells or macrophages.

[0269] Methods for targeted delivery In some embodiments, a method is provided herein for targeted delivery of a therapeutic agent to an organ or a cell therein in a subject in need thereof.The method may comprise administering to a subject a therapeutic agent assembled with a lipid composition such as those described herein.In some embodiments, the lipid composition comprises an ionizable cationic lipid; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid that is separate from the ionizable cationic lipid and the polymer-conjugated lipid.In some embodiments, the administering comprises intravenous administration.In some embodiments, the subject's body fluid (e.g., plasma or serum) comprises a plurality of target proteins.In some embodiments, the plurality of target proteins are a plurality of endogenous proteins of the subject.

[0270] In some embodiments of the methods described herein, upon administration, the surface of the lipid composition binds to a plurality of target proteins, as determined by incubation assay, the plurality of target proteins comprising a first target protein to a second target protein different from the first target protein in a weight or mass ratio of about 20:1, 15:1, or 10:1 or less, thereby delivering a therapeutic agent to a target organ or target cell in a subject. In some embodiments, the method provides an amount, expression, or activity of a therapeutic agent in an organ or cell in a subject that is greater than (e.g., at least about 2-fold) that is achieved with a corresponding reference lipid composition (e.g., without binding to the plurality of target proteins). In some embodiments, the method provides an amount, expression, or activity of a therapeutic agent in an organ or cell in a subject that is greater than (e.g., at least about 2-fold) that is achieved without the polymer-conjugated lipid. In some embodiments, the method provides an amount, expression, or activity of a therapeutic agent in an organ or cell in a subject that is greater than (e.g., at least about 2-fold) that is achieved with a reference organ or cell.

[0271] In some embodiments, the method described herein is for targeting delivery of therapeutic agent to liver or liver cells in a subject in need thereof.In some embodiments, when administered, the surface of the lipid composition binds to multiple target proteins as determined by incubation assay, and the multiple target proteins include apolipoprotein E (Apo E) and serum albumin, thereby delivering therapeutic agent to liver or liver cells in a subject.In some embodiments, Apo E is present in the multiple target proteins at a weight or mass ratio of about 6:1, 5:1, 4:1, or 3:1 or less to serum albumin as determined by incubation assay. In some embodiments, the plurality of target proteins further comprises complement C1q subcomponent subunit A, immunoglobulin heavy constant μ, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, or any combination thereof, as determined by incubation assay. In some embodiments, the plurality of target proteins comprises at least one, at least two, or at least three proteins listed in Table 10 (e.g., not listed in Table 9 or listed differently from Table 9). In some embodiments, the SORT lipid comprises an ionizable cationic moiety (e.g., a tertiary amine moiety). In some embodiments, the SORT lipid is an ionizable cationic lipid. In some embodiments, the lipid composition comprises a molar percentage of SORT lipid of about 5% to about 65%. In some embodiments, the method provides for an amount, expression, or activity of a therapeutic agent in a liver or liver cells in a subject that is greater (e.g., at least about 2-fold, 3-fold, 4-fold, 5-fold, or 6-fold) than that achieved with a corresponding reference lipid composition (e.g., no binding to multiple target proteins).

[0272] In some embodiments, the methods described herein are for targeted delivery of therapeutic agents to non-liver organs or non-liver cells therein in a subject in need thereof. In some embodiments, upon administration, the surface of the SORT lipid composition interacts with Apolipoprotein E (Apo E) to a lesser extent than with endogenous proteins in the subject that are not Apo E, as determined by incubation assay, and the endogenous proteins that are not Apo E are selected from beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H), immunoglobulin kappa constant, complement C1q subcomponent subunit A, vitronectin, and serum paraoxonase / arylesterase 1, thereby delivering therapeutic agents to non-liver organs or non-liver cells in the subject. In some embodiments, the non-liver organ comprises lung, spleen, bone marrow, or lymph node. In some embodiments, the non-liver cells comprise lung cells, spleen cells, or macrophages. In some embodiments, apolipoprotein E (Apo E) is not the most abundant protein in the plurality of target proteins. In some embodiments, upon administration, the surface of the lipid composition interacts with apolipoprotein C (Apo C) to a lesser extent than with apolipoprotein E (Apo E) in the subject as determined by incubation assay. In some embodiments, the method provides an amount or activity of a therapeutic agent in the liver or cells therein in the subject that is less than that achieved without the polymer-conjugated lipid. In some embodiments, the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of SORT lipid of about 5% to about 65%.

[0273] In some embodiments, the methods described herein are for targeted delivery of a therapeutic agent to the lung or lung cells therein in a subject in need thereof. In some embodiments, upon administration, the surface of the lipid composition binds to a plurality of target proteins, as determined by incubation assay, the plurality of target proteins including vitronectin (Vtn) and clusterin, thereby delivering the therapeutic agent to the lung or lung cells in the subject. In some embodiments, vitronectin is present in the plurality of target proteins at a weight or mass ratio of about 6:1, or 5:1 or less to clusterin, as determined by incubation assay. In some embodiments, the plurality of target proteins is serum paraoxonase / arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo E), as determined by incubation assay. H), immunoglobulin (Ig) μ chain C region, α-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen α chain, vitamin K-dependent protein Z, α-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein AI, immunoglobulin heavy constant alpha, immunoglobulin (Ig) gamma-2A chain C region, β-globin, complement C1q subcomponent subunit C, protein Z-dependent protease inhibitor, or any combination thereof. In some embodiments, the multiple target proteins include at least one, at least two, or at least three proteins listed in Table 12 (e.g., not listed in Table 9 or listed differently from Table 9). In some embodiments, the SORT lipid is a cationic lipid. In some embodiments, the SORT lipid is a permanent cationic lipid. In some embodiments, the SORT lipid is an ionizable cationic lipid.In some embodiments, the lipid composition comprises a molar percentage of SORT lipid of about 5% to about 65%. In some embodiments, the method provides for an amount, expression, or activity of a therapeutic agent in a lung or lung cell in a subject that is greater (e.g., at least about 2-fold, 5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold) than that achieved with a corresponding reference lipid composition (e.g., no binding to multiple target proteins).

[0274] In some embodiments, the method described herein is for targeted delivery of therapeutic agents to spleen, bone marrow, or lymph nodes, or cells therein, in a subject in need thereof.In some embodiments, when administered, the surface of the lipid composition binds to multiple target proteins, as determined by incubation assay, and the multiple target proteins comprise beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H) with a weight or mass ratio of about 20:1, 15:1, or 10:1 or less to a second target protein different from beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H), thereby delivering therapeutic agents to spleen, bone marrow, or lymph nodes, or cells in a subject.In some embodiments, the cells comprise spleen cells or macrophages. In some embodiments, the second target protein is selected from immunoglobulin kappa constant, complement C1q subcomponent subunit A, apolipoprotein E (Apo E), immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, vitronectin, complement C1q subcomponent subunit C, apolipoprotein CI, immunoglobulin (Ig) gamma-2A chain C region, immunoglobulin (Ig) μ chain C region, serum albumin, serum paraoxonase / arylesterase 1, immunoglobulin heavy constant alpha, and immunoglobulin kappa variable 6-13. In some embodiments, the SORT lipid is a permanent cationic lipid or an anionic lipid. In some embodiments, the multiple target proteins include at least one, at least two, or at least three proteins listed in Table 11 (e.g., not listed in Table 9 or listed differently from Table 9). In some embodiments, the SORT lipid is a permanent cationic lipid. In some embodiments, the SORT lipid is an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of SORT lipids from about 5% to about 65%.In some embodiments, the method provides an amount, expression, or activity of a therapeutic agent in the lung or lung cells in a subject that is greater (e.g., at least about 2-fold) than that achieved with a corresponding reference lipid composition (e.g., without binding to multiple target proteins).

[0275] In some embodiments, the method described herein is for targeted delivery of therapeutic agents to non-splenic organs or non-splenic cells in a subject in need thereof. In some embodiments, upon administration, the surface of the lipid composition binds to multiple target proteins, as determined by incubation assay, and the multiple target proteins include a first target protein to a second target protein different from the first target protein at a weight or mass ratio of about 20:1, 15:1, or 10:1 or less, thereby delivering therapeutic agents to non-splenic organs or non-splenic cells in the subject. In some embodiments, the non-splenic organ is not spleen, bone marrow, or lymph node. In some embodiments, the non-splenic cell is not a spleen cell or a macrophage. In some embodiments, beta-2 glycoprotein 1 (β2-GP1) or apolipoprotein H (Apo H) is not the most abundant protein in the multiple target proteins. In some embodiments, the multiple target proteins include clusterin. In some embodiments, the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid. In some embodiments, the lipid composition comprises a molar percentage of SORT lipid of about 5% to about 65%.

[0276] In some cases, in the methods described herein, the therapeutic agent is a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a microribonucleic acid (miRNA), a primary microribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a transactivating ribonucleic acid (TRNA), a ribonucleic acid (CRIC)-associated nucleic acid, ... The nucleic acid may include a nucleic acid sequence encoding a CRISPR-associated (Cas) protein, a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof.

[0277] formulation In some embodiments, in any of the methods or compositions provided herein, the therapeutic agent provided herein may be present in an intravenous composition. In some embodiments, the therapeutic agent provided herein may be present in an aerosol composition. In some embodiments, the lipid composition may be formulated as an aerosol. In some embodiments, the compositions provided herein may be formulated as an aerosol dosage form. In other embodiments, the compositions provided herein are formulated as an intravenous dosage form. In some embodiments, the lipid composition may be formulated as a nebulizer. In some embodiments, the compositions described herein are administered via a nebulizer. In some embodiments, the lipid composition may be administered as an aerosol. In some embodiments, the compositions described herein may be stored at -70°C or below.

[0278] In some embodiments, the compositions described herein may be formulated as a dispersion. In some embodiments, the concentration of the dispersion is about 0.5 mg / mL to about 5 mg / mL. In some embodiments, the concentration of the dispersion is about 0.5 mg / mL to about 1 mg / mL. In some embodiments, the concentration of the dispersion is about 0.5 mg / mL to about 2 mg / mL. In some embodiments, the concentration of the dispersion is about 0.5 mg / mL to about 3 mg / mL. In some embodiments, the concentration of the dispersion is about 2 mg / mL to about 3 mg / mL. In some embodiments, the concentration of the dispersion is about 2 mg / mL to about 4 mg / mL. In some embodiments, the concentration of the dispersion is 5 mg / mL or less. In some embodiments, the concentration of the dispersion is 1 mg / mL. In some embodiments, the compositions described herein are dispersed at pH 7.5.

[0279] In some embodiments, the compositions provided herein are administered to a human. In some embodiments, the compositions provided herein are administered to an adult. In other embodiments, the compositions provided herein are administered to a child. In some embodiments, the compositions provided herein are administered to a patient in the range of 18-35 kg / m 2 In some embodiments, the compositions provided herein are administered to patients with a body mass index of ≥ 50 kg. In other embodiments, the compositions provided herein are administered to patients with a total body weight of ≥ 50 kg. In some embodiments, the compositions described herein are administered intravenously. In some embodiments, the compositions described herein are delivered via inhalation. In some embodiments, the compositions described herein can include administration by nebulization. In some embodiments, the compositions described herein can include administration to the lungs by nebulization. In some embodiments, the compositions described herein are administered at least once a week. In some embodiments, the compositions described herein are administered at least twice a week.

[0280] In any of the compositions or methods provided herein, the composition is administered in any suitable dose. In some embodiments, the dose refers to the amount of the composition. In some embodiments, the dose refers to the amount of the therapeutic agent. In some embodiments, the dose administered is about 1 mg to about 30 mg. In some embodiments, the dose administered is about 1 mg to about 2.5 mg, about 1 mg to about 5 mg, about 1 mg to about 7.5 mg, about 1 mg to about 10 mg, about 1 mg to about 15 mg, about 1 mg to about 20 mg, about 1 mg to about 25 mg, about 1 mg to about 30 mg, about 2.5 mg to about 5 mg, about 2.5 mg to about 7.5 mg, about 2.5 mg to about 10 mg, about 2.5 mg to about 15 mg, about 2.5 mg to about 20 mg, about 2.5 mg to about 25 mg, about 2.5 mg to about 30 mg, about 5 mg to about 7.5 mg, about 5 mg to about 10 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 5 mg to about 25 mg, about 5 mg to about 30 mg, about 7.5 mg to about 10 mg, about 7.5 mg to about 15 mg, about 7.5 mg to about 20 mg, about 7.5 mg to about 25 mg, about 7.5 mg to about 30 mg, about 10 mg to about 15 mg, about 10 mg to about 20 mg, about 10 mg to about 25 mg, about 10 mg to about 30 mg, about 15 mg to about 20 mg, about 15 mg to about 25 mg, about 15 mg to about 30 mg, about 20 mg to about 25 mg, about 20 mg to about 30 mg, or about 25 mg to about 30 mg. In some embodiments, the administered dose is about 1 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg. In some embodiments, the administered dose is at least about 1 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, or about 25 mg. In some embodiments, the dose administered is at most about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, or about 30 mg. In some embodiments, the dose administered is about 2.5 mg. In some embodiments, the dose administered is about 5.0 mg.In some embodiments, the dose administered is about 10.0 mg. In some embodiments, the dose administered is about 20.0 mg.

[0281] In any of the compositions or methods provided herein, dosage can be determined by referring to body weight.In any of these compositions or methods, any suitable dosage can be used.In some embodiments, dosage is about 0.01 mg / kg body weight to about 1 mg / kg body weight. In some embodiments, the dose is from about 0.01 mg / kg body weight to about 0.05 mg / kg body weight, from about 0.01 mg / kg body weight to about 0.1 mg / kg body weight, from about 0.01 mg / kg body weight to about 0.5 mg / kg body weight, from about 0.01 mg / kg body weight to about 0.8 mg / kg body weight, from about 0.01 mg / kg body weight to about 1 mg / kg body weight, from about 0.05 mg / kg body weight to about 0.1 mg / kg body weight, from about 0.05 mg / kg body weight to about 0.5 mg / kg body weight, from about 0.05 mg / kg body weight to about 0.8 mg / kg body weight, from about 0.05 mg / kg body weight to about 1 mg / kg body weight, from about 0.1 mg / kg body weight to about 0.5 mg / kg body weight, from about 0.1 mg / kg body weight to abo...

Claims

1. 1. A composition for targeted delivery to the liver or liver cells in a subject in need thereof, comprising a lipid composition and a therapeutic agent assembled therewith, The lipid composition is ionizable cationic lipids; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. Including, when the therapeutic agent assembled with the lipid composition is administered to the subject, the surface of the lipid composition preferentially binds to a target protein relative to a lipid composition not assembled with the SORT lipids, wherein the binding is determined by an incubation assay, and the target protein comprises complement C1q subcomponent subunit A, immunoglobulin heavy constant μ, complement C1q subcomponent subunit B, immunoglobulin kappa constant, immunoglobulin heavy constant gamma 2B, beta-globin, immunoglobulin (Ig) gamma-2A chain C region, complement C1q subcomponent subunit C, immunoglobulin heavy constant alpha, fibrinogen beta chain, fibrinogen gamma chain, immunoglobulin kappa variable 17-127, alpha globin 1, fibrinogen alpha chain, or any combination thereof; The composition.

2. The composition of claim 1, wherein the administration provides an amount, expression, or activity of the therapeutic agent in the liver or liver cells of the subject that is at least about two times greater than that achieved with a corresponding reference lipid composition without the binding to the target protein.

3. 1. A composition for targeted delivery to a non-liver organ or cell in a subject in need thereof, comprising a lipid composition and a therapeutic agent assembled therewith, The lipid composition is ionizable cationic lipids; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. Including, When the therapeutic agent assembled with the lipid composition is administered to the subject, the surface of the lipid composition interacts with apolipoprotein E (Apo E) to a lesser extent than with endogenous proteins in the subject that are not Apo E, as determined by an incubation assay, and the endogenous proteins that are not Apo E are selected from the group consisting of immunoglobulin kappa constant, complement C1q subcomponent subunit A, serum paraoxonase / arylesterase 1, and any combination thereof. The composition.

4. The endogenous protein other than Apo E is immunoglobulin heavy constant gamma 2B, complement C1q subcomponent subunit B, complement C1q subcomponent subunit C, apolipoprotein CI, Ig gamma-2A chain C region, Ig μ chain C region, immunoglobulin heavy constant alpha, immunoglobulin kappa variable region 6-13, immunoglobulin heavy constant gamma 2C, fibrinogen beta chain, alpha-S1-casein, Ig heavy chain V region AC38 205.12, clusterin, immunoglobulin kappa variable region 1-110, protein Igkv12-41, beta-globin, fibrinogen alpha chain, apolipoprotein AI, fibrinogen gamma chain, lipopolysaccharide-binding protein, histidine-rich glycoprotein, Ig kappa chain VV region HP 124E, Ig kappa chain V-III region PC 2880 / PC 1229, Ig gamma-1 chain C region secretor, immunoglobulin kappa chain variable 8-30, apolipoprotein C-III, alpha globin 1, immunoglobulin heavy variable 5-9, complement C3, coagulation factor V, alpha-1-antitrypsin 1-3, carboxypeptidase N catalytic chain, immunoglobulin heavy constant gamma 3, or a combination thereof; The non-liver organ comprises a lung, a spleen, a bone marrow, or a lymph node. the non-liver cells include lung cells, spleen cells, or macrophages; Upon administration, the surface of the lipid composition interacts with apolipoprotein C (Apo C) to a lesser extent than with apolipoprotein E (Apo E) in the subject, as determined by an incubation assay; and / or the administration provides an amount or activity of the therapeutic agent in the liver or cells therein in the subject that is less than that achieved without the polymer-conjugated lipid. The composition of claim 3.

5. 1. A composition for targeted delivery to the lung or lung cells in a subject in need thereof, comprising a therapeutic agent assembled with a lipid composition, The lipid composition is ionizable cationic lipids; a polymer-conjugated lipid; and a selective organ targeting (SORT) lipid separate from the ionizable cationic lipid and the polymer-conjugated lipid. Including, when the therapeutic agent assembled with the lipid composition is administered to the subject, the surface of the lipid composition preferentially binds to the target protein over a lipid composition that is not assembled with the SORT lipids; The binding is determined by an incubation assay; The target protein may be clusterin, serum paraoxonase / arylesterase 1, apolipoprotein E (Apo E), serum albumin, immunoglobulin kappa constant, prothrombin, complement C1q subcomponent subunit A, fibrinogen beta chain, beta-2 glycoprotein 1 (β2-GP1), or apolipoprotein H (Apo E). H), immunoglobulin (Ig) μ chain C region, α-S1-casein, immunoglobulin heavy constant gamma 2B, fibrinogen gamma chain, fibrinogen α chain, vitamin K-dependent protein Z, α-1-antitrypsin 1-3, plasminogen, apolipoprotein C-III, complement C1q subcomponent subunit B, thrombospondin-1, coagulation factor X, apolipoprotein AI, immunoglobulin heavy constant alpha, immunoglobulin (Ig) gamma-2A chain C region, β-globin, complement C1q subcomponent subunit C, protein Z-dependent protease inhibitor, or any combination thereof, The composition.

6. The composition of claim 5, wherein the administration provides an amount, expression, or activity of the therapeutic agent in the lungs or lung cells of the subject that is at least about two times greater than that achieved with a corresponding reference lipid composition without the binding to the target protein.

7. the lipid composition is characterized by a zeta (ζ) potential of the lipid composition, as determined by dynamic light scattering (DLS), of about −10 millivolts (mV) to about 10 mV; Optionally, the lipid composition is characterized by a zeta (ζ) potential of the lipid composition, as determined by DLS, of about 0 millivolts (mV) to about 10 mV.

10. The composition of any one of claims 1, 3, and 5.

8. 6. The composition of any one of claims 1, 3, and 5, wherein the polymer-conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid.

9. A composition described in any one of claims 1, 3, and 5, wherein the polymer-conjugated lipid comprises one or more hydrocarbon chains each containing from about 8 to about 18 carbon atoms.

10. one hydrocarbon chain of the one or more hydrocarbon chains of the polymer-conjugated lipid comprises three or fewer unsaturated carbon-carbon bonds; 10. The composition of claim 9, wherein optionally, one hydrocarbon chain of the one or more hydrocarbon chains of the polymer-conjugated lipid contains no more than two unsaturated carbon-carbon bonds.

11. the polymer-conjugated lipid comprises a polymer having a molecular weight of about 100 Daltons (Da) to about 100,000 Da, and optionally the polymer-conjugated lipid comprises a polymer having a molecular weight of about 500 Da to about 100,000 Da; or the lipid composition comprises about 0.5% to about 20% molar percentage of the polymer-conjugated lipid, optionally the lipid composition comprises about 0.5% to about 10% molar percentage of the polymer-conjugated lipid; or the cationic ionizable lipid comprises a dendron or dendrimer comprising one or more branches, each of the one or more branches comprising two or more degradable functional groups; the cationic ionizable lipid is a dendron or dendrimer containing one or more diacyl groups; or The ionizable cationic lipid has the structural formula: or a pharmaceutically acceptable salt thereof, wherein: (a) The core has the structural formula (X コア ): Including, During the ceremony: Q, independently in each occurrence, is a covalent bond, -O-, -S-, or -NR 2 - or -CR 3a R 3b - and; R 2 is independently calculated for each occurrence by R 1g or -L 2 -NR 1e R 1f and R 3a and R 3b each occurrence independently represents hydrogen or an optionally substituted C 1 -C 6 is alkyl; R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (when present) independently at each occurrence represents a point of attachment to a branch, hydrogen, or an optionally substituted C 1 -C 12 is alkyl; L 0 , L 1 , and L 2 independently at each occurrence a covalent bond, C 1 -C 12 Alkylene, C 1 -C 12 Heteroalkylene, [C 1-C 6 alkylene]-[C 4 -C 6 Heterocycloalkyl]-[C 1 -C 6 alkylene], [C 1 -C 6 alkylene]-(arylene)-[C 1 -C 6 alkylene], C 4 -C 6 heterocycloalkyl, and arylene; or Alternatively, L 1 Part of R 1c and R 1d One of them, C 4 -C 6 forming a heterocycloalkyl; and x 1 is 0, 1, 2, 3, 4, 5, or 6; and (b) each branch of the plurality (N) of branches independently has the structural formula (X 分枝 ): Including, During the ceremony: * indicates the attachment point of the branch to the core; g is 1, 2, 3, or 4; Z＝2 (g-1) ； When g = 1, G = 0; or when g ≠ 1, is; (c) each diacyl group independently has the structural formula Including, During the ceremony: * indicates the point of attachment of the diacyl group at its proximal end; ** indicates the point of attachment of the diacyl group at its distal end; Y 3 is, independently at each occurrence, optionally substituted C 1 -C 12 Alkylene, optionally substituted C 1 -C 12 Alkenylene or optionally substituted C 1 -C 12 is allenylene; A 1 and A 2 are each independently at each occurrence -O-, -S-, or -NR 4 -where: R 4 is hydrogen or optionally substituted C 1 -C 6 is alkyl; m 1 and m 2 is each independently 1, 2, or 3 in each occurrence; and R 3c , R 3d , R 3e , and R 3f each occurrence independently represents hydrogen or an optionally substituted C 1 -C 8 is alkyl; and (d) each linker group independently has the structural formula Including, During the ceremony: ** indicates the point of attachment of the linker to the proximal diacyl group; *** indicates the point of attachment of the linker to the distal diacyl group; and Y 1 is, independently at each occurrence, optionally substituted C 1 -C 12 Alkylene, optionally substituted C 1 -C 12 Alkenylene or optionally substituted C 1 -C 12 is allenylene; and (e) each terminal group independently optionally substituted C 1 -C 18 Alkylthiols and optionally substituted C 1 -C 18 alkenyl thiols, 10. The composition of any one of claims 1, 3, and 5.

12. x 1 is 0, 1, 2, or 3, or R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (if present) each independently in each occurrence represents a point of attachment to a branch, hydrogen, or C 1 -C 12 alkyl, and the alkyl moiety is —OH, C 4 -C 8 Heterocycloalkyl, N-(C 1 -C 3 alkyl)-piperidinyl, piperazinyl, N-(C 1 -C 3 alkyl)-piperazidinyl, morpholinyl, N-pyrrolidinyl, pyrrolidinyl, N-(C 1 -C 3 alkyl)-pyrrolidinyl, C 6 -C 10 Aryl, C 3 -C 5 substituted with one or more substituents each independently selected from heteroaryl, heteroaryl, or pyridinyl; R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , and R 1g (if present) each independently in each occurrence is a point of attachment to a branch, hydrogen, or C 1 -C 12 alkyl, the alkyl moiety being optionally substituted with one substituent -OH; R 3a and R 3b is, independently at each occurrence, hydrogen or The plurality (N) of branches includes at least three branches, or (i) g=1; G=0; and Z=1, (ii) g=2; G=1; and Z=2, (iii) g=3; G=3; and Z=4, or (iv) g=4; G=7; and Z=8, 12. The composition of claim 11.

13. Each of the multiple branches has the structural formula Contains or Each of the multiple branches has the structural formula Contains or Each of the multiple branches has the structural formula Contains, or Each of the multiple branches has the structural formula Including, 13. The composition of claim 12.

14. The core has the structural formula: wherein Q' is -NR 2 -or-CR 3a R 3b - and;q 1 and q 2 are each independently 1 or 2, or The core has the structural formula: wherein ring A is an optionally substituted aryl or an optionally substituted C 3 -C 12 is heteroaryl, or The core has the structural formula Contains, or The core, and pharmaceutically acceptable salts thereof, wherein * indicates the point of attachment of the core to one branch of the multiple branches; A 1 is -O- or -NH-, A 2 is -O- or -NH-, Y 3 But C 1 -C 12 Is it alkylene? The diacyl group, independently at each occurrence, has the structural formula and optionally, wherein R 3c , R 3d , R 3e , and R 3f are each independently in each occurrence hydrogen or C 1 -C 3 Is it alkyl? L 0 , L 1 , and L 2 independently in each occurrence a covalent bond, C 1 -C 6 Alkylene, C 2 -C 12 Alkylene oxide, [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )heterocycloalkyl]-[(C 1 -C 4 ) alkylene], and [(C 1 -C 4 ) alkylene]-phenylene-[(C 1 -C 4 ) alkylene]; L 0 , L 1 , and L 2 However, each occurrence independently, C 1 -C 6 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)-; L 0 , L 1 , and L 2 However, each occurrence independently, C 1 -C 6 Is it alkylene? L 0 , L 1 , and L 2 However, each occurrence independently, C 2 -C 12 an alkylene oxide; L 0 , L 1 , and L 2 However, each occurrence is independent of the other, [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )heterocycloalkyl]-[(C 1 -C 4 ) alkylene], and [(C 1 -C 4 ) alkylene]-[(C 4 -C 6 )heterocycloalkyl]-[(C 1 -C 4 ) alkylene]; Each end group is independently C 1 -C 18 Alkenyl thiol or C 1 -C 18 alkylthiol, wherein the alkyl or alkenyl moiety is selected from the group consisting of halogen, C 6 -C 12 Aryl, C 1 -C 12 Alkylamino, C 4 -C 6 N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C 1 -C 3 alkyl)-(C 1 -C 6 alkylene)-(C 1 -C 12 alkylamino), -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 4 -C 6 The N-heterocycloalkyl moiety is C 1 -C 3 Alkyl or C 1 -C 3 may be substituted with hydroxyalkyl, Each end group is independently C 1 -C 18 alkylthiol, wherein the alkyl moiety is C 6 -C 12 Aryl, C 1 -C 12 Alkylamino, C 4 -C 6 N-heterocycloalkyl, -OH, -C(O)OH, -C(O)N(C 1 -C 3 alkyl)-(C 1 -C 6 alkylene)-(C 1 -C 12 alkylamino), -C(O)N(C 1 -C 3 alkyl)-(C 1 -C 6 alkylene)-(C 4 -C 6 N-heterocycloalkyl), and -C(O)-(C 4 -C 6 N-heterocycloalkyl), and C 4 -C 6 The N-heterocycloalkyl moiety is C 1 -C 3 Alkyl or C 1 -C 3 may be substituted with hydroxyalkyl, Each end group is independently C 1 -C 18 an alkylthiol, the alkyl moiety of which may be substituted with one substituent -OH; Each end group is independently C 1 -C 18 alkylthiol, wherein the alkyl moiety is C 1 -C 12 Alkylamino and C 4 -C 6 N-heterocycloalkyl, Each end group is independently C 1 -C 18 Alkenyl thiol or C 1 -C 18 an alkyl thiol, Each end group is independently C 1 -C 18 an alkyl thiol, Each end group independently represents or The dendrimer or dendron is and pharmaceutically acceptable salts thereof; 12. The composition of claim 11.

15. the lipid composition comprises about 5% to about 30% molar percentage of the ionizable cationic lipid; the lipid composition further comprises a phospholipid; the lipid composition comprises a molar percentage of phospholipids of about 8% to about 23%; the lipid composition further comprises a steroid or a steroid derivative; the molar ratio of the therapeutic agent to the total lipids in the lipid composition is about 1:1, 1:10, 1:50, or 1:100 or less; the therapeutic agent comprises a polynucleotide; and the molar ratio of nitrogen in the lipid composition to phosphate in the polynucleotide (N / P ratio) is about 20:1 or less; the lipid composition is characterized by a polydispersity index (PDI) of about 0.2 or less, as determined by dynamic light scattering (DLS); the lipid composition is characterized by an average diameter of about 200 nanometers (nm) or less, as determined by dynamic light scattering (DLS); the lipid composition is characterized by a lipid fusion percentage of at least about 5%, 6%, 7%, 8%, 9%, or 10%, as determined by a fluorescence resonance energy transfer (FRET)-based assay; or the therapeutic agent comprises a polynucleotide, a polypeptide, a protein, or a combination thereof; at least about 85% of the therapeutic agent is encapsulated in particles of the lipid composition; or the SORT lipid is present in the composition in an amount sufficient to achieve a therapeutic effect at a dose of the therapeutic agent lower than that required in a reference lipid composition; the therapeutic agent is present in the composition at a dose of about 2 milligrams per kilogram of subject body weight or less; the therapeutic agent is present in the intravenous composition at a dose of about 1.0, 0.5, 0.1, 0.05, or 0.01 mg / kg or less of the subject's body weight; the therapeutic agent is present in the aerosol composition at a dose of 1.0, 0.5, 0.1, 0.05, or 0.01 mg / kg body weight or less; the therapeutic agent is present in the intravenous dosage form at a concentration of about 5 or 2 milligrams per milliliter or less; the lipid composition comprises about 5% to about 65% molar percentage of the SORT lipids, optionally the lipid composition comprises about 20% to about 65% molar percentage of the SORT lipids, or the SORT lipid is a permanent cationic lipid, an ionizable cationic lipid, a zwitterionic lipid, or an anionic lipid; 10. The composition of any one of claims 1, 3, and 5.

16. The phospholipid is ethylphosphocholine, the lipid composition comprises about 15% to about 46% molar percentage of the steroid or steroid derivative; the lipid composition is characterized by an average diameter of about 150 nanometers (nm) or less as determined by dynamic light scattering (DLS), and optionally, the lipid composition is characterized by an average diameter of about 100 nanometers (nm) or less as determined by dynamic light scattering (DLS); or the steroid or steroid derivative is cholesterol, The SORT lipid has the structural formula: and During the ceremony: L is a bond or a linker; R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group; and R', R'', and R''' are each independently alkyl (C≦6) or substituted alkyl (C≦6) or The SORT lipid has the structural formula: and During the ceremony: R 1 and R 2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted version of either group; and R 3 , R 3 ', and R 3 '' are each independently an alkyl (C≦6) or substituted alkyl (C≦6) That is, 16. The composition of claim 15.

17. 16. The composition of claim 15, wherein the SORT lipid comprises a counterion to the permanent cationic moiety.

18. 16. The composition of claim 15, wherein the SORT lipid is an alkylated phosphocholine.

19. The SORT lipid has the structural formula: wherein L is a bond or a linker; Z + is a positively charged moiety; and X - 16. The composition of claim 15, wherein is a counterion.

20. The SORT lipid has the structural formula: wherein R 1 and R 2 each independently represents an optionally substituted C 6 -C 24 Alkyl or optionally substituted C 6 -C 24 16. The composition of claim 15, wherein the alkyl group is alkenyl.

21. The SORT lipid has the structural formula: and During the ceremony: R 1 and R 2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R', R'', and R''' are each independently C<6 alkyl or substituted C<6 alkyl; and X - is a monovalent anion, 16. The composition of claim 15.

22. L, and During the ceremony: p and q are each independently 1, 2, or 3; and R 4 may be substituted C 1 -C 6 is alkyl, 16. The composition of claim 15.

23. The SORT lipid has the structural formula: and During the ceremony: R 1 and R 2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R 3 , R 3 ', and R 3 each independently is C≦6 alkyl or substituted C≦6 alkyl; R 4 is C≦6 alkyl or substituted C≦6 alkyl; and X - is a monovalent anion, 16. The composition of claim 15.

24. The SORT lipid has the structural formula: and During the ceremony: R 1 and R 2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R 3 , R 3 ', and R 3 each independently is C≦6 alkyl or substituted C≦6 alkyl; X - is a monovalent anion, 16. The composition of claim 15.

25. The SORT lipid has the structural formula: and During the ceremony: R 4 and R 4 ' are each independently C6-C24 alkyl, C6-C24 alkenyl, or a substituted version of either group; R 4 '' is C ≦24 alkyl, C ≦24 alkenyl, or a substituted version of either group; R 4 "" is C1-C8 alkyl, C2-C8 alkenyl, or a substituted version of either group; and X 2 is a monovalent anion, 16. The composition of claim 15.

26. 16. The composition of claim 15, wherein the SORT lipid comprises a hydrophobically modified phosphate, sulfonate, or carboxylate anion.

27. The SORT lipid has the structural formula: and During the ceremony: R 1 and R 2 are each independently C8-C24 alkyl, C8-C24 alkenyl, or a substituted version of either group; R 3 is hydrogen, C ≦6 alkyl, or substituted C ≦6 alkyl, or -Y 1 -R 4 where: Y 1 is a C ≦6 alkanediyl or a substituted C ≦6 alkanediyl; and R 4 is C ≦8-24 acyloxy or substituted C ≦8-24 acyloxy; 27. The composition of claim 26.

28. 17. The composition of claim 16, wherein the therapeutic agent comprises a polypeptide or protein.

29. 6. The composition of any one of claims 1, 3, and 5, wherein the therapeutic agent comprises a small interfering ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a microribonucleic acid (miRNA), a primary microribonucleic acid (pri-miRNA), a long non-coding RNA (lncRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), an antisense ribonucleic acid (RNA), a guide ribonucleic acid, a deoxyribonucleic acid (DNA), a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a CRISPR-associated (Cas) protein, or a combination thereof.

30. 16. The composition of claim 15, wherein the N / P ratio is from about 5:1 to about 20:1.

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